Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 76(2): 51-57, 2023 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2300 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Fernando Tapia-Pastrana (2023). Cytogenetics of Diphysa americana (Mill.) M.Sousa (Leguminosae-Papil- ionoideae-dalbergioid clade), a rare species from the coast of Oaxaca, Mexico. Caryologia 76(2): 51-57. doi: 10.36253/caryologia-2300 Received: September 6, 2023 Accepted: October 27, 2023 Published: December 31, 2023 Copyright: © 2023 Fernando Tapia-Pas- trana. This is an open access, peer- reviewed article published by Firenze University Press (http://www.fupress. com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID FT-P: 0000-0003-0232-2110 Cytogenetics of Diphysa americana (Mill.) M.Sousa (Leguminosae-Papilionoideae- dalbergioid clade), a rare species from the coast of Oaxaca, Mexico Fernando Tapia-Pastrana Facultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma de México, Laboratorio de Genecología, Batalla 5 de mayo s/n esquina Fuerte de Loreto, Col. Ejército de Oriente, Iztapalapa, C.P. 09230, Ciudad de México, Mexico E-mail: pasfer@unam.mx Abstract. Diphysa Jacq. is an essentially Mexican and Central American genus that includes 21 species and only one cytogenetic report. In this work, a surface spread and air-drying method was used to obtain the karyotype of Diphysa americana (Mill) M.Sousa, a rare native tree that grows in a coastal town in the State of Oaxaca, Mexico. Metaphase cells showed a 2n = 20, consistent with the predominant diploid number in the dalbergioid clade. This number contrasts with a previously reported 2n = 16. The karyotypic formula 5m + 5sm, first proposed for a species of the genus, denotes a slightly asymmetric karyotype. The presence of secondary constrictions associated with satellites on the short arms of a pair of sm chromosomes and other cytogenetic parameters require studies in other species of the genus to verify their taxonomic util- ity. In addition, cells in prometaphase exhibited a circular fragment of unknown origin like that observed in a species of the genus Aeschynomene, also dalbergioid. This frag- ment could be related to extrachromosomal circular DNA (eccDNA) observed in oth- er plants. Diphysa is a small, cytogenetically favorable genus, and further studies will exhibit the karyotypic diversity that underlies its diversification. Keywords: basic number, dalbergioid clade, karyotype, SAT-chromosomes. INTRODUCTION Leguminosae is the third largest family within angiosperms and exhib- its enormous ecological, genomic, cytological, chemical, and morphologi- cal diversity (Doyle and Luckow 2003; Lewis et al. 2005). The Papilionoideae subfamily is the largest and most widespread of the three traditional Legu- minosae subfamilies, with an estimated 478 genera and 13,860 species (Lewis et al. 2005; Cardoso et al. 2013). Phylogenetic reconstructions point to it as a monophyletic group with highly specialized papilionate flowers that have a clearly distinctive standard petal, wings, and keel as well as partially fused stamens that wrap around the ovary, although there are unusual lineages with 52 Fernando Tapia-Pastrana marked radial floral symmetry (Pennington et al. 2001; Wojciechowski et al. 2004; Lavin et al. 2005; McMahon and Sanderson 2006; Cardoso et al. 2012, 2013; LPWG 2013). By using molecular data Lavin et al. (2001) detect- ed a pantropical monophyletic group of papilionate leg- umes called “dalbergioid” legumes, which is another example of a typical cryptic clade with an estimated age of 55.3 ± 0.5 million years (Lavin et al., 2005). All dalber- gioids belong to one of three well-supported subclades, the Adesmia, Dalbergia, and Pterocarpus clades. Circumscribed as up to now, the dalbergioids com- prise 54 genera and more than 1300 species of peren- nial and annual trees, shrubs, and herbs (Lavin et al. 2001; Wojciechowski et al. 2004; Cardoso et al. 2012; Moraes et al. 2020). Economically important species are included as hardwood species (Dalbergia spp. and Pterocarpus spp.), forage legumes (Stylosanthes spp. and Aeschynomene spp.), but also weeds of rice crops (Mar- tins et al. 2021), grown for consumption human (Arachis hypogaea, an allopolyploid) and some endangered taxa (Centrolobium paraense) and several Dalbergia species (Cervantes et al. 2019). Diphysa Jacq. (subclade Dalbergia) is an essentially Mexican and Central American genus with extensions to the southwestern United States and northern South America (Lavin et al. 2000; Lewis et al. 2012; Rzedows- ki et al. 2016) and includes 21 usually unarmed tree or shrub species (WFO 2023). Diphysa americana (Mill.) M.Sousa is a 4-15 m tall tree that grows mainly in the seasonally dry tropical biome (Sousa 1990; WFO 2023). It is characterized by a fissured bark and leaves 8-14 cm long, imparipinnate. Its leaflets (5-21) are alternate or sub-opposite, generally 1.5-3.5 cm long and 0.5-1 cm broad, oval, or obovate with entire margin, dark green above, paler below, and glabrous rachis. The inflores- cences with 6-7 yellow papilionate flowers (Fig. 1A), a standard with a macula with reddish edges (Fig. 1 B-C) and the turbinate calyx 6-9 mm (Fig. 1 D). Fruits 5.3-8.4 cm long and 1-1.8 cm wide, stipitate, reticulate, glabrous, veined margins and light brown seeds 6 x 3 mm (Fig. 1 E) (Martín et al. 2000; Rzedowski et al. 2016; Rojas-Rod- ríguez and Torres-Córdoba 2018). In Mexico, D. americana is an uncommon native species of tropical deciduous, seasonally dry, and ever- green forests. Its distribution encompasses both coast- lines as well as the center of the country at altitudes of 100 to 1000 m a.s.l. (Rzedowski et al. 2016; Villaseñor 2016). It is one of the first elements to colonize coastal dunes and is commonly found solitary in abandoned sites that were once used for agriculture (Acosta 1993; Ramírez-Pinero 2012). The trees have a high use value in agroforestry systems where the flowers are used as food cooked in salt or fried with egg (Manzanero-Medina et al. 2020), they can also be used as live fences to delimit cultivation areas and provide shade for coffee planta- tions. In addition, the stem is used as firewood or for the construction of houses (Pascual-Mendoza et al. 2020). It is also used in traditional medicine and is highly prized for the uses of its wood (Rzedowski et al. 2016; WFO 2023). Additionally, its influence on the structure and ecological dynamics of the dune vegetation describes it as a nucleating species (Ramírez-Pinero et al. 2018). It is known by the common names of Amarillo, Chilillo, Chipil, Chipilín, Flor de gallito, Cochipili, Cuachepil Guachipelín, Guachipilín, Macano, Palo amarillo, Que- bracho, Quiebracha (CONABIO 2023; Rzedowski et al. 2016). It is not considered a frequent plant, however, the IUCN (2021) places it in the category of least concern. The genus Diphysa records a 2n = 16 chromosome count for Diphysa robinioides Benth., a synonymy of D. americana (Atchison 1951; Sousa 1990; WFO 2023). However, this number is far from the basic number x = 10 and the diploid number 2n = 20 that predominate in genera belonging to the dalbergiod clade of Papilionoide- Figure 1. Morphological aspects in Diphysa americana. A. Inflores- cences. B-D, Three-quarter, frontal, and lateral views of individual flowers. E, Fruit, and seeds. Scale bars = 1 cm. 53Cytogenetics of Diphysa americana, a rare species from the coast of Oaxaca, Mexico ae (Goldblatt 1981; Lavin et al. 2001; Tapia-Pastrana et al. 2020) and therefore requires a cytogenetic reevaluation where, in addition to verifying its chromosome number, a detailed description of its karyotype is obtained. The objective of this work is to carry out a detailed analysis of the chromosomal characteristics of a Mexi- can population of D. americana, establish its karyotype and compare it with previous cytogenetic descriptions carried out in the genus and in other species belonging to the dalbergioid clade of the Papilionoideae subfamily. MATERIALS AND METHODS During June 2016, ripe fruits of Diphysa america- na were collected from two individuals separated by at least two kilometers in the vicinity of the municipality of Santiago Pinotepa Nacional in the coastal region of the state of Oaxaca, Mexico at 16°20'N, 98°03'W and 210 m a.s.l. The area is characterized by a warm sub-humid climate, with an average annual temperature of 26.2 ºC and a rainfall of 1,237.5 mm. The vouchers of the stud- ied specimens were deposited in the National Herbarium (MEXU) of the Instituto de Biología, UNAM. The mitotic cells were obtained from meristems of seeds germinated in Petri dishes lined with cotton moistened in distilled water. Chromosome preparations were made by surface spreading and air-drying (Tapia- Pastrana and Mercado-Ruaro 2001). All meristems were collected from 3-5 mm long roots pretreated with 2 mM 8-hydroxyquinolin for 5 h at room temperature and fixed in the fixative (ethanol: acetic acid=3:1). They were then treated with a mixture of 20% pectinase (Sigma) and 2% cellulase (Sigma) in 75 mM KCl for 60 min at 37 °C. After centrifugation at 1500 rpm for 10 min, the cell pellet was transferred to 75 mM KCl solution for 13 min at 37 °C. After two successive rinses with the KCl solution, they were again fixed in the fixative and subse- quently rinsed twice more. One or two drops of the sus- pension of pellet were placed on clean slides, air-dried, and stained in 10% Giemsa solution for 13 min for con- ventional karyotyping. At least ten metaphase and pro- metaphase plates with well-distributed chromosomes were photographed using a Carl Zeiss A1 axioscope. Five photographs of metaphases in which the chromosomes showed comparable degrees of contraction were used to determine: the diploid number (2n), the length differ- ence between the longest chromosome and the shortest chromosome (range), total haploid chromosome length (THC), average chromosome size (AC) and the longest chromosome/shortest chromosome ratio (Ratio, L/S). The asymmetry index (TF %) was obtained following Huziwara (1962). Chromosomes were classified as meta- centric (m), submetacentric (sm) according to their mor- phology and arm proportions (Levan et al. 1964). Chro- mosome size was estimated using a Mitutoyo Digimatic Caliper CD-G´́ BS digital caliper. Karyotypes were prepared from photomicrographs by cutting individual chromosomes, organizing them in descending order of length and matching according to their morphology. RESULTS A total of 184 cells in typical metaphase and well- distributed chromosomes were analyzed (Fig. 2 A-D), from which the Diphysa americana karyotype was pre- pared (Fig. 2 E). All of them exhibited a 2n = 20 and complements constituted by chromosomes m and sm, with a size that oscillated between 1.22 µm and 2.33 µm (Table 1). In some complements, the presence of one or two sm chromosomes carrying secondary constrictions and microsatellites on short arms was clearly visible (Fig. 2B-D). The observation of prometaphase cells revealed, on the one hand, the association between secondary constrictions and a single nucleolus (Fig. 3A) and, on Figure 2. Mitotic cells in metaphase 2n = 20 and karyotype of Diphysa americana. A-D, Metaphase chromosome plates with optimal distribution. Arrows point to secondary constrictions and satellites on short arms of submetacentric chromosomes. E, Karyo- type 5m + 5sm. Chromosomes are aligned by the centromere and arranged in decreasing order. Scale bars = 10 µm. 54 Fernando Tapia-Pastrana the other hand, the presence of a small circular-looking fragment that is not associated with the nucleolus or with any chromosome (Fig. 3B-G). This fragment can- not be confused with a secondary constriction or micro- satellite, and it stains with the same color and intensity as the rest of the chromosomes (Fig. 3F). The proposed karyotypic formula for D. americana, the position of the SAT chromosomes, and other characteristics of its kary- otype are summarized in Tables 1 and 2. DISCUSSION Diphysa americana is a species with a wide distribu- tion in the New World and the results of its cytogenetic analysis could be of importance in the understanding of chromosome evolution in the dalbergioid clade of Papil- ionoideae. It represents the only species of its genus so far studied karyologically. This is the first report of both diploid number and karyotype of Diphysa americana obtained in a Mexican population. The 2n = 20 con- firms on the one hand the predominance of x = 10 as the basic chromosome number of the dalbergioids and on the other hand Diphysa as a cytogenetically favora- ble taxon. The number of metaphases analyzed (184) and the absence of nuclei with a different set of chromo- somes allow us to state with certainty that the chromo- some number recorded here for D. americana is correct and consistent with what is expected for a genus includ- ed in the dalbergioid clade. A 2n = 20 (x= 10) recorded Figure 3. Prometaphase plates 2n = 20 of Diphysa americana. A. Arrows indicate satellites of NOR chromosomes associated with the nucleolus. B-G. Presence of circular fragments (arrows) of unknown origin not associated with the nucleolus or aligned with the chromosomes. In F is shown for comparison, one of these frag- ments and a secondary constriction indicated by an arrow. N = nucleolus. Scale bars = 10 µm. Table 1. Mean chromosome measures in D. americana. CP TCL (µm) LLA (µm) LSA (µm) r S 01 2.61±0.35 1.43±0.15 1.17±0.20 1.22 m 02 2.38±0.33 1.31±0.16 1.06±0.18 1.23 m 03 2.32±0.33 1.32±0.18 0.98±0.16 1.34 m 04 2.23±0.32 1.47±0.20 0.74±0.12 1.98 sm 05 2.19±0.32 1.23±0.17 0.95±0.15 1.29 m 06 2.15±0.36 1.48±0.29 0.66±0.07 2.24 sm* 07 2.08±0.29 1.17±0.18 0.90±0.11 1.30 m 08 2.05±0.30 1.44±0.21 0.60±0.09 2.40 sm 09 1.98±0.29 1.37±0.21 0.60±0.09 2.28 sm 10 1.82±0.23 1.26±0.17 0.54±0.06 2.33 sm CP=Chromosome pair; TCL=total chromosome length; LLA=length long arm; LSA=length short arm; ±=SD; r=arms ratio; S=shape after Levan et al. (1964). *Chromosomes with secondary constrictions on short arm. Table 2. Karyotype analysis of the taxon under study. Species NA 2n Karyotype formula Sat THC ± S.E (µm) AC ± S.E (µm) Range ± S.E (µm) L/S ± S.E TF% D. americana 184 20 5m + 5sm 2 21.81 ± 3.14 2.18 ± 0.21 0.79 ± 0.12 1.43 ± 0.02 37.59 NA = Nuclei analyzed; Sat = Number of satellites; THC = Total haploid chromosome length; AC = Average chromosome size; TF% = Asymmetry index. 55Cytogenetics of Diphysa americana, a rare species from the coast of Oaxaca, Mexico here differs from the 2n = 16 (x=8) shown by Atchison (1951) for a Central American population. In this regard, it is worth mentioning that Lewke Bandara et al. (2013) highlight the existence of variations in the number of chromosomes and the level of ploidy in some species of the genera Onobrychis, Hedysarum and Sulla (Hedys- areae: Papilioniodeae) and even information available in the IPCN database (tropicos.org, Missouri Botanical Garden) shows basic numbers far from x=10 in Acos- mium, Dalea and Ormocarpum (dalbergioid clade: Papil- ionoideae). Without overlooking the possible existence of cryptospecies with similar morphology and different chromosome numbers, the predominance of x = 10 rec- ognized in dalbergioids allows us to assume that num- bers as low as 2n = 16 in a diploid species can hardly be explained by decreasing aneuploidy. Clarifying this disa- greement requires broader population sampling. The karyotypic formula proposed here (5m + 5sm) shows that in the dalbergioid chromosome complements obtained so far, chromosomes with a medium (m) or slightly displaced (sm) centromere predominate (Table 1), and therefore they are symmetric or slightly asymmetric karyotypes (Tapia-Pastrana et al. 2020). Likewise, the pres- ence of secondary constrictions associated with microsat- ellites in short arms of sm chromosomes (SAT chromo- somes) corroborates a trend in plant species where second- ary constrictions are located preferentially in short arms (Lima de Faria 1976; Lim et al. 2001), particularly in leg- umes (Biondo et al. 2006; Tapia-Pastrana 2012; Tapia-Pas- trana and Tapia-Aguirre 2018; Tapia-Pastrana et al. 2020). Likewise, their role in the organization of the nucleolus is evident since they were observed associated with it in pro- metaphase cells (Fig. 3A) and considered nucleolar organ- izing regions (NOR) or at least part of them. In addition, its shape and behavior resemble those exhibited by species belonging to the genus Aeschynomene, Serie Americanae (Tapia-Pastrana et al. 2020). On the other hand, the values of THC, AC, Range, L/S and TF% (Table 2) are parameters obtained for the first time in the genus Diphysa and to demonstrate their taxonomic value, a greater number of species must be studied. However, with respect to THC (21.81 ± 3.14 µm) and AC (2.18 ± 0.21 µm), it can be stated that they differ little from the values obtained for species of the recent- ly reestablished genus Ctenodon (Tapia-Pastrana et al. 2020; Cardoso et al. 2020) and Dalbergia spinosa (Jena et al. 2004) also in the dalbergioid clade. Regarding the presence of small circular fragments, for the moment there is no information on their origin and function. It is noteworthy that similar structures in shape and size were also recorded in Aeschynomene americana var. glandulosa, another dalbergioid (Tapia-Pastrana et al. 2020). It remains to mention that this material is like extrachromosomal circular DNA (eccDNA) detected by electron microscopy in plants, which mainly con- tains repeated sequences derived from chromosomal DNA involved in the evolution of B chromosomes and rDNA mobility (Cohen et al. 2008). They also resemble the satellite-like structures recorded in the chromosomes of Giemsa-stained prometaphase cells of Nicotiana kawakamii Y. Ohashi (Nakamura et al. 2001). Decipher- ing this enigma will require molecular cytogenetic tech- niques and the analysis of a greater number of species. Circumscribed as so far, the dalbergioid clade is composed of more than 1300 species (Lavin et al. 2001; Wojciechowski et al. 2004; Cardoso et al. 2012; Moraes et al. 2020) and only about 300 (≈ 28%) are known cytogenetically. As shown here, the genus Diphysa, which includes few species, is cytogenetically favorable and represents an opportunity to corroborate not only the constancy of the basic chromosome number (x = 10) exhibited in dalbergioid taxa, but also to verify the kar- yotypic diversity that surely underlies their evolutionary and speciation processes. Characterizing the genome architecture of higher plants is an important scientific task. Its first approach is to visualize chromosomal domains by obtaining detailed karyotypes that reveal the physical organization of DNA in chromosomes. Comparative cytogenetic studies can be taxonomically relevant and complement phylogenies based on molecular data (Tapia-Pastrana et al. 2020; Cordeiro et al. 2020), so this task should continue. ACKNOWLEDGEMENTS The author is grateful for the support of the Division of Postgraduate Studies and Research, Faculty of Higher Studies Zaragoza. REFERENCES Acosta I. 1993. Lluvia de semillas en matorrales de dunas costeras en el Morro de La Mancha Veracruz. Bach- elor of Science Thesis. Facultad de Ciencias. Univer- sidad Nacional Autónoma de México, México, DF. Atchison E. 1951. Studies in the Leguminosae. VI. Chromosome numbers among tropical woody species. Am J Bot. 38:538-546. https://doi. org/10.1002/j.1537-2197.1951.tb14855.x Biondo E, Miotto STS, Schifino-Wittmann MT. 2006. Cytogenetics of species of Chamaecrista (Leguminos- ae-Caesalpinioideae) native to southern Brazil. Bot 56 Fernando Tapia-Pastrana J Linn Soc. 150(4):429-439. https://doi.org/10.1111/ j.1095-8339.2006.00480.x Cardoso D, de Queiroz LP, Pennington RT, de Lima HC, Fonty E, Wojciechowski MF, Lavin M. 2012. Revis- iting the phylogeny of papilionoid legumes: new insights from comprehensively sampled early-branch- ing lineages. Am J Bot. 99:1991-2013. http://www. jstor.org/stable/23321299. Cardoso D, Pennington RT, de Queiroz LP, Boatwright JS, Van Wyk B-E, Wojciechowski MF, Lavin M. 2013. Reconstructing the deep-branching relationships of the papilionoid legumes. S Afr J Bot. 89:58-75. htt- ps://doi.org/10.1016/j.sajb.2013.05.001 Cardoso D, Mattos CMJ, Filardi F, Delgado-Salinas A, Lavin M, de Moraes PLR, Tapia-Pastrana F, de Lima HC. 2020. A molecular phylogeny of the pantropi- cal papilionoid legume Aeschynomene supports rein- stating the ecologically and morphologically coher- ent genus Ctenodon. Neodiversity, 13:1-38. https://doi. org/10.13102/neod/131.1 Cervantes A, Linares J, Quintero E. 2019. An updated checklist of the Mexican species of Dalbergia (Legu- minosae) to aid in its conservation efforts. Rev Mex Biodivers. 90: e902528. https://doi.org/10.22201/ ib.20078706e.2019.90.2528 CONABIO (Comisión Nacional para el Conocimiento y uso de la Biodiversidad). [accessed 2023 March 23]. https://enciclovida.mx/especies/185459 Cohen S, Houben A, Segal D. 2008. Extrachromosom- al circular DNA derived from tandemly repeated genomic sequences in plants. Plant J. 53:1027-1034. https://doi.org/10.1111/j.1365-313X.2007.03394.x Cordeiro JMP, Kaehler M, Souza LG, Felix LP. 2020. Het- erochromatin and numeric chromosome evolution in Bignoniaceae, with emphasis on the Neotropical clade Tabebuia alliance. Genet Mol Biol. 43: e20180171. htt- ps://doi.org/10.1590%2F1678-4685-GMB-2018-0171 Doyle JJ, Luckow MA. 2003. The rest of the iceberg. Legume diversity and evolution in a phylogenet- ic context. Plant Physiol. 131:900-911. https://doi. org/10.1104%2Fpp.102.018150 Goldblatt P. 1981. Cytology and the phylogeny of Legu- minosae. In: Polhill RM, Raven PH, editors. Advanc- es in legume systematics. part 2. Royal Botanic Gar- den, London: Kew Publishing; p. 427-463. Huziwara Y. 1962. Karyotype analysis in some genera of Compositae. VIII. Further studies on the chromo- somes of Aster. Am J Bot. 49:116-119. https://doi. org/10.2307/2439026 IUCN (Red List of Threatened Species). Version 3.1. [accessed 2023 March 27]. https://www.iucnredlist. org/species/144264286/149030506 Jena S, Sahoo P, Mohanty S, Das AB. 2004. Identification of RAPD markers, in situ DNA content and struc- tural chromosomal diversity in some legumes of the mangrove flora of Orissa. Genetica. 122:217-226. http://dx.doi.org/10.1007/s10709-004-2040-5 Lavin M, Pennington RT, Klitgaard BB, Sprent JI, de Lima HC, Gasson PE. 2001. The Dalbergioid legumes (Fabaceae): Delimitation of a pantropical mono- phyletic clade. Am J Bot. 88:503-533. https://doi. org/10.2307/2657116 Lavin M, Herendeen PS, Wojciechowski MF. 2005. Evo- lutionary rates analysis of Leguminosae impli- cates a rapid diversification of lineages dur- ing the Tertiary. Syst Biol. 54:575-594. https://doi. org/10.1080/10635150590947131 Lavin M, Thulin M, Labat J-N, Pennington RT. 2000. Africa, the odd man out: molecular biogeogra- phy of dalbergioid legumes (Fabaceae) suggests otherwise. Syst Bot. 25(3):449-467. https://doi. org/10.2307/2666689 Levan A, Fredga K, Sandberg AA. 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52:201-219. https://doi.org/10.1111/j.1601-5223.1964. tb01953.x Lewis G, Schrire B, Mackinder B, Lock M, editors. 2005. Legumes of the world. Richmond, U. K.: Royal Botanic Gardens, Kew. Lewis GP, Wood JRI, Lavin M. 2012. Steinbachiella (Legu- minosae: Papilionoideae: Dalbergieae), endemic to Bolivia, is reinstated as an accepted genus. Kew Bull. 67:789-796. https://doi.org/10.1007/s12225-012-9415-z Lewke Bandara N, Papini A, Mosti S, Brown T, Smith LMJ. 2013. A phylogenetic analysis of genus Ono- brychis and its relationships within the tribe Hedys- areae (Fabaceae). Turk J Bot. 37:981-992. https://doi. org/10.3906/bot-1210-32 Lim KB, Wennekes J, de Jong JH, Jacobsen E, van Tuyl JM. 2001. Karyotype analysis of Lilium longiflo- rum and Lilium rubellum by chromosome band- ing and fluorescence in situ hybridisation. Genome, 44(5):911-918. https://doi.org/10.1139/g01-066 Lima de Faria A. 1976. The chromosome field. I. Predic- tion of the location of ribosomal cistrons. Hereditas, 83(1):1-22. https://doi.org/10.1111/j.1601-5223.1976. tb01565 . LPWG (Legume PhylogenyWorking Group 2013). Leg- ume phylogeny and classification in the 21st cen- tury: progress, prospects, and lessons for other spe- cies-rich clades. Taxon, 62:217-248. http://dx.doi. org/10.12705/622.8 Manzanero-Medina GI, Vásquez-Dávila MA, Lustre- Sánchez H, Pérez-Herrera A. 2020. Ethnobotany of 57Cytogenetics of Diphysa americana, a rare species from the coast of Oaxaca, Mexico food plants (quelites) sold in two traditional markets of Oaxaca, Mexico. S Afr J Bot. 130:215-233. https:// doi.org/10.1016/j.sajb.2020.01.002 Martín G, Milera M, Iglesias J, Simón L, Hernández H. 2000. Sistemas silvopastoriles para la producción ganadera en Cuba. Intensificación de la ganadería en Centroamérica, beneficios económicos y ambientales. CATIE-FAO. Costa Rica 247 p. Martins MB, Agostinetto D, Fogliatto S, Vidotto F, Andres A. 2021. Aeschynomene spp. Identification and weed management in rice fields in Southern Brazil. Agrono- my, 11:453. https://doi.org/10.3390/agronomy11030453 McMahon MM, Sanderson MJ. 2006. Phylogenetic super- matrix analysis of GenBank sequences from 2228 papilionoid legumes. Syst. Biol. 55:818-836. https:// doi.org/10.1080/10635150600999150 Moraes AP, Vatanparast M, Polido C, Marques A, Souza G, Fortuna-Perez AP, Forni-Martins ER. 2020. Chro- mosome number evolution in dalbergioid legumes (Papilionoideae, Leguminosae). Braz J Bot. 43:575- 587. https://doi.org/10.1007/s40415-020-00631-6 Nakamura R, Kitamura S, Inoue M, Ohmido N, Fukui K. 2001. Karyotype analysis of Nicotiana kawakamii Y. Ohashi using DAPI banding and rDNA FISH. Theor Appl Genet. 102(6-7):810-814. https://doi. org/10.1007/s001220100577 Pascual-Mendoza S, Manzanero-Medina GI, Saynes- Vásquez A, Vásquez-Dávila MA. 2020. Agroforestry systems of a Zapotec community in the Northern Sierra of Oaxaca, Mexico. Bot Sci. 98(1):128-144. htt- ps://doi.org/10.17129/botsci.2423 Pennington RT, Lavin M, Ireland H, Klitgaard B, Preston J, Hu JM. 2001. Phylogenetic relationships of basal papilionoid legumes based upon sequences of the chloroplast trnL intron. Syst. Bot. 26:537-556. http:// dx.doi.org/10.1043/0363-6445-26.3.537 Ramírez-Pinero M. 2012. Técnicas para la restauración de la selva baja caducifolia en el centro de Verac- ruz [master’s thesis]. Xalapa, Veracruz: Instituto de Ecología, A.C. México. Ramírez-Pinero M, Lira-Noriega A, Guevara S. 2018. Canopy asymmetry in solitary Diphysa americana trees: wind and landscape on the Mexican coast. J Coast Conserv. 23:163-172. https://doi.org/10.1007/ s11852-018-0648-3 Rojas-Rodríguez F, Torres-Córdoba G. 2018. Árbo- les del Valle Central de Costa Rica: reproducción guachipelín (Diphysa americana (Mill.) M. Sousa). RFMK. 16(38): 69-71. https://doi.org/10.18845/rfmk. v16i38.3998 Rzedowski J, Calderón de Rzedowski G, Torres Colín L, Grether R. 2016. Familia Leguminosae. Subfamilia Papilionoideae (Aeschynomene - Diphysa). In: Rze- dowski J, Calderón de Rzedowski G, editors. Flora del Bajío y regiones adyacentes. Fascículo 192. Insti- tuto de Ecología A.C., Michoacán, México; p.1-326. Sousa MS. 1990. Adiciones a las papilionadas de la Flora de Nicaragua y una nueva combinación para Oaxaca, México. Ann Mo Bot Gard. 77: 573-577. https://doi. org/10.2307/2399521 Tapia-Pastrana F. 2012. Karyological characterisation of four American species of Crotalaria (Legumi- nosae: Papilionoideae) by splash method. Kew Bull. 67(3):427-433. https://doi.org/10.1007/s12225-012- 9385-1 Tapia-Pastrana F, Delgado-Salinas A, Caballero J. 2020. Patterns of chromosomal variation in Mexican spe- cies of Aeschynomene (Fabaceae, Papilionoideae) and their evolutionary and taxonomic implica- tions. Comp Cytogenet. 14(1):157-182. https://doi. org/10.3897%2FCompCytogen.v14i1.47264 Tapia-Pastrana F, Mercado-Ruaro P. 2001. A combina- tion of the “squash” and “splash” techniques to obtain the karyotype and asses meiotic behavior of Prosopis laevigata L. (Fabaceae: Mimosoideae). Cytologia, 66:11-17. http://dx.doi.org/10.1508/cytologia.66.11 Tapia-Pastrana F, Tapia-Aguirre F. 2018. Localización de satélites y cromosomas NOR para la interpretación del cariotipo de Sesbania virgata (Papilionoideae, Sesbanieae) de dos poblaciones americanas. Bot Sci. 96(4):619-627. https://doi.org/10.17129/botsci.1972 Villaseñor JL. 2016. Checklist of the native vascular plants of Mexico. Rev Mex Biodivers. 87(3):559-902. https://doi.org/10.1016/j.rmb.2016.06.017 WFO (The World Flora Online). [accessed 2023 March 23]. http://www.worldfloraonline.org/taxon/wfo- 0000210419 Wojciechowski MF, Lavin M, Sanderson MJ. 2004. A phy- logeny of legumes (Leguminosae) based on analysis of the plastid MatK gene resolves many well-support- ed subclades within the family. Am J Bot. 91:1846- 1862. https://doi.org/10.3732/ajb.91.11.1846 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 76, Issue 2 - 2023 Firenze University Press Molecular classification of Barbeyaceae (Barbeya oleoides Schweinf.) using four different DNA barcodes Fatima Omari Alzahrani, Sami Asir Al-Robai Mitotic metaphase karyotype of the mosquito Anopheles arabiensis Patton (Diptera: Culicidae) from Kassala State, eastern Sudan Asma Mahmoud Hamza1,*, Sumaya Hussein Elboshra2 Karyotypic analysis of Crucian carp, Carassius carassius (Linnaeus, 1758) from cold waters of Kashmir Himalayas Gousia Jan1, Asim Iqbal Bazaz2, Azra Shah1, Saima Andleeb1, Irfan Ahmad1,*, Durdana Qazi1, Oyas Asimi3, Bilal A. Bhat5, Anayitullah Chesti4 Unfolding chromosomal uniqueness of the Scilloid ornamental Albuca virens by application of EMA based Giemsa- DAPI staining Biplab Kumar Bhowmick*, Sayani Nag Cytogenetic analysis of sympatric Trachelyopterus Valenciennes 1840 (Siluriformes, Auchenipteridae) species reveals highly conserved karyotypes despite the geographic distance Denise Felicetti1, Chrystian Aparecido Grillo Haerter2, Lucas Baumgärtner1, Leonardo Marcel Paiz1, Daniel Rodrigues Blanco3, Eliana Feldberg2, Vladimir Pavan Margarido1, Maelin da Silva4, Roberto Laridondo Lui1,* Cytogenetics of Diphysa americana (Mill.) M.Sousa (Leguminosae-Papilionoideae-dalbergioid clade), a rare species from the coast of Oaxaca, Mexico Fernando Tapia-Pastrana Cytogenetical studies of some Convolvulaceae members from the Western Ghats, India reveal uniformity in karyotypes R.N. Chougule, P.V. Deshmukh, P.E. Shelke, V.J. Patil, M.M. Lekhak* In vitro cytotoxic activity of phytosynthesized silver nanoparticles using Clematis vitalba L. (Ranunculaceae) aqueous decoction Nicoleta Anca Şuţan1, Diana Ionela Popescu (Stegarus)2, Oana Alexandra Drăghiceanu1, Carmen Topală3, Claudiu Şuţan3,*, Aurelian Denis Negrea4, Denisa Ştefania Vîlcoci4, Georgiana Cîrstea4, Sorin Georgian Moga4, Liliana Cristina Soare1