First cytogenetic register of an allopolyploid lineage of the genus Aeschynomene (Leguminosae, Papilionoideae) native to Mexico

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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-
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source are credited.
Data Availability Statement: All rel-
evant data are within the paper and its
Supporting Information les.
Competing Interests: The Author(s)
declare(s) no conict of interest.
ORCID
FTP: 0000-0003-0232-2110
First cytogenetic register of an allopolyploid
lineage of the genus Aeschynomene
(Leguminosae, Papilionoideae) native to Mexico
F T-P,*, A D-S
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 rst time an allopolyploid
lineage of the genus Aeschynomene in Mexico. e hybrid condition is conrmed aer
all the prometaphase and metaphase nuclei of the hybrids exhibited only one pair of
SAT-chromosomes, conrming the existence of nucleolar dominance and amphiplasty.
e 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
leaets of the rst 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” eects on owers 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 dierent ecological requirements. Several species con-
tribute to supplement nitrogen to the soil through the production of nodular
roots and stems in symbiosis with nitrogen xing 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 xing nodules
that enhance rice production (Arrighi et al. 2012, 2013).
is 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).
e 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 owers
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 basixed stipules and a campanulate calyx
(subgenus Ochopodium Vogel) are more related to the
genera Machaerium Persoon and Dalbergia Linnaeus f.
than to taxa with medxed stipules and a bilabiate calyx
(subgenus Aeschynomene Léonard) (Ribeiro et al. 2007;
Cardoso et al. 2012).
Currently Aeschynomene genus contains 170 (http://
www.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 diversication 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 diversication 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 infraspecic 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 owers, fruits and seeds generated suspicions
about a possible hybrid origin. In addition as the sam-
pled individuals exhibited oral 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 rst 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°01´W (Carleial s/n, MEXU). e climate
is semi-dry and warm. Mean temperature in the area is
25.2 °C, and there is a well-dened rainy season (average
annual precipitation: 1107mm) occurring from June to
October (García-Oliva et al. 20 02).
e 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. e predominant climate is warm subhumid
with rains from June to September (average annual
precipitation: 1100 to 1200 mm). e studied taxa are
assigned to the infrageneric classication 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).

19
First 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. e seeds were scaried and germinated in Petri
dishes lined with a moist lter 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 xed in the xative (ethanol: acetic acid=3:1).
ey were then treated with a mixture of 20% pectinase
(Sigma) and 2% cellulase (Sigma) in 75 mM KCl for 60
min at 37 °C. Aer centrifugation at 1500 rpm for 10
min, the cell pellet was transferred to 75 mM KCl solu-
tion for 13 min at 37 °C. Aer two successive rinses with
the KCl solution, they were again xed in the xative 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 dierence in length
between the longest chromosome and the shortest chro-
mosome (Range) and the longest/shortest chromosome
ratio (L/S). e shapes of chromosomes were classied
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. e 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 leaets 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. e
chromosomal range was 0.56 µm, the ratio 1.48 and
a TF = 42.46. e 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). e karyotype exhibits small
chromosomes (1.72-1.16 µm) clearly discernible, with
predominance of metacentric chromosomes (m) and
lacking subtelocentric chromosomes (st). is 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
e seedlings of the three taxa are illustrated in Fig.
2 A-C. Eophylls are stipulated, alternate, petiolate, pin-
nate, with alternate leaets, have elliptic to oblong leaf-
lets, a rounded apex, an entire margins, and one cen-
tral primary vein in the three taxa under study. e
leaflets did not present trichomes; both adaxial and
abaxial surfaces are glabrous. e number of leaets
in the rst 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 that the entire Dalbergioid 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 rst 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 owers 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. e 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 ve
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.

21
First 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). e origin of the polyploidy (auto
or allopolyploidy) were established easily from the num-
ber of SAT chromosomes unambiguously identied 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
oen invoked as a driver of diversication (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 leaets 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 leaets 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 leaets 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 eect 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 dierences in gene expression. Interspecic
hybrids oen 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.
e NOR competition is cytologically expressed as
amphiplasty: a term proposed to denote morphological
changes which occur in chromosomes following inter-
specic hybridization (Rieger et al. 1976). e 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). us, 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
rst examples of dierential gene expression discovered
in plant hybrids nearly a century ago (Navashin 1934;
Matyásӗk et al. 2007). In the present work, the repres-
sive eects 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).
e 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). ere 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). erefore we propose to A. evenia
and A. scabra as progenitors of the allopolyploid popula-
tion (2n = 4x = 40 = 34m + 6sm) registered in this work.
e reasoning is simple: if a diploid species is involved
in the origin of a tetraploid cytotype, its chromosomes
must be present in it. e 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).
is 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 identied 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 owers, fruits (hispidulous, verru-

23
First 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). e 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 conrm 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 leaets 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 leaets 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 leaets
Figure 3. Floral morphotypes (above), dissected owers 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 owers. ese zygomorphic owers
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. e fruits have similar characteristics and are mainly dierentiated 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 oen have novel pheno-
types that are not present in their diploid progenitors or
that exceed the range of parent species (“gigas” eects)
(Ramsey and Schemske 2002; Ramsey and Ramsey 2014).
In this sense, Fig. 3 shows oral morphotypes, dissected
owers and fruits of the populations studied here, where
similarities are observed, but the dierences in size of
such characters are highlighted. e results obtained in
this study conrm 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
is study is part of the doctoral thesis of the rst
author, F T-P, carried out at the Posgrado en Ciencias
Biológicas of the Universidad Nacional Autónoma de
México (UNAM). e authors thank to Dr. Samuel Car-
leial for seeds originally identied 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.
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