Length–weight relationships of two fish species from Yalong River,southwestern China

Length–weight relationships (LWRs) were evaluated for Jinshaia sinensis (Sauvage & Dabry de Thiersant, 1874) and Lepturichthys fimbriata (Günther, 1888) using a total of 289 specimens caught by gill nets (mesh size 5.0 cm) and angling in the Yalong River, southwestern China during February and March 2007. This study presents the first reference on LWR for these species from the Yalong River.     

Cytonuclear discordance among Southeast Asian black rats (Rattus rattus complex)

Black rats are major invasive vertebrate pests with severe ecological, economic and health impacts. Remarkably, their evolutionary history has received little attention, and there is no firm agreement on how many species should be recognized within the black rat complex. This species complex is native to India and Southeast Asia. According to current taxonomic classification, there are three taxa living in sympatry in several parts of Thailand, Cambodia and Lao People's Democratic Republic, where this study was conducted: two accepted species (Rattus tanezumi, Rattus sakeratensis) and an additional mitochondrial lineage of unclear taxonomic status referred to here as ‘Rattus R3’. We used extensive sampling, morphological data and diverse genetic markers differing in rates of evolution and parental inheritance (two mitochondrial DNA genes, one nuclear gene and eight microsatellite loci) to assess the reproductive isolation of these three taxa. Two close Asian relatives, Rattus argentiventer and Rattus exulans, were also included in the genetic analyses. Genetic analyses revealed discordance between the mitochondrial and nuclear data. Mitochondrial phylogeny studies identified three reciprocally monophyletic clades in the black rat complex. However, studies of the phylogeny of the nuclear exon interphotoreceptor retinoid‐binding protein gene and clustering and assignation analyses with eight microsatellites failed to separate R. tanezumi and R3. Morphometric analyses were consistent with nuclear data. The incongruence between mitochondrial and nuclear (and morphological) data rendered R. tanezumi/R3 paraphyletic for mitochondrial lineages with respect to R. sakeratensis. Various evolutionary processes, such as shared ancestral polymorphism and incomplete lineage sorting or hybridization with massive mitochondrial introgression between species, may account for this unusual genetic pattern in mammals.     

Multilocus phylogeny and taxonomy of East Asian voles Alexandromys (Rodentia,Arvicolinae)

Phylogenetic relationships, taxonomy and nomenclature issues within East Asian voles Alexandromys were addressed using comprehensive species samples, including all 12 valid species of the genus, and multilocus analysis. We examined the mitochondrial cytochrome b (cytb) gene and three nuclear genes in 36 specimens. Additionally, we examined a data set of 106 specimens using only the cytb gene. Our results did not confirm the aggregation of A. kikuchii, A. montebelli and A. oeconomus into a separate clade, namely the subgenus Pallasiinus. Analysis of incomplete lineage sorting using JML software highlighted both the cases of mitochondrial introgression and incomplete lineage sorting within the genus. Thus, the sister position of A. sachalinensis and A. maximowiczii in mitochondrial trees could be explained by mitochondrial introgression, while the sister position of A. limnophilus and A. fortis in mitochondrial trees could be successfully explained by incomplete lineage sorting. Very short genetic distances, together with an absence of monophyly, of the three species, A. evoronensis, A. mujanensis and A. maximowiczii, is supported by multiple morphological data, which indicates that these three taxa should be one species—A. maximowiczii. Analysis of genetic distances and tree topology revealed that three species of short‐tailed voles—A. middendorffii, A. mongolicus and A. gromovi—are more closely related to each other than to other established species of Alexandromys. The lacustrine vole, A. limnophilus, is closely related to the group of short‐tailed voles. Analysis of the type specimens of limnophilus and flaviventris confirmed that these taxa form one species together with A. l. malygini. Our results suggest that the mountains of western Mongolia are inhabited by a new taxon of short‐tailed voles of the same rank as middendorffii, mongolicus and gromovi—A. m. alpinus ssp. n.     

Fuzzy species limits in Mediterranean gorgonians (Cnidaria,Octocorallia): inferences on speciation processes

The study of the interplay between speciation and hybridization is of primary importance in evolutionary biology. Octocorals are ecologically important species whose shallow phylogenetic relationships often remain to be studied. In the Mediterranean Sea, three congeneric octocorals can be observed in sympatry: Eunicella verrucosa, Eunicella cavolini and Eunicella singularis. They display morphological differences and E. singularis hosts photosynthetic Symbiodinium, contrary to the two other species. Two nuclear sequence markers were used to study speciation and gene flow between these species, through network analysis and Approximate Bayesian Computation (ABC). Shared sequences indicated the possibility of hybridization or incomplete lineage sorting. According to ABC, a scenario of gene flow through secondary contact was the best model to explain these results. At the intraspecific level, neither geographical nor ecological isolation corresponded to distinct genetic lineages in E. cavolini. These results are discussed in the light of the potential role of ecology and genetic incompatibilities in the persistence of species limits.     

A mirage of cryptic species: Genomics uncover striking mitonuclear discordance in the butterfly Thymelicus sylvestris

Mitochondrial DNA (mtDNA) sequencing has led to an unprecedented rise in the identification of cryptic species. However, it is widely acknowledged that nuclear DNA (nuDNA) sequence data are also necessary to properly define species boundaries. Next generation sequencing techniques provide a wealth of nuclear genomic data, which can be used to ascertain both the evolutionary history and taxonomic status of putative cryptic species. Here, we focus on the intriguing case of the butterfly Thymelicus sylvestris (Lepidoptera: Hesperiidae). We identified six deeply diverged mitochondrial lineages; three distributed all across Europe and found in sympatry, suggesting a potential case of cryptic species. We then sequenced these six lineages using double‐digest restriction‐site associated DNA sequencing (ddRADseq). Nuclear genomic loci contradicted mtDNA patterns and genotypes generally clustered according to geography, i.e., a pattern expected under the assumption of postglacial recolonization from different refugia. Further analyses indicated that this strong mtDNA/nuDNA discrepancy cannot be explained by incomplete lineage sorting, sex‐biased asymmetries, NUMTs, natural selection, introgression or Wolbachia‐mediated genetic sweeps. We suggest that this mitonuclear discordance was caused by long periods of geographic isolation followed by range expansions, homogenizing the nuclear but not the mitochondrial genome. These results highlight T. sylvestris as a potential case of multiple despeciation and/or lineage fusion events. We finally argue, since mtDNA and nuDNA do not necessarily follow the same mechanisms of evolution, their respective evolutionary history reflects complementary aspects of past demographic and biogeographic events.     

Timing major conflict between mitochondrial and nuclear genes in species relationships of Polygonia butterflies (Nymphalidae: Nymphalini)

Background  

Major conflict between mitochondrial and nuclear genes in estimating species relationships is an increasingly common finding in animals. Usually this is attributed to incomplete lineage sorting, but recently the possibility has been raised that hybridization is important in generating such phylogenetic patterns. Just how widespread ancient and/or recent hybridization is in animals and how it affects estimates of species relationships is still not well-known.     

Positive and purifying selection in mitochondrial genomes of a bird with mitonuclear discordance

Diversifying selection on metabolic pathways can reduce intraspecific gene flow and promote population divergence. An opportunity to explore this arises from mitonuclear discordance observed in an Australian bird Eopsaltria australis. Across >1500 km, nuclear differentiation is low and latitudinally structured by isolation by distance, whereas two highly divergent, parapatric mitochondrial lineages (>6.6% in ND2) show a discordant longitudinal geographic pattern and experience different climates. Vicariance, incomplete lineage sorting and sex‐biased dispersal were shown earlier to be unlikely drivers of the mitonuclear discordance; instead, natural selection on a female‐linked trait was the preferred hypothesis. Accordingly, here we tested for signals of positive, divergent selection on mitochondrial genes in E. australis. We used codon models and physicochemical profiles of amino acid replacements to analyse complete mitochondrial genomes of the two mitochondrial lineages in E. australis, its sister species Eopsaltria griseogularis, and outgroups. We found evidence of positive selection on at least five amino acids, encoded by genes of two oxidative phosphorylation pathway complexes NADH dehydrogenase (ND4 and ND4L) and cytochrome bc₁ (cyt‐b) against a background of widespread purifying selection on all mitochondrial genes. Three of these amino acid replacements were fixed in ND4 of the geographically most widespread E. australis lineage. The other two replacements were fixed in ND4L and cyt‐b of the geographically more restricted E. australis lineage. We discuss whether this selection may reflect local environmental adaptation, a by‐product of other selective processes, or genetic incompatibilities, and propose how these hypotheses can be tested in future.     

Coalescent species delimitation of a Sumatran parachuting frog

We conduct species delimitation of the widespread parachuting frog species Rhacophorus catamitus using samples from across the island of Sumatra, Indonesia. We use mitochondrial, genomic and morphological data, and find that R. catamitus is composed of three lineages corresponding to northern, central and southern lineages. Mitochondrial and genomic data show admixture or incomplete lineage sorting between the central and southern lineages, but deep divergence from the northern lineage. Coalescent species delimitation supports a three species model for this complex, and we recommend that the northern lineage be described as a new species. Our study highlights the power of coalescent species delimitation in an integrative framework for identifying unrecognised diversity in understudied tropical species complexes. We also emphasise the evolutionary importance of northern Sumatra, a region that harboured montane refugia during Pliocene–Pleistocene climate change, but has also been heavily affected by volcanic activity.     

Nuclear and mitochondrial data reveal different evolutionary processes in the Lake Tanganyika cichlid genus <Emphasis Type="Italic">Tropheus</Emphasis>

Background  

Cichlid fishes are notorious for their wealth of intra- and interspecific colour pattern diversity. In Lake Tanganyika, the endemic genus Tropheus represents the most impressive example for geographic variation in the pattern and hue of integument colouration, but the taxonomy of the over 100 mostly allopatric colour morphs remains to a large degree unresolved. Previous studies of mitochondrial DNA sequence data revealed polyphyly of the six nominally described species and complex phylogeographic patterns influenced by lake level fluctuations and population admixture, and suggested the parallel evolution of similar colour patterns in divergent evolutionary lineages. A gene tree of a rapidly radiating group may be subject to incomplete and stochastic lineage sorting, and to overcome this problem we used multi-locus, nuclear AFLP data in comparison with mtDNA sequences to study diversification, migration and introgression in Tropheus colour morphs in Lake Tanganyika.     

Molecular evidence for hybridization between two Australian desert skinks, Ctenotus leonhardii and Ctenotus quattuordecimlineatus (Scincidae: Squamata)

Australian scincid lizards in the genus Ctenotus constitute the most diverse vertebrate radiation in Australia. However, the evolutionary processes that have generated this diversity remain elusive, in part because both interspecific phylogenetic relationships and phylogeographic structure within Ctenotus species remain poorly known. Here we use nucleotide sequences from a mitochondrial locus and two nuclear introns to investigate broad-scale phylogeographic patterns within Ctenotus leonhardii and C. quattuordecimlineatus, two geographically widespread species of skinks that were found to have a surprisingly close genetic relationship in a previous molecular phylogenetic study. We demonstrate that the apparent close relationship between these ecologically and phenotypically distinct taxa is attributable to mitochondrial introgression from C. quattuordecimlineatus to C. leonhardii. In the western deserts, Ctenotus leonhardii individuals carry mtDNA lineages that are derived from C. quattuordecimlineatus mtDNA lineages from that geographic region. Coalescent simulations indicate that this pattern is unlikely to have resulted from incomplete lineage sorting, implicating introgressive hybridization as the cause of this regional gene-tree discordance.     

Phylogeny and biogeography of the Poecilia sphenops species complex (Actinopterygii,Poeciliidae) in Central America

We inferred the phylogenetic relationships among members of the Poecilia sphenops species complex to resolve the colonization process and radiation of this group in Central America. We analyzed 2550 base pairs (bp) of mitochondrial DNA (mtDNA), including ATP synthase 6 and 8, cytochrome oxidase subunit I and NADH dehydrogenase subunit 2 genes, and 906 bp of the nuclear S7 ribosomal protein of 86 ingroup individuals from 61 localities spanning most of its distribution from Mexico to Panama. Our mitochondrial data rendered a well-supported phylogeny for the P. sphenops complex that differed with the nuclear data set topology, which did not recover the monophyly of the P. mexicana mitochondrial lineage. Coalescent-based simulations tests indicated that, although hybridization cannot be completely ruled out, this incongruence is most likely due to incomplete lineage sorting in this group, which also showed the widest geographic distribution. A single colonization event of Central America from South America was estimated to have occurred between the early Paleocene and Oligocene (53–22 million years ago). Subsequently, two largely differentiated evolutionary lineages diverged around the Early Oligocene–Miocene (38–13 million years ago), which are considered two separate species complexes: P. sphenops and P. mexicana, which can also be distinguished by their tricuspid and unicuspid inner jaw teeth, respectively. Ultimately, within lineage diversification occurred mainly during the Miocene (22–5 million years ago). All major cladogenetic events predated the final closure of the Isthmus of Panama. The allopatric distribution of lineages together with the long basal internodes suggest that vicariance and long term isolations could be the main evolutionary forces promoting radiation in this group, although dispersal through water barriers might also have occurred. Lastly, our results suggest the need to review the current species distribution and taxonomy of the P. sphenops complex sensu lato.     

Reproductive compatibility among Mexican populations of Anastrepha obliqua: theoretical and management implications

The fraterculus species group, composed of 34 species in the genus Anastrepha (Diptera: Tephritidae), includes the fraterculus cryptic species complex formed by eight reproductively isolated morphotypes. A previous study revealed six genetic mitochondrial types of Anastrepha obliqua, suggesting the existence of a second cryptic species complex. However, marked discrepancies between nuclear and mitochondrial loci rather suggest incomplete lineage sorting or introgression between A. obliqua and A. fraterculus. Such hybridization could nevertheless result in reproductive isolation, an outcome that could affect efficacy of area‐wide management for the most important pest of mangos in America. Two mitochondrial types occur in Mexico, and the limits of a third one, encompassing Central American populations, have not been clearly established. Here, we tested reproductive compatibility among three A. obliqua populations from the Pacific and a population from the Gulf of Mexico. No evidence of pre‐zygotic isolation was found. Flies from the Atlantic mated randomly for equal duration with individuals from three Pacific populations. Homotypic and heterotypic crosses produced similar numbers of eggs, with heterotypic crosses of Pacific males and Atlantic females hatching in lower proportions. Larvae of all cross types developed equally in mangos and exhibited no sex ratio distortion of hybrid F1. The three mitochondrial types identified in Mexico and Central America do not appear to be cryptic species and can be managed using one single strain for the sterile insect technique.     

Hybridization could be a common phenomenon within the highly diverse lizard genus Liolaemus

Hybridization is likely to occur more often between closely related taxa that have had insufficient time to diverge to the point of reproductive incompatibility; hybridization between deeply divergent lineages is rare. In squamate reptiles, hybridization has been proposed as a possible explanation for the extensive paraphyly observed in mitochondrial gene trees in several species complexes of the South American lizard genus Liolaemus. One of the best‐documented cases is within the L. boulengeri and L. rothi complexes, which diverged ~5.5 million years ago. Here, we describe a comprehensive study for approaching the hybridization hypothesis between these lizard species complexes. We explored the level of gene tree discordance using the novel ‘extra lineage contribution’ statistics (XLC, presented in this study) that quantifies the level of gene tree discordance contribution per individual within a species. We included molecular data (12 nuclear and two mitochondrial genes) from 127 individuals, and results of a coalescent model‐based analysis show that the most likely explanation for the gene tree‐species tree discordance is interspecific hybridization. Our best‐supported hypothesis suggests current and past hybridization between L. rothi (rothi complex) and L. tehuelche (boulengeri complex), and independently between L. rothi and L. boulengeri and L. telsen (boulengeri complex). The hybrid descendants are characterized by intermediate phenotypes between the parental species, but are more similar to L. rothi in body size. We discuss the possible role of hybridization in Liolaemus evolution.     

Molecular and plumage analyses indicate the incomplete separation of two woodpeckers (Aves,Picidae)

We evaluated the relationship between Celeus undatus and Celeus grammicus, with the objective of clarifying their evolutionary history. We analysed fragments of the mitochondrial and nuclear genes of 57 specimens. For comparative purposes, we inspected the plumage patterns of 77 skins. Our findings highlight the absence of reciprocal monophyly between the two taxa, given their reduced genetic divergence, and the lack of any clear separation of the two forms in the haplotype networks. A similar situation was found in the STRUCTURE analysis, with reciprocal contributions from the two taxa to the respective clusters, indicating that C. grammicus and C. undatus cannot be differentiated using the molecular markers. Corroborating the genetic data, our plumage analyses also failed to find any clear diagnostic characters between the polytypic C. undatus and C. grammicus, as they are defined at present. The genetic profile is consistent with either extensive historical gene flow between the species or, alternatively, incomplete lineage sorting, rather than recent secondary contact. The lack of monophyly between the two taxa impeded subspecies‐level phylogeographic inferences, with the subspecific variation being interpreted as a probable artefact of the phenotypic plasticity of the two forms. These findings indicate clearly that the two taxa form a single evolutionary unit, in which the morphological differentiation used to diagnose the species, combined with their geographic distribution, is at odds with the incomplete separation of the taxa. This may reflect disparities in the rates of differentiation between molecular and phenotypic markers, which is possibly due to the variation in selection pressures along a humidity gradient in Amazonia.     

Genetic drift or natural selection? Hybridization and asymmetric mitochondrial introgression in two Caribbean lizards (Anolis pulchellus and Anolis krugi)

Hybridization and gene introgression can occur frequently between closely related taxa, but appear to be rare phenomena among members of the species‐rich West Indian radiation of Anolis lizards. We investigated the pattern and possible mechanism of introgression between two sister species from Puerto Rico, Anolis pulchellus and Anolis krugi, using mitochondrial (ND2) and nuclear (DNAH3, NKTR) DNA sequences. Our findings demonstrated extensive introgression of A. krugi mtDNA (k‐mtDNA) into the genome of A. pulchellus in western Puerto Rico, to the extent that k‐mtDNA has mostly or completely replaced the native mtDNA of A. pulchellus on this part of the island. We proposed two not mutually exclusive scenarios to account for the interspecific matings between A. pulchellus and A. krugi. We inferred that hybridization events occurred independently in several populations, and determined that k‐mtDNA haplotypes harboured in individuals of A. pulchellus can be assigned to four of the five major mtDNA clades of A. krugi. Further, the spatial distribution of k‐mtDNA clades in the two species is largely congruent. Based on this evidence, we concluded that natural selection was the probable driving mechanism for the extensive k‐mtDNA introgression into A. pulchellus. Our two nuclear data sets yielded different results. DNAH3 showed reciprocal monophyly of A. pulchellus and A. krugi, indicating no effect of hybridization on this marker. In contrast, the two species shared nine NKTR alleles, probably due to incomplete lineage sorting. Our study system will provide an excellent opportunity to experimentally assess the behavioural and ecological mechanisms that can lead to hybridization in closely related taxa.     

Reconstructing the evolutionary history of the radiation of the land snail genus <Emphasis Type="Italic">Xerocrassa</Emphasis> on Crete based on mitochondrial sequences and AFLP markers

Background  

A non-adaptive radiation triggered by sexual selection resulted in ten endemic land snail species of the genus Xerocrassa on Crete. Only five of these species and a more widespread species are monophyletic in a mitochondrial gene tree. The reconstruction of the evolutionary history of such closely related species can be complicated by incomplete lineage sorting, introgression or inadequate taxonomy. To distinguish between the reasons for the nonmonophyly of several species in the mitochondrial gene tree we analysed nuclear AFLP markers.     

Incomplete lineage sorting or secondary admixture: disentangling historical divergence from recent gene flow in the Vinous‐throated parrotbill (Paradoxornis webbianus)

Although Pleistocene glaciations had a major impact on the population genetic patterns of many species in North America and Europe, it remains unclear how these climatic fluctuations contributed to species diversification in East Asia. One reason for this is the difficulty of distinguishing genetic admixture following secondary contact from incomplete lineage sorting, both of which can generate similar patterns of genetic variation. Using a combination of multilocus analyses and coalescent simulation, we explore how these two processes occurred in the Pleistocene evolutionary history of a widespread East Asian bird, the Vinous‐throated parrotbill, Paradoxornis webbianus. Maximum likelihood (ML) tree identified two major mitochondrial lineages, which are geographically separated in most parts of its range, but are sympatric at a few sampling sites. NJ tree and Structure analysis of microsatellite data set revealed an extensive level of admixture and little population structure, suggesting recent admixture between two formerly separated groups. Networks from nuclear DNA data sets, however, did not indicate any geographically isolated groups but rather a panmictic population, thus support incomplete lineage sorting. By using coalescent simulation approaches, we show that both processes did occur, although at different temporal scales. During the Pleistocene glaciations, probably around 0.1–0.5 Ma (the Marine Isotope Stage 6, MIS6), P. webbianus contracted into two separate refugia, and subsequently accumulated genetic divergence. During the interglacial MIS5, the species expanded into previously glaciated areas allowing the once separated groups to come into contact and become admixed. Taken together, our results indicate the current genetic variation within P. webbianus is a combination pattern of widespread distribution in pre‐Pleistocene, then contraction and fragmentation into separated refugia during glacial advance, followed by recently postglacial expansion and admixture.     

Morphology and nuclear markers reveal extensive mitochondrial introgressions in the Iberian Wall Lizard species complex

Mitochondrial markers are still often used alone to identify evolutionary units, despite widespread evidence for processes such as incomplete lineage sorting or introgressive hybridization that may blur past population history. The combination of mitochondrial DNA data with other sources of information (morphology, nuclear genes) is a powerful tool to reveal when and why mitochondrial markers are potentially misleading. In this study, we evaluate the performance of mtDNA markers to unravel the evolutionary history of Spanish lizards from the Podarcis hispanicus species complex. We first uncover several cases of discordance between morphological and mitochondrial data in delimitation of taxa. To assess the origin of these discordances, we analysed the same populations using several independent nuclear loci. Both morphological and nuclear markers identified the same three evolutionary units in the region, while mitochondrial data revealed four deeply divergent lineages. We suggest here that the most likely scenario to explain this discordance is ancient mitochondrial introgression originating from a fourth evolutionary unit presently absent from the study area. Notably, this resulted in a complete replacement of the original lineage in a large part of the distribution of one of the taxa investigated. We discuss the potential evolutionary scenarios leading to this complete mitochondrial replacement and suggest why the previous studies have failed to recover the correct history of this species complex.     

The effect of the Messinian salinity crisis on the early diversification of the Tettigettalna cicadas

The current distribution patterns of many Mediterranean species are often a consequence of large and impactful past geoclimatic events, such as the Messinian Salinity Crisis (MSC) and the Quaternary glacial cycles. Cicadas are flying insects with poor dispersal ability, which have experienced intense local differentiation in the Mediterranean, where the genus Tettigettalna has surfaced as a biogeographic model. The genus includes 10 species with species-specific calling songs but identical morphology. All Tettigettalna species are restricted to Southern Iberia, with the exception of T. estrellae (northwest Iberia), the widespread T. argentata (mainly Iberia, France and Italy), and T. afroamissa (Morocco). With an expanded genetic dataset involving nuclear (EF1α) and mitochondrial (5′ and 3′ COI and ATP) loci, we reconstructed the phylogeny of the genus and estimated divergence dates for Tettigettalna species under a Bayesian framework. Phylogeny with the new mitochondrial dataset was in agreement with previous studies, whereas the nuclear EF1α supported T. josei and T. afroamissa as monophyletic clades but lacked resolution to resolve the remaining taxa. Some sister taxa share mitochondrial haplotypes, hinting for incomplete lineage sorting. Estimates of divergence time settled T. josei as the earliest diverging lineage, likely as a pre- or early-MSC event. As for the origin of T. afroamissa in Morocco, though time estimates could not entirely rule out post-MSC dispersal, the most likely scenario points to isolation of African Tettigettalna after the reopening of the strait of Gibraltar. The Pleistocene glaciations that followed likely impacted on the diversification of the remaining species of the genus in southern Iberia refugia.     

Frequent mitochondrial gene introgression among high elevation Tibetan megophryid frogs revealed by conflicting gene genealogies

Historical mitochondrial introgression causes differences between a species' mitochondrial gene genealogy and its nuclear gene genealogy, making tree-based species delineation ambiguous. Using sequence data from one mitochondrial gene (cytochrome b ) and three nuclear genes (introns), we examined the evolutionary history of four high elevation Tibetan megophryid frog species, Scutiger boulengeri , Scutiger glandulatus , Scutiger mammatus and Scutiger tuberculatus . The three nuclear genes shared a similar history but the mitochondrial gene tree suggested a drastically different evolutionary scenario. The conflicts between them were explained by multiple episodes of mitochondrial introgression events via historical interspecific hybridization. 'Foreign' mitochondrial genomes might have been fixed in populations and extended through a large portion of the species' distribution. Some hybridization events were probably as old as 10 Myr, while others were recent. An F₁ hybrid was also identified. Historical hybridization events among the four species appeared to be persistent and were not restricted to the period of Pleistocene glaciation, as in several other well-studied cases. Furthermore, hybridization involved several species and occurred in multiple directions, and there was no indication of one mitochondrial genome being superior to others. In addition, incomplete lineage sorting resulting from budding speciation may have also explained some discrepancies between the mitochondrial DNA and nuclear gene trees. Combining all evidences, the former ' Scutiger mammatus ' appeared to be two species, including a new species. With the availability of a wide range of highly variable nuclear gene markers, we recommend using a combination of mitochondrial gene and multiple nuclear genes to reveal a complete species history.