Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(4): 17-26, 2020 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-949 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: F. Tapia-Pastrana, A. Delga- do-Salinas (2020) First cytogenetic register of an allopolyploid lineage of the genus Aeschynomene (Leguminosae, Papil- ionoideae) native to Mexico. Caryolo- gia 73(4): 17-26. doi: 10.13128/caryolo- gia-949 Received: May 24, 2020 Accepted: November 10, 2020 Published: May 19, 2021 Copyright: © 2020 F. Tapia-Pastrana, A. Delgado-Salinas. This is an open access, peer-reviewed article pub- lished by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, 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 FTP: 0000-0003-0232-2110 First cytogenetic register of an allopolyploid lineage of the genus Aeschynomene (Leguminosae, Papilionoideae) native to Mexico Fernando Tapia-Pastrana1,*, Alfonso Delgado-Salinas2 1 Facultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma de México, Laboratorio de Genecología, Batalla 5 de Mayo s/n esquina Fuerte de Loreto, Col. Ejér- cito de Oriente, Iztapalapa, C.P. 09230, Ciudad de México, Mexico 2 Instituto de Biología, Departamento de Botánica, Universidad Nacional Autónoma de México, Apartado Postal 70-233, 04510, Cd. de México, Mexico *Corresponding author. E-mail: pasfer@unam.mx Abstract. A conventional cytogenetics analysis revealed for first time an allopolyploid lineage of the genus Aeschynomene in Mexico. The hybrid condition is confirmed after all the prometaphase and metaphase nuclei of the hybrids exhibited only one pair of SAT-chromosomes, confirming the existence of nucleolar dominance and amphiplasty. The karyotype formula for this lineage was 2n = 4x = 40 = 34 m + 6 sm with a total diploid chromosome length (TDCL) = 28µm and an average chromosome size (AC) = 1.40 µm. Comparison of the karyotype and other chromosomal parameters with recent cytogenetics records for other species of the subgenus Aeschynomene included in the Nod-independent clade allows propose to Aeschynomene evenia and A. scabra as possi- ble progenitors. Furthermore, other comparison of seedlings focused at the number of leaflets of the first four eophylls of the proposed parents and of the hybrid individuals allowed to observe coincidences that support the proposal made from the cytogenetic analysis. Evidence of “gigas” effects on flowers and fruits of hybrids is also shown. Keywords: cryptic taxa, cytotype, karyotype, nucleolar dominance, SAT-chromo- somes, secondary constrictions, seedlings. I. INTRODUCTION Aeschynomene Linnaeus (Leguminosae, Tribe Dalbergieae s. l.) is a diverse genus of subfamily Papilionoideae (Papilionoid legumes) distributed in the tropics and subtropics of the world (Lavin et al. 2001, Klitgaard and Lavin 2005). It comprises herbaceous and woody species, annual, repetitive and perennial with different ecological requirements. Several species con- tribute to supplement nitrogen to the soil through the production of nodular roots and stems in symbiosis with nitrogen fixing bacteria, so they are eco- nomically important as green manure (Alazar and Becker 1987; Fernandes 1996; Souza et al. 2012) and recently, Aeschynomene evenia C. Wright has been proposed as a model species in genetics to develop new agronomic 18 Fernando Tapia-Pastrana, Alfonso Delgado-Salinas strategies in the engineering of nitrogen fixing nodules that enhance rice production (Arrighi et al. 2012, 2013). This taxon belongs to the group of 11 semi-aquatic spe- cies of Aeschynomene that have the property of being nodulated by photosynthetic Bradyrhizobium that lack the nodABC genes necessary for the synthesis of Nod factors and are grouped into the so-called Nod-inde- pendent clade (Chaintreuil et al. 2013; Brottier et al. 2018) and that correspond to the morphological series Indicae and Sensitivae (Rudd 1955). The genus Aeschynomene traditionally included in the Aeschynomeneae tribe (Polhill et al.1981) and cur- rently circumscribed in the Dalbergioid clade (Lavin et al. 2001; Wojciechowski et al. 2004) has evolved in dif- ferent ecological niches and includes herbaceous forms, annual and perennial shrubs and trees up to 8 meters, with compound pinnate leaves and papilionoid flowers that are generally self-pollinated, although there is cross- pollination by bees (Rudd 1955; Fernandes 1996; Arrighi et al. 2014, Carleial et al. 2015). Other studies indicate that the genus Aeschynomene is not monophyletic and taxa with basifixed stipules and a campanulate calyx (subgenus Ochopodium Vogel) are more related to the genera Machaerium Persoon and Dalbergia Linnaeus f. than to taxa with medfixed stipules and a bilabiate calyx (subgenus Aeschynomene Léonard) (Ribeiro et al. 2007; Cardoso et al. 2012). Currently Aeschynomene genus contains 170 (http:// w w w.theplantlist.org) to 180 species (Klitgaard and Lavin 2005) 231taxa and cytotypes at four ploidy levels: diploid (2x), tetraploid (4x), hexaploid (6x) and octoploid (8x) (Index to Plant Chromosome Numbers; Kawakami 1930; Bielig 1997; Arrighi et al. 2012, 2014; Chaintreuil et al. 2016, 2018; Brottier et al. 2018). America, where most of the taxa are 2n = 20 diploids, has been proposed as the center of origin of the genus, with a secondary distribution in Africa and Asia where polyploid species and some cases of aneuploidy predominate (Chaintreuil et al. 2018; Tapia-Pastrana et al. 2020). Although it is clear that in the Dalbergioid clade, diploid 2n = 20 genera predominate, with some poly- ploid and aneuploid species, in Aeschynomene there is currently a renewed interest in knowing to what extent polyploidy has contributed to the diversification and radiation of the group. In this respect Arrighi et al. (2014) revealed multiple hybridization/polyploidiza- tion events, highlighting the prominent role of allopoly- ploidy in the diversification of Nod-independent clade. In addition Chaintreuil et al. (2016) studied African Aeschynomene species and their data support the idea that the whole African group is fundamentally tetraploid and revealed the allopolyploid origin of A. afraspera J. Léonard (2n = 8x = 76) and A. schimperi Hochst. ex A.Rich. (2n = 8x = 56), where variations in the number of chromosomes also indicated possible dysploidy/ane- uploidy events. In Mexico, Aeschynomene is represented by 31 species and infraspecific taxa including several endemisms. An investigation about the patterns of chro- mosomal evolution in Mexican species, including six taxa of the Nod-independent clade, showed the predomi- nant of a basic 2n = 20 diploid structure and evolution- ary patterns related to the corresponding morphological series (Tapia-Pastrana et al. 2020). In the present research, a conventional cytogenetic study was carried out to obtain the karyotype and ana- lyze the level of ploidy in a Mexican population initially described as Aeschynomene scabra G. Don, where the size of the flowers, fruits and seeds generated suspicions about a possible hybrid origin. In addition as the sam- pled individuals exhibited floral morphotypes similar to those of A. evenia C. Wright and A. scabra, whose col- lection records in Mexico would support their partici- pation in the hybridization process, the growth pattern of the first four eophylls was also compared in putative hybrids and their parental assumptions. 2. MATERIAL AND METHODS 2.1 Collection sites Seeds of the putative hybrids were collected in the Municipio de la Huerta, Estado de Jalisco, Mexico, 19°29´N; 105°01́ W (Carleial s/n, MEXU). The climate is semi-dry and warm. Mean temperature in the area is 25.2 °C, and there is a well-defined rainy season (average annual precipitation: 1107  mm) occurring from June to October (García-Oliva et al. 2002). The seeds of Aeschynomene evenia and A. scabra were collected in the municipalities of Coyuca de Cat- alán (18° 19´ N; 100° 42´ W, JC Soto 15333 (MEXU)) and Arcelia (18°18´54´́ N; 100°17´02´́ W, JC Soto 15393 (MEXU)) respectively, in the State of Guerrero, Mexi- co. Both municipalities are part of the Tierra Caliente region. The predominant climate is warm subhumid with rains from June to September (average annual precipitation: 1100 to 1200 mm). The studied taxa are assigned to the infrageneric classification of Neotropi- cal Aeschynomene sensu Rudd (1955) series Indicae of subgenus Aeschynomene and are part of the Nod-inde- pendent monophyletic clade (Chaintreuil et al. 2013), whose taxa are nodulated on roots and stems by photo- synthetic Bradyrhizobium strains lacking the nod ABC genes necessary for the synthesis of Nod factors (Giraud et al. 2007). 19First cytogenetic register of an allopolyploid lineage of the genus Aeschynomene native to Mexico 2.2 Chromosome and karyotype procedures in putative hybrids Seeds were collected in summer 2014 and from at least six plants. Batches of 40 seeds from each plant were used. The seeds were scarified and germinated in Petri dishes lined with a moist filter paper at room tempera- ture and under natural light. Chromosomes at metaphase and prophase were obtained following the splash method (Tapia-Pastrana and Mercado-Ruaro 2001). All meris- tems were collected from 2-4 mm long roots pretreated with 2 mM 8-hydroxyquinolin for 5 h at room tempera- ture 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 solu- tion for 13 min at 37 °C. After two successive rinses with the KCl solution, they were again fixed in the fixative and subsequently rinsed twice more. One or two drops of the suspension of pellet were placed on clean slides, air-dried and stained in 10% Giemsa for 13 min. Preparations were made permanent using a synthetic resin. At least ten metaphase plates of intact cells with well-spread chromosomes, no chromosome overlap- ping, and same contraction and ten prophase plates were photographed from each collection, using a microscope (Axioscope, Carl Zeiss) and analyzed for chromosome number determinations. Five photographs of meta- phases with chromosomes having similar comparable degrees of contraction and centromeres clearly located were utilized to obtain the Total diploid chromosome length (TDCL), Total chromosome length (TCL), Aver- age chromosome length (AC), the difference in length between the longest chromosome and the shortest chro- mosome (Range) and the longest/shortest chromosome ratio (L/S). The shapes of chromosomes were classified according to Levan et al. (1964) and the TF was obtained following Huziwara (1962). Furthermore, prometaphase cells were analyzed to verify both the number of nucleo- li, and the behavior of the SAT chromosomes. The infor- mation thus obtained was compared with that recently recorded for Aeschynomene evenia and A. scabra in another cytogenetic study where the same method was used for karyotype analysis in Aeschynomene species and varieties (Tapia-Pastrana et al. 2020). 2.3 Seedlings and Eophylls In order to compare seedling morphology in indi- viduals of the supposedly hybrid population with those of Aeschynomene evenia and A. scabra, the development of 20 individuals grown in pots under greenhouse con- ditions was evaluated. Interest was particularly focused on the number of leaflets and the presence of hairs on their edges until the complete development of the fourth leaf. Eophylls at the first, second, third and fourth eophyllar nodes were referred to as E1, E2, E3 and E4, respectively following Schütz et al. (2019). Photographs of seedlings were taken with a Canon SX700 HS camera. 3. RESULTS 3.1 Karyotype analysis A total of 410 cells were analyzed in metaphase and 16 in prometaphase and all exhibited a 2n = 4x = 40 (Fig. 1 A-C). TDCL was 28 µm and AC 1.40 µm. The chromosomal range was 0.56 µm, the ratio 1.48 and a TF = 42.46. The karyotype formula was 2n = 4x = 34m + 6sm (Table 1). Consistently, in all prometaphase and metaphase nuclei, only one pair of submetacentric chromosomes was observed having lax secondary con- strictions and macrosatellites in short arms (SAT-chro- mosomes) (Fig. 1 A-C). The karyotype exhibits small chromosomes (1.72-1.16 µm) clearly discernible, with predominance of metacentric chromosomes (m) and lacking subtelocentric chromosomes (st). This arrange- ment is consistent with a TF that describes a slightly asymmetric karyotype (Fig. 1D and Table 1). Occasion- ally the SAT-chromosomes were observed immersed in a single nucleolus. 3.2 Seedlings and Eophylls The seedlings of the three taxa are illustrated in Fig. 2 A-C. Eophylls are stipulated, alternate, petiolate, pin- nate, with alternate leaflets, have elliptic to oblong leaf- lets, a rounded apex, an entire margins, and one cen- tral primary vein in the three taxa under study. The leaf lets did not present trichomes; both adaxial and abaxial surfaces are glabrous. The number of leaflets in the first four eophylls in seedlings of individuals of Aeschynomene evenia, A. scabra and putative hybrids are shown in Tables 2-4 respectively. 4. DISCUSSION It is clear t hat t he entire Da lbergioid clade (Adesmia, Dalbergia and Pterocarpus subclades) is dom- inated by 2n = 2x = 20 species, with scattered polyploids and aneuploids (Lavin et al. 2001). In addition an ances- 20 Fernando Tapia-Pastrana, Alfonso Delgado-Salinas tral state reconstruction performed in a phylogeny based on ITS + matK of the Aeschynomene genus and related genera indicated that diploidy is the ancestral condi- tion in the entire group reviewed (Brottier et al. 2018). However, the role of allopolyploid speciation events in the origin of new taxa is now recognized (Arrighi et al. 2014). As far as we know, the first assumption about of hybridization in Aeschynomene is attributed to Rudd (1955) who pointed out that the species with the widest distribution within the Indicae series (Nod-independent clade) tend to be more variable and intergrade with their neighbors. Later, Verdcourt (1971) suggested that speci- mens of Aeschynomene rudis Bentham (also into Nod- independent clade) with large flowers could be of poly- ploid origin, without pointing out the possible duplica- tion mechanism involved, auto or allopolyploidy. To date, several studies have shown that the clade of A. eve- nia is mainly diploid (2n = 2x = 20), however some spe- cies such as A. indica Linnaeus (2n = 4x = 40, 2n = 6x = 60) seem to be of recent allopolyploid origin (Arrighi et al. 2014; Chaintreuil et al. 2018; Tapia-Pastrana et al. 2020). Furthermore, it has been found that all species of the group A. afraspera are polyploid (2n = 4x = 28, 38, 40; 2n = 8x = 56, 76) and have a common AB genomic structure (Chaintreuil et al. 2016). In facts phylogenetic relationships between diploids and polyploids elucidated from ITS sequences show that in the Nod-independent clade, species such as A. evenia, A. scabra and A. rudis participate in the hybridization/polyploidization events and formation of polyploid complexes that have contrib- uted to the radiation of this group (Arrighi et al. 2014). Figure 1. Mitotic metaphase cells of hybrid Aeschynomene 2n = 4x = 40. A-C, Metaphase chromosome plates in optimal spread; D, Karyotype 34m + 6sm. The chromosomes are aligned in decreas- ing order. Arrows point to secondary constrictions and satellites on short arms of submetacentric chromosomes. Table 1. Average chromosome measurements obtained from five nuclei in metaphase of the hybrid population (2n = 4x = 40 = 34m + 6sm) under study. CP TCL (µm) LLA ( µm) LSA (µm) r CT 01 1.72 0.96 0.77 1.24 m 02 1.63 0.89 0.73 1.21 m 03 1.59 0.89 0.69 1.28 m 04 1.55 0.81 0.72 1.12 m 05 1.53 0.81 0.70 1.15 m 06 1.50 0.82 0.66 1.24 m 07 1.48 0.83 0.63 1.31 m 08 1.46 0.79 0.66 1.19 m 09 1.44 0.79 0.63 1.25 m 10 1.42 0.79 0.61 1.29 m 11 1.38 0.74 0.64 1.17 m 12 1.36 0.89 0.46 1.93 sm* 13 1.34 0.78 0.55 1.41 m 14 1.31 0.70 0.59 1.18 m 15 1.28 0.72 0.55 1.30 m 16 1.24 0.83 0.40 2.07 sm 17 1.23 0.69 0.52 1.32 m 18 1.19 0.67 0.54 1.24 m 19 1.19 0.66 0.49 1.34 m 20 1.16 0.78 0.36 2.16 sm TDCL 28.00 AC 1.40 Abbreviations: CP- chromosome pair; TCL- total chromosome length; LLA- length long arm; LSA- length short arm; r- arm ratio; CT-chromosome type; TDCL- Total diploid chromosome length; AC- Average chromosome length; m- metacentric; sm- submeta- centric; *- satellite. Abbreviations: CP- chromosome pair; TCL- total chromosome length; LLA- length long arm; LSA- length short arm; r- arm ratio; CT-chromosome type; TDCL- Total diploid chro- mosome length; AC- Average chromosome length; m- metacentric; sm- submetacentric; *- satellite. 21First cytogenetic register of an allopolyploid lineage of the genus Aeschynomene native to Mexico In the present investigation, the chromosomal num- ber obtained in all the nuclei analyzed from the individ- uals under study was 2n = 4x = 40, which undoubtedly shows that they are polyploid cells and that the individ- uals from which they come integrate a polyploid lineage not previously detected in Mexico (Rudd 1955; Tapia- Pastrana et al. 2020). The origin of the polyploidy (auto or allopolyploidy) were established easily from the num- ber of SAT chromosomes unambiguously identified both in nuclei in prometaphase and metaphase and by their position in relation to the nucleolus. Indeed, polyploidy, the process of genome dou- bling that gives rise to organism with multiple sets of chromosomes, is recognized as an important process in plant evolution, a major mechanism of adaptation and is often invoked as a driver of diversification (Ramsey and Figure 2. Seedling morphology of Aeschynomene under study until the complete development of the fourth eophyll A, Aeschynomene eve- nia; B, A. scabra; C, hybrid of Aeschynomene. Table 2. Number of leaflets up to the fourth eophyll in Aeschynomene evenia. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 E1 10 10 10 10 10 10 10 10 10 10 10 9 10 9 8 10 9 10 10 8 E2 12 12 12 12 11 12 14 12 10 14 12 12 14 12 10 13 12 11 10 11 E3 15 16 15 14 14 16 17 15 14 16 16 15 17 12 12 16 16 12 14 12 E4 16 18 16 16 16 16 18 18 14 16 18 16 18 16 15 16 16 14 16 15 Table 3. Number of leaflets up to fourth eophyll in Aeschynomene scabra.   1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 E1 10 8 10 10 10 10 10 8 10 9 10 10 10 10 10 8 8 8 8 10 E2 12 15 14 14 14 14 14 12 16 13 14 13 14 14 14 12 12 13 15 14 E3 20 20 22 18 18 18 19 19 20 17 17 18 18 16 18 19 19 19 20 16 E4 24 25 27 23 22 22 22 22 24 22 22 22 22 21 22 22 22 20 25 21 Table 4. Number of leaflets up to fourth eophyll in hybrids.   1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 E1 10 10 8 10 10 9 10 8 8 10 8 10 8 10 10 8 10 10 8 10 E2 14 14 12 14 13 14 14 14 14 14 14 14 14 16 14 14 14 14 14 10 E3 18 20 18 20 20 19 20 20 20 19 18 20 18 20 20 20 20 15 18 14 E4 21 25 22 23 23 26 23 24 23 24 23 26 22 27 22 22 23 22 23 14 22 Fernando Tapia-Pastrana, Alfonso Delgado-Salinas Schemske 1998; Soltis et al. 2009) and it is likely to be one of the most predominant mechanisms of sympat- ric speciation in plants (Otto and Whitton 2000). It can act alone, resulting in autopolyploidy, or in concert with hybridization, producing allopolyploids, and both modes lead to plant speciation. It should be mentioned that in the process of polyploidization by total gene duplica- tion (autopolyploidy) the number of satellites present in a diploid species is also doubled, since this does not involve loss or suppression of the nucleolar function, the NOR regions associated with secondary constrictions in SAT-chromosomes are they show lax and therefore satellites are clearly appreciated. It is known that NORs contain tandemly arranged highly reiterated riboso- mal rRNA genes coding for 18S-5.8S-26S rRNA whose expression is under epigenetic control (Pikaard 2000). For example, Medicago sativa Linnaeus, a recognized autotetraploid exhibits four macrosatélites in metaphase cells (Falistocco 1987). In contrast, plants of allopoly- ploid origin as cotton (Gossypium hirsutum Linnaeus 2n = 4x = 52 AADD, Endrizzi et al. 1985), wheat (Triti- cum aestivum Linnaeus 2n = 6x = 42, AABBDD, Laca- dena and Cermeño 1985; Friebre et al. 1995) and canola (Brassica napus Linnaeus 2n = 4x = 38 AACC; Xiong and Pires 2011) undergo inactivation of the regions of the nucleolar organizer (NOR) of one of the parental genomes, silenced by the effect of nucleolar dominance (Navashin 1934) and consequently a smaller num- ber of satellites is recorded (Doyle et al. 2008; Ge et al. 2013). It is, rDNA loci may be additive in number, but then exhibit differences in gene expression. Interspecific hybrids often have rRNA genes of one parent function- ally dominant over the rRNA of the other parent, and there are many examples of such regulation of rRNA gene activity in allopolyploids (Pikaard 2000; Pires et al. 2004). Comparative analyses of nucleolar organizer regions (NORs) of somatic metaphase chromosomes made by phase contrast, C-banding and silver staining have demonstrated that the activity of the NORs of cer- tain chromosomes can be suppressed or partially inhib- ited by the presence of other SAT-chromosomes. The NOR competition is cytologically expressed as amphiplasty: a term proposed to denote morphological changes which occur in chromosomes following inter- specific hybridization (Rieger et al. 1976). The second- ary constriction of the SAT-chromosome of one of the parental species is missing in the hybrid and the satellite is retracted onto the chromosome arm as a consequence (Lacadena and Cermeño 1985). Thus, in the Hordeum murinum Linnaeus complex (Poaceae, Triticeae), tetra- ploid and hexaploid cytotypes arising from hybridiza- tion exhibit only a pair of chromosomes with second- ary and satellite constrictions (Cuadrado et al. 2013). In fact, the inactivation or epigenetic silencing of ribosomal genes is one of the most common phenomena in hybrid and polyploid members of Triticeae Linnaeus (Cermeño and Lacadena 1985; Carmona et al. 2016) and one of the first examples of differential gene expression discovered in plant hybrids nearly a century ago (Navashin 1934; Matyásӗk et al. 2007). In the present work, the repres- sive effects on NORs from allopolyploid population are cytologically expressed (amphiplasty) as the suppression of a secondary constriction clearly observed in all their complements (Fig. 1 A-D). The karyotype exhibited in hybrid individuals (34m + 6sm) (Fig. 1D and Table 1) coincides in several respects with that expected at a cross between A. evenia (2n = 2x = 7m + 3sm) and A. scabra (2n = 2x = 10 m) (Fig. 2 in Tapia-Pastrana et al. 2020). For example, the num- ber of sm chromosomes in A. evenia agrees with the 6sm in hybrid individuals. In addition to submetacen- tric chromosomes, these individuals exhibit metacentric chromosomes whose predominance is consistent with the karyotype formulas described in their putative rela- tives, whose complements lack subtelocentric chromo- somes (Tapia-Pastrana et al. 2020). There is a coincidence between THC and AC and even the morphology of the SAT-chromosomes (submetacentrics with macrosatellites in short arms) and their position in the karyotype is very similar to that recently described in A. scabra (Tapia- Pastrana et al. 2020). Therefore we propose to A. evenia and A. scabra as progenitors of the allopolyploid popula- tion (2n = 4x = 40 = 34m + 6sm) registered in this work. The reasoning is simple: if a diploid species is involved in the origin of a tetraploid cytotype, its chromosomes must be present in it. The same is true if tetraploid forms are involved in the origin of hexaploid forms (Cuadrado et al. 2013). In Mexico, recent collection data shows that populations of both species occupy overlapping ranges in some central areas of the country where A. evenia is con- sidered an introduced species (Arrighi et al. 2013; Chain- treuil et al. 2018; Tapia-Pastrana et al. 2020). This new proposal is not surprising, since previously the Indicae series species grouped within Nod-inde- pendent clade, including A. evenia and A. scabra, have been identified as progenitors in allopolyploids and in the formation of polyploid complexes, although attempts at hybridization have failed to form fertile individu- als (Arrighi et al. 2014). Regarding the identity of the allopolyploid taxon recorded here, it can be argued that a detailed review of its complete morphological charac- ters (data not shown) suggests that it shares character- istics described for Aeschynomene rudis particularly in the shape and size of flowers, fruits (hispidulous, verru- 23First cytogenetic register of an allopolyploid lineage of the genus Aeschynomene native to Mexico coses, or muricate at the center) and seeds (Rudd 1955). However, it also recalls the robust version of A. scabra described by Rudd (1955). The existence of cryptic taxa in Aeschynomene as well as the need for broader sam- pling to detect new cytotypes has already been pointed out (Brottier et al. 2018, Chaintreuil et al. 2018) and the results of this study confirm this. Regarding the results obtained from the seedling comparison, these seem to support a close relationship between the individuals of the three populations studied (Fig. 2, Tables 2-4). In principle, the observed intervals in the number of leaflets per eophyll (E1-E4) show some uniformity, particularly E1, whose interval (8-10 leaf- lets) was repeated in the three populations. In interme- diate eophylls (E2-E4) a close concordance is observed between A. scabra and the hybrid population, while in A. evenia the number of leaflets was lower in corre- spondence with the taxonomic description of this spe- cies (Rudd 1955). Furthermore, the morphology of the eophylls was similar and in all populations the leaflets Figure 3. Floral morphotypes (above), dissected flowers and fruits (below) of the taxa under study. A, D and E, Aeschynomene evenia; B, F and G, A. scabra; C, H and I, hybrid of Aeschynomene. All three taxa exhibit typical pea or papilionoid flowers. These zygomorphic flowers comprise a standard (vexillum or banner) petal (adaxially placed), two lateral petals (wings) and two (usually partially fused and abaxially placed) keel petals, which conceal the androecium and gynoecium. The fruits have similar characteristics and are mainly differentiated by their size. Above scale bar = 0.5 cm, below = 1.0 cm. 24 Fernando Tapia-Pastrana, Alfonso Delgado-Salinas exhibited entire margins, without trichomes and with a central primary vein. Polyploids are known to often have novel pheno- types that are not present in their diploid progenitors or that exceed the range of parent species (“gigas” effects) (Ramsey and Schemske 2002; Ramsey and Ramsey 2014). In this sense, Fig. 3 shows floral morphotypes, dissected flowers and fruits of the populations studied here, where similarities are observed, but the differences in size of such characters are highlighted. The results obtained in this study confirm that in the Nod-independent lineage within the genus Aeschynomene, hybridization and poly- ploidization play a relevant role in the formation of spe- cies and those taxa such as the polymorphics A. evenia and A. scabra actively participate in it. ACKNOWLEDGEMENTS This study is part of the doctoral thesis of the first author, F T-P, carried out at the Posgrado en Ciencias Biológicas of the Universidad Nacional Autónoma de México (UNAM). The authors thank to Dr. Samuel Car- leial for seeds originally identified as A. scabra and to the Division of Postgraduate Studies and Research of the Faculty of Higher Studies, Zaragoza, UNAM for the sup- port provided during the development of this research. REFERENCES Alazar D, Becker M. 1987. Aeschynomene as green manure for rice. Plant and Soil 101: 141-143. Arrighi JF, Cartieaux F, Brown SC, Rodier-Goud M, Boursot M, Fardoux J, Patrel D, Gully D, Fabre S, Chaintreuil C, Giraud E. 2012. Aeschynomene evenia, a model plant for studying the molecular genetics of the Nod-independent rhizobium-legume symbiosis. Molecular Plant-Microbe Interactions 25: 851-861. Arrighi J-F, Cartieaux F, Chaintreuil C, Brown S, Bour- sot M, Giraud E. 2013. Genotype delimitation in the Nod-Independent model legume Aeschynomene eve- nia. PLoS ONE 8(5): e63836. doi:10.1371/journal. pone.0063836 Arrighi JF, Chaintreuil C, Cartieaux F, Cardi C, Rodier- Goud M, Brown SC, Boursot M, D´Hont A, Dreyfus B, Giraud E. 2014. Radiation of the Nod-independent Aeschynomene relies on multiple allopolyploid specia- tion events. New Phytologist 201: 1457-1468. Bielig LM. 1997. Chromosome numbers in the forage legume genus, Aeschynomene L. SABRAO Journal 29:33-39. Brottier L, Chaintreuil C, Simion P, Scornavacca C, Rival- lan R, Mournet P, Moulin L, Lewis GP, Fardoux J, Brown SC, Gomez-Pacheco M, Bourges M, Hervouet C, Gueye M, Duponnois R, Ramanankierana H, Ran- driambanona H, Vandrot H, Zabaleta M, DasGupta M, D’Hont A, Giraud E, Arrighi JF. 2018. A phyloge- netic framework of the legume genus Aeschynomene for comparative genetic analysis of the Nod-depend- ent and Nod-independent symbioses. BMC Plant Biology 18: 333. 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. American Journal of Botany 99: 1991- 2013. Carleial S, Delgado-Salinas A, Domínguez CA, Terrazas T. 2015. Reflexed flowers in Aeschynomene amor- phoides (Fabaceae: Faboideae): a mechanism promot- ing pollination specialization? Botanical Journal of the Linnean Society 177: 657-666. Carmona A, de Bustos A, Jouve N, Cuadrado A. 2016. Allopolyploidy and the complex phylogenetic rela- tionships within the Hordeum brachyantherum taxon. Molecular Phylogenetics and Evolution 97: 107-119. Cermeño MC, Lacadena JR. 1985. Nucleolar organizer competition in Aegilops–rye hybrids. Canadian Jour- nal of Genetics and Cytology 4: 479-483. Chaintreuil C, Arrighi JF, Giraud E, Miché L, Moulin L, Dreyfus B, Munive-Hernández J-A, Villegas-Hernán- dez MC, Béna G. 2013. Evolution of symbiosis in the legume genus Aeschynomene. New Phytologist 200: 1247-1259. Chaintreuil C, Gully D, Hervouet C, Tittabutr P, Ran- driambanona H, Brown SC, Lewis GP, Bourge M, Cartieaux F, Boursot M, Ramanankierana H, D´Hont A, Teaumroong N, Giraud E, Arrighi JF. 2016. The evolutionary dynamics of ancient and recent poly- ploidy in the African semiaquatic species of the leg- ume genus Aeschynomene. New Phytologist 211: 1077-1091. Chaintreuil C, Perrier X, Guillaume M, Fardoux J, Lew- is GP, Brottier L, Rivallan R, Gomez- Pacheco M, Bourges M, Lamy L, Thibaud B, Ramanankierana H, Randriambanona H, Vandrot H, Mournet P, Giraud E, Arrighi JF. 2018. Naturally occurring variations in the nod-independent model legume Aeschynomene evenia and relatives: a resource for nodulation genet- ics. BMC Plant Biology 18: 54. Cuadrado A, Carmona A, Jouve N. 2013. Chromosomal characterization of the three subgenomes in the poly- ploids of Hordeum murinum L.: New insight into the 25First cytogenetic register of an allopolyploid lineage of the genus Aeschynomene native to Mexico evolution of this complex. PLoS ONE 8(12),e81385. doi:10.1371/journal.pone.0081385 Doyle JJ, Flagel LE, Paterson AH, Rapp RA, Soltis DE, Soltis PS, Wendel JF. 2008. Evolutionary genetics of genome merger and doubling in plants. Annual Review of Genetics 42: 443-461. Endrizzi JE, Turcotte EL, Kohel RJ. 1985. Genetics, cytol- ogy, and evolution of Gossypium. Advances in Genet- ics 23: 271-375. Falistocco E. 1987. Cytogenetic investigations and karyo- logical relationships of two Medicago: M. sativa L. (Alfalfa) and M. arborea L. Caryologia 4: 339-346. Fernandes A. 1996. O táxon Aeschynomene no Brasil. - Fortaleza: Edições UFC. Brasil. Friebre B, Jiang J, Tulee N, Gill BS. 1995. Standard karyo- type of Triticum umbellulatum and the characteriza- tion of derived chromosome addition and transloca- tion lines in common wheat. Theoretical and Applied Genetics 90: 150-156. García-Oliva F, Camou A, Maass JM. 2002. El clima de la región central de la costa del Pacífico Mexicano. In: Noguera, F. A., Vega-Rivera, J. H., García-Aldrete, A. N., Quesada-Avendaño, M. eds. Historia natu- ral de Chamela. Mexico City: Universidad Nacional Autónoma de México, Instituto de Biología, 3-10. Ge X-H, Ding L, Li Z-Y. 2013. Nucleolar dominance and different genome behaviors in hybrids and allopoly- ploids. Plant Cell Reports 32: 1661-1673. Giraud E, Moulin L, Vallenet D, Barbe V, Cytryn E, Ava- rre JC, Jaubert M, Simon D, Cartieaux F, Prin Y, Bena G, Hannibal L, Fardoux J, Kojadinovic M, Vuil- let L, Lajus A, Cruveiller S, Rouy Z, Mangenot S, Segurens B, Dossat C, Franck WL, Chang W-S, Saun- ders E, Bruce D, Richardson P, Normand P, Drey- fus B, Pignol D, Stacey G, Emerich D, Vermeglio A, Medigue C, Sadowsky M. 2007. Legumes symbioses: absence of Nod genes in photosynthetic Bradyrhizo- bia. Science 316(5829): 1307-1312. Huziwara Y. 1962. Karyotype analysis in some genera of Compositae. VIII. Further studies on the chromo- somes of Aster. American Journal of Botany 49: 116- 119. Kawakami J. 1930. Chromosome numbers in Leguminos- ae. The Botanical Magazine, Tokyo 44: 319-328. Klitgaard BB, Lavin M. 2005. Tribe Dalbergieae sensu lato. In: Lewis GP, Schrire BD, Mackinder BA, Lock JM (Eds) Legumes of the world. Royal Botanic Gar- dens, Kew Publishing, London, 306-335. Lacadena JR, Cermeño MC. 1985. Nucleolus organizer competition in Triticum aestivum - Aegilops umbel- lulata chromosome addition lines. Theoretical and Applied Genetics 71: 278-283. Lavin M, Pennington RT, Klitgaard BB, Sprent JI, de Lima HC, Gasson PE. 2001. The dalbergioid legumes (Fabaceae): delimitation of a pantropical monophyl- etic clade. American Journal of Botany 88: 503-533. Levan A, Fredga K, Sandberg AA. 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52: 201-219. Matyásӗk R, Tate JA, Lim YK, Šrubařová H, Koh J, Leitch AR, Soltis DE, Soltis PS, Kovařík A. 2007. Concerted evolution of rDNA in recently formed Tragopogon allotetraploids is typically associated with an inverse correlation between gene copy number and expres- sion. Genetics 176: 2509-2519. Navashin M. 1934. Chromosome alterations caused by hybridization and their bearing upon certain general genetic problems. Cytologia 5: 169-203. Otto SP, Whitton J. 2000. Polyploid incidence and evolu- tion. Annual Review of Genetics 34: 401-437. Pikaard CS. 2000. The epigenetics of nucleolar domi- nance. Trends in Genetics 16: 495-500. Pires JC, Lim KY, Kovarík A, Matyásek R, Boyd A, Leitch AR, Leitch IJ, Bennett MD, Soltis PS, Soltis DE. 2004. Molecular cytogenetic analysis of recently evolved Tragopogon (Asteraceae) allopolyploids reveal a kar- yotype that is additive of the diploid progenitors. American Journal of Botany 91: 1022-1035. Polhill RM, Raven PH, Stirton CH. 1981. Evolution and systematics of the Leguminosae. In Polhill RM, Raven PH (eds.) Advances in Legume Systematics, part 1, 1-26. Royal Botanic Gardens, Kew, UK. Ramsey J, Schemske DW. 1998. Pathways, mechanisms, and rates of polyploid formation in flowering plants. Annu- al Review of Ecology and Systematics 29: 467-501. Ramsey J, Schemske DW. 2002. Neopolyploidy in flower- ing plants. Annual Reviews in Ecology and Systemat- ics 33: 589-639. Ramsey J, Ramsey TS. 2014. Ecological studies of poly- ploidy in the 100 years following its discovery. Philosophical Transactions of Royal Society B 369: 20130352. Ribeiro RA, Lavin M, Lemos-Filho JP, Mendonça Filho CV, Rodrigues dos Santos F, Lovato MB. 2007. The genus Machaerium (Leguminosae) is more closely related to Aeschynomene sect. Ochopodium than to Dalbergia: inferences from combined sequence data. Systematic Botany 32: 762-771. Rieger R, Michaelis A, Green MM. 1976. Glossary of genetics and cytogenetics: classical and molecular. Springer-Verlag, Berlin. 647 pp. Rudd VE. 1955. The American species of Aeschynomene. Contributions from the United States National Her- barium 32: 1-172. 26 Fernando Tapia-Pastrana, Alfonso Delgado-Salinas Schütz RR, da Silva HL, Silva FA. 2019. Seedling mor- phology of some Brazilian taxa of Aeschynomene (Leguminosae) and its systematic relevance. Flora 255: 69-79. Soltis DE, Albert VA, Leebens‐Mack J, Bell CD, Paterson AH, Zheng C, Sankoff D, de Pamphilis CW, Kerr Wall P, Soltis PS. 2009. Polyploidy and angiosperm diversi- fication. American Journal of Botany 96: 336-348. Souza MC, Vianna LF, Kawakita K, Miotto STS. 2012. O gênero Aeschynomene L. (Leguminosae, Faboideae, Dalbergieae) na planicie de inundação do alto rio Paraná, Brasil. Revista Brasileira de Biociências 10: 198-210. 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 implications. Com- parative Cytogenetics 14: 157-182. 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. T h e p l ant l i s t w w w. t h e p l ant l i s t . or g / tp l / s e arc h ? q = Aeschynomene Verdcourt B. 1971. Aeschynomene. In: Gillet JB, Polhill RM, Verdcourt B (Eds) Flora of Tropical East Africa, Leguminosae, Papilionoideae. Royal Botanic Gar- dens, Kew Publishing, London, 364-406. Wojciechowski MF, Lavin M, Sanderson MJ. 2004. A phylogeny of legumes (Leguminosae) based on analy- sis of the plastid matK gene resolves many well-sup- ported subclades within the family. American Journal of Botany 91: 1846-1862. Xiong Z, Pires JC. 2011. Karyotype and identification of all homoeologous chromosomes of allopolyploid Brassica napus and its diploid progenitors. Genetics 187: 37-49.