Springer-VerlagTokyo102650918-94401618-086030669031Journal of Plant Research J Plant Res007610.1007/s10265-003-0076-8 J Plant Res (2003) 116:115–132 Digital Object Identifier (DOI) 10.1007/s10265-003-0076-8 © The Botanical Society of Japan and Springer-Verlag Tokyo 2003 ORIGINAL ARTICLE M. Pfosser • W. Wetschnig • S. Ungar • G. Prenner Phylogenetic relationships among genera of Massonieae (Hyacinthaceae) inferred from plastid DNA and seed morphology Received: June 26, 2002 / Accepted: December 5, 2002 / Published online: February 22, 2003 Abstract The tribe Massonieae Baker (HyacinthaceaeHyacinthoideae) presently consists of about 19 genera and 230 species distributed from Africa (south of the Sahara) to Madagascar and India. Based on atpB and trnL-F DNA sequences the tribe is monophyletic only when the genus Pseudoprospero is excluded from Massonieae. In most trnL-F trees, this genus occupies a basal position within subfamily Hyacinthoideae and is sister to the rest of the subfamily. Molecular data suggest that the remaining genera of Massonieae do not share common ancestry with the Eurasian/North-African tribe Hyacintheae Dumort. (Scilla, Hyacinthus and allies), and thus a narrow concept of the essentially Eurasian genus Scilla is supported. Members of well-supported clades in Massonieae usually show similarities in seed characteristics as determined by scanning electron microscopy. Phylogenetic position and seed morphology indicate that Massonia angustifolia and M. zeyheri do not belong to the genus Massonia but fall into a clade together with Daubenya, Androsiphon and Amphisiphon. The genus Whiteheadia appears paraphyletic in the 50% majority rule trnL-F tree and occupies a basal position next to Massonia. However, in the strict consensus tree neither monophyly nor polyphyly can be excluded for this genus. Seed appendages are documented for members of the genera Ledebouria and Lachenalia. Within the genera of Massonieae there is a tendency towards bending of the seed axis. This phenomenon is most obvious within the genus Lachenalia. Delimitation of genera based on seed morphology largely agrees with the results of molecular studies. Correlation between number, size and color of seeds, geographical distribution and phylogenetic position of the genera are discussed. M. Pfosser (*) · S. Ungar Department of Higher Plant Systematics and Evolution, Institute of Botany, Rennweg 14, 1030, Vienna, Austria Tel. +43-1-427754150; Fax +43-1-42779541 e-mail: martin.pfosser@univie.ac.at W. Wetschnig · G. Prenner Institute of Botany, Karl-Franzens-University, Graz, Austria Key words Hyacinthaceae · Massonieae · Molecular phylogeny · Plastid DNA sequences · Scanning electron microscopy · Seed morphology Introduction Previous analyses using atpB, rbcL and trnL-F data have shown that the family Hyacinthaceae is monophyletic and is nested within Asparagales. Its closest relatives are the family Themidaceae and Aphyllanthes monspeliensis (Chase et al. 2000; Fay et al. 2000; Pfosser and Speta 1999). Based on molecular, morphological, karyological and chemotaxonomical data the family can be split into the four subfamilies Oziroeoideae, Urgineoideae, Ornithogaloideae and Hyacinthoideae (Pfosser and Speta 1999, 2001). Taxa from the fifth subfamily Chlorogaloideae previously included in Hyacinthaceae (Speta 1998a, 1998b) have been shown to have affinities to the families Anthericaceae, Funkiaceae and Agavaceae but not to Hyacinthaceae (Pfosser and Speta 1999, 2001). Within Hyacinthoideae a distribution of genera into the tribes Massonieae Baker and Hyacintheae Dumort. has been proposed (Speta 1998a). The two tribes show a strong geographic pattern. According to Speta, the Massonieae consist of genera with a distribution range from Africa (south of the Sahara) to the Arabian peninsula, Madagascar and India, whereas the tribe Hyacintheae is exclusively North Hemispheric and consists of genera with a predominantly Eurasian/North-African distribution. No phylogenetic investigations have been undertaken to infer relationships among genera of Massonieae so far. Recently, genetic diversity of South African species of Scilla sensu lato has been investigated by RAPD analysis (van Staden and Pan 2001). However, the absence of any genus outside of Massonieae makes it impossible to draw phylogenetic conclusions from their work. The inventory of species within Massonieae is not yet complete and delimitation of genera is still under dispute; for example, in 1997, Müller-Doblies and Müller-Doblies published a partial revision of Massonieae in which they 116 described the new genus Namophila and recognized 15 genera (Müller-Doblies and Müller-Doblies 1997). Speta (1998a) described the new genera Merwilla, Pseudoprospero and Avonsera and also recognized fifteen, partly different, genera. Pfosser and Speta (1999) questioned the independent generic status of Daubenya, Androsiphon and Amphisiphon. Goldblatt and Manning (2000) finally transferred the monotypic genera Androsiphon and Amphisiphon as well as Neobakeria namaquensis and Massonia angustifolia to Daubenya. Jessop (1970) and Stedje (1998) grouped several members of Massonieae within a broadly defined genus, Scilla. However, such a classification inevitably results in a highly polymorphic genus (Pfosser and Speta 1999, 2001, 2003). Morphology of seeds is rather diverse in Hyacinthaceae. Seed characteristics have been shown to be useful for subfamilial delimitation (Speta 1998a) and generic grouping (Jessop 1975). Recently, a survey of seed morphology among genera of Massonieae was presented (Wetschnig et al. 2002). The aim of the present study was to investigate the monophyly of the tribe Massonieae, the relationship among genera and to the sister tribe Hyacintheae. The study is based on plastid DNA and seed morphology using material from Africa, Madagascar, India, Eurasia, North Africa and East Asia. One coding region of plastid DNA (atpB) and two non-coding regions, the trnL(UAA) intron and the trnL(UAA)-trnF(GAA) intergenic spacer, were investigated. The atpB gene has been shown to be useful for higher level systematic studies in monocots (Chase et al. 2000; Savolainen et al. 2000), whereas the non-coding regions have been used successfully in Hyacinthaceae and many other groups to reveal subfamilial relationships (Pfosser and Speta 1999, 2001, 2003; Stedje 1998, 2000). Seed morphology data based on light and electron microscopic examinations were used as additional characters for generic delimitation within Massonieae. Correlation between seed morphology, geographical distribution and phylogenetic position of genera are discussed. Materials and methods Taxa sampled This analysis is based on material of Hyacinthoideae sampled from Africa, Madagascar, India, Eurasia, North Africa and East Asia. Voucher information for all plant accessions, geographic origin, and EMBL database accession numbers are provided in the Appendix. Nomenclature follows that of Speta (1998a, 1998b). DNA extraction Total genomic DNA was extracted from lyophilized and powdered leaf or seed material in 700 ml CTAB buffer (2% CTAB, 100 mM Tris, 1.4 M NaCl, 20 mM EDTA, 0.2% mercaptoethanol, pH 8.0) for 30 min at 60°C; 500 ml chloroform/isoamylalcohol (24/1) was added and the extraction mix incubated for 15 min at 4°C. After centrifugation, the DNA was precipitated with 500 ml isopropanol. The pellet was washed with 70% ethanol and dissolved in 100 ml TE buffer. DNA sequencing Two non-coding regions and one coding region of the plastid genome were sequenced. The trnL(UAA) intron and the intergenic spacer (IGS) between the trnL(UAA)-3¢exon and the trnF(GAA) gene were amplified together in a single PCR reaction (Pfosser and Speta 1999). The atpB gene was amplified using the primers S2 and 1493R (Hoot et al. 1995). Amplified double-stranded DNA fragments were sequenced directly on an ABI377 automated sequencer (Perkin Elmer, Beaconsfield, UK) following the DYEnamicET cycle sequencing protocol (Amersham Pharmacia, Piscataway, N.J.). Both strands were sequenced using the nested sequencing primers described for the trnL-F region (Pfosser and Speta 1999). Sequencing primers for the atpB gene were the same as those used by Hoot et al. (1995) except for two internal sequencing primers which were designed specifically for the sequencing of Asparagalean taxa (primer 385x: 5¢-GCG CAG ATC TAT GAA TAG GTG ATG T-3¢, primer 766x: 5¢-TAA CAT CCC GGA AAT ATT CCG CCA T-3¢). On average, less than 1% of data matrix cells were scored as missing data. Phylogenetic analysis Sequence manipulations were performed on a Digital Alpha 1000A 5/400 server under the operating system Digital Unix V.4.0D. DNA sequences were pre-aligned using the PileUp program of the GCG software package (Genetics Computer Group 1994). Final alignment of DNA sequences was performed by visual inspection. The sequences have been trimmed on both ends to exclude ambiguous positions in close proximity to the sequencing primers. All sequences have been deposited in the EMBL database (for accession numbers refer to Appendix). Phylogenetic analysis using the maximum parsimony method was performed with the computer program PAUP* version 4.0b10 (Swofford 2000). Most parsimonious trees were obtained by 1,000 replicates of random sequence addition using tree bisection-reconnection branch swapping under the Fitch criterion (Fitch 1971). Ten thousand fast bootstrap replicates (Felsenstein 1985) were used to assess confidence limits for the resulting tree topologies. Indels in the data matrix were coded as additional characters (indels longer than one nucleotide position were treated as a single evolutionary event), and tree searches were performed using the nucleotide data alone or together with the indel data. Since the combined data (nucleotide plus indels) yielded 117 similar tree topologies with higher bootstrap values than the nucleotide data alone, only the combined trees are presented here. To determine if the atpB and trnL-F sequence data are in significant conflict with regard to the phylogeny of Hyacinthaceae, we conducted a partition homogeneity test (Farris et al. 1994, 1995; Swofford 2000). Tree manipulations were performed using MacClade version 3.06 (Maddison and Maddison 1992). Morphological analysis Eighty-nine seed samples comprising 15 genera were analyzed morphologically. Out of 60 seed samples from species of the genus Lachenalia, 11 morphologically distinct samples were selected for analysis. All examinations were carried out on fully developed dry seeds. Weight and size (arithmetic mean) were determined from at least ten seeds. Dried seeds were mounted on aluminum stubs with “LeitTabs” and coated with gold in an Agar sputter coater. Electron micrographs were obtained with a Philips XL 30 ESEM scanning electron microscope operating at 20 kV. In total aspects the seeds were oriented with the chalazal pole pointing left and the micropylar pole pointing right. Lateral views were oriented with the raphe facing upwards. Results Sequence variation of atpB and trnL-F regions AtpB sequences were produced for 37 taxa of Hyacinthaceae (for a complete list of taxa and accession numbers see Appendix). The atpB sequence of Muilla maritima (Themidaceae; EMBL acc. no. AF209635) was used to root the phylogenetic trees. Except for a 6-bp in-frame insertion close to the 3¢ end of the atpB sequence of Dipcadi cf. heterocuspe no indels were found in the data matrix; 1,432 characters of the aligned sequences were used in phylogenetic analyses. TrnL-intron and trnL-trnF intergenic spacer sequences were analyzed for 123 taxa and 149 accessions of Hyacinthaceae (Table 1). In total, the combined trnL-F matrix was composed of 82 new plus 67 previously published sequences. The lengths of the trnL-F sequences were much more variable than those for the atpB locus. Within Massonieae, 25 parsimony-informative indels have been identified that were coded as additional characters and added to the data matrix. The aligned trnL-F data matrix yielded 1,497 characters. To exclude ambiguities in close proximity to the sequencing primers the sequences have been trimmed on both ends and only the unambiguous middle part consisting of 1,265 characters (including both informative and uninformative) was used for phylogenetic analyses. The trnL-F trees were rooted either with the sequence of Muilla maritima (EMBL acc. no. AF117019, AF117047) from the sister family Themidaceae or with Oziroe biflora and O. acaulis from subfamily Oziroeoideae of Hyacinthaceae. Subfamily Oziroeoideae has previously been shown to be the most basal subfamily within Hyacinthaceae (Pfosser and Speta 1999, 2001, 2003) and is therefore suited to serve as outgroup for inference of relationships among the remaining taxa of Hyacinthaceae. Phylogenetic analysis of trnL-intron and trnL-F IGS data Cladistic analysis of the combined trnL-intron and trnL-F IGS data yielded more than 500 equally parsimonious trees with a Fitch tree length of 722 steps and a consistency index (CI) of 0.697, a retention index (RI) of 0.917 and a rescaled consistency index (RC) of 0.639. A relatively low homoplasy index (HI = 0.303) indicates the suitability of the trnL-F region for inferring phylogenetic implications for the family Hyacinthaceae. One of the most parsimonious trees is shown in Fig. 1 and comparison of this tree with 50 randomly chosen, equally parsimonious, trees revealed no differences in the composition of clades. Delimitation of the subfamilies Oziroeoideae, Ornithogaloideae and Urgineoideae, which formed monophyletic groups in this analysis, was supported by high bootstrap values (100%). However, only a few representatives from these subfamilies have been included in the data matrix. A detailed study covering these subfamilies more completely will be presented elsewhere. The subfamily Hyacinthoideae was monophyletic and received 67% bootstrap support. Distribution of species into an essentially South African/ Madagascan/Arabian/Indian tribe Massonieae and an Eurasian/North African/Asian tribe Hyacintheae was not supported by monophyly. Instead, in the 50% majority rule consensus tree, the monotypic genus Pseudoprospero occupied the most basal position within Hyacinthoideae and thus splits off from the rest of Massonieae. The remaining species of Massonieae, however, formed a monophyletic group (68% bootstrap support) and appeared to be well separated from other members of Hyacinthoideae. Within Massonieae, the genera Merwilla, Schizocarphus, Drimiopsis, Resnova, Eucomis, Veltheimia and Lachenalia were monophyletic. The genus Ledebouria was paraphyletic with Drimiopsis and Resnova but formed a highly supported clade with these genera (99% bootstrap support). The Daubenya-Androsiphon-Amphisiphon clade received 100% bootstrap support. The two species Massonia angustifolia and M. zeyheri clearly fell into this group. Basal to this clade was Scilla cf. plumbea (96% bootstrap support). The affinity of this species to the clade DaubenyaAndrosiphon-Amphisiphon was also supported by a large, synapomorphic deletion of 64 bp in the IGS region. The genus Whiteheadia appeared paraphyletic and basal to a highly supported group of species of Massonia (99%) although neither monophyly nor polyphyly for this genus could be excluded based on the strict consensus tree. If M. angustifolia and M. zeyheri were excluded from Massonia, this genus was monophyletic. The genera Periboea, Polyxena and Lachenalia constituted the most derived clade (100% bootstrap support). The species Polyxena calcicola formed a clade together with Periboea (95% bootstrap 118 support). Low bootstrap support values for the remaining taxa of Polyxena (62%) as well as for Lachenalia (66%) may indicate either that generic delimitation is not yet optimal in this clade, or that they are more recently derived and therefore the number of base substitutions is insufficient to resolve them more clearly. The relationships within Hyacintheae will not be dealt with in this study but some representatives are included here because the delimitation of the genus Scilla cannot be discussed without inclusion of Eurasian members of Hyacinthoideae. Seed size and weight As a general rule, seed size and weight were higher in basal genera like Eucomis, Merwilla, Ledebouria and in the members of the Daubenya clade. The largest seeds were found in Veltheimia bracteata, at 0.056 g and with a length of 6.1 mm. The smallest seeds were found in the genus Lachenalia (L. angelica: 0.0003 g; 0.9 mm long). Seed shape Relationships among genera of Hyacinthaceae Representatives from all highly supported monophyletic groups as determined from the trnL-F analysis were included in a further analysis with the aim of studying relationships between clades. AtpB, trnL-F and combined atpB/ trnL-F trees were generated and the resulting tree topologies were compared (Fig. 2). Heuristic searches yielded 504 equally most parsimonious trees with a tree length of 317 steps for the atpB matrix, 396 trees (L = 485 steps) for the trnL-F matrix, and 15 most parsimonious trees (L = 806 steps) for the combined atpB + trnL-F matrix. The values for CI, RI and RC for the three trees were 0.751/0.794/0.596, 0.763/0.801/0.611 and 0.754/0.794/0.599, respectively. Overall tree topology was congruent for all three trees. A partition homogeneity test indicated no evidence that the trnL-F and atpB data sets contained significant incongruence (P = 0.81). The subfamilies Oziroeoideae, Ornithogaloideae and Urgineoideae received high bootstrap support values (92–100%). No bootstrap support was obtained for the clade consisting of Hyacinthoideae from atpB data. However, support values for Hyacinthoideae increased from 52% (trnL-F tree) to 74% in the combined analysis. In the atpB tree, monophyly of the tribe Hyacintheae was supported by 62% bootstrap value. No bootstrap support for the monophyly of Hyacintheae was found in the trnL-F data, but in the combined analysis it increased to 84%. Bootstrap support for the monophyly of the tribe Massonieae was found in the trnL-F and the combined analysis (63% vs 89%) only when Pseudoprospero firmifolium was excluded from Massonieae. This genus occupied labile positions in the three trees as did the genera Schizocarphus, Veltheimia and Whiteheadia. In all three trees, strong phylogenetic ties (bootstrap values >70%) within Massonieae were suggested between Ledebouria, Resnova and Drimiopsis, between Scilla cf. plumbea and Daubenya and between Lachenalia, Polyxena and Periboea. Morphological analysis of seed structure among genera of Massonieae Morphological characters of the seeds, like size and weight, shape, color and structure of the seed surface, proved to be highly variable within Massonieae, providing a set of characters diagnostic for most species and genera analyzed in this study (Fig. 3). Seed shape, as a single character, was most suitable for discriminating among genera of Massonieae. Shape of the endosperm, swelling of the seed coat – especially at the micropylar region – and folding of the seed coat at the chalazal pole are characters defining the shape of the seed. In most taxa, the form of the endosperm was more or less ellipsoid (e.g., Fig. 3, a-3 to a-8, b-3 to b-6), globose (c-1 to c-8, d-4, e-8) or ovate (b-7, b-8, d-1, d-5). Merwilla natalensis was the only species of our study with a compressed ellipsoid endosperm (a-1). Resnova showed an endosperm unique among Massonieae. In this genus the endosperm formed outgrowth along both sides of the raphe leading to a characteristic folding of the seed (b-1). The endosperm of the genera Whiteheadia (d-2), Massonia (d-3 to d-8), Periboea (e-1), Polyxena (e-2) and Lachenalia (e-3 to e-8, f-1 to f-5) showed a regular distinctive flattening or indentation at the chalazal pole sometimes even leading to depressions of the surrounding tissues in this region. Whereas the axis of the endosperm was straight in most genera of Massonieae, the genus Lachenalia was characterized by a distinctively bent axis (most obvious in e-5 and e-8). This feature was never found in seeds of Massonia. Swelling of the seed coat at the micropylar region was absent or inconspicuous in basal taxa like Merwilla natalensis (a-1, a-2), in most samples of Ledebouria (a-5, a-6) and in most species of Eucomis (b-5 to b-8). Species of the Daubenya clade were characterized by flat, elongated and compressed swellings at this region (c-1 to c-8), whereas Whiteheadia (d-2), Massonia (d3 to d-8), Periboea (e-1), Polyxena (e-2) and some species of Lachenalia (e-3 to e-5) showed acuminate swellings. In Lachenalia, many species were characterized by conspicuous appendages derived from prominent swellings of the seed coat in the micropylar region. In some species these swellings extended partly or totally across the entire raphe (e-6 to e-8, f-1 to f-5). Whereas in some species, such as L. mathewsii (e-6) or L. pusilla (e-7), these appendages were filled with cellular tissue, in the dry seeds of others, like L. aloides (e-8), L. rubida (f-1, f-2), L. undulata (f-4) or L. bulbifera (f-5), they were hollow (f-2). The exact function of these appendages, being either true elaiosomes or structures for enhancement of seed dispersal by hydrochory, remains unclear. A characteristic folding of the seed coat in the chalazal region (above the truncation or indentation of the endosperm) was obvious in Massonia (d-3 to d-8), in Periboea, Polyxena and in Lachenalia (e-1 to e-8). In some species of Lachenalia (e.g., L. rubida, L. undulata and L. bulbifera) the testa formed conspicuous structures in this 119 Fig. 1. Maximum parsimony tree based on trnL-F sequences showing the relationships within family Hyacinthaceae. The tree is rooted with representatives of subfamily Oziroeoideae. Bootstrap values >50% are indicated above branches. Nodes not present in the strict consensus tree are marked with arrows. Subfamilial and tribal assignments are indicated on the right 120 5 changes 62 99 c 99 Periboea paucifolia 100 Polyxena ensifolia 89 Lachenalia pusilla 5 changes MASSONIEAE HYACINTHEAE M MASSONIEAE Whiteheadia bifolia ´ Massonia depressa 1 Veltheimia bracteata 1 ´ 100 Daubenya aurea 56 Scilla cf. plumbea 53 100 Eucomis montana Eucomis bicolor 92 Schizocarphus nervosus 1 ´ 63 Drimiopsis sp. 89 100 Resnova humifusa Ledebouria sp. 1 Merwilla sp. 1 Pseudoprospero firmifolium ´ 77 Scilla spetana 65 Schnarfia messeniaca 74 Nectaroscilla hyacinthoides 100 Othocallis siberica 96 Hyacinthus orientalis 84 Hyacinthella heldreichii 83 Brimeura amethystina Hyacinthoides non-scripta 100 Barnardia scilloides 1 85 Barnardia scilloides 2 97 Charybdis hesperia 100 Charybdis aphylla 100 Charybdis undulata 100 Rhadamanthus mascarenensis 100 Bowiea sp. Bowiea volubilis 100 Stellarioides caudata/longebracteata 55 Stellarioides sp. 94 100 Pseudogaltonia clavata Dipcadi cf. heterocuspe Ornithogalum kochii 100 Oziroe biflora Oziroe acaulis OUTGROUP 77 58 MASSONIEAE Periboea paucifolia Polyxena ensifolia Lachenalia pusilla 54 Whiteheadia bifolia ´ 65 Massonia depressa 1 Veltheimia bracteata 1 ´ 89 Daubenya aurea Scilla cf. plumbea 100 Eucomis montana 70 Eucomis bicolor Drimiopsis sp. 63 100 Resnova humifusa 63 Ledebouria sp. 1 Schizocarphus nervosus 1 ´ Merwilla sp. 1 76 Scilla spetana 51 Schnarfia messeniaca Nectaroscilla hyacinthoides 95 Othocallis siberica 95 Hyacinthus orientalis Hyacinthella heldreichii 52 Brimeura amethystina Hyacinthoides non-scripta 93 Barnardia scilloides 1 Barnardia scilloides 2 Pseudoprospero firmifolium ´ 61 Charybdis hesperia 100 Charybdis aphylla 99 Charybdis undulata 100 Rhadamanthus mascarenensis 100 Bowiea sp. Bowiea volubilis 96 Stellarioides longebracteata Stellarioides sp. 76 100 Pseudogaltonia clavata Dipcadi cf. heterocuspe Ornithogalum kochii 100 Oziroe biflora Oziroe acaulis OUTGROUP 90 HYACINTHEAE b 98 Periboea paucifolia Polyxena ensifolia Lachenalia pusilla Massonia depressa 1 96 Daubenya aurea 60 Scilla cf. plumbea Whiteheadia bifolia ´ 60 Veltheimia bracteata 1 ´ 54 98 Eucomis montana Eucomis bicolor Schizocarphus nervosus 1 ´ 72 Drimiopsis sp. Resnova humifusa Ledebouria sp. 1 Merwilla sp. 1 Pseudoprospero firmifolium ´ Scilla spetana Schnarfia messeniaca Nectaroscilla hyacinthoides 98 Othocallis siberica 62 74 Hyacinthus orientalis Hyacinthella heldreichii 93 Brimeura amethystina Hyacinthoides non-scripta 96 Barnardia scilloides 1 Barnardia scilloides 2 95 Charybdis hesperia 100 Charybdis aphylla 89 Charybdis undulata 82 92 Rhadamanthus mascarenensis 100 Bowiea sp. Bowiea volubilis 99 Stellarioides caudata Stellarioides sp. 90 Pseudogaltonia clavata 98 Dipcadi cf. heterocuspe Ornithogalum kochii 99 Oziroe biflora Oziroe acaulis OUTGROUP 81 HYACINTHEAE a 10 changes Fig. 2a–c. Comparison of atpB, trnL-F and combined atpB + trnL-F analyses for major clades in Hyacinthaceae. Maximum parsimony trees are shown for the atpB matrix alone (a), the trnL-F matrix (b) and the combined atpB + trnL-F matrix (c). The trees are rooted with the sequences of Muilla maritima from the sister family Themidaceae. Bootstrap values >50% are indicated above branches. Nodes not present in the strict consensus tree are marked with arrows. Labile positions between the three trees of the genera Pseudoprospero, Schizocarphus, Veltheimia, and Whiteheadia are marked with asterisks region (f-1 to f-5). All seeds of Ledebouria investigated, with the exception of L. cf. concolor, showed small to conspicuous seed appendages at the chalazal region. In some species the appendage consisted of an expansion and increased folding of the seed coat at the chalazal region (a-5) whereas other species showed finger-shaped appendages reaching almost the length of the endosperm (a-8). Those appendages originated as conspicuous foldings at the raphe and continued along the axis of the endosperm at the chalazal pole. In dry seeds these appendages were filled with a spongy tissue. the otherwise brown seed. This region was also characterized by a polygonal cellular pattern with some dark-brownto-black cells scattered among the yellowish ones. This polygonal pattern was not visible in the micrographs. Seed color A light-brown to beige color of the dry seeds occurred only in Merwilla. All other species had brownish, dark-brown or black seeds. With the exception of Ledebouria cf. concolor, which had shiny black seeds (Fig. 3, a-7), all seeds from Schizocarphus, Ledebouria, Drimiopsis, Resnova, Eucomis up to the Daubenya clade (Fig. 1) were dark-brown or blackish-brown. All remaining taxa at derived positions in the phylogenetic tree from Whiteheadia to Lachenalia had black seeds. In a few species, e.g., Lachenalia bulbifera, the color of the seed was black but the prominent appendage was white. In some Ledebouria species (e.g., L. sp. 3) the yellowish-to-beige color of the appendage was in contrast to the brown color of other parts of the seed. In L. floribunda, a yellowish region at the hilum was in contrast to Seed surface Seed surface structures within Massonieae can be grouped into four morphological classes: 1. Patterns caused by inner seed coat layers. Wrinkled or rugose seeds characterize the genera Schizocarphus, Ledebouria, Resnova, Whiteheadia and Veltheimia. These wrinkles were most probably caused by differences in the amount of water loss in different tissues during desiccation. This was most obvious in Ledebouria floribunda (Fig. 3, a-5, a-6), the only taxon of our study where stomata were found in the epidermis of the seed coat. In dry seeds, these stomata were found at the base of cavities of the wrinkled seed coat (a-6). Wrinkled seeds also occurred in a few species of other genera like Eucomis (b-3), Massonia (d-3), and Lachenalia (e-4). 2. Cellular shape and size (primary sculpture): In general, all taxa of Massonieae showed a smooth primary sculpture. Only in basal genera like Merwilla and Schizocarphus was an alveolate sculpture of the dry seed coat observed (Fig. 3a-2, a-4). An alveolate sculpture has also been documented for Pseudoprospero firmifolium (Jessop 1975). Resnova showed a papillose primary 121 Fig. 3a. Ultrastructural comparison of seed morphology among genera of Massonieae. a1, a-2 Merwilla natalensis: raphe view, seed coat surface. a-3, a-4 Schizocarphus nervosus 1: raphe view, seed coat surface. a-5, a-6 Ledebouria floribunda: raphe view, seed coat surface showing stomata at the base of the cavities of the wrinkled seed coat. a-7 Ledebouria cf. concolor: lateral view. a-8 Ledebouria sp. 3: raphe view 3a-7 3a-8 3a-5 3a-6 3a-3 3a-4 3a-1 3a-2 sculpture (b-2) and Eucomis regia was the only species where a foveolate sculpture (b-6) was observed. 3. Fine relief of the outer cell wall (secondary sculpture) was smooth in all taxa of our study. 4. Epicuticular secretion (tertiary sculpture). The secretion of waxes is a rare phenomenon in seeds (Werker 1997). However, we found epicuticular waxes in taxa of the Daubenya clade, in Massonia and in some species of Lachenalia. Discussion We have shown that, based on atpB and trnL-F sequences, all sub-Saharan/Madagascan/Indian genera of subfamily Hyacinthoideae – with the exception of Pseudoprospero firmifolium (= Scilla firmifolia) – form a monophyletic group (tribe Massonieae). This tribe therefore represents an independent evolutionary lineage distinct from the 122 Fig. 3b. b-1, b-2 Resnova maxima: raphe view, seed coat surface showing collapsed papillae. b-3, b-4 Eucomis bicolor: lateral view, seed coat surface. b-5, b-6 Eucomis regia: raphe view, seed coat surface. b-7, b-8 Eucomis montana: raphe view, hilum 3b-7 3b-8 3b-5 3b-6 3b-3 3b-4 3b-1 3b-2 Eurasian/North African members (tribe Hyacintheae) of this subfamily. The monotypic genus Pseudoprospero occupies a labile position in the phylogenetic trees, being either the basal taxon within Massonieae (atpB data) or sister to Hyacintheae and Massonieae (trnL-F data). Nevertheless, the clear separation between Hyacintheae and Massonieae (when Pseudoprospero is excluded) seen in the atpB + trnL trees supports a narrow concept for the genus Scilla, confined to the Northern Hemisphere from northern Spain to Caucasus, Asia Minor and the Levant (Speta 1998a). The relationships among genera within Hyacintheae have already been analyzed in detail (Pfosser and Speta 1999) and will not be discussed here. Within Massonieae, species of the genera Merwilla, Ledebouria, Schizocarphus, Drimiopsis, Pseudoprospero and Resnova have, at least temporarily, been included within the large and heterogeneous genus Scilla (Baker 1873; Jessop 1975; Stedje 1998). For example, Jessop (1975) compared seed surface characters of P. firmifolium, Schizocarphus nervosus and M. natalensis (all of which he treated under the genus Scilla) and regarded the differences among them inconspicuous enough to maintain them within the 123 Fig. 3c. c-1, c-2 Androsiphon capense: lateral view, hilum. c-3, c-4 Daubenya aurea 2: lateral view, hilum. c-5 Massonia zeyheri 2: lateral view. c-6 Massonia angustifolia: lateral view. c-7, c-8 Amphisiphon stylosum 4: lateral view, hilum 3c-7 3c-8 3c-5 3c-6 3c-3 3c-4 3c-1 3c-2 same genus. In her combined analysis of molecular and morphological characters, Stedje (1998) came to the conclusion that Ledebouria and Drimiopsis should be treated as genera separate from the sub-Saharan species of Scilla. Although no support for monophyly was found in her data, she did not recommend a splitting of Merwilla lazulina and Schizocarphus nervosus from Mediterranean species of Scilla. In our analysis, the genera Merwilla and Schizocarphus appeared to be well separated by seed morphology, and the results of the phylogenetic analysis with the inclusion of many more taxa further supports the distribution of species of Scilla into smaller genera as suggested by Speta (1998a, 1998b). The phylogenetic trees supported no clear relationship of Schizocarphus to other genera, although it appeared in close vicinity to the Ledebouria-DrimiopsisResnova clade in the majority rule trnL-F tree. Similarities in seed surface characters among these genera also support this relationship. The exact generic treatment of Scilla cf. plumbea remains problematic. Our data have shown clearly that this taxon has common ancestry with species of the Daubenya clade (96% bootstrap support), and no direct relationship 124 Fig. 3d. d-1 Veltheimia bracteata 1: lateral view. d-2 Whiteheadia bifolia 1: lateral view. d-3 Massonia cf. echinata 2: lateral view. d-4 Massonia pustulata: lateral view. d-5 Massonia cf. tenella: lateral view. d-6 Massonia cf. pygmaea: lateral view. d-7, d-8 Massonia depressa 2: lateral view, hilum 3d-7 3d-8 3d-5 3d-6 3d-3 3d-4 3d-1 3d-2 to any species formerly included within Scilla is evident. Based on examination of the iconotype (Lindley 1830), Speta (1998a) has included this species within Merwilla, a decision not supported by our data. However, comparison of the iconotype with what is currently treated as Scilla plumbea (Goldblatt and Manning 2000; compare with Lewis 1947) revealed that two different plants are treated under the same species name. One species of Ledebouria (L. floribunda) showed regularly distributed openings in the cavities of the wrinkled testa. Whereas Jessop (1975) interpreted these structures as artifacts (“the round structure in the photograph is thought to be a result of damage”), the regular occurrence in each cavity led us to believe that these openings were stomata. Netolitzky (1926) reported stomata in the epidermis of the integument for the monocot families Liliaceae (Ornithogalum, Lilium, Tulipa and Fritillaria), Amaryllidaceae (Hymenocallis repanda, Ismene nutans, Nerine), Iridaceae (Iris germanica) and Cannaceae (Canna). Werker (1997) added Scoliopus (Calochortaceae) and Hymenocallis occidentalis (Amaryllidaceae) to the list. Our report of stomata in the seeds of L. floribunda thus documents the second 125 Fig. 3e. e-1 Periboea oliveri: lateral view. e-2 Polyxena ensifolia 7: lateral view. e-3 Lachenalia liliflora: lateral view. e-4 Lachenalia angelica: lateral view. e-5 Lachenalia zebrina: lateral view. e-6 Lachenalia mathewsii: lateral view. e-7 Lachenalia cf. pusilla: lateral view. e-8 Lachenalia aloides: lateral view 3e-7 3e-8 3e-5 3e-6 3e-3 3e-4 3e-1 3e-2 occurrence of such structures among members of Hyacinthaceae. Possible functions of such stomata have been related to gas exchange during seed growth, better desiccation of fully developed seeds, or improved water uptake during germination (Werker 1997). We have shown that in Massonieae, morphological characters of the seeds allow discrimination of most genera. A detailed analysis of morphological characteristics for each genus is given in Wetschnig et al. (2002). Mapping of seed morphology data onto a DNA-based cladogram revealed several trends (Fig. 4). In Massonieae, there seems to be a general tendency towards an increase in the number of ovules per locule. Most of the basal taxa have two (Pseudoprospero, Ledebouria, Drimiopsis, Resnova), or a few, ovules per locule (Merwilla, Schizocarphus, Eucomis, Veltheimia, Whiteheadia, Daubenya clade), whereas the number of ovules per locule was found to be 21 in Lachenalia aloides (data not shown), and as many as 30 have been reported in other species (Speta 1998b). Parallel to this tendency, there was a general decrease in size and weight of the seeds in more derived genera like Whiteheadia, Massonia, Periboea, Polyxena and Lachenalia. Whereas 126 Fig. 3f. f-1, f-2, f-3 Lachenalia rubida 1: lateral view, longitudinal section through the seed appendage, inner surface of the appendage (* in f-2 indicates collapsed inner tissue of the seed appendage). f-4 Lachenalia undulata: lateral view. f-5 Lachenalia bulbifera: lateral view; arrows indicate the position of the hilum in f-1, f-4 and f-5 3f-4 3f-5 3f-2 3f-1 most basal taxa had brown or blackish-brown seeds, all advanced taxa had black seeds. In Ledebouria, most of the taxa showed brown seeds, but black seeds were also evident within this big genus. Lachenalia, the most advanced genus in our study, showed the highest variability in form and structure of the micropylar swelling of the seed coat and in seed appendages. Although seed morphology could be used in most cases to delimit genera, taxa within the Daubenya clade could be distinguished only by differences in the micropylar and hilum regions and in the tertiary sculpture, whereas the whole clade was well separated from other genera by seed shape. This observation parallels the results of the phylogenetic analyses based on DNA sequences. In the trnL-F analysis, this group was separated from other groups by a high bootstrap support value and several synapomorphic indels. Based on these results there is no doubt that the species Massonia angustifolia and M. zeyheri belong to this group and not to the genus Massonia. Recently, these two taxa, as well as Androsiphon capense, Amphisiphon stylosum and Neobakeria namaquensis Schltr. were transferred to Daubenya by Goldblatt and Manning (2000). Unfortunately, the monotypic genus Neobakeria Schltr. (MüllerDoblies and Müller-Doblies 1997), which probably belongs to the same clade, was not available for our analysis. Our results substantiate a close relationship of the members of 3f-3 the Daubenya clade. However, there are considerable morphological differences between these taxa, thus the generic delimitation needs to be investigated in more detail. Within the Lachenalia clade, it was not possible to discriminate between the two Periboea seed samples and certain members of the polymorphic genus Lachenalia. Furthermore, the genera Periboea, Polyxena and Lachenalia were also closely related in the phylogenetic trees, and thus classification into the different subtribes Massoniinae and Lachenaliinae, respectively (Müller-Doblies and MüllerDoblies 1997), is not supported by our data. According to their classification, Veltheimia and Lachenalia form subtribe Lachenaliinae, whereas Massonia, Periboea, Polyxena and Eucomis belong to subtribe Massoniinae. Our data do not reflect any of these relationships. Instead, Veltheimia appears as the sister genus of a clade consisting of the Lachenalia clade and the Massonia/Whiteheadia clade. To our knowledge, seed appendages as seen in several species of Ledebouria have not been reported so far. However, the prominent seed appendages of Lachenalia have already been documented (Sernander 1906; Speta 1972) and have been interpreted as elaiosomes. However, longitudinal sections through seed appendages have shown that, in several species of Lachenalia, these appendages were empty, enclosing a large cavity resulting from the collapsed inner tissue. Preliminary investigations have shown Se e I bl s, (w) s n, m Polyxena several I bl s s n Periboea several I bl s s n Massonia many (10-30) I bl s, (w) s n Whiteheadia few I bl w s n Veltheimia 3-4 III bl w s n Daubenya clade few (I-) II br, (bl) s s n Eucomis 6-7 (I-) II-III br, (bl) s, (w) s, (f) n Resnova 2 III bl w p n Ledebouria Drimiopsis 2 I-II br, (bl) w s n, c 2 (-6) I br w a n 4-6 I lbr w a n O Lachenalia Se e fewmany vu Se ed d le s W pe ei gh rL oc ul e Fig. 4. Mapping of seed morphology data onto a DNAbased cladogram. A majorityrule consensus tree constructed from trnL-F data for major clades and genera of Massonieae (condensed tree from Fig. 2b) is used as a basis. Representative scanning electron micrographs, the number of ovules per locule, seed weight classes (I ≤ 0.009 g, II > 0.009 g and ≤ 0.018 g, III > 0.018 g), seed coat color (lbr light-brown, br brown, bl black), seed coat surface (s smooth, w wrinkled), seed coat sculpture (a alveolate, f foveolate, p papillose, s smooth), and seed appendages (c chalazal, m micropylar, n not developed) are mapped onto this tree. Morphological characters found only rarely within the same clade or genus are in brackets tC C la oa ss tC o d l o C r Se oat Su ed r C oa face Se tS ed cu Ap lp tu pe re nd ag es 127 99 62 90 65 54 70 100 63 Schizocarphus Merwilla Pseudoprospero (OUTGROUP) that the collapse of the inner tissue occurs before dehiscense of the capsules (data not shown). This makes myrmekochory unlikely and an alternative function, such as improvement of seed dispersal by hydrochory should be considered. Huber (1969) reported a tendency towards bending of the axis of the seeds in Liliiflorae. Although he mentioned seeds of Lachenalia on several occasions, he classified the seeds of Scilloideae (including Lachenalia) as being strictly anatropous, with straight or almost straight axis. This is in striking contrast to our data. In Lachenalia we exclusively found seeds with a bent axis of 45–80°. A slightly less pronounced bending was observed in Periboea, Polyxena and Whiteheadia. The major clades identified in the molecular phylogeny also show a strong geographical pattern. All basal genera occur exclusively, or with most of their species, in the summer rainfall areas (with higher precipitation) of eastern South Africa. The genera Ledebouria, Drimiopsis and Resnova have a tropical growth form with iterative innovation whereas an annual innovation is typical for most other species (Müller-Doblies and Müller-Doblies 1997). The latter growth form is better adapted to the seasonal fluctuating humid/arid climate prevailing in the region of western South Africa. Most of the advanced genera within Massonieae have the majority of their species in the winter rainfall areas of that region. 128 New findings in the present paper 1. The trnL-F and atpB trees largely agree in the placement of major clades of Hyacinthaceae. 2. Within subfamily Hyacinthoideae, the genera Pseudoprospero, Schizocarphus, Veltheimia and Massonia occupy labile positions in the atpB and trnL-F trees. 3. The South African members of Hyacinthoideae are monophyletic (tribe Massonieae) only when Pseudoprospero is excluded from Massonieae. 4. A clear separation between Massonieae (excl. Pseudoprospero) and Hyacintheae is seen in the combined atpB + trnL-F tree (89% and 84% bootstrap support, respectively). Thus, a narrow concept of the essentially Eurasian genus Scilla is supported. 5. Members of well-supported clades in Massonieae usually show similarities in seed characteristics. 6. In addition to their presence in Lachenalia, seed appendages are reported for members of the genus Ledebouria. 7. Delimitation of genera based on seed morphology largely agrees with the results of the molecular studies. Note added in proof The following taxa investigated in this study started to flower in our greenhouse and could now be determined as follows: Massonia sp. 1 = M. echinata L. f.; M. sp. 2 = M. pustulata Jacq.; M. sp. 3, 4, 5 = M. depressa Houtt.; M. cf. pygmaea = M. echinata L. f.; M. cf. echinata 1 = M. echinata L. f.; M. cf. echinata L. f. 2 = M. cf. sessiliflora (Dinter) U. & D. Müller-Doblies; M. cf. depressa Houtt. = M. sp.; M. cf. tenella = M. tenella Soland. ex Baker; Massonia angustifolia L. f. = Massonia marginata Willd. ex Kunth. Acknowledgements We thank all private collectors and botanical gardens that supplied us with living plant material and seeds. W.W. appreciates a grant given by the Republic of South Africa, which allowed him to spend half a year in South Africa to study and collect Hyacinthaceae. We are grateful to the Institute of Plant Physiology (University of Graz) for permission to use their SEM. References Baker JG (1873) Revision of the genera and species of Scilleae and Chlorogaleae. J Linn Soc Bot 13:209–292 Chase MW, Soltis D, Soltis P, Rudall PJ, Fay MF, Hahn WJ, Sullivan S, Joseph J, Molvray M, Kores PJ, Givinish TJ, Sytsma KJ, Pires JC (2000) Higher-level systematics of the monocotyledons: an assessment of current knowledge and a new classification. In: Wilson KL, Morrison DA (eds) Monocots: systematics and evolution. CSIRO, Melbourne, pp 3–16 Farris JS, Kallersjo M, Kluge AG, Bult C (1994) Testing significance of congruence. Cladistics 10:315–320 Farris JS, Kallersjo M, Kluge AG, Bult C (1995) Constructing a significance test for incongruence. Syst Biol 44:570–572 Fay MF, Rudall PJ, Sullivan S, Stobart KL, de Bruijn AY, Reeves G, Qamaruz-Zaman F, Hong W-P, Joseph J, Hahn WJ, Conran JG, Chase MW (2000) Phylogenetic studies of Asparagales based on four plastid DNA regions. In: Wilson KL, Morrison DA (eds) Monocots: systematics and evolution. CSIRO, Melbourne, pp 360–371 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791 Fitch WM (1971) Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20:406–416 Genetics Computer Group (1994) Program manual for the Wisconsin package, version 8. GCG, Madison, Wis. Goldblatt P, Manning JC (2000) Cape plants. A conspectus of the Cape flora of South Africa. Strelitzia 9:93–108 Hoot SB, Culham A, Crane PR (1995) The utility of atpB gene sequences in resolving phylogenetic relationships: comparison with rbcL and 18S ribosomal DNA sequences in the Lardizabalaceae. 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(Hyacinthaceae). Phyton (Horn, Austria) 38:1–141 Speta F (1998b) Hyacinthaceae. In: Kubitzki K (ed) The families and genera of vascular plants. Springer, Berlin Heidelberg New York, pp 261–285 Staden J van, Pan M (2001) Genetic diversity of blue-flowered Scilla species as determined by random amplified polymorphic DNA. S Afr J Bot 67:344–348 Stedje B (1998) Phylogenetic relationships and generic delimitation of sub-Saharan Scilla and allied African genera as inferred from morphological and DNA sequence data. Plant Syst Evol 211:1–11 Stedje B (2000) The evolutionary relationships of the genera Drimia, Thuranthos, Bowiea and Schizobasis discussed in the light of morphology and chloroplast DNA sequence data. In: Wilson KL, Morrison DA (eds) Monocots: systematics and evolution. CSIRO, Melbourne, pp 414–417 Swofford DL (2000) PAUP* Phylogenetic analysis using parsimony (*and other methods), version 4. Sinauer, Sunderland, Mass. Werker E (1997) Seed anatomy. Bornträger, Berlin Wetschnig W, Pfosser M, Prenner G (2002) Zur Samenmorphologie der Massonieae Baker 1871 (Hyacinthaceae) im Lichte phylogenetisch interpretierter molekularer Befunde. Stapfia 80:349–379 Appendix. List of taxa of Hyacinthaceae investigated in this study, with vouchers, citation information, and EMBL accession numbers. All vouchers are deposited in LI unless otherwise indicated. Two accession numbers for the trnL-Flocus indicate that the sequences for the trnLintron and thetrnL-F spacer have been deposited separately Species Voucher Origin EMBL accession numbers trnL-F atpB Citation (trnL-F/atpB) Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Wetschnig 1160 Wetschnig 1144 Wetschnig 1145 Wetschnig 1120 Wetschnig 1129 Klenner H160 S Africa S Africa S Africa S Africa S Africa Spain AJ507957 AJ507959 AJ507958 AJ507955 AJ232518/AJ232641 - This paper/This paper/This paper/-/This paper/Pfosser and Speta 1999/- Hyacinthoideae Vitek 1993 Spain AJ508001 - This paper/- Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Ehrendorfer H015 Pfosser H599 Pfosser H638 Jahn H214 Speta H052 Pfosser H225 Pfosser H230 Wetschnig 1162 Wetschnig 1107 A. Bjørnstad 1158 Nordal 1600 Pfosser L121 Speta H002 Pfosser H642 Schnabel M162 Schnabel M130 Speta H221 Schnabel 2001 Wetschnig M163 Pfosser H013 Fritsch H211 Speta H234 Gutermann H297 Markus H210 Schneider H065 Voglmayr H338 Pfosser H300 Koenen H066 Pfosser H235 Morocco Korea Japan Greece Greece cult. ex B. G. Tallinn Croatia S Africa S Africa Tanzania Tanzania cult. B. G. Vienna cult. LI Madagascar S Africa S Africa cult. LI S Africa ex Manchester Univ. cult. B. G. Vienna Tadschikistan Uzbekistan Croatia Turkey Algeria Spain France Tunisia France AJ232517/AJ232640 AJ507998 AJ507999 AJ232547/AJ232670 AJ232548/AJ232671 AJ232510/AJ232633 AJ232541/AJ232664 AJ507956 Z99137/Z99138 Z99139/Z99140 AJ507952 AJ232502/AJ232625 AJ507953 AJ507933 AJ507932 AJ232500/AJ232623 AJ507934 AJ232534/AJ232657 AJ232536/AJ232659 AJ232535/AJ232658 AJ232526/AJ232649 AJ232527/AJ232650 AJ232521/AJ232644 AJ232525/AJ232648 AJ232519/AJ232642 AJ232520/AJ232643 AJ232524/AJ232647 AJ508218 AJ508219 AJ508216 AJ508204 AJ508202 AJ508198 AJ508197 AJ508213 AJ508217 Pfosser and Speta 1999/This paper/this paper This paper/this paper Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/this paper Pfosser and Speta 1999/This paper/this paper -/Stedje 1998/Stedje 1998/This paper/Pfosser and Speta 1999/This paper/this paper This paper/this paper This paper/this paper Pfosser and Speta 1999/-/This paper/Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/this paper Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/this paper Hyacinthoideae Voglmayr H307 Spain AJ232523/AJ232646 - Pfosser and Speta 1999/- Hyacinthoideae Scheiblreiter H305 Portugal AJ232522/AJ232645 - Pfosser and Speta 1999/- Hyacinthoideae Hyacinthoideae Akhani H573 Speta H067 Iran Romania AJ508689 AJ232539/AJ232662 AJ508215 This paper/Pfosser and Speta 1999/this paper 129 Amphisiphon stylosa W.F. Barker 1 Amphisiphon stylosa W.F. Barker 2 Amphisiphon stylosa W.F. Barker 3 Amphisiphon stylosa W.F. Barker 4a Androsiphon capense Schltr.a Autonoe haemorrhoidalis (Webb & Berth.) Speta 1 Autonoe haemorrhoidales (Webb & Berth.) Speta 2 Autonoe latifolia (Willd.) Speta Barnardia scilloides Lindl. 1 Barnardia scilloides Lindl. 2 Bellevalia aff. brevipedicellata Turrill Bellevalia trifoliata Kunth Brimeura amethystina (L.) Chouard Chouardia litardierei (Breistr.) Speta Daubenya aurea Lindl. 1 Daubenya aurea Lindl. 2a Drimiopsis barteri Baker Drimiopsis botryoides Baker ssp. botryoides Drimiopsis kirkii Baker Drimiopsis maculata Lindley Drimiopsis sp. Eucomis bicolor Bak.a Eucomis montana Comptona Eucomis punctata (Thunb.) L’Herit. Eucomis regia (L.) L’Herit.a Eucomis zambesiaca Baker Fessia greilhuberi (Speta) Speta Fessia puschkinioides (E. Regel) Speta Fessia vvedenskyi (Pazij) Speta Hyacinthella dalmatica (Baker) Chouard Hyacinthella heldreichii (Boiss.) Chouard Hyacinthoides aristidis (Coss.) Rothm. Hyacinthoides hispanica (Mill.) Rothm. Hyacinthoides italica (L.) Rothm. Hyacinthoides lingulata (Poir.) Rothm. Hyacinthoides non-scripta (L.) Chouard ex Rothm. Hyacinthoides reverchonii (Degen & Hervier) Speta Hyacinthoides vincentina (Hoffmanns. & Link) Rothm. Hyacinthus litwinowii Czerniakowska Hyacinthus orientalis L. Subfamily 130 Appendix. Continued Species Hyacinthus orientalis L. var. alba Lachenalia aloides (L. f.) Engl. 1 Lachenalia aloides (L. f.) Engl. 2a Lachenalia angelica W. F. Barkera Lachenalia bulbifera (Cyr.) Engl.a Lachenalia cf. pusilla Jacq. 1a Lachenalia cf. pusilla Jacq. 2 Lachenalia contaminata Aiton Lachenalia liliflora Jacq.a Lachenalia mathewsii W. F. Barkera Lachenalia pallida Aiton 1 Lachenalia pallida Aiton 2 Lachenalia pusilla Jacq. Lachenalia rubida Jacq. 1a Lachenalia rubida Jacq. 2 Lachenalia sp. 1 Lachenalia undulata Masson ex Bak.a Lachenalia zebrina W. F. Barkera Ledebouria cf. concolor (Bak.) Jessopa Ledebouria cordifolia (Baker) Stedje & Thulin Ledebouria floribunda (Baker) Jessopa Ledebouria hyacinthina Roth. 1 Ledebouria hyacinthina Roth. 2 Ledebouria revoluta (L. f.) Jessop Ledebouria socialis (Baker) Jessop Ledebouria somaliensis (Baker) Stedje & Thulin Ledebouria sp. 1 Ledebouria sp. 2 Ledebouria sp. 3a Ledebouria sp. 4 Ledebouria sp. 5 Ledebouria sp. 6 Ledebouria sp. 7 Ledebouria sp. 8 Massonia angustifolia L. f.a Massonia cf. depressa Houtt. Massonia cf. echinata L. f. 1 Massonia cf. echinata L. f. 2a Massonia cf. pygmaea Kuntha Massonia cf. tenella Soland. ex Bakera Massonia depressa Houtt. 1 Massonia depressa Houtt. 2a Massonia pustulata Jacq.a Subfamily Voucher Origin EMBL accession numbers trnL-F atpB Citation (trnL-F/atpB) Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae J. Plass M178 Pfosser H159 Saunders 2002 Saunders 2003 Saunders 2004 Wetschnig 1146 Wetschnig 1164 Pfosser M141 Saunders 2005 Saunders 2006 Wetschnig 1168 Pfosser H021 Wetschnig 1115 Wetschnig 1131 Pfosser M140 Wetschnig 1110 Saunders 2007 Saunders 2008 Wetschnig 1412 Nordal & Stedje 2409 Syria cult. B. G. Vienna S Africa S Africa S Africa S Africa S Africa S Africa S Africa S Africa S Africa cult. B. G. Vienna S Africa S Africa S Africa S Africa S Africa S Africa S Africa Malawi AJ508002 AJ232508/AJ232631 AJ507988 AJ507987 AJ507985 AJ507981 AJ232507/AJ232630 AJ507986 AJ507982 AJ507983 AJ507984 AJ507946 Z99143/Z99144 AJ508207 - This paper/Pfosser and Speta 1999/-/-/-/This paper/This paper/This paper/-/-/This paper/Pfosser and Speta 1999/This paper/this paper This paper/This paper/This paper/-/-/This paper/Stedje 1998/- Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Wetschnig 1433 Jha H838 Jha H839 Nordal 2082 Pfosser H014 Nordal 2296 S Africa India India Zimbabwe cult. B. G. Vienna Ethiopia AJ507937 AJ507944 AJ507945 Z99146/Z99147 AJ232501/AJ232624 Z99150/Z99151 - -/This paper/This paper/Stedje 1998/Pfosser and Speta 1999/Stedje 1998/- Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Pfosser H641 Pfosser H795 Wetschnig 1421 Wetschnig 1419 Mucina LM201001/5 Mucina LM201001/9B Mucina LM201001/6 Mucina LM201001/9A Wetschnig 1101 Wetschnig 1136 Wetschnig 1135 Wetschnig 1137 Wetschnig 1161 Wetschnig 1145 Wetschnig 1142 Wetschnig 1140 Wetschnig 1148 Madagascar Guinea S Africa S Africa S Africa S Africa S Africa S Africa S Africa S Africa S Africa S Africa S Africa S Africa S Africa S Africa S Africa AJ507938 AJ507941 AJ507939 AJ507940 AJ507947 AJ507948 AJ507949 AJ507950 AJ507960 AJ507968 AJ507971 AJ507973 AJ507967 AJ507976 AJ507980 AJ507972 AJ507970 AJ508200 AJ508206 - This paper/this paper This paper/This paper/This paper/This paper/This paper/This paper/This paper/This paper/This paper/This paper/This paper/This paper/This paper/This paper/this paper This paper/This paper/- Species Massonia sp. 1 Massonia sp. 2 Massonia sp. 3 Massonia sp. 4 Massonia sp. 5 Massonia sp. 6 Massonia zeyheri Kunth 1 Massonia zeyheri Kunth 2a Merwilla natalensis (Planchon) Spetaa Merwilla sp. 1 Merwilla sp. 2 Muscari botryoides (L.) Mill. Muscari comosum (L.) Mill. Muscari macrocarpum Sweet Muscari parviflorum Desf. Nectaroscilla hyacinthoides (L.) Parl. Oncostema dimartinoi Raf. Oncostema peruviana (L.) Speta Oncostema villosa (Desf.) Raf. Othocallis siberica (Haw. in Andr.) Speta Othocallis sp. Periboea oliveri U. & D. Müller-Dobliesa Periboea paucifolia (W. F. Barker) U. & D. Müller-Doblies Pfosseria bithynica (Boiss.) Speta Polyxena calcicola U. & D. Müller-Doblies Polyxena ensifolia (Thunb.) Schönl. 1 Polyxena ensifolia (Thunb.) Schönl. 2 Polyxena ensifolia (Thunb.) Schönl. 3 Polyxena ensifolia (Thunb.) Schönl. 4 Polyxena ensifolia (Thunb.) Schönl. 5 Polyxena ensifolia (Thunb.) Schönl. 6 Polyxena ensifolia (Thunb.) Schönl. 7a Polyxena sp. Prospero elisae Speta 1 Prospero elisae Speta 2 Prospero haritonidae Speta Prospero obtusifolium (Poiret) Speta Pseudoprospero firmifolium (Baker) Speta Puschkinia scilloides Adams. var. libanotica Resnova humifusa (Baker) U. & D. MüllerDoblies Resnova maxima Merwea Schizocarphus nervosus (Burch.) Merwe 1a Schizocarphus nervosus (Burch.) Merwe 2 Schnarfia messeniaca (Boiss.) Speta Scilla albescens Speta Subfamily Voucher Origin EMBL accession numbers trnL-F atpB Citation (trnL-F/atpB) Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Wetschnig 1138 Wetschnig 1132 Wetschnig 1134 Wetschnig 1139 Wetschnig 1143 Wetschnig 1133 Wetschnig 1153 Wetschnig 1109 Wetschnig 1534 Puff H219 Puff H218 Kleesadl H011 Neuner H056 Fritsch H212 J. Plass M138 Scheiblreiter H016 Vezda H178 Pfosser H198 Gruber H217 Pfosser H982 Pasche H179 Wetschnig 0005 Wetschnig 1154 S Africa S Africa S Africa S Africa S Africa S Africa S Africa S Africa S Africa cult. B. G. Vienna cult. B. G. Vienna Austria Italy Turkey Syria Portugal Italy Portugal Tunisia cult. B. G. Vienna Turkey S Africa S Africa AJ507974 AJ507969 AJ507978 AJ507975 AJ507977 AJ507979 AJ507961 AJ507962 AJ507931 AJ232499/AJ232622 AJ232498/AJ232621 AJ232545/AJ232668 AJ232546/AJ232669 AJ232544/AJ232667 AJ508003 AJ232542/AJ232665 AJ232514/AJ232637 AJ232516/AJ232639 AJ232515/AJ232638 AJ232533/AJ232656 AJ507989 AJ507990 AJ508196 AJ508210 AJ508214 AJ508208 This paper/This paper/This paper/This paper/This paper/This paper/This paper/This paper/This paper/Pfosser and Speta 1999/this paper Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/This paper/Pfosser and Speta 1999/this paper Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/-/this paper Pfosser and Speta 1999/This paper/This paper/this paper Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Vasak H232 Müller-Doblies H216 Wetschnig 1113 Wetschnig 1157 Wetschnig 1150 Wetschnig 1104 Wetschnig 1156 Wetschnig 1147 Wetschnig 1114 Saunders M061 Speta H068 Ehrendorfer H155 Speta H027 HC H053 Wetschnig 1322–01 Pfosser H229 Wetschnig 1524 Bulgaria S Africa S Africa S Africa S Africa S Africa S Africa S Africa S Africa S Africa Greece Greece Greece Morocco S Africa Lebanon S Africa AJ232540/AJ232663 AJ232506/AJ232629 AJ507994 AJ507992 AJ507996 AJ507995 AJ507993 AJ507991 AJ507997 AJ232530/AJ232653 AJ232531/AJ232654 AJ232528/AJ232651 AJ232529/AJ232652 AJ507928 AJ508688 AJ507942 AJ508209 AJ508195 AJ508201 Pfosser and Speta 1999/Pfosser and Speta 1999/This paper/this paper This paper/This paper/This paper/This paper/This paper/-/This paper/Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/This paper/this paper This paper/This paper/this paper Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hankey M275 Saunders M132 Stedje 94/15 Pfosser H640 Speta H237 S Africa S Africa Zimbabwe cult. B. G. Vienna Greece AJ507943 AJ507936 Z99157/Z99158 AJ508004 AJ232553/AJ232676 AJ508199 AJ508212 - This paper/This paper/this paper Stedje 1998/This paper/this paper Pfosser and Speta 1999/131 132 Appendix. Continued Species Subfamily Scilla cf. bulgarica Speta Scilla cf. plumbea Lindl. Scilla cydonia Speta acc. 1 Scilla cydonia Speta acc. 2 Scilla nana (J.A. & J.H. Schultes) Speta Scilla siehei (Stapf) Speta cv. "Pink Giant" Scilla spetana Kereszty Scilla subnivalis (Halacsy) Speta Tractema lilio-hyacinthus (L.) Speta Tractema monophyllos (Link) Speta 1 Tractema monophyllos (Link) Speta 2 Tractema monophyllos (Link) Speta 3 Veltheimia bracteata Harv. ex Baker 1a Veltheimia bracteata Harv. ex Baker 2 Veltheimia bracteata Harv. ex Baker 3 Veltheimia bracteata Harv. ex Baker 4 Whiteheadia bifolia (Jacq.) Baker 1a Whiteheadia bifolia (Jacq.) Baker 2 Whiteheadia etesionamibensis Müller-Doblies Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Zagrosia persica (Hausskn.) Speta 1 Zagrosia persica (Hausskn.) Speta 2 Dipcadi cf. heterocuspe Baker Ornithogalum kochii Parl. Pseudogaltonia clavata (Masters) Phillips Stellarioides caudata (Jacq.) Speta Stellarioides longebracteata (Jacq.) Speta Stellarioides sp. Oziroe acaulis (Baker) Speta Oziroe biflora (Ruiz & Pavon) Speta Bowiea sp. Bowiea volubilis Harv. ex Hook. f. Charybdis aphylla (Forskål) Speta Charybdis hesperia (Webb & Berth) Speta Charybdis undulata (Desf.) Speta Rhadamanthus mascarenensis Baker Hyacinthoideae Hyacinthoideae Ornithogaloideae Ornithogaloideae Ornithogaloideae Ornithogaloideae Ornithogaloideae Ornithogaloideae Oziroeoideae Oziroeoideae Urgineoideae Urgineoideae Urgineoideae Urgineoideae Urgineoideae Urgineoideae a Taxa used for analysis of seed morphology Voucher Speta H158 Saunders M164 Jahn et al. H215 Speta H489 Speta H238 Speta H010 Speta H227 Speta H240 Hoog & Hoog H298 Raus H049 W. & S. Till H306 Ebert M156 Wetschnig 1535 Wetschnig 1525 Speta H060 Saunders 2009 Wetschnig 1130 Lavranos & Pehlemann H444 Leep H440 Stevens H500 Pfosser H603 Speta H717 Speta H220 Hahn 6928 (WIS) Pfosser H407 Pfosser H608 Weigend s. n. MWC 793 (K) Pfosser H600 Pfosser H222 Strauss H878 Speta H764 Raus M117 Pfosser H610 Origin EMBL accession numbers Citation (trnL-F/atpB) trnL-F atpB Romania S Africa Greece Greece Greece cult. LI Austria Greece Spain Spain Spain Spain S Africa S Africa cult. LI S Africa S Africa S Africa Namibia AJ232555/AJ232678 AJ507954 AJ232549/AJ232672 AJ232550/AJ232673 AJ232552/AJ232675 AJ232551/AJ232674 AJ232556/AJ232679 AJ232554/AJ232677 AJ232511/AJ232634 AJ232513/AJ232636 AJ232512/AJ232635 AJ508000 AJ507965 AJ507964 AJ232503/AJ232626 AJ507963 AJ507966 AJ232504/AJ232627 AJ508205 AJ508211 AJ508203 AJ417588 - Pfosser and Speta 1999/This paper/this paper Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/this paper Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/Pfosser and Speta 1999/This paper/This paper/this paper This paper/Pfosser and Speta 1999/This paper/This paper/-/Chase et al. 2000 Pfosser and Speta 1999/- Turkey Turkey Madagascar Slovenia cult. B. G. Vienna AJ232537/AJ232660 AJ232538/AJ232661 AJ507926 AJ507927 AJ232475/AJ232598 AJ232471/AJ232594 AJ507925 AJ507921 AJ232453/AJ232576 AJ507922 AJ232454/AJ232577 AJ426113 AJ426088 AJ507924 AJ507923 AJ508194 AJ508190/1 AJ508193 AF168935 AJ508192 AJ508183 AJ508182 AJ508184 AJ508185 AJ508189 AJ508188 AJ508187 AJ508186 Pfosser and Speta 1999/Pfosser and Speta 1999/This paper/this paper This paper/this paper Pfosser and Speta 1999/this paper -/Fay et al. 2000 Pfosser and Speta 1999/This paper/this paper This paper/this paper Pfosser and Speta 1999/this paper This paper/this paper Pfosser and Speta 1999/this paper Pfosser and Speta 2003/this paper Pfosser and Speta 2003/this paper This paper/this paper This paper/this paper cult. B. G. Vienna Madagascar Peru Chile Madagascar S Africa Jordan Spain Israel Madagascar