Morphological and Molecular Characterization of Cultivated Guinea
Yam Accessions and their Wild Relatives (Dioscorea cayenensis Lam.
complex) from South and Southwest Ethiopia.
Candidate: Wendawek Abebe Mengesha
A Thesis Submitted to the School of Graduate Studies of Addis Ababa
University in partial fulfilment of the requirement for the Degree of
Doctor of Philosophy in Biology (Applied Genetics Stream).
March, 2008
Morphological and Molecular Characterization of Cultivated Guinea
Yam Accessions and their Wild Relatives (Dioscorea cayenensis Lam.
complex) from South and Southwest Ethiopia.
Candidate: Wendawek Abebe Mengesha
A Thesis Submitted to the School of Graduate Studies of Addis Ababa
University in partial fulfilment of the requirement for the Degree of
Doctor of Philosophy in Biology (Applied Genetics Stream).
March, 2008
2
Acknowledgments
This work would not have been completed without the support and encouragement of
many people and Institutions. I would like to extend my deepest gratitude to my
supervisors: Prof. Sebsebe Demissew, Prof. Inger Nordal and Dr Paul Wilkin for their
constructive advice, guidance and support through out my study. My sincere thanks go to
Prof. Inger Nordal and Dr Paul Wilkin for their hospitality while I was away from my
home country in Oslo and London respectively, and Prof. Sebsebe Demissew for his
consistent encouragement through out the study period.
I would also like to thank my home institution Dilla University for granting the study leave
during my PhD training. My special thanks go to the Norwegian Programme for
Devolopment, Research and Education (NUFU) for supporting the Project 53/03
“Biodiversity of Eastern Africa (lilies, orchids and sedges) taxonomy, conservation and
use” a collaborative project between Addis Ababa University and the University of Oslo
that covered most expenses associated with my study and the Bentham-Moxon Trust for
funds to cover the laboratory costs while in London. The local people in all the collection
sites are also duly acknowledged for their willingness and kindness while collecting
samples from their home garden.
I am also very grateful to Dr Mike Fay for his valuable comments and guidance on AFLP
and Microsatellite studies and for allowing me to use his laboratory at Jodrell, Kew. I also
want to extend my deepest gratitude to Dr Rhian Smith for her guidance during the lab
work in the Jodrell. My sincere thanks also go to Mr Jeffrey Josseph (for technical support)
and Dr Christian Lexer and Mrs.Clarisse Palma-Silva (for their valuable comments on
microsatellite data analysis and interpretation). All the staffs at Jodrell lab (Conservation
Genetics and molecular systematic sections) also deserve special thanks as they were very
kind and helpful during my stay in London.
I would like to extend my sincere thank to Prof. Glenn-Peter Sætre, Dr Anne Krag
Brysting (both University of Oslo) and Dr Kifle Dagne (Addis Ababa University) for their
valuable comments on the draft thesis. I would also like to thank Dr Tesfaye Awas and his
family for their encouragement and hospitality during my stay in Oslo. Dr Tesfaye Awas
3
also needs acknowledgment for his contribution in converting the coordinates GPS data
into maps. Ato Abel Gizaw should also be acknowledged for his encouragement during the
write-up phase and for his contribution in editing and formatting tables and figures.
Technical support from Ato Samson Tilahun, Ato Anteneh Tesfaye, Ato Girmaye Atsbeha
and Ato Ejigu Belay during field work is highly appreciated. I would also like to express
my appreciation and sincere thank to my family for the love, determination and patience
they showed while I was focussing on my study.
My sincere thanks also go to staff members of the following institutions: Department of
Biology, Addis Ababa University; Institute of Biology, University of Oslo; National
Herbarium, Addis Ababa University, Royal Botanic Grden, Kew and the Ethiopian
Students Union in Oslo.
Last but not least I would like to thank the following persons who have helped me in one
way or another during the study period: Lakew Abebe (with his family), Kidane Fanta,
Worku Kassa, Yemisrach Adamu, Dr Mulugeta Kebede, Dion Devey, Messay Mulugeta,
Aklilu Ketema, Henoke Nugussie (with his family), Mesfin Tefera and Imalka
Kahandawala.
4
Table of contents
pages
TABLE OF FIGURES
................................................................................................................................ 7
LIST OF TABLES
................................................................................................................................ 8
LIST OF APPENDICES...................................................................................................................................9
ABSTRACT..................................................................................................................................................... 10
1. INTRODUCTION....................................................................................................................................... 12
2. BACKGROUND..........................................................................................................................................15
2.1 BRIEF DESCRIPTION OF THE FAMILY DIOSCOREACEAE..................................................................... 15
2.2 THE GENUS DIOSCOREA.......................................................................................................................16
2.3. ORIGIN AND DISTRIBUTION OF YAMS.................................................................................................. 20
2.4 DOMESTICATION OF YAMS................................................................................................................... 23
2.5. ECONOMIC IMPORTANCE OF YAMS.....................................................................................................26
2.6 CULTIVATION OF YAMS.........................................................................................................................29
2.7 REPRODUCTIVE BIOLOGY OF GUINEA YAMS...................................................................................... 32
2.8 CYTOGENETIC STUDIES......................................................................................................................... 34
2.9. TAXONOMIC AND PHYLOGENETIC RELATIONSHIPS BETWEEN THE SPECIES IN THE STUDY GROUP 35
2.9.1. The Cultivated Guinea Yams: D. rotundata and D. cayenensis................................................. 36
2.9.2. Wild Yams: Dioscorea praehensilis and Dioscorea abyssinica..................................................38
2.9.3 Phylogenetic relationships between wild and cultivated species..................................................41
2.10 STUDIES ON YAMS IN ETHIOPIA..........................................................................................................42
2.11. STATEMENT OF THE PROBLEM AND OBJECTIVES OF THE STUDY................................................... 45
2.11.1 Statement of the problem............................................................................................................. 45
2.11.2. Objectives of the study................................................................................................................ 47
2.11.2.1 General objectives.................................................................................................................................47
2.11.2.2 Specific objectives................................................................................................................................. 47
3. MATERIALS AND METHODS................................................................................................................48
3.1 MORPHOMETRY...................................................................................................................................48
3.1.1 The plant material..........................................................................................................................48
3.1.2 Morphological characters..............................................................................................................49
3.1.3 Data collection and analysis.......................................................................................................... 50
3.2 AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP)............................................................... 52
3.2.1 The plant material..........................................................................................................................52
3.2.2 DNA extraction and purification...................................................................................................54
3.2.3 AFLP analysis................................................................................................................................ 55
3.2.4. Data collection and statistical analysis........................................................................................ 56
3.3 MICROSATELLITES.............................................................................................................................. 57
3.3.1 Plant material.................................................................................................................................57
5
3.3.2 DNA extraction and microsattelite markers................................................................................. 57
3.3.3. PCR amplification........................................................................................................................ 57
3.3.4 Detection and analysis of PCR products......................................................................................58
3.3.5 Data collection and analysis..........................................................................................................59
3.3.5.1 Data collection......................................................................................................................................... 59
3.3.5.2 Taxonomic relationships between the taxa............................................................................................. 61
3.3.5.3 Genetic diversity.......................................................................................................................................61
3.3.5.4 Population structure............................................................................................................................... 62
4. RESULTS....................................................................................................................................................63
4.1 MORPHOMETRY.....................................................................................................................................63
4.2 AFLP ANALYSES................................................................................................................................... 67
4.2.1 Overall genetic structure of the populations.................................................................................67
4.2.2 Taxonomic delimitation and genetic relationships among the taxa............................................ 67
4. 3. MICROSATELLITE MARKERS..............................................................................................................73
4.3.1 Genetic diversity and population structure of Dioscorea species.................................................73
4.3.1.1. Allelic variation at microsattelite loci.................................................................................................. 73
4.3.1.2 Genetic variation within population......................................................................................................75
4.3.1.3 Population genetic structure.................................................................................................................. 76
4.3.1.4. Genotypic structure and deviation from Hardy-Weinberg equilibrium........................................ 78
4.3.1.5. Genetic relationships among the three populations............................................................................80
4.3.2. Genetic relationships of the wild and cultivated Guinea yam species........................................ 82
4.3.3. Taxonomic relationships among the three taxa.......................................................................... 83
5. DISCUSSION...............................................................................................................................................86
5.1 TAXONOMIC RELATIONSHIPS IN THE STUDY GROUP: ONE SPECIES, DIFFERENT SPECIES OR A
SPECIES COMPLEX?......................................................................................................................................86
5.2. TOTAL GENETIC DIVERSITY AND LEVEL OF POLYMORPHISM........................................................... 88
5.3 LEVEL OF HETEROZYGOSITY................................................................................................................90
5.4. POPULATION STRUCTURE....................................................................................................................92
5.5 THE IMPLICATIONS FOR YAM DOMESTICATION AND CONSERVATION IN ETHIOPIA......................... 94
6. CONCLUSIONS AND RECOMMENDATIONS.................................................................................... 96
6.1 CONCLUSIONS........................................................................................................................................ 96
6.2. RECOMMENDATIONS............................................................................................................................ 97
7. REFERENCE.............................................................................................................................................. 98
8. APPENDICES...........................................................................................................................................108
6
Table of figures
pages
Figure 1. Relationships of sections of Dioscorea (Burkhill, 1960)...............................................................19
Figure 2. Origin and distribution of Dioscorea species (Degras, 1993)..................................................... 22
Figure 3. Illustration of a typical Dioscorea leaf showing the measurements of leaf characters............. 51
Figure 4. Map of Ethiopia showing the collection sites of Dioscorea accessions used in this study.........54
Figure 5. The PCR products as revealed by electrophoreses on 1.5 % agarose gel..................................59
Figure 6. Microsatellite electropherogram for the locus Da1A01 as revealed by GENESCAN and
GENOTYPER 3.6 software............................................................................................................................ 60
Figure 7. UPGMA cluster derived from a similarity matrix of 40 accessions of Dioscorea based on
morphological characters............................................................................................................................... 64
Fig 8. Figure 8. Scatter plot showing the first and second axis of the PCA based on morphological data
for 40 individuals of Dioscorea....................................................................................................................... 65
Figure 9. 3-D plot of the first three axes obtained by principal PCA analysis of morphological data
among 40 individuals Dioscorea species........................................................................................................ 66
Figure 10. UPGMA cluster derived from a similarity matrix of 48 accessions of the Dioscorea
cayenensis complex from SW Ethiopia using AFLP …………………….………………………………67
Figure 11. Scatter plot showing the first and second axis of principal coordinate analysis of AFLP data
for 46 individuals of Dioscorea ……………………………………………………………………………68
Figure 12. Three principal axes of variation obtained by principal coordinate analysis of AFLP data
among 46 individuals of the Dioscorea cayenensis complex from SW…………………………………. 69
Figure 13. Scatter plot showing the first and second coordinate axis of principal coordinate analysis of
AFLP data from 43 individuals of Dioscorea excluding D. bulbifera and D. schemperiana…………..70
Figure 14. Distribution of rare and frequent AFLP fragments in each of the taxon as an estimate of
genetic
divergence
among
46
individuals
of
Dioscorea
cayenensis
complex
from
SW
Ethiopia …………………………………………………………………………………………………………
….71
Figure 15. The distribution of observed allele size (number of bp) at each locus and among the three
populations....................................................................................................................................................... 74
Figure 16. Allelic diversity per locus for each population based on the measure of allelic richness (R).75
Figure 17. Neighbour joining (NJ) tree inferred from allelic frequency data of microsatellite data for
59 individuals of Dioscorea species................................................................................................................ 81
Figure 18. A comparison of allelic diversity per locus in wild and cultivated accessions of SW Ethiopian
the D. cayenensis complex based on the measure of allelic richness...........................................................82
Figure 19. A dendrogram derived from a similarity matrix of 61 accessions of Dioscorea using
microsatellite markers.....................................................................................................................................84
Figure 20. Scatter plot showing the first and second axis of Principal Coordinate Analysis of
microsatellite data for 61 individuals of the D. cayenensis complex ……………………………………84
7
List of Tables
Table 1. The major sections of the genus Dioscorea based on morphological
characteristics of the
species............................................................................................................................................................... 19
Table 2. Mean annual production of yam for the period 1990 to 2005 (United Nations Food and
Agricultural Organization.............................................................................................................................. 26
Table 3. Range of nutritional values of yams (nutrients in 100 g of edible tuber) ................................... 28
Table 4. List of accessions of cultivated and wild yams sampled in the field and used for
mormphometric analysis.................................................................................................................................48
Table 5. List of morphological characters used in morphometric analysis............................................... 50
Table 6. List of accessions of cultivated and wild yams used in AFLP analysis........................................ 52
Table 7. Selective primer sequences used in the AFLP analyses................................................................ 56
Table 8. Characteristics of the 9 microsatellite loci used in this study with number of alleles and allele
size range observed in the study population ................................................................................................ 58
Table 9. Percentage of polymorphic fragments, total number of fragments analysed per individual and
per population in the three taxa..................................................................................................................... 72
Table 10. The observed number of alleles per locus and population..........................................................73
Table 11. Allelic variability at the seven SSR loci in the study populations ............................................. 76
Table 12. Relative measurements of genetic differentiation among populations in the study group......78
Table 13. Expected and observed heterozygosity (He and Ho) and Fixation indexes (FIS) per locus and
population at the seven microsattelite loci.................................................................................................... 79
Table 14. Gene diversity (expected level of heterozygosity) per locus and population of wild and
cultivated accessions........................................................................................................................................83
8
List of appendices
Appendix 1. List of the major morphological characters used to identify the voucher
specimens.
Appendix 2. Microsatellite electropherogram showing allelic distribution at each of
the 7 loci studied as revealed by GENESCAN and GENOTYPER 3.6
software.
Appendix 3. The raw data for morphometric analysis.
Appendix 4. The raw data for AFLP analyses.
Appendix 5. Microsatellite codominant data used to estimate population genetics
parameters.
Appendix 6. Microsatallite data used to infer population genetic structure.
Appendix 7. Microsatellite presence/absence data used to infer taxonomic
relationships among the taxa using NTYSYS.
9
Abstract
Yams (Dioscorea species L.) are among the most important of the tuber crops mainly
cultivated in the tropics. They are also an important source of diosgenein, a starting
material for the industrial production of sex hormones and steroidal drugs with
pharmaceutical properties. Despite their cultural and economic importance, there are
taxonomic confusions in the groups called Guinea yams that belong to the D. cayenensis
complex. Identification of living or dried specimens using the currently used classification
scheme can be extremely difficult. Establishing the taxonomic identity and understanding
the systematic and genetic relationships among the accessions of Guinea yams and their
wild relatives is vital to the conservation and management of the crop. Therefore, the major
objectives of this study were to evaluate the existing taxonomy and to determine the
amount and distribution of genetic variation within Dioscorea cayenensis complex in
Ethiopia. Collections of plant material were conducted between the months of July and
September 2005 and 2006 from different localities in South and Southwestern parts of
Ethiopia.
Morphometric analyses were carried out based on a similarity matrix constructed using 26
morphological characters on 40 accessions of Dioscorea cayenensis complex . The results
of the cluster and ordination analyses revealed that the wild and cultivated Guinea yams
are closely related. None of the UPGMA clusters entirely contained those accessions
considered as discrete taxa according to the existing classification system.
The three primer combinations used in the Amplified Fragment Length Polymorphism
(AFLP) analyses generated 158 scorable bands, with an overall polymorphism of 78%.
Ordination and cluster analyses of AFLP data failed to produce any clear species boundary
between the species within D. cayenensis complex. The average genetic similarity between
the accessions ranged from 60 % to 100 %. The first, second and third principal
coordinates axes cumulatively account 77.5 % of the total variation. AFLP analyses also
revealed a higher genetic divergence among cultivated Guinea yams accessions of the
Sheko cultivars.
10
Estimates of population parameters using microsatellite or simple sequence repeat (SSR)
markers were made by studying 7 loci. The total number of alleles amplified for the 7 loci
were found to be 60, with an average of 8.6 alleles per locus. Analyses of the data
indicated that Guinea yams and their wild relatives in the study area displayed a
tremendous genetic diversity. The wild forms exhibited greater allelic diversity than the
cultigens. Contrary to what is expected in vegetatively propagated crops, none of the seven
loci studied showed a significant excess of heterozygotes. The levels of heterozgosity
found in the study group were, in most cases lower than expected. Analyses of the
taxonomic status using microsattellite data also revealed comparable results with both
morphometry and AFLP. The accessions tended to group based on their geographical
origin rather than their supposed taxonomic identity.
In the present studies, the phenograms and scatter plots based on morphological, AFLP and
microsatellite markers failed to produce a clear partitioning of the study individuals studied
into discrete taxa according to the existing classification system. Therefore, we believe that
at least the wild or managed populations and cultivated Guinea yams of South and
Southwest Ethiopia form a single taxonomic entity. It also appears that the Sheko
population displayed the greatest genetic diversity. From a conservation perspective, it is
important that both the range of cultivars and the diversity within them is protected both
in-situ in the Sheko region, and perhaps also in ex-situ in selected areas in gardens. Future
studies must be undertaken at the population scale and in a broad range of ecosystems, so
as to take the diversity of each of the yams currently regarded as distinct species into
account.
Key words: Guinea yams, morphometry, AFLP, microsatellite, taxonomic status,
Genetic diversity.
11
1. Introduction
Yams rank as the world’s fourth most important tuber crop in economic terms (Mignouna
et al., 2005), after potatoes (Solanum tuberosum L.), Cassava (Manihot esculenta Crantz),
and Sweet potatoes (Ipomoea batatas (L.) Poir.). Yams are cultivated in most tropical
countries, but especially in West Africa, which produces over 90% of the world’s output
(Mignouna and Dansi, 2003) and they are the staple carbohydrate source of millions.
Worldwide at least 50-60 species of Dioscorea of the more than 600 known (Govaerts &
Wilkin, 2007) are recognized to be cultivated or wild-harvested, for food or pharmaceutical
purposes (Coursey, 1967; Craufurd et al., 2001)
The African domesticates known as Guinea yams (D. cayenensis Lam.-D. rotundata Poir.
complex) are one of the most important, preferred and widely planted tuber crops in the
tropics (Mignouna and Dansi, 2003), although D. alata L., a cultigen of Asian origin, is
also widely grown in Africa. They are the major source of carbohydrate in the”yam zone”
of West Africa (Coursey, 1967). Elsewhere in Africa, there are pockets of extensive yam
cultivation amid a widespread wild or semi-wild plants harvested as famine food when
cereal crops run short or fail. Due to their importance in the diet of people in Africa,
Guinea yams have been the subject of many studies by researchers in a number of
disciplines including systematics, plant breeding, pathology and genetics (see e.g. Coursey,
1967). The primary goal of these researchers has been to look for the ancestral species
from which the cultivated yams have originated, both to understand the patterns of
variation and processes of domestication, and to apply this knowledge to yam breeding and
improvement. Guinea yams have been proposed to result from a process of domestication
of wild yams of Dioscorea sect. Enantiophyllum Uline by African farmers (Mignouna and
Dansi, 2003; Dumont et al., 2006). Although currently practiced by relatively few farmers,
yam domestication is still ongoing, especially in places like Southwest Ethiopia.
However, there is still no agreement over the systematics or relationships within the
species complex; the number of species and the names which should be applied to them
have never been adequately clarified and determined. i.e., the delimitation of the D.
cayenensis-D. rotundata complex and the relationships within the complex are still far
from clear. Identification of living or dried specimens in the D. cayenensis.-D. rotundata
12
complex using the current taxonomic literature is extremely difficult, as was experienced
when trying to name Ethiopian material.
The taxonomy and evolution of the Guinea yams remain controversial, partly because of
the continuous variation in morphological descriptors observed in the cultivated and wild
species. These species exhibit considerable morphological polymorphism, high plasticity
and predominant vegetative reproduction (Terauchi et al., 1992). For example recent
studies made on Guinea yams from Southwest Ethiopia have indicated that leaf shapes
within an individual plant vary considerably (Hildebrand, 2003). There are also reports
which indicate that wild individuals identified as Dioscorea praehensilis Benth. or D.
abyssinica Hochst. ex Kunth or D. burkilliana Miège can directly “become” D. rotundata
or D. cayenensis following domestication without any genetic change (Mignouna et al.,
2005).
Conclusions drawn from earlier studies of relationships among Guinea yams based on
morphometry, chemotaxonomy, cytology, isozyme and molecular analysis were not
consistent (Ramser et al., 1997). For example, a numerical taxonomic analysis of 97
cultivars of Guinea yams (D. rotundata and D. cayenensis accessions), based on 75
morphological descriptors resulted in a phenetic tree with two main trunks interconnected
with many anastomosing branches. The authors suggested that all the 97 cultivars
investigated belong to a single highly evolved species (Martin and Rhodes, 1978). In
contrast, a similar study by Onyilagha and Lowe (1985) on 22 cultivars of D. rotundata
and D. cayenensis clearly separated the accessions as two distinct species. Using RFLP
(Restriction Fragment Length Polymorphism) markers Terauchi et al., (1992) failed to
evidently discriminate between the various species of Guinea yams. The authors proposed
that species referred to Guinea yams are all closely related. However, recent studies by
Scarcelli et al. (2005) using AFLP distinguished three groups of Guinea yams belonging to
D. cayenensis-D. rotundata complex, D. abyssinica and D. praehensilis in Benin.
Furthermore, cluster analysis based on RAPD (Randomly Amplified Polymorphic DNA)
and double stringency PCR (DS-PCR) data discriminate the cultivars classified as D.
cayenensis from D. rotundata (Mignouna et al., 2005). The authors proposed that D.
cayenensis should be considered as a taxon separate from D. rotundata.
13
Establishing the taxonomic identity of germplasm and understanding the systematic
relationships among crops are vital to the management of genetic resources and the
utilization of accessions (Bretting and Widrlechner, 1995). Yam cultivation systems in
Ethiopia and East Africa have not been studied as well as their West African counterparts.
Recent studies conducted in South and Southwest Ethiopia have revealed that the local
community has a strong tradition in cultivating and domesticating various species of yams
with wide genetic bases (Sebsebe Demissew et al., 2003; Hildebrand et al., 2002).
However, this excellent local knowledge of yam cultivation and domestication is beginning
to deteriorate as farming practice in the area reorients towards cash crops and coffee
plantation (Hildebrand et al., 2002).
The major objectives of this study were to evaluate the current taxonomic classification of
Guinea yams and to determine the amount and distribution of genetic variation in the
Ethiopian materials collected from south and southwestern part of the country, as a
prerequisite to devise a sound conservation strategy. Both morphological and molecular
(AFLP and Microsatellite) data were gathered using cultivated accessions of Guinea yams
and their wild relatives collected from South and Southwest Ethiopia. We adopted the “D.
cayenensis complex” (used by some authors e.g. Hildebrand et al., 2002) as a provisional
name for the set of sub-Saharan yam species whose relationships are currently being
examined: D. cayenensis, D. rotundata (in some literature both species recognized as
D.
cayenensis-D. rotundata complex), D. abyssinica, and D. praehensilis, plus D. sagittifolia
Pax, which occurs rarely in Ethiopia (Miège and Sebsebe Demissew, 1997) and was not
seen in the areas studied.
14
2. Background
2.1 Brief description of the family Dioscoreaceae
Members of the family Dioscoreaceae probably appeared 165 to 130 million years ago, i.e.
during the Cretaceous period, together with other primitive angiosperms (Coursey, 1976).
Recent studies based on analyses of rbcL sequence data reported 116 million years as the
stem node age of the family Dioscoreaceae (Jansen and Bremer, 2004). The genetic
isolation of the African Dioscoreaceae probably date back to Miocene (60-20 millions
years ago) when the desertification of what is now South Western Asia occurred (Coursey,
1976). The diversification of Dioscorea section Enantiophyllum in Africa took place
during the last 40 millions years. All representative members of this section occur in
mainland Africa, but not in Madagascar (except the introduced D. minutiflora Engl.)
(Burkill and Perier de la Bathie, 1950), which was separated from the African continent,
around 40 million years ago. Hence, the evolution of African yams of the section
Enantiophyllum, appears to be relatively recent, which may explain the incomplete
speciation and the large reserve of variability some of them still appear to have.
The family Dioscoreaceae is classified under the monocotyledons. However, some features
in yams such as the presence of a second non-emergent cotyledon and reticulate-venation
of the leaves are similar to those of certain dicotyledonous plants (Purseglove, 1972). This
has led to the suggestion that the genus Dioscorea might have been derived from plant
forms that occurred before the differentiation of monocots and dicots (Degras, 1993), or
that it was part of the first diverging lineage of monocot evolution (Dahlgren et al., 1995).
However, molecular analyses have confirmed that it is nested well within the monocots in
the Dioscoreales (Chase et al., 1993; Caddick et al., 2002a).
According to Méige and Sebsebe Demissew (1997) the family includes about seven
genera (Borderea Méige., Dioscorea L., Epipetrum Phil., Testudinaria Salisb. ex Burch,
Rajania L., Stenomeris Planch. and Tamus L.) with the greatest diversity occurring in
Central and South America, Indo-Malaysia, Micronesia and Madagascar. Representatives
also occur in Eastern Europe and Africa, but here the diversity is relatively low. Of the
seven genera recognized only one, Dioscorea, is represented in tropical Africa (Sebsebe
Demissew et al., 2003). A more recent classification (Caddick et al., 2002a) recognized
15
only 4 genera (Dioscorea, Stenomeris, Tacca J.R. & G. Frost (previously Taccaceae) and
Trichopus Gaertn.) within the family. The dioecious Dioscoreaceae genera; Borderea,
Epipetrum, Nanarepenta, Rajania, Tamus and Testudinaria Salisb. ex Burch. were shown
to be nested within Dioscorea and are therefore proposed sunk into it (Caddick et al.,
2002a; Caddick et al., 2002b).
Species of Dioscoreaceae are most frequently encountered as climbers which perennate by
rhizomes or tubers in forest margins and more open habitats (Wilkin, 2001).
2.2 The genus Dioscorea
The genus includes more than 600 species (Govaerts and Wilkin, 2007). In Ethiopia and
Eritrea ca 11 species of Dioscorea are recognized so far (D. quartiniana A. Rich, D.
dumetorum Pax., D. cochleari-apiculata De Wild., D. gillettii Milne-Redh., D. bulbifera L.,
D. schimperiana Kunth., D. alata, D. abyssinica, D. cayenensis-D. rotundata complex, D.
sagittifolia Pax. and D. praehensilis) (Sebsebe Demissew et al., 2003). One of the species,
D. gillettii., is a near-endemic occurring in Southeast Ethiopia and Northern Kenya
bordering Ethiopia. The remaining species are widespread in sub-Saharan Africa. Some of
the species, such as D. cayenensis-D. rotundata complex and D. abyssinica occur both in
the wild and in cultivations, and others such as D. quartiniana, D. dumetorum, D.
cochleari-apiculata and D. schimperiana occur only in the wild (Sebsebe Demissew et al.,
2003). Dioscorea alata has never been found in the wild. It is an introduction from
Southeast Asia and may have developed from crosses and domestication involving the
Asian species D. hamiltonii Hook. f. and D. persimilis Prain and Burkil (Purseglove,
1972). Dioscorea bulbifera is common in the wild in Asia and Africa, and the different
forms of D. bulbifera have been named as separate species by some authors (Coursey,
1967).
In tropical Africa the genus Dioscorea includes twining or climbing herbs, often prickly
below or sometimes unarmed. The flowers are small and unisexual, the plants are
dioecious with an extremely irregular production of male and female flowers, which are
pollinated by insects. The male inflorescences are spicate, racemose, or rarely cymose,
axillary or forming panicles at the ends of leafless branches. Male flowers have
campanulate to spreading tepals and six stamens, either all fertile or three reduced to
16
staminoides. The female inflorescences are spicate and axillary. Female flowers have
tepals similar to the male ones. The capsules are triangular or deeply three-lobed in crosssection dehiscing with three valves, and with 1-2 seeds in each locule (Méige and Sebsebe
Demissew, 1997). The seeds are most often winged and usually go through a dormancy
period of three to four months before germination can occur. In the cultivated forms seed
production is rare, so they are vegetatively propagated using the basal nodal region of the
tuber or the bulbils (Degras, 1993). The leaves are petiolate, often cordate at the base, and
entire to lobed (except for D. dumetorum and D. quartiniana which have trifoliate or
pentafoliate leaves), and of an arrangement either opposite or alternate with axillary buds
(Degras, 1993).
Members of the genus Dioscorea have usually a thin twining stem which allows the plants
to climb. The direction of the stem twining (clockwise or anticlockwise) is used as one of
the major taxonomic characters, to classify the species within the genus into different
sections (Table 1).
Dioscorea schimperiana and the members of the D. cayenensis complex usually produce a
single annual tuber, which varies in size, shape and weight, depending on species/cultivar
and growing conditions. The colour of the tuber flesh also varies between species and
cultivars. Some members of the genus, such as D. alata also produce bulbils (aerial tubers)
on the leaf axils which could weigh from ca 20 to 100g. Others such as D. bulbifera
produce a corresponding structure which can weigh up to one kg (Degras, 1993).
Tuber morphology, stem twining direction, dioecy, and fruit/seed wing
shape are among the most important characters in the systematics of
Dioscorea. The first taxonomic treatments of Dioscorea involving a large
number of species were those of Kunth (1850) and Uline (1898).
Knuth recognized ca. 600 species and divided them into four subgenera
based on seed wing position, and then into 60 sections. However, later
it was proposed that many of Knuth’s infrageneric taxa are clearly
17
para- or even polyphyletic. The taxonomic ideas of Knuth were to some
extent refined and improved by Burkill (1960). In his infrageneric
classification of the Old World taxa, based on seed characters,
underground organ morphology, and morphology and development of
male inflorescence, Burkill avoided the rank of subgenus, and divided
some 220 species into 23 sections (Fig. 1). Since 1960, the genus has
been the subject of piecemeal floristic studies (e.g., Méige 1968; MilneRedhead, 1975; Tellez and Schubert, 1994; N’Kounkou. 1993; Méige
and Sebsebe Demissew, 1997; Ding and Gilbert, 2000). The only
complete taxonomic treatment was that of Huber (1998), in which the
Knuth/Burkill system of classification was recapitulated, with all of the
dioecious taxa of Dioscoreaceae included in subfamily Dioscoreoideae as
‘‘genera and genus-equivalent sections’’ (in Wilkin et al., 2005)
18
Figure 1. Relationships of sections of Dioscorea (Burkhill, 1960)
Some of the characteristics and representative examples of the major sections of the genus
Dioscorea are presented in Table 1 (Alexander and Coursey, 1969). The section
Enantiophyllum is the largest in terms of the number of species
Table 1. The major sections of the genus Dioscorea based on morphological characteristics of the
species (Alexander and Coursey, 1969)
19
Sections
Major characteristics
Representative species
Enantiophyllum
• usually single tuber
• twine to the right
• winged stems
•occasional bulbils
Lasiphyton
•cluster of medium sized
D. alata
D. cayenensis
D. rotundata
D. preahensilis
D. abyssinica
D. hispida. Dennst.
tubers
• twine to the left
• large thorns on stems
D. dumetorum
D. pentaphylla L.
D. quartiniana
Opsophyton
•aerial bulbils
• twine to the left
D. bulbifera
Combilium
•large
number
of
individually small tubers
• twine to the left
•small and very toxic bulbils
• twine to the right
• small tubers
• twine to the left
• spineless stem
D. esculenta (Lour.)
Burkill
Macroura
Macrgynodium
D. sansibarensis. Pax.
D. trifida L.f.
2.3. Origin and distribution of yams
The generic name (Dioscorea) of yams and the family name Dioscoreaceae was given in
honour of Dioscorides, the 1st century Greek botanist and physician, who used yams for
medicine, as well as for food. The common name “yam” was a derivation of a word from a
West African language (“nyami”), picked up by Portuguese slave traders. While the
Portuguese were watching indigenous people digging up the yam tuber, they asked what
they were used for. Failing to understand the question fully, they replied that it was
"something to eat", nyami in the local dialect (Guinea). This became “inhame” in
Portuguese,
then
“igname”
in
French,
and
“yam”
in
English
(www.innvista.com/health/foods/vegetables/yams.htm).
Burkill (1960) thought that the family Dioscoreaceae arose in part of Laurasia which is
now in Southeast Asia between 160-130 million years ago (Burkhill, 1960). From the Far
East, the genus Dioscorea had spread worldwide by the end of the Cretaceous period,
approximately 75 million years ago (Alexander and Coursey, 1969). However, the major
cultivated Dioscorea species appear to have originated from tropical areas in three separate
continents: Africa (the D. cayenensis complex), South East Asia and south Pacific (D.
alata and D. esculenta (Lour.) Burkill), and South America (D. trifida L.f.) (Coursey, 1976;
Dergas, 1993) (Fig. 2). Dioscorea rotundata and D. cayenensis are believed to have
originated in eastern Nigeria and from land tracts adjoining the Niger and Benin rivers in
West Africa (Coursey, 1967). These species were slowly brought eastwards reaching as far
20
as East Africa. There was little or no cultivation of the African species in Asia. In India,
however, D. rotundata has been recently introduced from Africa by the International
Institute of Tropical Agriculture (IITA). The Asiatic yam, D. alata is believed to have
originated in tropical Myanmar and Thailand. Dioscorea alata probably spread from
Southeast Asia to India and across the Pacific Ocean to reach the east coast of Africa about
2000 years ago. Later during the time of the slave trade, both D. alata and D.
rotundata/D.cayenensis were taken from West Africa to the Caribbean and the Americas
where they are now established as important food crops (Craufurd et al., 2001). Dioscorea
bulbifera, characterized mainly by the production of bulbils, is native to both Asia and
Africa, where the wild forms still exist. The cush-cush yam (D. trifida) is the only yam of
tropical American origin to have attained significance as food crop; its production is
currently restricted to the West Indies (Onwueme and Charles, 1994).
In prehistoric times the most widely distributed of the starchy crops were various species
of the genus Dioscorea (Degras, 1993). The past importance of yams to many communities
is shown by the part they still play in socio-religious events (Alexander and Coursey,
1969). Some Dioscorea species are mentioned as supplying medical ingredients in the
earliest Chinese medical documents (before 2000 BC) (Coursey, 1967). The suitability of
yams as food on ships greatly facilitated their distribution through out the world (Coursey,
1967).
21
_______D. alata ______ D. dumetorum _______D. trifida_______ D. esculenta.
_______D. cayenensis/D. rotundata _________ D. bulbifera
Figure 2. Origin and distribution of Dioscorea species (Degras, 1993)
22
2.4 Domestication of yams
Domestication is a set of practices that are applied to edible (or potentially edible) wild
plants that involve their adaptation to the environmental conditions of agriculture. This is
achieved by changing the genetic equilibrium of the initial populations and enhancing their
valuable traits through selection. Many cultivars used for domestication purposes are
clones of edible wild forms and a few putative wild forms are probably feral plants that
have escaped from cultivation. Some cultivars are also clones of hybrids between wild
forms and feral or cultivated plants (Lebot et al., 2005)
According to Lebot et al., (2005) the practice of domestication of tuber crops, such as
yams, could be summarized as:
1. Selection of wild genotypes: the domesticator identifies a morphotype and tests its
“chemotype”. If it seems acceptable after chewing the flesh of the underground organ, a
propagule is collected.
2.
Improvement of the environment: the soil where the propagule is planted is well
prepared. Unlike the wild plants, the clone is planted into a considerably modified
environment. This improved environment contributes directly to the ennobled development
of the underground organs.
3. Rejuvenation of the plant: From a perennial and/or herbaceous wild form, regular
vegetative propagation induces a rejuvenation process which leads to an annual cultivar.
Farmers uproot their plants as soon as there is a consumable yield.
According to Coursey (1976) the pre-forest zone extending across Nigeria and Benin was
the first place for domestication of Guinea yams. However, it is not known exactly where
they were first domesticated for cropping under savannah agriculture conditions. Different
arguments indicate that domestication of yams in West Africa is a recent phenomenon
(Dumont et al., 2006).
The starting material for the domestication of Guinea yams are populations of wild
morphotypes that have well developed vegetative organs, sexual vigour, and a small
relatively bitter tuber which is difficult to harvest because it is often long and sometimes
branched or protected by spiny crown roots (Dumont et al., 2006). The physico-chemical
characteristics of their tubers are the most useful traits which are selected and domesticated.
23
In fact, the major differences between cultivated and wild forms are not morphological, but
rather chemical (Lebot et al., 2005). The tuber material or propagules of these wild yams
are usually collected either in the bush (most often near the village) or in the forest during
hunting. These clones are planted into a considerably modified environment. This
improved environment contributes directly to some phenotypic modifications including the
ennobled development of the underground organs (Lebot et al., 2005). Farmers in Benin
try to obtain the desired phenotypic modification in tuber form, size and taste by
maintaining and planting the predomesticated tuber in their home garden for at least three
years (Scarcelli et al., 2006b). During this period the aerial architecture of the plant is
substantially modified. i.e., stem internodes become shorter, with a concomitant equivalent
reduction in stem length. Changes mainly occur on the lower parts of the stem. The
reduction of primary branches at the stem base leads to the development of a large number
of secondary branches that are quickly covered with thick foliage. These transformations
condense the mass of the aerial vegetative organs, thus reducing or eliminating the need for
staking (Dumont et al., 2006). The tuber becomes shorter and thicker, with fewer roots.
The selected wild forms produce a tuber morphologically identical or similar to the
cultivated forms after 3-6 years of cultivation (Scarcelli et al., 2006a). A study in
southwest Ethiopia reported that wild growing yams transplanted to domestic gardens
retain their wild traits for up to 4 years, before they begin to take on traits of the cultivated
forms (Hildebrand, 2003). There are also reports which indicate that the domesticated
plants return to their wild state if they are abandoned (Dumont et al., 2006).
The mechanism underlying the phenotypic modifications observed during the process of
domestication is unknown. A phenotype is the result of the interaction of the genotype and
the environment. A change in the latter can lead to profound modifications in the
phenotype. A genotype can thus have various phenotypes. Transferring wild yams to a
cropping environment subject them to a drastic change of habitat, and these changes might
induce phenotypic modifications observed during the process of domestication (Dumont et
al., 2006).
Different authors consider phenotypic plasticity, epigenetic modifications
(Tostain et al., 2003) or somatic mutations (e.g. Scarcelli et al., 2006a) as possible
explanation for the changes encountered during the domestication practice in yams.
Tubers of the predomesticate obtained after 3-6 years of cultivation in the home garden are
evaluated for their agronomic quality and then multiplied if the farmers are satisfied with
24
the evaluation (Scarcelli et al., 2006b). The aerial morphological traits of the
predomesticate are not of particular importance for selection. If they are used, it is for
identifying a familiar morphotype which is known to present underground organs with an
acceptable chemotype. Since farmers propagate yams vegetatively, they are likely to select
genotypes that allocate more resources to tuber development than to sexual reproduction.
Thus, a decrease in flowering ability could also be considered as one domestication
syndrome trait (Scarcelli et al., 2006a). The farmer also take into consideration some
physiological traits, such as production period, storage, seed potential, and sometimes the
ability to produce large number of tubers. More general criteria relating to the plant’s
biological plasticity and yield potential are also taken into account for selection (Dumont et
al., 2006).
During the process of domestication some farmers collect different tubers with different
genotypes and put them simultaneously into the domestication process. Consequently after
a while, farmers are unable to identify each clone separately. This practice leads to the
cultivation of different clones from unknown origin under the same cultivar. When the
process of domestication is considered as completed by the farmers, they generally mix the
tubers obtained through the process with those cultivars that are found to be alike. It is only
when the shape of the newly domesticated yams tuber does not correspond to existing
varieties that the farmers give them a new name (Chaír et al., 2005).
The process of domestication, in yams could be described as the adaptation of spontaneous
(wild) plants to cultivation constraints without any genetic changes (Scarcelli et al., 2006b).
Since only vegetative propagation is used in yams no genetic changes are expected during
the process of domestication. The genotypes are not modified and so there is no barrier to
sexual reproduction. There is a possibility that reciprocal gene flow would occur between
the cultivated yams and their wild relatives (Dumont et al., 2006). According to Mignouna
and Dansi (2003), although D. praehensilis is the most exploited (in Benin), three species
of wild yams namely: D. abyssinica, D. burkilliana and D. praehensilis are used for
domestication purpose. Although, D. burkilliana is not found in Ethiopia, Hildebrand et al.
(2003) have reported the same findings regarding the species most commonly used for
domestication purposes in Ethiopia. Since the wild yams, D. abyssinica and D.
preahensilis, are principally the result of sexual reproduction (Ayensu and Coursey, 1972),
25
sexual reproduction indirectly contributes to the evolutionary dynamics of yams through
the domestication of wild species of the D.cayenensis complex (Scarcelli et al., 2006a).
2.5. Economic importance of Yams
Worldwide 50-60 species of Dioscorea are known to be cultivated or at least gathered for
food or pharmaceutical purposes. There are however, ca 12 species of economic
significance as food (Coursey, 1967). The most important of these are: D. rotundata
(White Guinea yam), D. alata (Water yam, Winged yam or Greater yam), D. cayenensis
(Yellow yam or Yellow Guinea yam), D. esculenta (Lesser yam, Potato yam or Chinese
yam), D. dumetorum (Bitter yam or Trifoliate yam), D. bulbifera (Aerial potato yam), D.
trifida (Cush-cush yam), D. opposita Thunb. also known as D. batatas Decaisne.
(Cinnamon yam), D. nummularia Lam., D. pentaphylla L., and D. hispida (Craufurd et al.,
2001).
Yams are an important staple food and source of carbohydrate for millions of people in
tropics and subtropics (Craufurd et al., 2001; Hochu et al. 2006). They are also important
medicinally and have ritual and socio-cultural significance (Craufurd et al., 2001). A study
by IFPRI (International Food Policy Research Institute, Washington) indicated that
production of food yams increased by 183% between 1983 and 1996 (in Dumont et al.,
2006). The field performance of yams lags behind demographic growth and the supply has
been increased mainly through an expansion of the cultivation area (Tschannen et al.,
2005). According to the 2005 report by FAO, Africa accounts for nearly 96% of the
world’s yam production. Almost all of the African output is confined to West Africa, with
D. rotundata/ D. cayenensis yams, representing nearly 91% of all yams cultivated (FAO,
2005; Dumont et al., 2006). The total production of yam in Ethiopia was estimated to be
277 metric tons from an area of ca 68000 ha, corresponding to a yield of about 4 tons per
hectare (FAO, 2005) (Table. 2).
Table 2. Mean annual production of yam for the period 1990 to 2005 (United Nations Food and
Agricultural Organization (FAO) (2005)
World
Africa
West Africa
Ethiopia*
Area harvested
(x1000 ha)
3572
3418
3149
68
yield
(Kg/ha)
9694
9708
10088
4065
total production
(x1000 MT)
34355
32874
31388
277
* Figures are mean values for the years 1992 to 2005
26
In West Africa tubers of food yams are consumed in different ways, they are processed
into pounded yam, boiled yam, roasted or grilled yam, fried yam slices, yam balls, yam
chips or yam flakes. Fresh yam tubers could also be peeled, chipped, dried and milled into
flour that can be used to prepare dough (Mahalakshmi et al., 2007). In Ethiopia tubers are
mostly consumed boiled without processing. In some areas however, boiled yam tubers
are pounded and mixed with butter and fermented milk before they are served. The
practice of roasting tubers has also been reported in some localities (Muluneh Tamiru,
2006).
The tubers of food yams, which have a high capacity to store food reserves are regarded
mainly as a source of carbohydrate, some species are nearly as rich in protein as rice or
maize (Hahn et al., 1987). Typically, yam tubers contain 65 to 81% moisture, 16 to 31%
carbohydrate, 1.4 to 3.5 % protein, 0.03 to 1.2 % lipid, (all % of fresh weight) and
important quantities of amino acids (aspartic acid, glutamic acid alanine and
phenylalanine), minerals (Calcium, Phosphorus and Magnesium) and vitamins (Ascorbic
acid, Beta carotene, Thiamine and Riboflavin) (Table 3). Recent study by Muluneh Tamiru
(2006) revealed that the starch content of yam tubers collected from South Ethiopia ranged
from 65.2% to 76.6% of the dry matter, while protein content varied between 6.4% and
13.4%.
27
Table 3. Range of nutritional values of yams (nutrients in 100 g of edible tuber) (FAO, 2005)
Nutrient
Calories
Moisture
Protein
Fat
Carbohydrate
Fiber
Ash
Calcium
Phosphorus
Iron
Sodium
Potassium
Beta carotene
Thiamine
Riboflavin
Niacin
Ascorbic acid
Unit
calories
(%)
(g)
(g)
(g)
(g)
(g)
(mg)
(mg)
(mg)
(mg)
(mg)
(mg)
(mg)
(mg)
(mg)
(mg)
Composition
71-135
65-81
1.4-3.5
0.2-0.4
16.4-31.8
0.1-0.4
0.6-1.7
12-69
17-61
0.7-5.2
8-12
294-397
0.0-0.1
0.01-0.11
0.01-0.04
0.3-0.8
4-18
Yam tubers have been suggested to have nutritional superiority compared to other tropical
root crops. They are reported as a good source of essential dietary nutrients. A few yam
species (mainly the wild forms), however, produce toxic compounds that can cause serious
health complications. In some species poisonous substances, such as oxalic acid, are found
just beneath the skin of the tubers, and could be destroyed by peeling and boiling
(http://www.kew.org/information). In general the tubers with toxic compounds usually
taste bitter and cause vomiting and diarrhoea when large amount are ingested without
proper processing or even eaten raw. In some species of yam (D. dumetorum and D.
hispida), the toxic component has been reported as dioscorine, a toxic alkaloid that could
triggers fatal paralysis of the nervous system, when even fragments of the tuber are
ingested. Similarly, histamine was reported as principal allergen in some plants of the
family Dioscoreaceae, causing mild inflammation and itching. The bitter substances have
been reported to be saponins (in many species of yams) and furanoid-norditerpene group
compounds (in some) (Rajabhandari and Kawabata, 2005).
Yams are not cultivated exclusively for their role as food alone; they are also a rich source
of diosgenin such as sapogenins, the primary precursor of corticosteriods and anabolic
28
drugs (Purseglove, 1985; O'Hair, 1990; Twyford et al., 1990; Craufurd et al., 2001).
Diosgenin is a steroidal sapogenin which is the starting compound for the synthesis of sex
hormones and steroidal drugs with pharmaceutical properties. Diosgenin is known from
several species of Dioscorea (Adam et al., 2002). Approximately 50 species of Dioscorea
are considered to have medicinal value. Among these 50 species only 5 to 7 species (D.
composita Hemsl., Dioscorea deltoidea Wall., Dioscorea elephantipes Engl., Dioscorea
floribunda Mart & Gall., Dioscorea sylvatica Ecklon.) are found cultivated in Asia and
Central America providing diosogenin, which accounts two-thirds of steroid production
(Niño et al., 2006).
Over 50 different steriodal saponins have been discovered and characterized so far. Most
species of Dioscorea contain steroid saponins and sapogenins, such as diosgenin.
Diosgenin is used in the in the synthesis of many steroids which are on the market as antiinflammatory, androgenic, estrogenic and contraceptive drugs. Several pharmacological in
vitro and in vivo assays allowed researchers to characterize various pharmacologically
active steroid saponins in Dioscorea species having cytotoxic, immunomodulating,
antimicrobial, anabolizing, hormonal, anti-osteoportic, anti-inflammatory and anti-allergic
activities (Sautour et al., 2007). Some species of Dioscorea are used in traditional Chinese
Medicine as anticancer agents, cardiocerebrovascular, gastrophaty-protective, curative
agent and anti-rheumatism agents (Sautour et al., 2007).
2.6 Cultivation of yams
All cultivars of root crops are vegetatively propagated and they share a narrow within
clone genetic base. Unlike most crops, root crops are not cultivated for the characteristics
of their sexual organs (fruits and seed), and their flowering is erratic. They have variable
ploidy levels, are predominantly allogamous, highly heterozygous. They are usually
cultivated for the interesting chemical compositions of their underground organs. Some of
these biological characters are not specific to root crops but these species present all of
them together (Craufurd et al., 2001).
Yam tubers have traditionally been classified as root tubers rather than stem tubers.
However, they are modified stems which develop from the hypocotyle, i.e. a short region
of meristematic cells below the cotyledons (Conlan et al., 1995; Craufurd et al., 2001).
29
Cultivated yams are propagated vegetativelly from whole tubers (seed yams), large pieces
of tubers (setts) or increasingly, from minisetts. They can also be propagated from trueseeds though this practice is largely limited to breeding programmes (Craufurd et al.,
2001). The sett cut from apical tuber parts emerge earlier and yield better than setts cut
from the lower part of the tuber. The key sett characteristics that play an important role in
yam crops are its size and its physiological condition, which could in turn be influenced by
storage conditions. Although the storage period varies largely because of climatic and
cultural conditions, the generally quoted vegetation period of 7 to 12 months suggests
storage of seed tubers from 0 to 5 months. Biologically this period corresponds to the post
harvest component of the dormant period, which allows the yam to overcome periods
unfavourable to growth (Tschannen et al., 2005).
Cultivated yams are grown as annuals with tubers being planted between February (in the
humid forest) and April (in the savanna area). In West Africa flowering usually occurs
between June and September. Harvesting can take place 180 days after planting, i.e., in
August in the humid forest agroecological zone, but mostly it is carried out when the shoot
senesces at about 180 to 270 days later in the savanna and humid forest, respectively (i.e.,
October and November). The harvest season is usually celebrated with special rituals with
D. alata having the most important social significance (Alexander and Coursey, 1969).
After harvest, tubers are sun dried to prevent fungal infections. They are commonly stored
in barns, which provide good ventilation, and protection from termite attack and flooding
(Degras, 1993). Yam tubers can also be stored well in a dry, dark, cool, and ventilated
place such as storage huts. Tubers are stored for several months for consumption, and for
provision of planting materials for the following season (Coursey, 1967).
Harvested tubers remain dormant (incapable of developing an internal or external shoot
bud) for 30 to 150 days, depending on the date of harvest and growing and storage
conditions (Ile et al., 2006). Dormancy in yam tubers prevents precocious sprouting,
prolongs storability and maintains food quality (Ile et al., 2006). Dormant yam tubers,
uniquely and in contrast to potatoes (Solanum tuberosum) do not have apical buds. Instead,
dormant yam tubers have meristematic cells below the surface of the tuber (Ile et al.,
2006). Once the dormancy period is over, sprouting tubers are planted at the start of the
rainy season. The new yam plant draws on material of the mother tuber until the eighth
30
week (Tschannen et al., 2005). The plants are usually grown in ridges or mounds with
stakes or live support, which allow the vines to climb. Yams are frequently grown with
other types of plant species. Yam intercropping with grain legume is a common practice,
because it is an economical method for weed management (Coursey, 1967; Onwueme,
1988).
Yams exhibit the sigmoidal growth pattern that is common to most annual plants. This is a
period of slow growth during establishment followed by a phase of rapid exponential
growth as the canopy reaches its maximum area and finally a declining growth rates as the
canopy senesces. In brief, following the breaking of dormancy (sprouting), the following
four distinct phases of development are commonly recognized (Craufurd et al., 2001):
1. Tuber germination and sprout emergence: dormancy ends when the tubers
germinate and the growing shoots or vines emerge. The duration of this phase
is typically between 30 and 50 days, but can be protracted if the conditions
are unfavourable.
2.
Canopy establishment and tuber initiation: typically, this phase lasts between
20 and 70 days. The vines elongate, cataphylls and then true leaves are
initiated and expand and the plant becomes autotrophic.
3. Maximum canopy development and maximum tuber growth rate: this third
phase is the most critical period for growth of the yam tuber; it is
characterized by maximum canopy development and tuber growth rate. It has
a typical duration of 60 to 90 days. During this period plant growth is highly
plastic in response to both positive and negative elements, such as
management inputs, weeds, fertilizers and pests.
4.
Canopy senescence and tuber maturity: during this phase of development
leaves senesce and dry matter accumulation declines. Tubers attain their
maximum volume and weight. The combined duration of phase 3 and 4 varies
from 80 to more than 150 days.
31
2.7 Reproductive biology of Guinea yams
Guinea yams are usually dioecious, rarely monoecious individuals have also been observed.
Monoecious individuals are often encountered when plants grown from seeds (Hamon,
1987; Zoundjihèkpon, 1993). For centuries, cultivated yams have as mentioned been
vegetatively propagated from tubers of local cultivars. This continued vegetative
propagation and lack of hybridization have precluded the possibility of genetic
improvement of yams through breeding programs (Senou et al., 1992). In West Africa no
direct use of seeds by farmers has been reported for the main cultivated species (D.
rotundata). The farmers use tubers from the previous harvest. The two wild relatives, D.
abyssinica and D. praehensilis, mainly reproduce by sexual means, an insect called Thrips
(Larothrips dentipes) being the major pollinator. Other species such as, Acantolepsis spp.,
Chirothrips spp. and Haplothrips spp. may also be involved in the pollination (Scarcelli et
al., 2006). According to Senou et al. (1992), insects belonging to five different families
were found entering the open, receptive flowers, and their presence on yams coincides with
the duration of the flowering period.
Flowers, especially the male ones, are small, difficult to handle (e.g. in crossing
experiments) and often have sticky pollen (Zoundjihékpon et al., 1994). Mature male
flowers start opening in the morning and reach peak number late in the afternoon. Up to
five male flowers open daily depending on the spike length. Female flowers open
acropetally any time after attaining maximum size and remain open or partially closed after
pollination. Usually 2-3 female flowers open per spike daily (Akoroda, 1983).
Studies from different countries regarding variation in sex ratio revealed some differences.
For example the study by Dansi et al., (1999) indicated that the two sexes are almost
equally represented in Benin. A higher male to female ratio has been reported from the
Ivory Coast (Hamon, 1987), whereas the reverse has been reported from Togo (Kassmada,
1982 in Dumont et al., 2006). According to Dumont et al. (2006) sex might not be fully
genetically determined and might be influenced by yet unknown environmental factors.
Sex reversal has also been reported in juvenile hybrids of early-maturing Ivorian cultivars
(Zoundjihèkpon, 1993).
32
Based on the success of controlled crosses in wild and cultivated yams, pollen obtained
from the wild forms was found to be more efficient compared to the cultivated forms
(Zoundjihèkpon et al., 1994). The authors suggested a high quantity of relatively fertile
pollen produced by the wild forms, as a possible reason to explain the observed results.
The success of cross-pollination not only requires the presence of pollen on the stigma but
also the absence of genetic incompatibility and sterility (Zoundjihèkpon et al., 1997)
Adequate knowledge of the floral biology of a crop is a prerequisite for overcoming the
morphological and genetic barrier to successful hybridization (Akoroda, 1983). Cultivated
Guinea yams have a highly variable flowering capacity which might be associated with
different genetic and environmental factors. Some of the environmental factors are related
to climate (rainfall quantity and distribution) and agronomic conditions, such as soil
fertility, planting density, staking practice, weed control etc. Photoperiod is also an
important factor for flowering. The photosensitivity of D. rotundata has been demonstrated
by Okezie et al. (1993). Some studies link flowering and fruiting abilities of the different
cultivars to the maturity period. Thus, the late maturing cultivars were reported to exhibit
less fertility compared to the early maturing cultivars (Dumont et al., 2006). The
maturation period has also been linked to the fertility of cultivated Guinea yams
(Zoundjihèkpon, 1993). According to the results of this study, fertility was found to be low
in late maturing D. rotundata cultivars, with several male cultivars producing abnormally
small pollen grains with low germination rate. The author attributed the low fertility to lack
or deficiency in the mechanism controlling the tepal opening during floral development.
Efforts to improve yam production are hampered by the low rate of flowering, the very low
rate of fruit setting and poor seed germination (Senou et al., 1992). With hand pollination
Akorda (1983) was able to increase fruit set in Guinea yams threefold and proposed that
low fruit set is primarily due to the less efficient mechanism of pollination by insects.
Some have attributed low fruiting in D. rotundata to pistil sterility, while others suggested
poor pollen release or poor pollen germination (Zoundjhekpon et al., 1997). Poor
reproductive capacity in yams has often been attributed to the polyploid nature of the crop
(Egesi et al., 2002).
Earlier studies on flowering pattern in D. rotundata indicated that there is difference in the
time of flower initiation and maturation in male and female clones (Ayensu and Coursey,
33
1972; Akoroda, 1983; Senou et al., 1992). According to the results of these studies
staminate plants flowered earlier than pistilate flowers. However, an overlap in the timing
of flower opening has been reported by Zoundjhèkpon et al. (1997).
2.8 Cytogenetic studies.
At present, yams are widely regarded as being polyploids. The basic chromosome number
for polyploid yams has been reported to be x = 9 or x = 10. All the Asian Dioscorea, 52%
of the African species, and 13% of the American species have the basic chromosome
number x = 10, whereas most of the American clones display a basic chromosome number
of x = 9 (Essad, 1984). However, recent data have challenged the previous report and
revealed new basic chromosome numbers for yams, x = 6 (Segarra-Moragues et al., 2004)
and x = 20 (Scarcelli et al., 2005; Bousalem et al., 2006). These results should lead us to
reconsider the ploidy level of some species of yams, including Guinea yams (Bousalem et
al., 2006).
The existence of various ploidy levels and the lack of diploid relatives to the cultivated
polyploid yams have complicated the study on yams (Bousalem et al., 2006). Reports
about the ploidy level in the wild species are rare and often contradictory. Thus using
flowcytometry Hamon (1992) reported that D. abyssinica, D. mangenotiana Méige and
D. praehensilis have the same DNA content. However, Essad (1984) described the former
as tetraploid and both D. mangenotiana and D. praehensilis as octoploids. According to
Zoundjihèkpon (1993), D. mangenotiana and D. burkilliana were considered to be
hexaploid and tetraploid, respectively. Using the conventional methods of chromosome
counting and flow cytometry, Gamiette et al. (1999) depicted the ploidy of levels of
Guinea yams (including their wild relatives) fitted in a 4x, 6x and 8x ploidy series. These
authors also proposed that D. cayenensis-D. rotundata cultigens and their wild relatives
might belong to the same gene pool as most of the clones tested have the same DNA
content. The ploidy levels described by Gamiette et al. (1999) were later supported by
Dansi et al. (2001).
The results of segregation analyses for three enzyme systems in the progeny from a
controlled cross between two cultivars of yams from Ivory Coast matched the theoretical
1-2-1 segregation expected in diploid individuals from a cross involving heterozygote
34
parents (Zoundjihèkpon, 1993). The author described the cultivars as tetraploids
segregating like diploids. Another study involving population of F1 crosses between two
presumed heterozygous parents of D. rotundata by Mignouna et al. (2002a) produced a
similar result. The authors proposed that D. rotundata genome is an allotetraploid with 2n
= 4x = 40. Although both studies revealed the segregation pattern expected in diploids, the
authors did not use these results to challenge the ploidy status of the cultivated Guinea
yams. By monitoring enzymatic traits in the progeny of monoecious clones of a cultivar in
Benin, Daïnou et al. (2002) were the first to propose the diploid status of D. rotundata (2n
= 40). Later the analysis of
the segregation pattern of two isozyme loci and six
microsattellite markers in the progeny of a self-fertilized monoecious plant by Scarcelii et
al. (2005) supported diplody of D. rotundata (2n = 40). The latter authors also suggested
both D. abyssinica and D. praehensilis to be diploids with 2n = 40.
2.9. Taxonomic and phylogenetic relationships between the species in the study group
The taxonomy of the species Dioscorea cayenensis and D. rotundata has been the subject
of much confusion (Coursey, 1967). They were first described as separate species: D.
cayenensis by Lamarck (1789) based on specimens from French Guiana (hence the name
Cayenne) and D. rotundata by Poiret (1813) based on samples from Puerto Rico long
before their African origin was established. Grisebach (1864) reduced D. rotundata to
subspecfic status within D. cayenensis (Onyilagha and Lowe, 1985). This was accepted by
Prain and Burkill (1919) and subsequently maintained by Francophone writers such as
Chevalier (1936) and Méige (1952, 1968). Burkill (1921) restored D. rotundata to species
status, and this was accepted by Hutchinson and Dalziel (1936). Chevalier (1936) created a
new taxon; subsection Cayenensis Chev., under the section Enantiophyllum Uline, which
includes all the Guinea yams and their wild relatives (Terauchi et al., 1992). A numerical
taxonomic analysis by Martin and Rhodes (1978) on 97 cultivars of Guinea yams (D.
rotundata -D. cayenensis complex accessions), based on 75 morphological descriptors,
resulted in a phenetic tree with two main trunks interconnected with many anastomosing
branches. The authors suggested that all the 97 cultivars investigated belong to a single
highly evolved species. Based on an agro-botanical study of the West African cultivars,
Akoroda and Chheda (1983) proposed that D. cayenensis and D. rotundata should be
considered as distinct species and referred the taxonomic confusion to the existence of
35
some intermediate forms which are presumably hybrids. This idea was later supported by
Onyilagha and Lowe (1985). Using 76 morphological and ecological characters on eight
cultivars, the authors conducted the study for three successive years.
The concept of the D. cayenensis species complex, first proposed by Ayensu and Coursey
(1972) was discussed in 1978 at a seminar on yams conducted in Cameroon. This concept
was later supported by Hamon (1987) as a way of pooling all West African cultivated
yams that are not bulbiferous and have entire leaves under the same name (Dumont et al.,
2006). Some authors use D. cayenensis complex as a provisional name for the set of subSaharan yam species whose taxonomic relations are currently being examined:
D.
cayenensis, D. rotundata, D. abyssinica, D. praehensilis and D. sagittifolia (Wilkin and
Caddick, 2000; Wilkin, 2001; Wilkin et al., in prep). Member species are indigenous to
Ethiopia and occur all over sub-Saharan Africa from 500 m to 1800 m altitude, especially
in seasonally hot and moist areas (Miège and Sebsebe Demissew, 1997).
2.9.1. The Cultivated Guinea Yams: D. rotundata and D. cayenensis
According to Dumont et al. (2006), there has been considerable confusion regarding the
yams D. rotundata and D. cayenensis. In general, in English speaking West Africa,
particularly Nigeria, they are known as white yams and yellow yams respectively, and
pooled under the term Guinea yams. Farmers in French speaking Africa, on the other hand
do not make a clear distinction between D. rotundata and D. cayenensis where the generic
name is accordingly used for all the cultivated yams. For the African farmer, a yam
cultivar is identified by its common name, which often contains technical or historical
information. Yam cultivars are best differentiated on the basis of their tuber traits like
colour or taste of the flesh. The characteristics of the vegetative organs are sometimes, but
not always, also used as distinctive markers. Farmers, in general, define yam cultivars by
sets of technical criteria consisting of agronomic requirements, harvesting time, cooking
quality and storage life (Dumont et al., 2006).
Dioscorea cayenensis and D. rotundata are believed to be domesticated from wild African
Dioscorea species of the section Enantiophylum. The two taxa differ to some extent with
respect to some traits. But none of the studies conducted so far have clearly established the
36
identity of each taxon as a separate species (Dumont et al., 2006). It is difficult to
consistently differentiate the two taxa using morphology. This is because the original
diagnoses are not complete enough to define them precisely and many forms are
intermediate between the two (Méige and Sebsebe Demissew, 1997). However,
D.
rotundata could be described as a group of cultivated yams of African origin, with a short
annual vegetative cycle (6-8 months), tubers with a long dormancy period (3-5 months)
and with slightly to non-pigmented creamy or white flesh (Dumont et al., 2006). It is
harvested twice a year, prefers a short rainy season, has ovate leaves and 4, 8 or 12
vascular bundles (Coursey, 1967; Méige, 1968; Ayensu, 1970; Hamon and Toure, 1990a).
In Dioscorea cayenensis the vegetative cycle ranges from 8 to 12 months and the tuber
flesh is usually yellow (Terauchi et al., 1992). It is harvested annually, prefers a long rainy
season, has orbicular leaves, and 8 vascular bundles (Coursey, 1967; Méige, 1968; Ayensu,
1970; Hamon and Toure, 1990a).
Hamon (1987) suggested that D. cayenensis might be the product of interspecific
hybridization and emphasized the likely involvement of D. burkilliana.
Other authors
claimed that D. cayenensis is phyletically close to or the same as the domesticated form of
D. burkilliana (Akoroda and Chheda, 1983; Onyilgha and Lowe, 1985; Mignouna et al.,
1998; Dansi et al., 2000). According to Terauchi et al. (1992), D. rotundata yams are
usually cultivated in both the savanna and rainforest zone, whereas D. cayenensis is
restricted to the rainforest zone.
Conclusions drawn from earlier studies of relationships among members of the D.
cayenensis complex based on morphometry, chemotaxonomy, cytology, isozyme and
molecular analyses, have not been consistent (Ramser et al., 1997). In the last two decades
different studies using various techniques have been conducted. Based on the results from
RFLP markers and analyses of chloroplast and ribosomal DNA, Terauchi et al. (1992)
proposed the species name D. rotundata to encompass all the cultivated Guinea yams.
Using isozyme analyses Hamon et al. (1997) supported the idea of Terauchi et al. (1992).
However, using different molecular techniques (RAPD, MP-PCR and RAMPO and cpDNA sequencing) on 42 accessions of cultivated Guinea yams and their wild relatives,
Ramser et al. (1997) were able to differentiate D. rotundata from D.
cayenensis,
supporting the idea that both should be treated as separate species. Based on analyses of
isozymic and morphological characters, Dansi et al. (2000) argued that D. rotundata and D.
37
cayenensis represent different genetic entities. Morphological and isozyme analyses by
Mignouna et al. (2002b) and Mignouna and Dansi (2003) distinguished between D.
cayenensis and D. rotundata cultivars, but they failed to demonstrate that they are distinct
species. Recent studies by Scarcelli et al. (2006) using AFLP data delimitated three groups
of Guinea yams (and their wild relatives) from Benin belonging to D. cayenensis-D.
rotundata complex, D. abyssinica and D. praehensilis, respectively. Cluster analysis of
molecular data, generated by using RAPD and DS-PCR discriminated the cultivars
classified as D. cayenensis from D. rotundata (Mignouna et al., 2005), and these authors
proposed that D. cayenensis should be considered as a taxon separate from D. rotundata.
Molecular markers have also been used to characterize Guinea yams at cultivar levels.
Isozyme marker analysis of D. rotundata from Ivory Coast and Benin indicated that the
cultivars in both countries appear to have the same genetic structure (Dumont et al., 2006)
2.9.2. Wild Yams: Dioscorea praehensilis and Dioscorea abyssinica
Evolution within the section Enantiophyllum has produced D. alata in Asia and D.
cayenensis and D. rotundata in Africa. These domestication products, with their high
cultivar diversity, account for virtually all yam production worldwide. The wild species of
this section, such as D. abyssinica and D. praehensilis, are considered to be the major
source of the variability (Dumont et al., 2006).
It is hard to accurately define the
morphological boundaries between D. abyssinica and D. praehensilis. Moreover, in West
Africa the latter is often regarded as the same species as D. lecardii, which in turn is poorly
separated from D. sagittifolia (Dumont et al., 2006 ).
Most of the criteria set for the separation of D. abyssinica from D. praehensilis are based
on taxonomic studies of dried herbarium plant material. There are little or no detailed
reports regarding studies on these wild species at population level. Therefore, the range of
variability of the two species and any taxonomic and genetic relationships between them
still remain obscure. Nevertheless, both have several traits in common. They usually
produce one tuber, and tubers and aerial vegetative parts are renewed annually. Both
reproduce sexually and they are propagated mainly by seeds, in contrast to the cultivated
yams, which are vegetatively propagated (Scarcelli et al., 2006a). Studies conducted in
West Africa have indicated that most of the wild plants belonging to D. abyssinica and
38
D.
praehensilis are male plants (Dumont et al., 2006). This phenomenon has been explained
in terms of the biological characteristics and population structure of wild Dioscorea
species. In the wild, the plants are often found very scattered and they are pollinated by
insects such as thrips (Larothrips dentipes) (Zoundjihèkpon, 1993). The population size of
these insects is largely determined by climatic conditions. Therefore, as a means of
ensuring survival, the wild plants need to produce surplus pollen. The female plants in the
wild usually produce several dozen to several thousand flowers, each with six ovules, so
each plant can be pollinated by a large number of male parents. The products of these
fertilizations are half sibs as they all have the same maternal genetic heritage. Thus the less
represented sex has reproductive advantage, as it has higher probability of passing its genes
(Dumont et al. 2006).
Dioscorea abyssinica and D. praehensilis also share some ecological and morphological
characters. Firstly, their preferred ecosystem is regenerating plant environments. Both
species are dependent on fallows or windfall areas, where they grow while the climax plant
population become re-established. Secondly, the aerial architecture of D. abyssinica and D.
praehensilis is typical of wild yams overall. The stems grow to a considerable height
before branching, as they need to rise above the supporting shrub vegetation before
exposing leaves and flowers. Lastly, both species are extremely polymorphic; the
variability being linked to the age of the plant, which can be viewed in two levels: 1.
Variation associated with the annual renewal of the vegetative organs and tubers. Some
traits vary during the annual vegetative cycle, for example leaf shape and size. 2. Variation
associated to the genotype age, which corresponds to the number of annual vegetative
cycles since the plant first grew from seed. Some morphological traits are modified as a
result of inter-annual variations. For example, in West Africa, the elongated leaves and
vine colour at the leaf base are linked to the juvenility of the genotype. The morphological
diversity of a genotype is generally reduced with ageing (Dumont et al., 2006).
Dioscorea praehensilis has a wide geographical range in Africa, being found throughout
the Western, Central and Eastern parts of the continent to as far South to Zimbabwe. This
species is regarded as a forest yam in West Africa. According to Dumont et al., (2006), it
grows abundantly in post fire regeneration areas within dense communities of semideciduous trees. It is common in the bimodal rainfall zone, but under drier climatic
conditions it takes refuges in the rare remnants of mesophyll forests that have survived the
39
combined effects of annual fire and anthropogenic pressure. Dioscorea liebrechtsiana,
which ranges from central Africa to Cameroon, is morphologically very close to D.
praehensilis. Some authors (e.g. Wilkin, 2001) found no difference between them.
No comprehensive studies are conducted on the genetic diversity of D. praehensilis, except
a few studies that have been undertaken in West Africa. The study conducted by Tostain et
al. (2002), which involved 46 accessions of D. praehensilis revealed that grouping of the
genotypes correlated to their geographical location.
Dioscorea abyssinica has long been regarded as the same species as D. togoensis (Miège
1952). Based on morphological characters Miège (1982) proposed that D. abyssinica, D.
lecardii, and D. sagittifolia are morphologically so similar that their status as distinct
species is questionable.
Dioscorea abyssinica grows mainly north of the Equator in sub-Saharan Africa in the
climatic belt roughly ranging from latitudes 80 to 120 N. Dioscorea abyssinica is a savanna
yam appearing to prefer a unimodal rainfall regime (Dumont et al., 2006). In West Africa,
D. abyssinica is distributed throughout the area where D. rotundata yams are domesticated
(Miège, 1968). In Ethiopia D. abyssinica is found widely distributed in the southern,
western and northern part of the country in woodlands or wooded grasslands between 1000
m and 1800 m above sea level (Miège and Sebsebe Demissew, 1997).
Several scientific studies have been conducted on the genetic diversity of D. abyssinica.
Ramser et al. (1997) placed D. abyssinica in an intermediate position among D.
praehensilis, D. liebrechtsiana and D. rotundata, on the basis of four types of molecular
markers. Another study by Tostain et al. (2002), using AFLP, has shown genetic continuity
between D. abyssinica and D. praehensilis. A study in Benin (Dansi et al., 1999) indicated
that a variety domesticated from wild D. praehensilis yams genetically resembled D.
abyssinica. The authors proposed that wild yams identified as D. abyssinica might actually
be D. praehensilis adapted to the forest-savanna transition or an escape from cultivated
fields in the form of seeds. Furthermore, study on D. abyssinica materials from Benin,
Togo and Guinea using AFLP, revealed a geographically structured genetic diversity
(Tostain et al., 2002).
40
2.9.3 Phylogenetic relationships between wild and cultivated species
The phylogenetic relationships between cultivated and wild yams have long been the focus
of scientific investigations.
According to Dumont et al. (2006)Chevalier linked one
cultivar of D. rotundata from Benin first (Chevalier, 1920) to D. praehensilis and later
(Chevalier, 1936) to D. lecardii. Burkill (1939) proposed that D. rotundata is derived from
D. abyssinica or from another wild yam of the same type (possibly D. lecardii). Miège
(1952) proposed D. abyssinica, D. sagittifolia, D. preahensilis, D. liebrechstiana, D.
mangenotiana and D. lecardii as possible ancestors of Guinea yams, whereas Coursey
(1976) suggested D. praehensilis as the possible predecessor for the cultivated Guinea
yams.
Studies based on morphology, ecology and chemotaxonomy have suggested a
polyphyletic origin of Guinea yams involving one or multiple hybridization events
(Ramser et al., 1997). Several scientific works (Hamon, 1987; Terauchi et al., 1992;
Zoundjihèkpon, 1993; Ramser et al, 1997; Dansi et al., 1999; Tostain et al , 2002) based
on different molecular markers have supported that the wild yams D. praehensilis and D.
abyssinica are possible ancestors of the cultivated Guinea yams (Dumont et al. 2006), but
they may be part of a single biological species as discussed above.
Terauchi et al. (1992) proposed D. abyssinica, D. liebrechtsiana, D. praehensilis or their
hybrids as possible predecessors for D. rotundata (all characterized by annual replacement
of tubers and stems). According to those authors D. cayenensis is possibly an interspecific
hybrid between D. rotundata, D. praehensilis, D. liebrechtsiana or D. abyssinica (as
possible female parents) and the perennial species D. minutiflora, D. burkilliana. or D.
smilacifolia De Wild. (as possible male parents). Morphological and ecological data
support the hypotheses of the hybrid nature of D. cayenensis. It has a longer growth period
(8-12) months, some cultivars have perennial nature, with relatively a large corm, thick and
flat leaves, its main habitat being in the rain forest zone (Terauchi et al., 1992).
Terauchi et al. (1992) used the RFLP technique to analyze chloroplast DNA and ribosomal
DNA, but failed to separate D. rotundata and D. cayenensis from their putative wild
parents. Similar results were obtained by Chaïr et al. (2005) with chloroplast DNA data
and accessions from Benin. However, Chaïr et al. (2005) found a unique haplotype for
41
some of the accessions of D. abyssinica. The authors proposed that cultivars sharing
identical haplotypes with the cultivated Guinea yams (D. cayenensis-D. rotundata complex)
might be considered as morphotypes within the complex. They might be escapes found in
forests or ancient forest bushes, originating from either seed germination or sprouting from
remains of tuber fragments after harvesting. Morphotypes with a different haplotype might
represent the true wild type of D. abyssinica. Tostain et al. (2002) and Scarcelli et al.
(2006a) used AFLP to compare wild (D. praehensilis, D. abyssinica) and cultivated (D.
cayenensis-D. rotundata complex) Guinea yams that had been domesticated in the past or
were in the course of domestication. According to the authors, cluster and ordination
analyses partially separated the wild forms from the cultivated Guinea yams. Further more,
the degree of relatedness of the wild to the cultivated forms was found to depend on the
geographical distance between the wild and the cultivated yams.
Successful interspecific hybridization between wild and cultivated Guinea yams has been
carried out at the International Institute of Tropical Agriculture (IITA) in Ibadan, Nigeria
(Mignouna et al., 2005). There are some genetic evidence for spontaneous hybridizations
between wild yams and cultivated yams (Scarcelli et al., 2006b). The sympatric situation
of wild and cultivated species, field introduction of wild plants during domestication, and
to some extent synchronization of flowering time could favour inter-specific hybridization
in nature (Scarcelli et al., 2006a).
2.10 Studies on yams in Ethiopia
Ethiopia is considered to be the center of origin for D. abyssinica, which is found widely
distributed in the savanna region of West Africa (Coursey 1967). Generally the country is
regarded as an isolated center of yam cultivation outside the yam belt of West Africa
(Norman et al., 1995).
Recent studies made on Dioscorea species in South Ethiopia (Muluneh Tamiru, 2006) and
Southwest Ethiopia (Hildebrand, 2003; Sebsebe Demisew et al., 2003) have revealed that
people in the study area have a strong tradition in cultivating and domesticating various
yam cultivars with a wide genetic base. However, this excellent knowledge of yam
cultivation and domestication is as mentioned beginning to deteriorate as farming reorients
towards cash crops and coffee plantations (Hildebrand et al., 2002). These studies reported
42
a total of 60 named cultivars (landraces) in the study area of which the majority have
limited distribution and abundance. The number of landraces per farmer in southern
Ethiopia ranged from 1 to 6 (Muluneh Tamiru, 2006), even if the local classification not
always is consistent.
Yam is exclusively cultivated by subsistence farmers in the densely populated areas of
Southern, Southwestern and Western parts of Ethiopia, where it has considerable
importance in the local livelihood. Yam tubers are the preferred food product for honored
guests, and traditional meals made of yams are served during the main traditional and
religious festivals. Accordingly, farmers sell yam tubers at relatively high prices compared
to other root and tuber crops. Hence, yam is important not only for household food security,
but also as a source of cash income (Muluneh Tamiru, 2006).
According to Muluneh Tamiru (2006), farmers in the southern part of Ethiopia recognize
two major categories of yams which differ in their maturation period. The early maturing
group consists of male yams, which grow vigorously and tolerate drought. These are the
most popular, as they fit well into the local subsistence agriculture and, are the preferred
choice for yam production. The late maturing group are all female plants. They grow less
vigorously and produce poorly under suboptimal conditions. Within each group,
morphological attributes such as stem color, presence or absence of spines, leaf color, leaf
shape, and tuber flesh color are the major criteria for identifying individual landraces.
There are very few reports dealing with aspects of yam production in Ethiopia (Muluneh
Tamiru, 2006). Yam requires 7 to 11 months from planting to harvesting. The supply of
fresh tubers to local markets in Ethiopia is limited to the periods from May to September.
The planting of yams usually starts in October (in most parts of Southern Ethiopia),
November and December (in South Western and Western part of the country) (Muluneh
Tamiru, 2006; Hildebrand 2003). Factors such as soil moisture content, intensity of the dry
season and anticipated harvesting time are considered in timing of field planting. There is
no formal seed supply system nor do farmers specialize in producing yam planting
materials. Farmers mostly rely on seed tubers saved from the preceding cropping season.
Some partly meet their demand for seed tubers through purchases from local markets or
exchanges with neighbors. At the end of each cropping cycle, healthy tubers are selected
and stored in shallow pits under shade for one to three months or until required for field
43
planting. For single-harvested landraces that normally produce a single tuber per plant, the
head region (proximal end) of each tuber is retained while the remaining part is consumed.
With the double-harvested landraces, a single plant produces many tubers used as for
propagation (Muluneh Tamiru, 2006). The decision as to type and number of cultivars to
be planted on a farm, is mainly influenced by environmental factors (such as altitude), time
of maturation and market demand. Selection for desirable agro-morphological traits, as
well as socio-cultural factors, appear to be the major forces behind the dynamics of yam
diversity in South Ethiopia (Muluneh Tamiru, 2006)
The genetic structure and diversity of both cultivated and wild yams in Ethiopia is poorly
understood (Hildebrand et al., 2003).
The studies by Hildebrand et al., (2003) and
Muluneh Tamiru (2006) revealed that there are a large numbers of cultivars grown in small
farm holds in south and southwest Ethiopia. According to Hildebrand (2003) wild and
domestic yams appear to vary significantly in time and space of their availability. Wild
yams are harvested from September to February, whereas the cultivated forms from June to
October. Wild yams thrive in lowland wooded grasslands, whereas cultivated yams do
better in upland settings with more rainfall.
A study conducted on 48 accessions of Ethiopian materials (Muluneh Tamiru, 2006) from
South Ethiopia and 8 cultivars of D. cayenensis and D. rotundata from West Africa based
on AFLP, revealed that although the Ethiopian materials are genetically closer to the West
African D. cayenensis and D. rotundata (compared to their genetic distance to D. bulbifera
and D. alata), they show some degree of distinctiveness. The author suggested that the
distinctiveness of the Ethiopian materials may represent a divergent evolutionary pathway
isolated from the widely known center of diversity in West Africa. Based on the results of
morphometric analyses, Muluneh Tamiru (2006) divided the Ethiopian accessions into six
groups with two major clusters that mainly differ in their maturity time.
44
2.11. Statement of the problem and objectives of the study
2.11.1 Statement of the problem
The genus Dioscorea includes ca 600 species (Govaerts and Wilkin, 2007), occurring
mainly in the Old and New World tropics; the highest levels of species diversity per unit
area occur in mainland tropical Asia, Madagascar, South Africa, the Caribbean, Mexico
and central South America. There are ca 11 species are found in Ethiopia and Eritrea
(Sebsebe Demissew et al., 2003). Among the species of Dioscorea section Enantiophylum,
there is a high degree of phenotypic plasticity in morphology and identification based on
morphological characters is sometimes difficult (Lay et al., 2001). The currently used
classification scheme which relies on identification keys constructed mainly using
vegetative and male inflorescence characters do not delimit species boundaries consistently
between D. cayenensis, D. rotundata, D. abyssinica, D. praehensilis and D. sagittifolia
(Wilkin, 2001; Sebsebe Demisew et al. 2003). For example recent studies made on
Dioscorea species of Southwest Ethiopia have indicated that leaves of members of the
D.
cayenensis complex vary considerably in shape within one individual plant (Hildebrand,
2003).
Yam cultivation systems in Ethiopia and East Africa have not been studied as well as their
West African counterparts, and the delimitation of the D. cayenensis complex and
relationships within it are still far from clear. Studies of both morphological and molecular
patterns of variations in the wild and cultivated D. cayenensis complex in Ethiopia may
shed new light on the classification and domestication history of this complex. In particular,
such research may test whether the current species boundaries, which are based on some
rather cryptic characters, have any grounding in biological fact (Hildebrand et al,. 2002).
The study conducted in Southwest Ethiopia has also revealed that the local community has
a strong tradition in cultivating and domesticating various species of yams with a wide
genetic base. However, the removal of vegetation cover by human activities (for
agricultural expansion and settlement) undoubtedly reduces the genetic base of these
important cultivated and semi-cultivated crops leading to genetic erosion (Hildebrand et al.
2002; Sebsebe Demisew et al., 2003).
45
Plant genetic resources are one of the most valuable assets to mankind. Protection and
conservation of these resources for future generations, therefore, assume great significance.
Reliable information on the distribution of genetic variation is a prerequisite for sound
selection, breeding and conservation programs. Genetic variation of a species or population
can be assessed by measuring morphological and quantitative characters in the field or by
studying molecular markers in the laboratory. The uses of DNA based markers are
increasingly playing an important role in conservation and use of plant genetic resource
(Rao, 2004). Thus determination of the extent of variation at the genetic level within and
among populations is of value in guiding genetic conservation activities, which are aimed
at maintaining genetic diversity, and molecular marker data have been widely used in
taxonomic evaluations particularly in the identification of genotypes (Ford-Lyold, 2001).
Good taxonomy is fundamental to conservation and crop improvement programs.
The usefulness, reliability and potential of molecular markers in identification, assessment
of genetic diversity and establishment of genetic relatedness have been well documented
(Graham et al., 1996). Reports on identification or characterization of cultivars of
Dioscorea species from West Africa using the isozyme electrophoresis method and the
morphological methods have been published (Twyford et al,. 1990; Lebot et al. 1998;
Dansi et al. 2000; Mignouna et al. 2002b). Recently molecular based techniques, such as
the randomly amplified polymorphic DNA (RAPD), AFLP, and microsatellites or simple
sequence repeats (SSRs) have been recognized as powerful and efficient tools to detect
genetic diversity and assess phylogenetic relationships (Lay et al. 2001; Mignouna et al.,
2003).
A comparative study by Mignouna et al. (2003) using three different molecular techniques
indicated that AFLP showed the highest efficiency
in detecting polymorphism and
revealing genetic relationships that most closely reflected the morphological classification.
AFLP has also been used to asses the genetic relationships between D. alata and other
edible Dioscorea species from different section including Guinea yams. The study revealed
that members of the sections Enantiophyllum are distinguished from each other and are
genetically distant from species of other sections. They also proposed that AFLP can be
used to characterize Dioscorea species at a varietal level (Malapa et al., 2005).
46
2.11.2. Objectives of the study
2.11.2.1 General objectives
The main objectives of this study are:
-
To evaluate and improve the taxonomy and species delimitation within the D.
cayenensis complex by using both morphological and molecular techniques.
-
To investigate the amount and distribution of genetic variation among and within
populations of the D. cayenensis complex, as a prerequisite for devising
conservation strategy.
2.11.2.2 Specific objectives
-
To determine the taxonomic status of the speices within in the D. cayenensis
complex in Ethiopia
-
To determine the genetic diversity of the wild and cultivated species within the D.
cayenensis complex.
-
To determine level of population differentiation and population structure among the
populations of the D. cayenensis complex in Ethiopia.
-
To identify sites or areas where priority should be given for in-situ conservation of
Guinea yams in Ethiopia.
47
3. Materials and Methods
3.1 Morphometry
3.1.1 The plant material
The plant material used for morphometry consisted of 40 dried herbarium specimens of
wild and cultivated accessions of the D. cayenensis complex, collected in different
localities in South and Southwestern parts of Ethiopia (Table 4) from a field trip conducted
in August 2006. Collections were first named using the folk taxonomy as the field
identification and formal taxonomic identification to species level was made later using the
voucher specimens at Royal Botanic Gardens, Kew. D. cayenensis and D. rotundata were
treated as a single species (under the former earlier name) because they are
indistinguishable based on the morphology of the above-ground organs. The major
morphological characters used for identification of the specimens to species level are
presented in appendix 1.Voucher specimens were housed at the Royal Botanic Gardens,
Kew, London, UK. During the field work, flowers from the live specimens of each of the
individual plants were collected and preserved in 70 % alcohol.
Table 4. List of accessions of cultivated and wild yams sampled in the field and used for
mormphometric analysis
Serial
No
Species name
Accession
number
1 D. abyssinica
Daby-20
2 D. abyssinica
Daby-22
3 D. abyssinica
Daby-59
4 D. abyssinica
Daby-69
5 D. abyssinica
Daby-77
6 D. abyssinica
Daby-8
7 D .bulbifera
Dbul-28
8 D. cayenensis
Dcay-1
9 D. cayenensis
Dcay-17
10 D. cayenensis
Dcay-18
11 D. cayenensis
Dcay-19
12 D. cayenensis
13 D. cayenensis
Dcay-21
Dcay-35
Place of collection, locality
Bench- Magi zone Sheko area , Selalea locality,13 km along the
Mizan- Sheko road
Bench- Magi zone Sheko area , Selalea locality,13 km along the
Mizan- Sheko road
Areka agricultural institute, Welaita zone, at the outskirt of Areka
town
Areka agricultural institute, Welaita zone, at the outskirt of Areka
town
Welayita zone, Sodo Zuria district, Wejekerea locality, 3 km
along the Sodo- Areka road
Bench- Magi zone Sheko area , Selalea locality,13 km along the
mizan- sheko road
Ilubabor, 28 km along the Bedelea-Metu road near dedesa state
farm
Bench- Magi zone Temenja yadj area ,25 km along the Chenamizan road
Bench- Magi zone Sheko area , Shekoka locality, 8-10 km along
the mizan- sheko road
Bench- Magi zone Sheko area , Selalea locality,13 km along the
mizan- sheko road
Bench- Magi zone Sheko area , Selalea locality,13 km along the
mizan- sheko road
Bench- Magi zone Sheko area , Selalea locality,13 km along the
mizan- sheko road
Wellega, Nedjo area 5 km along Nedjo-Ghmbi road
48
14
15
16
17
18
19
20
D. cayenensis
D. cayenensis
D. cayenensis
D. cayenensis
D. cayenensis
D. cayenensis
D. cayenensis
Dcay-38
Dcay-47
Dcay-48
Dcay-49
Dcay-50
Dcay-56
Dcay-57
21 D. cayenensis
Dcay-61
22 D. cayenensis
Dcay-62
23 D. cayenensis
Dcay-67
24 D. cayenensis
Dcay-68
25 D. cayenensis
Dcay-7
26 D. cayenensis
Dcay-70
27 D. cayenensis
Dcay-71
28 D. cayenensis
Dcay-73
29 D. cayenensis
Dcay-74
30 D. cayenensis
Dcay-75
31 D. cayenensis
Dcay-76
32 D. praehensilis
Dprh-13
33 D. praehensilis
Dprh-16
34 D. praehensilis
Dprh-25
35 D. praehensilis
Dprh-26
36
37
38
39
D. praehensilis
D. praehensilis
D. praehensilis
D. praehensilis
Dprh-27
Dprh-30
Dprh-31
Dprh-32
40 D. praehensilis
Dprh-53
Wellega, Nedjo area 5 km along Nedjo-Ghmbi road
Gedeo, Kocherea district, Hama locality
Gedeo, Kocherea district, Hama locality
Gedeo, Yirgachefea district, Konga locality
Gedeo, Yirgachefea district, Konga locality
Sidama, Aleta wondo district, Debi locality
Sidama, Aleta wondo district, Debi locality
Areka agricultural institute, Welaita zone, at the outskirt of Areka
town
Areka agricultural institute, Welaita zone, at the outskirt of Areka
town
Areka agricultural institute, Welaita zone, at the outskirt of Areka
town
Areka agricultural institute, Welaita zone, at the outskirt of Areka
town
Bench- Magi zone sheko area , 13 km along the mizan- sheko
road
Areka agricultural institute, Welaita zone, at the outskirt of Areka
town
Areka agricultural institute, Welaita zone, at the outskirt of Areka
town
Areka agricultural institute, Welaita zone, at the outskirt of Areka
town
Areka agricultural institute, Welaita zone, at the outskirt of Areka
town
Areka agricultural institute, Welaita zone, at the outskirt of Areka
town
Welayita zone, Sodo Zuria district, Wejekerea locality, 3 km
along the Sodo- Areka road
Bench- Magi zone Sheko area , 13 km along the mizan- sheko
road 8 km along the Mizan-Sheko road
Bench- Magi zone Sheko area , Shekoka locality, 8-10 km along
the mizan- sheko road.
Ilubabor, Yayu area, ca 13 km along the road from Yayu to
Bedele
Ilubabor, Yayu area, ca 25 km along the road from Yayu to
Bedele
Ilubabor, Yayu area, ca 25 km along the road from Yayu to
Bedele
Wellega, ca 28 kms along the Nekemt-Ghimbi road
Wellega, ca 28 kms along the Nekemt-Ghimbi road
Wellega, ca 28 kms along the Nekemt-Ghimbi road
Sidama, zone, at the outskirts of Dilla town, ca 3 km form Dilla
town, near the main road from Dilla to Awassa
3.1.2 Morphological characters
Leaf and floral characters were used in the morphometric analyses. Two mature leaves
(one in few individuals) from each of the individuals were randomly selected to be used for
measurements using an ordinary ruler and a protractor. Measurements of floral
characteristics were made using only the male inflorescence (because more than 75 % of
49
the specimens are male); female flowers and fruits are too infrequent in populations to
obtain a sufficiently large sample. Two male flowers from the same inflorescence were
picked randomly. As the flowers are very small in size, measurement of floral characters
was made by using a binocular microscope (American Optical Corporation Mod 570) with
a magnification power of 60X. The morphological characters used in this study are listed
in Table 5 (see Fig. 3 for illustration of leaf characters).
Table 5. List of morphological characters used in morphometric analysis.
Leaf characters
Male floral characters
1. Blade length (L)
2. Tip base width (TBW)
3. Basal width (through point of petiole insertion)
(BW)
4. Sinus base width (SBW) (between apices of
auricles either side of basal sinus).
5. Width at ¼ length from the base (14W).
6. Width at ½ length from the base (12W)
7. Width at ¾ length from the base (34W)
8. Distance from midrib to first vein at ¼ W(M1)
9. Distance from midrib to second vein at ¼ W (M2)
10. Distance from midrib to margin at ¼ W (MM)
11. Ratio of BW to SBW
12. Ratio of 14W to 12W
13. Ratio of 14W to 34W
14. Ratio of L to BW
15. Ratio of L to 14W
16. Ratio of L to 12W
17. Ratio of L to 34W
18. Ratio of M2-M1 to MM
19. Ratio of MM-M2 to MM
20. Ratio of MM-M1 to MM
1. Outer tepal length (OTL)
2. Outer tepal width (OTW)
3. Inner tepal length (ITL)
4. Inner tepal width (ITW)
5. Anther length (AL)
6. Anther width (AW)
7. Filament length (FL)
3.1.3 Data collection and analysis
Only quantitative characters were used in the morphometric analyses. The leaf
measurements were log transformed before they were used in the final data analysis. These
characters were also used to estimate leaf shape parameters by transforming them into
ratios (Table 5).
50
Figure 3. Illustration of a typical Dioscorea leaf showing the measurements of leaf characters
A data matrix was constructed using 68 individuals and 26 attributes. Each individual used
in the study was considered as one OTU (operational taxonomic unit). The data matrices
were subjected to multivariate analyses such as cluster analysis (Unweighed Pair Group
method of Analysis, UPGMA, Sneath and Sokal, 1973) and ordination analysis (Principal
Component Analysis, PCA) using NTSYSpc version 2.2 (Rohlf 2004). For the cluster
analysis (UPGMA,), the SIMQUAL module with simple matching coefficients as a
measure of similarity was used to construct a similarity matrix. Phenograms were
constructed by using the SHAHN option. The same similarity matrix was also used for
PCA.
51
3.2 Amplified Fragment Length Polymorphism (AFLP)
3.2.1. The plant material
The plant material of D. cayenensis complex consisted of 43 accessions belonging to 16
farmers’ varieties (Table 6). Three accessions of D. schimperiana and D. bulbifera were
included for comparative purposes. The plant materials were collected in July 2005 from a
fieldtrip conducted in Sheko and its environs (Sh population, see Fig 4) of Southwest
Ethiopia. Collections and identification to species level was done using the same procedure
as used in morphometric analyses. The voucher specimens, listed in Table 6, have been
deposited at the herbarium ETH (http://sciweb.nybg.org/science2/IndexHerbariorum.asp).
Table 6. List of accessions of cultivated and wild yams used in AFLP analysis, giving their Sheko
name, collection site and identification (all collected from Bench-Maji zone expept the first two ).
Serial
No
1
Species name
D. schmperiana
Accession
number
14333A
Local
name
-
(Sheko)
2
D. praehensilis
1577B
-
3
4
5
6
7
8
9
D. cayenensis
D. cayenensis
D. cayenensis
D. cayenensis
D. abyssinica
D. cayenensis
D. abyssinica
CA1
CBD1
CBD2
CBD5
CC1
CC10
CC2
Addis Kachi
10
11
D. abyssinica
D. abyssinica
CC6
CC8
12
13
D. cayenensis
D. cayenensis
CD2
CD3
14
D. abyssinica
CDB3
15
D. praehensilis
CDB4
16
D. cayenensis
CDK1
17
D. cayenensis
CDK10
18
D. cayenensis
CDK12
19
D. cayenensis
CDK13
20
D. cayenensis
CDK14
21
D. cayenensis
CDK16
22
D. cayenensis
CDK3
23
D. cayenensis
CDK6
24
D. cayenensis
CDK7
Banda
Chebja
Don
Donbai
site of collection
90 km along the road from Nekemte to
Bure, Oromia region
142 km along the road from Chagni to
Mankush, Beneshangul Gumuz
Sheko district, Gaizika village.
Ediget Behibret School yard ,
Mizan Teri town
Sheko district, Mehal Sheko, Serer
village and from outskirts of Sheko
town near the health center.
Sheko district, Gaizika village.
Sheko district, Mehal Sheko, Serer
village.
Dizu kechi
Sheko district, Gaizika village and
outskirts of Sheko town near the health
center.
Dizu kechi
Bench District, Gabuka Village
Dizu kechi
Sheko district, Gaizika village and
outskirts of Sheko town near the health
center.
52
25
D. abyssinica
CDK9
26
D. cayenensis
CE1
27
D. cayenensis
CE2
28
D. cayenensis
CE3
29
D. cayenensis
CE4
30
D. abyssinica
CKR2
31
D. cayenensis
CS1
32
D. cayenensis
CS2
33
D. cayenensis
CS4
34
D. abyssinica
CSKB2
35
D. cayenensis
CSKB4
36
D. cayenensis
CT1
37
D. abyssinica
CT3
38
D. cayenensis
CTS1
39
D. cayenensis
CTS2
40
D. abyssinica
CTs4
41
D. praehensilis
WC8
42
D. praehensilis
WK4
43
D. praehensilis
WK5
44
45
D. cayenensis
D.bubifera
WY3
D.bul
46
D. schimperiana
D.sch
Esintie
Sheko District, outskirts of Sheko town
near the health center.
Kerkebat
Bench District, Gabuka Village
Surkachi
Sheko district, Mehal Sheko, Serer
village
Surkechibai
Sheko District, outskirts of Sheko town
near the health center.
Torbai
Sheko District, outskirts of Sheko town
near the health center.
Tsanu
Sheko district, Mehal Sheko, Serer
village.
Chebja (Wild)
Sheko District, Gaizika village, Onta
Kakeb (wild)
Sheko District, Gaizika village, Onta
Yasint (wild)
Ama
Bench District, near Woshikit School.
Bench district, 10 km from Kitea town
near forest coffee plantation
Bench district, 10 km from Kitea town
near forest coffee plantation
-
53
Figure 4. Map of Ethiopia showing the collection sites of Dioscorea accessions used in this study.
(▲= Or population, all collected from Oromia region ● = Sh population from Benchi-Maji zone of
SNNPRS (Southern Nations, Nationalities and Peoples Regional State) and
= Sn population, from
SNNPRS excluding Bench-Maji zone).
3.2.2. DNA extraction and purification
Total DNA was extracted from silica gel dried leaf materials using a modified CTAB
procedure from Doyle and Doyle (1987). 10 ml of isolation buffer (100 mM Tris HCl pH
8.0, 1.4 M NaCl, 20 mM EDTA, 2% CTAB) was preheated in 50 ml of Blue Cap tubes to
65 oC. Dried leaf material (0.1- 0.3 g) was ground into powder using a preheated mortar
and pestle. While grinding, small portions of the buffer were added until uniform slurry
was obtained. Further extraction of total DNA was carried out by adding equal volume of
SEVAG (24:1 chloroform: isoamayl alcohol) and rocking the mixture for about 30 minutes.
This was followed by spinning the tubes at 8000 rpm at 25 oC for 10 minutes. The aqueous
top phase containing the DNA was removed using Pasteur pipettes and transferred to 50 ml
54
Yellow Cap tubes. Then about twice the volume of ethanol (absolute) cooled to
-20 oC
was added to the extract by mixing it gently to precipitate the DNA. The mixture was
stored over night at -20 oC. The next day DNA pellets were collected by centrifuging the
mixture at 3200 rpm for 5 minutes. The pellets were allowed to dry in a fume hood at room
temperature for about 2-3 hours. Finally the dried pellets were resuspended in 500 μl of TE
buffer (10 mM Tris HCl pH 8, 0.25 mM EDTA) and stored at -20 oC until use.
Purification of nuclear DNA was carried out using spine column chromatography. Thus,
about 600 μl of binding buffer (NT buffer, 140 mM NaCl, 6 mM KCl, 1mM MgCl2, 2 mM
CaC12, 10 mM glucose) and 120 μl of the DNA extract were added into a mini spin
column (NucleoSpin®). The mixture was centrifuged at 12000 rpm for one minute
followed by addition of 750 μl of wash buffer (PB buffer, 0.15 M Na2HPO4 (anhydrous)
and 0.04 M NaH2PO4). After spinning the mixture at 12000 rpm for 1 minute, 50 μl of
elution buffer (TE buffer, 10 mM Tris base, 1 mM EDTA•Na2 , pH 7.5) was added and the
mixture was allowed to stand for 30 minutes at room temperature. Finally, the dissolved
DNA was collected in 0.5 ml Eppendorf tubes and stored at -20 oC. The quality of the
DNA was visually assessed by electrophoresis on a 1% agarose gel. The DNA
concentration was quantified using a spectrophotometer (Eppendorf Biophotometer) at 260
nm wave length, according to the manufacturer’s instructions.
3.2.3. AFLP analysis
AFLP analysis was performed according to the method of Vos et al. (1995), with a slight
modification in the restriction and ligation of the genomic DNA. Briefly, 0.5 µg DNA was
cleaved by restriction enzymes and ligated simultaneously in a mixture containing 10X T4
ligase buffer, 0.5 M NaCl, 1 mg/ml BSA, 50 pmol MseI adaptor pair, 5 pmol EcoRI
adaptor pairs (Applied Biosystems AFLP®) , 1 U MseI, 5U EcoRI and 1 U T4 ligase .
The mixture was incubated in a PCR machine (Applied Biosystems GeneAmp® PCR
System 9700) for a period of 2 hours at 37 oC. The products of the restriction ligation were
diluted with 95.5 ml of TE buffer and subsequently used for preselective amplification.
Preselective amplification was performed via 20 PCR cycles (94 oC for 30s, 56 oC for 30 s,
72 oC for
1 min) using 7.5 µl Core mix (Applied Biosystems AFLP®) and 0.5 µl
preselective primers for small genomes (Applied Biosystems AFLP®). The efficiency of
55
both the restriction and the preselective amplification reactions was assessed by visualizing
the banding pattern on a 1.5 % agarose gel. The products of preselective amplification
were diluted and subsequently used for selective amplification. Three selective primer
combinations were used; the selective primers sequences are given in Table 7.
The
products of selective amplification were then diluted with 10 µl formamide and 0.2 µl Rox
size standard, denatured and loaded on ABI Prism 3100 Genetic Analyzer for fragment
analysis.
Table 7. Selective primer sequences used in the AFLP analyses.
Primer combinations
Mse
Labeled with
EcoRI
5'-GATGAGTCCTGAGTAACAC-3'
5'-GACTGCGTACCAATTCTT-3'
5'-GATGAGTCCTGAGTAACAC-3'
5'-GACTGCGTACCAATTCAG
5’-GATGAGTCCTGAGTAACTT-3'
5'-GAC TGCGTACCAATTCAT-3'
FAM
-3'
JOE
NED
3.2.4. Data collection and statistical analysis
The presence/absence of unequivocally scorable bands was transformed in to a binary
character matrix (1 for presence and 0 for absence of a band at a particular position). For
cluster analysis and Principal Coordinate Analysis (PCO), pair wise distance matrices were
compiled by the NTYSYSpc 2.2 software packages, using the DICE coefficient of
similarity. A dendrogram was constructed by UPGMA.
The genetic diversity of the
populations and genetic divergence among the taxa were estimated by comparing the
frequency of rare fragments and the percentage of polymorphism.
56
3.3. Microsatellites
3.3.1. Plant material
Plant materials for the microsatellite analyses consisted of 58 accessions of wild and
cultivated yams collected during the months July, August and September of 2005 and 2006,
from a field trip conducted to South and Southwest Ethiopia (Fig 4). As in the case of
AFLP studies, the accessions were first named using the folk taxonomy and formal
taxonomic identification to species level was made later using the voucher specimens at
Royal Botanic Gardens, Kew. All the accessions used for microsatellite analyses belong to
the D. cayenensis complex (D. abyssinica, D. praehensilis and D. rotundata-D.
cayenensis).
3.3.2. DNA extraction and microsatellite markers
Total DNA extraction and purification nuclear DNA was carried out using the same
procedure as for the AFLP study. Variable microsatellite loci previously identified for
Dioscorea species by Tostain et al. (2006) were surveyed and 9 loci with strong,
unambiguous banding patterns were selected for use in this study (Table 8). These loci are
all composed of six different dinucleotide repeats (GT, TG, AC, GA, CT, and AG) with
repeat motifs ranging from 8-23.
3.3.3. PCR amplification
Polymerase chain reactions (PCRs) were carried out in a total volume of 10 μl , containing
50 ng of genomic DNA, 0.5 μl forward primer (10 pmol/ μl), 1 μl reverse primer (10
pmol/ μl), 1 μl M 13 primer with dye (2 pmol/ μl), 1 μl 10x NH4 reaction buffer (160mM
(NH4)2SO4, 670mM Tris-HCl (pH 8.8 at 25°C), 0.1% Tween-20), 0.2 μl MgCl2 (25 mM),
0.1 μl dNTPs (100 mM), and 0.1 μl BiotaqTM DNA polymerase (5 u/μl, Bioline). PCR was
performed on a GenAmp® PCR system 9700 thermocycler (AB, Applied Biosystems).
The PCR program involved denaturation at 94 oC for 5 min, followed by 35 cycles at 94 oC
for 30 sec, 51 oC (annealing temperature) for 1 min and 72 oC for 1 min, with a final
extension step at 72 oC for 8 min.
57
Table 8. Characteristics of the 9 microsatellite loci used in this study with number of alleles and allele
size range observed in the study population (where F = forward primer sequence and R =
reverse primer sequence)
Locus
Primer sequences (5’-3’)
Da1A01
F: TATAATCGGCCAGAGG
Repeat motif
Size (bp)
No. of alleles
Allele size range
(GT)8
204
8
212–260
(CA)8
309
12
335-400
(TG)13
177
11
165-220
(AC)12
305
5
285-340
(GA)19
190
13
175-220
(CT)19
174
7
170-205
(CT)23
152
7
105-150
(GA)15
151
7
160-190
(AG)15
128
5
120-145
R: TGTTGGAAGCATAGAGAA
Da1D08
F: GATGCTATGAACACAACTAA
R: TTTGACAGTGAGAATGGA
Da1F08
F: AATGCTTCGTAATCCAAC
R: CTATAAGGAATTGGTGCC
Da3G04
F: CACGGCTTGACCTATC
R: TTATTCAGGGCTGGTG
Dab2C05
F: CCCATGCTTGTAGTTGT
R: TGCTCACCTCTTTACTTG
Dab2D06
F: TGTAAGATGCCCACATT
R: TCTCAGGCTTCAGGG
Dab2E07
F: TTGAACCTTGACTTTGGT
R: GAGTTCCTGTCCTTGGT
Dpr3D06
F: ATAGGAAGGCAATCAGG
R: ACCCATCGTCTTACCC
Dpr3F04
F: AGACTCTTGCTCATGT
R: GCCTTGTTACTTTATTC
3.3.4. Detection and analysis of PCR products
Detection of amplification products was carried out by running the samples on a 1.5%
agarose gel containing 4 μl Ethidium Bromide solution. The procedure was as follows: 5 μl
of the PCR products were mixed with 5 μl of loading buffer (0.025 g Bromophenol blue
and 40 g Sucrose in 100 Milli Q water), the mixture was loaded on to the gel which was
subjected to electrophoresis in 1 X TBE buffer (45 mM Tris base, 45 mM Boric acid, 1
mM EDTA pH 8.0). The bands were revealed on a radiography film (Fig 5). The PCR
products were then diluted with 10 µl formamide and 0.2 µl of Rox size standard,
denatured and loaded on to ABI Prism 3100 Genetic Analyzer for fragment analysis.
58
Figure 5. The PCR products as revealed by electrophoreses on 1.5 % agarose gel
3.3.5
Data collection and analysis
3.3.5.1 Data collection.
Using GENESCAN and GENOTYPER 3.6 software (Applied Biosystems), loci were
initially scored as codominant marker data. Presence/absence data were then generated
form the codominant marker data matrices. Both types of data matrices were subjected to
various multivariate analyses using different softwares (see below). For parameters related
to population genetic analyses such as, estimation of the level of population differentiation
we used only 7 of the 9 loci as the alleles for two of the loci (DA1D08 and Dpr3F04) failed
to be amplified for the some of the study individuals (hence, to avoid inclusion of too
many missing data in the analyses). For each of the loci studied the genotyping data
observed produced only one or two alleles per sample (Fig 6).
59
Figure 6. Microsatellite electropherogram for the locus Da1A01 as revealed by GENESCAN and
GENOTYPER 3.6 software.
60
3.3.5.2 Taxonomic relationships between the taxa
In order to examine the relationships among the three taxa, distance trees were inferred
from the presence/absence data matrix constructed using 58 individuals and 91 attributes
(alleles) from the 9 loci studied. The similarity matrix generated was subjected to
multivariate analyses such as, UPGMA and ordination analysis (PCO) using NTSYSpc
version 2.2. A dendrogram was constructed by UPGMA using the option DICE coefficient
as a measure of similarity. The same similarity matrix was also used for PCO analyses.
3.3.5.3 Genetic diversity
Three populations (Or, Sn and Sh) were defined based on their geographical location
(Refer to map-Fig 4). Genetic polymorphism for each population was assessed by
calculating the number of alleles per locus (A), allelic richness (R), the observed
heterozygosity (Ho) and the expected heterozygosity (He) using the programs GENEPOP
version 3.1 (Raymound and Rousset, 1995), Microsatellite analysis (MSA) (Dieringer and
Schlotter, 2003) and FSTAT 2.9.3.2 (Goudet, 2001). The average expected heterozgosity
(He) within a population is the best general measure of genetic variation. There are a
variety of characteristics of average heterozygosity that makes it a valuable for measuring
genetic variation. It can be used for genes of different ploidy level and in organisms with
different reproductive systems. It is also a good measure of the response of a population to
natural selection. It can further provide the inbreeding coefficients of individuals. The total
number of alleles at a locus has also been used as a measure of genetic variation. This is a
valuable complementary measure of genetic variation because it is more sensitive to the
loss of genetic variation due to small population size, than heterozygosity. Accordingly, it
is an important measure of the long term evolutionary potential of populations. However,
unlike heterozygosity, it is highly dependent on sample size. Therefore, comparisons
among samples are not meaningful unless sample sizes are similar because of the presence
of many low frequency alleles in natural populations. This problem can be avoided by
using allelic richness, which is a measure of allelic diversity that takes into account sample
size. This measure uses a rarefaction method to estimate the allelic richness at a locus for
fixed sample size, usually the smallest sample size if a series of populations are sampled.
The effective number of alleles, defined as the number of alleles that, if equally frequent,
would infer the observed hetrozygosity, is also used to describe genetic variation at a locus.
61
However, this parameter provides no more information about the number of alleles present
at a locus than does heterozygosity (Allendorf and Luikart, 2007).
For each population-locus combination, departure from Hardy-Weinberg expectation was
assessed using exact tests (Guo and Thompson, 1992), with unbiased p-values estimated
through a Markov-chain method (Guo and Thompson, 1992). A global test across loci and
populations was constructed using Fisher’s method (Raymound and Rousset, 1995). The
comparisons of genetic diversity and population structure of wild and cultivated accessions
of the D. cayenensis complex were also carried out using some of the population genetic
parameters listed above.
3.3.5.4 Population structure
The level of population differentiation among the three subpopulations (Or, Sn and Sh)
mentioned above were estimated using unbiased estimated p-value for a log likelihood (G)
base exact test (Goudet, et al., 1996). Genetic differentiation was quantified using Fstatistics (Weir and Cockerham, 1984) by the computer program FSTAT2.9.3.2 (Goudet,
2001). Genetic relationships among the populations were assessed using distance trees
inferred from allelic frequency data. The distance matrix based on proportion of shared
alleles (Dps) was generated using the program MSA (Dieringer and Schlotter, 2003). The
distance matrix was then imported into PHYLIP computer package version 3.66
(Felsenstein, 1995) from which a neighbor joining phenogram was generated using the
program NEIGHBOR. The distance tree was then viewed using TREEVIEW.
62
4. Results
4.1 Morphometry
Figures 7 and 8 show the results of the cluster and ordination analyses based on the data
matrix generated from morphological characters. In the UPGMA analysis two of the
Dioscorea bulbifera accession included for comparisons represented branches outside the
study group. Within the study group the cultivated and wild accessions of D. cayenensis
complex were intermixed within the different phenon group (Fig.7) indicating that they are
closely related. Accordingly none of the clusters contained entirely those accessions treated
as the same species according to the existing species concepts. The first split within the
study group separates a single accession of D. abyssinica from the rest of the group.
However, all the remaining accessions in the study group are found scattered in the
phenogram. On the next level two accessions of D. praehensilis are separated from the rest,
but again several accessions of D. praehensilis are found scattered on the succeeding level.
The two phenons on the succeeding levels are separated by a very short distance and their
internal structure show no pattern in relation to the predelimitated taxa
The principal component analysis (PCA) indicated that the first 23 axes totally account for
100% of the observed variation between the accessions. The Eigen values of the first,
second and third components correspond to 51, 26 and 9% of the variability, respectively.
Accordingly, the first three axes explain cumulatively 86% of the total diversity within
Dioscorea species included in this study. A plot showing the first and second axes
clustered all three species of the D. cayenensis complex into one group (Fig 8). The two
individuals belonging to D. bulbifera were found to be well separated from the rest of the
group. This has also been demonstrated by the three dimensional plot of the first three PCA
axes accounting 86% of the total variation (Fig 9).
63
Figure 7. UPGMA cluster derived from a similarity matrix of 40 accessions of Dioscorea based on morphological
characters (Dcay, Daby, Dprh and Dbul refer to D. cayenesis, D. abyssinica, D. praehensilis and D. bulbifera
respectively)
64
1.50
1.00
0.50
PCA2
0.00
Dab
Dcay
Dprh
Dbul
-0.50
-1.00
-1.50
-2.00
-2.50
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
PCA1
Fig 8. Figure 8. Scatter plot showing the first and second axis of the PCA based on morphological data
for 40 individuals of Dioscorea (Dcay, Dab, Dprh and Dbul refer to D. cayenesis, D. abyssinica, D.
praehensilis and D. bulbifera, respectively
65
Figure 9. 3-D plot of the first three axes obtained by principal PCA analysis of morphological data
among 40 individuals Dioscorea species (Dcay, Daby, Dprh and Dbul refer to D. cayenesis, D.
abyssinica, D. praehensilis and D. bulbifera, respectively).
66
4.2 AFLP Analyses
4.2.1 Overall genetic structure of the populations
From the three primer combinations used a total of 245 different fragments ranging from
53 bp to 496 bp were generated. Although, amplification using the three primer pairs
generated a total of 245 fragments, only 158 of the AFLP fragments were scored for the
purpose of data analysis. Thus, 87 fragments were discarded from the analysis because
they either showed weak signal or because they were not reproducible for some of the
study individuals. Out of the selected 158 fragments 35 (22%) were found to be
monomorphic and 123 (78%) were found to be polymorphic. The number of fragments
generated per individual (and used for data analysis) ranged from 39 to 74. Altogether, five
fragments were found to be common to all the individuals belonging to the cultivated and
wild accessions of the D. cayenensis complex. These fragments could potentially be used
as diagnostic markers for the group.
4.2.2 Taxonomic delimitation and genetic relationships among the taxa
The results of the cluster and ordination analyses failed to produce any partitioning among
the taxa under study (Figs 10-13). The UPGMA tree shows no patterns of clustering of the
accessions into groups in relation to the existing species concepts (Fig. 10). The UPGMA
tree produced two major clusters, the level of similarity between these two clusters being
32%. The first split separated all the wild and cultivated accessions of D. cayenensis
complex (sharing 60-100% of the average genetic similarity) from D. bulbifera and D.
schmeperiana. The phenogram indicates that the genetic distance between individuals of
Guinea yam accessions and their wild relatives varies from 0 to 40%. Two accessions were
found to display identical AFLP profiles (Dcay13 and Dcay25) with 100% similarity.
However, these two accessions might not necessarily represent a single genetic entity due
to the dominant nature of AFLP markers. In the study group, the first split separates one
accession of D. abyssinica from the rest. On the next level five accessions of Guinea yams
identified as D. cayenensis are separated from the rest, however, several accessions of the
same species are found scattered on the succeeding levels.
67
Figure 10. UPGMA cluster derived from a similarity matrix of 48 accessions of the Dioscorea
cayenensis complex from SW Ethiopia using AFLP markers (Daby, Dsch, Dcay, Dprh and Dbul, refer
to D abyssinica, D. schemperiana, D. cayenensis, D. praehensilis and D. bulbifera, respectively).
68
Principal Coordinates Analysis (PCO) revealed that the first, second and third principal
coordinates axes account for 70%, 4% and 3% of the total variation, respectively. A plot
showing the first and second axes (Fig 11.) produced a scatter plot where the three species
within the D. cayenensis complex again were intermixed. These results are supported by
the three dimensional plot of the first three PCO axis which cumulatively account 77 % of
the total variation (Fig 12). As in the UPGMA analysis the species identified as D.
schimperiana and D. bulbifera are well separated from the accessions belonging to the D.
cayenensis complex. Another PCO plot excluding this two species produced a more
dispersed plot where no structuring can be elucidated (Fig 13).
0.30
0.20
0.10
0.00
PCO2
Daby
-0.10
Dsch
Dcay
Dprh
-0.20
Dbul
-0.30
-0.40
-0.50
-0.60
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
PCO1
Figure 11. Scatter plot showing the first and second axis of principal coordinate analysis of AFLP data
for 46 individuals of Dioscorea (Daby, Dsch, Dcay, Dprh and Dbul, refer to D abyssinica, D.
schemperiana, D. cayenensis, D. praehensilis and D. bulbifera, respectively).
69
Figure 12. Three principal axes of variation obtained by principal coordinate analysis of AFLP data
among 46 individuals of the Dioscorea cayenensis complex from SW Ethiopia (Daby, Dsch, Dcay, Dprh
and Dbul, refer to D abyssinica, D. schemperiana, D. cayenensis, D. praehensilis and D. bulbifera,
respectively).
70
Figure 13. Scatter plot showing the first and second coordinate axis of principal coordinate analysis of
AFLP data from 43 individuals of Dioscorea excluding D. bulbifera and D. schemperiana (Daby, Dcay
and Dprh refer to D abyssinica, D. cayenensis, and D. praehensilis, respectively.
As far as their genetic relationships are concerned, no diagnostic fragments were recorded
for any of the taxa in the study group. However, some fragments were found to be common
to all individuals of some pairs of taxa, while they are absent in some members of the other
taxon. For example, in addition to the 5 fragments shared with all memebers of the study
population all individuals of D. abyssinica and D. praehensilis share five more fragments,
while both D. abyssinica and D. praehensilis share only one more each with D. cayenensis.
This might be associated to the fact that both the former are wild s or at least managed
species native to the Sheko region, whereas the cultivated D. cayenensis may have been
transported or traded there from elsewhere.
Used as estimate of the diversity, the percentage of AFLP fragments (% Ptax) that are
polymorphic within each taxon were calculated. The lowest % Ptax was recorded for D.
praehensilis (59 %) and the highest (86 %) for D. cayenensis (Table 9).
71
Table 9. Percentage of polymorphic fragments, total number of fragments analysed per individual and
per population in the three taxa.
Name of the species
%
of
fragment
86
64
59
Dioscorea cayenensis
Dioscorea abyssinica
Dioscorea praehensilis
Polymorphic
Total number of fragments
per an individual
39-73
54-73
46-74
Total
number
of
fragments per taxon.
106
97
93
The distribution of rare and frequent fragments in each of the taxa was also compared as an
estimate of genetic divergence among individuals within the taxa, as an indirect way of
estimating genetic diversity. The results indicated that the rarest and the most frequent
fragments for the D. cayenensis accessions were found to be at lower frequencies (less than
0.3) compared to D. abyssinica and D. praehensilis. This indicates that higher genetic
divergence was observed among accessions of D. cayenensis (Fig. 14). However, the
sample size for both D. abyssinica and D. praehensilis is small. A common consequence of
small sample size is that low frequency polymorphism may remain undetected, so
increasing the number of individuals sampled per each taxa usually leads to better
assessment
of
the
overall
genetic
structure
of
the
group.
0.7
proportion of AFLP fragments
0.6
0.5
0.4
Daby
Dcay
Dprh
0.3
0.2
0.1
0
<25 %
25-50%
51-75%
>75%
Percent population
Figure 14. Distribution of rare and frequent AFLP fragments in each of the taxon as an estimate of
genetic divergence among 46 individuals of Dioscorea cayenensis complex from SW Ethiopia (Daby,
Dcay and Dprh refer to D abyssinica, D. cayenensis and D. praehensilis, respectively).
72
4. 3. Microsatellite Markers
4.3.1 Genetic diversity and population structure of Dioscorea species
4.3.1.1. Allelic variation at microsatellite loci
All the microsatellite (SSRs) used in the study are based on dinucleotide repeats, with
allele size ranging from 120 to 329 bp. The smallest difference between the highest and the
lowest allele size length was 12 bp at locus Da3Ga4 and the highest difference was found
to be 31 bp at locus Da1F08. When the alleles detected at each locus were sorted in
ascending order by their size, 62 % of adjacent alleles differed by one dinucleotide repeat
unit. However, 6 % of the adjacent alleles were separated by one base pair and 32 % by
more than two base pairs.
A total of 60 different alleles were recorded for the 7 loci studied, with the mean number of
alleles per locus equalling 9.2 (Table 11). Out of the 60 different alleles 27 were found to
be private alleles present only in one of the three population samples (16 alleles for Sh, 9
for Or and 2 for Sn populations). The frequency of such private alleles ranges from 0.01 to
0.048. On the other hand the allelic frequency in the study population was found to be
between 0.009 (at locus Da1F08) and 0.93 (at locus Da3G04).
At all the loci, a higher number of alleles were detected in the Sh population (44 alleles)
followed by Or (37 alleles) and Sn populations (31) (Table 10). Altogether 18 alleles were
found to be shared among the three populations in the study group. When the shared
number of alleles are compared, the Sh population shared 22 alleles with Or and 23 alleles
with the Sn population, whereas the Or and Sn population shared 18 alleles.
Table 10. The observed number of alleles per locus and population
Population
Dba2D06
Da3G04
Da1F08
Dpr3D06
Da1A01
Dab2E07
Dab2C05
total
or
5
1
8
5
3
6
9
37
sh
6
5
7
3
6
8
9
44
sn
4
1
6
5
5
3
7
31
Total
7
5
11
7
8
9
13
60
Six of the seven loci studied (except Da3G04) were polymorphic across all the three
populations sampled, with the number of alleles per locus ranging from 5 (at Da3G04) to
73
13 (at Dab2C05). At most of the SSR loci, alleles were detected at lower frequencies. Thus,
60 % of the alleles in all the loci were found to be at frequencies less than 0.1. The
distribution of observed allele size (number of bp) at each locus and among the three
populations were irregular except for loci Da3G04 and Da1F08, which showed
unimodality (Fig 15).
Da3G04
Dab2D0 6
1.2
0.6
0.5
0r
0.4
0.3
Sh
0.2
average
sn
0.1
0
177
179
181
183
191
193
Allele frequency
Allele frequency
0.7
1
Or
0.8
Sh
0.6
Sn
0.4
average
0.2
0
195
317
319
Allele size
321
Or
Sh
Sn
Average
161
165
167
169
171
175
0.6
Allele frquency
Allele frquency
0.7
0.5
Or
0.4
Sh
0.3
Sn
0.2
Average
0.1
0
179
179 181 187 189 193 195 199 203 205 209 210
Allele size
Allele Size
Dab2E07
0.8
0.7
0.6
0.5
0.4
0.3
Allele frequency
Da1D08
Allele frequency
329
Da1F08
Dpr3D06
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
328
Allele Size
Or
Sh
Sn
Average
0.2
0.1
0
221
227
229
231
234
236
238
0.6
0.5
Or
0.4
Sh
0.3
Sn
0.2
Average
0.1
0
248
120
Allele size
124
131
132
134
136
138
140
142
Allele size
Allele frequency
Dab2C05
0.35
0.3
0.25
Or
0.2
Sh
0.15
Sn
0.1
average
0.05
0
1
184 86 188 190 192194 196 198 201 203 207 211 213
Allele Size
Figure 15. The distribution of observed allele size (number of bp) at each locus and among the three
populations
74
The Sh population displayed the highest level of allelic diversity in 5 of the 7 loci studied,
compared to Or (2 loci) and Sn population (none). However, for one locus (Dpr3D06) the
Sh population showed the lowest level of allelic diversity compared to both Or and Sn
populations (Fig 16). Pooling together all the 7 loci the Sh population displayed the highest
level of allelic diversity followed by the Or populations.
Figure 16. Allelic diversity per locus for each population based on the measure of allelic richness (R).
4.3.1.2 Genetic variation within population
Genetic diversity parameters based on allelic frequencies are shown in Table 11. In
individual populations the mean number of alleles per locus (A) varied from 4.43 (Sn
population) to 6.29 (Sh population) with an average of 6.09. While effective number of
alleles per locus (Ae) varied from 5.86 (Sn population) to 6.45 (Sh population) with an
average of 6.09. Allelic richness (R) which is a measure of allelic diversity taking into
account sample size, ranges from 3.28 (Sn population) to 5.65 (Sh population) with an
average of 4.47. The observed heterozygosity (Ho) ranged from 0.457 to 0.507 with an
average of 0.481. The average expected heterozgosity (He) equalled 0.590 and varied from
0.539 to 0.636. All the above results indicated that the Sh population displayed greater
allelic or genetic diversity compared to both the Or and the Sn populations. When pooling
together all the 7 loci, the mean number of alleles per locus, effective number of alleles per
75
locus, allelic richness, and expected and observed heterozygosity for the meta population
were found to be 9.20, 8.57, 5.1, 0.49 and 0.64, respectively.
The percentage of polymorphic loci (P) in the three populations studied ranges from 85.7%
to 100% corresponding to a mean polymorphism of 90.47%. The Sh population displayed
the highest level of polymorphism (100%), while both the Sn and the Or population
possessed one monomorphic locus (Da3Ga4) with an overall allelic polymorphism of
85.7%. One of the loci (Da3Ga4) was found to be fixed for the Or and Sn populations.
Table 11. Allelic variability at the seven SSR loci in the study populations (N=population size, A= mean
number of alleles per locus, Ae=effective number of alleles per locus, R=allelic richness, P= percentage
polymorphic loci and Ho, He , average observed and expected heterozygosity respectively)
Pop
N
A
Ae
R
P
Ho
He
Or
Sh
Sn
14
18
26
5.29
6.29
4.43
5.96
6.45
5.86
4.49
5.65
3.28
85.71
100
85.71
0.457
0.480
0.507
0.595
0.636
0.539
Mean
19.3
5.34
6.09
4.472
90.47
0.481
0.590
Meta pop
58
8.57
9.20
5.1
90.47
0.490
0.640
Comparison of the genetic within population diversity of the three populations based on
the average expected heterozgosity indicated the highest level of diversity for the Sh
population (He = 0.636), while the lowest value corresponds to the Sn population (He =
0.539). In relation to the other populations the Sh population was collected from localities
in close geographical vicinity.
4.3.1.3 Population genetic structure
The genetic analyses revealed moderate differentiation among the three populations (Or,
Sn, Sh). The FST value which reflect the proportion of the observed genetic variation that
can be explained by partitioning among populations, ranged from 0.005 for locus DA1F08
to 0.16 for locus Dpr3DO6, with an average value of 0.088. This showed that only 8.8% of
the genetic diversity is found among the three populations, indicating a moderate
76
differentiation among them. In other words the heterozygosity in the entire population of
the Dioscorea cayenensis complex sampled in South and Southwest Ethiopia decreased by
ca 8.8% as a result of the partition among the three populations. Although, the FST value
recorded for these populations was found to be small, it is highly significant (P = 0.0001).
Wright’s F- statistic for each locus is summarized in Table 12. Altogether 5 of the 7 loci
showed a statistically significant FST values ranging from 0.035 to 0.15 (P ≤ 0.004). Two of
the loci (Dab2D06 and Dpr3D06) possessed the highest FST value (0.15 and 0.16,
respectively). Thus the level of heterozygosity in the entire population of the study group
was found to be lower than we would expect (if the whole population is panmictic) by
15% (Dab2D06) and 16% (Dpr3D06) due to the partitioning between the three populations.
These values are highly significant (P ≤ 0.0001) and with respect to these two loci the three
populations are highly differentiated. Loci Da1A01 and DA1F08 exhibited the lowest FST
values (0.001 and 0.005, respectively) thus, contributing small share (0.1 and 0.5%,
respectively) in reducing of the level of heterozygosity in the entire population as a result
of subdivision. Accordingly, with respect to these loci the there is no differentiation among
the three populations.
Pairwise comparison of genetic differentiation among the three populations indicated that
the Sh and the Sn populations are genetically closest with an FST of only 4%, whereas Or
population differ from both (Sh and Sn) with FST values of 13 and 12%, respectively. All
the three pairwise comparisons are highly significant (P ≤ 0.0003).
77
Table 12. Relative measurements of genetic differentiation among populations in the study group
Locus
global FST
P-value:
global FIT
global FIS
Dab2D06
0.151
0.0001
0.103
-0.056
Da3Ga4
0.120
0.0001
0.859
0.840
Da1F08
0.005
0.233
-0.022
-0.027
Dpr3D06
0.164
0.0001
0.242
0.092
Da1A01
0.001
0.2757
-0.070
-0.071
Dab2E07
0.139
0.0001
0.570
0.500
Dab2C05
0.036
0.0035
0.326
0.300
Over all loci
0.088
0.0001
0.235
0.161
4.3.1.4. Genotypic structure and deviation from Hardy-Weinberg equilibrium
Global tests for the departure from Hardy-Weinberg equilibrium showed a statistically
significant deviation in the study populations (P ≤ 0.0001). The departure from HardyWeinberg equilibrium was primarily due to heterozygote deficit.
FIT is the over all
inbreeding coefficient of an individual relative to the whole set of populations, while FIS is
the inbreeding coefficient relative to its own population. The global FIT value 0.235
indicated that overall, there is a heterozygote deficit in the study populations. Among the
study population, 3 of the 7 loci showed a significant deficit in heterozygotes (DA3G04,
Dab2E07 and Dab2C05, P ≤ 0.0003) and 3 loci showed excess of the heterozygotes
(negative FIS value) relative to Hardy-Weinberg expectation (Tables 12 and 13), with the
average FIS value equalling 0.16. None of the 7 loci showed a significant excess of
heterozygotes.
78
Results from multi locus tests for deviation from Hardy-Weinberg equilibrium
expectations showed that the Sh and Or populations exhibit a significant deficit of
heterozygotes (FIS = 0.22 and 0.17 respectively, P ≤ 0.0001). However, for the Sn
population the results of the test for heterozygote deficit was not found to be significant
(FIS = 0.007 P = 0.49).
Only 0.7% of the Sn population deviates from Hardy-Weinberg
expectations compared to the 17% in Or and 22% in Sh.
Table 13. Expected and observed heterozygosity (He and Ho) and Fixation indexes (FIS) per locus and
population at the seven microsatellite loci.
Locus/Pop.
Dab2D06
Ho (He)
FIS
Da3Ga4
Ho (He)
FIS
Da1F08
Ho (He)
FIS
Dpr3D06
Ho (He)
FIS
Da1A01
Ho (He)
FIS
Dab2E07
Ho (He)
FIS
Dab2C05
Ho (He)
FIS
Mean
Ho (He)
FIS
Or
Sh
Sn
Mean/ FIS global
0.46 (0.60)
0.29
0.69 (0.64)
-0.065
0.8 (0.67)
-0.24
0.65 (0.64)
-0.056
0 (0)
NA
0. 07 (0.45)
0.84
0 (0)
NA
0.02 (0.15)
0.84
0.83 (0.73)
-0.15
0.72 (0.69)
-0.07
0.58 (0.61)
-0.002
0.71 (0.67)
-0.027
0.5 (0.62)
0.21
0.3 (0.48)
0.39
0.75 (0.67)
-0.26
0.51 (0.59)
0.092
0.45(0.58)
0.23
0.65 (0.57)
-0.17
0.58 (0.50)
-0.24
0. 56 (0.55)
-0.071
0.30 (0.72)
0.42
0.56 (0.78)
0.29
0.18 (0.53)
0.65
0.35 (0.67)
0.5
0.64 (0.89)
0.14
0.44 (0.85)
0.45
0.65 (0.79)
0.15
0.58 (0.84)
0.3
0.46 (0.60)
0.17
0.49 (0.64)
0.22
0.51 (0.54)
0.007
0.48 (0.59)
0.161
79
4.3.1.5. Genetic relationships among the three subpopulations
Genetic relationships among the populations were assessed using distance trees inferred
from allelic frequency data. The distance matrix based on proportion of shared alleles (Dps)
was used to generate a neighbor joining phenogram (Fig 17). The results indicated that
there is no clear partitioning among the three subpopulations. However, in some of the
clusters individuals collected from same locality tend to cluster together. For example the
individuals or00 8, or009, or011, or012 and or013 and the cluster containing sn002, sn020,
sn009, sn001, sn018 and sn006 are collected from geographically close areas, the former
along the Nedjo Ghimbi road and the latter from the Areka area.
80
.
Figure 17. Neighbour joining (NJ) tree inferred from allelic frequency data of microsatellite data for 59
individuals of Dioscorea species (Sn, sh and or refer to the three populations defined based on their geographical
areas) ( N.. B. Or003=sn003)
81
4.3.2. Genetic relationships of the wild and cultivated Guinea yam species
For most of the seven loci examined a higher number of alleles were detected in the
cultivated accessions compared to the wild forms. Considering the entire set of germplasm
under study, 17 alleles were present only in the cultivated accessions and 8 alleles were
found to be unique to the wild forms. Altogether, 36 alleles were shared between the
cultivated and wild accessions. However, based on the measure of allelic richness (R), the
wild forms exhibited a greater diversity in all the 7 loci studied (Fig. 18)
Figure 18. A comparison of allelic diversity per locus in wild and cultivated accessions of SW Ethiopian
the D. cayenensis complex based on the measure of allelic richness
Using expected heterozygosity as a measure of genetic diversity, the wild accessions of
Dioscorea cayenensis complex displayed a greater diversity (He = 0.79) compared to the
cultivated forms (He = 0.60). The same result was obtained when comparison was made at
a locus level. Thus, in all the 7 loci studied the wild forms displayed greater gene diversity
(Table 14).
82
Table 14. Gene diversity (expected level of heterozygosity) per locus and population of wild and
cultivated accessions.
Loci
Growth
habit
Cultivated
Wild
Dba2D06
Da3G04
Da1F08
Dpr3D06
Da1A01
Dab2E07
Dab2C05
mean
0.72
0.82
0.14
0.41
0.63
0.85
0.64
0.86
0.52
0.73
0.72
0.86
0.84
0.97
0.60
0.79
mean
0.77
0.28
0.74
0.75
0.63
0.79
0.91
0.69
The FST value which reflects, the proportion of the observed genetic variation that can be
explained by partitioning between populations was found to be low (FST = 0.03) but
significant (P = 0.006) demonstrating little differentiation between the wild and cultivated
species sampled in the study sites.
4.3.3. Taxonomic relationships among the three taxa inferred from microsatellite data.
The UPGMA tree shows no patterns of clustering of the accessions into groups that could
be interpreted in terms of the existing species concepts (Fig 19.). Instead the distance tree
grouped the accessions broadly according to their geographical region in which they were
collected. The average genetic similarity among the Guinea yam accessions and their wild
relatives used in this study ranged from 77% to 100%. The first split of UPGMA tree
separated one wild accession (DprhsH71) from the rest.
The second and third split
contained two accessions each identified as different species. Although, they are different
species the accessions in each phenon belong to the same population (the first to the Sh and
the second to the Or populations). Similarly, the succeeding levels group the accessions
based on mainly their geographical location rather than taxonomic identity Altogether ten
duplication groups with identical allelic profiles in all the loci studied were obtained from
the cluster analysis (Fig 19.). Most of the duplicates observed include accessions collected
from the same geographical area. In one of the duplicates two accessions identified as
different species were found to exhibit the same profile (Dcaysh58 and Dprhsh33). They
were found to be cultivated accessions collected from the same farm in the Sheko locality.
The farmer who owns them identified the accessions as two different cultivars.
83
Figure 19. A dendrogram derived from a similarity matrix of 61 accessions of Dioscorea using
microsatellite markers [labels indicate species name and geographical location or the population where
that particular accession belongs, for example DabyOR refers to D. abyssinica belonging to Or
populations (Note: DabyOR50=DabySH50)].
84
Associations among the 59 Dioscorea cayenensis complex accessions revealed by
principal coordinate analysis (PCO) calculated from the microsatellite based similarity
matrix are presented in Figure 18. The first principal coordinate axis (PCO1) and the
second axis (PCO2) accounted 83.8% and 2.8% of the total variation, respectively. Neither
of the two axes separated the three taxa into distinct groups (Fig. 20).
0,40
0,30
PCO2 (2.8%)
0,20
0,10
Dab
0,00
Dcay
Dprh
-0,10
-0,20
-0,30
-0,40
0,80
0,82
0,84
0,86
0,88
0,90
0,92
0,94
0,96
0,98
PCO1 (83.8 %)
Figure 20. Scatter plot showing the first and second axis of Principal Coordinate Analysis of
microsatellite data for 61 individuals of the D. cayenensis complex in the study population (Dab, Dcay
and Dprh refer to D. abyssinica, D. cayenensis and D. praehensilis, respectively).
85
5. Discussion
5.1 Taxonomic relationships in the study group: one species, different species or a
species complex?
Relatively few studies have been conducted to understand the taxonomic relationships
among the various species of yams (Hamon and Toure 1990b; Terauchi et al. 1992,
Mignouna and Dansi, 2003; Mignouna et al. 2005; Scarcelli et al. 2006a). Different
authors consider Guinea yams (and their wild relatives) to be represented either by one or
two species or even by a species complex (Ramser et al. 1997). In the present study the
phenogram and PCO/PCA scatter plot based on morphological, AFLP and microsatellite
markers failed to produce a clear partitioning of the individuals into discrete taxa that could
be interpreted according to the existing classification system. Other clear taxonomic
structures were neither revealed.
Our results stand in agreement with Martine and Rhodes (1978), Terauchi et al. (1992),
and Hamon et al. (1997), who all emphasized the lack of genetic structure within the
different species within D. cayenensis complex. However, our results are in contrast with
Akoroda and Chheda (1983), Ramser et al. (1997), Dansi et al. (2000), Mignouna et al.
(2005) and Scarcelli et al. (2006a) who claimed that the different species of Guinea yams
represent different genetic entities. All the mentioned publications are based on West
African materials. These studies argued that isozyme and chloroplast DNA analyses by
Terauchi et al. (1992) and Hamon et al. (1997) did not show significant differentiation
between the putative species, probably because of the low levels of polymorphism these
markers exhibit.
Ramser et al. (1997), Mignouna et al. (2005) and Scarcelli et al. (2006a) used PCR based
methods such as RAPD and AFLP and proposed that the species of Guinea yams should be
treated as separate taxa. The discrepancy between our results and that of Ramser et al.
(1997); Mignouna et al. 2005; Scarcelli et al. (2006a) could be viewed in terms of sample
size used or validity of sampling, the difficulty in determining taxonomic status based on
morphology, differences in the methodology employed and handling of the data during the
analyses. Ramser et al. (1997) and Mignouna et al. (2005) basically used RAPD markers
which solely depend on comigrating bands for estimating relatedness among taxa. The
86
general validity of this approach has been challenged, because certain percentage of
comigrating bands may in fact be non-homologous, producing a random background noise
that can influence the result (Allendorf and Luikart, 2007). The algorithm used to calculate
genetic distance based on RAPD markers may vastly over or under estimate true similarity.
Thus the assumption of 100% similarity at a locus or allele where a band is shared between
two accessions is not necessarily valid. The problems are even worse in a polyploid
genome, where the marker may be present in any of several dosage states. Thus, a
dominant marker present at a single, double, triple or quadruple dose in one accession and
in the same possible range of dosage state in another accession will result in similarity
estimates of 100 % where as the actual similarity estimated could be as little as 25 %
(Mignouna et al., 2003). Further more, Mignouna et al. (2005) used accessions with 12
duplication groups with identical RAPD profiles (comprising more than 50 % of the
individuals in the study group). This might indicate that most of the accessions used in the
study represent clones with identical within population genetic background.
Ramser et al. (1997) employed a small sample size representing a wide range of
geographical area, with all the plants grown at ITTA. Since cultivated Dioscorea species
are propagated vegetatively, each of the individuals collected from wide range of
geographical area are expected to show different pattern of heterozygosity and hence
appear as genetically distinct groups. According to Ramser et al., (1997), in view of the
500-2500 agricultural varieties that probably exist, a conclusive definition of the
taxonomical status of Guinea yams would be difficult without analysing the majority of
these varieties.
Scarecelli et al. (2006) employed a different approach to study the taxonomic status of
Guinea yams and their wild relatives. Thus, they grouped the different plants under
cultivation into two classes: predomesticates and cultivated varieties. Those considered as
predomesticates were accessions which were under cultivation, but in the course of
domestication. In the cluster and PCO analysis, this group was found to cluster with any of
the three species identified as D. cayenensis-D. rotundata, D abyssinica, and D.
praehensilis. In this study individual plants showing intermediate characteristics and
accessions that did not fit into their morphological identification were also discarded from
the analyses. Such an approach would clearly bias the study towards detecting discrete taxa.
Furthermore, the authors scored 91 AFLP markers from 4 primer combinations (in contrast
87
to 158 markers from 3 primer combinations in our analysis). Some of the discrepancies
observed could also be attributed to the difficulties in determining the taxonomic status of
the germplasm on the basis of morphology. There are some reports which indicate that
individuals with identical morphotype might display different genotypes (Mignouna and
Dansi, 2003; Mignouna et al., 2003). In our study we encountered the converse, i.e. two
individuals (Dcaysh58 and Dpr3sh53) with different morphotypes displayed identical
microsatellite allelic profiles.
Based on the results from morphometric and molecular markers (AFLP and microsatellite)
analyses, the variation in the study group was found to be continuous. Therefore, at least
the wild or managed populations and cultivated plants of South and Southwestern Ethiopia,
the D. cayenensis complex is a single taxonomic entity. In a book summarizing the studies
on West African Guinea yams, Dumont et al., (2006) reached a similar conclusion and
proposed that D. praehensilis, D abyssinica and D rotundata-D.cayenensis should be
regarded as a single taxonomic entity. There are no discrete morphological characters to
separate the “species” in the study group into distinct taxa at specific or subspecific rank. If
regarded as a single species or species complex the correct name will have to be
D. cayenensis (Lamark, 1789). Morphological, AFLP and microsatellite markers used in
this study indicated no consistent genetic differentiation as far as three taxa are concerned.
Rather a largely geographically structured clustering pattern was observed, when using the
microsatellite markers. A comparative study on Ethiopian materials and a few West
African accessions by Muluneh Tamiru (2006), based on AFLP revealed a similar result.
Thus, the Ethiopian accessions were found to be genetically close to each other than they
were to their putative conspecific West African population. However, they showed some
degree of distinctiveness compared to the West African accessions (Muluneh Tamiru
2006). The author suggested that the distinctiveness of the Ethiopian Guinea yams may
represent a divergent evolutionary path way isolated from the widely known centre of
diversity in West Africa.
5.2. Total genetic diversity and level of polymorphism.
Both the molecular markers (AFLP and microsatellites) used in this study detected a high
degree of intraspecific variation and a low degree of interspecific variation. Comparable
results have been reported in most of the studies on Guinea yams of West Africa (e.g.
88
Ramser et al. 1997; Scarecelli et al., 2006). A higher level of polymorphism was obtained
for both AFLP (78 %) and microsatellite (90.47 %) markers. The level of polymorphism
detected by AFLP in this study was found to be less than the reports from previous studies
on Giunea yams from West Africa by Mignouna et al. (2003) (90.2 %) and Scarcelii et al.
(2006) (94.9 %) using the same marker. In both studies however, 4 pairs of primer
combinations were employed compared to 3 pairs of primer combinations, used in this
study. Furthermore, in our study, AFLP analyses were carried out using accessions
collected from the Sheko area only. On the other hand, the level of polymorphism
determined by microsatellite markers (90.47 %) was found to be greater than in previous
reports, 80.5 % was reported by Mignouna et al. (2003) which were based on analyses of 9
loci.
AFLP analyses revealed a high degree of similarities among the D. cayenensis complex
accessions from Sheko area (coefficients of similarity ranging from 0.6 to 0.94). This
indicates that the individuals included in this study are genetically closely related. The
values of the coefficient of similarity from AFLP analyses are comparable with those
obtained from West African accessions of Guinea yams by Mignouna et al., (2005).
Compared to the wild forms, the AFLP markers used also revealed a high genetic
divergence between the accessions in the cultivated forms. This might indicate that the
farmers in the study area (Sheko and its environs) cultivate different cultivars of yams with
a broad genetic basis. Similar results were also reported by Mignouna et al., (2005) for D.
rotundata accessions from Nigeria. The author associated the observed high diversity in
the cultivated forms with the availability of the wild yams with cropping potential,
different selection pressures, successive domestication and somatic mutations. Although,
high genetic divergence was obtained between the accessions of cultivated Guinea yams,
comparison of wild yams and cultivated Guinea yams using SSR markers based on the
measure of allelic richness indicated that the wild yams exhibited the greatest allelic
diversity. Wild yams are, therefore, important for yam breeding because they could act as
reservoirs of useful genes for agronomic characteristics such as yield, storability, tolerance
to drought and weeds, organoleptic qualities, and tolerance to pest and diseases.
The total number of alleles amplified for the 7 microsatellite markers was found to be 60,
with an average of 8.6 alleles per locus. A similar study by Tostain et al., (2005) on 156
accessions of Dioscorea species from Benin using 17 SSR markers revealed a total of 124
89
alleles with an average of 7.3 alleles per locus. In our study the observed heterozygosity
values were found to vary from 0.15 to 0.67 with an average of 0.48. A similar study by
Tostain et al. (2005) reported an average observed heterozgosity value of 0.58 (0.0 to 0.94).
The average expected heterozgosity (He) within a population is the best general measure of
genetic variation. The expected heterozygosity for the accessions within D. cayenensis
complex used in this study varied between 0.018 (for Da3Ga4) and 0.86 (Dab2C05) with
an overall value of 0.64 for the metapopulation. This indicates that there is considerable
genetic diversity in the wild and cultivated accessions of Dioscorea species from Ethiopia.
Although, the species in the study group have long been considered as polyploidys, the
genotyping data observed produced only one or two alleles per sample for each of the loci
studied. This might indicate that all the accessions in the study group are actually diploids.
Such results have also been reported by Scarcelli et al. (2006) form studies on West
African accessions of Guinea yams and their wild relatives.
5.3 Level of heterozygosity
Out crossing plants with dioecious floral morphology are expected to have a high level of
genetic heterozygosity within populations (Avise, 1994). In principle the Dioscorea
species evaluated in this study fit into this group of plants but in contrast to the theory, the
observed number of heterozygotes (Ho) was less than the expected (He) in 11 of the 21
locus specific comparisons. The FIS values for these locus specific comparisons were found
to be greater than zero, and according to Gibbs et al. (1997) this could be associated with
non-random association of alleles in the three populations tested (Gibbs et al., 1997).
Studies on Dioscorea species form Benin by Tostain et al. (2005), revealed a significant
excess of heterozygotes in 9 of the 15 polymorphic loci studied. In our study, however,
none of the seven loci showed a significant excess of heterozygotes. The levels of
heterozgosity found in the study group were, in most cases lower than expected. The
deficit of heterozygotes relative to Hardy-Weinberg proportions for microsatellite markers
could be explained by a) presence of an unrecognized genetic structure within populations
(Wahulund effect) b) inbreeding, that is the tendency for related individuals to mate.
c) Presence of null allele such that many apparent homozygotes are, in reality
heterozygotes
90
The most general cause an excess of homozygotes is non-random mating or population
subdivision. The presence of multiple demes within a single population sample will
produce an excess of homozygotes at all loci for which the demes differ in allelic
frequency. Inbreeding within a single deme will produce a similar genotypic effect.
However, large proportion of the study individuals are cultivated accessions of Guinea
yams, which are propagated vegetatively.
The best way to discriminate between non-random mating (either inbreeding or including
multiple populations in a single sample) and null alleles to explain an excess of
homozygotes is to examine if the effect appears to be locus-specific or population-specific.
All loci that differ in allele frequency between demes will have a tendency to show an
excess of homozygotes (Allendorf and Luikart, 2007). In our study out of the 21 (7 loci x 3
subpopulations) possible tests, heterozygote deficiency was detected in 11 of the tests (5
positive FIS value out of the 7 possible for the Or population, 4 for the Sh population and 2
for the Sn population). It seems that there is an unrecognized population structure in both
the Or and the Sh populations, i.e. both populations might include more than one deme.
This may also be demonstrated by the observed allelic frequencies in all loci within and
among populations. Fine scale differentiation within and among the study populations
could be reflected in a substantial difference between observed and expected
heterozygosity. In our study 60 % of the observed alleles in all loci were found to be at
frequencies lower than 0.1 (most of the alleles are rare alleles). Such a high proportion of
rare alleles COULD BE a good indicator of high genetic divergence among the accessions
in each population. This may explain the significant difference in the observed and
expected heterozygosity within the study populations. And hence unrecognized (fine scale)
genetic differentiations within the study populations might have been the cause of the
observed heterozygote deficiency.
A homozygote excess due to null alleles should be locus-specific. When the 7 loci used in
our study are compared 3 (Da3Ga4, Dab2E07, and Dab2C05) of them showed a significant
excess of homozygotes for all the three different populations studied, whereas, 3 other loci
(Dab2D06, Da1F08, and Da1A01) displayed an excess of heterozygotes (but not
significant) relative to Hardy-Weinbergs expectation, in at least one of the study
populations. Further comparison of the FIS values for each of the 60 alleles at a locus level
indicated that at least one allele from each of the 7 loci studied showed excess of the
91
heterozygote or homozygote deficit (negative FIS value). This demonstrates that null alleles
might be ruled out as a possible explanation for the observed deficit of heterozygotes in the
study population. However, Tostain et al. (2005) associated the significant excess of
homozygotes estimated at loci Da3G04 for Guinea yams of West Africa, with the presence
of null alleles.
5.4. Population structure
Understanding the patterns of genetic differentiation among populations is crucial for
protecting species and developing effective conservation plans. In addition developing
priorities for conservation of a species requires an understanding of adaptive genetic
differentiation among populations. Perhaps most importantly, an understanding of
population genetic structure is essential for identifying units to be conserved (Allendorf
and Luikart, 2007).
Methods of quantifying genetic differentiation from microsatellite data are an area of
significant debate (Goldstein and Pollock, 1997). Some measures have been developed
specifically for these markers, which takes into account variation in allele size under the
stepwise mutation model (SMM) (Kimura and Ohta, 1978). In microsatellites, a majority
of mutations may be caused by slipped strand mispairing during replication resulting in
small gains or losses of repeat copy number rather than in large changes. This type of
mutation behaviour is better explained by SMM. The basic idea behind SMM is that
mutations predominantly differ from their previous state by the change of a single repeat
unit. This type of mutational process results in a unimodal distribution of allele size
(Weising et al., 2005). Hence, under the SMM model alleles of similar size are assumed to
be more closely related to each other than those of very different size. The theoretical
framework for allozyme-based population genetics assumes that any new allele created by
mutation is unrelated to ancestral alleles (Infinite allele model, IAM). On the basis of the
SMM, Goldstein et al. 1995 and Slatkin, (1995) independently proposed a method to
evaluate the genetic distance between microsatellite loci that includes allelic repeat scores.
Goldstein et al. (1995) showed that these distances are a linear function of time. As a result,
SMM based measures of population differentiation are expected to be most accurate for
populations (taxa) that diverged long enough ago that current genetic differentiation
reflects mutations accumulated since divergence (Goldstein and Pollock, 1997). In contrast
92
traditional measures based on the IAM should be more appropriate for intraspecific
comparison (Weising et al., 2005).
In this study all the measures used to quantify
population differentiation are based on the IAM model.
The genetic structure of a population has been characterized as the non-random distribution
of alleles and genotypes in space. The presence of variability within species (among
populations and also between individuals within populations) is essential for their ability to
survive and to successfully respond to environmental changes. In all the comparisons made
in our study a low mean FST (but significant) has been observed, indicating that the
majority of microsatellite diversity in the Dioscorea cayenensis complex populations under
study was found within rather than among populations. Pairwise comparison of the three
subpopulations (Or, Sh and Sn) defined based on their geographical location indicated that
the Sh and Sn populations are genetically close to each other, both compared to the Or
population. This could probably be associated with gene flow mediated by exchange of
planting materials between farmers. Gene flow reduces the genetic differences between
populations and increases the genetic variation within populations (Allendorf and Luikart,
2007). Gene flow among populations is the cohesive force that holds together
geographically separated populations into a single evolutionary unit. Comparison of
populations of the cultivated Guinea yams and their wild relatives also resulted an FST
value of only 3 %, even lower than the FST values obtained by pairwise comparisons of the
three subpopulations defined based on geographical location, but highly significant (P =
0.006). This indicates that there is some degree of differentiation between the wild and
cultivated accession of Dioscorea cayenensis complex populations under study. Gene flow
between wild and cultivated species is mainly manifested by the still ongoing
domestication practice by farmers in the study area. Spontaneous Gene flow between wild
and cultivated Guinea yams has also been reported based on studies of West African
material (Scarcelli et al., 2006b). In general, the observed high allelic diversity and
heterozygosity within the study populations and low FST estimates might suggest that
genetic drift has not yet had a major influence on the Dioscorea cayenensis complex
populations in the study area. In addition, as a crop plant selection by humans might have
played a great role in shaping the genetic structure of Dioscorea cayenensis complex
accessions under study.
93
5.5 The implications for yam domestication and conservation in Ethiopia
Cultivated yams of the D. cayenensis complex are vegetatively propagated. In West Africa,
yam fields in traditional agroecosystems are seeded with tuber fragments from the previous
harvest (e.g. Scarcelli et al. 2006a). No direct seed use by farmers has been reported. In
contrast, wild yam species reproduce sexually (Ayensu & Coursey, 1972; Coursey 1976;
Hildebrand et al., 2002, pers. obs.). In South West Ethiopia, it is common practice for
farmers to collect “wild” or managed yam tubers in the forest as food and to plant them
under trees in their home garden (Hildebrand et al., 2002). This process means that
domestication is a constantly repeating process. If these or other yams in cultivation
produce sexual organs, then gene exchange between wild and cultivated plants is possible
(Hildebrand et al., 2002, pers. obs). Indeed, the results above suggest that it is likely that it
is a regular occurrence. It is probable that wild and cultivated plants of the D. cayenensis
complex represent a spectrum based on degrees of management rather than distinct taxa.
The morphological diversity encountered (tuber colour, time taken to mature, leaf shape,
number and size of male inflorescences) is a result of differing degrees of domestication,
human selection and the local environment. A good example of the ability of these plants
to vary under human selection is possession of a spiny root. This has been thought to be a
key character of D. praehensilis, especially in West Africa, although it is usually spineless
in East Africa. This difference led Milne-Redhead (1975) to separate the non-spiny forms
as the species D. odoratissima Pax. However, as we have been told by farmers (also
reported by Hildebrand et al., 2002) that “wild” plants having spiny roots lose this
character and become spineless within a few years of being taken into a garden context.
We have also encountered that a cultivar which was collected in the field as a non-spiny
individual, but produced massive root spines when it was planted in a glasshouse (in Oslo).
It is probable that other morphological features which farmers would select against such as
stem spines, leaf shape and inflorescence number can be changed just as quickly when the
environment or management regime changed. This suggests that a considerable proportion
of such variation may be phenotypic. It would be expected that tuber characters (shape,
colour, taste) would be stabilised by positive human selection in cultivars, in contrast to the
characters mentioned above.
.
94
One of the main aims of this study was to consider the conservation of yams in South and
South Western Ethiopia, because they are in danger of being replaced by cash crops. From
a conservation perspective, it appears that the vernacular names should be viewed as
corresponding with cultivars. It is important that both the range of cultivars and the
diversity within them is protected both in situ, and perhaps also in a local garden as ex-situ.
The greater genetic divergence (from AFLP analysis) encountered in the cultivated forms
over the “wild” plants of the Sh population suggests that there is indeed diversity which it
will be important to protect to ensure local food security. However, the managed forest
plants are also an important source of diversity and need protection, ideally by conserving
the native vegetation of the Sheko region. The results of microsatellite data analyses also
confirmed that the Sh population (collected from relatively a small geographical area)
displayed a higher allelic diversity.
95
6. Conclusions and recommendations
6.1 Conclusions
In general, this study has revealed that:
1. There is no clear taxonomic boundaries among cultivated and wild accessions of D.
cayenensis complex included in this study.
2. There is a considerable genetic diversity within the D. cayenensis complex in
country with the Sheko cultivars (Sh population) displaying the greatest diversity,
where as the Sn displayed the least. However, unlike both Or and Sh populations,
the Sn population are at Hardy-Weinberg equilibrium.
3. Pairwise comparison of the different subpopulation (defined based on their
geographical isolation and growth habit) indicated that there is some degree of
genetic differentiation among the study population.
4. The wild yams exhibited the greatest allelic diversity (as measured by allelic
richness) in all the loci studied compared to the cultivated forms and hence through
domestication they contribute to the gene pool of cultivated Guinea yams.
5. High genetic divergence was observed between the cultivated accessions of Guinea
yams collected from Sheko area. Thus, the farmers in the Sheko area posses different
cultivars with a wide genetic basis.
6. Contrary to what is expected in vegetatively propagated crops, none of the 7
microsatellite loci displayed a significant excess of heterozygote.
7. The Sh population displayed the highest allelic diversity in 5 of the 7 SSR loci
studied with the over all allelic polymorphism of 100%. The Sn and Or population
displayed a fixed allele for one SSR locus (Da3Ga4), with an overall allelic
polymorphism of 87.5%.
8. Results of SSR analysis indicated that, the difference in the observed and expected
heterozygosity in Sh population highly significant compared to both Or and Sn
populations.
96
6.2. Recommendations
1. Studies must be undertaken at the population scale and in a broad range of
geographical regions, so as to take the diversity within each member of the Guinea
yams.
2. Comparative studies involving Ethiopian and West African material must be
undertaken to clarify the taxonomic confusion and observed differences in genetic
structure.
3. Conservation activities aiming to conserve Dioscorea species in the country should
primarily focus on the Sheko accessions, as they exhibit the highest genetic
diversity.
97
7. Reference
Adam, L., Edwards, Ronald, L. and Davenport, L. J. (2002). Presence of diosgenin in
Dioscorea batata (Dioscoreaceae), Economic Botany. 56:204-206
Akoroda,
M.O. and Chheda, H.R. (1983). Agro-botanical and species relationships of
Guinea yams, Tropical Agriculture. 60 (4):242-248
Akoroda, M.O. (1983). Floral biology in relation to hand pollination of white yams,
Euphytica. 32:831-838.
Alexander, J. and Coursey, D. G. (1969). The origins of yam cultivation. In: The
domestication and exploitation of plants and animals (Ucko P.J., Dimbleby G.W.,
editors), Aldine Publishing Company. Chicago. pp. 405-425.
Allendorf, F. W. and Luikart, G. (2007). Conservation and the genetics of populations,
Blackwell publishing, Malden USA. 641p
Anonymous.
(2006).
Yam,
ñame
blanco
(Spanish),
igname
(French).
www.innvista.com/health/foods/vegetables/yams.htm
Avise, J. C. (1994). Molecular markers, natural history and evolution. Chapman and Hall,
New York. pp 1-65
Ayensu, E. S. (1970). Dioscoreales. In: Anatomy of the monocotyledons (Metcalfe C. R,
editor), Vol, 6, Clarendon Press, Oxford, UK.
Ayensu, E. S. and Coursey, D. G. (1972). Guinea yams. The botany, ethnobotany, use and
possible future of yams in West Africa., Economic Botany. 26 (4):301–318
Bousalem, M., Arnau, G., Hochu, I., Arnolin, R., Viader, V., Santoni, S. and David, J.
(2006). Microsatellite segregation analysis and cytogenetic evidence for tetrasomic
inheritance in the American yam Dioscorea trifida and new basic chromosome
number in the Dioscorea, Theoretical and App. Gen.113:439-451
Bretting, P. K. and Widerlechner, M. P. (1995). Genetic markers and plant genetic resource
management, Plant Breed.Rev. 31:11-86
Burkhill, I. H. (1985). The useful plants of West tropical Africa. Vol. 1, Families A-D,
Royal Botanic Garden, Kew, UK. 960 p
Burkhill, I. H. (1939). Notes on the genus Dioscorea in the Belgian Congo, Bull. Jard. Bot.
Etat. 15 (4):345-392
Burkhill, I. H. (1960). The organography and the evolution of the Dioscoreaceae, the family
of the yams, Botanical Journal of Linnean Society. 56:319-420
98
Burkill, I. H. and Perier de la Bathie, H. (1950). Dioscoreacea In: Flore de Madagascar et
des Comores. Muséum national d’histoire naturelle, Paris, France, 78 p
Caddick, L. R., Wilkin, P., Rudall, P. J., Hedderson, T.A.J. and Chase, M.W. (2002b). Yams
reclassified: a recircumscription of Dioscoreaceae and Dioscoreales, Taxon. 51(1):
103 –114.
Caddick, L.R., Rudall, P.J., Wilkin, P., Hedderson, T.A.J. and Chase. M.W. (2002a).
Phylogenetics of Dioscoreales based on Combined Analysis of Morphological and
Molecular Data, Botanical Journal of Linnaean Society. 138:123-144.
Chaír, H., Agbangala, C., Marchand, J. L., Daïnou, O. and Noyer, J. L., (2005). Use of
cpSSRs for the characterization of yam phylogeny in Benin, Genome. 48 (4):674-684
Chase, M.W., Soltis, D.E., Olmstead, R.G., Morgan, D., Les, D.H., Mischler,
B.D., Duvall, M.R., Price, R.A., Hills, H.G., Qiu, Y.L., Kron, K.A.,
Rettig, J.H., Conti, E., Palmer, J.D., Manhart, J.R., Systma, K.J.,
Michael, H.J., Kress, W.J., Karol, K.G., Clark, W.D., Hedren, M.,
Gaut, B.S., Jansen, R.K., Kim, K.J., Wimpee, C.F., Smith, J.F.,
Furnier, G.R.S.H, Strauss, Xiang, Q.Y., Plunkett, G.M., Soltis, P.S.,
Swensen, S.M., Williams, S.E., Gadek, P.A., Quinn, C.J., Eguiarte,
L.E., Golenberg, E., Learn, G.H., JrGraham, S.W., Barrett, S.C.H.,
Dayanandan, S. and
Albert, V.A. (1993). Phylogenetics of seed
plants: an analysis of nucleotide sequences from the plastid gene
rbcl, Annals of the Missouri Botanical Garden. 80:528–580.
Chevalier, A. (1936). Contribution a l'étude de quelques espèces africaines du genre
Dioscorea. Bull. Mus.Natn. Hist. nat., Ser. 2, 8(6):52O-551.
Conlan, R. S., Griffiths, L .A., Napier, J.A., Shewry, P.S. and Mantell, S. (1995). Isolation
and characterization of cDNA clones representing the genes encoding the major
tuber storage protein (Dioscorin) of yams (Dioscorea cayenensis Lam.), Plant
Molecular Biology. 28:369-380
99
Coursey, D. G. (1967). Yams: an account of the nature, origins, cultivation and utilisation
of the useful members of the Dioscoreaceae. London: Longmans.
Coursey, D.G. (1976). The origins and domestication of yams in Africa. In: Origin of
African Plant Domestication (eds Harlan, J. R., De Wet, J. M. J.), Stemler Mouton
A.B.L Publishers, Hague, the Netherlands. pp. 383–408
Craufurd, P. Q., Summerfield, R. J., Asiedu, R. and. Vara Prasad, P. V. (2001). Dormancy
in Yams, Expl Agric. 37:147-181
Dahlgren, R.M.T., Clifford, H.T. and Yeo, P.F. (1985). The families of the
monocotyledons. Berlin: Springer.
Daïnou, O., Agbangala,
C., Berthaud,
J.
and Tostain, S. (2002). Le nombre
chromosomiqie de base des espèces de Dioscorea constituent la section
Enanthiophylum pourrait étre égal à X=20.Quelques preuves, Annales des sciences
agronomiques du Bénin, 3:(2) special edition
Dansi, A., Mignouna, H. D, Zoundjihékpon, J., Sangare, A., Asiedu, R. and Quin, F. M.
(1999). Morphological diversity, cultivar groups and possible descent in the
cultivated yams (Dioscorea cayenensis/D. rotundata complex in Benin Republic,
Genetic Resources and Crop Evolution. 46:371-388
Dansi A., Mignouna H. D., Zoundjihékpon J., Sangare A., Asiedu R. and Ahoussou N.
(2000). Using isozyme polymorphism to assess genetic variation with cultivated
yams (Dioscorea cayenensis /Dioscorea rotundata complex) of the republic of Benin,
Genetic Resources and Crop Evolution, 47:371-383
Dansi, A., Mignouna, H.D., Pillay, M. and Zok, S. (2001). Ploidy variation in the cultivated
yams (Dioscorea cayenesis complex) from Cameroon as determined by Flow
cytometry, Euphytica. 119:301-307
Degras, L. (1993). The Yam: A Tropical Root Crop, Macmillan Press Ltd, London, UK .
Dieringer, D. and Schlotterer, C. (2003). Microsatellite Analysis (MSA): a plat form
independent analysis tool for large microsatellite data sets, Molecular Ecology Notes.
3:167-169
Doyle, J. J. and. Doyle, J.L. (1987). A rapid DNA isolation procedure from small quantities
of fresh leaf tissues, Phytochem Bull. 19:11-15.
Dumont, R., Dansi, A., Vernier, P. and Zoundjihékpon, J.
(2006). Biodiversity and
Domestication of yams in West Africa: Traditional practices leading to Dioscorea
rotundata Poir. CIRAD, IPGRI. 95p
100
Egesi, C.N., Pillay, M., Asiedu, R. and Egunjobi, J. K. (2002). Ploidy analysis in water yam,
Dioscorea alata L. germplasm, Euphytica. 128:225–230
Essad, S. and Maunoury, C. (1984). Variation géographique des nombres chromosomiques
de base et polyploïdie dans le genre Dioscorea à propos du dénombrement des
espèces transversa Brown, pilosiuscula Bert. et trifida L., Agromonei. 4(7): 611-617
FAO, (2005). Food and Agricultural Organization of the United Nations, Statistical data
base, http:/faostat.fao.org/faostat/collections?subset=agriculture
Felsenstein, J. (1995). PHYLIP (Phylogeny inference packages). Version 3.57c. University
of Washington, Seattle, Washington, USA
Ford-Lloyd, B.V. (2001). Genotyping of Plant Genetic Resources. In: plant genotyping: the
DNA Fingerprinting of Plants (Henery, R. J. editor). CABI publishing, New York,
USA.
Gamiette, F., Bakry, F. and Ango, G.. (1999). Ploidy determination of some yam species
(Dioscorea spp) by flow cytometry and conventional chromosome counting, Genetic
Resources and Crop Evolution. 46:19-27
Gibbs, H. L., Kent, A. P., Patrick, J. W. and Glenn, J. (1997). Genetic structure of
populations of the threatened eastern massasauga rattlesnake, Sistrurus c. catenatus:
evidence from microsatellite DNA markers, Molecular Ecology. 6 (12):1123-1132,
Goldstein, D.B., Linares, A.R., Cavalli-Sforza, L. L and Feldman, M. (1995). Genetic
absolute dating based on microsatellites and the origin of modern humans.
Proceedings of the National Academy of Sciences of the USA. 92: 6723-6727.
Goldstein, D. B and Pollock, D. D. (1997) Launching microsatellites: a review of mutation
processes and methods of phylogenetic inferences. Journal of Heredity, 88: 335-342.
Goudet, J. (2001). FSTAT, A Program to Estimate and Test Gene Diversities and Fixation
Indices (Version 2.9.3). Institute of Ecology, Biology building, Lausanne,
Switzerland.
Goudet, J. M., Raymond, T., De Meeüs and Rousset, F. (1996). Testing differentiation in
diploid populations, Genetics. 144:1933-1940
Govaerts, R. and Wilkin, P. (2007). World Checklist of Dioscoreaceae. The Board of
Trustees of the Royal Botanic Gardens, Kew. Published on the Internet;
http://www.kew.org/wcsp/monocots. Accessed 30 Aug. 2007.
Graham, J., McNicol, R.J. and McNicol, J. W. (1996). A comparison of methods for the
estimates of genetic diversity in strawberry cultivars. Theor. App. Genet. 93:402-406
101
Guo, S. W. and Thompson, E. A. (1992). Performing the exact test of Hardy- Weinberg
proportion for multiple alleles, Biometrics. 48:361-372
Hahn, S. K., Osiru, D.S.O, Akoroda, M. O. and Otoo, A. (1987). Yam production and its
future prospects, Outlooks on Agriculture. 16:105-110
Hamon, P. (1987). Structure, origine genetique des ignames cultivees du complexe
Dioscorea cayenesis-rotundata et domestication des ignames en Afrique de l’Ouest.
These de Doctorat _es-Sciences, Universit_e Paris XI, Centre d’Orsay, 203 p.
Hamon, P., Brizard, J.P., Zoundjihékpon, J., Duperray, C. and Borgel, A. (1992). Etude des
index d’ADN de huit espèces d’ignames (Dioscorea sp.) par cytométrie en flux. Can.
J. Bot. 70: 996–1000.
Hamon, P. & Toure, B.(1990b). Characterization of traditional yam varieties belonging to
the Dioscorea cayenensis-rotundata complex by their isozymic patterns, Euphytica.
46:101–107.
Hamon, P. and Toure, B. (1990a). The classification of the cultivated yams (Dioscorea
cayenensis–rotundata complex) of West Africa, Euphytica. 47:179–187.
Hamon, P., Dumont, E., Zoundjihékpon, J., Ahoussou, N., Tio-Toure´, B (1997) Les
ignames. In: L’ame´ lioration des plantes tropicales (Charrier A, Jacquot M, Hamon
S, Nicolas D eds). CIRADORSTOM Repe` res, Paris, pp 385–400
Hildebrand, E., Sebsebe Demissew and Wilkin, P. (2002). Local and Regional Landrace
Disappearance in Species of Dioscorea L. (Yams) in Southwest Ethiopia. In:
Ethnobiology and Biocultural Diversity (Stepp J.R.; Wyndham F.S. and Zarger R. K.
eds). Proceeding of the 7th International Congress of Ethnobiology, University of
Georgia press. 717 PP.
Hildebrand, E.A. (2003). Enset, Yams, and Honey: Ethnoarchacological approaches to the
origins of horticulture in Southwest Ethiopia. A dissertation presented to the graduate
school of Arts and Sciences of Washington University, in partial fulfilment of the
requirements for the degree of Doctor of Philosophy.
Hochu, I., Santoni, S. and Bousalem. (2006). Isolation, Characterization and Cross-species
Amplification of Microsatellite DNA loci in the tropical American yam Dioscorea
trifida, Molecular Ecology Notes. 6:137-140
Ile, E. I., Craufurd, P. Q., Battey, N. H. and Asiedu, R. (2006). Phase of dormancy in yam
tubers (Dioscorea rotundata), Annals of Botany. 97:497-504.
Janssen, T. and Bremer, K. (2004). The age of major monocot groups inferred from 800+
rbcL sequences, Botanical Journal of the Linnean Society. 146 (4):385-398
102
Kimura, M and
Otha, T (1978) Stepwise mutation model and distribution of allelic
frequencies in a finite population, Proceedings of the National Academy of Sciences
of the USA, 75:2868-2872.
Lay, H-L., Liu, H-J, Liao, M-H., Chen, C-C., Liu, S-Y and Sheu, B-W. (2001). Genetic
identification of Chinese drug materials in yams (Dioscorea spp.) by RAPD analysis,
Journal of Food and Drug Analysis. 9 (3):132 – 138.
Lebot, V., Ivancic, A. and Abraham, K. (2005). The geographical distribution of allelic
diversity, a practical means of preserving and using minor root crop genetic
resources, Expl. Agric. 41:475-489
Lebot, V., Trilles, B., Nayer, J. L and Modesto, J. (1998). Genetic relationship between
Dioscorea alata cultivars, Genetic Resources and Crop Evolution. 45:499-509.
Mahalakshim V., Atalobhor Q., Ng. J., Ogunsola D., Lawson M. and Ortiz R. (2007).
Development of West African yam Diocorea spp. core collection, Genetic Resources
and Crop Evolution. 58 (8): 1827-1835
Malapa, R., Arnau, G., Noyer, J.L. and Lebot, V. (2005). Genetic Diversity of the greater
yam (Dioscorea alata L.) and relatedness to D. nummularia and D. transversa Br as
revealed with AFLP markers, Euphytica. 65:113-122
Martin, F.W. and Rhodes, A.M. (1978). The relationship of Dioscorea cayenensis and D.
rotundata, Trop Agric (Trinidad). 55:193-206
Miège, J.(1982). Notes sur les especes Dioscorea cayenensis Lamk.et Dioscorea rotundata
Poir. In: Yams (Ignames) (Eds, Miège, J. & Lyonga, S. N.). Oxford University Press,
Oxford UK, pp. 367–375
Miège, J. (1952) Contribution a` l’e´ tude syste` matique des Dioscorea d’Afrique
occidentale. The` se de doctorat, Universite´ de Paris, Paris, France, p 266
Miège, J. (1968). Dioscoreaceae In: Flora of tropical West Africa (eds. Hutchison J. Dalizel,
M. and Hepper ,F.N.) Crown Agents, London, UK V- 3, pp 144-154
Miège, J. and Sebsebe Demissew. (1997). Dioscoreaceae. In: Flora of Ethiopia and Eritrea
(Edwards, S. Sebsebe Demissew and Hedberg, I. eds). A Joint publication of the
National Herbarium, Biology Department, AAU, Ethiopia and the Department of
Systematic Botany, Uppsala University, Sweden .
Mignouna,
H. D., Aseidu, R. and Thottappilly, G. (1998). Molecular taxonomy of
cultivated and wild yams in West Africa. In: Proceedings of the symposium of
international society for tropical root crops- Africa branch (ISTRC-AB) (Akoroda,
103
M. O. and Ekanayake, I. J. eds), Lilongwe, Malawi, 22-28 October 1995. ISTRCAB, IITA, Ibadan, Nigeria, p 429
Mignouna, H.D. and Dansi, A. (2003). Yam (Dioscorea spp.) domestication by the Nago
and Fon ethnic groups in Benin, Genet. Resources and Crop Evol. 50:519–528.
Mignouna, H.D., Abang, M.M. and Fagbemi, S.A. (2003a). A comparative assessment of
molecular marker assays (AFLP, RAPD and SSR) for white yam (Dioscorea
rotundata Poir.) germplasm characterisation, Ann. Appl. Biol. 142:269–276.
Mignouna, H.D., Abang, M.M., Wanyera, M.W., Chikaleke, V.A., Asiedu, R. &
Thottappilly, G. (2005). PCR marker-based analysis of wild and cultivated yams
(Dioscorea spp.) from Nigeria: genetic relationships and implications for ex situ
conservation, Genetic Resources and Crop Evolution. 52:755–763.
Mignouna, H.D., Dansi, A. and Zok, S. (2002b). Morphological and isozymic diversity of
the cultivated yams (Dioscorea cayenensis /Dioscorea rotundata complex) of
Cameroon, Genetic Resources and crop Evolution. 49:21-29
Mignouna, H.D., Mank, R.A., Ellis, T.H.N., Bosh van den, N., Asiedu, R., Ng, S.Y.C. and
Peleman, J. (2002a). A genetic linkage map of Guinea yam (Dioscorea rotundata
Poir.) based on AFLP markers, Theor. Appl. Genet. 105:716–725.
Niño, J., Díez, M.J. and Nuez, F. (2006). Molecular variability of collection of Dioscorea
polygonoides (Wild) Humb and Bonpl. (wild yam) of the Gran Caldas region
(Colombia) using AFLPs, Journal of Horticultural Science and Biotechnology. 81
(5):803-808
Norman, M. J. T, Pearson, C. J. and Searle, P.G .E. (1995). The ecology of tropical food
crops, second edition. Cambridge University Press, UK, pp 305-318
O'Hair, S. K. (1990). Tropical Root and Tuber Crops. In: Advances in New Crops (Janick, J.
and Simon, J.E, eds.). Timber Press, Portland, Oregon, USA, pp 424-428
Okezie, C. E. A., Okonkwo, S. N, and Nwoke, F. I. (1993). The effect of day length on
growth and tuberization of Dioscorea rotundata propagated by seed, Nigerian
Journal of Botany. 6:53-59
Onwueme, I. C. and Charles, W. B. (1994). Tropical root and tuber crops; production,
perspective and future prospects. FAO, Rome, Italy. FAO Plant Production and
Protection paper no 126, 112p
Onwueme, I. C. (1988). The Tropical Tuber Crops: Yam, Cassava, Sweet Potato and
Cocoyams. Chichester: John Wiley & Sons
104
Onyilagha, J.C. and Lowe, J. (1985). Studies on the relationships of D. cayenesis and D.
rotundata cultivars, Euphytica. 35:733-739
Purseglove, J. W. (1972). Dioscoreaceae. In: Tropical Crops. Monocotyledons, Longman .
man Group Limited, Harlow, UK. pp 97-117.
Purseglove J.W. (1985) Tropical crops: Monocotyledons, Volume 1 and 2 combined. Long
man Group Limited, England.
Rajabhandari, M.
and Kawabata, J. (2005).
Bitterness and toxicity in wild yams
(Dioscorea spp.) tubers in Nepal, Plant Foods for Human Nutrition. 60:129-135
Ramser, J., Weising, K., Lopez-Peralta, C., Terhalle, W., Terauchi, R., and Kahl, G.
(1997). Molecular marker based taxonomy and phylogeny of Guinea yam (Dioscorea
rotundata D-cayenensis), Genome. 40: 903-915
Rao, N. K. (2004). Plant Genetic Resources: Advancing Conservation and use through
Biotechnology, African Journal of Biotechnology. 3(2): 136-145
Raymond, M. & Rousset, F., (1995). GENEPOP (version 1.2): population genetics software
for exact tests and ecumenicism, J. Heredity. 86:248-249
Rholf , F.J. (2004), NTSYSpc: Numerical Taxonomy and Multivariate Analysis System,
version 2.2. Exter software, New York, USA
Sautour, M., Mitaine-offer, A.C. and Lacaille-Dubois, M.A. (2007). The Dioscorea genus: a
review of bioactive steroid saponins, J Nat Med. 61:91-101
Scarcelli, N., Tostain ,S., Vigouroux, Y., Agbangla, C., Daïnou, O. and Pham, J.L. (2006b).
Farmers’ use of wild relative sexual reproduction in a vegetatively propagated crops:
The case of Yam, Molecular Ecology. 15:2421-2431
Scarcelli, N., Daïnou, O., Tostain, S., Agbangla, C. and Pham, J.L. (2005). Segregation
patterns of isozyme loci and microsatellite markers show the diploidy of African yam
Dioscorea rotundata (2n=40), Theoretical and App. Gen. 111:226-232.
Scarcelli, N., Tostain, S., Mariac, C., Agbangla, C., Berthaud, J & Pham, J. L. (2006a),
Genetic nature of yams (Dioscorea sp.) domesticated by farmers in Benin (West
Africa), Genetic Resources and Crop Evolution. 53:121–130.
Sebsebe Demissew, Nordal, I. and Stabbetorp, P.O.E. (2003). Flowers of Ethiopia and
Eritrea: Aloes and other Lilies. 1st edition. Shama Books, Addis Ababa Ethiopia
Segarra-Moragues, J.G., Palop-Esteban, M., Gonzalez –Candelas, F. and Catalán, P. (2004).
Characterization of seven (CTT)n microsatellite loci in the Pyrenean Endemic
Borderea pyrenaica (Dioscoreaceae): remarks on ploidy level and hybrid origin
assessed through allozyme and microsatellite analyses. J Heredity. 97:177-183
105
Segnou, C.A., Fatokun, Akoroda, M.O. and Hahn, S.K. (1992). Studies on reproductive
biology of white yam (Dioscorea rotundata Poir), Euphytica. 64:197-203
Slatkin, M (1995) A measure of population subdivision based on microsatellite allele
frequencies, Genetics. 139:457-462
Sneath, P. H. A. and Sokal, R. R. (1973). Numerical Taxonomy. Freeman, San Francisco.
Muluneh Tamiru. (2006). Assessing Diversity in Yams (Dioscorea spp.) from Ethiopia
based on Morphology, AFLP markers and Tuber Quality and Farmers’ Management
of Landraces. A dissertation submitted for the degree of doctor of Agricultural
Sciences, Georg-August University, Germany. Cuvillier Verlag, Göttingen. 155 pp
Terauchi, R., Chikaleke, V., Thottappilly, G. and Hahn, S.K. (1992). Origin and phylogeny
of Guinea yams as revealed by RFLP analysis of chloroplast DNA and nuclear
ribosomal DNA, Theor. Appl. Genet. 83:743–751.
Tostain, S., Agbangla, C. and Da O. (2002). Diversite´ AFLP des ignames sauvages D.
abyssinica et D. praehensilis. Annales des sciences agronomiques du Benin. 3: 1–20.
Tostain, S., Okry, F.K., Baco, M.N., Mongbo, R.L., Agbangla, C. and Danou, O. (2003). La
domestication’ des ignames Dioscorea abyssinica dans les sous-pre´ fectures de
Sinende´ et de Bante´ auBe´ nin (Afrique de l’Ouest), Annales des sciences
agronomiques du Bénin. 4: 33–53.
Tostain, S., Scarcelli, N., Brottier, .P, Marchand, J.L., Pham, J.L., Noyer J.L. (2006).
Development of DNA microsatellite markers in tropical yam (Dioscorea sp.), Mol
Ecol Notes. 6:173–175
Tschannen, A.B., Escher, F. and Stamp, P. (2005). Post-harvest treatment of seed tubers
with Gibberallic acid and field performance of yam (Dioscorea cayenesisisrotundata) in Ivory Coast, Expl. Agric. 41:175-186
Twyford, C.T., Viana, A.M., James, A.C. and Menatell, S.H. (1990). Characterization of
species of vegetative clones of Dioscorea food yams using isoelectric focusing of
peroxidase and acid phosphatase isoenzymes, Trop. Agric. (Trinidad). 67(4):337 –
341.
Voss, P., Hogers, R., Bleeker, M., Rijans, M., van der Lee, T., Hornes, M., Frijters, A., Pot,
J., Peleman, J., Kuiper, M. and Zabeau, M. (1995). A new technique for DNA
fingerprinting, Nucl Acid Res. 23:4407–4414
Weir, B.S and Cockerham, C.C. (1984). Estimating F-statistics for the analysis of
population structure, Evolution. 38:1358–1370.
106
Weising, K.; Nybom, H.; Wolff, K. and Kahl, G. (2005). DNA finger printing in plants:
principles, Methods and Applications, 2nd edition ACRC press book.
Wilkin, P. (2001). Dioscoreaceae of South and Central Africa, Kew Bulletin. 56:361-404.
Wilkin, P., Hildebrand, E., Hladik, A., Sebsebe Demissew, Wendawek Abebe & Mayo, S.
(in prep). African Guinea Yams and their wild relatives (the Dioscorea cayenensis
Lam. complex): morphology, taxon delimitation and South-West Ethiopia.
Wilkin, P., Schols, P., Chase, M., Chayamari,t K., Furness, C.A., Husymas, S.,
Rakotonasolo, F., Smets, E. and Thapyai. (2005). A plastid phylogeny of the Yam
genus Dioscorea: roots, fruits and Madagascar, Sysetemic Botany. 30(4): 736-749
Wilkin, P. and. Caddick, L. R. (2000). Palaeotropical compound-leaved yams (Dioscorea;
Dioscoreaceae): monophyly and relationships. Pp. 494–500 in Monocots: systematics
and evolution, eds. K. L. Wilson and D. A. Morrison. Melbourne: CSIRO.
Zoundjihékpon, J., Hamon, P., Noirot, M., Tio-Ture, B. and Hamon, S. (1997). Flowering
synchronization between male and female West African cultivated yams (Dioscorea
cayenesis-rotundata complex), Euphytica. 95:371-375
Zoundjihékpon,
J., Hamon, S., Tio-Touré,
B. & Hamon, P. (1994). First controlled
progenies checked by isozymic markers in cultivated yams Dioscorea cayenensis–
rotundata, Theor. Appl. Genet. 88:1011–1016.
Zoundjihékpon, J. (1993). Biologie de la reproduction et g´en´etique des ignames cultiv´ees
de l’Afrique de l’Ouest,Dioscorea cayenensis-rotundata). Doctoral Thesis,
Universit´e Nationale de Cˆote d’Ivoire.
107
8. Appendices
Appendix 1. List of the major diagnostic morphological characters used to identify
the voucher specimens (Compiled by Dr Paul Wilkin)
Characters
1. Thorny roots
2. Stem prickles
3. Leaf base shape
4. Number of male
infls/axil
5. Male
inflorescence
inter-floral
distance
6. Leaf blade (L/W)
ratio
7. Leaf texture
8. Tepal texture
9. Tuber form
D. rotundata/D.caynensis
Absent
Absent or present (few to
many)
Deep sinus with a deltoid
petiolar attachment
D. abyssinica
Absent
Absent
D. praehensilis
Present
Present
Cordate-ovate
with round
basal lobes
2-6(-8)
Shortly cordate
Flowers less than
their own diameter
apart
Less than 1.8
Flowers at
least their
own diameter
apart
Less than 1.8
Herbaceous
Herbaceous
Variable not deeply
buried
Herbaceous
herbaceous
Cylindrical
deeply buried
Herbaceous
Basal half scarious
Cylindrical deeply
buried
1-2 (-3)
Flowers at least their own
diameter apart
108
2-6 (-8)
Less than 1.8
Appendix 2. Microsatellite electropherograms showing allelic distribution at each of the loci studied as revealed by GENESCAN and GENOTYPER 3.6 software.
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
Appendix 3. The raw data for morphometric analysis (measurements were in mm for floral and cm for leaf characters)
Acc.NO
Daby-20
Daby-20
Daby-22
Daby-22
Daby-59
Daby-59
Daby-69
Daby-69
Daby-77
Daby-77
Daby-8
Daby-8
Dbul-28
Dbul-28
Dcay-1
Dcay-1
Dcay-17
Dcay-17
Dcay-18
Dcay-18
Dcay-19
Dcay-19
Dcay-21
Dcay-21
Dcay-35
Dcay-35
Dcay-38
Dcay-38
Dcay-47
Dcay-47
Dcay-48
Dcay-48
Dcay-49
Dcay-49
Dcay-50
Dcay-56
Dcay-57
Dcay-57
Dcay-61
Dcay-61
Dcay-62
Dcay-62
Dcay-67
Dcay-68
Dcay-68
Dcay-7
Dcay-7
Dcay-70
Dcay-70
Dcay-71
OTL OTW
1.89
0.42
1.78
0.40
1.78
0.40
1.78
0.40
1.78
0.40
1.78
0.40
1.78
0.40
1.56
0.35
2.00
0.44
2.00
0.44
2.00
0.44
2.00
0.44
2.11
0.47
2.22
0.49
1.78
0.40
1.78
0.40
1.78
0.40
1.89
0.42
1.89
0.42
1.89
0.42
1.89
0.42
1.89
0.42
2.22
0.49
1.78
0.40
1.67
0.37
1.78
0.40
1.78
0.40
1.67
0.37
2.22
0.49
2.00
0.44
2.00
0.44
2.00
0.44
2.00
0.44
2.00
0.44
1.78
0.40
2.00
0.44
2.00
0.44
2.00
0.44
1.78
0.40
1.56
0.35
1.56
0.35
1.56
0.35
1.78
0.40
1.78
0.40
1.78
0.40
1.78
0.40
1.78
0.40
1.78
0.40
1.78
0.40
2.00
0.44
ITL ITW
1.78 1.56
1.78 1.33
1.56 1.11
1.78 1.33
1.78 1.56
1.44 1.33
1.78 1.51
1.33 1.22
1.78 1.33
1.56 1.22
1.78 1.33
1.78 1.33
1.78 1.22
2.00 1.22
1.56 1.31
1.56 1.33
1.89 1.33
1.78 1.11
1.78 1.11
1.67 1.33
1.78 1.11
2.00 1.33
1.56 1.33
1.67 1.33
1.56 1.07
1.56 1.11
1.56 1.33
1.67 1.22
2.00 1.11
2.00 0.89
2.00 1.11
2.00 1.22
1.78 1.33
1.78 1.33
1.33 1.11
1.56 1.33
1.89 1.44
1.78 1.33
1.56 1.33
1.56 1.44
1.33 1.11
1.33 1.11
1.56 1.11
1.56 1.33
1.33 1.33
1.78 1.33
1.78 1.33
1.78 1.22
1.78 1.22
1.56 0.89
AL
0.40
0.40
0.44
0.33
0.44
0.44
0.44
0.44
0.53
0.44
0.56
0.49
0.44
0.44
0.44
0.44
0.33
0.44
0.44
0.44
0.44
0.44
0.44
0.44
0.44
0.42
0.38
0.44
0.44
0.44
0.44
0.44
0.44
0.44
0.44
0.44
0.44
0.47
0.40
0.36
0.44
0.42
0.44
0.44
0.33
0.33
0.33
0.22
0.44
0.40
AW
0.22
0.18
0.22
0.27
0.22
0.22
0.22
0.22
0.22
0.22
0.27
0.22
0.22
0.22
0.22
0.22
0.22
0.22
0.27
0.27
0.22
0.29
0.22
0.29
0.22
0.22
0.33
0.22
0.22
0.22
0.36
0.22
0.22
0.22
0.27
0.22
0.22
0.22
0.18
0.22
0.22
0.22
0.33
0.22
0.22
0.29
0.33
0.22
0.22
0.33
FL LEN 14W 12W 34W Bw L/BW SBW L/14W L/12W L/34W
0.89
8.00
2.22 1.20
1.78
2.16
8.00
4.8
4
2.3 3.60
0.89
8.30
1.89 1.50
1.80
2.18
8.30
4.6
4.1
2.2 4.40
0.67
6.50
2.41 1.30
1.91
2.50
4.06
4.2
3.6
1.8 2.70
1.00
6.20
2.07 1.10
1.94
2.38
5.17
4.3
3.7
1.9 3.00
0.89
5.00
1.79 1.00
1.25
1.47
3.57
7
6.8
4.2 2.80
0.89
5.20
1.93 1.00
1.30
1.41
2.89
4.8
4.6
3 2.70
1.11
6.30
1.58 1.10
1.37
1.58
2.63
5.1
5.1
2.8 4.00
1.00
6.60
1.65 1.40
1.50
1.65
2.75
6.8
6.3
3 4.00
0.89
8.00
2.50 1.00
1.86
2.11
4.00
6
5.7
3.2 3.20
0.67
8.00
2.50 1.00
1.86
2.11
4.00
6.1
6
4.3 3.20
0.89
9.00
1.55 2.00
1.29
1.32
2.14
6.9
6.2
3.2 5.80
0.89
8.00
3.00
2.67
1.00
1.67
1.74
2.67
7
6.3
3.4
0.89 14.00
15.00
0.93
3.50
0.90
1.17
2.15
6.8
6.2
4
0.89
9.00
8.80
1.02
3.00
0.90
1.06
1.73
6.2
5.5
3.1
1.11
9.50
4.40
2.16
1.30
1.98
2.38
4.13
6
6
4
1.11
8.00
4.00
2.00
1.40
1.74
1.95
3.64
6
6
4
0.67 12.00
5.00
2.40
1.50
1.74
1.94
3.75
4.5
3.7
1
0.78 11.00
5.00
2.20
1.70
1.57
1.75
3.24
4.6
3.8
1
0.89 10.00
5.50
1.82
1.80
1.47
1.61
2.50
4.5
4
2.5
0.89
8.50
5.00
1.70
1.60
1.37
1.55
2.74
4.2
3.9
2
0.67
8.20
1.91 1.50
1.37
1.37
2.05
3.4
2.6
1.6 4.30
0.89
9.00
2.00 1.00
1.50
1.50
2.25
3.2
2.6
1.2 4.50
0.89
7.50
1.97 1.00
1.67
1.88
3.00
4.8
4.4
2.8 3.80
0.89
8.00
2.22 1.00
1.90
2.05
4.00
6.5
5.8
4.2 3.60
0.78
8.80
2.32 1.60
1.87
1.91
3.14
6
5
2 3.80
0.78
7.50
2.50 1.00
1.97
2.27
3.75
6.1
4.9
2 3.00
0.89
7.00
2.80 0.90
2.33
2.26
3.50
4
3.6
2.2 2.50
0.78
6.80
2.72 0.90
2.27
2.19
3.40
4
3.7
2.6 2.50
0.89
5.50 15.5
2.29 0.70
1.83
1.96
3.06
12
6.5 2.40
0.89
6.00
2.14 1.00
1.88
2.00
3.33
10
8.5
5.2 2.80
0.78
7.80
2.23 1.00
1.63
1.66
2.60
3.8
3.2
1.7 3.50
0.78
8.00
2.00 1.20
1.74
1.82
2.86
4
3.6
1.5 4.00
0.67
7.60
3.00
2.53
1.40
1.81
1.90
3.17
4.6
4
2
0.67
7.00
3.80
1.84
1.30
1.75
1.79
3.50
4.7
3.8
2
0.67
7.00
4.80
1.46
1.50
1.40
1.75
2.92
5.2
5.2
4.2
0.78
7.50
3.00
2.50
1.20
2.08
2.14
3.13
5.3
5.3
4
0.89
8.00
3.60
2.22
1.60
1.90
2.00
3.81
4.7
4.6
2.8
0.67
7.00
3.60
1.94
1.50
1.56
1.75
3.68
3.8
3.3
2
0.67
8.00
5.00
1.60
1.50
1.45
1.63
2.67
3
3.1
2
0.67
7.00
3.60
1.94
1.40
1.56
1.84
3.50
3
3.1
2
0.89
6.80
1.45 1.50
1.36
1.55
2.62
3
2.8
1.8 4.70
0.78
7.40
1.76 1.50
1.45
1.64
2.85
3.2
3
1.8 4.20
0.89
6.20
1.55 1.20
1.24
1.32
1.88
4.8
4.7
3 4.00
0.89
6.50
1.91 1.20
1.55
1.71
2.32
4.6
4.4
2.8 3.40
0.67
6.50
1.63 1.10
1.48
1.71
2.71
4.2
4
2.4 4.00
0.67
7.00
1.94 1.00
1.67
1.94
3.89
4
3.9
2 3.60
0.67
7.00
1.89 1.00
1.63
1.89
3.68
5
4
2.4 3.70
0.89
9.50
1.67 1.80
1.42
1.53
2.38
5.4
5.7
4.5 5.70
0.67
6.70
1.68 0.80
1.46
1.72
2.91
6
6
4.2 4.00
0.89
6.00
1.71 1.00
1.40
1.67
2.50
3.6
3.5
2.4 3.50
M-1 M2 BW/SBW 14W/12W 12W/34W 14W/34W M2-M1 MM-M2 MM-M1 M2-M1/M M MM-M2/M M MM-M1/M M
1.00 2.2
3.00
1.22
3.70
4.50
0.80
0.20
1.00
0.40
0.10
0.50
0.70
2.93
1.21
3.80
4.60
1.00
0.70
1.70
0.42
0.29
0.71
2
0.80 1.7
2.08
1.31
1.63
2.13
0.70
0.20
0.90
0.41
0.12
0.53
0.80
2.73
1.23
2.17
2.67
0.60
0.20
0.80
0.38
0.13
0.50
2
1.00 3.3
2.80
1.18
2.43
2.86
0.60
0.30
0.90
0.32
0.16
0.47
0.70 2.1
2.70
1.08
2.06
2.22
0.90
1.40
2.30
0.30
0.47
0.77
0.70
3.64
1.15
1.67
1.92
1.10
0.50
1.60
0.48
0.22
0.70
2
0.80 2.5
2.86
1.10
1.67
1.83
1.00
0.40
1.40
0.45
0.18
0.64
0.90
3.20
1.13
1.90
2.15
0.90
0.30
1.20
0.43
0.14
0.57
2
0.90 1.8
3.20
1.13
1.90
2.15
0.90
0.30
1.20
0.43
0.14
0.57
1.60 2.9
2.90
1.03
1.62
1.67
1.70
0.50
2.20
0.45
0.13
0.58
1.00 3.2
3.00
1.04
1.53
1.60
1.10
0.30
1.40
0.46
0.13
0.58
2.20 2.5
4.29
1.29
1.85
2.38
2.80
2.50
5.30
0.37
0.33
0.71
1.20 2.5
2.93
1.18
1.63
1.92
1.30
2.50
3.80
0.26
0.50
0.76
1.20 1.7
3.38
1.20
1.74
2.09
1.00
0.20
1.20
0.42
0.08
0.50
1.00 1.7
2.86
1.12
1.86
2.09
1.00
0.20
1.20
0.45
0.09
0.55
1.50 1.8
3.33
1.11
1.94
2.16
1.40
0.60
2.00
0.40
0.17
0.57
1.40 1.7
2.94
1.11
1.85
2.06
1.80
0.50
2.30
0.49
0.14
0.62
1.00 1.5
3.06
1.10
1.55
1.70
1.50
0.80
2.30
0.45
0.24
0.70
0.90 1.8
3.13
1.13
1.77
2.00
1.60
0.70
2.30
0.50
0.22
0.72
0.60 1.5
2.87
1.00
1.50
1.50
1.10
1.30
2.40
0.37
0.43
0.80
0.70 1.4
4.50
1.00
1.50
1.50
1.00
1.30
2.30
0.33
0.43
0.77
0.60
3.80
1.13
1.60
1.80
0.90
0.70
1.60
0.41
0.32
0.73
2
0.80 2.9
3.60
1.08
1.95
2.10
1.00
0.30
1.30
0.48
0.14
0.62
1.20 2.5
2.38
1.02
1.64
1.68
0.80
0.40
1.20
0.33
0.17
0.50
0.80 2.6
3.00
1.15
1.65
1.90
0.90
0.30
1.20
0.45
0.15
0.60
0.80 1.8
2.78
0.97
1.55
1.50
0.70
0.10
0.80
0.44
0.06
0.50
0.80 1.8
2.78
0.97
1.55
1.50
1.10
0.20
1.30
0.52
0.10
0.62
0.60
3.43
1.07
1.56
1.67
0.80
0.20
1.00
0.50
0.13
0.63
5
0.60 2.5
2.80
1.07
1.67
1.78
0.90
0.20
1.10
0.53
0.12
0.65
0.80 1.9
3.50
1.02
1.57
1.60
1.00
0.70
1.70
0.40
0.28
0.68
0.60
3.33
1.05
1.57
1.64
1.20
0.50
1.70
0.52
0.22
0.74
2
0.80 1.5
2.14
1.05
1.67
1.75
0.90
0.50
1.40
0.41
0.23
0.64
0.70 1.5
2.92
1.03
1.95
2.00
1.00
0.30
1.30
0.50
0.15
0.65
0.70 1.2
3.20
1.25
1.67
2.08
0.30
1.50
1.80
0.12
0.60
0.72
0.80 1.8
2.50
1.03
1.46
1.50
0.90
0.30
1.20
0.45
0.15
0.60
0.90
2.25
1.05
1.90
2.00
1.00
0.40
1.40
0.43
0.17
0.61
2
0.80 1.7
2.40
1.13
2.11
2.37
1.10
0.50
1.60
0.46
0.21
0.67
1.10 1.5
3.33
1.12
1.63
1.83
1.00
0.40
1.40
0.40
0.16
0.56
1.00 1.9
2.57
1.18
1.90
2.25
0.90
0.30
1.20
0.41
0.14
0.55
1.00 1.4
3.13
1.14
1.69
1.92
1.10
0.60
1.70
0.41
0.22
0.63
1.00 1.5
2.80
1.13
1.73
1.96
1.20
0.50
1.70
0.44
0.19
0.63
1.00 1.8
3.33
1.06
1.42
1.52
1.00
0.50
1.50
0.40
0.20
0.60
0.80 1.8
2.83
1.11
1.36
1.50
1.00
0.40
1.40
0.45
0.18
0.64
0.80 1.7
3.64
1.16
1.58
1.83
1.00
0.30
1.30
0.48
0.14
0.62
1.00 1.7
3.60
1.17
2.00
2.33
0.70
0.40
1.10
0.33
0.19
0.52
1.00
3.70
1.16
1.95
2.26
1.00
0.20
1.20
0.45
0.09
0.55
1
1.60 1.7
3.17
1.08
1.55
1.68
1.20
0.60
1.80
0.35
0.18
0.53
1.00 1.7
5.00
1.18
1.70
2.00
0.90
0.40
1.30
0.39
0.17
0.57
1.00 1.7
3.50
1.19
1.50
1.79
0.80
0.40
1.20
0.36
0.18
0.55
158
Acc.NO OTL OTW
Dcay-73
2.00
0.44
Dcay-73
2.11
0.47
Dcay-74
1.78
0.40
Dcay-74
1.78
0.40
Dcay-75
1.78
0.40
Dcay-75
1.78
0.40
Dcay-75
1.78
0.40
Dcay-75
1.78
0.40
Dcay-76
1.78
0.40
Dcay-76
2.00
0.44
Dprh-13
1.78
0.40
Dprh-13
2.00
0.44
Dprh-16
2.00
0.44
Dprh-16
1.67
0.37
Dprh-25
1.78
0.40
Dprh-25
2.00
0.44
Dprh-26
1.78
0.40
Dprh-26
1.89
0.42
Dprh-27
2.00
0.44
Dprh-27
1.78
0.40
Dprh-30
2.00
0.44
Dprh-30
1.89
0.42
Dprh-31
1.67
0.37
Dprh-31
2.00
0.44
Dprh-32
1.56
0.35
Dprh-32
1.56
0.35
Dprh-53
1.78
0.40
Dprh-53
2.00
0.44
ITL ITW
2.11 1.33
2.11 1.11
1.56 1.33
1.56 1.44
1.33 1.11
1.33 1.33
1.56 1.11
1.78 1.33
1.67 0.89
1.56 1.22
1.89 1.56
1.78 1.56
1.56 1.56
1.78 1.44
1.78 1.33
1.89 1.33
1.78 1.22
1.78 1.22
1.78 1.56
1.56 1.38
1.67 1.33
1.56 1.33
1.67 1.44
1.56 1.11
1.33 1.22
1.44 1.11
1.78 1.44
1.78 1.44
AL
0.44
0.44
0.44
0.44
0.40
0.33
0.44
0.44
0.44
0.22
0.56
0.44
0.44
0.44
0.44
0.44
0.38
0.44
0.33
0.47
0.44
0.42
0.33
0.44
0.44
0.42
0.44
0.47
AW
0.33
0.33
0.33
0.22
0.22
0.22
0.22
0.22
0.22
0.22
0.29
0.31
0.22
0.22
0.44
0.22
0.22
0.31
0.44
0.22
0.22
0.22
0.22
0.22
0.22
0.22
0.22
0.22
FL LEN 14W 12W 34W Bw
LBW SBW L/14W L/12W L/34W
0.89
9.50
4.80
1.98 2.00
1.58
1.79
3.17
4.2
4
2.1
1.11
8.60
4.00
2.15
1.60
1.39
1.65
2.32
4.5
4
1.9
1.11
8.50
5.00
1.70
2.00
1.42
1.63
2.36
4
3.4
1.4
0.89
9.00
5.00
1.80
2.00
1.36
1.53
2.31
4
3.7
1.8
0.67
6.30
3.00
2.10
1.00
1.62
1.70
2.25
5.5
4.9
3
0.67
8.00
3.60
2.22
1.20
1.78
2.00
2.96
4.5
3.8
2
0.89
6.30
2.10 1.00
1.62
1.70
2.25
5
4.4
2.6 3.00
0.67
8.00
2.22 1.20
1.78
2.00
2.96
5.1
4.5
2.6 3.60
0.67
9.10
1.82 2.00
1.98
2.28
3.79
5
4.7
3.3 5.00
0.67
9.10
1.82 2.00
1.98
2.28
3.79
4.2
3.8
2.8 5.00
0.78
8.50
2.07 1.00
1.67
1.67
3.04
4.4
3.8
2.4 4.10
0.67
9.50
2.02 1.20
1.40
1.51
3.17
4.6
4
2.4 4.70
0.56
8.80
2.51 1.00
1.47
1.54
2.75
4.4
4
2.4 3.50
0.67
9.50
2.26 1.50
1.56
1.58
2.21
6.7
6.2
4 4.20
1.11
8.40
2.40 1.00
1.75
1.91
3.00
4.6
3.9
2.3 3.50
0.89
9.50
1.73 1.50
1.46
1.64
2.26
4.3
3.6
2.4 5.50
0.67 10.00
1.92 1.50
1.67
2.00
5.00
6
5.3
3 5.20
0.89 10.00
1.96 1.50
1.64
2.04
5.00
6.2
5.2
3.7 5.10
0.89
7.70
2.20
3.50
0.60
1.93
2.14
3.50
6
5.2
3.6
0.89
7.50
1.80
4.17
0.70
1.88
2.03
2.88
6.6
5.9
3.9
0.67
9.30
3.60
2.58
1.50
2.45
2.91
5.47
3.9
3.7
2.8
0.67 10.50
3.80
2.76
1.60
2.63
2.92
7.00
4.5
4
2.7
0.67
9.50
4.00
2.38
1.50
2.07
2.38
4.75
3.9
3.7
2.8
0.67
9.50
4.00
2.38
1.60
2.02
2.50
4.75
4.5
4
2.7
0.67
6.50
4.00
1.63
1.20
1.25
1.25
1.55
4.6
4
2.4
0.78
6.70
4.10
1.63
1.00
1.26
1.26
1.68
4.6
4
2.4
0.67
6.00
1.71 1.00
1.11
1.05
1.33
4.3
3.8
2 3.50
0.67
7.40
1.64 1.30
1.23
1.23
1.76
4.3
3.8
2 4.50
M-1 M2 BW/SBW 14W/12W 12W/34W 14W/34W M2-M1 MM-M2 MM-M1 M2-M1/M M MM-M2/M M MM-M1/M M
1.10 1.9
2.40
1.13
1.77
2.00
1.30
0.60
1.90
0.43
0.20
0.63
1.00 1.9
2.50
1.19
1.41
1.68
1.40
0.60
2.00
0.47
0.20
0.67
1.30 1.6
2.50
1.15
1.44
1.67
1.20
0.60
1.80
0.39
0.19
0.58
1.40 1.6
2.50
1.12
1.51
1.69
1.20
0.80
2.00
0.35
0.24
0.59
0.90 2.1
3.00
1.05
1.32
1.39
0.90
0.20
1.10
0.45
0.10
0.55
1.10 1.9
3.00
1.13
1.48
1.67
0.90
0.20
1.10
0.41
0.09
0.50
0.90 2.1
3.00
1.05
1.32
1.39
0.90
0.20
1.10
0.45
0.10
0.55
1.10 2.2
3.00
1.13
1.48
1.67
0.90
0.20
1.10
0.41
0.09
0.50
1.40
2.50
1.15
1.67
1.92
1.00
0.20
1.20
0.38
0.08
0.46
2
1.40 1.8
2.50
1.15
1.67
1.92
1.00
0.20
1.20
0.38
0.08
0.46
0.90 1.8
4.10
1.00
1.82
1.82
1.10
0.80
1.90
0.39
0.29
0.68
0.90 1.8
3.92
1.08
2.10
2.27
1.60
1.10
2.70
0.44
0.31
0.75
0.80 1.8
3.50
1.05
1.78
1.88
1.20
1.00
2.20
0.40
0.33
0.73
0.70 2.8
2.80
1.02
1.40
1.42
1.10
1.20
2.30
0.37
0.40
0.77
1.20 1.9
3.50
1.09
1.57
1.71
0.80
0.20
1.00
0.36
0.09
0.45
1.50 1.8
3.67
1.12
1.38
1.55
1.40
0.50
1.90
0.41
0.15
0.56
1.20 2.4
3.47
1.20
2.50
3.00
1.30
0.40
1.70
0.45
0.14
0.59
1.40 2.4
3.40
1.24
2.45
3.05
1.20
0.50
1.70
0.39
0.16
0.55
0.80 2.5
3.67
1.11
1.64
1.82
1.00
0.30
1.30
0.48
0.14
0.62
0.60 2.6
2.57
1.08
1.42
1.54
1.20
0.20
1.40
0.60
0.10
0.70
1.00 1.8
2.40
1.19
1.88
2.24
0.90
0.10
1.00
0.45
0.05
0.50
1.00
2.38
1.11
2.40
2.67
1.00
0.10
1.10
0.48
0.05
0.52
2
0.50 1.8
2.67
1.15
2.00
2.30
1.00
0.50
1.50
0.50
0.25
0.75
0.50
2.50
1.24
1.90
2.35
1.00
0.90
1.90
0.42
0.38
0.79
2
0.60 2.4
3.33
1.00
1.24
1.24
0.60
1.10
1.70
0.26
0.48
0.74
0.80 2.4
4.10
1.00
1.33
1.33
1.00
0.90
1.90
0.37
0.33
0.70
0.70 1.8
3.50
0.95
1.27
1.20
1.00
1.00
2.00
0.37
0.37
0.74
0.50 1.8
3.46
1.00
1.43
1.43
1.20
1.50
2.70
0.38
0.47
0.84
159
Appendix 4. The raw data for AFLP analyses
Category
56
57
58
60
61
64
65
67
71
72
75
77
78
79
80
86
87
90
91
98
106
107
108
109
110
111
112
123
124
133
137
138
139
140
141
152
155
162
163
167
170
175
176
177
178
179
181
183
184
185
186
188
193
206
216
219
221
235
236
D. prh
0
0
1
1
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
D. cay
0
1
0
1
1
0
1
0
0
0
0
1
1
0
1
1
0
0
0
1
0
1
1
0
1
0
0
0
1
1
0
0
0
0
0
1
0
1
0
1
0
0
1
1
0
1
0
0
0
1
0
0
0
1
1
0
0
1
1
D. cay
0
1
0
1
1
1
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
1
1
0
0
0
0
1
0
0
0
0
0
0
0
1
0
1
0
0
0
1
1
1
0
1
0
0
0
1
0
0
0
0
1
0
0
0
0
D. cay
0
0
0
1
1
0
1
0
0
0
1
1
0
0
1
1
0
0
0
0
0
0
0
0
0
1
0
0
1
1
0
0
0
0
0
1
0
1
0
1
0
1
0
1
0
1
0
0
0
1
0
0
1
0
0
0
0
0
1
D. cay
0
0
1
1
1
0
1
0
1
0
0
1
0
0
1
1
0
0
0
0
0
1
1
0
0
0
0
0
1
1
0
0
0
0
0
1
0
0
1
1
0
0
1
1
0
1
0
0
0
1
0
0
1
1
1
0
0
0
1
D. cay
0
0
1
1
1
0
1
0
0
0
0
1
0
0
1
1
0
1
0
0
0
1
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
1
0
0
1
1
0
1
0
0
0
1
0
0
0
1
0
0
0
1
0
D. aby
0
1
0
1
1
0
1
0
1
0
1
1
0
1
1
1
0
0
0
1
1
1
1
0
1
1
0
0
1
1
0
0
0
0
0
1
0
0
1
1
0
0
1
1
0
1
0
0
0
1
0
0
1
1
1
0
0
1
1
D. aby
0
0
1
1
1
0
1
0
1
0
0
1
0
0
1
1
0
0
1
1
0
1
1
0
1
0
0
0
1
1
0
0
0
0
0
0
0
0
1
1
0
0
1
1
0
1
0
0
0
1
0
0
1
1
1
0
0
1
0
D. aby
0
0
1
1
1
0
1
0
1
0
0
1
0
0
1
1
0
0
1
1
0
1
1
0
1
0
0
0
1
1
0
1
0
0
0
1
0
0
1
1
0
0
1
1
0
1
0
0
0
1
0
0
0
0
1
0
0
1
0
D. aby
0
1
0
0
1
1
0
0
0
1
0
0
0
1
0
0
0
1
0
0
1
1
1
1
0
1
1
1
0
1
0
1
0
0
0
1
0
1
0
1
0
1
1
0
0
1
0
0
0
1
0
0
1
1
1
0
0
1
0
D. cay
0
1
0
0
1
1
0
0
0
0
0
1
0
1
0
1
0
0
0
0
1
0
1
0
1
0
1
1
0
0
0
1
0
0
0
1
0
1
0
1
0
1
1
0
0
0
0
0
0
1
0
0
0
0
1
0
0
1
0
D. cay
0
0
0
1
1
0
1
0
1
0
0
1
0
0
1
1
0
0
0
1
0
1
1
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
1
1
0
0
1
1
0
1
0
0
0
1
0
0
0
1
1
0
0
1
0
D. aby
0
1
0
0
1
1
0
0
1
0
0
0
0
1
0
0
0
0
0
0
1
0
1
0
0
0
1
1
1
1
0
0
0
0
0
1
0
1
0
1
0
1
1
0
0
1
0
0
0
1
0
0
1
1
0
0
0
0
1
D. cay
0
1
0
0
1
0
1
0
0
0
0
0
1
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
1
1
0
1
0
0
0
1
0
0
0
0
1
0
0
1
0
160
D. cay
0
1
0
1
1
1
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
1
1
0
1
1
0
1
0
1
0
0
0
0
0
1
0
1
0
1
0
0
1
1
0
1
0
0
0
1
1
0
0
0
0
0
0
0
0
D. cay
0
0
1
1
1
0
1
0
1
0
1
1
0
0
1
0
0
0
0
1
1
0
1
0
1
0
0
0
1
1
0
0
0
0
0
1
0
1
0
1
0
0
0
1
0
1
0
0
0
1
0
0
1
1
1
0
0
1
0
D. cay
0
0
1
1
1
0
1
0
1
0
0
1
0
0
1
1
0
0
0
1
0
0
1
0
0
0
0
0
1
1
0
0
0
0
0
0
0
1
1
0
0
0
1
1
0
1
0
0
0
1
0
0
0
0
1
0
0
1
0
D. cay
0
0
1
1
1
0
1
0
1
0
1
1
0
0
1
1
0
0
0
0
1
1
1
0
1
1
0
0
1
1
0
0
0
0
0
1
0
0
1
1
0
0
1
1
0
1
0
0
0
1
0
0
1
1
1
0
0
1
1
D. cay
0
1
0
0
1
1
0
0
1
1
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
1
1
0
0
1
0
0
0
1
0
0
1
0
1
0
0
1
1
D. cay
0
1
0
1
1
1
0
0
1
1
0
0
0
1
0
0
0
0
0
0
1
1
1
0
0
1
1
1
0
1
0
0
0
0
0
1
0
1
0
1
0
0
1
1
0
1
0
0
0
1
0
0
1
1
1
0
0
0
1
D. aby
0
0
1
1
1
0
1
0
1
0
0
1
1
0
1
1
0
0
0
1
0
1
1
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
1
1
0
0
1
1
0
1
0
1
0
1
0
0
1
0
1
0
0
1
1
D. cay
0
1
0
1
1
1
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
1
1
0
1
0
0
0
0
0
1
0
1
0
0
0
1
1
1
0
1
0
0
0
1
0
0
0
0
1
0
0
0
0
D. cay
0
0
1
1
1
0
1
0
1
0
0
1
0
1
1
1
0
0
0
1
0
1
1
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
1
1
0
0
1
1
0
1
0
0
0
1
0
0
0
1
0
0
0
1
0
D. cay
0
0
1
1
1
0
1
0
1
0
0
1
0
1
1
1
0
0
0
1
1
1
1
0
1
0
0
0
1
1
0
0
0
0
0
1
0
0
1
1
0
0
1
1
0
1
0
0
0
1
0
0
0
1
0
0
0
1
0
D. cay
0
0
1
1
1
0
1
0
1
0
0
1
0
1
1
1
0
0
0
1
1
1
1
0
1
1
0
0
1
1
0
1
0
0
0
1
0
0
1
1
0
0
1
1
0
1
0
0
0
1
0
0
1
1
1
0
0
1
0
D. aby
0
1
0
0
1
1
0
0
1
1
0
0
0
1
0
0
0
1
0
0
1
1
1
1
1
0
1
1
0
0
1
0
0
0
0
1
0
1
0
1
0
1
1
0
0
1
0
0
0
1
0
0
1
1
0
0
0
1
0
D. cay
0
0
1
1
1
0
1
0
0
0
0
1
1
0
1
0
0
0
0
1
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
1
0
1
0
1
0
1
1
1
0
1
0
0
0
1
0
0
0
0
1
0
0
1
0
D. cay
0
1
0
0
1
1
0
0
1
0
0
0
0
1
0
0
0
0
0
0
1
1
1
1
0
0
1
1
0
0
0
0
0
0
0
1
1
1
0
1
0
1
1
0
0
1
0
0
0
1
0
0
1
1
1
0
0
1
1
D. cay
0
0
1
1
1
0
1
0
1
0
0
1
1
0
1
1
0
0
1
1
1
1
1
0
1
0
0
0
1
1
0
1
0
0
0
0
0
1
1
1
0
0
1
1
0
1
0
0
0
1
0
0
1
0
1
0
0
1
0
Daby
0
0
1
1
1
0
1
0
1
0
1
1
0
0
1
1
0
0
0
1
0
1
1
1
0
1
0
0
1
1
0
0
0
0
0
0
0
0
1
1
0
1
1
1
0
1
0
0
0
1
0
0
1
0
1
0
0
1
0
Dcay
0
0
1
1
1
0
1
0
0
0
0
1
0
0
1
1
0
0
0
1
0
1
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
1
1
0
1
0
0
0
1
0
0
1
1
1
0
0
1
0
D. aby
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
1
0
0
0
0
0
1
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
Category
237
239
241
242
243
244
245
246
256
258
261
281
283
300
301
302
307
309
310
311
312
313
321
322
326
327
350
351
357
366
369
385
388
53
56
59
64
65
66
68
74
76
78
79
81
82
84
86
90
91
94
95
102
106
107
111
112
121
122
126
131
133
D. prh
0
0
0
1
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
1
1
1
1
1
1
0
0
1
0
0
0
0
0
0
0
1
1
1
1
0
0
0
1
1
0
0
1
0
1
0
0
1
D. cay
1
0
0
1
0
1
0
1
0
0
0
1
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
1
1
1
1
1
1
1
0
0
1
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
1
1
0
1
0
1
0
0
0
0
D. cay
0
0
0
1
0
1
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
0
1
0
1
1
1
1
1
0
0
0
0
1
0
0
0
0
0
0
0
1
0
1
1
0
0
0
1
1
0
0
1
0
1
0
0
0
D. cay
1
0
0
1
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
1
1
1
1
1
1
0
1
0
0
1
0
0
0
1
0
0
0
1
0
1
0
0
0
0
1
0
1
0
1
0
1
0
0
0
D. cay
0
0
0
1
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
1
1
1
1
1
1
0
1
1
0
1
0
0
0
0
0
0
0
1
0
1
1
1
1
0
1
0
1
0
1
0
1
0
0
0
D. cay
1
0
0
1
0
0
0
1
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
1
0
1
1
1
0
1
0
1
1
0
1
0
0
0
0
0
0
0
1
0
1
1
1
0
0
1
0
1
0
1
0
1
0
0
0
D. aby
0
0
0
1
0
0
1
1
0
0
1
0
0
1
0
0
1
0
0
0
0
0
1
0
1
0
0
1
1
1
1
1
1
1
1
0
0
1
0
0
0
0
0
0
0
1
0
1
1
1
0
0
1
0
1
0
1
0
1
0
0
0
D. aby
1
0
0
1
0
1
1
1
0
0
0
1
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
1
1
1
1
1
1
0
1
1
0
1
0
0
0
0
0
0
0
1
0
1
1
1
0
0
1
0
1
0
1
0
1
0
0
0
D. aby
1
0
0
1
0
1
1
1
0
0
0
0
0
0
1
0
1
0
0
0
0
0
1
0
1
0
0
1
1
1
1
1
1
0
1
1
0
0
1
0
0
0
0
0
0
1
0
1
1
1
0
0
1
0
1
0
1
0
1
0
0
0
D. aby
1
0
0
1
0
1
1
1
0
0
0
0
0
0
1
0
1
0
0
0
0
0
1
0
1
0
1
0
1
1
1
1
1
1
1
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
1
0
0
1
1
0
0
0
0
D. cay
1
0
0
1
0
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
1
1
1
1
1
1
0
0
1
0
0
1
1
0
0
0
0
1
1
0
0
0
0
1
0
0
1
0
1
0
0
0
0
D. cay
1
0
0
1
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
0
1
1
0
1
0
0
0
0
0
0
0
1
0
1
1
1
0
0
1
0
1
0
1
0
1
0
0
0
D. aby
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
1
1
0
1
1
1
0
1
1
0
1
0
0
0
1
0
0
0
1
0
1
0
0
0
0
1
1
0
0
1
1
0
0
0
0
D. cay
0
0
0
1
0
1
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
1
1
1
1
0
1
0
0
0
1
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
1
0
0
1
0
0
1
0
0
0
161
D. cay
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
1
0
1
1
1
1
1
1
1
0
0
1
0
0
0
0
0
0
0
1
0
1
1
1
0
0
1
1
0
0
1
0
1
0
0
0
D. cay
1
0
0
1
0
0
1
1
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
1
1
0
0
1
1
1
1
1
1
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
1
0
1
0
1
0
1
0
0
0
D. cay
1
0
0
1
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
1
1
1
1
1
1
0
1
1
0
1
0
0
0
0
0
0
0
1
0
1
1
1
0
0
1
0
1
0
1
0
1
0
0
0
D. cay
1
0
0
1
0
1
1
1
0
0
0
0
0
1
0
0
1
0
0
1
0
0
0
1
1
0
0
1
1
1
1
1
1
0
1
0
0
1
0
0
0
0
0
0
0
1
0
1
1
1
0
0
1
0
1
0
1
0
1
0
0
0
D. cay
1
0
1
1
0
1
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
0
1
1
0
1
0
0
0
0
0
0
0
0
0
1
1
1
0
0
1
0
1
0
1
0
1
0
0
0
D. cay
1
0
1
1
1
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
1
1
0
1
1
1
1
1
1
1
1
0
1
0
0
1
1
0
1
1
0
0
1
0
1
1
0
1
1
0
0
1
0
0
0
0
0
D. aby
1
0
0
1
0
1
1
1
0
0
0
0
0
1
0
0
0
0
0
1
0
0
1
0
0
0
0
1
1
1
1
1
1
0
1
1
0
1
0
0
0
0
0
0
0
1
0
1
1
1
0
0
1
0
1
0
1
0
1
0
0
0
D. cay
0
0
0
1
0
1
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
1
1
1
1
0
0
0
0
1
0
0
0
1
0
0
0
0
1
0
0
0
0
0
1
1
0
1
0
0
1
0
0
0
D. cay
1
0
0
1
0
1
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
1
1
0
0
0
0
1
1
1
1
0
1
1
1
1
0
1
0
0
0
0
0
0
0
1
0
1
1
0
0
0
1
0
1
0
1
0
1
0
0
0
D. cay
1
0
0
1
0
1
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
1
1
0
0
0
0
1
1
1
1
1
1
1
1
0
0
1
0
0
1
0
0
0
0
1
0
1
1
0
0
0
1
0
1
0
1
0
1
0
0
0
D. cay
0
0
0
1
0
0
1
1
0
0
1
1
0
0
1
0
0
0
0
1
0
1
1
0
1
0
0
1
1
1
1
1
1
1
1
1
0
1
0
0
0
0
0
0
0
1
0
1
1
0
0
0
1
1
0
0
1
0
1
0
0
1
D. aby
1
0
0
1
0
1
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
1
1
1
1
1
1
0
0
1
0
0
1
0
0
0
0
1
0
1
1
1
1
0
1
0
1
0
1
1
1
0
0
0
D. cay
1
0
0
1
1
1
0
1
0
0
0
1
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
1
1
0
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
1
0
0
0
D. cay
1
0
1
1
1
1
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
1
0
1
0
1
1
1
1
1
1
1
1
0
1
0
0
0
1
0
1
1
0
1
1
0
1
0
0
1
1
0
0
1
1
0
0
0
0
D. cay Daby
0
1
0
0
0
0
1
1
0
1
1
1
0
1
1
1
0
0
0
0
0
0
1
1
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
1
1
1
1
0
0
1
1
1
1
1
1
1
0
1
1
1
1
1
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
1
1
1
1
0
1
0
0
0
0
1
1
0
0
1
1
0
0
1
1
0
0
1
1
0
0
0
0
0
1
Dcay
1
0
1
1
0
1
1
1
0
0
0
1
0
0
1
0
0
1
0
0
0
0
1
0
1
0
0
1
1
0
1
1
1
1
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
1
0
1
0
1
0
0
0
D. aby
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
1
0
1
0
0
0
1
0
0
0
0
1
1
1
1
0
1
0
0
1
0
0
1
0
0
0
0
0
0
1
0
1
1
0
0
0
1
0
1
0
1
0
1
0
0
0
Category
134
141
149
150
151
161
168
186
187
188
189
190
191
194
201
206
209
217
222
223
225
230
239
246
250
253
264
284
285
287
295
296
297
303
304
313
389
422
437
438
442
478
496
53
54
59
70
73
74
76
82
85
95
97
113
119
120
121
122
125
126
145
D. prh
1
0
1
0
0
1
1
1
1
1
0
0
1
0
0
1
0
0
1
0
0
0
1
0
0
0
0
1
0
0
0
1
0
1
0
0
0
1
0
1
0
0
0
0
1
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
D. cay
1
0
1
0
0
0
1
0
1
0
0
1
1
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
1
0
0
1
0
0
0
1
1
0
0
1
1
0
1
0
1
0
0
0
0
1
0
0
1
0
0
0
0
0
0
1
D. cay
1
0
1
0
0
1
1
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
1
0
1
1
0
1
0
0
0
0
0
0
0
0
0
1
D. cay
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
1
0
1
0
1
0
0
0
0
0
0
0
0
0
0
1
0
1
0
1
0
0
1
0
0
0
0
0
0
0
0
0
1
D. cay
1
0
1
0
1
1
1
1
0
0
0
0
1
0
0
0
0
1
0
1
0
0
1
0
0
0
0
1
0
0
0
1
0
1
0
0
0
1
1
0
0
1
0
0
1
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
D. cay
1
0
1
0
1
1
1
1
1
0
1
1
0
0
1
0
0
1
0
1
0
0
1
0
0
0
0
1
0
1
0
1
0
1
0
0
0
0
1
0
0
1
0
0
1
0
1
0
0
0
0
0
0
0
1
0
1
0
0
0
0
1
D. aby
1
0
1
0
1
1
1
1
1
0
0
0
1
0
0
0
0
1
1
0
0
0
1
0
0
0
0
1
0
0
0
1
0
1
0
0
0
1
1
0
0
1
1
0
1
0
1
0
0
0
0
0
0
0
1
0
0
1
0
0
1
1
D. aby
1
0
1
0
1
1
1
1
1
0
1
0
1
0
0
0
0
1
0
1
1
0
1
0
0
0
0
1
0
0
0
1
0
1
0
0
0
1
1
0
0
1
1
0
1
0
1
0
0
0
0
1
0
0
1
0
1
1
0
0
1
1
D. aby
1
0
1
0
1
1
1
1
1
0
1
0
1
0
0
0
0
1
0
1
0
0
1
0
0
0
0
1
0
0
0
1
0
1
0
0
0
1
1
0
0
1
0
0
1
0
1
0
0
0
0
0
0
0
1
0
1
0
0
0
1
1
D. aby
1
0
1
0
0
0
1
0
0
0
0
1
0
0
0
0
0
1
0
1
0
0
1
0
0
0
0
1
0
0
0
1
0
1
0
0
0
0
0
1
0
0
0
0
1
0
1
1
0
1
0
1
0
1
0
1
1
0
0
0
0
1
D. cay
1
0
1
0
0
0
1
0
0
1
0
1
0
0
0
0
0
1
1
0
0
0
1
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
1
0
0
1
0
0
1
0
1
1
0
0
0
1
0
1
0
0
0
0
0
0
0
1
D. cay
1
0
1
0
1
1
1
1
1
0
1
0
1
0
0
0
0
1
0
1
1
0
1
0
0
0
0
1
0
1
0
1
0
1
0
0
0
0
1
0
0
1
0
0
1
0
1
0
0
0
0
0
0
0
1
0
0
1
0
0
1
1
D. aby
1
0
1
0
1
0
1
0
0
0
0
1
1
0
0
0
0
1
1
0
0
0
1
0
0
0
0
1
0
0
0
0
0
1
0
0
1
1
1
0
0
0
0
0
1
0
1
1
0
1
0
1
0
0
0
0
0
0
0
0
0
1
D. cay
1
0
1
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
162
D. cay
1
0
1
0
1
1
1
1
1
0
0
1
0
0
0
0
0
1
1
0
0
0
1
0
0
0
0
1
0
0
0
1
0
1
0
0
0
1
1
0
0
1
1
0
1
0
1
1
1
1
0
1
0
0
1
0
1
0
0
0
0
1
D. cay
1
0
1
0
1
1
1
0
0
0
0
0
1
0
0
0
0
0
0
1
1
0
1
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
1
1
D. cay
1
0
1
0
1
1
1
0
1
0
0
0
1
0
0
0
0
0
0
1
0
0
1
0
0
0
0
1
0
0
0
1
0
1
0
0
0
1
1
0
0
1
0
0
1
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
1
1
D. cay
1
0
1
0
1
1
1
0
0
0
0
0
1
0
0
0
0
0
0
1
1
0
1
0
0
0
0
1
0
0
0
1
0
1
0
0
1
1
1
0
0
0
0
0
1
0
1
0
1
0
0
0
0
0
1
0
0
1
0
0
0
1
D. cay
1
0
1
0
1
1
1
1
1
0
1
0
0
0
0
0
0
1
0
1
0
0
1
0
0
0
0
1
0
0
0
1
0
1
0
0
0
1
1
0
0
0
0
0
1
0
1
1
0
0
0
1
0
0
0
0
0
0
0
0
0
1
D. cay
1
0
1
0
1
1
1
1
0
0
0
0
1
0
0
0
0
1
0
1
1
0
1
0
0
0
0
0
1
0
0
0
1
0
1
1
1
1
1
0
1
1
0
0
1
0
1
1
0
1
0
1
0
0
0
0
1
0
0
0
0
1
D. aby
1
0
1
0
1
1
1
1
1
0
0
0
1
0
0
0
0
1
0
1
0
0
1
0
0
0
0
1
0
0
0
1
0
1
0
0
0
1
1
0
0
1
1
0
1
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
D. cay
1
0
1
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
1
0
0
1
0
1
0
0
0
0
0
0
0
0
0
1
D. cay
1
0
1
0
1
1
1
0
0
1
0
1
0
0
0
0
0
1
0
1
0
0
1
0
0
0
0
0
0
0
0
1
0
1
0
0
0
1
1
0
0
1
0
0
1
0
1
0
0
0
0
1
0
0
1
0
0
0
0
0
1
1
D. cay
1
0
1
0
1
1
1
0
0
1
0
0
1
0
0
0
0
1
1
0
0
0
1
0
0
1
0
1
0
0
0
1
0
1
0
0
0
1
0
1
0
1
0
0
1
0
1
0
0
0
0
1
0
0
1
0
0
0
0
0
1
1
D. cay
1
0
1
0
1
0
1
1
0
1
0
1
0
0
0
0
0
1
1
0
1
0
1
0
0
0
0
1
0
0
0
1
0
1
0
0
0
1
0
1
0
1
0
0
1
0
1
0
0
0
0
1
0
0
1
0
0
0
0
0
1
1
D. aby
1
0
1
0
1
1
1
1
0
1
0
0
1
0
0
0
0
0
0
1
1
1
1
1
0
0
0
1
0
1
0
1
0
1
0
0
0
0
1
0
0
1
1
0
1
0
1
0
0
1
0
1
0
1
0
1
0
0
0
1
0
1
D. cay
1
0
1
0
0
0
1
0
1
0
0
1
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
1
0
0
1
0
0
0
0
0
0
1
D. cay
1
0
1
0
1
0
1
1
1
0
0
1
0
0
0
0
0
1
1
0
0
0
1
0
0
0
0
1
0
0
0
1
0
1
0
0
0
1
1
0
0
1
1
0
1
0
1
0
1
1
0
1
0
1
0
0
1
0
0
0
0
1
D. cay Daby
1
1
0
0
1
1
0
0
1
1
1
1
1
1
0
0
0
1
0
0
1
0
1
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
1
0
1
0
0
1
1
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
1
0
0
0
1
0
0
0
0
0
0
0
1
1
1
0
0
0
0
0
1
1
0
0
0
1
1
0
0
1
1
0
0
0
0
0
0
0
0
1
1
0
0
0
0
1
1
1
0
0
1
0
1
0
0
0
0
1
1
1
1
Dcay
1
0
1
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
1
0
0
0
0
1
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
D. aby
1
0
1
0
1
1
1
0
0
0
0
0
1
0
0
0
0
1
0
1
0
0
1
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
Category
148
149
153
180
182
204
210
212
213
217
229
277
54
55
58
60
63
73
77
80
85
87
88
89
97
101
103
108
109
113
114
128
129
132
140
159
160
167
169
171
179
181
182
192
195
210
221
227
236
237
242
256
257
266
267
282
283
301
312
394
404
444
D. prh
0
0
0
0
0
0
0
1
0
0
0
1
0
1
0
0
0
0
1
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
0
1
D. cay
0
0
0
0
0
0
0
1
0
0
0
1
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
1
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
1
0
1
D. cay
0
0
0
0
0
0
0
1
0
0
0
1
1
1
0
0
0
0
1
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
D. cay
0
0
0
0
0
0
0
1
1
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
D. cay
0
0
0
0
0
0
0
1
0
0
0
1
1
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
1
D. cay
0
0
1
0
0
0
0
1
1
1
0
1
1
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
0
1
D. aby
0
0
0
0
1
0
0
1
0
0
0
1
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
0
1
D. aby
0
0
1
0
1
0
0
1
0
1
0
1
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
0
1
D. aby
0
0
0
1
0
0
0
1
1
1
0
1
1
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
0
1
D. aby
0
0
0
0
0
0
0
1
1
1
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
D. cay
0
0
0
0
0
0
0
1
0
1
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
1
1
0
1
D. cay
0
0
1
0
0
0
0
1
0
1
0
1
1
1
0
0
0
0
1
1
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
1
D. aby
0
0
0
0
0
0
0
1
0
0
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
0
1
D. cay
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
163
D. cay
0
0
0
0
0
0
0
1
0
1
0
1
1
1
0
0
0
0
1
0
0
0
0
0
0
0
0
1
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
0
1
D. cay
0
0
0
0
0
0
0
1
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
D. cay
0
0
1
0
0
0
0
1
0
0
0
1
1
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
1
D. cay
0
0
0
0
0
0
0
1
0
0
0
1
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
1
D. cay
0
0
0
0
0
0
0
1
0
0
0
1
1
1
0
0
0
0
1
1
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
0
1
D. cay
0
0
0
0
0
0
0
1
0
0
0
1
0
1
0
0
0
0
0
0
0
1
0
1
0
0
0
1
1
1
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
1
0
0
D. aby
0
0
1
0
0
0
0
1
0
0
0
1
1
1
0
0
0
0
1
0
0
0
0
1
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
0
1
D. cay
0
0
0
0
0
0
0
1
0
0
0
1
0
1
1
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
D. cay
0
0
1
0
0
0
0
1
0
0
0
1
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
1
D. cay
0
0
0
0
0
0
0
1
0
1
0
1
0
1
1
0
0
0
1
0
1
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
1
D. cay
0
0
0
0
0
0
0
1
0
1
0
1
1
1
0
0
0
0
1
0
0
1
0
0
0
0
0
1
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
0
1
D. aby
0
0
0
0
0
0
0
1
1
1
0
1
1
1
0
0
0
0
1
1
1
1
0
0
0
0
0
1
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
1
D. cay
0
0
0
0
1
0
0
1
0
0
0
1
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
D. cay
0
0
0
0
0
0
0
1
0
0
0
1
0
1
0
0
0
0
0
0
0
1
0
1
0
0
0
1
1
1
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
1
D. cay Daby
0
0
0
0
1
1
1
0
1
1
0
0
0
0
1
1
0
0
1
0
0
0
1
1
1
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
1
1
1
0
0
1
0
Dcay
0
0
1
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
0
D. aby
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
Category
D. cay
D. cay
D. cay
D. aby
D. cay
D. aby
D.bul.
D.shc.
D. prh
D. prh
D. prh
D. prh
D. cay
56
0
0
0
0
0
0
1
0
0
0
0
0
0
57
1
0
1
0
0
0
0
0
1
0
1
0
1
58
0
1
0
1
1
1
0
1
0
1
0
1
0
60
0
1
0
1
1
1
1
0
1
1
1
1
1
61
1
1
1
1
1
1
0
0
1
1
1
1
1
64
1
0
1
0
0
0
0
0
0
0
0
0
1
65
0
1
0
1
1
1
1
0
1
1
1
1
0
67
0
0
0
0
0
0
0
1
0
0
0
0
0
71
1
1
0
0
0
1
1
1
0
1
1
1
0
72
0
0
0
1
0
0
0
1
1
0
1
0
0
75
0
0
0
0
1
1
1
0
0
1
0
1
0
77
0
1
0
1
1
1
0
0
1
1
0
1
0
78
0
0
0
0
0
0
0
0
0
0
0
0
0
79
1
0
1
0
1
1
0
0
0
0
1
1
1
80
0
1
0
1
1
1
1
0
1
1
1
1
0
86
0
1
0
0
1
1
1
1
1
1
1
1
0
87
0
0
0
0
0
0
0
1
0
0
1
0
0
90
0
0
0
1
1
0
0
0
0
0
0
0
0
91
0
0
0
0
0
0
0
0
0
0
1
0
0
98
1
0
0
1
1
1
0
0
1
1
1
1
1
106
0
0
0
0
1
1
0
0
0
0
0
1
1
107
0
1
1
0
1
1
0
0
1
0
1
1
0
108
1
1
1
1
1
1
0
0
1
1
1
1
0
109
0
0
0
0
0
0
0
0
0
0
0
0
1
110
0
1
0
0
1
1
0
0
1
1
0
1
0
111
0
0
0
0
1
1
0
0
0
0
0
0
0
112
1
0
0
0
0
0
0
0
0
0
0
0
1
123
0
0
1
1
0
0
0
1
1
0
0
0
1
124
0
1
0
0
1
1
1
1
0
1
1
1
0
133
1
1
1
1
1
1
0
0
1
0
0
1
1
137
0
0
0
0
0
0
0
0
0
0
0
0
0
138
0
0
0
0
0
0
0
0
0
1
0
1
0
139
0
0
0
0
0
0
1
1
0
0
0
0
0
140
0
0
0
0
0
0
1
0
0
0
0
0
0
141
0
0
0
0
0
0
1
0
0
0
0
0
0
152
1
1
1
1
1
1
1
0
1
0
1
1
1
155
0
0
0
0
0
0
0
1
0
0
0
0
0
162
0
1
1
1
0
0
0
0
1
0
1
0
1
163
0
0
0
0
1
1
0
0
0
1
1
1
0
167
0
1
0
0
1
1
0
0
1
1
1
1
1
170
0
0
0
0
0
0
1
0
0
0
0
0
0
175
1
0
1
1
1
0
0
0
1
0
1
0
1
176
1
1
1
1
1
1
0
1
1
1
1
1
1
177
0
1
0
0
1
1
1
1
1
1
0
1
1
178
1
0
0
0
0
0
0
0
0
0
1
0
1
179
0
1
1
1
1
1
1
0
1
1
1
1
0
181
0
0
0
0
0
0
0
1
0
0
0
0
0
183
0
0
0
0
0
0
1
0
0
0
0
0
0
164
Category
D. cay
D. cay
D. cay
D. aby
D. cay
D. aby
D.bul.
D.shc.
D. prh
D. prh
D. prh
D. prh
D. cay
184
0
0
0
0
0
0
1
0
0
0
0
0
0
185
1
1
1
1
1
1
0
0
1
1
1
1
1
186
0
0
0
0
0
0
1
0
0
0
0
0
0
188
0
0
0
0
0
0
0
1
0
0
0
0
0
193
0
0
1
0
1
1
0
1
0
1
0
1
0
206
1
1
1
0
1
1
0
0
0
0
0
0
0
216
1
0
0
0
1
1
1
0
1
1
1
1
1
219
0
0
0
0
0
0
1
0
0
0
0
0
0
221
0
0
0
0
0
0
0
1
0
0
0
0
0
235
1
1
1
1
1
1
0
0
1
1
1
1
1
236
0
0
0
0
1
1
0
0
1
1
0
1
1
237
0
1
1
0
1
1
0
0
0
0
0
0
0
239
0
0
0
0
0
0
1
0
0
0
0
0
0
241
0
0
0
0
0
0
0
0
0
0
0
0
0
242
1
1
1
1
1
1
0
0
1
1
1
1
1
243
0
0
0
0
0
0
0
0
0
0
1
0
0
244
0
1
0
1
1
0
0
0
1
1
1
0
1
245
0
1
0
0
0
1
0
0
1
1
0
1
0
246
1
1
1
1
1
1
0
1
1
1
1
1
1
256
0
0
0
0
0
0
0
1
0
0
0
0
0
258
0
0
0
0
0
0
0
1
0
0
0
0
0
261
0
0
0
0
1
1
0
0
0
0
0
0
0
281
0
0
0
0
1
0
1
0
0
1
1
1
0
283
0
0
0
0
0
0
1
0
0
0
0
0
0
300
1
0
1
1
1
1
0
0
0
0
0
0
0
301
0
0
0
0
0
0
0
0
0
1
1
1
1
302
0
0
0
0
0
0
0
0
0
0
0
0
0
307
0
0
0
0
0
0
0
0
0
0
0
1
0
309
0
0
0
0
0
0
0
0
0
0
0
0
0
310
0
0
0
0
0
0
0
0
0
0
0
0
0
311
0
1
0
0
0
0
0
0
0
0
0
0
0
312
0
0
0
0
0
0
1
0
0
0
0
0
0
313
0
0
0
0
0
0
0
1
0
0
0
0
0
321
1
1
0
1
1
1
1
0
1
1
1
1
1
322
0
0
0
0
0
0
0
1
0
0
0
0
0
326
0
0
0
0
0
0
0
0
0
0
0
1
0
327
0
0
0
0
0
0
0
0
0
0
0
0
0
350
1
0
1
0
0
0
0
0
0
0
0
0
0
351
0
1
0
1
1
1
0
0
1
1
1
1
0
357
1
1
1
0
1
1
0
0
1
1
1
1
1
366
1
1
1
1
1
1
0
0
0
0
0
1
0
369
1
1
1
1
1
1
0
0
1
1
1
1
1
385
0
1
0
1
1
1
0
0
0
1
1
1
0
388
1
1
1
1
1
1
0
0
1
1
1
1
1
53
1
1
1
1
1
1
0
0
1
0
1
1
1
56
0
1
1
1
1
1
0
0
1
1
0
1
0
59
0
0
1
1
0
0
0
0
0
1
0
1
0
64
0
0
0
0
0
0
0
0
0
0
0
0
1
65
0
1
1
1
1
1
0
1
1
0
1
1
0
66
0
0
0
0
0
0
0
0
0
1
0
0
0
165
Category
D. cay
D. cay
D. cay
D. aby
D. cay
D. aby
D.bul.
D.shc.
D. prh
D. prh
D. prh
D. prh
D. cay
68
0
0
0
0
0
0
0
1
0
0
0
0
0
74
0
0
1
0
0
0
0
0
1
0
1
1
0
76
1
1
1
0
0
0
1
0
0
0
1
0
1
78
0
0
0
0
0
0
0
1
0
0
0
0
0
79
0
0
1
0
0
0
0
0
0
0
0
0
0
81
0
0
1
0
0
0
0
0
0
0
0
0
0
82
1
0
0
1
1
1
0
0
1
1
0
1
0
84
0
0
0
0
0
0
1
0
0
0
0
0
0
86
0
1
1
1
1
1
1
1
1
1
1
1
1
90
0
0
0
1
1
1
0
0
1
0
0
0
0
91
0
0
0
1
1
1
1
0
1
1
0
1
0
94
0
0
0
0
0
0
0
0
0
0
0
0
0
95
0
0
0
0
0
0
0
1
0
0
0
0
0
102
1
1
1
1
1
1
1
1
1
1
1
1
1
106
1
1
1
0
0
0
0
0
1
0
1
0
1
107
0
0
0
1
1
1
0
0
0
1
0
1
0
111
0
1
0
0
0
0
0
0
0
0
1
0
1
112
0
1
1
1
1
1
0
0
1
1
0
1
0
121
0
1
0
0
0
0
1
0
0
0
0
0
1
122
1
0
1
1
1
1
0
0
1
1
1
1
0
126
0
0
0
0
0
0
0
0
0
1
0
0
0
131
0
0
0
0
0
0
1
0
0
0
0
0
0
133
0
0
0
0
0
0
1
1
0
0
0
0
0
134
0
1
1
1
1
1
0
0
1
1
1
1
1
141
0
0
0
0
0
0
0
1
0
0
0
0
0
149
0
1
1
1
1
1
0
0
1
1
1
1
0
150
0
0
0
0
0
0
0
1
0
0
0
0
0
151
1
0
1
1
1
1
0
0
1
1
0
0
0
161
0
0
1
1
1
1
1
1
1
1
0
1
0
168
0
1
1
1
1
1
0
1
1
1
1
1
0
186
0
0
1
1
1
1
0
0
1
1
0
1
0
187
0
1
1
1
1
1
0
0
1
1
1
0
0
188
0
1
1
1
0
0
0
0
1
0
0
1
0
189
0
0
0
0
0
0
0
0
0
0
0
0
0
190
1
1
1
1
1
1
0
0
1
0
1
1
1
191
0
0
0
0
0
0
0
0
0
1
0
0
0
194
0
0
0
0
0
0
0
1
0
0
0
0
0
201
0
0
0
0
0
0
0
0
0
0
0
0
0
206
0
0
0
0
0
0
0
0
0
0
1
1
0
209
0
0
0
0
0
0
1
0
0
0
0
0
0
217
0
1
0
1
1
1
0
0
1
1
1
1
0
222
0
1
1
1
1
1
0
0
1
0
1
1
1
223
0
0
0
0
0
0
0
0
0
1
0
0
0
225
0
0
1
0
0
0
0
0
0
0
0
0
0
230
0
0
0
0
0
0
1
1
0
0
0
0
0
239
0
1
1
1
1
1
1
0
1
1
1
1
1
246
0
0
0
0
0
0
0
0
0
0
0
0
0
250
0
0
0
0
0
0
0
0
0
1
0
0
0
253
0
0
0
0
0
0
0
0
0
0
0
0
0
264
0
0
0
0
0
0
0
1
0
0
0
0
0
166
Category
D. cay
D. cay
D. cay
D. aby
D. cay
D. aby
D.bul.
D.shc.
D. prh
D. prh
D. prh
D. prh
D. cay
284
0
0
1
1
1
1
0
0
1
1
0
1
0
285
0
0
0
0
0
0
0
0
0
0
0
0
0
287
0
0
0
0
0
0
0
0
0
0
0
0
0
295
0
1
0
0
0
0
0
0
0
0
1
0
0
296
0
0
1
1
1
1
0
0
1
1
0
1
0
297
0
0
0
0
0
0
0
0
0
0
0
0
0
303
0
1
1
1
1
1
0
0
1
1
0
1
0
304
1
0
0
0
0
0
0
0
0
0
0
0
0
313
0
0
0
0
0
0
0
0
0
0
0
0
0
389
0
0
0
0
0
0
0
0
0
0
0
0
0
422
0
1
0
1
1
1
0
0
1
0
0
1
0
437
0
1
1
1
1
1
0
0
1
1
1
0
0
438
0
0
0
0
0
0
0
0
0
0
0
1
0
442
0
0
0
0
0
0
0
0
0
0
0
0
0
478
0
1
0
1
1
1
0
0
0
0
0
0
0
496
0
1
0
0
1
0
0
0
0
0
0
0
0
53
0
0
0
0
0
0
1
1
0
0
0
0
0
54
1
1
1
1
1
1
1
1
1
1
1
1
1
59
0
0
0
0
0
0
0
0
0
0
0
0
0
70
1
1
1
1
1
1
0
1
1
1
1
1
1
73
0
0
1
0
0
0
0
1
0
0
0
0
1
74
1
1
0
0
0
0
0
1
0
0
0
0
0
76
0
0
1
0
0
0
0
1
0
0
1
0
1
82
0
0
0
0
0
0
0
1
0
0
0
0
0
85
1
0
1
0
0
0
0
1
0
0
1
1
1
95
0
0
0
0
0
0
1
1
0
0
0
0
0
97
0
0
0
0
0
0
0
0
0
0
0
0
0
113
0
1
1
1
1
1
0
1
1
1
1
1
0
119
0
0
0
1
0
0
0
0
0
0
0
0
0
120
0
0
0
0
0
0
0
1
1
1
1
1
0
121
0
0
0
0
1
1
0
0
1
0
0
1
0
122
0
0
0
0
0
0
0
0
0
0
0
0
0
125
0
0
0
0
0
0
1
0
0
0
0
0
0
126
0
0
0
0
1
0
0
1
0
0
0
1
0
145
1
1
1
1
1
1
0
1
1
1
1
1
1
148
0
0
0
0
0
0
1
1
0
0
0
0
0
149
0
0
0
0
0
0
1
1
0
0
0
0
0
153
0
0
0
0
0
0
0
1
0
1
0
0
0
180
0
0
0
0
0
0
0
1
1
0
1
1
0
182
0
0
0
0
0
0
0
1
0
0
1
1
1
204
0
0
0
0
0
0
0
1
0
0
0
0
0
210
0
0
0
0
0
0
0
1
0
0
0
0
0
212
1
1
1
1
1
1
0
0
1
1
1
1
1
213
0
0
1
1
0
0
0
1
0
0
0
1
0
217
0
0
1
0
0
0
0
1
0
1
1
1
1
229
0
0
0
0
0
0
0
1
0
0
0
0
0
277
1
1
1
1
1
1
0
1
1
1
1
1
1
54
1
1
0
0
1
1
1
0
0
1
0
0
0
55
0
1
1
1
1
1
1
1
1
0
1
1
0
58
1
1
0
0
0
0
0
0
1
0
1
0
0
167
Category
D. cay
D. cay
D. cay
D. aby
D. cay
D. aby
D.bul.
D.shc.
D. prh
D. prh
D. prh
D. prh
D. cay
60
0
0
0
0
0
0
1
0
0
0
0
0
0
63
0
0
0
0
0
0
1
1
0
0
0
0
0
73
0
0
0
0
0
0
1
0
0
0
0
0
0
77
0
0
0
1
1
1
0
0
1
1
0
1
0
80
0
0
0
0
0
0
0
1
0
0
0
0
0
85
0
0
0
0
0
0
0
0
0
1
0
1
0
87
0
0
1
0
0
0
1
0
1
0
0
0
0
88
0
0
0
0
0
0
0
0
0
0
0
0
0
89
0
0
0
0
0
0
0
0
1
1
1
1
0
97
0
0
0
0
0
0
1
0
0
0
0
0
0
101
0
0
0
0
0
0
1
0
0
0
0
0
0
103
0
0
0
0
0
0
0
1
0
0
0
0
0
108
0
1
1
0
0
1
0
1
1
0
1
0
0
109
1
1
1
1
1
1
1
0
1
1
1
1
0
113
1
1
1
1
1
1
1
0
1
1
1
1
0
114
0
0
0
0
0
0
0
0
0
0
0
0
0
128
0
0
0
0
0
0
1
0
0
0
0
0
0
129
0
0
0
0
0
0
1
0
0
0
0
0
0
132
0
0
0
0
0
0
0
0
0
0
0
0
0
140
0
0
0
0
0
0
1
1
0
0
0
0
0
159
0
1
0
0
1
0
0
0
0
0
1
0
0
160
1
1
1
1
0
0
0
0
1
1
1
0
0
167
0
0
0
0
0
0
0
0
0
0
0
0
1
169
0
0
0
0
0
0
0
0
0
0
0
0
0
171
0
0
0
0
0
0
1
0
0
0
0
0
0
179
0
0
0
0
0
0
0
0
0
0
0
0
0
181
0
0
0
0
0
0
1
0
0
0
0
0
0
182
0
0
0
0
0
0
1
0
0
0
0
0
0
192
0
0
1
0
0
0
1
0
0
0
1
0
0
195
0
0
0
0
0
0
1
1
0
0
0
0
0
210
0
0
0
0
0
0
1
0
0
0
0
0
0
221
0
0
0
0
0
0
1
0
0
0
0
0
0
227
0
0
0
0
0
0
1
0
0
0
0
0
0
236
0
0
0
0
0
0
1
0
0
0
0
0
0
237
0
0
0
0
0
0
0
0
0
0
0
0
0
242
0
0
0
0
0
0
0
0
0
0
0
0
0
256
0
0
0
0
0
0
1
0
0
0
0
0
0
257
0
0
0
0
0
0
0
0
0
0
0
0
0
266
0
0
1
1
0
1
1
0
0
1
0
0
0
267
0
0
0
0
0
0
0
0
0
0
0
0
0
282
0
0
0
0
0
0
1
0
0
0
0
0
0
283
0
1
0
0
0
0
0
0
0
0
1
0
0
301
0
0
0
0
0
0
1
0
0
0
0
0
0
312
0
1
0
1
1
1
1
1
1
1
1
1
0
394
0
1
1
1
1
1
1
0
1
1
1
1
0
404
0
0
0
0
0
0
1
0
0
0
0
0
0
444
0
1
1
1
1
1
0
0
1
1
1
1
0
168
Appendix 5. Microsatellite codominant data used to estimate population genetics parameters
Species name
DabOR12
DabOR13
DabOR38
DabyOR31
DabyOR56
DabySH23
DabySN32
DabySN47
DabySN49
DabySN79
Dalata246
Dalata26
DabySN25
DabySN41
DabySN42
DabySN66
D/prhSN62
DcayOR15
DcayOR78
DcaySN65
DcayOr11
DcayOR37
DcayOR69
DcaySh14
DcaySh16
DcaySh20
DcaySh24
DcaySh27
DcaySH30
DcaySh39
DcaySH4
DcaySH43
DcaySh5
DcaySH54
DcaySh57
DcaySH58
DcaySH6
DcaySH61
DcaySH68
DcaySh72
DcaySH74
DcaySh8
DcaySH82
DcaySH9
DcaySN1
DcaySN17
DcaySN18
DcaySN19
DcaySN2
DcaySN21
Extraction# Dba2D06 Dba2D06 Da3G04 Da3G04 Da1F08 Da1F08 Dpr3D06 Dpr3D06 Da1D08 Da1D08 Dpr3F04
154.12
177
181
321
321
195
210
165
167
132
154.13
177
181
321
321
210
210
165
167
132
155.24
177
181
321
321 195?
209
165
167
?
155.16
203
203
122
155.46
181
181
321
321
181
195
165
165
367
367
132
155.7
177
177
329
329
195
195
167
167
155.17
177
191
321
321
195
195
165
167
356
356
130
155.35
177
183
189
203
155.37
195
195
179
179
155.76
?
155.34
177
183
321
321
181
195
165
169
346
380
129
155.10
177
181
308
320
195
197
336
336
115
155.9
183
183
321
321
193
203
167
167
356
390
130
155.29
183
183
321
321
193
203
167
167
356
356
130
155.30
191
199
155.59
183
183
321
321
193
203
167
167
356
390
130
155.55
177
183
321
321
181
195
165
167
356
356
130
154.15
181
181
321
321
195
195
165
165
367
367
132
155.75
181
181
321
321
181
195
165
165
367
367
132
155.58
177
183
321
321
181
195
165
169
346
380
130
154.11
155.23
181
181
321
321
195
195
165
165
367
367
132
155.64
154.14
177
177
321
321
195
203
167
169
346
380
132
154.16
339
356
155.4
177
183
195
195
169
169
346
381
155.8
177
177
321
321
187
195
167
167
339
346
129
155.12
177
183
193
205
167
169
155.15
317
317
199
203
127
155.26
177
177
321
321
187
195
167
167
339
346
130
154.4
195
195
167
167
155.31
177
191
321
321
195
195
167
167
339
356
130
154.5
177
191
321
321
195
205
167
169
346
356
129
155.43
155.47
181
191
155.49
321
321
195
203
167
175
154.6
?
155.54
177
177
321
321
187
195
167
167
339
346
130
155.67
177
183
328
328
195
195
167
167
346
352
129
155.69
177
191
321
321
181
195
167
167
369
356
130
154.8
177
191
321
321
181
195
167
167
339
356
155.81
181
191
154.9
154.1
195
195
155.1
195
195
356
356
155.2
177
191
195
195
165
167
356
356
130
155.3
177
191
321
321
195
195
165
167
356
356
154.2
177
191
321
321
181
195
165
167
356
356
130
155.5
177
177
321
321
195
195
165
169
346
380
155.13
177
183
321
321
195
195
165
169
346
380
130
169
Dpr3F04 Da1A01 Da1A01 Dab2E07 Dab2E07 Dab2C05 Dab2C05
132
248
248
124
140
194
211
132
248
248
124
140
194
211
?
248
248
140
140
192
211
122
118
129
132
231
248
140
140
192
213
248
248
203
203
130
231
248
136
136
192
207
136
136
184
184
221
234
184
190
?
129
248
248
124
124
190
190
127
236
236
124
124
192
211
130
248
248
124
136
186
207
130
248
248
124
136
186
207
136
136
130
248
248
124
136
186
207
130
231
248
136
136
192
207
132
124
124
190
213
132
231
248
140
140
190
213
130
248
248
124
124
188
188
136
136
132
231
248
140
140
190
213
184
196
132
136
136
190
190
129
130
130
130
132
231
231
238
227
231
248
248
236
248
248
248
236
248
248
248
248
136
123
120
120
124
136
136
120
131
136
134
124
118
134
136
118
227
248
231
248
248
248
227
248
248
248
248
248
136
127
124
136
136
136
124
120
142
127
136
140
136
136
124
132
?
130
129
130
130
130
130
231
231
231
231
248
248
248
248
248
248
248
248
136
136
136
136
124
124
124
124
188
192
186
190
190
192
190
192
188
207
201
211
207
192
190
192
194
184
194
192
190
203
190
190
184
207
203
190
190
192
192
190
192
192
192
190
190
192
207
207
207
207
190
190
Species name
DcaySN28
DcaySN34
DcaySN35
DcaySN36
DcaySN45
DcaySN59
DcaySN63
DcaySN64
DcaySN67
DcaySN75
DcaySN76
DcaySN77
DcaySN81
DcaySN85
DcaySN86
DprhOR22
DabySh50
DprhOR29
DprhOR40
DprhOR48
DprhOR51
DprhOR52
DprhOR53
DprhOr60
DprhOR70
DprhSH33
DprhSh44
DprhSH55
DprhSH7
DprhSH71
Daby84
DcayOR73
DcaySN83
Dsch3
Dsch80
DschSh10
Extraction# Dba2D06 Dba2D06 Da3G04 Da3G04 Da1F08 Da1F08 Dpr3D06 Dpr3D06 Da1D08 Da1D08 Dpr3F04 Dpr3F04 Da1A01 Da1A01 Dab2E07 Dab2E07 Dab2C05 Dab2C05
155.19
177
191
321
321
195
195
165
167
356
356
130
130
231
248
136
136
192
207
155.21
177
183
321
321
195
195
165
169
346
380
129
129
248
248
124
124
190
190
155.22
183
183
193
203
167
167
186
207
155.33
177
191
321
321
195
195
165
167
356
356
130
130
231
248
136
136
192
207
155.50
177
191
321
321
179
193
167
171
365
376
127
130
227
231
124
138
188
203
155.56
177
191
321
321
181
195
165
169
346
380
130
130
248
248
124
124
190
190
155.57
181
191
124
136
155.60
177
191
321
321
181
195
165
167
356
356
231
248
136
136
192
207
155.70
155.73
177
179
321
321
181
195
167
167
356
380
130
130
221
248
136
136
190
190
155.74
177
183
321
321
181
195
165
169
346
380
130
130
248
248
123
123
190
190
155.80
177
191
321
321
181
195
165
167
356
356
231
248
136
136
192
207
155.84
177
191
321
321
181
195
165
167
356
356
130
130
231
248
124
136
190
207
155.85
177
183
321
321
195
195
165
169
248
248
123
123
190
190
155.61
177
191
321
321
195
195
231
248
192
207
155.6
181
181
321
321
189
203
161
167
370
367
227
248
124
138
188
188
155.38
321
321
181
195
165
169
346
380
129
129
248
248
124
124
190
190
155.14
190
190
155.27
177
177
169
169
120
120
155.36
231
248
198
198
155.39
177
183
142
142
198
198
155.40
177
191
136
136
196
196
155.42
142
142
188
192
155.51
177
177
321
321
195
199
171
171
130
130
231
231
132
138
188
201
155.65
184
184
155.18
181
191
321
321
195
203
167
175
346
356
129
129
227
248
136
142
184
192
155.32
195
203
184
201
155.45
331
331
120
120
190
211
154.7
181
195
203
205
169
169
346
356
229
248
124
124
186
186
155.66
177
179
319
321
193
203
169
169
127
132
236
248
124
138
184
203
155.83
183
193
321
321
199
205
167
171
227
227
194
194
155.68
181
181
321
321
181
195
165
165
367
367
132
132
231
248
140
140
190
213
155.82
132
138
194
194
154.3
195
195
155.79
177
177
321
321
175
195
215
215
124
124
154.10
177
191
302
302
173
195
348
348
132
132
124
140
178
178
170
Appendix 6. microsatallite data used to infer population structure
Pop.
or1
or2
or4
or5
or6
or7
or8
or9
or10
or11
or12
or13
or14
or15
sh1
sh2
sh3
sh4
sh5
sh6
sh7
sh8
sh9
sh10
sh11
sh12
sh13
sh14
sh15
sh16
sh17
sh18
sn (or3)
sn1
sn2
sn3
sn4
sn5
sn6
sn7
sn8
sn9
sn10
sn11
sn12
sn13
sn14
place of coll
ilubabor
ilubabor
Ghibe
Ghibe
wellega
wellega
wellega
wellega
wellega
wellega
wellega
wellega
Ghibe
gojam
sheko
sheko
sheko
sheko
sheko
sheko
sheko
sheko
sheko
sheko
sheko
sheko
sheko
sheko
sheko
sheko
sheko
sheko
areka
areka
areka
areka
gedeo
sidamo
areka
gedeo
areka
areka
welaiyta
gedeo
areka
areka
gedeo
Dba2D06
181
181
177
177
177
177
181
181
177
181
181
181
183
177
181
183
177
177
177
177
183
191
181
181
181
181
181
181
181
181
193
177
191
195
179
183
177
183
177
177
177
177
181
177
177
177
177
177
177
177
177
177
177
177
177
177
177
183
177
177
177
177
177
177
191
191
191
177
183
191
191
177
177
183
191
191
191
177
183
191
183
183
191
191
191
191
179
183
Da3G04
Da1F08
Dpr3D06
Da1A01
Dab2E07
321
321
321
321
189
181
203
195
161
165
167
169
227
248
248
248
321
321
321
321
321
321
321
321
321
321
321
321
321
321
321
321
321
321
321
321
321
321
321
321
165
165
165
165
165
165
165
165
167
171
167
169
169
169
167
167
167
167
165
165
167
165
165
165
171
171
175
169
169
169
167
169
248
248
321
317
321
317
321
321
321
321
321
328
321
321
329
321
321
321
321
321
321
328
321
321
329
321
321
321
321
321
321
321
321
321
321
321
321
321
321
167
167
167
169
175
167
167
167
167
167
169
167
167
167
167
169
169
167
169
167
167
171
169
167
167
169
248
248
248
248
248
227
231
248
248
248
248
248
248
236
248
248
248
248
248
248
248
248
248
248
321
321
321
321
321
321
167
167
167
167
167
167
167
167
167
167
167
165
165
165
165
165
165
165
165
167
165
167
165
165
167
165
231
248
231
231
231
227
231
227
229
236
231
231
238
227
231
248
248
236
227
231
248
248
248
248
321
321
321
321
321
321
210
210
195
195
209
195
195
195
205
199
203
205
203
195
195
205
203
195
195
195
205
203
195
195
195
195
195
203
195
195
195
195
195
195
195
195
203
195
193
195
195
195
195
248
248
319
195
195
195
195
195
181
181
181
199
195
195
203
193
195
187
193
199
187
195
195
195
195
187
195
181
181
195
195
181
195
195
181
195
195
195
195
193
195
179
181
181
181
181
231
231
231
231
248
248
231
248
248
248
248
248
248
248
248
248
231
227
248
231
221
248
248
231
248
248
248
248
171
124
124
142
136
124
124
124
140
140
140
140
140
138
124
142
136
140
140
124
140
140
140
140
140
132
136
124
124
136
124
120
120
124
138
142
124
138
136
136
120
131
136
134
124
136
124
136
134
136
134
136
142
136
140
134
136
136
136
136
136
136
136
136
136
124
124
136
124
124
124
136
124
136
124
124
136
136
124
136
138
124
136
136
124
Dab2C05
188
190
198
196
194
194
190
190
192
192
190
190
194
188
184
186
184
188
192
186
190
190
192
190
192
184
190
203
190
190
203
190
192
192
192
192
190
190
192
190
186
192
188
190
192
190
190
188
190
198
196
211
211
213
213
211
213
213
213
194
201
192
186
203
188
207
201
211
207
192
190
192
192
207
203
190
190
203
190
207
207
207
207
190
190
207
190
207
207
203
190
207
190
190
sn15
sn16
sn17
sn18
sn19
sn20
sn21
sn22
sn23
sn24
sn25
areka
areka
areka
areka
gamugofa
gamugofa
areka
areka
areka
gedeo
areka
177
177
177
177
183
177
183
177
191
191
183
191
183
191
183
183
321
321
321
321
321
321
321
321
321
321
321
321
321
321
177
183
183
183
321
321
321
321
181
181
195
195
193
195
193
189
195
181
193
195
195
195
195
203
195
203
203
195
195
203
165
165
165
167
167
169
167
167
167
231
231
248
231
248
231
248
248
248
248
248
248
248
248
167
165
167
179
165
167
179
169
167
221
248
248
234
248
248
172
136
124
124
136
136
124
124
136
124
136
136
136
136
136
124
124
124
124
192
190
190
192
186
192
186
184
184
188
186
207
207
190
207
207
207
207
184
190
188
207
Appendix 7. Microsattelite presence/absence data used to infer taxonomic relationships among the taxa using NTYSYS
catagory DabyOR DabyOR DabyOR DabyOR DabySH DabySN DabySN DabySN DabySN DabySN DprhSN6 DcayOR DcayOR Dcaysh Dcaysh DcaySh DcaySh DcaySh DcaySH DcaySh DcaySH DcaySH DcaySh DcaySH DcaySH DcaySH DcaySh
178
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
184
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
186
0
0
0
0
0
0
0
1
1
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
188
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
190
0
0
0
0
0
0
0
0
0
0
0
1
1
0
1
0
0
0
1
1
0
1
0
0
1
1
1
192
0
0
1
1
0
1
0
0
0
0
1
0
0
0
0
0
1
0
0
0
1
0
1
1
0
0
0
194
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
196
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
198
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
201
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
203
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
205
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
207
0
0
0
0
0
1
0
1
1
1
1
0
0
0
0
0
1
0
0
1
0
0
0
0
1
0
0
211
1
1
1
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
213
0
0
0
1
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
115
0
0
-9
0
-9
0
-9
0
0
0
0
0
0
0
0
-9
0
-9
0
0
-9
0
0
-9
0
0
-9
122
0
0
-9
0
-9
0
-9
0
0
0
0
0
0
0
0
-9
0
-9
0
0
-9
0
0
-9
0
0
-9
127
0
0
-9
0
-9
0
-9
0
0
0
0
0
0
0
0
-9
0
-9
1
0
-9
0
0
-9
0
0
-9
129
0
0
-9
0
-9
1
-9
0
0
0
0
0
0
1
0
-9
1
-9
1
1
-9
1
1
-9
1
0
-9
130
0
0
-9
0
-9
0
-9
1
1
1
1
0
0
0
0
-9
0
-9
0
0
-9
0
0
-9
0
1
-9
132
1
1
-9
1
-9
0
-9
0
0
0
0
1
1
0
1
-9
0
-9
0
0
-9
0
1
-9
0
0
-9
215
0
0
0
0
0
0
-9
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
221
0
0
0
0
0
0
-9
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
227
0
0
0
0
0
0
-9
0
0
0
0
-9
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
229
0
0
0
0
0
0
-9
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
231
0
0
0
1
0
1
-9
0
0
0
1
-9
1
0
1
1
1
0
0
1
0
0
0
0
1
0
0
234
0
0
0
0
0
0
-9
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
236
0
0
0
0
0
0
-9
0
0
0
0
-9
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
238
0
0
0
0
0
0
-9
0
0
0
0
-9
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
248
1
1
1
1
1
1
-9
1
1
1
1
-9
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
118
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
0
0
120
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
-9
0
0
0
0
0
0
124
1
1
0
0
-9
0
0
1
1
1
0
1
0
1
0
0
1
0
0
1
-9
0
1
0
1
0
0
127
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
0
0
129
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
0
0
132
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
-9
0
0
0
0
0
0
134
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
1
0
0
0
0
0
136
0
0
0
0
-9
1
1
1
1
1
1
0
0
0
0
1
1
0
0
1
-9
0
1
1
1
1
1
138
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
0
0
140
1
1
1
1
-9
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
-9
0
0
0
0
1
0
142
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
1
0
0
0
177
1
1
1
0
1
1
1
0
0
0
1
0
0
1
0
1
1
1
-9
1
-9
1
1
0
1
1
1
179
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
-9
0
0
0
0
0
0
181
1
1
1
1
0
0
0
0
0
0
0
1
1
0
1
0
0
0
-9
0
-9
0
0
1
0
0
0
183
0
0
0
0
0
0
1
1
1
1
1
0
0
1
0
1
0
1
-9
0
-9
0
0
0
0
0
0
191
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
-9
1
1
1
0
1
1
193
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
-9
0
0
0
0
0
0
195
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
-9
0
0
0
0
0
0
302
0
0
0
0
0
0
-9
0
0
0
0
0
0
0
0
-9
0
-9
0
0
-9
0
0
0
0
0
0
308
0
0
0
0
0
0
-9
0
0
0
0
0
0
0
0
-9
0
-9
0
0
-9
0
0
0
0
0
0
317
0
0
0
0
0
0
-9
0
0
0
0
0
0
0
0
-9
0
-9
1
0
-9
0
0
0
0
0
0
173
catagory
319
320
321
328
329
331
173
175
179
181
187
189
191
193
195
197
199
203
205
210
336
339
346
348
352
356
365
360
367
370
376
380
390
161
165
167
169
171
175
179
DabOR
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
0
1
1
0
0
0
0
DabOR
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
0
1
1
0
0
0
0
DabOR
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
0
1
1
0
0
0
0
DabyOR
DabySH
0
0
1
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
0
0
1
0
0
0
0
DabySN
DabySN
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
1
0
0
0
0
-9
-9
-9
-9
-9
-9
0
0
0
0
0
1
0
0
0
0
0
1
0
0
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
DabySN DabySN DabySN DprhSN6
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
1
0
0
0
0
DcayOR
0
0
1
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
1
0
0
0
0
DcayOR
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
DcaySh DcaySh DcaySh
0
0
1
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
174
0
0
1
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
1
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
DcaySh
-9
-9
-9
-9
-9
-9
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
DcaySh
0
0
1
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
DcaySH
-9
-9
-9
-9
-9
-9
0
0
0
0
0
0
0
1
0
0
0
0
1
0
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
0
0
1
1
0
0
0
DcaySh
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
DcaySH
0
0
1
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
DcaySH
-9
-9
-9
-9
-9
-9
0
0
0
0
0
0
0
0
1
0
0
0
0
0
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
0
0
1
0
0
0
0
DcaySh
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
DcaySH
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
0
DcaySH
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
-9
0
0
1
0
0
1
0
DcaySH
0
0
1
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
DcaySh
0
0
1
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
catagory DcaySH DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DabyOR DprhOR DprhOR DprhOR DprhOr DprhSH DprhSh DprhSH DprhSH DprhSH DabySh DcayO
R
178
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
0
184
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
1
1
0
0
1
0
0
186
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
0
0
0
1
0
0
0
188
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
0
-9
0
0
0
1
0
0
0
0
0
0
0
190
0
0
0
0
1
1
0
1
0
0
1
0
0
1
1
0
1
1
0
0
-9
1
0
0
0
0
0
1
0
0
0
1
192
1
1
1
1
0
0
1
0
1
0
0
0
1
0
0
1
0
0
1
0
-9
0
0
0
0
1
0
0
0
0
0
0
194
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
1
0
196
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
1
0
0
0
0
0
0
0
0
198
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
1
0
0
0
0
0
0
0
0
0
201
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
1
0
1
0
0
0
0
0
203
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
-9
0
0
0
0
0
0
0
0
1
0
0
205
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
0
207
0
1
1
1
0
0
1
0
1
0
0
0
1
0
0
1
1
0
1
0
-9
0
0
0
0
0
0
0
0
0
0
0
211
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
0
0
1
0
0
0
0
213
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
1
115
-9
0
-9
0
-9
0
0
0
-9
0
0
-9
-9
0
0
-9
0
-9
-9
0
-9
0
-9
-9
0
0
-9
-9
-9
0
-9
0
122
-9
0
-9
0
-9
0
0
0
-9
0
0
-9
-9
0
0
-9
0
-9
-9
0
-9
0
-9
-9
0
0
-9
-9
-9
0
-9
0
127
-9
0
-9
0
-9
0
0
0
-9
1
1
-9
-9
0
0
-9
0
-9
-9
0
-9
0
-9
-9
0
0
-9
-9
-9
1
-9
0
129
-9
1
-9
0
-9
1
1
1
-9
1
0
-9
-9
1
1
-9
0
-9
-9
1
-9
1
-9
-9
0
1
-9
-9
-9
0
-9
0
130
-9
0
-9
1
-9
0
0
0
-9
0
1
-9
-9
0
0
-9
1
-9
-9
0
-9
0
-9
-9
1
0
-9
-9
-9
0
-9
0
132
-9
0
-9
0
-9
0
0
0
-9
0
0
-9
-9
0
0
-9
0
-9
-9
0
-9
0
-9
-9
0
0
-9
-9
-9
1
-9
1
215
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
-9
-9
0
0
-9
-9
0
0
0
0
221
0
0
0
0
0
0
0
0
0
0
0
-9
0
1
0
0
0
0
0
0
0
0
-9
-9
0
0
-9
-9
0
0
0
0
227
1
0
0
0
0
0
0
0
0
1
0
-9
0
0
0
0
0
0
0
0
1
0
-9
-9
0
1
-9
-9
0
0
1
0
229
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
-9
-9
0
0
-9
-9
1
0
0
0
231
0
1
1
1
0
0
1
0
1
1
0
-9
1
0
0
1
1
0
1
0
0
0
-9
-9
1
0
-9
-9
0
0
0
1
234
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
-9
-9
0
0
-9
-9
0
1
0
0
236
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
-9
-9
0
0
-9
-9
0
0
0
0
238
0
0
0
0
0
0
0
0
0
0
0
-9
0
0
0
0
0
0
0
0
0
0
-9
-9
0
0
-9
-9
0
0
0
0
248
1
1
1
1
1
1
1
1
1
0
1
-9
1
1
1
10
1
1
1
1
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175
catagory DcaySH DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DcaySN DabyOR DprhOR DprhOR DprhOR DprhOr DprhSH DprhSh DprhSh DprhSh DprhSH DabySh DcayO
R
308
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339
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390
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161
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165
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176
Declaration
This thesis is my original work and has not been presented for a degree in any other
University, and that all sources of material used for the thesis have been duly
acknowledged.
___________________
Wendawek Abebe Mengesha
(PhD Candidate)
________________________
Prof. Sebsbe Demissew
_________________________
Prof. Inger Nordal
__________________________
Dr Paul Wilkin
(Supervisors)
177
wendawek Abebe