The systematic relationships of the snake genus Anomochilus

Phylogenetic analysis of 38 skeletal characters, 12 muscular characters and 15 visceral characters in 17 major snake clades plus Anomochilus suggests that Anomochilus is the sister taxon of all other living alethinophidian snakes. However, skeletal, muscular and visceral character sets analysed separately or in pairs give four groups of nonconcordant tree topologies. Based on the cladogram derived from the total evidence, two families are erected to prevent the existing family Uropeltidae from becoming paraphyletic: Anomochilidae, for the Malaysian and Indonesian genus Anomochilus , and Cylindrophiidae, for the Sri Lankan, Southeast Asian and Indonesian genus Cylindrophis and the Upper Eocene fossil Eoanilius.     

Complete Mitochondrial DNA Sequences of Six Snakes: Phylogenetic Relationships and Molecular Evolution of Genomic Features

Complete mitochondrial DNA (mtDNA) sequences were determined for representative species from six snake families: the acrochordid little file snake, the bold boa constrictor, the cylindrophiid red pipe snake, the viperid himehabu, the pythonid ball python, and the xenopeltid sunbeam snake. Thirteen protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 control regions were identified in these mtDNAs. Duplication of the control region and translocation of the tRNA^Leu gene were two notable features of the snake mtDNAs. The duplicate control regions had nearly identical nucleotide sequences within species but they were divergent among species, suggesting concerted sequence evolution of the two control regions. In addition, the duplicate control regions appear to have facilitated an interchange of some flanking tRNA genes in the viperid lineage. Phylogenetic analyses were conducted using a large number of sites (9570 sites in total) derived from the complete mtDNA sequences. Our data strongly suggested a new phylogenetic relationship among the major families of snakes: ((((Viperidae, Colubridae), Acrochordidae), (((Pythonidae, Xenopeltidae), Cylindrophiidae), Boidae)), Leptotyphlopidae). This conclusion was distinct from a widely accepted view based on morphological characters in denying the sister-group relationship of boids and pythonids, as well as the basal divergence of nonmacrostomatan cylindrophiids. These results imply the significance to reconstruct the snake phylogeny with ample molecular data, such as those from complete mtDNA sequences.[Reviewing Editor: Dr. Bill Ballard]     

Molecular Systematics and Evolution of Regina and the Thamnophiine Snakes

Snakes of the tribe Thamnophiini represent an ecologically important component of the herpetofauna in a range of habitats across North America. Thamnophiines are the best-studied colubrids, yet little is known of their systematic relationships. A molecular phylogenetic study of 32 thamnophiine species using three complete mitochondrial genes (cytochrome b, NADH dehydrogenase subunit 2, and 12S ribosomal DNA) recovered a well-supported phylogeny with three major clades: a garter snake group, a water snake group, and a novel semifossorial group. The historically contentious genus Regina, which contains the crayfish-eating snakes, is polyphyletic. The phylogeographic pattern of Thamnophis is consistent with an hypothesis of at least one invasion of northern North America from Mexico.     

Shaking the Tree: Multi-locus Sequence Typing Usurps Current Onchocercid (Filarial Nematode) Phylogeny

During the past twenty years, a number of molecular analyses have been performed to determine the evolutionary relationships of Onchocercidae, a family of filarial nematodes encompassing several species of medical or veterinary importance. However, opportunities for broad taxonomic sampling have been scarce, and analyses were based mainly on 12S rDNA and coxI gene sequences. While being suitable for species differentiation, these mitochondrial genes cannot be used to infer phylogenetic hypotheses at higher taxonomic levels. In the present study, 48 species, representing seven of eight subfamilies within the Onchocercidae, were sampled and sequences of seven gene loci (nuclear and mitochondrial) analysed, resulting in the hitherto largest molecular phylogenetic investigation into this family. Although our data support the current hypothesis that the Oswaldofilariinae, Waltonellinae and Icosiellinae subfamilies separated early from the remaining onchocercids, Setariinae was recovered as a well separated clade. Dirofilaria, Loxodontofilaria and Onchocerca constituted a strongly supported clade despite belonging to different subfamilies (Onchocercinae and Dirofilariinae). Finally, the separation between Splendidofilariinae, Dirofilariinae and Onchocercinae will have to be reconsidered.     

Diversity of teiid lizards from Gran Chaco and Western Cerrado (Squamata: Teiidae)

The Gran Chaco dry forest ecoregion corresponds to the southern portion of the South America diagonal belt of open formations, being one of the most threatened subtropical woodland savannas in the world. The area is still poorly known biologically and has been suffering with impressively high forest cover loss in the last 10 years. Integrating morphological and molecular data, we detected and describe a cryptic new species of lizard genus Ameivula endemic from the eastern part of this ecoregion, the called Humid Chaco. Ameivula apipensis sp nov. is characterised by a whitish brown vertebral stripe in adults and juveniles, a lateral field without ocelli and with overlapping spot, presence of an interfrontoparietal scale in 46.2% of the specimens, 12–17 femoral pores, an hemipenis without lateral sac, five xiphisternal ribs, and by a combination of meristic features as confirmed by discriminant analysis. The new species was recovered sister to a clade from Western Cerrado in our analysis, the first phylogenetic hypothesis for the Ameivula and Glaucomastix genera based on 1977 base pairs of three mitochondrial (12S, 16S and cyt‐b) and one nuclear (c‐mos) genes, including all the recognised species at the moment. Maximum parsimony and Bayesian inference recovered the monophyly of Ameivula and Glaucomastix with strong support. Reinforcing previous studies, our results suggest the presence of additional cryptic species in Ameivula from the Western Cerrado.     

Genus-level phylogeny of snakes reveals the origins of species richness in Sri Lanka

Snake diversity in the island of Sri Lanka is extremely high, hosting at least 89 inland (i.e., non-marine) snake species, of which at least 49 are endemic. This includes the endemic genera Aspidura, Balanophis, Cercaspis, Haplocercus, and Pseudotyphlops, which are of uncertain phylogenetic affinity. We present phylogenetic evidence from nuclear and mitochondrial loci showing the relationships of 40 snake species from Sri Lanka (22 endemics) to the remaining global snake fauna. To determine the phylogenetic placement of these species, we create a molecular dataset containing 10 genes for all global snake genera, while also sampling all available species for genera with endemic species occurring in Sri Lanka. Our sampling comprises five mitochondrial genes (12S, 16S, cyt-b, ND2, and ND4) and five nuclear genes (BDNF, c-mos, NT3 RAG-1, and RAG-2), for a total of up to 9582 bp per taxon. We find that the five endemic genera represent portions of four independent colonizations of Sri Lanka, with Cercaspis nested within Colubrinae, Balanophis in Natricinae, Pseudotyphlops in Uropeltidae, and that Aspidura + Haplocercus represents a distinct, ancient lineage within Natricinae. We synonymize two endemic genera that render other genera paraphyletic (Haplocercus with Aspidura, and Cercaspis with Lycodon), and discover that further endemic radiations may be present on the island, including a new taxon from the blindsnake family Typhlopidae, suggesting a large endemic radiation. Despite its small size relative to other islands such as New Guinea, Borneo, and Madagascar, Sri Lanka has one of the most phylogenetically diverse island snake faunas in the world, and more research is needed to characterize the island’s biodiversity, with numerous undescribed species in multiple lineages.     

Phylogenetic relationships of the spider family Tetragnathidae (Araneae, Araneoidea) based on morphological and DNA sequence data

The monophyly of Tetragnathidae including the species composition of the family (e.g., Are Nephila and their relatives part of this lineage?), the phylogenetic relationships of its various lineages, and the exact placement of Tetragnathidae within Araneoidea have been three recalcitrant problems in spider systematics. Most studies on tetragnathid phylogeny have focused on morphological and behavioral data, but little molecular work has been published to date. To address these issues we combine previous morphological and behavioral data with novel molecular data including nuclear ribosomal RNA genes 18S and 28S, mitochondrial ribosomal RNA genes 12S and 16S and protein‐coding genes from the mitochondrion [cytochrome c oxidase subunit I (COI)] and from the nucleus (histone H3), totaling ca. 6.3 kb of sequence data per taxon. These data were analyzed using direct optimization and static homology using both parsimony and Bayesian methods. Our results indicate monophyly of Tetragnathidae, Tetragnathinae, Leucauginae, the “Nanometa clade” and the subfamily Metainae, which, with the exception of the later subfamily, received high nodal support. Morphological synapomorphies that support these clades are also discussed. The position of tetragnathids with respect to the rest of the araneoid spiders remains largely unresolved but tetragnathids and nephilids were never recovered as sister taxa. The combined dataset suggests that Nephilidae is sister to Araneidae; furthermore, the sister group of Nephila is the clade composed by Herennia plus Nephilengys and this pattern has clear implications for understanding the comparative biology of the group. Tetragnathidae is most likely sister to some members of the “reduced piriform clade” and nephilids constitute the most‐basal lineage of araneids.     

Phylogenetic relationships of Steinernema Travassos, 1927 (Nematoda: Cephalobina: Steinernematidae) based on nuclear, mitochondrial and morphological data

Entomopathogenic nematodes of the genus Steinernema are lethal parasites of insects that are used as biological control agents of several lepidopteran, dipteran and coleopteran pests. Phylogenetic relationships among 25 Steinernema species were estimated using nucleotide sequences from three genes and 22 morphological characters. Parsimony analysis of 28S (LSU) sequences yielded a well-resolved phylogenetic hypothesis with reliable bootstrap support for 13 clades. Parsimony analysis of mitochondrial DNA sequences (12S rDNA and cox 1 genes) yielded phylogenetic trees with a lower consistency index than for LSU sequences, and with fewer reliably supported clades. Combined phylogenetic analysis of the 3-gene dataset by parsimony and Bayesian methods yielded well-resolved and highly similar trees. Bayesian posterior probabilities were high for most clades; bootstrap (parsimony) support was reliable for approximately half of the internal nodes. Parsimony analysis of the morphological dataset yielded a poorly resolved tree, whereas total evidence analysis (molecular plus morphological data) yielded a phylogenetic hypothesis consistent with, but less resolved than trees inferred from combined molecular data. Parsimony mapping of morphological characters on the 3-gene trees showed that most structural features of steinernematids are highly homoplastic. The distribution of nematode foraging strategies on these trees predicts that S. hermaphroditum, S. diaprepesi and S. longicaudum (US isolate) have cruise forager behaviours.     

Molecular systematics of the Middle American genus Hypopachus (Anura: Microhylidae)

We present the first phylogenetic study on the widespread Middle American microhylid frog genus Hypopachus. Partial sequences of mitochondrial (12S and 16S ribosomal RNA) and nuclear (rhodopsin) genes (1275 bp total) were analyzed from 43 samples of Hypopachus, three currently recognized species of Gastrophryne, and seven arthroleptid, brevicipitid and microhylid outgroup taxa. Maximum parsimony (PAUP), maximum likelihood (RAxML) and Bayesian inference (MrBayes) optimality criteria were used for phylogenetic analyses, and BEAST was used to estimate divergence dates of major clades. Population-level analyses were conducted with the programs NETWORK and Arlequin. Results confirm the placement of Hypopachus and Gastrophryne as sister taxa, but the latter genus was strongly supported as paraphyletic. The African phrynomerine genus Phrynomantis was recovered as the sister taxon to a monophyletic Chiasmocleis, rendering our well-supported clade of gastrophrynines paraphyletic. Hypopachus barberi was supported as a disjunctly distributed highland species, and we recovered a basal split in lowland populations of Hypopachus variolosus from the Pacific versant of Mexico and elsewhere in the Mesoamerican lowlands. Dating analyses from BEAST estimate speciation within the genus Hypopachus occurred in the late Miocene/early Pliocene for most clades. Previous studies have not found bioacoustic or morphological differences among these lowland clades, and our molecular data support the continued recognition of two species in the genus Hypopachus.     

Snake evolution in Melanesia: origin of the Hydrophiinae (Serpentes,Elapidae), and the evolutionary history of the enigmatic New Guinean elapid Toxicocalamus

The venomous snake subfamily Hydrophiinae includes more than 40 genera and approximately 200 species. Most members of this clade inhabit Australia, and have been well studied. But, because of poor taxon sampling of Melanesian taxa, basal evolutionary relationships have remained poorly resolved. The Melanesian genera Ogmodon, Loveridgelaps, and Salomonelaps have not been included in recent phylogenetic studies, and the New Guinean endemic, Toxicocalamus, has been poorly sampled and sometimes recovered as polyphyletic. We generated a multilocus phylogeny for the subfamily using three mitochondrial and four nuclear loci so as to investigate relationships among the basal hydrophiine genera and to determine the status of Toxicocalamus. We sequenced these loci for eight of the 12 described species within Toxicocalamus, representing the largest molecular data set for this genus. We found that a system of offshore island arcs in Melanesia was the centre of origin for terrestrial species of Hydrophiinae, and we recovered Toxicocalamus as monophyletic. Toxicocalamus demonstrates high genetic and morphological diversity, but some of the molecular diversity is not accompanied by diagnostic morphological change. We document at least five undescribed species that all key morphologically to Toxicocalamus loriae (Boulenger, 1898), rendering this species polyphyletic. Continued work on Toxicocalamus is needed to document the diversity of this genus, and is likely to result in the discovery of additional species. Our increased taxon sampling allowed us to better understand the evolution and biogeography of Hydrophiinae; however, several unsampled lineages remain, the later study of which may be used to test our biogeographic hypothesis.     

Genetic diversity and phylogenetic analyses of ixodid ticks infesting cattle in northeast Thailand: the discovery of Rhipicephalus microplus clade C and the rarely detected R. haemaphysaloides

In total, 160 ticks infesting cattle in the northeast region of Thailand were collected and used for molecular investigation. Three tick species—Rhipicephalus microplus Canestrini, Rhipicephalus haemaphysaloides Supino and Haemaphysalis bispinosa Neumann—were identified based on morphology and DNA sequences of mitochondrial cytochrome c oxidase subunit 1 (CO1) and 16S ribosomal RNA (16S rRNA). In total, 26 and seven unique haplotypes of the CO1 and 16S rRNA genes, respectively, were recovered. Phylogenetic analysis using the CO1 sequence revealed that the R. microplus from northeastern Thailand were grouped into the previously described clades A and C, whereas the 16S rRNA phylogenetic tree assigned all isolates of R. microplus from Northeast Thailand into the previously described clade B. Clade C of the CO1 phylogenetic tree is a new genetic assemblage recently discovered from India and Malaysia, which has now been detected in our study. The haplotype network also demonstrated that R. microplus is divided into two haplogroups corresponding to the assemblage of the CO1 phylogenetic tree. Our findings strongly support the previous genetic assemblage classification and evidence that R. microplus from Northeast Thailand is a species complex comprising at least two genetic assemblages, i.e., clades A and C. However, further investigation is needed and should involve more comprehensive genetic and morphological analyses and cover a larger part of their distributional range throughout Southeast Asia.

     

Molecular phylogenetic analysis of tropical freshwater mussels (Mollusca: Bivalvia: Unionoida) resolves the position of Coelatura and supports a monophyletic Unionidae

In previous molecular phylogenetic analyses of the freshwater mussel family Unionidae (Bivalvia: Unionoida), the Afrotropical genus Coelatura had been recovered in various positions, generally indicating a paraphyletic Unionidae. However that result was typically poorly supported and in conflict with morphology-based analyses. We set out to test the phylogenetic position of Coelatura by sampling tropical lineages omitted from previous studies. Forty-one partial 28S nuclear rDNA and partial COI mtDNA sequences (1130 total aligned nucleotides) were analyzed separately and in combination under both maximum parsimony and likelihood, as well as Bayesian inference. There was significant phylogenetic incongruence between the character sets (partition homogeneity test, p < 0.01), but a novel heuristic for comparing bootstrap values among character sets analyzed separately and in combination illustrated that the observed conflict was due to homoplasy rather than separate gene histories. Phylogenetic analyses robustly supported a monophyletic Unionidae, with Coelatura recovered as part of a well-supported Africa–India clade (= Parreysiinae). The implications of this result are discussed in the context of Afrotropical freshwater mussel evolution and the classification of the family Unionidae.     

Phylogenetic estimation of Mytilidae in the East China Sea inferred from mitochondrial genes and nuclear DNA sequence variation

We performed a phylogenetic estimation of the family Mytilidae in the East China Sea based on nuclear internal transcribed spacer (ITS) genes and two mitochondrial genes (COI and 16S RNA). Analysis of five mytilid species based on each of the three genes resulted in mostly congruent trees, although there were some discrepancies in the classification of these species. We combine the results obtained from the three separate analyses to provide a phylogenetic estimation of Mytilidae. We found that the Mytilidae was divided into two major lineages: in one clade, Mytilus galloprovincialis was grouped with Mytilus coruscus; in the second clade, Septifer bilocularis was placed at the basal position in an individual clade, and Perna viridis and Musculista senhousia were recovered as a monophyletic group. Although these finding provide important insights into the taxonomic relationships among the Mytilidae, many aspects of Mytilidae phylogeny remain unresolved. Further analysis based on more molecular information and extensive taxon sampling is necessary to elucidate the phylogenetic relationships among the major lineages within the Mytilidae.     

Family ties: molecular phylogeny of crab spiders (Araneae: Thomisidae)

The first quantitative phylogenetic analysis of three sequenced genes (16S rRNA, cytochrome c oxidase subunit I, histone 3) of 25 genera of crab spiders and 11 outgroups supports the monophyly of Thomisidae. Four lineages within Thomisidae are recovered. They are informally named here as the Borboropactus clade, Epidius clade, Stephanopis clade and the Thomisus clade, pending detailed morphology based cladistic work. The Thomisus clade is recovered as a strongly supported monophyletic group with a minimal genetic divergence. Philodromidae previously widely considered a subfamily of Thomisidae do not group within thomisids and is excluded from Thomisidae. However, Aphantochilinae previously generally considered as a separate family falls within the Thomisus clade and is included in Thomisidae. The recently proposed new family Borboropactidae is rejected, as it is paraphyletic. © The Willi Hennig Society 2008.     

The phylogeny of leaf beetles (Chrysomelidae) inferred from mitochondrial genomes

The high-level classification of Chrysomelidae (leaf beetles) currently recognizes 12 or 13 well-established subfamilies, but the phylogenetic relationships among them remain ambiguous. Full mitochondrial genomes were newly generated for 27 taxa and combined with existing GenBank data to provide a dataset of 108 mitochondrial genomes covering all subfamilies. Phylogenetic analysis under maximum likelihood and Bayesian inference recovered the monophyly of all subfamilies, except that Timarcha was split from Chrysomelinae in some analyses. Three previously recognized major clades of Chrysomelidae were broadly supported: the ‘chrysomeline’ clade consisting of (Chrysomelinae (Galerucinae + Alticinae)); the ‘sagrine’ clade with internal relationships of ((Bruchinae + Sagrinae) + (Criocerinae + Donaciinae)), and the ‘eumolpine’ clade comprising (Spilopyrinae (Cassidinae (Eumolpinae (Cryptocephalinae + Lamprosomatinae)))). Relationships among these clades differed between data treatments and phylogenetic algorithms, and were complicated by two additional deep lineages, Timarcha and Synetinae. Various topological tests favoured the PhyloBayes software as the preferred inference method, resulting in the arrangement of (chrysomelines (eumolpines + sagrines)), with Timarcha placed as sister to the chrysomeline clade and Synetinae as a deep lineage splitting near the base. Whereas mitogenomes provide a solid framework for the phylogeny of Chrysomelidae, the basal relationships do not agree with the topology of existing molecular studies and remain one of the most difficult problems of Chrysomelidae phylogenetics.     

Phylogenetic utility of the first two introns of the S7 ribosomal protein gene in African electric fishes (Mormyroidea: Teleostei) and congruence with other molecular markers

A series of recent molecular systematic studies of the African electric fishes (Mormyroidea) have challenged many aspects of their traditional taxonomy and precladistic hypotheses of their phylogeny. However, poor resolution of some interrelationships within the subfamily Mormyrinae in these studies highlights the need for additional data and analyses. Here we evaluate the phylogenetic information content of nucleotide sequences from the first two introns of the low‐copy nuclear S7 ribosomal protein gene in 40 mormyroid species. Alignment of S7 sequences from 38 taxa within the subfamily Mormyrinae is non‐problematic, but these are difficult to align with sequences of Petrocephalus bovei (Petrocephalinae) and Gymnarchus niloticus (Gymnarchidae), which we exclude from our analysis. There are no significant differences in base frequencies among these sequences and base compositional bias is low. Maximum parsimony (MP) analysis on the S7 dataset, designating Myomyrus macrops as the outgroup, generates a phylogenetic hypothesis for these taxa with a low level of homoplasy (RI = 0.87). We examine agreement between the S7 data with previously published mitochondrial (12S/16S, cytochrome b) and nuclear (rag 2) datasets for the same taxa by means of incongruence length difference tests and partitioned Bremer support (decay) analysis. While we find significant agreement between the S7 dataset and the others, MP analysis of the S7 data alone and in combination with the other datasets indicates two novel relationships within the Mormyrinae: (1) Mormyrus is the sister group to Brienomyrus brachyistius and Isichthys henryi, and (2) Hippopotamyrus pictus is the sister group of a clade, previously recovered, containing Marcusenius senegalensis. S7 data provide additional support for a number of clades recovered in the earlier molecular studies, some of which conflict with current mormyrid taxonomy. Inferred indels and a single inversion in the S7 fragment provide supplemental character support for many of these relationships. These phylogenetic results strengthen recent hypotheses concerning the evolution of electric organ structure in these fishes. The evolutionary characteristics of this nuclear marker and its phylogenetic utility in this group suggests that it could be widely useful for systematic studies at the subfamilial level in teleost fishes. © 2003 The Linnean Society of London. Biological Journal of the Linnean Society, 2003, 78 , 273–292.     

Molecular Evidence for the Last Survivor of an Ancient Kangaroo Lineage

The Australasian marsupial family Macropodidae includes potoroos and bettongs (Potoroinae) as well as larger kangaroos, wallabies, and pademelons (Macropodinae). Perhaps the most enigmatic macropodid is the banded hare wallaby, Lagostrophus fasciatus, a taxon listed as vulnerable by the IUCN. Lagostrophus had traditionally been grouped as a sister-taxon to hare wallabies (Lagorchestes), in a clade with hypsodont macropodines, or intercalated in some other fashion within Macropodinae. Flannery (1983, 1989) proposed a radically different hypothesis wherein Lagostrophus is outside of Macropodinae and is more closely related to extinct sthenurine (short-faced) kangaroos. Given this controversy, we addressed the phylogenetic placement of the banded hare wallaby using molecular sequences for three mitochondrial genes (12S rRNA, valine tRNA, 16S rRNA) and one nuclear gene (protamine P1). Diverse phylogenetic methods all provided robust support for a macropodine clade that excludes the banded hare wallaby. The split between macropodines and the banded hare wallaby was estimated at approximately 20 million years ago (mya) using the Thorne/Kishino relaxed molecular clock method. Whereas our molecular results neither corroborate nor refute the sthenurine hypothesis, since all short-faced kangaroos and their immediate ancestors are extinct, the overriding implication of molecular phylogenetic analyses is manifest: the banded hare wallaby is the only living relict of an ancient kangaroo lineage. Regardless of its precise relationships, special efforts should be directed at conserving this unique and endangered taxon, which has not been recorded from mainland Australia since 1906 and is now restricted to two tiny islands off the coast of Western Australia.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1023/A:1022697300092     

Scorpions of the genus Odontobuthus Vachon, 1950 (Scorpiones: Buthidae) from Iran: Phylogenetic relationships inferred from mitochondrial DNA sequence data

The molecular phylogeny of the genus Odontobuthus Vachon, 1950 (Scorpiones: Buthidae) in Iran was evaluated using two mitochondrial DNA genes, cytochrome c oxidase, subunit I (COI) and 16S ribosomal RNA (16S rRNA). The molecular phylogenetic analyses were performed using Maximum Parsimony, Maximum Likelihood and Bayesian inference methods. The resulting topologies supported two main clades: the clade comprising Odontobuthus doriae, O. bidentatus, and O. tavighiae, and another one which is the O. tirgari clade. The results clearly presented additional support for the taxonomic validity of the recently described species, O. tirgari and O. tavighiae. In addition, the monophyly of two previously described species O. doriae and O. bidentatus was confirmed. According to the data presented here, three taxonomically valid species belonging to the genus Odontobuthus occur in Iran.     

Phylogenetic and diversity patterns of Blanus worm lizards (Squamata: Amphisbaenia): insights from mitochondrial and nuclear gene genealogies and species tree

Phylogenetic and phylogeographic patterns of amphisbaenians are poorly known. Molecular data from mitochondrial and nuclear loci are particularly needed for amphisbaenian phylogeny and taxonomy because their specializations to subterranean habits make morphology poorly informative and the occurrence of cryptic species probable. The Mediterranean genus Blanus includes five species – three of them have been recently studied mainly at the mitochondrial level. In this study, we collected mitochondrial (16S and nd4) and nuclear (mc1r and pomc) sequences from 49 specimens, including multiple individuals for each of the five species. We used multilocus coalescent‐based species‐tree inference and single‐gene analyses to estimate phylogenetic relationships among Blanus and to assess patterns of intraspecific differentiation within all the five species. Species‐tree and single‐gene phylogenies provided strong support for the Anatolian worm lizard B. strauchi lying outside a clade comprising all other congeners, with a sister relationship between the Iberian clade (B. cinereus and B. mariae) and the North African clade (B. tingitanus and B. mettetali). Mitochondrial and nuclear data supported the genetic distinctiveness of the recently described B. mariae and also indicated that the distribution of this species is wider than previously known and overlaps with B. cinereus in central Portugal. Blanus tingitanus showed two phylogeographic groups, from the northern and the southern portion of the range, respectively, having high mitochondrial and nuclear divergence and a possible contact zone in northwestern Morrocco. Finally, high genetic variation was found within B. mettetali and B. strauchi, suggesting in the latter case, the occurrence of cryptic taxa to be tested by further research.