Prior studies have been carried out to locate heterochromatin rich in AT and GC among species from the Caesalpinia group using the fluorochromes CMA3 and DAPI. Previous studies in the Senna and Chamaecrista genera revealed the presence of CMA3+/DAPI− and small CMA3−/DAPI+ terminal or subterminal blocks [14]. In Copaifera, only CMA3+/DAPI− blocks were observed [24], similar to the terminal pattern observed in the present study, for which the analyzed species showed heterochromatin blocks that were rich in GC and poor in AT. These CMA3+/DAPI− bands have been observed only in the terminal regions of chromosomes and have ranged from two to four pairs of chromosomes in the analyzed species. Additionally, CMA3+ blocks have been observed in the metaphase chromosomes of L. ferrea and Po. microphylla, indicating either the existence of GC-rich pericentromeric sequences in related genera (suggesting a shared trait with a common ancestor) or changes related to the composition of centromeric satellite DNA, which is qualitatively rich in GC in these species.
Fluorochrome CMA3−/DAPI+ blocks have already been observed in Senna obtusifolia (L.) H.S. Irwin & Barneby and in one population of Chamaecrista nictitans Moench [14]. However, these bands were not reported in the present study. The absence of this type of heterochromatin may be a typical characteristic of species in the analyzed genera of Poincianella, Libidia, Erytrostemon, Paubrasilia, Caesalpinia and Cenostigma. However, the karyotype characterization on more population can relieve the interspecific variation in cytogenetic markers. In the Leguminosae family, various patterns of AT-rich and GC-poor heterochromatin were also observed for the Mimosa [25] and Erythrina [26] genera. These variations reinforce the need to characterize heterochromatin in other species so as to understand the distribution pattern and evolution of this class of DNA, which is variously colored within the Caesalpinia group.
In the present study, CMA3+/DAPI− terminal blocks coincided with certain 45S rDNA hybridization sites, reinforcing the fact that these CMA3+ blocks relate to these 45S rDNA, which in turn are rich in GC bases [27,28,29]. However, the application of base-specific fluorochromes did not reveal rDNA sites with few repetitions, as the small heterochromatic block made detection and photographic documentation unviable for the epifluorescence microscope [30].
Molecular cytogenetics techniques have been widely used to localize specific in situ DNA sequences [31]. Genetically related species tend to have karyotypes with similar characteristics in terms of sequence localization and are useful in studies of plant systematics, taxonomy and evolution mainly contributing to groupings of species or cytotypes that share common characteristics, thus suggesting primitiveness or deactivation for a given cytological marker that is shared among a group of plants [32,33,34,35,36,37,38].
The hybridization sites of 45S rDNA probes in species of the Caesalpinia group have shown variations in both the number and the location of these sequences. The differences in the number of such sequences generally occur due to chromosomal rearrangements such as translocations, inversions, duplications and deletions [39], whereas variations in the signal intensity of hybridization are observed between sites with different numbers of rDNA replicates. Any changes in these sites’ patterns of distribution are levels of speciation; this may assist in determining how evolution has occurred within a group of taxonomically complex plants [31, 40]. Lower quantitative variation has been observed in the Poincianella genus, suggesting greater stability in the number of 45S rDNA sites (a total of eight). Conservation in the location of the rDNA genes (as revealed using FISH) was observed for species from the genus Trifolium (Leguminosae: Papilionoideae), which may indicate that some Leguminosae have great stability in this region [41]. This stability, which is based on the number and location of a chromosomal marker, is a good characteristic for species identification and delimitation through karyotype analysis.
The species in this study showed significant variations in 2C DNA and, consequently, in the size of the genomes for the evaluated species and genera. In addition, the low coefficient of variation among the replicates indicates the precision of the sample, as analyzed using flow cytometry. The variation in the amount of DNA across species can be attributed to the loss or gain of DNA sequences, which usually consist of repetitive DNA; this may occur due to evolutionary changes in accumulation and/or loss of repeating monomers in the micro and macro environments during the species’ evolution [42, 43]. This suggests that such losses or additions to the genome become stabilized during microevolution and selection [43].
In this work, only Ca. pulcherrima had a lower estimated amount of 2C DNA (1.63 pg) than its previous estimate (1.80 pg) [16]. This may be due to the variation in number of chromosomes for the two analyzed populations of Ca. pulcherrima, as the population evaluated in this study presented 2n = 24, but the population evaluated by OHRI et al. [16] presented 2n = 28. The estimated DNA nuclear content for diploid Ca. crista (2n = 24) indicated a too-high value of 0.707 pg per chromosome, which leads to 17.67 pg for the 2C DNA. This value, which is much higher than our result, can be attributed to the different cytophotometric methods, as Allium cepa L.’s DNA value was computed as a DNA size pattern [17]. The 2C value that we found for Ca. crista was also considerably higher than those that OHRI et al. [16] found for the species in the genus Caesalpinioideae. Analyzing the DNA content and chromosomal differences observed for taxa from the Caesalpinia genus, together with the results from the literature, requires an interdisciplinary mode in order to indicate the species’ taxonomy, delimitation and clustering.
Many species in the Caesalpinia group exhibit a high degree of phenotypic plasticity, especially in foliage and leaflets. This has resulted in multiple nomenclature for the species, with each leaf-size variant having a specific condition, thus resulting in taxonomic problems [1, 5, 7, 9]. This fact can be observed in Pa. echinata, which was previously arranged in the Caesalpinia genus and which has three morphotypes that were previously characterized using chloroplast DNA sequences [44]. The three morphotypes (leaf-size variants) presented small variations in 2C DNA, with values of 2.76, 2.81 and 2.82 pg for the SV, MV and LV types, respectively. The Scott-Knott test separated these morphotypes into two groups, one with only Pa. echinata SV and one composed of Pa. echinata MV and LV; this shows that, although the variations among Pa. echinata leaf-size morphotypes are not large, the values are sufficient to separate the SV morphotype from the other two variants, with this type’s low DNA content acting as a differentiating feature.
In legume species, a positive correlation has been observed between leaf size and nuclear DNA content [45]. This relationship was also observed in this study, wherein the variants with the relatively large leaves (MV and LV) had more DNA than the SV variants. Therefore, diversification of genome size results from speciation, which, along with phenotypic changes in quantitative descriptors, is an adaptation response such as the ones observed in polyploid plants [46]. Thus, plants’ 2C DNA can be used to estimate the taxonomic differentiation between species, as seen here for the variants of Pa. echinata.
The data obtained in our studies corroborate the new classification of the species that were initially placed in the Caesalpinia group [5, 9,10,11], showing that the species that had at least 1.87 pg of 2C DNA in this study should actually be grouped in the Poincianella genus. Among the species analyzed in the present study, the only representative of the Erythrostemon genus was E. calycina, which had the least amount of 2C DNA (1.54 pg) and which was also the only species to present five pairs of chromosomes with the presence of 45S rDNA sites; these were unique characteristics of this genus. In previous analyses, similarities in chromosome morphology have been reported for the six species studied herein, and karyotype formulas have shown the predominance of metacentric chromosomes [47].
Only one species was evaluated for the Cenostigma genus, C. macrophyllum; this species which was initially collected under the belief that it belonged to the Caesalpinia genus. The similarities between species of these two genera have also been visualized previously, as the species Cenostigma sclerophyllum Tul. was later described as a synonym for Caesalpinia marginata Tul. [8, 48]. The analyses of C. macrophyllum enabled us to observe that it was the only species to present only six CMA3+/DAPI− bands, showing that the amount of GC-rich heterochromatin was lower than that of the other species evaluated herein; this could be a feature exclusive to the Cenostigma genus.
In this work, the distribution pattern for heterochromatin, the physical location of 45S rDNA regions and the amount of DNA were all useful to corroborate studies of systematics and of evolution in Caesalpinia-group species. Although the quantity of species evaluated herein is only a small fraction of the diversity already described as belonging to the Caesalpinia group, and although some species were relocated within new genera, it was possible to observe a distinctive pattern for individual cytogenetic characteristics in the genera currently specified as Poncianella. This shows that the karyotypic analysis and the quantification of 2C DNA are valid methods to support taxonomic and biosystematics studies.