Divergent Mitochondrial Lineages Research Articles

Unravelling the maternal evolutionary history of the African leopard (Panthera pardus pardus).

The African leopard (Panthera pardus pardus) has lost a significant proportion of its historical range, notably in north-western Africa and South Africa. Recent studies have explored the genetic diversity and population structure of African leopards across the continent. A notable genetic observation is the presence of two divergent mitochondrial lineages, PAR-I and PAR-II. Both lineages appeared to be distributed widely, with PAR-II frequently found in southern Africa. Until now, no study has attempted to date the emergence of either lineage, assess haplotype distribution, or explore their evolutionary histories in any detail. To investigate these underappreciated questions, we compiled the largest and most geographically representative leopard data set of the mitochondrial NADH-5 gene to date. We combined samples (n = 33) collected in an altitudinal transect across the Mpumalanga province of South Africa, where two populations of leopard are known to be in genetic contact, with previously published sequences of African leopard (n = 211). We estimate that the maternal PAR-I and PAR-II lineages diverged approximately 0.7051 (0.4477-0.9632) million years ago (Ma). Through spatial and demographic analyses, we show that while PAR-I underwent a mid-Pleistocene population expansion resulting in several closely related haplotypes with little geographic structure across much of its range, PAR-II remained at constant size and may even have declined slightly in the last 0.1 Ma. The higher genetic drift experienced within PAR-II drove a greater degree of structure with little haplotype sharing and unique haplotypes in central Africa, the Cape, KwaZulu-Natal and the South African Highveld. The phylogeographic structure of PAR-II, with its increasing frequency southward and its exclusive occurrence in south-eastern South Africa, suggests that this lineage may have been isolated in South Africa during the mid-Pleistocene. This hypothesis is supported by historical changes in paleoclimate that promoted intense aridification around the Limpopo Basin between 1.0-0.6 Ma, potentially reducing gene flow and promoting genetic drift. Interestingly, we ascertained that the two nuclear DNA populations identified by a previous study as East and West Mpumalanga correspond to PAR-I and PAR-II, respectively, and that they have come into secondary contact in the Lowveld region of South Africa. Our results suggest a subdivision of African leopard mtDNA into two clades, with one occurring almost exclusively in South Africa, and we identify the potential environmental drivers of this observed structure. We caution that our results are based on a single mtDNA locus, but it nevertheless provides a hypothesis that can be further tested with a dense sample of nuclear DNA data, preferably whole genomes. If our interpretation holds true, it would provide the first genetic explanation for the smaller observed size of leopards at the southernmost end of their range in Africa.

A preliminary taxonomic revision of the girii clade of South Asian Cnemaspis Strauch, 1887 (Squamata: Gekkonidae) with the description of four new species from southern Maharashtra, India

The girii clade is the most diverse clade of South Asian Cnemaspis in the Northern Western Ghats, including ten valid species. Recent collections of four divergent lineages from southern Maharashtra prompted us to examine the type material of all species in the girii clade, which led to the discovery of numerous errors and discrepancies in the literature. We take this opportunity to provide an updated and expanded morphological dataset including diagnoses and comparisons for all species of the girii clade and to build an almost complete species-level phylogeny including the first ND2 sequences of C. rajgadensis and C. uttaraghati, as well as the four new species described herein (but excluding C. fortis). We describe the four divergent mitochondrial lineages as new species using morphological data, and provide a key to species of the girii clade. Finally, we list some inconsistences in previous publications and data for the girii clade.

Global species delimitation of the cosmopolitan marine littoral earthworm Pontodrilus litoralis (Grube, 1855)

The marine littoral earthworm Pontodrilus litoralis (Grube, 1855) is widely distributed and is reported as a single species. This study utilized an integrative taxonomic approach based upon morphological examination, phylogenetic reconstruction, and molecular species delimitation, to test whether the taxon is a single species or a species complex. For this, a total of 114 P. litoralis specimens collected from North America, Africa, Australia and Oceania, Europe and Asia were used. The phylogenetic analyses revealed deeply divergent mitochondrial lineages and a high level of genetic diversity among P. litoralis populations. Both single and multi-locus species delimitation analyses yielded several molecular operational taxonomic units. Therefore, due to the homogeneity of morphological characteristics, it is likely that the morphospecies P. litoralis is a complex of four or more cryptic species, suggesting that more sampling is required and that the population structure genetic data and gene flow need to be investigated.

Genetics, Age Demographics, and Shell Size of Atlantic Surfclams from the Southern Edge of Their Range

The Atlantic surfclam (Spisula solidissima solidissima) is an economically valuable clam species that supports a major US fishery. Until recently, fishery efforts along the southern edge of the surfclam range were low due to clam mortalities there in the 1990s. Recent surfclam fishing efforts off Virginia raised questions of whether the surfclam population has returned in the southern region or if a single cohort is supporting the fishery there. Questions have also arisen about whether S. s. similis is among the population fished off the coast of VA. Spisula solidissima similis is a warm-water cryptic subspecies of the Atlantic surfclam. Although morphologically indistinguishable, S. s. similis grows to a smaller size and is genetically distinct. Atlantic surfclams (n = 103) were collected from the fishing grounds off the coast of VA. Each surfclam was aged, and shell length and tissue weight recorded for comparison to surfclams of the same age from the center of the population. Analyses of mitochondrial (mtCOI) sequences suggests that the two groups sampled off VA are genetically homogeneous, both groups contain two divergent mitochondrial lineages, and one surfclam sampled shares the S. s. similis mtCOI sequence. There are multiple cohorts of surfclams, suggesting that environmental conditions may have improved for surfclams in the south, or that this population has acclimated to altered conditions. Further research should investigate the potential for subspecies hybridization.

Genomics Reveals Complex Population History and Unexpected Diversity of Eurasian Otters (Lutra lutra) in Britain Relative to Genetic Methods.

Conservation genetic analyses of many endangered species have been based on genotyping of microsatellite loci and sequencing of short fragments of mtDNA. The increase in power and resolution afforded by whole genome approaches may challenge conclusions made on limited numbers of loci and maternally inherited haploid markers. Here, we provide a matched comparison of whole genome sequencing versus microsatellite and control region (CR) genotyping for Eurasian otters (Lutra lutra). Previous work identified four genetically differentiated "stronghold" populations of otter in Britain, derived from regional populations that survived the population crash of the 1950s-1980s. Using whole genome resequencing data from 45 samples from across the British stronghold populations, we confirmed some aspects of population structure derived from previous marker-driven studies. Importantly, we showed that genomic signals of the population crash bottlenecks matched evidence from otter population surveys. Unexpectedly, two strongly divergent mitochondrial lineages were identified that were undetectable using CR fragments, and otters in the east of England were genetically distinct and surprisingly variable. We hypothesize that this previously unsuspected variability may derive from past releases of Eurasian otters from other, non-British source populations in England around the time of the population bottleneck. Our work highlights that even reasonably well-studied species may harbor genetic surprises, if studied using modern high-throughput sequencing methods.

Assessment of genetic and morphological differentiation among populations of the Diederik Cuckoo Chrysococcyx caprius

The Diederik Cuckoo Chrysococcyx caprius is an African species widely distributed south of the Sahara, which migrates seasonally between breeding and nonbreeding sites. It is currently unknown whether the species consists of a single panmictic population or if it is genetically structured. To investigate this, we analysed sequence variation in three mitochondrial and two nuclear gene regions in combination with morphological measurements in specimens from four localities. Phylogenetic relationships were estimated using maximum-likelihood methods and included samples of Klaas’s Cuckoo Chrysococcyx klaas, Red-chested Cuckoo Cuculus solitarius, and African Cuckoo Cuculus gularis. Haplotype networks and analysis of molecular variance were used to characterise the spatial distribution of genetic diversity. A principal component analysis was performed to investigate morphological variation among localities. Molecular analysis identified two divergent mitochondrial lineages, which were found to occur in sympatry in one South African locality (Limpopo Province). The magnitude of divergence between versus within these lineages was low (0.4–1%) yet significant (F ST: 0.84–0.88). Lack of apparent phylogeographic structure provides support for the absence of physical barriers to gene flow in this species. The divergent mitochondrial lineages did not differ in morphological measurements. The emergence and persistence of shallow mitochondrial divergence among sympatric lineages in the Diederik Cuckoo could be linked to maternal divergence in host selection of these brood parasites — a hypothesis requiring additional data to be tested.

Multiple concordant cytonuclear divergences and potential hybrid speciation within a species complex in Asia

Various environmental factors impact the distribution, population structure, demography and evolutionary trajectory of a bird species, leading to genetic and morphological divergences between populations across its distribution. The Paradoxornis webbianus species complex is found throughout much of East Asia, where its geographically distinct populations exhibit dramatic morphological variation. This has resulted in a hotly debated taxonomy. This study intended to identify genetic divergence patterns and their underlying contributing factors for this species complex. We collected 243 birds, whose data was combined with those available in GenBank to perform phylogeographic analyses using one mitochondrial and six nuclear loci. Six mitochondrial clades were observed in the species complex, while individual-based Bayesian clustering using nuclear markers showed multiple congruent breaks. Overall, the six molecular lineages could be recognized as independent species under the lineage species concept in view of genetic divergence, clade-specific morphological changes and distribution: P. webbianus, P. w. bulomachus, P. alphonsianus, P. a. ganluoensis, P. brunneus brunneus and P. b. ricketti. The estimated divergence times range from 0.46 to 3.36 million years ago, suggesting it was likely impacted by paleoclimatic changes. Interestingly, P. alphonsianus carries two divergent mitochondrial lineages shared with P. webbianus and P. a. ganluoensis, respectively, and analyses based on nuclear loci found a similar pattern. We discussed the various hypotheses for this pattern and argued that P. alphonsianus was likely the result of hybridization between P. webbianus and P. a. ganluoensis. Further data on genome, transcriptome and breeding ecology are needed to address the hypothesis of hybrid speciation and its underlying mechanisms.

Towards a DNA barcode library for Madagascar's threatened ichthyofauna.

In order to improve the molecular resources available for conservation management of Madagascar’s threatened ichthyofauna, we elaborated a curated database of 2860 mitochondrial sequences of the mitochondrial COI, 16S and ND2 genes of Malagasy fishes, of which 1141 sequences of freshwater fishes were newly sequenced for this data set. The data set is mostly composed of COI (2015 sequences) while 16S and ND2 sequences from partly the same samples were used to match the COI sequences to reliably identified reference sequences of these genes. We observed COI uncorrected pairwise genetic distances of 5.2‒31.0% (mean 20.6%) among species belonging to different genera, and 0.0‒22.4% (mean 6.4%) for species belonging to the same genus. Deeply divergent mitochondrial lineages of uncertain attribution were found among Malagasy freshwater eleotrids and gobiids, confirming these groups are in need of taxonomic revision. DNA barcodes assigned to introduced cichlids (tilapias) included Coptodon rendallii, C. zillii, Oreochromis aureus (apparently a new country record), O. cf. mossambicus, O. niloticus, and one undetermined species of Oreochromis, with sequences of up to three species found per location. In aplocheiloid killifishes of the genus Pachypanchax, most species from northern Madagascar had only low mitochondrial divergences, three of these species (P. omalonotus, P. patriciae, and P. varatraza) were not reciprocally monophyletic, and one genetically deviant lineage was discovered in a northern locality, suggesting a need for partial taxonomic revision of this genus. While the lack of voucher specimens for most of the samples sequenced herein precludes final conclusions, our first step towards a DNA barcoding reference library of Madagascar’s fishes already demonstrates the value of such a data set for improved taxonomic inventory and conservation management. We strongly suggest further exploration of Madagascar’s aquatic environments, which should include detailed photographic documentation and tissue sampling of large numbers of specimens, and collection of preserved voucher specimens as well as of living fish for the buildup of ex situ assurance populations of threatened species complying with the One Plan Approach proposed by the IUCN SSC Conservation Breeding Specialist Group (CBSG).

Assessing diversity of King Crab Lithodes spp. in the south-eastern pacific using phylogeny and molecular species delimitation methods.

The purpose of this study was to test the hypothesis that the genetic diversity of commercially significant species of King Crabs (Lithodes spp.) along the south‐eastern Pacific (SEP) comprises different independent evolutionary units (IEUs) with spatially isolated distribution. Nine localities from inner and open waters along the SEP Chilean coast (39°S‐55°S) were sampled. We analyzed sequences from 173 individuals for the mitochondrial gene Cytochrome oxidase I (COX‐I), 151 individuals for the Internal Transcribed Spacer 1 (ITS) and 135 for the structural ribosomal RNA (28S). Genetic delimitation was performed through three analytical methods: ABGD, GMYC, and its Bayesian implementation, bGMYC. Bayesian phylogenetic analyses and haplotype networks were also performed. Divergence time between clades was assessed for the COX‐I marker and estimated from known evolutionary rates for this marker in other crustacean species and fossil calibration from other Anomuran species. Delimitation analyses, phylogenetic analyses, and mitochondrial haplotype networks suggested the presence of two deeply divergent mitochondrial lineages of Lithodes in the SEP, referred to as Clade1 and Clade 2. Nuclear markers showed low phylogenetic resolution and therefore were unsuitable for molecular species delimitation. Divergence time analysis of the mitochondrial lineages suggests a separation between Clades of approximately 2.3 Mya. The divergence time obtained suggested that Pliocene glaciations and deglaciations cycles could be involved in hybridization events between Lithodes IEUs at southern tip of South American coasts. The different frequencies of Lithodes haplotypes in inner and open water environments along SEP coasts could be explained by events such as the last glacial maximum or by differences in the adaptation of each clade to different environments. These findings support the necessity of evaluating the taxonomic status of Lithodes individuals found along SEP coasts under an integrative taxonomy approach or through markers with other evolution rates than those already used.

African climate and geomorphology drive evolution and ghost introgression in sable antelope.

The evolutionary history of African ungulates has been explained largely in the light of Pleistocene climatic oscillations and the way these influenced the distribution of vegetation types, leading to range expansions and/or isolation in refugia. In contrast, comparatively fewer studies have addressed the continent's environmental heterogeneity and the role played by its geomorphological barriers. In this study, we performed a range-wide analysis of complete mitogenomes of sable antelope (Hippotragus niger) to explore how these different factors may have contributed as drivers of evolution in southcentral Africa. Our results supported two sympatric and deeply divergent mitochondrial lineages in west Tanzanian sables, which can be explained as the result of introgressive hybridization of a mitochondrial ghost lineage from an archaic, as-yet-undefined, congener. Phylogeographical subdivisions into three main lineages suggest that sable diversification may not have been driven solely by climatic events affecting populations differently across a continental scale. Often in interplay with climate, geomorphological features have also clearly shaped the species' patterns of vicariance, where the East Africa Rift System and the Eastern Arc Mountains acted as geological barriers. Subsequent splits among southern populations may be linked to rearrangements in the Zambezi system, possibly framing the most recent time when the river attained its current drainage profile. This work underlines how the use of comprehensive mitogenomic data sets on a model species with a wide geographical distribution can contribute to a much-enhanced understanding of environmental, geomorphological and evolutionary patterns in Africa throughout the Quaternary.

Molecular taxonomic identification and species-level phylogeny of the narrow-mouthed frogs of the genus Rhombophryne (Anura: Microhylidae: Cophylinae) from Madagascar

The study of diamond frogs (genus Rhombophryne, endemic to Madagascar) has been historically hampered by the paucity of available specimens, because of their low detectability in the field. Over the last 10 years, 13 new taxa have been described, and 20 named species are currently recognized. Nevertheless, undescribed diversity within the genus is probably large, calling for a revision of the taxonomic identification of published records and an update of the known distribution of each lineage. Here we generate DNA sequences of the mitochondrial 16S rRNA gene of all specimens available to us, revise the genetic data from public databases, and report all deeply divergent mitochondrial lineages of Rhombophryne identifiable from these data. We also generate a multi-locus dataset (including five mitochondrial and eight nuclear markers; 9844 bp) to infer a species-level phylogenetic hypothesis for the diversification of this genus and revise the distribution of each lineage. We recognize a total of 10 candidate species, two of which are identified here for the first time. The genus Rhombophryne is here proposed to be divided into six main species groups, and phylogenetic relationships among some of them are not fully resolved. These frogs are primarily distributed in northern Madagascar, and most species are known from only few localities. A previous record of this genus from the Tsingy de Bemaraha (western Madagascar) is interpreted as probably due to a mislabelling and should not be considered further unless confirmed by new data. By generating this phylogenetic hypothesis and providing an updated distribution of each lineage, our findings will facilitate future species descriptions, pave the way for evolutionary studies, and provide valuable information for the urgent conservation of diamond frogs.

A new lineage of Galapagos giant tortoises identified from museum samples

The Galapagos Archipelago is recognized as a natural laboratory for studying evolutionary processes. San Cristóbal was one of the first islands colonized by tortoises, which radiated from there across the archipelago to inhabit 10 islands. Here, we sequenced the mitochondrial control region from six historical giant tortoises from San Cristóbal (five long deceased individuals found in a cave and one found alive during an expedition in 1906) and discovered that the five from the cave are from a clade that is distinct among known Galapagos giant tortoises but closely related to the species from Española and Pinta Islands. The haplotype of the individual collected alive in 1906 is in the same clade as the haplotype in the contemporary population. To search for traces of a second lineage in the contemporary population on San Cristóbal, we closely examined the population by sequencing the mitochondrial control region for 129 individuals and genotyping 70 of these for both 21 microsatellite loci and >12,000 genome-wide single nucleotide polymorphisms [SNPs]. Only a single mitochondrial haplotype was found, with no evidence to suggest substructure based on the nuclear markers. Given the geographic and temporal proximity of the two deeply divergent mitochondrial lineages in the historical samples, they were likely sympatric, raising the possibility that the lineages coexisted. Without the museum samples, this important discovery of an additional lineage of Galapagos giant tortoise would not have been possible, underscoring the value of such collections and providing insights into the early evolution of this iconic radiation.

Phylogenetic and multivariate analyses of Gekko smithii Gray, 1842 recover a new species from Peninsular Malaysia and support the resurrection of G. albomaculatus (Giebel, 1861) from Sumatra

AbstractAn integrative taxonomic analysis of Sundaic populations ofGekko smithiifrom the Thai-Malaya Peninsula, Sumatra, and Borneo recovered four deeply divergent mitochondrial lineages that are separated by major geographic barriers (mountains and seaways). Furthermore, they bear a number of concordant statistically significant differences in meristic and morphometric features, morphospatial separation in multivariate space, and discrete differences in color pattern.Gekko smithiisensu strictois restricted to southern Thailand south of the Isthmus of Kra and Peninsular Malaysia west of the Banjaran (mountain range) Titiwangsa, being that the type locality is on Penang Island, Penang.Gekko hulksp. nov.is a new species from extreme southern Thailand and Peninsular Malaysia east of the Banjaran Titiwangsa and five east coast islands—the type locality being Pulau (island) Tioman, Pahang. Gekko cf. albofasciolatus is tentatively used to include Bornean populations west of the Iran Mountains in Sabah and Sarawak which, in the absence of molecular data, cannot unequivocally be separated morphologically fromG. albofasciolatusfrom the type locality at Banjarmasin, Kalimantan, Indonesia east of the Iran Mountains. In the absence of molecular data,G. albomaculatusis resurrected to include mainland Sumatran, Nias Island, and Banyak Islands populations which, based on their morphology, cannot be separated from descriptions ofG. albomaculatusfrom the type locality of Bangka Island, 15 km off the southeast coast of mainland Sumatra. Further integrative analyses of all Sumatran and Bornean populations are currently underway as well as the enigmatic Wallacean populations from Sulawesi. Data are presented that strongly suggest all references toG. smithiifrom Java stem from a 151 year-old misidentification of a specimen ofG. geckoof unknown provenance. Additionally, there are no vouchered records ofG. smithiifrom Myanmar. The phylogeographic patterns of Sundaic populations of theG. smithiicomplex are concordant with those of a plethora of other Sundaic lineages.

Geographic Variation in the Green Rat Snake Senticolis triaspis (Squamata: Colubridae): Evidence from Mitochondrial DNA, Morphology, and Niche Modeling

Senticolis triaspis is a widespread polytypic colubrid snake that ranges from the southwestern United States southward along the Pacific Coast and part of the Atlantic Coast of Mexico to Costa Rica. Three subspecies have been described based on differences in color pattern and scutellation: S. t. intermedia, S. t. mutabilis, and S. t. triaspis. The last taxonomic revision was completed in 1960. Here, we present an integrative taxonomic analysis of geographic variation using multiple sources of evidence: mitochondrial DNA (817 bp ND4 from 62 samples), traditional morphology (meristic and mensural characters), geometric morphometrics of head shape, and bioclimatic estimates of environmental niche similarity. Molecular data revealed two deeply divergent mitochondrial lineages corresponding to northern and southern clades and eight well-supported clades within these two main groups. Head shape and niche models also showed north–south differences, while traditional morphological analyses did not discriminate among mtDNA lineages. Thus, our study suggests there are at least two candidate species within Senticolis triaspis based on the mtDNA divergence, differences in head shape, and non-overlapping ecological niches. However, we refrain from making taxonomic recommendations pending the addition of nuclear DNA sequence data, finer sampling at contact zones, and additional morphological data; these data are warranted to provide a more robust, stable species delimitation.

The mitochondrial phylogeography of the Crimean endemic lizard Darevskia lindholmi (Sauria, Lacertidae): Hidden diversity in an isolated mountain system

AbstractThe Lindholm rock lizard,Darevskia lindholmi, is the only member of the genusDarevskiawhose range is restricted solely to Europe, representing a local endemism found only in the Crimean Mountains. In our study, we investigated the cytochromebgene (mtDNA) of 101D. lindholmisequences from 65 Crimean localities, representing its entire range. We found thatD. lindholmiis highly genetically structured, and its range is divided into populations belonging to three mitochondrial lineages. The Lindholm rock lizard populations inhabiting the middle part of the Crimean Mountains (further referred to as the Central lineage) are sharply differentiated from the other two lineages (the Common and the Southwestern lineages), which are present in most of the species range. The genetic distance between the Central lineage and the other two taken together is 4.6%, according to our results, suggesting that the divergence occurred during the Early Pleistocene. The narrowly distributed Southwestern lineage and the widespread Common lineage, on the other hand, are differentiated by 1%. Field observations on the representatives of the main evolutionary groups show that their ecology is also different: the Central lineage is a mesophilic and cold-resistant form, while the other two closely related lineages are more xerophilic and thermophilic. Results of the potential ranges modeling and ecological niche analysis confirm that the genetic lineages occupy different niches of the Crimea. Furthermore, the area of inhabitation of the Central lineage splits the western and eastern parts of the Common lineage range, while the Southwestern lineage is restricted along the coast of the southwestern coast of the peninsula. The long-term co-existence of deeply divergent sister mitochondrial lineages in a relatively small (circa 7,000 km2) isolated mountain system serves as a mesocosm for understanding the speciation process. Our data suggest that the Central lineage warrants further taxonomic investigation.

Gene flow between island populations of the malaria mosquito, Anopheles hinesorum, may have contributed to the spread of divergent host preference phenotypes.

Anopheles hinesorum is a mosquito species with variable host preference. Throughout New Guinea and northern Australia, An. hinesorum feeds on humans (it is opportunistically anthropophagic) while in the south‐west Pacific's Solomon Archipelago, the species is abundant but has rarely been found biting humans (it is exclusively zoophagic in most populations). There are at least two divergent zoophagic (nonhuman biting) mitochondrial lineages of An. hinesorum in the Solomon Archipelago representing two independent dispersals. Since zoophagy is a derived (nonancestral) trait in this species, this leads to the question: has zoophagy evolved independently in these two populations? Or conversely: has nuclear gene flow or connectivity resulted in the transfer of zoophagy? Although we cannot conclusively answer this, we find close nuclear relationships between Solomon Archipelago populations indicating that recent nuclear gene flow has occurred between zoophagic populations from the divergent mitochondrial lineages. Recent work on isolated islands of the Western Province (Solomon Archipelago) has also revealed an anomalous, anthropophagic island population of An. hinesorum. We find a common shared mitochondrial haplotype between this Solomon Island population and another anthropophagic population from New Guinea. This finding suggests that there has been recent migration from New Guinea into the only known anthropophagic population from the Solomon Islands. Although currently localized to a few islands in the Western Province of the Solomon Archipelago, if anthropophagy presents a selective advantage, we may see An. hinesorum emerge as a new malaria vector in a region that is now working on malaria elimination.

Sympatric occurrence of deeply diverged mitochondrial DNA lineages in Siberian geometrid moths (Lepidoptera: Geometridae): cryptic speciation, mitochondrial introgression, secondary admixture or effect ofWolbachia?

AbstractThe divergent sympatric mitochondrial lineages within traditionally recognized species present a challenge regularly faced by taxonomists and evolutionary biologists. We encountered this problem when studying the Siberian geometrid moths, Alcis deversata and Thalera chlorosaria. Within each of these species we found two deeply diverged mitochondrial lineages that demonstrated a level of genetic differentiation exceeding the standard interspecific DNA barcode threshold. Using analyses of nuclear genes, morphology, ecological preferences and Wolbachia endosymbionts, we tested five hypotheses that might explain the mitochondrial pattern observed: cryptic speciation, ancestral polymorphism, interspecific mitochondrial introgression, secondary admixture of allopatrically evolved populations and an effect of intracellular Wolbachia endosymbionts. We demonstrate that in A. deversata and Th. chlorosaria the mitochondrial differences are not correlated with differences in nuclear genes, morphology, ecology and Wolbachia infection status, thus not supporting the hypothesis of cryptic species and an effect of Wolbachia. Mitochondrial introgression can lead to a situation in which one species has both its own mitochondrial lineage and the lineage obtained from another species. We found this situation in the species pair Alcis repandata and Alcis extinctaria. We conclude that the mitochondrial heterogeneity in A. deversata and Th. chlorosaria is most likely to be attributable to the secondary admixture of allopatrically evolved populations.

Not the Cryptic Species: Diversity of Hipposideros gentilis (Chiroptera: Hipposideridae) in Indochina

We present here the result of phylogenetic analysis for Vietnamese Hipposideros gentilis specimens using 7 nuclear genes and one mitochondrial gene. The complex distribution of divergent mitochondrial DNA lineages contradicts, at least in part, nuclear and morphological data. The most likely explanation for this discordance is the historical hybridization between ancestral populations of H. gentilis and H. rotalis/H. khaokhouayensis. Our data supports the species status of H. gentilis, while only partially corroborating its previously proposed subspecies delimitation. We suggest the lowland forest populations from south Vietnam may correspond to their own subspecies. At the same time, the close phylogenetic relationship and morphological similarity of mountain forms from south and central Vietnam to the north Vietnamese populations make doubtful the subspecies status of H. gentilis sinensis.

Completing a taxonomic puzzle: integrative review of geckos of the Paroedura bastardi species complex (Squamata, Gekkonidae)

The Paroedura bastardi clade, a subgroup of the Madagascan gecko genus Paroedura, currently comprises four nominal species: P. bastardi, supposedly widely distributed in southern and western Madagascar, P. ibityensis, a montane endemic, and P. tanjaka and P. neglecta, both restricted to the central west region of the island. Previous work has shown that Paroedura bastardi is a species complex with several strongly divergent mitochondrial lineages. Based on one mitochondrial and two nuclear markers, plus detailed morphological data, we undertake an integrative revision of this species complex. Using a representative sampling for seven nuclear and five mitochondrial genes we furthermore propose a phylogenetic hypothesis of relationships among the species in this clade. Our analyses reveal at least three distinct and independent evolutionary lineages currently referred to P. bastardi. Conclusive evidence for the species status of these lineages comes from multiple cases of syntopic occurrence without genetic admixture or morphological intermediates, suggesting reproductive isolation. We discuss the relevance of this line of evidence and the conditions under which concordant differentiation in unlinked loci under sympatry provides a powerful approach to species delimitation, and taxonomically implement our findings by (1) designating a lectotype for Paroedura bastardi, now restricted to the extreme South-East of Madagascar, (2) resurrecting of the binomen Paroedura guibeae Dixon & Kroll, 1974, which is applied to the species predominantly distributed in the South-West, and (3) describing a third species, Paroedura rennerae sp. nov., which has the northernmost distribution within the species complex.

Completing a taxonomic puzzle: integrative review of geckos of the Paroedura bastardi species complex (Squamata, Gekkonidae)

The Paroedura bastardi clade, a subgroup of the Madagascan gecko genus Paroedura, currently comprises four nominal species: P. bastardi, supposedly widely distributed in southern and western Madagascar, P. ibityensis, a montane endemic, and P. tanjaka and P. neglecta, both restricted to the central west region of the island. Previous work has shown that Paroedura bastardi is a species complex with several strongly divergent mitochondrial lineages. Based on one mitochondrial and two nuclear markers, plus detailed morphological data, we undertake an integrative revision of this species complex. Using a representative sampling for seven nuclear and five mitochondrial genes we furthermore propose a phylogenetic hypothesis of relationships among the species in this clade. Our analyses reveal at least three distinct and independent evolutionary lineages currently referred to P. bastardi. Conclusive evidence for the species status of these lineages comes from multiple cases of syntopic occurrence without genetic admixture or morphological intermediates, suggesting reproductive isolation. We discuss the relevance of this line of evidence and the conditions under which concordant differentiation in unlinked loci under sympatry provides a powerful approach to species delimitation, and taxonomically implement our findings by (1) designating a lectotype for Paroedura bastardi, now restricted to the extreme South-East of Madagascar, (2) resurrecting of the binomen Paroedura guibeae Dixon & Kroll, 1974, which is applied to the species predominantly distributed in the South-West, and (3) describing a third species, Paroedura rennerae sp. nov., which has the northernmost distribution within the species complex.