DNA EVOLUTION AND COLONIZATION SEQUENCE OF ISLAND LIZARDS IN RELATION TO GEOLOGICAL HISTORY: MTDNA RFLP,CYTOCHROME B,CYTOCHROME OXIDASE, 12S RRNA SEQUENCE,AND NUCLEAR RAPD ANALYSIS

A novel source of nuclear DNA information from random amplified polymorphisms (RAPD) and a wide-range mitochondrial DNA information (cytochrome b, cytochrome oxidase, and 12s rRNA sequence, RFLP from 4-base and 6-base recognition endonucleases) are used to reconstruct the population phylogeny of the western Canary Island lizard, Gallotia galloti, which, for geological reasons, has been subject to dispersal but not vicariance. Interpretation of DNA phylogenies in terms of colonization sequence indicates that G. galloti arose in Tenerife and dispersed westward in two independent pathways: north from north Tenerife to La Palma, and south from south Tenerife to Gomera to Hierro. The direction and timing of colonization by DNA divergence is entirely compatible with geological time and sequence of island origin.     

Differences Between two Subspecies of Dolly Varden, Salvelinus malma, Revealed by RFLP—PCR Analysis of Mitochondrial DNA

Genetic differences between two subspecies of Dolly Varden, northern Salvelinus malma malma and southern Salvelinus malma krascheninnikovi, from rivers of eastern Russia were studied. Mitochondrial DNA was analyzed by restriction fragment length polymorphisms (RFLP) performed on products amplified with polymerase chain reaction. Three adjacent segments (approximately 7670 bp), comprising 47% of the mitochondrial genome were used: two encoding the five complete NADH dehydrogenase subunits and the other the cytochrome b gene and the control region (D-loop). Total composite haplotypes 46 were found among 136 fishes using RFLP analysis with 14 restriction enzymes. The amount of nucleotide divergence between haplotypes of two subspecies of Dolly Varden was estimated to be approximately 4%. The differences in the level of nucleotide diversity, mismatch distribution between haplotypes, and population-genetic structure of two subspecies of Dolly Varden suggest that these two forms have existed separately for a long time.     

Genetic diversity and population divergence in fragmented habitats: Conservation of Idaho ground squirrels

The Idaho ground squirrel, which consists of a northern (Spermophilus brunneus brunneus) and a southern subspecies (S. b. endemicus), has suffered from habitat loss and fragmentation, resulting in a reduction in both numbers and geographic range of the species. The northern Idaho ground squirrel (NIDGS) is listed as a threatened subspecies under the Endangered Species Act, and the southern Idaho ground squirrel (SIDGS) is a candidate. Because Idaho ground squirrel populations are small and often isolated, they are susceptible to inbreeding and loss of genetic diversity through drift. This research evaluates levels of genetic diversity and patterns of population divergence in both subspecies of Idaho ground squirrels. We hypothesized that NIDGS would exhibit lower genetic diversity and greater population divergence due to a longer period of population isolation relative to most SIDGS populations. Genetic diversity and divergence were quantified using 8 microsatellite loci. Contrary to expectations, SIDGS populations exhibited consistently lower levels of microsatellite diversity. Additionally, NIDGS exhibited only modest divergence among populations, while divergence levels among SIDGS populations were highly varied. Preliminary evaluations of mitochondrial DNA diversity and structure revealed lower diversity in NIDGS and some differences in gene flow that warrant further study. Based on our results, we suggest different management strategies for the two subspecies. Habitat restoration appears to be the most desirable conservation strategy for NIDGS populations. In contrast, low genetic diversity observed in SIDGS may warrant supplementation of isolated populations through translocations or captive breeding to mitigate further loss of genetic variability.     

Ecological divergence combined with ancient allopatry in lizard populations from a small volcanic island

Population divergence and speciation are often explained by geographical isolation, but may also be possible under high gene flow due to strong ecology‐related differences in selection pressures. This study combines coalescent analyses of genetic data (11 microsatellite loci and 1 Kbp of mtDNA) and ecological modelling to examine the relative contributions of isolation and ecology to incipient speciation in the scincid lizard Chalcides sexlineatus within the volcanic island of Gran Canaria. Bayesian multispecies coalescent dating of within‐island genetic divergence of northern and southern populations showed correspondence with the timing of volcanic activity in the north of the island 1.5–3.0 Ma ago. Coalescent estimates of demographic changes reveal historical size increases in northern populations, consistent with expansions from a volcanic refuge. Nevertheless, ecological divergence is also supported. First, the two morphs showed non‐equivalence of ecological niches and species distribution modelling associated the northern morph with mesic habitat types and the southern morph with xeric habitat types. It seems likely that the colour morphs are associated with different antipredator strategies in the different habitats. Second, coalescent estimation of gene copy migration (based on microsatellites and mtDNA) suggest high rates from northern to southern morphs demonstrating the strength of ecology‐mediated selection pressures that maintain the divergent southern morph. Together, these findings underline the complexity of the speciation process by providing evidence for the combined effects of ecological divergence and ancient divergence in allopatry.     

Population systematics of the snake genus Naja (Reptilia: Serpentes: Elapidae) in Indochina: Multivariate morphometrics and comparative mitochondrial DNA sequencing (cytochrome oxidase I)

We analyze the population systematics of Asiatic cobras in Indochina, China and the Andaman Islands by means of comparative sequencing of the cytochrome oxidase subunit I gene of the mitochondrial DNA molecule and multivariate analysis of morphological characters. Canonical variate analysis and mtDNA sequence information reveal that the cobras of this region comprise four distinct species: Naja atra from China and northern Vietnam, Naja kaouthia from Burma, central Thailand, Cambodia and southern Vietnam, Naja siamensis from Thailand, Cambodia and southern Vietnam, and Naja sagittifera from the Andaman Islands. The subspecies N. kaouthia suphanensis Nutaphand 1986 shows no mtDNA sequence difference from typical N. kaouthia from central Thailand, and multivariate analysis does not reveal differences in general phenotypic profile; the subspecies is therefore synonymised with Naja kaouthia. The cytochrome oxidase subunit I gene, little used in molecular taxonomy, is shown to be well suited for studies at the species level, as it shows taxonomically useful levels of interspecific divergence but low levels of intraspecific variation; this is particularly relevant for studies of rare species, where sample size is a problem. The combination of multivariate morphometrics and molecular systematics can be particularly powerful in resolving systematic problems in such cases.     

Road edge effect on the abundance of the lizard <Emphasis Type="Italic">Gallotia galloti</Emphasis> (Sauria: Lacertidae) in two Canary Islands forests

Transportation infrastructure is a main cause of environmental change in forest landscapes worldwide. In the Canary Islands, a dense road system fragment the native Canarian pine and laurel forests causing potential changes in population densities of endemic lacertid lizards (genus Gallotia). Our aim was to assess road edge effects on relative abundance patterns of the endemic Gallotia galloti in both forests. We also explored the species–habitat relationships in this road-fragmentation context. We found that lizard relative density in relation to road edges differed between forests. Lizards were more abundant along edges and leeward interior, but virtually absent from the interior of the windward laurel forest. In the pine forest, lizards were present at three distances from edge, with a net decrease in abundance from edge to interior. These patterns may be explained partly by differences in vegetation structure regarding road proximity in each forest that potentially affect the helio- and thigmothermic character of G. galloti, and thus its habitat use. A general suggestion of this study is that road margins create corridors that may be used by native lizards for dispersal through inhospitable forest matrix. The high road density in Tenerife may have negative implications for the conservation of the genetic variability of G. galloti. At the island scale, increased communication between lizard populations through road corridors might increase homogenization of the gene pool. Ecological processes in which this lizard plays important roles may also experience changes along road edges.     

Genetic divergence of three freshwater isopod species from southern New Zealand

Aim We examined the biogeography of three freshwater isopod species (Austridotea annectens, A. lacustris, A. benhami), and tested the hypotheses that genetic differences would: (1) exist between geographic locations; and (2) correspond to known geological events (e.g. appearance of islands leading to the availability of habitat). Location Southern New Zealand, including South Island, Stewart Island, Campbell Island and Chatham Islands. Methods We examined specimens throughout the known species range from 12 populations of A. lacustris, five populations of A. annectens, and three populations of A. benhami, using mitochondrial DNA (cytochrome c oxidase I) sequence analyses. Results We resolved three main clades corresponding to the three species, with 16% sequence divergence between A. annectens and A. benhami, and 31% divergence between these species and A. lacustris. Divergence within A. benhami was < 2.0%. However, divergence within A. lacustris reached up to 10% with four main groupings: (1) Chatham Islands; (2) Campbell Island; (3) Fiordland; and (4) east coast South Island and Stewart Island. Divergence within A. annectens reached up to 4.4%, with two main groupings: (1) Chatham Islands and (2) east coast South Island and Stewart Island. Patterns of genetic divergence were most likely the result of geographical isolation among A. lacustris and A. annectens populations. In particular, the divergence of A. lacustris and A. annectens on Chatham Islands may correspond to the availability of this habitat c. 4 Ma, whereas the divergence of A. lacustris on the much older Campbell Island and in Fiordland may indicate either a rare founder event or a change in ocean circulation that resulted in their isolation from a once more widespread gene pool. Main conclusions The three New Zealand species of Austridotea are genetically distinct, with up to 31% divergence between species. Genetic variability was highest between populations of the two most widely distributed species, and divergence was greatest on islands distant from mainland New Zealand and in the discrete Fiordland region. The magnitude of genetic divergence of isopods on the Auckland and Chatham Islands is consistent with these populations having been founded in the Pliocene via oceanic dispersal from mainland New Zealand.     

Temperature,energy metabolism,and adaptive divergence in two oyster subspecies

Comparisons of related species that have diverse spatial distributions provide an efficient way to investigate adaptive evolution in face of increasing global warming. The oyster subjected to high environmental selections is a model species as sessile marine invertebrate. This study aimed to detect the adaptive divergence of energy metabolism in two oyster subspecies from the genus Crassostrea—C. gigas gigas and C. gigas angulata—which are broadly distributed along the northern and southern coasts of China, respectively. We examined the effects of acute thermal stress on energy metabolism in two oyster subspecies after being common gardened for one generation in identical conditions. Thermal responses were assessed by incorporating physiological, molecular, and genomic approaches. Southern oysters exhibited higher fluctuations in metabolic rate, activities of key energetic enzymes, and levels of thermally induced gene expression than northern oysters. For genes involved in energy metabolism, the former displayed higher basal levels of gene expression and a more pronounced downregulation of thermally induced expression, while the later exhibited lower basal levels and a less pronounced downregulation of gene expression. Contrary expression pattern was observed in oxidative stress gene. Besides, energy metabolic tradeoffs were detected in both subspecies. Furthermore, the genetic divergence of a nonsynonymous SNP (SOD‐132) and five synonymous SNPs in other genes was identified and validated in these two subspecies, which possibly affects downstream functions and explains the aforementioned phenotypic variations. Our study demonstrates that differentiations in energy metabolism underlie the plasticity of adaptive divergence in two oyster subspecies and suggest C. gigas angulata with moderate phenotypic plasticity has higher adaptive potential to cope with exacerbated global warming.     

Microsatellite genetic variation of Asian populations of Dolly Varden char

Genetic variation at eight microsatellite loci was examined in 21 populations of Dolly Varden charrs, Salvelinus malma, representing five geographical regions (Kamchatka Peninsula, Sea of Okhotsk coast, Sea of Japan coast, Sakhalin Island, and Kuril islands). Hierarchical analysis of molecular variance showed that 11% (58% in terms of R-statistics) of the variation was distributed among of northern subspecies of Dolly Varden, Salvelinus malma malma and southern Asian Dolly Varden, S. m. krascheninnikovi while similar values were attributed to the among-regional level within northern Dolly Varden 9% (7%) and southern Asian Dolly Varden 11% (14%). Permutation-based tests indicated a mutational component to genetic differentiation based on allelic size variance and suggested that divergence of the two subspecies had occurred at least 3,000 generations ago. On large spatial scales (within the Asian range of the species), populations clustered according to their geographical location. On smaller scales (within regions and subregions) correlation between genetic and geographic distances was not significant. Northern Dolly Varden has higher allelic diversity and more private alleles than southern subspecies, this probably indicating differences in demographic history.     

Sex‐specific responses of phenotypic diversity to environmental variation

Identifying the factors generating ecomorphological diversity within species can provide a window into the nascent stages of ecological radiation. Sexual dimorphism is an obvious axis of intraspecific morphological diversity that could affect how environmental variation leads to ecological divergence among populations. In this paper we test for sex‐specific responses in how environmental variation generates phenotypic diversity within species, using the generalist lizard Gallotia galloti on Tenerife (Canary Islands). We evaluate two hypotheses: the first proposes that different environments have different phenotypic optima, leading to shifts in the positions of populations in morphospace between environments; the second posits that the strength of trait‐filtering differs between environments, predicting changes in the volume of morphospace occupied by populations in different environments. We found that intraspecific morphological diversity, provided it is adaptive, arises from both shifts in populations’ position in morphospace and differences in the strength of environmental filtering among environments, especially at high elevations. However, effects were found only in males; morphological diversity of females responded little to environmental variation. These results within G. galloti suggest natural selection is not the sole source of phenotypic diversity across environments, but rather that variation in the strength of, or response to, sexual selection may play an important role in generating morphological diversity in environmentally diverse settings. More generally, disparities in trait–environment relationships among males and females also suggest that ignoring sex differences in studies of trait dispersion and clustering may produce misleading inferences.     

Primate Genus Miopithecus: Evidence for the Existence of Species and Subspecies of Dwarf Guenons Based on Cellular and Endogenous Viral Sequences

Sequence data from the mitochondrial 12S rRNA gene were combined with endogenous retrovirus sequences to study the position of the genus Miopithecus in the primate tree. The mitochondrial sequences indicated that Miopithecus is a true genus distinct from Cercopithecus, although talapoin monkeys are commonly referred to as dwarf guenons. The existence of two species of dwarf guenons, suggested by differences in coat color, pigmentation, and geographic location, was supported by substantial mitochondrial 12S rRNA gene divergence. In line with the informal proposal of J. Kingdon (1997, “The Kingdon Field Guide to African Mammals,” Academic Press, London), we use the names Miopithecus talapoin for the southern, darker species and Miopithecus ougouensis for the northern, lighter-colored monkeys. Different 12S rRNA gene haplotypes found in M. ougouensis individuals suggest the possible existence of additional subspecies. Simian endogenous retrovirus (SERV) strain 23.1 proviruses were introduced in the primate germ-line after the Cercopithecinae split from the Colobinae, estimated at around 9–14 million years ago. SERV sequences were used for timing of divergence events in Cercopithecinae and confirmed the close relationship between the genera Cercopithecus and Miopithecus, which was only weakly supported by the more variable mtDNA sequences in a distance analysis, demonstrating the utility of these pseudogenes in phylogenetic grouping.     

Reflectance of sexually dichromatic UV‐blue patches varies during the breeding season and between two subspecies of Gallotia galloti (Squamata: Lacertidae)

Body coloration is sexually dimorphic in many vertebrate species, including lizards, in which males are often more conspicuous than females. A detailed analysis of the relative size of coloured patches and their reflectance, including the ultraviolet (UV) range, has rarely been performed. In the present work we quantified sexual dimorphism in body traits and surface area of all lateral patches from adult females and males of two subspecies of Gallotia galloti (G. g. galloti and G. g. eisentrauti). We also analysed the magnitude of sexual dichromatism in the UV‐visible reflectance of such patches and the changes in patch size and brightness during the reproductive season (April–July). Males had significantly larger patch areas (relative to their snout‐vent length) and higher brightness (mainly in the UV‐blue range) than did females in both subspecies. The comparison of relative patch areas among months did not reach statistical significance. However, patch brightness significantly changed during the breeding season: that of the UV‐blue (300–495 nm) range from lizards of the two subspecies was significantly larger in June than in April, while brightness in the 495–700 nm range in G. g. galloti was larger in May, June, and July than in April. A different pattern of dichromatism was also detected in the two populations, with G. g. eisentrauti being more sexually dichromatic than G. g. galloti. We discuss the results in terms of possible evolutionary causes for the sexual dichromatism related to different ecological characteristics of the habitats where each subspecies live. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 556–569.     

Bioacoustic and genetic divergence in a frog with a wide geographical distribution

The study of intraspecific variation of acoustic signals and its relationship with genetic divergence is important for understanding the origin of divergence in communication systems. We studied geographical variation in the acoustic structure of advertisement calls from five populations of the four‐eyed frog, Pleurodema thaul, and its relationship with the genetic divergence among these populations. By analyzing temporal and spectral parameters of the advertisement calls, we report that the signals of northern, central, and southern populations have remarkable differences between them. A phylogeographical analysis from a mitochondrial DNA fragment demonstrated three phylogenetic groups coincident with those found with the bioacoustics analysis. Furthermore, bioacoustic and genetic distances show significant correlations after controlling for geographical distance. These results suggest that behavioural divergence among populations of P. thaul has a phylogenetic basis, supporting three evolutionary units within this species, as well as prompting the exploration of divergence processes in the sound communication system of this species. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 142–155.     

Mitochondrial DNA Introgression in the European Abalone Haliotis tuberculata tuberculata: Evidence for Experimental mtDNA Paternal Inheritance and a Natural Hybrid Sequence

Two subspecies of the European abalone have been morphologically recognized: Haliotis tuberculata tuberculata, present in the North Atlantic, and Haliotis tuberculata coccinea, present in the Canary Islands. Among the different nuclear markers used to differentiate these two subspecies, the sperm lysin gene was the most reliable, leading to a 2.2% divergence. Concerning the subunit I of the mitochondrial cytochrome oxydase gene (COI), we observed a difference of 3.3% between the two subspecies. In the North Atlantic, an introgression of mitochondrial DNA from H. tuberculata coccinea to H. tuberculata tuberculata was evident in around 30% of individuals. Due to this difference, we were able to experimentally detect the transfer of paternal mitochondrial DNA (mtDNA) by specific quantitative polymerase chain reaction measurements. The presence of the two mtDNA signatures was also detected in 20% of individuals tested in the field. Moreover, one mt DNA hybrid sequence was identified. The sequencing of this mitochondrial DNA hybrid revealed a mosaic structure with many specific mutations. The origin of this hybrid sequence is discussed.     

Phylogeny and biogeography of the Anderson's crocodile newt, Echinotriton andersoni (Amphibia: Caudata), as revealed by mitochondrial DNA sequences

The Anderson's crocodile newt, Echinotriton andersoni, is considered a relic and endangered species distributed in the Central Ryukyus. To elucidate phylogenetic relationships and detailed genetic structures among populations, we analyzed variation in the mitochondrial cytochrome b gene. Results strongly support a primary dichotomy between populations from the Amami and Okinawa Island Groups with substantial genetic divergence, favoring a primary divergence between the two island groups. Within the latter, populations from the southern part of Okinawajima Island are shown to be more closely related to those from Tokashikijima Island than to those from the northern and central parts of Okinawajima. The prominent genetic divergence between the two island groups of the Central Ryukyus seems to have initiated in the Miocene, i.e., prior to formation of the strait that has consistently separated these island groups since the Pleistocene. The ancestor of the southern Okinawajima-Tokashikijima is estimated to have migrated from the northern and central parts of Okinawajima into southern Okinawajima at the Pleistocene, and dispersed into Tokashikijima subsequently.     

Genetic divergence of Connecticut Melanoplus femurrubrum populations

We surveyed Melanoplus femurrubrum populations within the state of Connecticut for genetic diversity at multiple genetic markers, including three mitochondrial [cytochrome oxidase subunit 1 (COI), reduced form of nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2), and AT rich] and one nuclear [internal transcribed spacers of the ribosomal DNA cluster (ITS1)] gene regions. All markers were variable, and the AT-rich gene showed the highest sequence divergence. Analysis of molecular variance (AMOVA), fixation index (Fst) analysis, and phylogeographic patterns showed little divergence between northern and southern regions. Estimates of genetic diversity (pi) showed higher mitochondrial diversity in the northern region but nearly equal diversity for the ITS1 gene. This study shows for the first time in Melanoplus genetic variation for the ND2, AT rich, and ITS genes within a small geographic area. Our methods and results should be useful for other researchers interested in conducting population-level studies on closely related species.     

Complete lack of mitochondrial divergence between two species of NE Atlantic marine intertidal gastropods

Some mitochondrial introgression is common between closely related species, but distinct species rarely show substantial introgression in their entire distribution range. In this study, however, we report a complete lack of mitochondrial divergence between two sympatric species of flat periwinkles (Littorina fabalis and Littorina obtusata) which, based on previous allozyme studies, diverged approximately 1 Ma. We re‐examined their species status using both morphology (morphometric analysis) and neutral genetic markers (microsatellites) and our results confirmed that these species are well separated. Despite this, the two species shared all common cytochrome‐b haplotypes throughout their NE Atlantic distribution and no deep split between typical L. fabalis and L. obtusata haplotypes could be found. We suggest that incomplete lineage sorting explains most of the lack of mitochondrial divergence between these species. However, coalescent‐based analyses and the sympatric sharing of unique haplotypes suggest that introgressive hybridization also has occurred.     

Geological history and within-island diversity: a debris avalanche and the Tenerife lizard Gallotia galloti

Several processes have been described that could explain geographical variation and speciation within small islands, including fragmentation of populations through volcanic eruptions. Massive landslides, or debris avalanches, could cause similar effects. Here we analyse the potential impact of the 0.8 million-year-ago (Ma) Güimar valley debris avalanche on the phylogeography of the lizard Gallotia galloti on the Canary Island of Tenerife. Distributions of mitochondrial DNA lineages (based on cytochrome b sequences) were analysed on a 60-km southeastern coast transect centred on this area. Three main clades were detected, which can be divided into northern (one clade) and southern (two clades) groups that introgress across the valley. Maximum-likelihood estimates of migration rates (scaled for mutation rate) revealed highly asymmetric patterns, indicating that long-term gene flow into this region from both the northern and the southern populations greatly exceeded that in the opposite directions, consistent with recolonization of the area. The ancestral Tenerife node on the G. galloti tree is estimated at 0.80 Ma, matching closely with the geological estimate for the debris avalanche. Morphological variation (body dimensions and scalation) was also analysed and indicated a stepped cline in female scalation across the valley, although the patterns for male scalation and male and female body dimensions were not as clear. Together these findings provide support for the hypothesis that the debris avalanche has shaped the phylogeography of G. galloti and may even have been a primary cause of the within-island cladogenesis through population fragmentation and isolation. Current estimates of timing of island unification mean that the original hypothesis that within-island diversity is explained by the secondary contact of populations from the two ancient precursor islands of Teno and Anaga is less plausible for this and some other Tenerife species. Large-scale landslides have occurred on many volcanic islands, and so may have been instrumental in shaping within-island diversities.     

Extensive genetic differentiation in Gobiomorphus breviceps from New Zealand

Partial mitochondrial DNA sequences for parts of the cytochrome b gene and control region were obtained for 89 upland bullies Gobiomorphus breviceps from 19 catchments in New Zealand. There were two highly distinctive mtDNA clades: a northern clade corresponding to the North Island, northern South Island and west coast South Island, and a south‐east clade, in the southern and eastern South Island. Within these major clades there were further distinct clades that correlated with geographic sub‐regions and catchments. The marked genetic differentiation has occurred in the absence of obvious morphological divergence. Based on cytochrome b sequence divergences and the molecular clock hypothesis, the northern and southeastern clades correspond with the uplift of the Southern Alps during the Pliocene, while populations in the North Island and northern South Island were estimated to have diverged during the Pleistocene. The widescale geographic divergences were similar to those observed in the galaxiids, Galaxias vulgaris and Galaxias divergens , but biogeographic management boundaries may not be the same, reflecting different evolutionary histories for non‐diadromous species occupying the same areas.     

Comparison of whole mitochondrial genome sequences of northern and southern white rhinoceroses (<Emphasis Type="Italic">Ceratotherium simum</Emphasis>): the conservation consequences of species definitions

The probable extinction of the last confirmed population of northern white rhinoceros (Ceratotherium simum cottoni) in the world has ignited debate regarding its species status compared to the southern white rhinoceros (Ceratotherium simum simum). Previous studies, based on partial mitochondrial sequences, have reported conflicting results regarding the species status of the northern white rhinoceros. We use whole mitochondrial genomes obtained using Next Generation Sequencing of four northern white rhinoceros and three southern white rhinoceros using novel primers in three overlapping fragments. Phylogenetic relationships were constructed, using Maximum Likelihood, and recovered monophyletic clades for northern white rhinoceros and southern white rhinoceros. The divergence time between the two mitochondrial DNA lineages was estimated to be between 0.46 and 0.97 million years ago using Bayesian inferences. Since there are currently only three surviving northern white rhinoceros individuals these results put into sharp focus the ongoing debate regarding the methods of species definition, and in particular the consequences of such definitions in conservation management of endangered species and subspecies. We conclude that the designation of sub-species status is more applicable to northern and southern white rhinoceros.