Cytogenetics of Mimosa bimucronata (DC.) O. Kuntze (Mimosoideae, Leguminosae): chromosome number, polysomaty and meiosis

Chromosome numbers (somatic and/or gametic) were determined in 50 populations of M. bimucronata (DC.) O. Kuntze collected in the species area of distribution in Rio Grande do Sul, south Brazil. All populations were diploid (2n = 2x = 26, n = 13). Polysomatic (mostly tetraploid) cells were detected in the seedlings root-tip cells in 39 out of the 41 populations examined, ranging from 3.0 to 28.2 % among populations, but were absent in the root-tips of grown plants. Polysomaty was as well absent in pollen-mother cells. In M. bimucronata pollen-mother cells are joined two-by-two before the onset of meiosis, remaining attached during all the meiotic division until the formation of pollen grain polyads, composed of two sets of four pollen grains each, that are dispersed in this way , which, accor ding to previous suggestions would be an adaptation to ensur high seed set after a single pollination event.


INTRODUCTION
The genus Mimosa L. (Mimosoideae, Leguminosae) comprises around 530 species, most of them native to the Americas (Barneby 1991).Many of Mimosa species are multipurpose trees, and among them is M. bimucronata (DC.)O. Kuntze, which occurs in Paraguay, Argentina and Brazil (from the State of Alagoas in the northeast to the State of Rio Grande do Sul in the south).Two varieties of the species are described, M. bimucronata (DC.)O. Kuntze var.bimucronata, the most widespread variety, and M.ibimucronata var.adenocarpa Hasll, restricted to Paraguay, which differs only by the more abundant trichomas in M bimucronata var.adenocarpa.
Polysomaty in root tip meristematic cells, the presence of cells with higher ploidy levels, have been reported in some Mimosa species by Witkus and Berger (1947) and Seijo (1993Seijo ( , 1999)).According to Witkus and Berger (1947) polysomaty could be a way to assure a quick seedling establishment.Seijo and Neffa (2004) described the formation of polyads, compound pollen grains, in M. bimucronata.Compound pollen grains are found in ca.15 % of the angiosperm families (Kenrick and Knox 1982) and would be a mechanism to reinforce pollination efficiency.
The aim of this paper was to analyze chromosome numbers and meiotic behaviour in M. bimucronata populations from the species distribution area in the State of Rio Grande do Sul, South Brazil, to study frequency and distribution of polysomaty.

MATERIAL AND METHODS
All populations analyzed were collected (fruits with mature seeds and/or inflorescences) along the area of distribution of the species in the State of Rio Grande do Sul during the years of 2008 and 2009.Taxonomic vouchers are deposited at the ICN Herbarium (Departamento de Botânica, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil) (Table 1).
For somatic chromosome number determination, the seeds were scarified with sand paper or by a small cut in the testa and germinated in petri dishes lined with moist filter paper.Following Dahmer et al. (2009) with some modifications, root-tips around 1 cm length were pretreated with a saturated solution of paradichlorobenzene for 24 h at 4 ºC, fixed in 6: 3: 1 (ethanol: chloroform: acetic acid) for 12-24 h and stored in 70 % alcohol below 0 ºC until slide preparation.For that, the roots were hydrolyzed with 1 N HCl at 60 o C for 8-10 min, stained by Feulgen for around 2 h, treated with 2% pectinase for 2 min, and squashed in 2% propionic carmine.At least ten cells with good chromosome spreading and with chromosomes in equivalent degree of contraction were analysed per plant.The best cells were photographed and/or registered by a digital image capturing system.
Percentage of polysomatic cells were determined in the same slides used for chromososome number counting.As a cross check to eliminate the possible cause of polysomaty being a result of pretreatment, slides of the same populations were prepared by the same technique as described above, but with no pretreatment.Additionaly, individuals of the five populations with higher frequency of polysomatic cells in meristematic root-tip cells (4, 9, 11, 24 and 37) were grown in pots with commercial garden soil in a greenhouse.After ca.11 months, root-tips were collected from these plants and went to the same technical procedures (fixation, pretreatment, staining and squashing) as the seedlings root-tips.
For gametic chromosome number determination and meiotic analysis, young inflorescences, collected at the field, were immediately fixed in Carnoy 6: 3: 1 (ethanol: clorophorm: acetic acid) for 24 h at room temperature and stored in 70% alcohol below 0 ºC until slide preparation.Slides were prepared by squashing the anthers of one flower bud per slide in 2 % propionic carmine.All available cells in all meiotic stages were analyzed.The best cells were photographed and/or registered by a digital image capturing system.

RESULTS AND DISCUSSION
This is the first work where a great number of M bimucronata populations (50) were cytogenetically analyzed.Previous works were restricted to a few populations from Paraguay and Argentina (Seijo 1999(Seijo , 2000)).
Somatic chromosome numbers were determined in 193 individuals from 41 populations and gametic chromosome numbers in 49 individuals of 12 populations (Table 1).All individuals were diploid with 2n = 2x= 26 (Figure 1 a to c) or n = 13 (Figure 2b) confirming the diploid nature of M. bimucronata, as already reported for two populations of de M. bimucronata var.bimucronata from Argentina (Seijo 1999) and for one population of M. bimucronata var.adenocarpa from Paraguay (Seijo 2000) No intraspecific variability for chromosome number was detected among the analyzed populations.
Polysomatic cells were detected in the seedlings roottip cells, with and without pretreatment, in all the 41 populations examined except populations 13 and 21

(Tableo1
). Almost all of polysomatic cells were tetraploid (Figure 1d) and a few triploid.Percentage of polysomatic cells (in root-tips with pretreatment) among populations ranged from 3.0 to 28.2 among populations.However polysomaty was absent from the root-tip cells of adult plants as well as in pollen-mother cells.Witkus and Berger (1947) working with Mimosa pudica L., detected polysomaty in seedling root-tips from 1 to 4 mm length, but in 10 mm long root-tips no polysomatic cells were found.Seijo (1993Seijo ( , 1999) ) commented that polysomatic cells up to four times the diploid number of chromosomes were found in root-tips of some Mimosa species.Therefore, the results for M. bimucronata support Witkus and Berger (1947) suggestion that polysomaty in Mimosa pudica L. would be a mechanism for a rapid seedling establishment.On the other hand, absence of polysomatic cells in the germinative line would avoid the potential formation of polyploid gametes (Galbraith et al. 1991).
In M. bimucronata pollen-mother cells are joined two-by-two even before the onset of meiosis and remain linked during all the meiotic division (Figure 2a to 2e) until the formation of pollen grain polyads (Figure 2f).The sporogenic cells undergo mitosis and, the two resulting cells (Figure 2a) remain attached during meiotic division, that is synchronous in both sister cells (Figure 2b to 2e).At the end of meiosis the microspores of each set of two sister cells remain linked, originating the polyad, in fact a bitetrad of two groups of four pollen grains, each from one of the sister cells, that are dispersed in that form (Figure 2f).Seijo and Neffa (2004) described the formation of polyads in M. bimucronata and proposed that the number of pollen grains per polyad is determined by the number of cells that remain attached after the last mitotic divisions of the sporogenic cell, that are two in M. bimucronata.In Acacia Mill., also a Mimosoideae genus, the mechanism of polyad formation was studied in detail (Kenrick and Knox 1979, 1982, Fitzgerald et al. 1993).In Acacia, polyads of eight, 16 and up to 32 pollen grains are formed.Polyads with 12 and 16 pollen grains are found in other Mimosa species.According to Seijo and Neffa (2004), the number of pollen grains per polyad would correlate to the numbers of ovules per ovary and, due to the dimensions of M. bimucronata stigma, only one polyad would fit per stigma.The occurrence of polyads in M. bimucronata would be an adaptation to ensure high seed set after a single pollination event (Seijo and Neffa 2004) Concluding, all the 50 M. bimucronata populations analyzed were diploid (2n = 2x = 26, n = 13) and no intraspecific variability for chromosome number was detected.Polysomaty occurred in varied degrees but only in seedlings root-tips and was absent in root-tips of established plants as well as in pollen-mother-cells, supporting the theory that, in Mimosa, polysomaty is a way to assure seedling establishment.During all the meiotic process pollen-mother cells remain joined two-by-two, leading to the formation of polyads with eight pollen grains (two sets of four) each,    (2n = 2x = 26, n = 13).Células polissomáticas (em geral tetraplóides) foram detectadas em ponta de raiz de plântulas em 39 das 41 populações analisadas, variando de 3 a 28,2% entre populações.mas estavam ausentes nas pontas de raiz de plantas adultas.Polissomatia também estava ausente nas células-mães-de-pólen.Em M. bimucronata as células-mães-depólen estão juntas duas a duas antes do início da meiose, permanecendo unidas durante toda a divisão meiótica até a formação de políades de graõs de pólen.As políades são compostas de dois conjuntos de quatro grãos cada, que são dispersos desta forma o que, de acordo com sugestões prévias, seria uma adaptação para assegurar alta formação de sementes após um único evento de polinização.

Table 1 .
continuation a Number of individuals and cells (between brackets) analyzed.b Percentage of polysomatic cells in seedling root-tips.