Ecological strategies in the ancient asexual animal group Darwinulidae (Crustacea, Ostracoda)

• 1 We investigated the relationship between geographical distribution and ecological tolerance within the ancient asexual family Darwinulidae. Distribution maps were compiled based on data from the literature, the Non‐marine Ostracod Distribution in Europe database and personal collections. Ecological tolerance was assessed experimentally by exposing individual ostracods to a combination of eight different salinities (range from 0 to 30 g L^?1) and three different temperatures (10, 20 and 30 °C).
• 2 The type species of the family, Darwinula stevensoni, is ubiquitous and cosmopolitan; the two species Penthesilenula brasiliensis and Microdarwinula zimmeri also have an intercontinental distribution. Two other darwinulid species tested here (Vestalenula molopoensis and P. aotearoa) are known only from their type localities. The latter is also true for most extant darwinulids.
• 3 Darwinula stevensoni and P. brasiliensis had a broad salinity tolerance, tolerating distilled water and also salinity up to 25–30 g L^?1, whereas the maximum salinity tolerance of V. molopoensis was 12 g L^?1 and of P. aotearoa, 20 g L^?1.
• 4 The results indicate that both ecological specialists and generalists, as well as intermediate forms, exist in the Darwinulidae and that taxa with the broadest ecological tolerance also have the widest distribution.

     

Slow molecular evolution in an ancient asexual ostracod

Genetic variability of the non-marine ostracod species Darwinula stevensoni was estimated by sequencing part of the nuclear and the mitochondrial genome. As Darwinulidae are believed to be ancient asexuals, accumulation of mutations should have occurred, both between alleles within lineages and between lineages, during the millions of years of parthenogenetic reproduction. However, our sequence data show the opposite: no variability in the nuclear ITS1 region was observed within or among individuals of D. stevensoni, despite sampling a geographical range from Finland to South Africa. Lack of allelic divergence might be explained by concerted evolution of rDNA repeats. Homogeneity among individuals may be caused either by slow molecular evolution in ITS1 or by a recent selective sweep. Variability of mitochondrial cytochrome oxidase (COI) was similar to intraspecific levels in other invertebrates, thus weakening the latter hypothesis. Calibrating interspecific, genetic divergences among D. stevensoni and other Darwinulidae using their fossil record enabled us to estimate rates of molecular evolution. Both COI and ITS1 evolve half as fast, at most, in darwinulids as in other invertebrates, and molecular evolution has significantly slowed down in ITS1 of D. stevensoni relative to other darwinulids. A reduced ITS1 mutation rate might explain this inconsistency between nuclear and mitochondrial evolution in D. stevensoni.     

A general purpose genotype in an ancient asexual

Many parthenogenetic species are geographically more widely distributed than their sexual relatives. Their success has been partly attributed to the existence of general purpose genotypes (GPGs). Darwinula stevensoni is an ostracod species, which has persisted for >25 million years without sex, and is both ubiquitous and cosmopolitan. Here, we test the hypothesis that this ancient asexual species may possess a highly generalised (or general purpose) genotype. The ecological tolerance of D. stevensoni was compared with asexual populations of Heterocypris incongruens, a common cypridinid species with mixed reproduction, as well as with that of another ancient asexual darwinulid species with a limited geographic and ecological distribution, Vestalenula molopoensis. The unusually wide tolerance range for both salinity (0-30 g/l) and temperature (10°C, 20°C and 30°C) of the freshwater species D. stevensoni, supports the hypothesis that this ancient asexual has indeed developed a GPG. This coincides with its wide geographic and ecological distribution and might explain its persistence as an obligate asexual in its long-term evolution. The more restricted salinity tolerance of V. molopoensis (maximum at 12 g/l) shows that not all species of the ancient asexual family Darwinulidae have a GPG. D. stevensoni has a much broader tolerance than the asexuals of H. incongruens. We argue why a GPG is most likely to develop in long-term asexuals.     

Exceptional cryptic diversity and multiple origins of parthenogenesis in a freshwater ostracod

The persistence of asexual reproduction in many taxa depends on a balance between the origin of new asexual lineages and the extinction of old ones. This turnover determines the diversity of extant asexual populations and so influences the interaction between sexual and asexual modes of reproduction. Species with mixed reproduction, like the freshwater ostracod (Crustacea) morphospecies Eucypris virens, are a good model to examine these dynamics. This species is also a geographic parthenogen, in which sexual females and males co-exist with asexual females in the circum-Mediterranean area only, whereas asexual females occur all over Europe. A molecular phylogeny of E. virens based on the mitochondrial COI and 16S fragments is presented. It is characterised by many distinct clusters of haplotypes which are either exclusively sexual or asexual, with only one exception, and are often separated by deep branches. Analysis of the phylogeny reveals an astonishing cryptic diversity, which indicates the existence of a species complex with more than 40 cryptic taxa. We therefore suggest a revision of the single species status of E. virens. The phylogeny indicates multiple transitions from diverse sexual ancestor populations to asexuality. Although many transitions appear to be ancient, we argue that this may be an artefact of the existence of unsampled or extinct sexual lineages.     

Evaluating the Ribosomal Internal Transcribed Spacer (ITS) as a Candidate Dinoflagellate Barcode Marker

Background

DNA barcoding offers an efficient way to determine species identification and to measure biodiversity. For dinoflagellates, an ancient alveolate group of about 2000 described extant species, DNA barcoding studies have revealed large amounts of unrecognized species diversity, most of which is not represented in culture collections. To date, two mitochondrial gene markers, Cytochrome Oxidase I (COI) and Cytochrome b oxidase (COB), have been used to assess DNA barcoding in dinoflagellates, and both failed to amplify all taxa and suffered from low resolution. Nevertheless, both genes yielded many examples of morphospecies showing cryptic speciation and morphologically distinct named species being genetically similar, highlighting the need for a common marker. For example, a large number of cultured Symbiodinium strains have neither taxonomic identification, nor a common measure of diversity that can be used to compare this genus to other dinoflagellates.

Methodology/Principal Findings

The purpose of this study was to evaluate the Internal Transcribed Spacer units 1 and 2 (ITS) of the rDNA operon, as a high resolution marker for distinguishing species dinoflagellates in culture. In our study, from 78 different species, the ITS barcode clearly differentiated species from genera and could identify 96% of strains to a known species or sub-genus grouping. 8.3% showed evidence of being cryptic species. A quarter of strains identified had no previous species identification. The greatest levels of hidden biodiversity came from Scrippsiella and the Pfiesteriaceae family, whilst Heterocapsa strains showed a high level of mismatch to their given species name.

Conclusions/Significance

The ITS marker was successful in confirming species, revealing hidden diversity in culture collections. This marker, however, may have limited use for environmental barcoding due to paralogues, the potential for unidentifiable chimaeras and priming across taxa. In these cases ITS would serve well in combination with other markers or for specific taxon studies.     

Cryptic species in tropic sands--interactive 3D anatomy, molecular phylogeny and evolution of meiofaunal Pseudunelidae (Gastropoda, Acochlidia)

Background

Towards realistic estimations of the diversity of marine animals, tiny meiofaunal species usually are underrepresented. Since the biological species concept is hardly applicable on exotic and elusive animals, it is even more important to apply a morphospecies concept on the best level of information possible, using accurate and efficient methodology such as 3D modelling from histological sections. Molecular approaches such as sequence analyses may reveal further, cryptic species. This is the first case study on meiofaunal gastropods to test diversity estimations from traditional taxonomy against results from modern microanatomical methodology and molecular systematics.

Results

The examined meiofaunal Pseudunela specimens from several Indo-Pacific islands cannot be distinguished by external features. Their 3D microanatomy shows differences in the organ systems and allows for taxonomic separation in some cases. Additional molecular analyses based on partial mitochondrial cytochrome c oxidase subunit I (COI) and 16S rRNA markers revealed considerable genetic structure that is largely congruent with anatomical or geographical patterns. Two new species (Pseudunela viatoris and P. marteli spp. nov.) are formally described integrating morphological and genetic analyses. Phylogenetic analysis using partial 16S rRNA, COI and the nuclear 18S rRNA markers shows a clade of Pseudunelidae species as the sister group to limnic Acochlidiidae. Within Pseudunela, two subtypes of complex excretory systems occur. A complex kidney already evolved in the ancestor of Hedylopsacea. Several habitat shifts occurred during hedylopsacean evolution.

Conclusions

Cryptic species occur in tropical meiofaunal Pseudunela gastropods, and likely in other meiofaunal groups with poor dispersal abilities, boosting current diversity estimations. Only a combined 3D microanatomical and molecular approach revealed actual species diversity within Pseudunela reliably. Such integrative methods are recommended for all taxonomic approaches and biodiversity surveys on soft-bodied and small-sized invertebrates. With increasing taxon sampling and details studied, the evolution of acochlidian panpulmonates is even more complex than expected.     

Did Pleistocene glaciations shape genetic patterns of European ostracods? A phylogeographic analysis of two species with asexual reproduction

Nested clade analyses (NCA) and likelihood mapping were applied to DNA sequence data of the ribosomal ITS1 and mitochondrial COI region from two non-marine ostracod species. The aim was to test whether Pleistocene glaciations may have shaped genetic and geographic patterns. According to the results from both types of analyses, evidence was lacking for any kind of geographic grouping of European (and one African) population from the putatively ancient asexual ostracod, Darwinula stevensoni. This counters the possibility that a recent selective sweep could have caused the low intraspecific, genetic diversity observed in this species. One of the most cited hypotheses to explain geographic parthenogenesis invokes faster, postglacial colonization by asexual lineages. However, no evidence for northern ‘range expansion’ of asexual haplotypes was found for Eucypris virens, a species with geographic parthenogenesis. Rather, the outcome of the NCA reveals that phylogeographic relationships are characterized by ‘restricted dispersal with isolation by (geographic) distance’. This result suggests that either no faster, postglacial range expansion of asexuals occurred in E. virens or, that patterns of subsequent colonization became ‘overwritten’ by more recent dispersal events. Likelihood mapping provides evidence for the second scenario because genetic instead of geographic clustering was statistically supported. Handling editor: K. Martens     

The ontogeny of two species of Darwinuloidea (Ostracoda, Crustacea)

The ontogeny of two species of the ostracod superfamily Darwinuloidea, Darwinula stevensoni (Brady and Robertson, 1870) and Vestalenula sp., is documented. The development of the appendages of the two species is very similar, with the exception of the antennule, which shows some variation. The general appearance of appendages during ontogeny of the two species is very similar to that of species of other podocopid superfamilies, such as the Cytheroidea, Terrestricytheroidea, Bairdioidea and Cypridoidea, and different from that of the Platycopida. Using the development of the antenna and the appearance of antennal features, a revised model of darwinulid antennal terminology is presented that homologizes these features with those of the Cypridoidea.     

Evolution in the slow lane: molecular rates of evolution in sexual and asexual ostracods (Crustacea: Ostracoda)

Parthenogenetic lineages within non-marine ostracods can occur either in mixed (with sexual and asexual females) or exclusively asexual taxa. The former mode of reproduction is associated with a high intraspecific diversity at all levels (genetic, morphological, ecological) and, at least in the Cypridoidea, with geographical parthenogenesis. Obligate asexuality is restricted to the Darwinuloidea, the strongest candidate for an ancient asexual animal group after the bdelloid rotifers, and is characterized by low diversity. We have compared rates of molecular evolution for the nuclear ITS1 region and the mitochondrial COI gene amongst the three major lineages of non-marine ostracods with sexual, mixed and asexual reproduction. Absolute rates of molecular evolution are low for both regions in the darwinulids. The slow-down of evolution in ITS1 that has been observed for Darwinula stevensoni (Brady & Robertson) apparently does not occur in other darwinulid species. ITS1 evolves more slowly than COI within non-marine ostracod families, including the darwinulids, but not between superfamilies. The ancient asexuals might have a higher relative substitution rate in ITS1, as would be expected from hypotheses that predict the accumulation of mutations in asexuals. However, the speed-up of ITS could also be ancient, for example through the stochastic loss of most lineages within the superfamily after the Permian–Triassic mass extinction. In this case, the difference in rate would have occurred independently from any effects of asexual reproduction.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 79 , 93–100.     

Alternative oxidase mediates pathogen resistance in Paracoccidioides brasiliensis infection

Background

Paracoccidioides brasiliensis is a human thermal dimorphic pathogenic fungus. Survival of P. brasiliensis inside the host depends on the adaptation of this fungal pathogen to different conditions, namely oxidative stress imposed by immune cells.

Aims and Methodology

In this study, we evaluated the role of alternative oxidase (AOX), an enzyme involved in the intracellular redox balancing, during host-P. brasiliensis interaction. We generated a mitotically stable P. brasiliensis AOX (PbAOX) antisense RNA (aRNA) strain with a 70% reduction in gene expression. We evaluated the relevance of PbAOX during interaction of conidia and yeast cells with IFN-γ activated alveolar macrophages and in a mouse model of infection. Additionally, we determined the fungal cell''s viability and PbAOX in the presence of H₂O₂.

Results

Interaction with IFN-γ activated alveolar macrophages induced higher levels of PbAOX gene expression in PbWt conidia than PbWt yeast cells. PbAOX-aRNA conidia and yeast cells had decreased viability after interaction with macrophages. Moreover, in a mouse model of infection, we showed that absence of wild-type levels of PbAOX in P. brasiliensis results in a reduced fungal burden in lungs at weeks 8 and 24 post-challenge and an increased survival rate. In the presence of H₂O₂, we observed that PbWt yeast cells increased PbAOX expression and presented a higher viability in comparison with PbAOX-aRNA yeast cells.

Conclusions

These data further support the hypothesis that PbAOX is important in the fungal defense against oxidative stress imposed by immune cells and is relevant in the virulence of P. brasiliensis.     

Testing a short nuclear marker for inferring staphylinid beetle diversity in an African tropical rain forest

Background

The use of DNA based methods for assessing biodiversity has become increasingly common during the last years. Especially in speciose biomes as tropical rain forests and/or in hyperdiverse or understudied taxa they may efficiently complement morphological approaches. The most successful molecular approach in this field is DNA barcoding based on cytochrome c oxidase I (COI) marker, but other markers are used as well. Whereas most studies aim at identifying or describing species, there are only few attempts to use DNA markers for inventorying all animal species found in environmental samples to describe variations of biodiversity patterns.

Methodology/Principal Findings

In this study, an analysis of the nuclear D3 region of the 28S rRNA gene to delimit species-like units is compared to results based on distinction of morphospecies. Data derived from both approaches are used to assess diversity and composition of staphylinid beetle communities of a Guineo-Congolian rain forest in Kenya. Beetles were collected with a standardized sampling design across six transects in primary and secondary forests using pitfall traps. Sequences could be obtained of 99% of all individuals. In total, 76 molecular operational taxonomic units (MOTUs) were found in contrast to 70 discernible morphospecies. Despite this difference both approaches revealed highly similar biodiversity patterns, with species richness being equal in primary and secondary forests, but with divergent species communities in different habitats. The D3-MOTU approach proved to be an efficient tool for biodiversity analyses.

Conclusions/Significance

Our data illustrate that the use of MOTUs as a proxy for species can provide an alternative to morphospecies identification for the analysis of changes in community structure of hyperdiverse insect taxa. The efficient amplification of the D3-marker and the ability of the D3-MOTUs to reveal similar biodiversity patterns as analyses of morphospecies recommend its use in future molecular studies on biodiversity.     

Identification and characterization of expressed retrotransposons in the genome of the Paracoccidioides species complex

Background

Species from the Paracoccidioides complex are thermally dimorphic fungi and the causative agents of paracoccidioidomycosis, a deep fungal infection that is the most prevalent systemic mycosis in Latin America and represents the most important cause of death in immunocompetent individuals with systemic mycosis in Brazil. We previously described the identification of eight new families of DNA transposons in Paracoccidioides genomes. In this work, we aimed to identify potentially active retrotransposons in Paracoccidioides genomes.

Results

We identified five different retrotransposon families (four LTR-like and one LINE-like element) in the genomes of three Paracoccidioides isolates. Retrotransposons were present in all of the genomes analyzed. P. brasiliensis and P. lutzii species harbored the same retrotransposon lineages but differed in their copy numbers. In the Pb01, Pb03 and Pb18 genomes, the number of LTR retrotransposons was higher than the number of LINE-like elements, and the LINE-like element RtPc5 was transcribed in Paracoccidioides lutzii (Pb01) but could not be detected in P. brasiliensis (Pb03 and Pb18) by semi-quantitative RT-PCR.

Conclusion

Five new potentially active retrotransposons have been identified in the genomic assemblies of the Paracoccidioides species complex using a combined computational and experimental approach. The distribution across the two known species, P. brasiliensis and P. lutzii, and phylogenetics analysis indicate that these elements could have been acquired before speciation occurred. The presence of active retrotransposons in the genome may have implications regarding the evolution and genetic diversification of the Paracoccidioides genus.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1564-7) contains supplementary material, which is available to authorized users.     

Statistical Parsimony Networks and Species Assemblages in Cephalotrichid Nemerteans (Nemertea)

Background

It has been suggested that statistical parsimony network analysis could be used to get an indication of species represented in a set of nucleotide data, and the approach has been used to discuss species boundaries in some taxa.

Methodology/Principal Findings

Based on 635 base pairs of the mitochondrial protein-coding gene cytochrome c oxidase I (COI), we analyzed 152 nemertean specimens using statistical parsimony network analysis with the connection probability set to 95%. The analysis revealed 15 distinct networks together with seven singletons. Statistical parsimony yielded three networks supporting the species status of Cephalothrix rufifrons, C. major and C. spiralis as they currently have been delineated by morphological characters and geographical location. Many other networks contained haplotypes from nearby geographical locations. Cladistic structure by maximum likelihood analysis overall supported the network analysis, but indicated a false positive result where subnetworks should have been connected into one network/species. This probably is caused by undersampling of the intraspecific haplotype diversity.

Conclusions/Significance

Statistical parsimony network analysis provides a rapid and useful tool for detecting possible undescribed/cryptic species among cephalotrichid nemerteans based on COI gene. It should be combined with phylogenetic analysis to get indications of false positive results, i.e., subnetworks that would have been connected with more extensive haplotype sampling.     

Barcoding bugs: DNA-based identification of the true bugs (Insecta: Hemiptera: Heteroptera)

Background

DNA barcoding, the analysis of sequence variation in the 5′ region of the mitochondrial cytochrome c oxidase I (COI) gene, has been shown to provide an efficient method for the identification of species in a wide range of animal taxa. In order to assess the effectiveness of barcodes in the discrimination of Heteroptera, we examined 344 species belonging to 178 genera, drawn from specimens in the Canadian National Collection of Insects.

Methodology/Principal Findings

Analysis of the COI gene revealed less than 2% intra-specific divergence in 90% of the taxa examined, while minimum interspecific distances exceeded 3% in 77% of congeneric species pairs. Instances where barcodes fail to distinguish species represented clusters of morphologically similar species, except one case of barcode identity between species in different genera. Several instances of deep intraspecific divergence were detected suggesting possible cryptic species.

Conclusions/Significance

Although this analysis encompasses 0.8% of the described global fauna, our results indicate that DNA barcodes will aid the identification of Heteroptera. This advance will be useful in pest management, regulatory and environmental applications and will also reveal species that require further taxonomic research.     

Geographically widespread swordfish barcode stock identification: a case study of its application

Background

The swordfish (Xiphias gladius) is a cosmopolitan large pelagic fish inhabiting tempered and tropical waters and it is a target species for fisheries all around the world. The present study investigated the ability of COI barcoding to reliably identify swordfish and particularly specific stocks of this commercially important species.

Methodology

We applied the classical DNA barcoding technology, upon a 682 bp segment of COI, and compared swordfish sequences from different geographical sources (Atlantic, Indian Oceans and Mediterranean Sea). The sequences of the 5′ hyper-variable fragment of the control region (5′dloop), were also used to validate the efficacy of COI as a stock-specific marker.

Case Report

This information was successfully applied to the discrimination of unknown samples from the market, detecting in some cases mislabeled seafood products.

Conclusions

The NJ distance-based phenogram (K2P model) obtained with COI sequences allowed us to correlate the swordfish haplotypes to the different geographical stocks. Similar results were obtained with 5′dloop. Our preliminary data in swordfish Xiphias gladius confirm that Cytochrome Oxidase I can be proposed as an efficient species-specific marker that has also the potential to assign geographical provenance. This information might speed the samples analysis in commercial application of barcoding.     

Paracoccidioides lutzii Plp43 Is an Active Glucanase with Partial Antigenic Identity with P. brasiliensis gp43

Background

Paracoccidioides brasiliensis and P. lutzii cause paracoccidioidomycosis (PCM). P. brasiliensis main diagnostic antigen is glycoprotein gp43, and its peptide sequence is 81% identical with a P. lutzii ortholog here called Plp43. P. lutzii (“Pb01-like”) apparently predominates in Midwestern/Northern Brazil, where high percentages of false-negative reactions using P. brasiliensis antigens have recently been reported. The aim of this work was to produce recombinant Plp43 to study its antigenic identity with gp43.

Methodology

We expressed rPlp43 as a secreted major component in Pichia pastoris and studied its reactivity in immunoblot with PCM patients'' sera from Southwestern and Midwestern Brazil.

Principal Findings

We showed that rPlp43 is not glycosylated and bears glucanase activity. The protein did not react with anti-gp43 monoclonal antibodies in immunoblot, suggesting absence of the corresponding gp43 epitopes. Nevertheless, common epitope(s) might exist, considering that gp43-positive PCM sera recognized rPlp43 in immunoblot, while gp43-negative sera (33 out of 51) from patients resident in Midwestern Brazil were also rPlp43-negative. Two genotyped P. lutzii were from patients with gp43-negative sera, suggesting that non-reactive sera are from patients infected with this species.

Conclusion

Our data suggest that gp43 and Plp43 bear one or only a few common epitopes and that gp43 cannot be used in diagnosis of PCM patients infected with P. lutzii probably because Plp43 is poorly expressed during infection.     

The life-cycle of the asexual ostracod Darwinula stevensoni (Brady & Robertson, 1870) (Crustacea,Ostracoda) in a temporate pond

The life-cycle of the ancient asexual ostracod Darwinula stevensoni was studied during 1 year in a eutrophic pond in Belgium. The reproductive period of this species started in March and was effectively completed by September of the same year. All changes in population structure took place during the spring and summer months and a rapid turnover of the instars was observed. The life-cycle of Darwinula stevensoni appears to take one year or less in Belgium and this is considerably shorter than the 4 years which had been reported previously from subarctic populations. The difference to the present study is most likely temperature-related. Maximal densities of D. stevensoni were observed in June and July and attained 10⁵ ind. m^–2. During winter, densities were lower with a mean of 10⁴ ind. m^–2. Consequently, the calculated population size of each month was high throughout the year. Together with the low mutation rate, such a large population size could effectively counteract the stochastic loss of mutation-free genotypes as predicted by Muller's ratchet. D. stevensoni is a brooder; the maximum number of embryos and juvenile instars (up to third stage) found within a single female was 11.     

Early release of eggs and embryos in a brooding ancient asexual ostracod: brood selection or a gambling strategy to increase fecundity?

Asexual lineages lack the means to purge their genomes of (deleterious) mutations through recombination. Evolutionary theory thus predicts that such lineages will be prone to early extinction. In brooding animals, brood selection might provide a mechanism to counter the accumulation of mutations. Of the three putative ancient asexual animal groups, only the darwinulid ostracods are brooders. Here, we test the incidence of egg and juvenile abortion in a darwinulid species, Penthesilenula brasiliensis, under two temperature treatments. Part of the offspring is released without brooding (close to 30% in one treatment). The majority of these aborted eggs hatches and develops. As it is unlikely that females are such bad judges of offspring quality, either the surviving animals will present deficiencies later on in development (brood selection) or early egg release can be a (bet-hedging) strategy to increase fecundity in favourable conditions.     

Cytogenetic and molecular evidence suggest multiple origins and geographical parthenogenesis in Nothoscordum gracile (Alliaceae)

Background and Aims

Nothoscordum gracile is an apomitic tetraploid widely distributed throughout the Americas and naturalized in many temperate regions of other continents. It has been suggested to form a species complex with sexual and apomictic N. nudicaule and N. macrostemon. Tetraploids of these species also share a structurally heterozygous chromosome complement 2n = 19 (13M + 6A). In this work, the origin of N. gracile and its relationships with its related species was investigated based on cytological and molecular data.

Methods

Cytogenetic analyses were based on meiotic behaviour, CMA bands, localization of 5S and 45S rDNA sites, and genomic in situ hybridization (GISH). Nuclear ITS and plastidial trnL-trnF sequences were also obtained for most individuals.

Key Results

Proximal CMA bands were observed in the long arms of all acrocentrics of 2x and 4x N. macrostemon but not in diploid and some tetraploid cytotypes of N. nudicaule. Samples of N. gracile showed a variable number of CMA bands in the long arms of acrocentrics. Analysis of ITS sequences, dot-blot, GISH, and 5S and 45S rDNA sites, revealed no differentiation among the three species. The trnL-trnF cpDNA fragment showed variation with a trend to geographical structuring irrespective of morphospecies and fully congruent with karyotype variation.

Conclusions

The 2n = 19 karyotype was probably formed by a centric fusion event occurring in N. nudicaule and later transmitted to tetraploid cytotypes of N. macrostemon. Diploids of N. nudicaule and N. macrostemon appeared as consistent recently diverged species, whereas tetraploid apomicts seem to constitute an assemblage of polyploid hybrids originating from multiple independent hybridization events between them, part of which are morphologically recognizable as N. gracile.