Coalescent-based Methods Research Articles

Estimating Variable Effective Population Sizes from Multiple Genomes: A Sequentially Markov Conditional Sampling Distribution Approach

Throughout history, the population size of modern humans has varied considerably due to changes in environment, culture, and technology. More accurate estimates of population size changes, and when they occurred, should provide a clearer picture of human colonization history and help remove confounding effects from natural selection inference. Demography influences the pattern of genetic variation in a population, and thus genomic data of multiple individuals sampled from one or more present-day populations contain valuable information about the past demographic history. Recently, Li and Durbin developed a coalescent-based hidden Markov model, called the pairwise sequentially Markovian coalescent (PSMC), for a pair of chromosomes (or one diploid individual) to estimate past population sizes. This is an efficient, useful approach, but its accuracy in the very recent past is hampered by the fact that, because of the small sample size, only few coalescence events occur in that period. Multiple genomes from the same population contain more information about the recent past, but are also more computationally challenging to study jointly in a coalescent framework. Here, we present a new coalescent-based method that can efficiently infer population size changes from multiple genomes, providing access to a new store of information about the recent past. Our work generalizes the recently developed sequentially Markov conditional sampling distribution framework, which provides an accurate approximation of the probability of observing a newly sampled haplotype given a set of previously sampled haplotypes. Simulation results demonstrate that we can accurately reconstruct the true population histories, with a significant improvement over the PSMC in the recent past. We apply our method, called diCal, to the genomes of multiple human individuals of European and African ancestry to obtain a detailed population size change history during recent times.

Identification of species in the <i>Cladia aggregata</i> group using DNA barcoding (Ascomycota: Lecanorales)

The DNA barcode approach is using a short genetic marker for rapid identification of a particular species. The internal transcribed spacer (ITS) region has been chosen as barcode marker for fungi. Here we tested the potential of ITS to identify distinct lineages in the Cladia aggregata complex, a group of lichenized fungi exhibiting remarkable morphological and chemical diversity. Our recent studies using multilocus DNA sequence data and coalescent-based species delimitation methods supported a 12 species delimitation scenario. In this study, we evaluated the ratio of the intra- and interspecific genetic distances of ITS in these 12 putative species. All 12 putative species showed a lower ratio of intraspecific variation than interspecific variation, supporting the hypothesis that these represent distinct lineages. Consequently, these lineages are here accepted at species level and three new species, viz. Cladia blanchonii Parnmen & Lumbsch, C. cryptica Parnmen & Lumbsch and C. tasmanica Parnmen & Lumbsch are described and the new combinations Cladia gorgonea (Eschw.) Parnmen & Lumbsch, C. neocaledonica (Räs.) Parnmen & Lumbsch, and C. terebrata (Laurer) Parnmen & Lumbsch proposed.

Spatiotemporal dynamics of the HIV-1 CRF06_cpx epidemic in western Africa

To investigate the origin and spatiotemporal dynamics of dissemination of the HIV-1 CRF06_cpx clade in western Africa. A total of 180 HIV-1 CRF06_cpx-like pol sequences isolated from 12 different countries from west and west-central Africa over a period of 16 years (1995-2010) were analyzed. Evolutionary, phylogeographic and demographic parameters were jointly estimated from sequence data using a Bayesian coalescent-based method and combined with molecular epidemiology and spatial accessibility data. The CRF06_cpx most probably emerged in Burkina Faso in 1979 (1970-1985). From Burkina Faso, the virus was first disseminated to Mali and Nigeria during the 1980s and later to other countries from west and west-central Africa. Demographic reconstruction indicates that the CRF06_cpx epidemic grew exponentially during the 1980s, with a median growth rate of 0.82 year (0.60-1.09 year), and after stabilize. We found a negative correlation between CRF06_cpx prevalence and the geographical distance to Burkina Faso's capital. Regional accessibility information agrees with the overall geographical range of the CRF06_cpx, but not fully explains the highly heterogeneous distribution pattern of this CRF at regional level. The CRF06_cpx epidemic in western Africa probably emerged at the late 1970s and grew during the 1980s at a rate comparable to the HIV-1 epidemics in the United States and Europe. Burkina Faso seems to be the most important epicenter of dissemination of the HIV-1 CRF06_cpx strain at regional level. The explanation for the current geographical distribution of CRF06_cpx is probably multifactorial.

DNA barcode identification of lichen-forming fungal species in the Rhizoplaca melanophthalma species-complex (Lecanorales, Lecanoraceae), including five new species

Recent studies using sequence data from multiple loci and coalescent-based species delimitation have revealed several species-level lineages within the phenotypically circumscribed taxon Rhizoplaca melanophthalma sensu lato. Here, we formally describe five new species within this group, R. occulta, R. parilis, R. polymorpha, R. porterii, and R. shushanii, using support from the coalescent-based species delimitation method implemented in the program Bayesian Phylogenetics and Phylogeography (BPP) as the diagnostic feature distinguishing new species. We provide a reference DNA sequence database using the ITS marker as a DNA barcode for identifying species within this complex. We also assessed intraspecific genetic distances within the six R. melanophthalma sensu lato species. While intraspecific genetic distances within the five new species were less than or equal to the lowest interspecific pairwise comparison values, an overlap in genetic distances within the R. melanophthalma sensu stricto clade suggests the potential for additional phenotypically cryptic lineages within this broadly distributed lineage. Overall, our results demonstrate the potential for accurately identifying species within the R. melanophthalma group by using molecular-based identification methods.

The Confounding Effect of Population Structure on Bayesian Skyline Plot Inferences of Demographic History

Many coalescent-based methods aiming to infer the demographic history of populations assume a single, isolated and panmictic population (i.e. a Wright-Fisher model). While this assumption may be reasonable under many conditions, several recent studies have shown that the results can be misleading when it is violated. Among the most widely applied demographic inference methods are Bayesian skyline plots (BSPs), which are used across a range of biological fields. Violations of the panmixia assumption are to be expected in many biological systems, but the consequences for skyline plot inferences have so far not been addressed and quantified. We simulated DNA sequence data under a variety of scenarios involving structured populations with variable levels of gene flow and analysed them using BSPs as implemented in the software package BEAST. Results revealed that BSPs can show false signals of population decline under biologically plausible combinations of population structure and sampling strategy, suggesting that the interpretation of several previous studies may need to be re-evaluated. We found that a balanced sampling strategy whereby samples are distributed on several populations provides the best scheme for inferring demographic change over a typical time scale. Analyses of data from a structured African buffalo population demonstrate how BSP results can be strengthened by simulations. We recommend that sample selection should be carefully considered in relation to population structure previous to BSP analyses, and that alternative scenarios should be evaluated when interpreting signals of population size change.

Species tree reconstruction of a poorly resolved clade of salamanders (Ambystomatidae) using multiple nuclear loci

The analysis of diverse data sets can yield different phylogenetic estimates that challenge systematists to explain the source of discordance. The mole salamanders (family Ambystomatidae) are a classic example of this phylogenetic conflict. Previous attempts to resolve the ambystomatid species tree using allozymic, morphological, and mitochondrial sequence data have yielded different estimates, making it unclear which data source best approximates ambystomatid phylogeny and which ones yield phylogenetically inaccurate reconstructions. To shed light on this conflict, we present the first multi-locus DNA sequence-based phylogenetic study of the Ambystomatidae. We utilized a range of analyses, including coalescent-based methods of species-tree estimation that account for incomplete lineage sorting within a locus and concordance-based methods that estimate the number of sampled loci that support a particular clade. We repeated these analyses with the removal of individual loci to determine if any locus has a disproportionate effect on our phylogenetic results. Collectively, these results robustly resolved many deep and relatively shallow clades within Ambystoma, including the placement of A. gracile and A. talpoideum as the sister clade to a clade containing all remaining ambystomatids, and the placement of A. maculatum as the sister lineage to all remaining ambystomatids excluding A. gracile and A. talpoideum. Both Bayesian coalescent and concordance methods produced similar results, highlighting strongly supported branches in the species tree. Furthermore, coalescent-based analyses that excluded loci produced overlapping species-tree posterior distributions, suggesting that no particular locus – including mtDNA – disproportionately contributed to our species-tree estimates. Overall, our phylogenetic estimates have greater similarity with previous allozyme and mitochondrial sequence-based phylogenetic estimates. However, intermediate depths of divergence in the ambystomatid species tree remain unresolved, potentially highlighting a region of rapid species radiation or a hard polytomy, which limits our ability to comment on previous morphologically-based taxonomic groups.

Present genetic structure revealed by microsatellites reflects recent history of the Finnish moose (Alces alces)

Genetic structures of Holarctic species are largely formed by Pleistocene colonisation history, dispersal capacity and interactions between biotic and abiotic factors, even though the human impact can also be significant. The Holarctic moose (Alces alces) arrived in Fennoscandia around 9,000–8,000 years ago, and it has been exploited by humans ever since. During the last 400 years, the Finnish moose population has suffered from several population declines, and even local and regional extirpations have occurred. The purpose of the present study is to describe the genetic variation and population structure of the Finnish moose in order to clarify how historical events and human exploitation have influenced the present-day genetic patterns. Altogether 130 moose individuals from seven sampling sites in Finland were analysed at ten microsatellite loci. A variety of population genetic and coalescent-based methods was applied. The Finnish moose population was found to be divided into southern and northern subpopulations with additional lower hierarchical genetic structure. The estimated time of divergence between these two subpopulations was about 96–238 years ago. In addition, an isolation-by-distance pattern was discovered.

Population structure and gene flow among anadromous arctic lamprey (Lethenteron camtschaticum) populations deduced from polymorphic microsatellite loci

Information on migration ecology is important for effective conservation and management of fishery resources, particularly diadromous animals. We investigated the genetic population structure and gene flow pattern of anadromous Arctic lamprey Lethenteron camtschaticum by analyzing polymorphic microsatellite loci. Bayesian population structure analysis was used to estimate the existence of two genetically divergent ancestral sources and latitudinal gradual changes of their frequencies among the present populations. When hypotheses of historical migration models were tested using coalescent-based methods, the best model was found to be an n-island model assuming equal population size and reciprocal migration among all populations. These results suggest that L. camtschaticum has considerable ability to migrate long distances in the sea and has lower homing abilities. For resource management of L. camtschaticum, unconstrained gene flow across all populations should be maintained. The present findings might be beneficial for conservation and contribute the sustainable use of L. camtschaticum resource.

The integration of multiple independent data reveals an unusual response toPleistocene climatic changes in the hard tickIxodes ricinus

In the last few years, improved analytical tools and the integration of genetic data with multiple sources of information have shown that temperate species exhibited more complex responses to ice ages than previously thought. In this study, we investigated how Pleistocene climatic changes affected the current distribution and genetic diversity of European populations of the tick Ixodes ricinus, an ectoparasite with high ecological plasticity. We first used mitochondrial and nuclear genetic markers to investigate the phylogeographic structure of the species and its Pleistocene history using coalescent-based methods; then we used species distribution modelling to infer the climatic niche of the species at last glacial maximum; finally, we reviewed the literature on the I.ricinus hosts to identify the locations of their glacial refugia. Our results support the scenario that during the last glacial phase, I.ricinus never experienced a prolonged allopatric divergence in separate glacial refugia, but persisted with interconnected populations across Southern and Central Europe. The generalist behaviour in host choice of I.ricinus would have played a major role in maintaining connections between its populations. Although most of the hosts persisted in separate refugia, from the point of view of I.ricinus, they represented a continuity of 'bridges' among populations. Our study highlights the importance of species-specific ecology in affecting responses to Pleistocene glacial-interglacial cycles. Together with other cases in Europe and elsewhere, it contributes to setting new hypotheses on how species with wide ecological plasticity coped with Pleistocene climatic changes.

Phylogeographic analysis of the true lemurs (genus Eulemur) underlines the role of river catchments for the evolution of micro-endemism in Madagascar

IntroductionDue to its remarkable species diversity and micro-endemism, Madagascar has recently been suggested to serve as a biogeographic model region. However, hypothesis-based tests of various diversification mechanisms that have been proposed for the evolution of the island’s micro-endemic lineages are still limited. Here, we test the fit of several diversification hypotheses with new data on the broadly distributed genus Eulemur using coalescent-based phylogeographic analyses.ResultsTime-calibrated species tree analyses and population genetic clustering resolved the previously polytomic species relationships among eulemurs. The most recent common ancestor of eulemurs was estimated to have lived about 4.45 million years ago (mya). Divergence date estimates furthermore suggested a very recent diversification among the members of the “brown lemur complex”, i.e. former subspecies of E. fulvus, during the Pleistocene (0.33-1.43 mya). Phylogeographic model comparisons of past migration rates showed significant levels of gene flow between lineages of neighboring river catchments as well as between eastern and western populations of the redfronted lemur (E. rufifrons).ConclusionsTogether, our results are concordant with the centers of endemism hypothesis (Wilmé et al. 2006, Science 312:1063–1065), highlight the importance of river catchments for the evolution of Madagascar’s micro-endemic biota, and they underline the usefulness of testing diversification mechanisms using coalescent-based phylogeographic methods.

Recipient of the 2012 Molecular Ecology Prize: Craig Moritz

Recipient of the 2012 <i>Molecular Ecology</i> Prize: Craig Moritz

Cryptic speciation in the field vole: a multilocus approach confirms three highly divergent lineages in Eurasia

Species are generally described from morphological features, but there is growing recognition of sister forms that show substantial genetic differentiation without obvious morphological variation and may therefore be considered 'cryptic species'. Here, we investigate the field vole (Microtus agrestis), a Eurasian mammal with little apparent morphological differentiation but which, on the basis of previous sex-linked nuclear and mitochondrial DNA (mtDNA) analyses, is subdivided into a Northern and a Southern lineage, sufficiently divergent that they may represent two cryptic species. These earlier studies also provided limited evidence for two major mtDNA lineages within Iberia. In our present study, we extend these findings through a multilocus approach. We sampled 163 individuals from 46 localities, mainly in Iberia, and sequenced seven loci, maternally, paternally and biparentally inherited. Our results show that the mtDNA lineage identified in Portugal is indeed a distinct third lineage on the basis of other markers as well. In fact, multilocus coalescent-based methods clearly support three separate evolutionary units that may represent cryptic species: Northern, Southern and Portuguese. Divergence among these units was inferred to have occurred during the last glacial period; the Portuguese lineage split occurred first (estimated at c. 70 000 bp), and the Northern and Southern lineages separated at around the last glacial maximum (estimated at c. 18 500 bp). Such recent formation of evolutionary units that might be considered species has repercussions in terms of understanding evolutionary processes and the diversity of small mammals in a European context.

Molecular-based rapid inventories of sympatric diversity: A comparison of DNA barcode clustering methods applied to geography-based vs clade-based sampling of amphibians

Molecular markers offer a universal source of data for quantifying biodiversity. DNA barcoding uses a standardized genetic marker and a curated reference database to identify known species and to reveal cryptic diversity within wellsampled clades. Rapid biological inventories, e.g. rapid assessment programs (RAPs), unlike most barcoding campaigns, are focused on particular geographic localities rather than on clades. Because of the potentially sparse phylogenetic sampling, the addition of DNA barcoding to RAPs may present a greater challenge for the identification of named species or for revealing cryptic diversity. In this article we evaluate the use of DNA barcoding for quantifying lineage diversity within a single sampling site as compared to clade-based sampling, and present examples from amphibians. We compared algorithms for identifying DNA barcode clusters (e.g. species, cryptic species or Evolutionary Significant Units) using previously published DNA barcode data obtained from geography-based sampling at a site in Central Panama, and from clade-based sampling in Madagascar. We found that clustering algorithms based on genetic distance performed similarly on sympatric as well as clade-based barcode data, while a promising coalescent-based method performed poorly on sympatric data. The various clustering algorithms were also compared in terms of speed and software implementation. Although each method has its shortcomings in certain contexts, we recommend the use of the ABGD method, which not only performs fairly well under either sampling method, but does so in a few seconds and with a user-friendly Web interface.

PHYLOGENETIC INFERENCE OF NUPTIAL TRAIT EVOLUTION IN THE CONTEXT OF ASYMMETRICAL INTROGRESSION IN NORTH AMERICAN DARTERS (TELEOSTEI)

Introgressive hybridization and incomplete lineage sorting complicate the inference of phylogeny, and available species-tree methods do not simultaneously account for these processes. Both hybridization and ancestral polymorphism have been invoked to explain divergent phylogenies inferred from different datasets for Stigmacerca, a clade of 11 North American darter species. Species of Stigmacerca are characterized by a mating system involving parental care with males guarding nesting territories and fertilized eggs. Males of four species of Stigmacerca develop egg-mimic nuptial structures on their second dorsal fins during the breeding season. Previous phylogenies suggest contrasting scenarios for the evolution of this nuptial trait. Using a combination of coalescent-based methods, we analyzed a dataset comprising a mitochondrial gene and 15 nuclear loci to estimate relationships and simultaneously test for introgressive hybridization. Our analyses identified several instances of interspecific gene flow involving both cytoplamsmic haplotypes and nuclear alleles. The new phylogeny was used to infer a single origin and recent loss of egg-mimic structures in Stigmacerca and led to the discovery of a phylogenetically distinct species. Our results highlight the limited strategies available to account for introgressive hybridization in the inference of species relationships and the likely effects of this process on reconstructing trait evolution.

Evolutionary and molecular analysis of the emergent severe fever with thrombocytopenia syndrome virus

In 2009, a novel Bunyavirus, called severe fever with thrombocytopenia syndrome virus (SFTSV) was identified in the vicinity of Huaiyangshan, China. Clinical symptoms of this zoonotic virus included severe fever, thrombocytopenia, and leukocytopenia, with a mortality rate of ∼10%. By the end of 2011 the disease associated with this pathogen had been reported from eleven Chinese provinces and human-to-human transmission suspected. However, current understanding of the evolution and molecular epidemiology of SFTSV before and after its identification is limited. To address this we undertake phylogenetic, evolutionary and structural analyses of all available SFTSV genetic sequences, including a new SFTSV complete genome isolated from a patient from Henan in 2011. Our discovery of a mosaic L segment sequence, which is descended from two major circulating lineages of SFTSV in China, represents the first evidence that homologous recombination plays a role in SFTSV evolution. Selection analyses indicate that negative selection is predominant in SFTSV genes, yet differences in selective forces among genes are consistent between Phlebovirus species. Further analysis reveals structural conservation between SFTSV and Rift Valley fever virus in the residues of their nucleocapsids that are responsible for oligomerisation and RNA-binding, suggesting the viruses share similar modes of higher-order assembly. We reconstruct the epidemic history of SFTSV using molecular clock and coalescent-based methods, revealing that the extant SFTSV lineages originated 50–150 years ago, and that the viral population experienced a recent growth phase that concurs with and extends the earliest serological reports of SFTSV infection. Taken together, our combined structural and phylogenetic analyses shed light into the evolutionary behaviour of SFTSV in the context of other, better-known, pathogenic Phleboviruses.

Testing hypotheses for genealogical discordance in a rainforest lizard

Genealogical discordance, or when different genes tell distinct stories although they evolved under a shared history, often emerges from either coalescent stochasticity or introgression. In this study, we present a strong case of mito-nuclear genealogical discordance in the Australian rainforest lizard species complex of Saproscincus basiliscus and S. lewisi. One of the lineages that comprises this complex, the Southern S. basiliscus lineage, is deeply divergent at the mitochondrial genome but shows markedly less divergence at the nuclear genome. By placing our results in a comparative context and reconstructing the lineages' demography via multilocus and coalescent-based approximate Bayesian computation methods, we test hypotheses for how coalescent variance and introgression contribute to this pattern. These analyses suggest that the observed genealogical discordance likely results from introgression. Further, to generate such strong discordance, introgression probably acted in concert with other factors promoting asymmetric gene flow between the mitochondrial and nuclear genomes, such as selection or sex-biased dispersal. This study offers a framework for testing sources of genealogical discordance and suggests that historical introgression can be an important force shaping the genetic diversity of species and their populations.

Phylodynamics of HIV-1 Subtype C Epidemic in East Africa

The HIV-1 subtype C accounts for an important fraction of HIV infections in east Africa, but little is known about the genetic characteristics and evolutionary history of this epidemic. Here we reconstruct the origin and spatiotemporal dynamics of the major HIV-1 subtype C clades circulating in east Africa. A large number (n = 1,981) of subtype C pol sequences were retrieved from public databases to explore relationships between strains from the east, southern and central African regions. Maximum-likelihood phylogenetic analysis of those sequences revealed that most (>70%) strains from east Africa segregated in a single regional-specific monophyletic group, here called CEA. A second major Ethiopian subtype C lineage and a large collection of minor Kenyan and Tanzanian subtype C clades of southern African origin were also detected. A Bayesian coalescent-based method was then used to reconstruct evolutionary parameters and migration pathways of the CEA African lineage. This analysis indicates that the CEA clade most probably originated in Burundi around the early 1960s, and later spread to Ethiopia, Kenya, Tanzania and Uganda, giving rise to major country-specific monophyletic sub-clusters between the early 1970s and early 1980s. The results presented here demonstrate that a substantial proportion of subtype C infections in east Africa resulted from dissemination of a single HIV local variant, probably originated in Burundi during the 1960s. Burundi was the most important hub of dissemination of that subtype C clade in east Africa, fueling the origin of new local epidemics in Ethiopia, Kenya, Tanzania and Uganda. Subtype C lineages of southern African origin have also been introduced in east Africa, but seem to have had a much more restricted spread.

Genetic variation and population decline of an endangered hoverfly Blera fallax (Diptera: Syrphidae)

Genetic diversity is one of several factors affecting extinction risk in vulnerable populations. In addition to informing conservation management strategies, data on genetic variability can also shed light on the recency and magnitude of historic bottlenecks. The pine hoverfly Blera fallax is one of the rarest invertebrates in the UK, known from just two sites in Scotland. It belongs to an often overlooked, species-rich community that is fundamental to forest function, the saproxylics (that depend on dead wood). To assist current conservation management for B. fallax, including captive breeding and translocations, it is important to know whether genetic factors will limit the success of recovery. Using 12 microsatellite loci, we compared the genetic variation in Scottish and Swedish specimens (Swedish populations are thought to represent a more outbred B. fallax population). As expected, the Scottish population showed significantly lower levels of polymorphism, expected heterozygosity and allelic richness than the Swedish population. Furthermore, significant genetic differentiation was found between the two B. fallax populations (FST = 0.134). We then used an allele frequency-based approach and a Bayesian coalescent-based method to assess genealogical history and detect recent changes in population size. Unexpectedly, data from not only the Scottish but also the Swedish population indicated a strong and relatively recent decline that was more pronounced in Scotland. We discuss the implications of our findings for future conservation management planning, the first undertaking of its kind for saproxylic species in Britain.

A multigene species tree for Western Mediterranean painted frogs (Discoglossus)

Painted frogs (Discoglossus) are an anuran clade that originated in the Upper Miocene. Extant species are morphologically similar and have a circum-Mediterranean distribution. We assembled a multilocus dataset from seven nuclear and four mitochondrial genes for several individuals of all but one of the extant species and reconstructed a robust phylogeny by applying a coalescent-based species-tree method and a concatenation approach, both of which gave congruent results. The earliest phylogenetic split within Discoglossus separates D. montalentii from a clade comprising all other species. Discoglossus montalentii is monophyletic for haplotype variation at all loci and has distinct morphological, bioacoustic and karyotypic characters. We find moderate support for a sister-group relationship between the Iberian taxa and the Moroccan D. scovazzi, and high support for a D. pictus –D. sardus clade distributed around the Tyrrhenian basin. Topological discordance among gene trees during the speciation of D. galganoi, D. scovazzi, D. pictus and D. sardus is interpreted as the consequence of nearly simultaneous, vicariant diversification. The timing of these events is unclear, but possibly coincided with the final geotectonic rearrangement of the Western Mediterranean in the Middle Miocene or later during the Messinian salinity crisis. The Iberian taxa D. galganoi galganoi and D. g. jeanneae are reciprocally monophyletic in mitochondrial DNA but not in nuclear gene trees, and are therefore treated as subspecies of D. galganoi.

Species tree of a recent radiation: The subfamily Delphininae (Cetacea, Mammalia)

Lineages undergoing rapid radiations provide exceptional opportunities for studying speciation and adaptation, but also represent a challenge for molecular systematics because retention of ancestral polymorphisms and the occurrence of hybridization can obscure relationships among lineages. Dolphins in the subfamily Delphininae are one such case. Non-monophyly, rapid speciation events, and discordance between morphological and molecular characters have made the inference of phylogenetic relationships within this subfamily very difficult. Here we approach this problem by applying multiple methods intended to estimate species trees using a multi-gene dataset for the Delphininae (Sousa, Sotalia, Stenella, Tursiops, Delphinus and Lagenodelphis). Incongruent gene trees obtained indicate that incomplete lineage sorting and possibly hybridization are confounding the inference of species history in this group. Nonetheless, using coalescent-based methods, we have been able to extract an underlying species-tree signal from divergent histories of independent genes. This is the first time a molecular study provides support for such relationships. This study further illustrates how methods of species-tree inference can be very sensitive both to the characteristics of the dataset and the evolutionary processes affecting the evolution of the group under study.