Intra-and interspecifi c karyotypic variations of the genus Senna Mill . ( Fabaceae , Caesalpinioideae )

Although the chromosome number 2n = 28 predominates in most species of the genus Senna, variations are often observed, resulting from either polyploidy (2n = 42, 56, 112) or disploidy (2n = 22, 24, 26) events. To better understand the karyotypic variations in Senna, we examined heterochromatin patterns of 10 species of that genus using chromomycin A3 (CMA) and 4’6-diamidino-2-phenylindole (DAPI) staining, and reviewed information on the chromosome counts of 72 species of that genus. Th e CMA/DAPI banding patterns were relatively variable among the 10 species, both in terms of the numbers of bands (from two to 26) and their locations on the chromosomes (terminal or proximal regions). Our review indicated that 2n = 28 is the most common ploidy among species of Senna, although other numbers were observed, apparently due to polyploidy or disploidy events; polysomy and aneusomy were also observed. Karyotype variations appear to have contributed to the diversifi cation and wide distribution of Senna.


Introduction
Senna is one of the most diverse genera within the family Fabaceae, with approximately 350 species of trees, shrubs, and sub-shrubs distributed throughout the American, African, and Australian continents, with occurrences also in Asia and on Pacifi c islands (Irwin & Barneby 1982;Marazzi et al. 2006).Species occupy an extremely wide range of habitats, varying from humid forests, dry forests, rock outcrops, dry or cold deserts to anthropized areas (Irwin & Barneby 1982;Acharya et al. 2011).
Phylogenetic analyses have demonstrated that Senna is monophyletic and occupies a position near Cassia senso stricto and Chamaecrista (Marazzi et al. 2006;Acharya et al. 2011).Th ose three genera form the subtribe Cassiinae Irwin & Barneby, and they are morphologically distinguished in relation to characteristics of their androceu, corolla, fl oral architecture, bracteoles, and fruits.Senna has traditionally been divided into six sections: Astroites, Chamaefi stula, Paradictyon, Peiranisia, Psilorhegma, and Senna, based on their fl oral morphologies and fruit and extrafl oral nectary structures (Irwin & Barneby 1982), although examinations of the DNA sequences of diff erent chloroplast gene regions (rpS16, rpL16, matK) have demonstrated that most of those sections are polyphyletic (Marazzi et al. 2006).
Karyotypic analyses of representatives of Senna using fluorochromes have been relatively scarce, with the exception of work by Souza & Benko-Iseppon (2004).Those authors reported the occurrence of two chromosome pairs with terminal or subterminal CMA + /DAPI -bands in most of the species analyzed, with the exception of a population of Senna obtusifolia (which demonstrated two chromosome pairs with terminal DAPI + /CMA -bands).Studies of heterochromatin patterns can be important tools for understanding the taxonomic relationships between different plant species, especially those that are morphologically very similar (Pessoa et al. 2014;Almeida et al. 2016;Cordeiro et al. 2016).
In order to understand the role of karyotype differentiation and evolutionary trends in this genus, we analyzed the heterochromatin patterns of 10 species of Senna (Fabaceae, Caesalpinioideae) using the fluorochromes chromomycin A3 (CMA) and 4'6-diamidino-2-phenylindole (DAPI) and reviewed the chromosome numbers of 72 species of that genus (and performed first counts for two species) for evidence of polyploidy, disploidy, and intra-and interspecific variations.

Taxonomic sampling
We examined variations in the chromosome numbers of 74 species of the genus Senna based on chromosome counts published in the literature and on work presented here.The species names, authors, and references are listed in Table 1.For those species whose counts were exclusively obtained from the Chromosome Counts Database (CCDB, Rice et al. 2015), we present only the predominant chromosome count.
Cytogenetic analyses were performed on ten species of the genus Senna growing in areas of Caatinga (dryland) and humid forest vegetation in the Agreste region of Paraíba State, northeastern Brazil, to determine their heterochromatin patterns.Prepared samples of collected specimens were deposited in the Professor Jayme Coelho de Moraes Herbarium (EAN) of the Federal University of Paraíba.Detailed information concerning the species and their main karyological features are listed in Table 2. Seeds from the collected species were sown to germinate in Petri dishes.After their roots had grown to approximately 1.5 cm in length, they were excised and treated as described below.At least 10 roots per species were analyzed.

Cytogenetic and CMA/DAPI banding pattern analyses
To analyze heterochromatin patterns, the root tips were pretreated with 0.002 M 8-hydroxyquinoline (8-HQ) for 24 hours, fixed in 3 : 1 (v/v) absolute ethanol/glacial acetic acid for 30 minutes, and then stored in a freezer.For slide preparation, the roots were digested in a solution of 2 % cellulase and 20 % pectinase at 37 °C for 40 minutes.
The root tips were squashed in 45 % acetic acid, the slides frozen in liquid nitrogen to remove the coverslip.Slides were aged for three days and then stained for one hour with 10 µL of CMA (0.1 mg/mL) followed by staining with 10 µL DAPI (1 µg/mL) for 30 min., with subsequent mounting in glycerin/McIlvaine buffer (pH 7.0) (1:1, v/v).Slides were held for three days in the dark to stabilize the fluorochromes (Guerra & Souza 2002).Metaphases were recorded using a Zeiss microscope equipped with a Axio Cam MRC5 video camera, using Axiovision 4.8 software.At least three slides were analyzed for each species, generally photographing 10 cells per slide.Chromosome measurements were performed using Image Tool version 3.0 software (Donald et al. 2008).Chromosome morphologies were characterized using the centromeric index, following Guerra (1986).The images were edited using Adobe Photoshop CS3 Extended Version 10.0 software.
CMA/DAPI banding analysis showed an occurrence of GC-rich base pairs (CMA + /DAPI -) preferentially located in the terminal chromosome regions of the chromosomes, corresponding to Nucleolar Organizing Regions (NORs).Heterochromatic bands located in proximal regions were observed in some species, such as S. obtusifolia, S. georgica, and S. rizzinii.There were also differences band numbers, which varied between two and six bands in the terminal chromosome regions, and between one and 24 bands in proximal regions (Fig. 1, Tab. 2).Two species, S. siamea and S. splendida, showed heteromorphic bands, with only one of the homologous chromosomes of each species showing CMA + / DAPI -bands in their proximal region (Fig. 1H, J, arrows).
The consistent records of 2n = 28 in most species of Senna subjected to karyological analysis, especially in the basal clades (Marazzi et al. 2006), allied to that same number in diverse species of closely related genera (such as Cassia, Apuleia and Delonix [Biondo et al. 2005b;Rice et al. 2015]), confirms x = 14 as the basic ancestral number of Senna,  corroborating the positions of various authors (Goldblatt 1981;Biondo et al. 2005a).Some species, such as S. occidentalis and S. obtusifolia, demonstrated intraspecific karyotypic variations, with karyotypes of 2n = 24, 26 and 28 (Chaulagain & Sakya 2002;Biondo et al. 2005a;Rice et al. 2015).Diverse karyological phenomena may be involved in the intraspecific variations observed in different plant groups, especially neopolyploidy (species with diploid and polyploid cytotypes) and disploidy (centric fusions and fissions) (Guerra 2008).Additionally, polysomy and aneusomy (intra-individual variations in somatic chromosome numbers caused by polyploidy or aneuploidy respectively [Nirmala & Rao 1996;Rodrigues et al. 2009]) have likely occurred in certain species of Senna (Chaulagain & Sakya 2002;Matos et al. 2011), contributing to chromosome number variability in the genus.Intraspecific variations in chromosome numbers are quite common in other plant groups, such as Epidendrum secundum (Orchidaceae) (Assis et al. 2013), Rutidosis leptorrhynchoides (Asteraceae) (Murray & Young 2001) and Zephyranthes sylvatica (Amaryllidaceae) (Felix et al. 2008).In those cases, intraspecific variations could be related to factors such as distance or geographic isolation, together with hybridization in natural populations.Intraspecific variations were also observed in Senna spectabilis, with records of 2n = 26 and 28 (Resende et al. 2013;Rice et al. 2015), although those variations appear to be distinct at the variety level, as S. spectabilis var.excelsa shows 2n = 28 in all of its karyotypic descriptions (including the present work), while S. spectabilis var.spectabilis generally shows 2n = 26 (Rice et al. 2015).
Chromosome records of the genus Senna also demonstrated variations in ploidy levels, especially for S. rugosa In addition to intraspecific and ploidy-level variations, interspecific chromosome variations were also observed in Senna.Although 2n = 28 predominated (68.9 % of the species), there are significant numbers of records of 2n = 26 (14.8 %) and 2n = 24 (13.5 %) among its species (Goldblatt 1981;Souza & Benko-Iseppon 2004;Biondo et al. 2005a;Matos et al. 2011;Resende et al. 2013;Rice et al. 2015).Interspecific variations were observed in the present work, with five species showing 2n = 28 but four species showing 2n = 26.These numbers most likely represent disploidy events during the evolution of the genus, although they are generally treated as random phenomena (Goldblatt 1981;Biondo et al. 2005a;Resende et al. 2013).
Phylogenetic analyses undertaken by Marazzi et al. (2006) demonstrated that the genus Senna can be divided into seven monophyletic clades.Chromosome count comparisons with the results of those phylogenetic analyses can facilitate our understanding of interspecific variations.The prevalence of 2n = 28 is observed in essentially all of the seven clades of Senna, especially in the most primitive and most derived clades.Most variations (x = 11, 13, 21 and 28), on the other hand, occur in clade IV.That clade corresponds to the monophyletic series Bacillaris (section Chamaefistula), which comprises approximately 50 species of shrubs and small trees whose leaves are exclusively composed of two pairs of folioles (Irwin & Barneby 1982;Marazzi et al. 2006).Most chromosome records of x = 13 occur in clade IVb.Phylogenetic analyses point to a large polytomy among the species that compose that clade, indicating its probable recent radiation (Marazzi et al. 2006); the clade probably experienced a disploidy event (x = 13) in a common ancestor, with most of the descendent species then conserving that cytological characteristic.Other variations, such as x = 12 in S. atomaria and x = 13 in S. spectabilis (clade III), x = 12 in S. multijuga (clade VI), and x = 12 in S. hirsuta var.hirta (clade VIIb) were also apparently important for the diversification of the genus, although with lesser evolutive significance in terms of their respective clades.
In relation to heterochromatin patterns, significant differences were observed among most of the species of the genus Senna analyzed, whether in terms of the numbers of bands (2-26) or their localizations on the chromosomes (terminal or proximal).Only S. georgica and S. rizzinii demonstrated the same banding pattern (four terminal CMA + /DAPI -and 22 proximal CMA + /DAPI -bands).Senna martiana and S. aversiflora demonstrated the same numbers of bands (four terminal CMA + /DAPI -bands), although the latter demonstrated a polyploid karyotype (2n = 52).Variations in heterochromatic banding patterns are quite common in plant groups and can vary among species, populations, or even individuals (Guerra 2000;Dobigny et al. 2004).Those variations can reflect the results of various cytological phenomena, especially satellite DNA amplification (Guerra 2000;Silva et al. 2010;Ribeiro et al. 2016).Differences in heterochromatic banding patterns are quite useful for cytotaxonomic characterizations of plant species, especially among those having karyotypes formed by morphologically similar chromosomes that are numerically stable (Guerra 2000;Scaldaferro et al. 2012;Cordeiro et al. 2016).The observed differences in the CMA/ DAPI banding patterns in Senna ratified the effectiveness of that technique for facilitating karyological differentiation of its species.
Among the 10 species analyzed, only three (S.alata, S. obtusifolia, and S. siamea) had previously been examined in terms of their CMA/DAPI banding patterns (Souza & Benko-Iseppon 2004).Those species did, however, demonstrate differences when compared to the analyses performed here.Senna alata, S. obtusifolia, and S. siamea had previously been reported to have two terminal CMA + /DAPI -bands (Souza & Benko-Iseppon 2004), while we found those same species to have six terminal CMA + /DAPI -bands, two terminal CMA + / DAPI -+ 24 proximal CMA + /DAPI -bands, and six terminal CMA + /DAPI -bands plus one proximal heteromorphic band respectively.Differences in heterochromatin patterns within the same species can occur in distinct populations, as was previously reported by Souza & Benko-Iseppon (2004) in S. obtusifolia; those authors found one population having two terminal CMA + /DAPI -bands and another showing two terminal DAPI + /CMA -bands.Variations in the patterns of heterochromatic bands in different individuals of the same species are often observed in plants, including Oziroë argentinensis (Dematteis et al. 2006), Capsicum (Scaldaferro et al. 2012), Allium pulchellum (Vosa 1996), and Pinus nigra (Bogunić et al. 2011).Variations in heterochromatin bands between individuals of the same species appear to be an intraspecific cytological characteristic of the genus Senna that is worthy of further investigation, calling for the analysis of distinct populations from different ecosystems and different geographic regions.
The present study allowed us to put forward the following general considerations: a) CMA/DAPI banding patterns in the genus Senna are quite useful for cytotaxonomically differentiating its species, although possible variations between different populations of a given species will need to be closely examined; b) although most species of Senna show 2n = 28, records of polyploidy (2n = 52, 56, 104 and 112) and disploidy (2n = 22, 24 and 26) were observed in numerous species of that genus; c) intraspecific variations observed in certain species, such as S. obtusifolia and S. occidentalis, appeared to be result of cytological phenomena such as disploidy, or polysomy and aneusomy.
The karyotypic variations observed in Senna, whether interspecific or intraspecific, probably contributed to the diversification of that genus, making it one of the most

Table 1 .
Chromosome records of species of the genus Senna, and their respective references.

Table 2 .
Principal karyological information concerning species of the genus Senna.Legend: T = terminal region of the chromosome, P = proximal region, H = heteromorphic band.