Molecular genetics of Rhabdomys pumilio subspecies boundaries: mtDNA phylogeography and karyotypic analysis by fluorescence in situ hybridization

The phylogeography of the African four-striped mouse, Rhabdomys pumilio, was investigated using complete sequences of the mtDNA cytochrome b gene (1140 bp) and a combination of fluorescence in situ hybridization (FISH) and conventional cytogenetic banding techniques (G- and C-banding). Two cytotypes (2n=46 and 2n=48) were identified by cytogenetic analysis. There is no evidence of diploid number variation within populations, difference in gross chromosome morphology or of subtle interchromosomal rearrangements at levels detected by ZOO-FISH. Analysis of the mtDNA cytochrome b resulted in two major lineages that correspond roughly to the xeric and mesic biotic zones of southern Africa. One mtDNA clade comprises specimens with 2n=48 and the other representatives of two cytotypes (2n=48 and 2n=46). The mean sequence divergence (12%, range 8.3–15.6%) separating the two mtDNA clades is comparable to among-species variation within murid genera suggesting their recognition as distinct species, the prior names for which would be R. dilectus and R. pumilio. Low sequence divergences and the diploid number dichotomy within the mesic lineage support the recognition of two subspecies corresponding to R. d. dilectus (2n=46) and R. d. chakae (2n=48). Our data do not support subspecific delimitation within the nominate, R. pumilio. Molecular dating places cladogenesis of the two putative species at less than five million years, a period characterised by extensive climatic oscillations which are thought to have resulted in habitat fragmentation throughout much of the species range.     

Biome specificity of distinct genetic lineages within the four-striped mouse Rhabdomys pumilio (Rodentia: Muridae) from southern Africa with implications for taxonomy

Within southern Africa, a link between past climatic changes and faunal diversification has been hypothesized for a diversity of taxa. To test the hypothesis that evolutionary divergences may be correlated to vegetation changes (induced by changes in climate), we selected the widely distributed four-striped mouse, Rhabdomys, as a model. Two species are currently recognized, the mesic-adapted R. dilectus and arid-adapted R. pumilio. However, the morphology-based taxonomy and the distribution boundaries of previously described subspecies remain poorly defined. The current study, which spans seven biomes, focuses on the spatial genetic structure of the arid-adapted R. pumilio (521 specimens from 31 localities), but also includes limited sampling of the mesic-adapted R. dilectus (33 specimens from 10 localities) to act as a reference for interspecific variation within the genus. The mitochondrial COI gene and four nuclear introns (Eef1a1, MGF, SPTBN1, Bfib7) were used for the construction of gene trees. Mitochondrial DNA analyses indicate that Rhabdomys consists of four reciprocally monophyletic, geographically structured clades, with three distinct lineages present within the arid-adapted R. pumilio. These monophyletic lineages differ by at least 7.9% (±0.3) and these results are partly confirmed by a multilocus network of the combined nuclear intron dataset. Ecological niche modeling in MaxEnt supports a strong correlation between regional biomes and the distribution of distinct evolutionary lineages of Rhabdomys. A Bayesian relaxed molecular clock suggests that the geographic clades diverged between 3.09 and 4.30Ma, supporting the hypothesis that the radiation within the genus coincides with paleoclimatic changes (and the establishment of the biomes) characterizing the Miocene-Pliocene boundary. Marked genetic divergence at the mitochondrial DNA level, coupled with strong nuclear and mtDNA signals of non-monophyly of R. pumilio, support the notion that a taxonomic revision of the genus is needed.     

Rapid chromosomal evolution in the mesic four‐striped grass rat Rhabdomys dilectus (Rodentia,Muridae) revealed by mtDNA phylogeographic analysis

The mesic four‐striped grass rat Rhabdomys dilectus De Winton, 1897 is distributed in mesic regions of southern and eastern Africa. We carried out a molecular and chromosomal study of the northernmost populations of the species to provide insight into the subspecific boundaries identified within the species and to describe its genetic structure in Eastern Africa. Maximum likelihood, maximum parsimony and neighbour‐joining methods were used to construct phylogenetic relationships among all the haplotypes belonging to the large part of the species range. Times of divergences were estimated assuming a relaxed molecular clock with two calibration points. We identified three well‐supported clades within R. dilectus. One basal clade corresponding to Rhabdomys d. chakae (2n = 48) is found in South Africa. Two additional sister clades corresponding to R. d. dilectus (2n = 48 and 2n = 46) are allopatrically distributed in southern and northern parts of the species range. Genetic divergence among the three clades is relatively high (ranges 4.2–5.7%). A very divergent new karyotype 2n = 38, FNa = 60 was found in two high‐altitude populations on Mt. Meru and Mt. Kilimanjaro. The karyotype differences consist in three Robertsonian fusions and one whole‐arm reciprocal translocation. Interestingly, the mtDNA phylogeny does not match with the diploid numbers. In fact, the 2n = 38 specimens form a monophyletic group within a clade that includes specimens with the 2n = 46 karyotype that appears as paraphyletic. We estimated the new karyotype originated in peripatric condition during the last phases of the Pleistocene. This study confirms the importance of chromosomal analysis in detecting taxonomic units and cryptic diversity in rodents.     

Chromosome polymorphism in Astyanax fasciatus (Teleostei, Characidae). 1. Karyotype analysis, Ag-NORs and mapping of the 18S and 5S ribosomal genes in sympatric karyotypes and their possible hybrid forms

Astyanax fasciatus may be characterized as a chromosomally diversified 'species' presenting distinct cytotypes, each with its specific variants. The sympatric and syntopic occurrence of different cytotypes reinforces the hypothesis in which A. fasciatus may represent a group of species currently placed under a single common designation. Specimens from three collection points spread along the Mogi-Gua?u River in southeast Brazil were examined in the present work: (1) near its headwaters (Ouro Fino--MG), (2) in the middle region of the river (Cachoeira de Emas, Pirassunun ga--SP) and (3) close to its confluence with the Pardo River (Barrinha--SP). The 2n = 48 chromosomes cytotype was found in all sampling points, while cytotype 2n = 46 was only encountered in Barrinha and Cachoeira de Emas. In the latter locality, cytotype 2n = 46 predominated; nevertheless, other karyotype forms with 2n = 45 and 47 chromosomes also occurred, besides a structural variant of cytotype 2n = 46. One specimen with 2n = 47 chromosomes was also found in Ouro Fino. The Ag-NOR analysis, as well as the location of the 18S and 5S ribosomal genes, were conserved in all cytotypes. The data indicate that the variant karyotypes are a consequence of interbreeding between the standard cytotypes (2n = 46 and 48) and/or its descendants. This suggests a karyotype plasticity for this species, where at least a few variant karyotypes would not have deleterious effects on their bearers.     

Comparative chromosome analysis (R- and C-bands) of two South African murid species, Lemniscomys rosalia and Rhabdomys pumilio (Rodentia, murinae)

We carried out a comparative chromosome analysis (R- and C-bands) on two South African murid rodent species, Lemniscomys rosalia and Rhabdomys pumilio, whose banded karyotypes are reported here for the first time. The study revealed that, in spite of minor differences in diploid number and the number of autosomal arms (48 vs. 46 and 58 vs. 60, respectively), these species differ by at least 10 structural rearrangements, comprising seven Robertsonian translocations (two of which share monobrachial homology), two tandem translocations, and one pericentric inversion. Despite the high level of chromosomal differentiation observed, almost complete homologous banding patterns were detected between the two species. The present study therefore strengthens the hypothesis about the close phylogenetic affinities between L. rosalia and R. pumilio and their belonging to a set of genera referred to as "arvicanthine" rodents.     

Geographical variations in heat production and dissipation within two populations of Rhabdomys pumilio (Muridae)

A comparative study of heat production and dissipation was carried out on Rhabdomys pumilio from two extremely different climatic zones. Our results show higher oxygen consumption (Vo2) values for the desert population at Ta = 5 degrees C when compared to the mesic population. The overall thermal conductance is also higher for the desert population. It seems as though penetration into mesic areas of R. pumilio, which originated in an arid zone, was accompanied by an increase in body mass.     

Comparative Study of the Ploidy Series Bromus sterilis, B. diandrus and B. rigidus (Poaceae) Based on Chromosome Numbers, Morphology and Isozymes

Abstract: Chromosome numbers, morphological characters and isozymes of seven enzymes were studied to assess relationships between species of the Bromus diandrus-rigidus polyploid complex and the closely related species, B. sterilis. The four different cytotypes detected, 2n = 14, 28, 42 and 56, could be divided into two species: B. sterilis (2n = 14, 28) and B. diandrus (2n = 42, 56) by morphological features and isozymes. The shape of the scar of rachilla segments in the floret proved to be a suitable character for distinguishing the two species, but not the chromosomal races within species. The tetraploid shared homozygous isozyme phenotypes with diploid B. sterilis at all loci, except one, suggesting that it is autopolyploid. No diagnostic isozymes could be found to distinguish between hexa- and octoploid cytotypes in the B. diandrus-rigidus complex.     

Cytogenetics, geographical distribution, and pollen fertility of diploid and tetraploid cytotypes of Santolina pectinata Lag. (Asteraceae: Anthemideae)

A cytogenetic study of 62 populations of Santolina pectinata in Spain shows the existence of two ploidy levels. The diploid cytotypes with 2 n  = 18 occupy the eastern Betic mountains, and the tetraploid cytotypes with 2 n  = 36 are located on the spurs of the Iberian System. The former show a much wider ecological spectrum than the latter. Mixed cytotypes were observed in two diploid populations, with one tetraploid in each, showing different karyotypes. Three trisomic individuals were detected, one in a diploid population and the other two in a tetraploid population. Also, three hypotetraploid individuals were detected in a tetraploid population. Polyploidy is shown to be spontaneous and recurrent, promoting partial sterility in the pollen. Structural chromosomal changes, principally translocations, and local speciation through autopolyploidy are the principal factors in the evolution and diversification of this species.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 657–667.     

Cytotype segregation on regional and microgeographic scales in snow buttercups (Ranunculus adoneus: Ranunculaceae)

Polyploid speciation is an important source of angiosperm diversity. Insights into the origin and establishment of new polyploid species may be gained by studying the distributions of ancestral and derivative cytotypes at multiple spatial scales. Diploid (2n = 16) and tetraploid (2n = 32) snow buttercups (Ranunculus adoneus: Ranunculaceae) occur in the alpine of the central and southern Rocky Mountains. Root-tip squashes and flow cytometry were used to determine the ploidy of 1618 individuals from 35 populations. Samples from 31 of the 35 sites were entirely of one cytotype, either diploid or tetraploid. Diploid and tetraploid snow buttercups have nonoverlapping regional distributions. Where both cytotypes occur on the same site, the two are spatially segregated despite no apparent change in habitat. Triploid snow buttercups were only found at a diploid/tetraploid contact zone, while two hexaploid plants were found in tetraploid populations. Tetraploid establishment once or twice in the history of the species complex could account for the regional distribution of the two cytotypes. Habitat differentiation between cytotypes or reproductive exclusion of minority cytotypes may explain the observed segregation at both microgeographic and regional scales.     

Sponge cytogenetics - mitotic chromosomes of ten species of freshwater sponge

Porifera (sponges) are the most basal phylum of extant metazoans. To gain insight into sponge genome construction, cytogenetic analysis was performed for ten freshwater sponge species of six genera, using conventional Giemsa staining, chromosome banding, and fluorescence in-situ hybridization. The karyotypes were very similar among the ten species, exhibiting a diploid chromosome number of 2n=46 or 48, and usually consisted of microchromosomes with one or two pairs of large chromosomes. The 18S-28S rRNA genes were localized to a single pair of microchromosomes in two Ephydatia species. Hybridization signals of the telomere (TTAGGG)n sequences were observed at the ends of metaphase chromosomes. The genome sizes of Ephydatia fluviatilis and Ephydatia muelleri were estimated by flow cytometric analysis as about 0.7 pg per diploid complement. These freshwater sponge species appear to represent a fairly homogeneous group with respect to karyotypes.     

Cytotypes of Kirk's dik-dik (Madoqua kirkii,Bovidae) show multiple tandem fusions

Madoqua kirkii, a miniature African antelope, is noted for extensive chromosomal variation that has been categorized in four distinct cytotypes (A-D). In this investigation, we analyzed the A cytotype (2n = 46, FN = 48) using a suite of molecular cytogenetic approaches that entailed (i) whole chromosome and subchromosomal painting by fluorescence in situ hybridization (FISH), (ii) the study of Madoqua centromeric-specific DNA derived from pooled DNA obtained from the centromeric regions of the acrocentric chromosomes, and (iii) DNA from the telomere:centromere junctions of tandemly fused chromosomes. DNA from these sources was used to probe for the persistence of interstitial satellite DNA and residual centromeric sequences in the tandem and centric fusion junctions by PCR and FISH. The analyses show centromeric sequences at two of the six tandem fusion junctions. These data, and those of hybrid specimens (A × B cytotypes) in conjunction with published information permitted an interpretation of the probable sequence of chromosomal rearrangements among the M. kirkii cytotypes. We discuss the findings in the context of chromosomal evolution in these antelopes, and the implications that these hold for ex-situ breeding programs of the species.     

Mitochondrial DNA polymorphisms in subterranean mole-rats of the Spalax ehrenbergi superspecies in Israel, and its peripheral isolates

Patterns of mitochondrial DNA (mtDNA) variation were examined in 133 mole-rats constituting all four chromosomal species (2n = 52, 2n = 54, 2n = 58, and 2n = 60) of the Spalax ehrenbergi superspecies in Israel, as well as the peripheral isolates of 2n = 60. In the main range of the complex, a total of 28 mtDNA haplotypes were found in 64 mole-rats, with most haplotypes being unique to either a single chromosomal species or population. mtDNA divergence increased from low to high diploid number in a north-to-south direction in Israel. Overall levels of mtDNA diversity were unexpectedly the highest in the 2n = 60, the youngest species of the complex. The mtDNA haplotypes can be separated into two major groups, 2n = 52-54 and 2n = 58-60, and a phylogenetic analysis for each group revealed evidence of a few haplotypes not sorted by diploid number. The overall patterns of mtDNA divergence seen within and among the four chromosomal species are consistent with the parapatric mode of speciation as suggested from previous studies of allozyme and DNA hybridization. In a separate data set the patterns of mtDNA variation were examined across the main geographic range and across peripheral semi-isolates and isolates of the 2n = 60 chromosomal species. Fifteen haplotypes were found in 69 mole-rats. High levels of mtDNA diversity characterized the main range, semi-isolated, and even some desert isolated populations. The peripheral isolates contain much mtDNA diversity, including novel haplotypes.      

Cytogenetic characterization of six species of flatfishes with comments to karyotype differentiation patterns in Pleuronectiformes (Teleostei)

The karyotypes and cytogenetic characteristics of flatfishes species Paralichthys orbignyanus , Paralichthys patagonicus , Citarichthys spilopterus and Etropus crossotus (Paralichthyidae), Bothus ocellatus (Bothidae) and Symphurus tessellatus (Cynoglossidae) were investigated by conventional [Giemsa staining, C-banding, Ag- and chromomycin (CMA₃)-stainings] and molecular [ in situ hybridization (ISH)] cytogenetic techniques. The results showed 2n = 46 and FN = 48 (2msm + 46sta) in P. orbignyanus , 2n = 46 and FN = 46 (46sta) in P. patagonicus , 2n = 26 and FN = 44 (18msm + 8sta) in C. spilopterus , 2n = 38 and FN = 64 (26msm + 12sta) in E. crossotus , 2n = 32 and FN = 50 (18msm + 14sta) in B. ocellatus , and 2n = 46 and FN = 62 (46msm + 62sta) in S. tessellatus . All species exhibited weak C-band positive segments in terminal and centromeric positions of some chromosome pairs. Silver staining of the nucleolus organizer regions (Ag-NOR) technique showed a single Ag-NOR-bearing chromosome pair in all species except E. crossotus . All these sites were CMA₃ positive and showed clear ISH signals after probing with a 18S rRNA probe. Etropus crossotus presented until seven chromosomes with Ag-NORs and CMA₃ positively stained segments in five chromosome pairs. Conversely only one chromosome pair was identified with the ISH experiments in this species. The available results show that the fishes of the order Pleuronectiformes experienced a marked chromosome evolution that included reduction in diploid number, mainly due to Robertsonian rearrangements, and several chromosome inversions.     

Distribution and ecology of cytotypes of the Aster amellus aggregates in the Czech Republic

BACKGROUND AND AIMS: Polyploidy is viewed as an important mechanism of sympatric speciation, but only a few studies have documented patterns of distribution and ecology of different cytotypes in their contact zone. Aster amellus agg. (Asteraceae) is one of the species with documented multiple ploidy levels. The aim of this study was to determine spatial distribution and ecology of two cytotypes, diploid (2n = 18) and hexaploid (2n = 54), of Aster amellus agg. at their contact zone in the Czech Republic. METHODS: Root-tip squashes and flow cytometry were used to determine the ploidy of 2175 individuals from 87 populations. To test whether some differences in ecology between the two ploidy levels exist, in each locality relevés were recorded and abiotic conditions of the sites were studied by estimating potential direct solar radiation, Ellenberg indicator values and above-ground biomass. KEY RESULTS: Together with diploid and hexaploids, minorite cytotypes (triploid, pentaploid and nonaploid) were found. No significant ecological differences between diploid and hexaploid cytotypes were found. In spite of this, no population consisting of both of the two basic cytotypes was found. CONCLUSIONS: The results of this study show that the contact zone of diploid and hexaploid cytotypes in the Czech Republic is much more diffuse than indicated in previous records. Although populations of both cytotypes occur in close proximity (the closest populations of different cytotypes were 500 m apart), each individual population consists of only one basic ploidy level. This was unexpected since there are no clear differences in abiotic conditions between populations. Taken together with the absence of an intermediate tetraploid cytotype and with reference to published world distributional patterns of different ploidy levels, this suggests a secondary contact zone. Detailed genetic study is, however, necessary to confirm this.     

The species complex Astyanax fasciatus Cuvier (Teleostei, Characiformes) – a multidisciplinary approach

Cytogenetic data have provided important clues that the Astyanax fasciatus populations from the Upper Paraná River basin could be a part of a more diverse fish group, usually included on the same taxa. Samples collected in Cachoeira de Emas, SP, in Mogi-Guaçu River basin, show two major cytotypes presenting 2 n = 46 and 2 n = 48 chromosomes, with distinct karyotypic formula, despite the fact that the molecular data suggested some degree of gene flow between these cytotypes. Cytogenetic and morphometric analyses were performed in this species, aiming to contribute to the understanding of the natural history from such fish group. Two allopatric populations with distinct standard cytotypes were analysed, and the data obtained suggest the separation into two groups.     

Evidence of environmental niche differentiation in the striped mouse (Rhabdomys sp.): inference from its current distribution in southern Africa

The aim of this study was to characterize environmental differentiation of lineages within Rhabdomys and provide hypotheses regarding potential areas of contact between them in the Southern African subregion, including the Republic of South Africa, Lesotho, and Namibia. Records of Rhabdomys taxa across the study region were compiled and georeferenced from the literature, museum records, and field expeditions. Presence records were summarized within a 10 × 10 km grid covering the study area. Environmental information regarding climate, topography, land use, and vegetation productivity was gathered at the same resolution. Multivariate statistics were used to characterize the current environmental niche and distribution of the whole genus as well as of three mitochondrial lineages known to occur in southern Africa. Distribution modeling was carried out using MAXENT in order to generate hypotheses regarding current distribution of each taxa and their potential contact zones. Results indicate that the two species within Rhabdomys appear to have differentiated across the precipitation/temperature gradient present in the region from east to west. R. dilectus occupies the wettest areas in eastern southern Africa, while R. pumilio occupies the warmer and drier regions in the west, but also penetrates in the more mesic central part of the region. We provide further evidence of environmental differentiation within two lineages of R. dilectus. Contact zones between lineages appear to occur in areas of strong environmental gradients and topographic complexity, such as the transition zones between major biomes and the escarpment area where a sharp altitudinal gradient separates coastal and plateau areas, but also within more homogeneous areas such as within grassland and savannah biomes. Our results indicate that Rhabdomys may be more specialized than previously thought when considering current knowledge regarding mitochondrial lineages. The genus appears to have differentiated along two major environmental axes in the study region, but results also suggest dispersal limitations and biological interactions having a role in limiting current distribution boundaries. Furthermore, the projection of the potential geographic distribution of the different lineages suggests several contact zones that may be interesting study fields for understanding the interplay between ecological and evolutionary processes during speciation.     

Unusual chromosome numbers in Paspalum L. (Poaceae: Paniceae) from Brazil

Somatic chromosome numbers were determined for 20 new germplasm accessions of Paspalum, belonging to 17 species collected in Brazil. Chromosome number is reported for the first time for P. reduncum (2n = 18), P. cinerascens (2n = 20), P. cordatum (2n = 20), P. filgueirasii (2n = 24), P. ammodes (2n = 36), P. bicilium (2n = 40), P. heterotrichon (2n = 40), and P. burmanii (2n = 48). New cytotypes were confirmed for two germplasm accessions of P. carinatum (2n = 30) and P. trachycoleon (2n = 36), one of P. clavuliferum (2n = 40) and one of P. lanciflorum (2n = 40), indicating variability in these species. The remaining chromosome numbers reported here confirm previous counts. The unexpected chromosome numbers 2n = 18, 24, 36, and 48 in Paspalum species, which are usually shown to be multiples of 10, suggest that much more collection and cytogenetic characterization are necessary to assess the whole chromosomal and genomic multiplicity present in the genus, which seems to be much more diverse than currently thought to be.     

Chromosome evolution by robertsonian translocation in Gibasis (Commelinaceae)

The plant species Gibasis schiedeana (Kunth) D. R. Hunt sens. lat. contains two cytotypes viz. a self-sterile diploid with 2n=10 (x=5) and a selffertile cytological autotetraploid with 2n=16 (x=4). Single chromosome sets of these plants consist of 2 metacentrics +3 acrocentrics, and 3 metacentrics +1 acrocentric chromosomes respectively suggesting a Robertsonian relationship between them. Their artificial F₁ hybrids show the pairing of acrocentrics with metacentric arms confirming the supposed nature of the chromosome affinities. Both breeding systems and ploidy levels show that the direction of the change has been from x=5 to x=4 by a translocation of the Robertsonian type.     

The chromosome banding pattern in two cytotypes (2n = 36 and 38) of blind mole rats from Turkey (Mammalia: Spalaxidae)

Two cytotypes (2n = 36 and 38) of blind mole rats, Nannospalax xanthodon (Nordmann, 1840), from the Ayd?n and Manisa provinces in Turkey were investigated. Conventional chromosome staining, Ag-NOR staining and C-banding analysis were carried out. From the cytogenetic point of view, the particular phylogenetic position of these populations is supported by their low diploid numbers only, and the Cbanding pattern and the NORs distribution seem generally similar to populations with higher chromosome numbers. Several autosomal pairs with centromeric dark Cbands were observed in the 2n=36 cytotype. One autosomal pair possessed an interstitial dark C-band on the short arm; another pair possessed an interstitial dark Cband on the long arm. Whole C-heterochromatic short arms were observed in three subtelocentric autosomal pairs in the 2n=38 cytotype. Most of the other autosomal pairs possessed centromeric dark C-bands. Distinct dark C-bands were observed also in the presumed X chromosomes of both the cytotypes. The Ag-NOR regions were found on three autosomal pairs of both the cytotypes. These sites were located in telomeric areas of the short arms of two subtelocentric and one submetacentric pair.     

Genomic relationships among Nicotiana species with different ploidy levels revealed by 5S rDNA spacer sequences and FISH/GISH

We used the intergenic spacer sequences of the 5S ribosomal RNA genes (5S rDNA) to obtain insights into the genomic origin of putative amphidiploid/tetraploid species with 2n = 48 and their descendants in Nicotiana. Amplification of the spacer sequences and subsequent multiple alignment using the consensus sequences from each species, showed that two Australian species shared common large deletions, suggesting that the origin of the 5S rDNA is closely related in these species. Comparison of the spacer sequences with those from diploid (2n = 24) Nicotiana species made it possible to detect some groups consisting of the sequences from the 2n = 24 and 2n = 48 level species. Chromosomal localizations of the 5S rDNA arrays were similar in most groups. The relationships suggested by the 5S rDNA were also assessed at the genome level by using genomic in situ hybridization. We showed that the grouping based on the 5S rDNA spacer sequence reflects high genomic homology between 2n = 24 and 2n = 48 level species. As a result, the putative polyploid species such as N. debneyi, N. quadrivalvis, and N. africana were suggested to involve the close relatives of the diploid species such as N. glauca, N. obtusifolia and N. sylvestris, and N. langsdorffii, respectively, in their speciation. Our results are generally in agreement with the relationships previously suggested by morphological and cytogenetic observations, and some novel relationships were also revealed.