151? 271277 Original Article CHROMOSOME NUMBERS IN BRAZILIAN CROTALARIA A. S. FLORES ET AL. Botanical Journal of the Linnean Society, 2006, 151, 271–277. With 12 figures Chromosome numbers in Brazilian species of Crotalaria (Leguminosae, Papilionoideae) and their taxonomic significance Departamento de Botânica, Instituto de Biologia, CP 6109, Universidade Estadual de Campinas, 13083– 970, Campinas, SP, Brazil Received March 2005; accepted for publication August 2005 Chromosome numbers were counted for 23 species of Crotalaria native to Brazil. Among these data there were new counts for 15 taxa, and some confirmed previous reports or represented numbers that were different from those cited previously. The chromosome numbers most frequently found were 2n = 16 and 2n = 32. Only C. incana L. had 2n = 14 and C. tweediana Benth. had 2n = 54. The counts 2n = 32 and 54 were found in species of section Calycinae and 2n = 16 and 14 in species of section Chrysocalycinae. The data revealed the importance of chromosomal parameters in the characterization of sections Calycinae and Chrysocalycinae in Brazil. We discuss the systematic significance and evolutionary aspects for the genus, comparing the results with the two sections that are native in Brazil. © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 151, 271–277. ADDITIONAL KEYWORDS: Calycinae – Chrysocalycinae – evolution – mitosis – polyploidy – systematics. INTRODUCTION The genus Crotalaria L. comprises herbaceous and shrubby species with simple, one-foliolate or digitately three (four to five)-foliolate leaves and inflated legumes. It is considered to be the third largest genus of Papilionoideae, with approximately 600 species distributed throughout the tropics and subtropics (Polhill, 1982). The centres of diversity are eastern and southern tropical Africa and India, with two additional centres in Mexico and Brazil (Palomino & Vásquez, 1991). The genus was subdivided into eight sections by Polhill (1968) and Bisby & Polhill (1973), based on the floral morphology of African species. In Brazil there are 31 native and 11 introduced or naturalized species of Crotalaria (Flores, 2004). The native ones are grouped into two sections, section Chrysocalycinae (Benth.) Baker f. and section Calycinae Wight & Arn. These have a west–east distribution, the former mainly in western Africa and tropical *Corresponding author. E-mail: anatozzi@unicamp.br America, and the latter extending from there to Asia, Australia and the New World (Polhill, 1982). The classification of Brazilian Crotalaria species in the sections had needed a review or extension of the circumscription originally proposed for these taxonomic levels. Chromosome numbers of Leguminosae have long been studied (e.g. Bandel, 1974; Goldblatt, 1981) and can provide useful characters in delimiting groups at different taxonomic levels (Maciel & Schifino-Wittmann, 2002; Mendonça Filho, Forni-Martins & Tozzi, 2002; Sede et al., 2003). According to the literature, 195 species of Crotalaria have been studied cytologically, which represents approximately 28% of the total species of the genus (Mangotra & Koul, 1991; Palomino & Vásquez, 1991). Chromosome numbers of n = 8, 16, 7 and 21 have been reported, of which n = 8 is found most frequently. This suggests that x = 8 and 7 are basic for the genus (Palomino & Vásquez, 1991). Although chromosomal data on African and Indian species of Crotalaria are abundant, they are not for the South American species. Cytotaxonomical studies © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 151, 271–277 271 Downloaded from https://academic.oup.com/botlinnean/article/151/2/271/2420359 by guest on 07 May 2023 ANDRÉIA SILVA FLORES, ANDRÉA M. CORRÊA, ELIANA R. FORNI-MARTINS and ANA M. G. AZEVEDO TOZZI* 272 A. S. FLORES ET AL. of Crotalaria native to Brazil. The cytotaxonomical evaluation of two sections represented by taxa native to Brazil was investigated. Here, the evolutionary aspects of chromosome numbers in relation to levels of ploidy are discussed. MATERIAL AND METHODS Seeds of 23 species and one subspecies were collected from wild populations in several regions of Brazil. The species’ names, with their sections and populations studied, are listed in Table 1. Vouchers are kept at the Table 1. Sections and vouchers of the Crotalaria taxa analysed in the present work Taxa Voucher Crotalaria section Calycinae Wight & Arn. C. balansae Micheli C. breviflora DC. C. flavicoma Benth. C. grandiflora Benth. C. hilariana Benth. C. martiana ssp. martiana Benth. C. martiana ssp. mohlenbrockii (D.R. Windler & S.G. Skinner) Planchuelo C. otoptera Benth. C. paulina Schrank C. pilosa Mill. C. stipularia Desv. C. subdecurrens Mart. ex Benth. C. tweediana Benth. C. vespertilio Benth. Crotalaria section Chrysocalycinae (Benth.) Baker f. C. clausseni Benth. C. harleyi D.R.Windler & S.G. Skinner C. holosericea Nees & Mart. C. incana L. C. laeta Mart. ex Benth. C. maypurensis Kunth C. micans Link C. miottoae A.S. Flores & A.M.G. Azevedo C. rufipila Benth. C. vitellina Ker-Gawler Rio Grande do Sul. Arroio do Sal. Flores 905 & Rodrigues São Paulo. Itararé. Flores 909 & Rodrigues Minas Gerais. Carrancas. Flores et al. 668 São Paulo. Atibaia. Flores 603 & Rodrigues Minas Gerais. Diamantina. Flores 1034 & Rodrigues Goiás. Catalão. Flores 970 & Rodrigues Distrito Federal. Brasília. Flores 976 & Rodrigues Rio Grande do Sul. São Francisco de Paula. Flores 448 & Rodrigues Santa Catarina. Lages. Flores 908 & Rodrigues Minas Gerais. Gouveia. Flores et al. 1069 Minas Gerais. Diamantina. Flores et al. 1064 Mato Grosso. Rondonópolis. Flores 541 & Rodrigues Goiás. Alto Paraíso. Flores 1007 & Rodrigues Goiás. Caldas Novas. Flores 1005 & Rodrigues São Paulo. Valinhos. Flores 601 & Rodrigues Minas Gerais. Botumirim. Flores et al. 798 São Paulo. Campinas. Flores 414 Goiás. Goiás. Flores 897 & Rodrigues Rio Grande do Sul. Canoas. Flores 907 & Rodrigues Santa Catarina. Criciúma. Flores 222 & Rodrigues (ICN) São Paulo. Cunha. Handro s.n. (UEC 105700) Bahia. Rio de Contas. Flores 1026 & Rodrigues Bahia. Lençóis. Flores 763 & Rodrigues Bahia. Palmeiras. Flores 759 & Rodrigues Minas Gerais. Porteirinha. Flores 1022 & Rodrigues Bahia. Rio de Contas. Flores 786 & Rodrigues Mato Grosso. Rondonópolis. Flores 540 & Rodrigues Goiás. Alto Paraíso de Goiás. Flores 993 & Rodrigues Goiás. Caldas Novas. Flores 1004 & Rodrigues Rio Grande do Sul. Osório. Flores 906 & Rodrigues Goiás. Jataí. Flores 558 & Rodrigues Minas Gerais. Camanducaia. Flores 1036 & Rodrigues Minas Gerais. Serra do Cipó. Flores et al. 586 Minas Gerais. Camanducaia. Flores 615 & Rodrigues Minas Gerais. Serra do Cipó. Flores 1009 & Rodrigues Ceará. Cumbuco. Taroda s.n. (UEC 139675) © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 151, 271–277 Downloaded from https://academic.oup.com/botlinnean/article/151/2/271/2420359 by guest on 07 May 2023 seeking to understand the relationships between the South American taxa are scarce, but notable are those of Windler (1974) who reported chromosome counts for nine taxa of the section Calycinae, Oliveira & AguiarPerecin (1999) who made a study of the comparison and morphometric analysis of the karyotypes of five species, and Mondin (2003) who presented a karyotypic study involving chromosomal banding procedures and fluorescent in situ hybridization. However, these papers included few Brazilian species. In order to provide additional chromosomal information, this paper reports chromosome counts of taxa CHROMOSOME NUMBERS IN BRAZILIAN CROTALARIA RESULTS AND DISCUSSION The somatic chromosome numbers (2n) are presented in Table 2. The chromosomal counts for 15 taxa of the Table 2. Chromosome numbers (2n) and levels of ploidy of the Crotalaria taxa studied Taxa 2n Ploidy level Crotalaria section Calycinae C. balansae* C. breviflora C. flavicoma* C. grandiflora* C. hilariana* C. martiana ssp. martiana* C. martiana ssp. mohlenbrockii* C. otoptera* C. paulina C. pilosa C. stipularia C. subdecurrens* C. tweediana* C. vespertilio* 32 32 32 32 32 32 32 32 32 32 32 32 54 32 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 4x 7x − 2/6x** 4x Crotalaria section Chrysocalycinae C. clausseni* C. harleyi* C. holosericea C. incana C. laeta C. maypurensis C. micans C. miottoae* C. rufipila* C. vitellina* 16 16 16 16 14 16 16 16 16 16 16 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x *New records; **see discussion in text. genus are new records. The most frequent chromosome number was 2n = 32, occurring in 12 species, followed by 2n = 16, found in nine species, C. incana had 2n = 14 chromosomes, whereas C. tweediana had 2n = 54. The polyploid species belong to the Calycinae (Figs 1–6) and species with 2n = 16 and 14 to the Chrysocalycinae (Figs 7–12). The number 2n = 32 confirms previous findings in C. pilosa, C. paulina and C. stipularia (Atchinson, 1950; Turner & Fearing, 1960; Verma, Kesavacharyulu & Raina, 1984; Oliveira & Aguiar-Perecin, 1999; Mondin, 2003). A polyploid 2n = 32 was observed for C. martiana ssp. mohlenbrockii. This is the first mitotic record for this species and agrees with the haploid report of n = 16 published under C. foliosa Benth. by Coleman & DeMenezes (1980), but renamed following examination of their voucher specimen. Our data for three accessions of C. breviflora, 2n = 32, do not agree with an earlier report of 2n = 16 by Verma et al. (1984). This species is native to South America, occurring mainly in Brazil, and is assigned to section Calycinae, which has shown only polyploid reports in this study. Voucher specimens were apparently not kept by Verma et al. (1984), and we could not determine whether their report corresponds to C. breviflora. The number 2n = 16 for C. micans, C. laeta and C. maypurensis confirms previous findings obtained from meiotic and mitotic studies (Raina & Verma, 1979; Coleman & DeMenezes, 1980). The observed chromosome number 2n = 16 for C. holosericea agrees with one previous report (Oliveira & Aguiar-Perecin, 1999), but not with another, that of 2n = 14 by Soares, Guerra & Gallindo (1988). Crotalaria incana was the only species with 2n = 14. Our record agrees with the many chromosome counts that have been published for this species, which is widespread in tropical and subtropical regions (Atchinson, 1950; Boulter et al., 1970; Raina & Verma, 1979; Oliveira & Aguiar-Perecin, 1999). Most tetraploidy occurs in Crotalaria section Calycinae, but it also occurs in section Hedriocarpae Wight & Arn., only in C. massaiensis Taub. (Boulter et al., 1970) and in some Neotropical species of section Crotalaria, which is considered more closely related to Calycinae. Species of sections Crotalaria and Hedriocarpae are not native in Brazil, although there are Asian and African taxa there. In this study, we found the first case of 2n = 54 in the genus, in C. tweediana, which is the highest chromosome number to date. Until now, the highest ploidy level was in the Asian C. ferruginea Graham, with two different counts, 2n = 42 (Bhaumik, 1975) and 2n = 48 (Mangotra & Koul, 1979). Despite the anomalous chromosome number reported for C. tweediana, there is no unusual morphological feature that could distin- © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 151, 271–277 Downloaded from https://academic.oup.com/botlinnean/article/151/2/271/2420359 by guest on 07 May 2023 herbarium of the Universidade Estadual de Campinas, São Paulo, Brazil (UEC). The seeds were scarified and germinated on wet filter paper in Petri dishes. Root tips of some species were pretreated with a saturated solution of paradichlorobenzene (PDB) at 10 °C for 6 h and others were pretreated with 0.002 M 8-hydroxyquinoline at 10 °C for 6 h. All tips were fixed in absolute ethanol and glacial acetic acid (3 : 1) for 12 h and stored in 70% ethanol at 4 °C. Root tips were stained using the HCl/Giemsa method (Guerra, 1983). Chromosome number determination was based on the observation of approximately ten cells for each species. An Olympus photomicroscope was used to photograph suitable chromosome spreads. Permanent slides are kept at the Biosystematic Laboratory of the Botany Department. 273 274 A. S. FLORES ET AL. 2 3 4 5 6 Figures 1–6. Mitotic chromosomes of Crotalaria, section Calycinae. Fig. 1. C. balansae (2n = 32). Fig. 2. C. breviflora (2n = 32). Fig. 3. C. grandiflora (2n = 32). Fig. 4. C. pilosa (2n = 32). Fig. 5. C. subdecurrens (2n = 32). Fig. 6. C. tweediana (2n = 54). Arrows indicate satellites. Scale bar = 10 µm. guish it from the other Calycinae species. This species has a restricted distribution in subtropical South America, occurring mostly in Rio Grande do Sul State (Brazil), Paraguay, Argentina and Uruguay (Flores & Miotto, 2001). It is difficult to establish the ploidy level in C. tweediana, but we can suggest at least two inter- pretations. First, we might consider this species as an aneuploid polyploid with 7x − 2. This interpretation is founded on the basic number x = 8 registered for all species of Calycinae. However, species with odd ploidy levels are sterile or they have problems in sexual reproduction (Singhal, Gill & Bir, 1985), reproducing vegetatively. We have no indications that C. tweediana © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 151, 271–277 Downloaded from https://academic.oup.com/botlinnean/article/151/2/271/2420359 by guest on 07 May 2023 1 275 CHROMOSOME NUMBERS IN BRAZILIAN CROTALARIA 8 9 10 11 12 Figures 7–12. Mitotic chromosomes of Crotalaria, section Chrysocalycinae. Fig. 7. C. clausseni (2n = 16). Fig. 8. C. holosericea (2n = 16). Fig. 9. C. vitellina (2n = 16). Fig. 10. C. micans (2n = 16). Fig. 11. C. miottoae (2n = 16). Fig. 12. C. incana (2n = 14). Arrows indicate satellites. Scale bar = 10 µm. shows problems in sexual reproduction, but the presence of a rhizome, which is unusual in Brazilian species of Crotalaria, can be associated with vegetative reproduction. In the second and more plausible interpretation, we could consider that this species is a hexaploid of x = 9 (2n = 6x = 54). In this case, this record would be a neobasic number for Crotalaria. It should be noted that x = 9 is considered a possible synapomorphy of the clade Genistoid, in which Crotalaria has been included (Pennington et al., 2001). Moreover, a prevalence of x = 9 is evident in Crotalarieae (Goldblatt, 1981). Further karyological approaches, especially including molecular procedures and breeding system studies, appear to be in order to reveal the © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 151, 271–277 Downloaded from https://academic.oup.com/botlinnean/article/151/2/271/2420359 by guest on 07 May 2023 7 276 A. S. FLORES ET AL. not contribute to the distinction between subsections Glaucae and Stipulosae, but they might be relevant to the characterization of subsection Incanae in Brazil. Cytological knowledge must be used in conjunction with other sources of data to achieve a better understanding of the phylogenetic relationship of Crotalaria species, and consequently to reveal their natural classification. ACKNOWLEDGEMENTS We are grateful to Yara R. Bressan for laboratory assistance, Rodrigo Schütz Rodrigues for help in collecting Crotalaria species and the Fundação de Amparo à Pesquisa do estado de São Paulo (FAPESP, proc. 00/11674–2) and CNPq (proc. 301892/82) for grants to support the research. We thank P. E. Brandham for valuable suggestions that improved our final version of this paper. REFERENCES Atchinson E. 1950. Studies in the Leguminosae V. Cytological observations on Crotalaria. Journal of the Elisha Mitchell Science Society 66: 7–75. Bandel G. 1974. Chromosome numbers and evolution in the Leguminosae. Caryologia 27: 17–32. Bhaumik GH. 1975. Chromosome studies of some Indian species of Crotalaria. Science and Culture 41: 521–523. Bisby FA, Polhill RM. 1973. The role of taximetrics in angiosperm taxonomy II. Parallel taximetric and orthodox studies in Crotalaria L. New Phytologist 72: 727–742. Boulter D, Derbyshire E, Frahm-Leliveld JA, Polhill RM. 1970. Observations on the cytology and seed proteins of various African species of Crotalaria L. (Leguminosae). New Phytologist 69: 117–131. Coleman JR, DeMenezes EM. 1980. Chromosome numbers in Leguminosae from the state of São Paulo, Brazil. Rhodora 82: 475–481. Flores AS. 2004. Taxonomia, números cromossômicos e química de espécies de Crotalaria L. (Leguminosae-Papilionoideae) no Brasil. DPhil Thesis, Universidade Estadual de Campinas. Flores AS, Miotto STS. 2001. O Gênero Crotalaria L. (Leguminosae-Faboideae) na Região Sul do Brasil. Iheringia, Série Botânica 55: 189–247. Goldblatt P. 1981. Cytology and phylogeny of Leguminosae. In: Polhill RM, Raven PH, eds. Advances in legume systematics, part 2. Kew: Royal Botanic Gardens, 427–463. Guerra MS. 1983. O uso do corante Giemsa na citogenética vegetal-comparação simples e o bandeamento. Ciência e Cultura 35: 190–193. Maciel HS, Schifino-Wittmann MT. 2002. First chromosome number determinations in south-eastern South American species of Lupinus L. (Leguminosae). Botanical Journal of the Linnean Society 139: 395–400. © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 151, 271–277 Downloaded from https://academic.oup.com/botlinnean/article/151/2/271/2420359 by guest on 07 May 2023 ploidy level and the mechanism of its origin in C. tweediana. Neotropical species of section Calycinae have some features, e.g. the presence of internodal wings, which are unusual in Asian species. Polyploidy is another feature of those New World species, in agreement with evidence of a polyploid origin for American Calycinae, as suggested by Polhill (1968), Windler (1974) and Oliveira & Aguiar-Perecin (1999). Otherwise, the Asian species of Calycinae are often diploid, except C. ferruginea, which is c. hexaploid. These data suggest the existence of two distinct groups in Calycinae, which can be established from morphological, chromosomal and geographical data. A phylogenetic analysis of Crotalaria, using molecular as well as nonmolecular data, would perhaps clarify whether the Neotropical elements of Calycinae might deserve sectional or subsectional status. The chromosome numbers 2n = 16 and 2n = 14 were found for Chrysocalycinae species. This section has been subdivided into five subsections, which present overlapping morphological characters. It makes it difficult to place the species in these subsections. In Brazil, C. incana is positioned in subsection Incanae (Benth.) F.A. Bisby & Polhill, but the other species of Chrysocalycinae form an intermediate group that is close to subsections Glaucae (Benth.) F.A. Bisby & Polhill and Stipulosae (Baker f.) F.A. Bisby & Polhill. The chromosome number 2n = 14 is unusual for Crotalaria, occurring in approximately 3% of the investigated species (Oliveira & Aguiar-Perecin, 1999). This number occurs in some species of subsection Incanae, suggesting a cytological characterization for this subsection. However, little cytological information is available for this subsection, and furthermore C. polysperma Kotschy was found to have both 2n = 14 and 16 (Boulter et al., 1970). There is no obvious value of chromosome number data in terms of distinguishing the other subgroups of Brazilian Chrysocalycinae species from each other, as all have 2n = 16. Polyploidy has played an important role in the evolution of Leguminosae (Bandel, 1974), but it is not considered to be the major evolutionary force (Mangotra & Koul, 1991). For Crotalaria species from the New World, cytological studies have indicated that polyploidy is at least one of the evolutionary strategies of this genus in this area. 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Polyploidy in genus Crotalaria. Cytologia 56: 293–296. Mendonça Filho CV, Forni-Martins ER, Tozzi AMGA. 2002. New chromosome counts in Neotropical Machaerium Pers. species (Fabaceae) and their taxonomic significance. Caryologia 55: 111–114. Mondin M. 2003. Estudo da evolução cariotípica do gênero Crotalaria L. (Leguminosae-Papilionoideae) com o emprego de técnicas de bandeamento cromossômico e hibridização in situ fluorescente (FISH). DPhil Thesis, ESALQ – Universidade de São Paulo. Oliveira ALPC, Aguiar-Perecin LR. 1999. Karyotype evolution in the genus Crotalaria (Leguminosae). Cytologia 64: 165–174. Palomino G, Vásquez R. 1991. Cytogenetic studies in Mexican populations of species of Crotalaria L. (LeguminosaePapilionoideae). Cytologia 56: 343–351. Pennington RT, Lavin M, Ireland H, Klitgaard B, Preston J, Hu J. 2001. Phylogenetic relationships of basal Papilionoid legumes based upon sequences of the chloroplast trnL intron. Systematic Botany 26: 537–556. Polhill RM. 1968. 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