Birds perching on bushes: Networks to visualize conflicting phylogenetic signals during early avian radiation

Hybridization is increasingly seen as an important source of adaptive genetic variation and biotic diversity. Recent phylogenetic studies on the early evolution of birds suggest that the early diversification of neoavian orders perhaps involved a period of extensive hybridization or incomplete lineage sorting. Phylogenetic error, saturation, long-branch attraction, and convergence make it difficult to detect ancient hybridization events and differentiate them from incomplete lineage sorting using sequence data. We used recently published retroposon marker data to visualize the early radiation of Neoaves within a phylogenetic network approach, and found that the most basal neoavian taxa indeed show a complex pattern of reticulated relationships. Moreover, the reticulation levels of different parts of the network are consistent with the insertion pattern of the retroposon elements. The use of network-based analyses on homoplasy-free data shows true conflicting signals and the taxa involved that are not represented in trees.     

The Dynamics of Incomplete Lineage Sorting across the Ancient Adaptive Radiation of Neoavian Birds

The diversification of neoavian birds is one of the most rapid adaptive radiations of extant organisms. Recent whole-genome sequence analyses have much improved the resolution of the neoavian radiation and suggest concurrence with the Cretaceous-Paleogene (K-Pg) boundary, yet the causes of the remaining genome-level irresolvabilities appear unclear. Here we show that genome-level analyses of 2,118 retrotransposon presence/absence markers converge at a largely consistent Neoaves phylogeny and detect a highly differential temporal prevalence of incomplete lineage sorting (ILS), i.e., the persistence of ancestral genetic variation as polymorphisms during speciation events. We found that ILS-derived incongruences are spread over the genome and involve 35% and 34% of the analyzed loci on the autosomes and the Z chromosome, respectively. Surprisingly, Neoaves diversification comprises three adaptive radiations, an initial near-K-Pg super-radiation with highly discordant phylogenetic signals from near-simultaneous speciation events, followed by two post-K-Pg radiations of core landbirds and core waterbirds with much less pronounced ILS. We provide evidence that, given the extreme level of up to 100% ILS per branch in super-radiations, particularly rapid speciation events may neither resemble a fully bifurcating tree nor are they resolvable as such. As a consequence, their complex demographic history is more accurately represented as local networks within a species tree.     

Retroposed elements as archives for the evolutionary history of placental mammals

Reconstruction of the placental mammalian (eutherian) evolutionary tree has undergone diverse revisions, and numerous aspects remain hotly debated. Initial hierarchical divisions based on morphology contained many misgroupings due to features that evolved independently by similar selection processes. Molecular analyses corrected many of these misgroupings and the superordinal hierarchy of placental mammals was recently assembled into four clades. However, long or rapid evolutionary periods, as well as directional mutation pressure, can produce molecular homoplasies, similar characteristics lacking common ancestors. Retroposed elements, by contrast, integrate randomly into genomes with negligible probabilities of the same element integrating independently into orthologous positions in different species. Thus, presence/absence analyses of these elements are a superior strategy for molecular systematics. By computationally scanning more than 160,000 chromosomal loci and judiciously selecting from only phylogenetically informative retroposons for experimental high-throughput PCR applications, we recovered 28 clear, independent monophyly markers that conclusively verify the earliest divergences in placental mammalian evolution. Using tests that take into account ancestral polymorphisms, multiple long interspersed elements and long terminal repeat element insertions provide highly significant evidence for the monophyletic clades Boreotheria (synonymous with Boreoeutheria), Supraprimates (synonymous with Euarchontoglires), and Laurasiatheria. More importantly, two retropositions provide new support for a prior scenario of early mammalian evolution that places the basal placental divergence between Xenarthra and Epitheria, the latter comprising all remaining placentals. Due to its virtually homoplasy-free nature, the analysis of retroposon presence/absence patterns avoids the pitfalls of other molecular methodologies and provides a rapid, unequivocal means for revealing the evolutionary history of organisms.     

Incomplete Lineage Sorting and Hybridization Statistics for Large-Scale Retroposon Insertion Data

Ancient retroposon insertions can be used as virtually homoplasy-free markers to reconstruct the phylogenetic history of species. Inherited, orthologous insertions in related species offer reliable signals of a common origin of the given species. One prerequisite for such a phylogenetically informative insertion is that the inserted element was fixed in the ancestral population before speciation; if not, polymorphically inserted elements may lead to random distributions of presence/absence states during speciation and possibly to apparently conflicting reconstructions of their ancestry. Fortunately, such misleading fixed cases are relatively rare but nevertheless, need to be considered. Here, we present novel, comprehensive statistical models applicable for (1) analyzing any pattern of rare genomic changes, (2) testing and differentiating conflicting phylogenetic reconstructions based on rare genomic changes caused by incomplete lineage sorting or/and ancestral hybridization, and (3) differentiating between search strategies involving genome information from one or several lineages. When the new statistics are applied, in non-conflicting cases a minimum of three elements present in both of two species and absent in a third group are considered significant support (p<0.05) for the branching of the third from the other two, if all three of the given species are screened equally for genome or experimental data. Five elements are necessary for significant support (p<0.05) if a diagnostic locus derived from only one of three species is screened, and no conflicting markers are detected. Most potentially conflicting patterns can be evaluated for their significance and ancestral hybridization can be distinguished from incomplete lineage sorting by considering symmetric or asymmetric distribution of rare genomic changes among possible tree configurations. Additionally, we provide an R-application to make the new KKSC insertion significance test available for the scientific community at http://retrogenomics.uni-muenster.de:3838/KKSC_significance_test/.     

Evolutionary origins of retroposon lineages of Mhc class II Ab alleles

Major histocompatibility complex (Mhc) class II Ab genes have evolved into three distinct lineages. While lineage 2 alleles differ from lineage 1 alleles by the insertion of a retroposon in intron 2, the basis for the extremely large intron 2 in lineage 3 alleles has heretofore been undetermined. In this report, we demonstrate by nucleotide sequencing that the genomic sequences of prototypic alleles from all three lineages diverge significantly and that lineage 3 is derived from lineage 2 by two insertional events in intron 2. One insert, composed of a member of B1 short interspersed repetitive elements (SINEs), occurs 508 base pairs (bp) 3 of exon 2, and the other, 1141 bp 3 of exon 2 within the retroposon that distinguishes lineage 2 from lineage 1. To assess the evolutionary stability of these lineages and the extent of ancestral polymorphisms of Ab within Mus species, we extended our restriction site polymorphism analysis to include 86 alleles from 120 independently derived H2 haplotypes from 12 separate species and subspecies of Mus. A phylogenetic tree revealing the relationships of these Ab alleles with respect to restriction site polymorphisms, but excluding the retroposon insertions, demonstrated that these lineages have distinctive genomic structures beyond the retroposon polymorphisms. In summary, these mouse Ab genes were produced from successive retroposon insertion events. Lineage 1 and 2 were detected in a variety of Mus species, including Mus caroli, indicating that these lineages diverged more than 2 million years ago. Lineage 3 alleles were found only in the Mus musculus subspecies, suggesting that it diverged from lineage 2 more recently. These results indicate that all three lineages of Ab have persisted through several speciation events in the genus Mus.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M60562     

BOOM AND BUST: ANCIENT AND RECENT DIVERSIFICATION IN BICHIRS (POLYPTERIDAE: ACTINOPTERYGII), A RELICTUAL LINEAGE OF RAY‐FINNED FISHES

Understanding the history that underlies patterns of species richness across the Tree of Life requires an investigation of the mechanisms that not only generate young species‐rich clades, but also those that maintain species‐poor lineages over long stretches of evolutionary time. However, diversification dynamics that underlie ancient species‐poor lineages are often hidden due to a lack of fossil evidence. Using information from the fossil record and time calibrated molecular phylogenies, we investigate the history of lineage diversification in Polypteridae, which is the sister lineage of all other ray‐finned fishes (Actinopterygii). Despite originating at least 390 million years (Myr) ago, molecular timetrees support a Neogene origin for the living polypterid species. Our analyses demonstrate polypterids are exceptionally species depauperate with a stem lineage duration that exceeds 380 million years (Ma) and is significantly longer than the stem lineage durations observed in other ray‐finned fish lineages. Analyses of the fossil record show an early Late Cretaceous (100.5–83.6 Ma) peak in polypterid genus richness, followed by 60 Ma of low richness. The Neogene species radiation and evidence for high‐diversity intervals in the geological past suggest a “boom and bust” pattern of diversification that contrasts with common perceptions of relative evolutionary stasis in so‐called “living fossils.”     

Ancient forest fragmentation or recent radiation? Testing refugial speciation models in chameleons within an African biodiversity hotspot

Aim East Africa is one of the most biologically diverse regions, especially in terms of endemism and species richness. Hypotheses put forward to explain this high diversity invoke a role for forest refugia through: (1) accumulation of new species due to radiation within refugial habitats, or (2) retention of older palaeoendemic species in stable refugia. We tested these alternative hypotheses using data for a diverse genus of East African forest chameleons, Kinyongia. Location East Africa. Methods We constructed a dated phylogeny for Kinyongia using one nuclear and two mitochondrial markers. We identified areas of high phylogenetic diversity (PD) and evolutionary diversity (ED), and mapped ancestral areas to ascertain whether lineage diversification could best be explained by vicariance or dispersal. Results Vicariance best explains the present biogeographic patterns, with divergence between three major Kinyongia clades (Albertine Rift, southern Eastern Arc, northern Eastern Arc) in the early Miocene/Oligocene (> 20 Ma). Lineage diversification within these clades pre‐dates the Pliocene (> 6 Ma). These dates are much older than the Plio‐Pleistocene climatic shifts associated with cladogenesis in other East African taxa (e.g. birds), and instead point to a scenario whereby palaeoendemics are retained in refugia, rather than more recent radiations within refugia. Estimates of PD show that diversity was highest in the Uluguru, Nguru and East Usambara Mountains and several lineages (from Mount Kenya, South Pare and the Uluguru Mountains) stand out as being evolutionarily distinct as a result of isolation in forest refugia. PD was lower than expected by chance, suggesting that the phylogenetic signal is influenced by an unusually low number of extant lineages with long branch lengths, which is probably due to the retention of palaeoendemic lineages. Main conclusions The biogeographic patterns associated with Kinyongia are the result of long evolutionary histories in isolation. The phylogeny is dominated by ancient lineages whose origins date back to the early Miocene/Oligocene as a result of continental wide forest fragmentation and contraction due to long term climatic changes in Africa. The maintenance of palaeoendemic lineages in refugia has contributed substantially to the remarkably high biodiversity of East Africa.     

A Universal Method for the Study of CR1 Retroposons in Nonmodel Bird Genomes

Presence/absence patterns of retroposon insertions at orthologous genomic loci constitute straightforward markers for phylogenetic or population genetic studies. In birds, the convenient identification and utility of these markers has so far been mainly restricted to the lineages leading to model birds (i.e., chicken and zebra finch). We present an easy-to-use, rapid, and cost-effective method for the experimental isolation of chicken repeat 1 (CR1) insertions from virtually any bird genome and potentially nonavian genomes. The application of our method to the little grebe genome yielded insertions belonging to new CR1 subfamilies that are scattered all across the phylogenetic tree of avian CR1s. Furthermore, presence/absence analysis of these insertions provides the first retroposon evidence grouping flamingos + grebes as Mirandornithes and several markers for all subsequent branching events within grebes (Podicipediformes). Five markers appear to be species-specific insertions, including the hitherto first evidence in birds for biallelic CR1 insertions that could be useful in future population genetic studies.     

Retrotransposon insertion polymorphisms in six rice genes and their evolutionary history

Retrotransposons are abundant in higher plant genomes. Although retrotransposons associated with plant genes have been identified, little is known about their evolutionary conservation at the level of species and subspecies. In the present study, we investigated the phylogenetic distribution of long terminal repeat (LTR) retrotransposon, long interspersed nuclear element (LINE) and short interspersed nuclear element (SINE) insertions in six genes in 95 cultivated and wild rice genotypes. These six genes are likely to be functional based on nonsynonymous (Ka) to synonymous (Ks) substitution ratios which were found to be significantly <1. Different conservation patterns of these retrotransposons in genes were observed in cultivated and wild rice species. Four out of seven retrotransposon insertions appear to predate the ancestral Oryza AA genome. Two of these insertions in genes 4 and 5 occurred early in the evolutionary history of Oryza. Two retrotransposon insertions in gene 1 arose after the divergence of Asian cultivated rice from its wild ancestor. Furthermore, the retrotransposon insertion in gene 3 appears to have occurred in the ancestral lineage leading to temperate japonicas. Conservation of retrotransposon insertions in genes in specific groups, species, and lineages might be related to their specific function.     

Evolutionary History of Assassin Bugs (Insecta: Hemiptera: Reduviidae): Insights from Divergence Dating and Ancestral State Reconstruction

Assassin bugs are one of the most successful clades of predatory animals based on their species numbers (∼6,800 spp.) and wide distribution in terrestrial ecosystems. Various novel prey capture strategies and remarkable prey specializations contribute to their appeal as a model to study evolutionary pathways involved in predation. Here, we reconstruct the most comprehensive reduviid phylogeny (178 taxa, 18 subfamilies) to date based on molecular data (5 markers). This phylogeny tests current hypotheses on reduviid relationships emphasizing the polyphyletic Reduviinae and the blood-feeding, disease-vectoring Triatominae, and allows us, for the first time in assassin bugs, to reconstruct ancestral states of prey associations and microhabitats. Using a fossil-calibrated molecular tree, we estimated divergence times for key events in the evolutionary history of Reduviidae. Our results indicate that the polyphyletic Reduviinae fall into 11–14 separate clades. Triatominae are paraphyletic with respect to the reduviine genus Opisthacidius in the maximum likelihood analyses; this result is in contrast to prior hypotheses that found Triatominae to be monophyletic or polyphyletic and may be due to the more comprehensive taxon and character sampling in this study. The evolution of blood-feeding may thus have occurred once or twice independently among predatory assassin bugs. All prey specialists evolved from generalist ancestors, with multiple evolutionary origins of termite and ant specializations. A bark-associated life style on tree trunks is ancestral for most of the lineages of Higher Reduviidae; living on foliage has evolved at least six times independently. Reduviidae originated in the Middle Jurassic (178 Ma), but significant lineage diversification only began in the Late Cretaceous (97 Ma). The integration of molecular phylogenetics with fossil and life history data as presented in this paper provides insights into the evolutionary history of reduviids and clears the way for in-depth evolutionary hypothesis testing in one of the most speciose clades of predators.     

Heterogeneous Histories of Recombination Suppression on Stickleback Sex Chromosomes

How consistent are the evolutionary trajectories of sex chromosomes shortly after they form? Insights into the evolution of recombination, differentiation, and degeneration can be provided by comparing closely related species with homologous sex chromosomes. The sex chromosomes of the threespine stickleback (Gasterosteus aculeatus) and its sister species, the Japan Sea stickleback (G. nipponicus), have been well characterized. Little is known, however, about the sex chromosomes of their congener, the blackspotted stickleback (G. wheatlandi). We used pedigrees to obtain experimentally phased whole genome sequences from blackspotted stickleback X and Y chromosomes. Using multispecies gene trees and analysis of shared duplications, we demonstrate that Chromosome 19 is the ancestral sex chromosome and that its oldest stratum evolved in the common ancestor of the genus. After the blackspotted lineage diverged, its sex chromosomes experienced independent and more extensive recombination suppression, greater X–Y differentiation, and a much higher rate of Y degeneration than the other two species. These patterns may result from a smaller effective population size in the blackspotted stickleback. A recent fusion between the ancestral blackspotted stickleback Y chromosome and Chromosome 12, which produced a neo-X and neo-Y, may have been favored by the very small size of the recombining region on the ancestral sex chromosome. We identify six strata on the ancestral and neo-sex chromosomes where recombination between the X and Y ceased at different times. These results confirm that sex chromosomes can evolve large differences within and between species over short evolutionary timescales.     

Night and day: the comparative study of strepsirrhine primates reveals socioecological and phylogenetic patterns in olfactory signals

Studies of chemical signals in vertebrates typically target single species; however, a broader understanding of olfactory communication may derive from comparative studies. We collected urine from 12 species representing most families of strepsirrhine primates--an excellent model clade because of variation in scent marking and socioecology. Using SPDE/GC-MS, we identified the volatile chemical composition of male and female urine from six 'urine marking' species and six glandular or 'non-urine marking' species. We found no sex differences, but as predicted, urine markers expressed the most chemically complex and distinctive urine. More distantly related species had more dissimilar urinary profiles, suggesting gradual signal evolution. Reconstructing ancestral chemical profiles revealed different evolutionary trajectories for urine and non-urine markers. We suggest that urine marking is an ancestral behaviour related to solitary, nocturnal living and that parallel evolutionary shifts towards greater reliance on derived glandular marking occurred in a family (Lemuridae) characterized by diurnality and sociality.     

North American origin and recent European establishments of the amphi-Atlantic peat moss Sphagnum angermanicum

Genetic and morphological similarity between populations separated by large distances may be caused by frequent long-distance dispersal or retained ancestral polymorphism. The frequent lack of differentiation between disjunct conspecific moss populations on different continents has traditionally been explained by the latter model, and has been cited as evidence that many or most moss species are extremely ancient and slowly diverging. We have studied intercontinental differentiation in the amphi-Atlantic peat moss Sphagnum angermanicum using 23 microsatellite markers. Two major genetic clusters are found, both of which occur throughout the distributional range. Patterns of genetic structuring and overall migration patterns suggest that the species probably originated in North America, and seems to have been established twice in Northern Europe during the past 40,000 years. We conclude that similarity between S. angermanicum populations on different continents is not the result of ancient vicariance and subsequent stasis. Rather, the observed pattern can be explained by multiple long-distance dispersal over limited evolutionary time. The genetic similarity can also partly be explained by incomplete lineage sorting, but this appears to be caused by the short time since separation. Our study adds to a growing body of evidence suggesting that Sphagnum, constituting a significant part of northern hemisphere biodiversity, may be more evolutionary dynamic than previously assumed.     

Transposable elements reveal the impact of introgression,rather than transposition,in Pisum diversity,evolution, and domestication

The genetic structure and evolutionary history of the genus Pisum were studied exploiting our germplasm collection to compare the contribution of different mechanisms to the generation of diversity. We used sequence-specific amplification polymorphism (SSAP) markers to assess insertion site polymorphism generated by a representative of each of the two major groups of LTR-containing retrotransposons, PDR1 (Ty1/copia-like) and Cyclops (Ty3/gypsy-like), together with Pis1, a member of the En/Spm transposon superfamily. The analysis of extended sets of the four main Pisum species, P. fulvum, P. elatius, P. abyssinicum, and P. sativum, together with the reference set, revealed a distinct pattern of the NJ (Neighbor-Joining) tree for each basic lineage, which reflects the different evolutionary history of each species. The SSAP markers showed that Pisum is exceptionally polymorphic for an inbreeding species. The patterns of phylogenetic relationships deduced from different transposable elements were in general agreement. The retrotransposon-derived markers gave a clearer separation of the main lineages than the Pis1 markers and were able to distinguish the truly wild form of P. elatius from the antecedents of P. sativum. There were more species-specific and unique PDR1 markers than Pis1 markers in P. fulvum and P. elatius, pointing to PDR1 activity during speciation and diversification, but the proportion of these markers is low. The overall genetic diversity of Pisum and the extreme polymorphism in all species, except P. abyssinicum, indicate a high contribution of recombination between multiple ancestral lineages compared to transposition within lineages. The two independently domesticated pea species, P. abyssinicum and P. sativum, arose in contrasting ways from the common processes of hybridization, introgression, and selection without associated transpositional activity.     

Ecological Specialization and Evolutionary Reticulation in Extant Hyaenidae

During the Miocene, Hyaenidae was a highly diverse family of Carnivora that has since been severely reduced to four species: the bone-cracking spotted, striped, and brown hyenas, and the specialized insectivorous aardwolf. Previous studies investigated the evolutionary histories of the spotted and brown hyenas, but little is known about the remaining two species. Moreover, the genomic underpinnings of scavenging and insectivory, defining traits of the extant species, remain elusive. Here, we generated an aardwolf genome and analyzed it together with the remaining three species to reveal their evolutionary relationships, genomic underpinnings of their scavenging and insectivorous lifestyles, and their respective genetic diversities and demographic histories. High levels of phylogenetic discordance suggest gene flow between the aardwolf lineage and the ancestral brown/striped hyena lineage. Genes related to immunity and digestion in the bone-cracking hyenas and craniofacial development in the aardwolf showed the strongest signals of selection, suggesting putative key adaptations to carrion and termite feeding, respectively. A family-wide expansion in olfactory receptor genes suggests that an acute sense of smell was a key early adaptation. Finally, we report very low levels of genetic diversity within the brown and striped hyenas despite no signs of inbreeding, putatively linked to their similarly slow decline in effective population size over the last ∼2 million years. High levels of genetic diversity and more stable population sizes through time are seen in the spotted hyena and aardwolf. Taken together, our findings highlight how ecological specialization can impact the evolutionary history, demographics, and adaptive genetic changes of an evolutionary lineage.     

Unusual signatures of highly adaptable R-loci in closely-related Arabidopsis species

Plant resistance genes (R-genes) evolve rapidly in response to changing environments. What are the most remarkable signatures of fast adaptive genes, besides the commonly revealed rapid divergence and high non-synonymous substitution rate? Here we investigated these changes in five R-loci following recent differentiation between Arabidopsis thaliana and Arabidopsis lyrata. Extreme differences in evolutionary rates were observed: e.g., an overall 5.46-9.83-fold different nucleotide diversity at two R-loci between species, ten-fold higher non-synonymous substitution rates within one species versus the other, significantly different Ka/Ks ratios between species for the same R-gene, and high interspecific divergence at one R-locus. Particularly, we observed an elevated level of trans-specific polymorphism at one R-locus and a differentially maintained presence/absence polymorphism at another. The high frequency of ancestral polymorphisms amongst R-genes suggests that the persistence of some functional variation is an important evolutionary mechanism shaping genetic variation in R-genes, while the variation of presence/absence polymorphisms provides a potential mechanism for malleable activation of adaptive resistance response pathways. The distinct patterns among R-genes suggest that the same R-gene ortholog can be quickly shaped by different evolutionary processes, e.g., purifying selection in one species but positive selection in a closely-related species.     

Deep-sea squat lobster biogeography (Munidopsidae: Leiogalathea) unveils Tethyan vicariance and evolutionary patterns shared by shallow-water relatives

The ecology, abundance and diversity of galatheoid squat lobsters make them an ideal group to study deep-sea diversification processes. Here, we reconstructed the evolutionary and biogeographic history of Leiogalathea, a genus of circum-tropical deep-sea squat lobsters, in order to compare patterns and processes that have affected shallow-water and deep-sea squat lobster species. We first built a multilocus phylogeny and a calibrated species tree with a relaxed clock using StarBEAST2 to reconstruct evolutionary relationships and divergence times among Leiogalathea species. We used BioGeoBEARS and a DEC model, implemented in RevBayes, to reconstruct ancestral distribution ranges and the biogeographic history of the genus. Our results showed that Leiogalathea is monophyletic and comprises four main lineages; morphological homogeneity is common within and between clades, except in one; the reconstructed ancestral range of the genus is in the Atlantic and Indian oceans (Tethys). They also revealed the divergence of the Atlantic species around 25 million years ago (Ma), intense cladogenesis 15–25 Ma and low levels of speciation over the last 5 million years (Myr). The four Leiogalathea lineages showed similar patterns of speciation: allopatric speciation followed by range expansion and subsequent stasis. Leiogalathea started diversifying during the Oligocene, likely in the Tethyan. The Atlantic lineage then split from its Indo-Pacific sister group due to vicariance driven by closure of the Tethys Seaway. The Atlantic lineage is less speciose compared with the Indo-Pacific lineages, with the Tropical Southwestern Pacific being the current centre of diversity. Leiogalathea diversification coincided with cladogenetic peaks in shallow-water genera, indicating that historical biogeographic events similarly shaped the diversification and distribution of both deep-sea and shallow-water squat lobsters.     

Diversity of tuco-tucos (Ctenomys, Rodentia) in the Northeastern wetlands from Argentina: mitochondrial phylogeny and chromosomal evolution

Tuco-tucos (small subterranean rodents of the genus Ctenomys) that inhabit sandy soils of the area under the influence of the second largest wetland of South America, in Northeastern Argentina (Corrientes province), are a complex of species and forms whose taxonomic status were not defined, nor are the evolutionary relationships among them. The tuco-tuco populations of this area exhibit one of the most ample grades of chromosomal variability within the genus. In order to analyze evolutionary relationships within the Corrientes group and its chromosomal variability, we completed the missing karyotypic information and performed a phylogenetic analysis. We obtained partial sequences of three mitochondrial markers: D-loop, cytochrome b and cytochrome oxidase I. The Corrientes group was monophyletic and split into three main clades that grouped related karyomorphs. The phylogeny suggested an ancestral condition of the karyomorph with diploid number (2n) 70 and fundamental number (FN) 84 that has evolved mainly via reductions of the FN although amplifications occurred in certain lineages. We discuss the relationship between patterns of chromosomal variability and species and groups boundaries. From the three main clades the one named iberá exhibited a remarkable karyotypic homogeneity, and could be considered as an independent and cohesive evolutionary lineage. On the contrary, the former recognized species C. dorbignyi is a polyphyletic lineage and hence its systematic classification should be reviewed.