Mitochondrial DNA phylogeography and population history of Meladema diving beetles on the Atlantic Islands and in the Mediterranean basin (Coleoptera,Dytiscidae)

The phylogeny and population history of Meladema diving beetles (Coleoptera, Dytiscidae) were examined using mitochondrial DNA sequence from 16S ribosomal RNA and cytochrome oxidase I genes in 51 individuals from 22 populations of the three extant species (M. imbricata endemic to the western Canary Islands, M. lanio endemic to Madeira and M. coriacea widespread in the Western Mediterranean and on the western Canaries), using a combination of phylogenetic and nested clade analyses. Four main lineages are observed within Meladema, representing the three recognized species plus Corsican populations of M. coriacea. Phylogenetic analyses demonstrate the sister relationship of the two Atlantic Island taxa, and suggest the possible paraphyly of M. coriacea. A molecular clock approach reveals that speciation within the genus occurred in the Early Pleistocene, indicating that the Atlantic Island endemics are not Tertiary relict taxa as had been proposed previously. Our results point to past population bottlenecks in all four lineages, with recent (Late-Middle Pleistocene) range expansion in non-Corsican M. coriacea and M. imbricata. Within the Canary Islands, M. imbricata seems to have independently colonized La Gomera and La Palma from Tenerife (although a colonization of La Palma from La Gomera cannot be discarded), and M. coriacea has independently colonized Tenerife and Gran Canaria from separate mainland lineages. In the Mediterranean basin this species apparently colonized Corsica on a single occasion, relatively early in its evolutionary history (Early Pleistocene), and has colonized Mallorca recently on multiple occasions. On the only island where M. coriacea and M. imbricata are broadly sympatric (Tenerife), we report evidence of bidirectional hybridization between the two species.     

Population genetics of the roan antelope (Hippotragus equinus) with suggestions for conservation

The roan antelope (Hippotragus equinus) is the second largest African antelope, distributed throughout the continent in sub-Saharan savannah habitat. Mitochondrial DNA (mtDNA) control region sequencing (401 bp, n = 137) and microsatellite genotyping (eight loci, n = 137) were used to quantify the genetic variability within and among 18 populations of this species. The within-population diversity was low to moderate with an average mtDNA nucleotide diversity of 1.9% and average expected heterozygosity with the microsatellites of 46%, but significant differences were found among populations with both the mtDNA and microsatellite data. Different levels of genetic resolution were found using the two marker sets, but both lent strong support for the separation of West African populations (samples from Benin, Senegal and Ghana) from the remainder of the populations studied across the African continent. Mismatch distribution analyses revealed possible past refugia for roan in the west and east of Africa. The West African populations could be recognized together as an evolutionarily significant unit (ESU), referable to the subspecies H. e. koba. Samples from the rest of the continent constituted a geographically more diverse assemblage with genetic associations not strictly corresponding to the other recognized subspecies.     

Phylogeographic history of the land snail Candidula unifasciata (Helicellinae,Stylommatophora): fragmentation,corridor migration,and secondary contact

Abstract.— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.     

Dispersal, habitat differences, and comparative phylogeography of Southeast Asian seahorses (Syngnathidae: Hippocampus)

Four distinct phylogeographical patterns across Southeast Asia were observed for four species of seahorse (genus Hippocampus) with differing ecologies. For all species, genetic differentiation (based on cytochrome b sequence comparisons) was significantly associated with sample site (Phi(ST) = 0.190-0.810, P < 0.0001) and with geographical distance (Mantel's r = 0.37-0.59, P < 0.019). Geographic locations of genetic breaks were inconsistent across species in 7/10 comparisons, although some similarities across species were also observed. The two shallow-water species (Hippocampus barbouri and Hippocampus kuda) have colonized the Sunda Shelf to a lesser degree than the two deeper-water species (Hippocampus spinosissimus and Hippocampus trimaculatus). In all species the presence of geographically restricted haplotypes in the Philippines could indicate past population fragmentation and/or long-distance colonization. A nested clade analysis (NCA) revealed that long-distance colonization and/or fragmentation were likely the dominant forces that structure populations of the two shallow-water species, whereas range expansion and restricted dispersal with isolation by distance were proportionally more important in the history of the two deeper-water species. H. trimaculatus has the most widespread haplotypes [average clade distance (D(c)) of nonsingleton haplotypes = 1169 km], indicating potentially high dispersal capabilities, whereas H. barbouri has the least widespread haplotypes (average D(c) = 67 km) indicating potentially lower dispersal capabilities. Pleistocene separation of marine basins and postglacial flooding of the Sunda Shelf are extrinsic factors likely to have contributed to the phylogeographical structure observed, whereas differences among the species appear to reflect their individual ecologies.     

Beringian origins and cryptic speciation events in the fly agaric (Amanita muscaria)

Amanita muscaria sensu lato has a wide geographic distribution, occurring in Europe, Asia, Africa, Australia, New Zealand, and North, Central and South America. Previous phylogenetic work by others indicates three geographic clades (i.e. 'Eurasian', 'Eurasian-alpine' and 'North American' groups) within A. muscaria. However, the historical dispersal patterns of A. muscaria remained unclear. In our project, we collected specimens from arctic, boreal and humid temperate regions in Alaska, and generated DNA sequence data from the protein-coding beta-tubulin gene and the internal transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA repeat. Homologous sequences from additional A. muscaria isolates were downloaded from GenBank. We conducted phylogenetic and nested clade analyses (NCA) to reveal the phylogeographic history of the species complex. Although phylogenetic analyses confirmed the existence of the three above-mentioned clades, representatives of all three groups were found to occur sympatrically in Alaska, suggesting that they represent cryptic phylogenetic species with partially overlapping geographic distributions rather than being allopatric populations. All phylogenetic species share at least two morphological varieties with other species, suggesting ancestral polymorphism in pileus and wart colour pre-dating their speciations. The ancestral population of A. muscaria likely evolved in the Siberian-Beringian region and underwent fragmentation as inferred from NCA and the coalescent analyses. The data suggest that these populations later evolved into species, expanded their range in North America and Eurasia. In addition to range expansions, populations of all three species remained in Beringia and adapted to the cooling climate.     

Mitochondrial DNA phylogeography of the Mesoamerican spiny-tailed lizards (Ctenosaura quinquecarinata complex): historical biogeography, species status and conservation

Through the examination of past and present distributions of plants and animals, historical biogeographers have provided many insights on the dynamics of the massive organismal exchange between North and South America. However, relatively few phylogeographic studies have been attempted in the land bridge of Mesoamerica despite its importance to better understand the evolutionary forces influencing this biodiversity 'hotspot'. Here we use mitochondrial DNA sequence data from fresh samples and formalin-fixed museum specimens to investigate the genetic and biogeographic diversity of the threatened Mesoamerican spiny-tailed lizards of the Ctenosaura quinquecarinata complex. Species boundaries and their phylogeographic patterns are examined to better understand their disjunct distribution. Three monophyletic, allopatric lineages are established using mtDNA phylogenetic and nested clade analyses in (i) northern: México, (ii) central: Guatemala, El Salvador and Honduras, and (iii) southern: Nicaragua and Costa Rica. The average sequence divergence observed between lineages varied between 2.0% and 3.7% indicating that they do not represent a very recent split and the patterns of divergence support the recently established nomenclature of C. quinquecarinata, Ctenosaura flavidorsalis and Ctenosaura oaxacana. Considering the geological history of Mesoamerica and the observed phylogeographic patterns of these lizards, major evolutionary episodes of their radiation in Mesoamerica are postulated and are indicative of the regions' geological complexity. The implications of these findings for the historical biogeography, taxonomy and conservation of these lizards are discussed.     

Phylogeographical history of the sponge Crambe crambe (Porifera, Poecilosclerida): range expansion and recent invasion of the Macaronesian islands from the Mediterranean Sea

We studied sequence variation in the nuclear ribosomal internal transcribed spacers (ITS-1 and ITS-2) in 111 individuals from 11 populations/localities of the sponge Crambe crambe across the core species range in the western Mediterranean Sea and Atlantic Ocean. We report the first confirmed instance of intragenomic variation in sponges. Phylogeographical, nested clade and population genetic analyses were used to elucidate the species' evolutionary history. The study revealed highly structured populations affected by restricted gene flow and isolation-by-distance. A contiguous range expansion in the whole distribution area of the sponge was inferred. Phylogenetic analyses indicate a recent origin of most sequence types that could be explained by a recent origin of the species or a by recent bottleneck event in the studied area. A recent expansion of the distribution range to the Macaronesian region from the Mediterranean Sea was also detected, suggesting that C. crambe was recently introduced from the Mediterranean Sea to the Atlantic Ocean via human-mediated transport, and that the pattern observed is not the result of a natural biogeographical relationship between these zones.     

Pan-European phylogeography of the aquatic snail Theodoxus fluviatilis (Gastropoda: Neritidae)

Investigating the geographical distribution of genetic lineages within species is critical to our understanding of how species evolve. As many species inhabit large and complex ranges, it is important that phylogeographical research take into account the entire range of widespread species to clarify how myriad extrinsic variables have affected their evolutionary history. Using phylogenetic, nested clade, and mismatch distribution analyses on a portion of the mitochondrial COI gene, I demonstrate that the wide-ranging freshwater snail Theodoxus fluviatilis possesses in parallel many of the phylogeographical patterns seen in less widespread freshwater species of Europe. Fragmentary forces play a major part in structuring the range of this species, with 12 of 14 geographically structured nested clades displaying a distribution consistent with fragmentation or restricted dispersal. Certain regions of southern Europe harbour the majority of genetic diversity (total haplotype diversity, H = 0.87), particularly Italy (H = 0.87) and areas surrounding the Black Sea (H = 0.81). Post-Pleistocene range expansion is pronounced, with the majority of northern European populations (95% of sample sites) having arisen from northern Italian individuals that initially colonized northern Germany. Additionally, two highly divergent haplotype lineages present in northern Germany imply that there were at least two postglacial recolonization routes. Estuaries may also provide a means of dispersal given that no genetic differentiation was found between estuarine populations and neighbouring freshwater populations. Taken together, these data reveal a species with a complex genetic history resulting from the fragmentary effects of European geology as well as continuous and discrete range expansion related to their aquatic biology.     

Phylogeography and conservation genetics of the Columbia spotted frog (Rana luteiventris; Amphibia, Ranidae)

The Columbia spotted frog (Rana luteiventris) has a widespread distribution in western Canada and the western US, although the southern reach of its range is highly fragmented into several isolated populations. Threats from various factors have raised concerns regarding the long-term survival of many small, isolated populations. Here, we report a study designed to determine the phylogeographic and conservation genetic parameters of R. luteiventris in the western US. Mitochondrial DNA (mtDNA) sequences were examined for phylogeographic structuring using phylogenetic reconstruction methods, coupled with networking and nested clade analyses. These methods permitted a distinction to be made between historic and demographic forces acting to generate geographical patterning of genetic variation. Phylogenetic analysis revealed four geographically correlated monophyletic clades. Three of these clades correspond to well-defined, nonoverlapping geographical locations in the fragmented portion of the range. The other is comprised of all samples collected from the contiguous range and includes one isolate from northern Wyoming. Networking and nested clade analyses confirmed these results and revealed that historical processes, such as range expansion and vicariance, rather than recurrent gene flow are likely responsible for observed patterns of genetic variation. A measure of genetic variation (theta = 4N(e)mu) revealed that R. luteiventris populations in Utah have a relatively low amount of genetic variation compared with populations in the continuous portion of the range.     

Nested cladistic analysis, phylogeography and speciation in the Timarcha goettingensis complex (Coleoptera, chrysomelidae)

The Timarcha goettingensis complex is a monophyletic assemblage of closely related leaf beetles (Chrysomelidae), distributed from the north half of the Iberian Peninsula to Central Europe. Oligophagy, mountainous habitat and apterism are factors which are assumed to promote speciation in these beetles. We have used cytochrome oxidase subunit II mitochondrial DNA genealogies obtained from 31 sampling localities and a nested geographical distance analysis to assess the population structure and demographic factors explaining the geographical distributions of the mtDNA haplotypes in the T. goettingensis complex. The results show that there is a significant association between genetic structuring and geography. Inferences about the historical population processes in the species complex are discussed, being in general in accordance with contiguous range expansions and past fragmentations. The use of the cohesion species concept approach suggests the existence of several systematic ranks among the different T. goettingensis populations, which is in part supported by ecological traits such as trophic selection and altitudinal distribution.     

Pigmentation and tunic cells in Cystodytes dellechiajei (Urochordata, Ascidiacea)

The tunic of Cystodytes dellechiajei (Poly- citoridae), a colony-forming species of the Ascidiacea that contains biologically active alkaloids, was investigated using light microscopy, laser-scanning microscopy and nuclear magnetic resonance techniques. The colonies contain numerous individual zooids, which are embedded in a common tunic. Each zooid is protected by a firm capsule of overlapping calcareous spicules. The colonies lack blood vessels in the tunic, but six morphologically different types of tunic cells were found: pigment cells, bladder cells, vacuolated filopodial cells, granular filopodial cells, morula cells and granular cells. Rod-like bacteria were found in the tunic matrix. Bladder cells and pigment cells could be identified as storage units for acid and pyridoacridine alkaloids, making the tunic inedible and repelling predators. Filopodial cells have long filopodia, which probably are connected to each other. They may be involved in transportation processes within the tunic tissue. The functions of the morula cells and the granular cells are unknown as yet. With its several specialised cells, the tunic of C. dellechiajei represents a dynamic living tissue containing biologically active compounds. Accepted: 20 September 2000     

Comparative phylogeography of Zacco platypus and Opsariichthys bidens (Teleostei, Cyprinidae) in China based on cytochrome b sequences

We compared the phylogenetic relationships and the population structure of the sympatric cyprinids Opsariichthys bidens and Zacco platypus across the Yangzte, Pearl and Hai He drainages in Mainland China. We used the mitochondrial cytochrome b gene to evaluate their phylogeographical structure and to discuss further systematic implications. We sequenced 71 new individuals of Z. platypus to add to a previous study and to construct a geographically comparable data set with O. bidens (Mol. Phylogenet. Evol. 31, 2004, 192; Mol. Phylogenet. Evol. 37, 2005, 920). Phylogeographic analysis of Z. platypus identified two additional mtDNA lineages (Zacco E and F) in addition to the previously recovered lineages (Zacco A–D). The geographical range of the Zacco A was extended across the Pearl drainage, broadening considerably the previous distribution that was circumscribed to the Yangtze drainage. Our results show high molecular divergence between all Zacco A–F mtDNA lineages (mean TrN + I + G distance 17.7%, range 4.4–30.8%), and the population analyses showed that most of the genetic variation was found among major river tributaries (90.2%). Values of θ_ST higher than 0.97 corroborated this interruption of gene flow between Zacco A–F mtDNA lineages. Nested clade analysis results were also concordant with the phylogenetic relationships recovered. We found similar phylogeographic patterns in Z. platypus and O. bidens: they exhibited two widespread and common lineages (Zacco A and Opsariichthys 1) ranging across the Yangtze and Pearl drainages, and both taxa had Northern lineages (Zacco F and Opsariichthys 5) clearly differentiated. Similar phylogeographic patterns suggest that common historical factors have shaped current Zacco and Opsariichthys diversity. High genetic distances and θ_ST values suggested that differentiation of Zacco A–F mtDNA lineages and Opsariichthys 1–5 was not recent and was probably favoured by old geological events. However, population parameters and low genetic differentiation inside Zacco A and Opsariichthys 1 suggest a recent gene flow across river drainages. Mitochondrial‐based phylogenies for the combined data sets of Zacco A–F and Opsariichthys 1–5 mtDNA lineages always recovered both taxa as paraphyletic, highlighting the notion that their current systematics need a revision.     

Mitochondrial phylogeography of the moor frog, Rana arvalis

The moor frog Rana arvalis is a lowland species with a broad Eurasiatic distribution, from arctic tundra through forest to the steppe zone. Its present-day range suggests that glacial refugia of this frog were located outside southern European peninsulas. We studied the species-wide phylogeographical pattern using sequence variation in a 682 base pairs fragment of mtDNA cytochrome b gene; 223 individuals from 73 localities were analysed. Two main clades, A and B, differing by c. 3.6% sequence divergence were detected. The A clade is further subdivided into two subclades, AI and AII differing by 1.0%. All three lineages are present in the Carpathian Basin (CB), whereas the rest of the species range, including huge expanses of Eurasian lowlands, are inhabited solely by the AI lineage. We infer that AII and B lineages survived several glacial cycles in the CB but did not expand, at least in the present interglacial, to the north. The geographical distribution and genealogical relationships between haplotypes from the AI lineage indicate that this group had two glacial refugia, one located in the eastern part of the CB and the other probably in southern Russia. Populations from both refugia contributed to the colonization of the western part of the range, whereas the eastern part was colonized from the eastern refugium only. The effective population size as evidenced by theta(ML) is an order of magnitude higher in the AI lineage than in the AII and B lineages. Demographic expansion was detected in all three lineages.     

Determining the origin and age of the Westland beech (Nothofagus) gap, New Zealand, using fungus beetle genetics

The formation and maintenance of the Nothofagus beech gap in the South Island, New Zealand, has been the focus of biogeographical debate since the 1920s. We examine the historical process of gap formation by investigating the population genetics of fungus beetles: Brachynopus scutellaris (Staphylinidae) inhabits logs and is absent from the beech gap, and Hisparonia hystrix (Nitidulidae) is contiguous through the gap and is found commonly on sooty mould growing on several plant species. Both species show distinctive northern and southern haplotype distributions while H. hystrix recolonized the gap as shown by definitive mixing. B. scutellaris shows two major haplotype clades with strong geographical concordance, and unlike H. hystrix, has clearly defined lineages that can be partitioned for molecular dating. Based on coalescence dating methods, disjunct lineages of B. scutellaris indicate that the gap was formed less than 200 000 years ago. Phylogenetic imprints from both species reveal similar patterns of population divergence corresponding to recent glacial cycles, favouring a glacial explanation for the origin of the gap. Post-gap colonization by H. hystrix may have been facilitated by the spread of Leptospermum scoparium host trees to the area, and they may be better at dispersing than B. scutellaris which may be constrained by fungal host and/or microhabitat. The gap-excluded species B. scutellaris is found in both beech and podocarp-broadleaf forests flanking the Westland gap and its absence in the gap may be related to incomplete recolonization following glacial retreat. We also discuss species status and an ancient polymorphism within B. scutellaris .     

Mitochondrial DNA phylogeography of Lissotriton boscai (Caudata, Salamandridae): evidence for old, multiple refugia in an Iberian endemic

In Europe, southern peninsulas served as refugia during cold periods in the Pleistocene, acting both as centres of origin of endemisms and as sources from which formerly glaciated areas were recolonized during interglacial periods. Previous studies have revealed that within the main refugial areas, intraspecific lineages often survived in allopatric refugia. We analysed two mitochondrial markers (nad4, control region, approximately 1.4 kb) in 103 individuals representing the entire distribution of Lissotriton boscai, a newt endemic to the western Iberian Peninsula. We inferred the evolutionary history of the species through phylogenetic, phylogeographic and historical demographic analyses. The results revealed unexpected, deep levels of geographically structured genetic variability. We identified two main evolutionary lineages, each containing three well-supported clades. The first historical split involved populations from central-southwestern coastal Portugal and the ancestor of all the remaining populations around 5.8 million years ago. Both lineages were subsequently fragmented into different population groups between 2.5 and 1.2 million years ago. According to nested clade analysis, at lower hierarchical levels the patterns suggest restricted gene flow with isolation by distance, whereas at higher levels the clades exhibit signatures of contiguous range expansion. Bayesian Skyline Plots show recent bottlenecks, followed by demographic expansions in all lineages. The significant genetic structure found is consistent with long-term survival of populations in allopatric refugia, supporting the 'refugia-within-refugia' scenario for southern European peninsulas. The comparison of our results with other co-distributed species highlights the generality of this hypothesis for the Iberian herpetofauna and suggests that Mediterranean refuges had more relevance for the composition and distribution of present biodiversity patterns than currently acknowledged. We briefly discuss the taxonomic and conservation implications of our results.     

Genetic structure of the star sea squirt, Botryllus schlosseri, introduced in southern European harbours

The introduction of new genetic variants or species is often caused by maritime transport between harbours. Botryllus schlosseri is a cosmopolitan ascidian species that is found in both harbours and open shore habitats. In order to determine the influence of ship traffic on the genetic structure and phylogeography of B. schlosseri in southern Europe, we analyzed the variability of a fragment of the mitochondrial gene cytochrome c oxidase subunit I (COI). We sampled seven Atlanto-Mediterranean harbour populations and three open-shore populations. In addition, we sequenced some colonies from the US-Atlantic coast and from other Mediterranean localities to perform phylogenetic analyses. Although the number of polymorphic sites recorded (25.8%) was within the range observed in other population studies based on ascidian COI sequences, the haplotypic diversity (16 haplotypes out of 181 sequences) was much lower. Moreover, a lack of intermediate haplotypes was observed. This pattern of high nucleotide diversity and low haplotype diversity was consistent with introduction events of a few divergent haplotypes. We found a strong genetic structure in the study populations. Gene flow was only appreciable between some harbour populations. Harbour- and open-shore populations were well differentiated, although there was no evidence for isolation by distance. A nested clade analysis pointed to long-distance colonization, possibly coupled with subsequent fragmentation, as the underlying process. Our results suggest that B. schlosseri entered the study area via harbour-hopping, possibly through recurrent introduction events. The haplotypes from North America and most of the European ones were grouped in the same phylogenetic clade. This suggests occasional gene flow between both continents, probably through ship transport.     

Statistical hypothesis testing in intraspecific phylogeography: nested clade phylogeographical analysis vs. approximate Bayesian computation

Nested clade phylogeographical analysis (NCPA) and approximate Bayesian computation (ABC) have been used to test phylogeographical hypotheses. Multilocus NCPA tests null hypotheses, whereas ABC discriminates among a finite set of alternatives. The interpretive criteria of NCPA are explicit and allow complex models to be built from simple components. The interpretive criteria of ABC are ad hoc and require the specification of a complete phylogeographical model. The conclusions from ABC are often influenced by implicit assumptions arising from the many parameters needed to specify a complex model. These complex models confound many assumptions so that biological interpretations are difficult. Sampling error is accounted for in NCPA, but ABC ignores important sources of sampling error that creates pseudo-statistical power. NCPA generates the full sampling distribution of its statistics, but ABC only yields local probabilities, which in turn make it impossible to distinguish between a good fitting model, a non-informative model, and an over-determined model. Both NCPA and ABC use approximations, but convergences of the approximations used in NCPA are well defined whereas those in ABC are not. NCPA can analyse a large number of locations, but ABC cannot. Finally, the dimensionality of tested hypothesis is known in NCPA, but not for ABC. As a consequence, the 'probabilities' generated by ABC are not true probabilities and are statistically non-interpretable. Accordingly, ABC should not be used for hypothesis testing, but simulation approaches are valuable when used in conjunction with NCPA or other methods that do not rely on highly parameterized models.     

Cryptic speciation in the southern African vlei rat Otomys irroratus complex: evidence derived from mitochondrial cyt b and niche modelling

The vlei rat Otomys irroratus has a wide distribution in southern Africa with several datasets indicating the presence of two putative species (O. irroratus and O. auratus). In the present study we use mitochrondrial cyt b data (～950 bp) from 98 specimens (including museum material) collected throughout the range of the species to determine the geographical limits of the two recognized species, and we link this to niche modelling to validate these species. Phylogenetic analysis of the DNA sequence data, using maximum parsimony, neighbour joining and Bayesian inference, retrieved two divergent statistically well‐supported clades. Clade A occurs in the Western and Eastern Cape while Clade B occurs in the Free State, KwaZulu‐Natal, Northern Cape and Mpumalanga provinces of South Africa and Zimbabwe. Mean sequence divergence between the two clades (A and B) was 7.0% and between sub‐clades comprising clade B it was 4.8%; the two clades diverged during the Pleistocene. Within Clade A the mean sequence divergence among specimens was 1.91%. Niche modelling revealed that the incipient species occupy distinct bioclimatic niches associated with seasonality of precipitation. Our data allow insightful analysis into the factors that could have led to cladogenesis within this rodent. More significantly, the new data enable us to pinpoint the Eastern Cape province as a contact zone for the divergent species. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 192–206.