Conservation Genetics of Kangaroo Mice, Genus Microdipodops

The two currently recognized species of kangaroo mice, Microdipodops megacephalus and M. pallidus, inhabit sandy soils of the Great Basin Desert in western North America. Given their habitat specificity and the fluctuating climate throughout the Pleistocene, kangaroo mice likely endured a turbulent biogeographic history that resulted in disjunct distributions and isolation of genetic lineages. Recent phylogenetic investigations using mitochondrial data have revealed several mitochondrial clades within this genus that may represent cryptic species. These mitochondrial clades are genetically unique, occupy relatively small distributions, and, as such, may be at an increased risk of extinction due to climate change and extensive recent habitat alteration. Herein, we apply haplotype network, population genetic, and historical demographic analyses to mitochondrial data of each Micropdipodops species and mitochondrial clade to assess conservation genetics within kangaroo mice. Results indicate that each mitochondrial clade is a distinct lineage with little to no gene flow occurring among clades. Additionally, historical demographic analyses support past population expansions and identify locations of past refugium for each distinct lineage. Although mitochondrial data indicate that the clades appear to be in approximate genetic equilibrium and have not suffered any extreme bottlenecks over time, there is still concern for the survival of smaller and more vulnerable Microdipodops subpopulations due to impending habitat threats in the Great Basin Desert.     

Are solitary and gregarious Mormon crickets (Anabrus simplex, Orthoptera, Tettigoniidae) genetically distinct?

Phase polyphenisms are usually thought to reflect plastic responses of species, independent of genetic differences; however, phase differences could correlate with genetic differentiation for various reasons. Mormon crickets appear to occur in two phases that differ in morphology and behaviour. Solitary individuals are cryptic and sedentary whereas gregarious individuals form bands, migrate, and are aposematically coloured. These traits have been thought to be phenotypically plastic and induced by environmental conditions. However, there has been no previous investigation of the extent of genetic differences between solitary and gregarious populations of this widespread North American species. We sequenced two mitochondrial genes, COII and COIII, in samples of Mormon crickets from gregarious populations west of the continental divide and solitary mountain populations primarily east of the divide. Sequencing revealed two genetically distinct clades that broadly correspond with the solitary eastern populations and the mainly gregarious western populations. We used coalescent modelling to test the hypothesis that the species consists of two deep genetic clades, as opposed to a series of equally distinct populations. Results allowed us to reject the null hypothesis that a radiation independent of phase produced these clades, and molecular clock estimates indicate the time of divergence to be approximately 2 million years ago. This work establishes that the solitary populations found in the mountains on the eastern slope are part of a clade that is genetically distinct from the western populations, which are primarily gregarious, and the implications of this apparent correlation between phase and genetic differentiation are discussed.     

A phylogenetic analysis of the Orchidaceae: evidence from rbcL nucleotide

Cladistic parsimony analyses of rbcL nucleotide sequence data from 171 taxa representing nearly all tribes and subtribes of Orchidaceae are presented here. These analyses divide the family into five primary monophyletic clades: apostasioid, cypripedioid, vanilloid, orchidoid, and epidendroid orchids, arranged in that order. These clades, with the exception of the vanilloids, essentially correspond to currently recognized subfamilies. A distinct subfamily, based upon tribe Vanilleae, is supported for Vanilla and its allies. The general tree topology is, for the most part, congruent with previously published hypotheses of intrafamilial relationships; however, there is no evidence supporting the previously recognized subfamilies Spiranthoideae, Neottioideae, or Vandoideae. Subfamily Spiranthoideae is embedded within a single clade containing members of Orchidoideae and sister to tribe Diurideae. Genera representing tribe Tropideae are placed within the epidendroid clade. Most traditional subtribal units are supported within each clade, but few tribes, as currently circumscribed, are monophyletic. Although powerful in assessing monophyly of clades within the family, in this case rbcL fails to provide strong support for the interrelationships of the subfamilies (i.e., along the spine of the tree). The cladograms presented here should serve as a standard to which future morphological and molecular studies can be compared.     

Systematics of Mesocestoides (Cestoda: Mesocestoididae): evaluation of molecular and morphological variation among isolates

A hypothesis-based framework was used to test if 3 genetic strains of Mesocestoides (clades A, B, and C) are distinct evolutionary lineages, thereby supporting their delimitation as species. For comparative purposes, 3 established cestode species, Taenia pisiformis, Taenia serialis, and Taenia crassiceps were assessed using the same methods. Sequence data from mitochondrial rDNA (12S) and the second internal transcribed spacer of nuclear rDNA (ITS-2) revealed derived (autapomorphic) characters for lineages representing clade A (n = 6 autapomorphies), clade B (n = 4), and clade C (n = 9) as well as T. pisiformis (n = 15) and T. serialis (n = 12). Furthermore, multivariate analysis of morphological data revealed significant differences among the 3 genetic strains of Mesocestoides and between T. pisiformis and T. serialis. The level of phenotypic variation within evolutionary lineages of Mesocestoides and Taenia spp. tapeworms was similar. Results from this study support recognizing Mesocestoides clades A, B, and C as separate species, and provide evidence that clade B and Mesocestoides vogae are conspecific.     

GENETIC VARIATION OF KOGIA SPP. WITH PRELIMINARY EVIDENCE FOR TWO SPECIES OF KOGIA SIMA

Concordance between mitochondrial DNA (mtDNA) markers and morphologically based species identifications was examined for the two currently recognized Kogia species. We sequenced 406 base pairs of the control region and 398 base pairs of the cytochrome b gene from 108 Kogia breviceps and 47 K. sima samples. As expecred, the two sister species were reciprocally monophyletic to each other in phylogenetic reconstructions, but within K. sima , we unexpectedly observed another reciprocally monophyletic relationship. The two K. sima clades resolved were phylogeographically concordant with all of the haplotypes in one clade observed solely among specimens sampled from the Atlantic Ocean and with those in the other clade observed solely among specimens sampled from the Indo-Pacific Ocean. These apparently allopatric clades were observed in all phylogenetic reconstructions using the maximum parsimony, maximum likelihood, and neighborjoining algorithms, with the mtDNA gene sequences analyzed separately and combined. The nucleotide diversity for the combined gene sequence haplotypes of the two K. sima clades resolved in our analyses was 0.58% and 1.03% for the Atlantic and Indo-Pacific, respectively, whereas for the two recognized sister species, nucleotide diversity was 1.65% and 4.02% for K. breviceps and K. sima , respectively. The combined gene sequence haplotypes have accumulated 44 fixed base pair differences between the two K. sima clades compared to 20 fixed base pair differences between the two recognized sister species. Although our results are consistent with species-level differences between the two K. sima clades, recognition of a third Kogia species awaits supporting evidence that these two apparently allopatric clades represent reproductively isolated groups of animals.     

Specific pollinator attraction and the diversification of sexually deceptive Chiloglottis (Orchidaceae)

Evidence indicates that sexually deceptive Chiloglottis R.Br. (Orchidaceae) taxa specifically attract their thynnine wasp (Tiphiidae) pollinators through the floral odour mimicry of female wasp sex pheromones. We use amplified fragment length polymorphisms (AFLPs) to reconstruct the species-level phylogeny of Chiloglottis, make a preliminary evaluation of genetic distinctions between species, and compare the historical association among orchids and their pollinators using wasp sequence data from a previous study. AFLPs show large differences between three sub-generic clades relative to that found among species within each clade. Interspecific genetic barriers are indicated by AFLP discontinuities among species unlike in previously reported DNA sequence data. However, such barriers are demonstrated clearly in only one of the two pairs of sympatric species sampled more intensively. We interpret these patterns as indicating either (i) a rapid and recent radiation of species within each clade following histories of stasis or extinction, or (ii) alternating cycles of divergence and gene flow acting to homogenize genetic differences among species within each of the three clades.     

Population genetic structure of Tomicus piniperda L. (Curculionidae: Scolytinae) on different pine species and validation of T. destruens (Woll.)

Genetic diversity and population structure of Tomicus piniperda was assessed using mitochondrial sequences on 16 populations sampled on 6 pine species in France. Amplifications of Internal transcribed space 1 (ITS1) were also performed. Our goals were to determine the taxonomic status of the Mediterranean ecotype T. piniperda destruens, and to test for host plant or geographical isolation effect on population genetic structure. We showed that T. piniperda clusters in two mtDNA haplotypic groups. Clade A corresponds to insects sampled in continental France on Pinus sylvestris, P. pinaster and P. uncinata, whereas clade B gathers the individuals sampled in Corsica on P. pinaster and P. radiata and in continental France on P. pinea and P. halepensis. Insects belonging to clade A and clade B also consistently differ in the length of ITS1. Individuals belonging to both clades were found once in sympatry on P. pinaster. Genetic distances between clades are similar to those measured between distinct species of Tomicus. We concluded that clade B actually corresponds to the destruens ecotype and forms a good species, T. destruens. Analyses of molecular variance ( amova ) were conducted separately on T. destruens and T. piniperda to test for an effect of either geographical isolation or host species. Interestingly, the effect of host plant was significant for T. piniperda only, while the effect of geographical isolation was not. Pine species therefore seems to act as a significant barrier to gene flow, even if host race formation is not observed. These results still need to be confirmed by nuclear markers.     

Random interbreeding between cryptic lineages of the Common Raven: evidence for speciation in reverse

DNA sequence studies frequently reveal evidence of cryptic lineages in morphologically uniform species, many of which turn out to be evolutionarily distinct species. The Common Raven (Corvus corax) includes two deeply divergent mtDNA lineages: one lineage seems restricted to western North America and the other is Holarctic in distribution. These deep clades hint of the possibility of cryptic species in the western United States. We tested this hypothesis in a population consisting of an equal proportion of both mtDNA clades, by quantifying mating patterns and associated fitness consequences with respect to mtDNA. We also tested for morphological, behavioural and ecological correlates of sex and mtDNA clade membership. Mate pairings were random with respect to mtDNA clades, and there were no differences in reproductive success between assortatively and nonassortatively mated pairs. We found no differences in survival or resource use between clades. There were no differences in morphological or behavioural characters between mtDNA clades, except one clade trended towards greater mobility. These results suggest there are no barriers to gene flow between mtDNA clades and argue that the mtDNA clades have remerged in this population, likely due to a lack of ecological or signal differentiation between individuals in each lineage. Hence, in Common Ravens, phylogeographic structure in mtDNA is a reflection of likely past isolation rather than currently differentiated species.     

Multiple tandem gene duplications in a neutral lipase gene cluster in Drosophila

We have examined a highly dynamic section of the Drosophila melanogaster genome which contains neutral lipase family genes that have undergone multiple tandem duplication events. We have identified the orthologous clusters, encoding between five and eight apparently functional lipases, in other Drosophila genomes: yakuba, ananassae, pseudoobscura, virilis, mojavensis, persimilis, grimshawi and willistoni. We examined their gene structure, duplication and pseudogene formation, and the presence of transposable elements. Based on phylogenetic comparisons, the lipase genes contained in each of the clusters fall into four distinct clades. Clades I and II have distinct evolutionary constraints to clades III and IV. Multiple gene duplications have occurred in different lineages of clades I and II while clades III and IV contain a single lipase gene from each species. Compared with lipases from other clades, clade IV genes contain an additional 3' domain of tandemly repeated sequence of varying length and composition, and a substitution in the residue adjacent to the key catalytic serine in the encoded proteins. A comparison of non-synonymous to synonymous nucleotide substitution (dN/dS) rates within each clade showed the highest rate of divergence was between paralogous lipase gene pairs suggesting selection pressure on duplicated genes. Analysis of the encoded lipase protein sequences within each species using PAML identified positively selected sites; structure homology modeling based on human pancreatic lipase indicated many of these residues formed part of the active site of the enzyme. As some of the cluster lipase genes are known to be expressed in the insect midgut and respond to changes in dietary components, we propose that the lipase cluster has undergone dynamic evolutionary changes to maximize absorption of lipid nutrients from the diet.     

Phylogenetic relationships and biogeography of Podarcis species from the Balkan Peninsula, by bayesian and maximum likelihood analyses of mitochondrial DNA sequences

Wall lizards of the genus Podarcis (Sauria, Lacertidae) comprise 17 currently recognized species in southern Europe, where they are the predominant nonavian reptile group. The taxonomy of Podarcis is complex and unstable. Based on DNA sequence data, the species of Podarcis falls into four main groups that have substantial geographic coherence (Western island group, southwestern group, Italian group, and Balkan Peninsula group). The Balkan Peninsula species are divided into two subgroups: the subgroup of P. taurica (P. taurica, P. milensis, P. gaigeae, and perhaps P. melisellensis), and the subgroup of P. erhardii (P. erhardii and P. peloponnesiaca). In the present study, the question of phylogenetic relationships among the species of Podarcis encountered in the Balkan Peninsula was addressed using partial mtDNA sequences for cytochrome b (cyt b) and 16S rRNA (16S). The data support the monophyly of Podarcis and suggest that there are three phylogenetic clades: the clade A (P. taurica, P. gaigeae, P. milensis, and P. melisellensis); the clade B (P. erhardii and P. peloponnesiaca), and the clade C (P. muralis and P. sicula). By examining intraspecific relationships it was found that extant populations of P. erhardii are paraphyletic. Furthermore, subspecies previously defined on the basis of morphological characteristics do not correspond to different molecular phylogenetic clades, suggesting that their status should be reconsidered. The distinct geographic distribution of the major clades of the phylogenetic tree and its topology suggest a spatial and temporal sequence of phylogenetic separations that coincide with some major paleogeographic separations during the geological history of the Aegean Sea. The results stress the need for a reconsideration of the evolutionary history of Balkan Podarcis species and help overcome difficulties that classical taxonomy has encountered at both the species and subspecies level.     

Plio-Pleistocene history of West African Sudanian savanna and the phylogeography of the Praomys daltoni complex (Rodentia): the environment/geography/genetic interplay

Rodents of the Praomys daltoni complex are typical inhabitants of the Sudanian savanna ecosystem in western Africa and represent a suitable model for testing the effects of Quaternary climatic oscillations on extant genetic variation patterns. Phylogeographical analyses of mitochondrial DNA sequences (cytochrome b) across the distribution range of the complex revealed several well‐defined clades that do not support the division of the clade into the two species currently recognized on the basis of morphology, i.e. P. daltoni (Thomas, 1892) and Praomys derooi ( Van der Straeten & Verheyen 1978 ). The observed genetic structure fits the refuge hypothesis, suggesting that only a small number of populations repeatedly survived in distinct forest‐savanna mosaic blocks during the arid phases of the Pleistocene, and then expanded again during moister periods. West African rivers may also have contributed to genetic differentiation, especially by forming barriers after secondary contact of expanding populations. The combination of three types of genetic markers (mtDNA sequences, microsatellite loci, cytogenetic data) provides evidence for the presence of up to three lineages, which most probably represent distinct biological species. Furthermore, incongruence between nuclear and mtDNA markers in some individuals unambiguously points towards a past introgression event. Our results highlight the importance of combining different molecular markers for an accurate interpretation of genetic data.     

A complete species-level phylogeny of the Hylobatidae based on mitochondrial ND3-ND4 gene sequences

The Hylobatidae (gibbons) are among the most endangered primates and their evolutionary history and systematics remain largely unresolved. We have investigated the species-level phylogenetic relationships among hylobatids using 1257 bases representing all species and an expanded data set of up to 2243 bases for select species from the mitochondrial ND3-ND4 region. Sequences were obtained from 34 individuals originating from all 12 recognized extant gibbon species. These data strongly support each of the four previously recognized clades or genera of gibbons, Nomascus, Bunopithecus, Symphalangus, and Hylobates, as monophyletic groups. Among these clades, there is some support for either Bunopithecus or Nomascus as the most basal, while in all analyses Hylobates appears to be the most recently derived. Within Nomascus, Nomascus sp. cf. nasutus is the most basal, followed by N. concolor, and then a clade of N. leucogenys and N. gabriellae. Within Hylobates, H. pileatus is the most basal, while H. moloch and H. klossii clearly, and H. agilis and H. muelleri likely form two more derived monophyletic clades. The segregation of H. klossii from other Hylobates species is not supported by this study. The present data are (1) consistent with the division of Hylobatidae into four distinct clades, (2) provide the first genetic evidence for all the species relationships within Nomascus, and (3) call for a revision of the current relationships among the species within Hylobates. We propose a phylogenetic tree as a working hypothesis against which intergeneric and interspecific relationships can be tested with additional genetic, morphological, and behavioral data.     

Phylogenetic analyses of mtDNA sequences corroborate taxonomic designations based on cuticular hydrocarbons in subterranean termites

Cuticular hydrocarbons (CHCs) are valuable characters for the analysis of cryptic insect species with few discernible morphological characters. Yet, their use in insect systematics, specifically in subterranean termites in the genus Reticulitermes (Isoptera: Rhinotermitidae), remains controversial. In this paper, we show that taxonomic designations in Reticulitermes from California (USA) suggested in light of differences among CHC phenotypes are corroborated by phylogenetic analyses using mtDNA sequences. Analyses based on CHC phenotypes and supported, in part, by behavioral and ecological differences have suggested the presence of more species than the two currently recognized: R. hesperus Banks and R. tibialis Banks. We analyze a 680 base pair fragment of the mitochondrial DNA cytochrome oxidase (COII) gene from 45 new (21 collection localities) and two previously recorded samples of Reticulitermes from California using parsimony and maximum likelihood methods. Both methods result in trees with highly similar topologies. Bootstrapping indicates support for six clades of Reticulitermes, and corroborates groupings based on cuticular hydrocarbons. One of the clades, R. hesperus, is already recognized in California, while four clades appear to be previously undescribed taxa. Although identification of the final clade is inconclusive, it includes a sample putatively identified as R. tibialis. Therefore, using phylogenetic analyses we corroborate chemical characters used to identify taxa, associate a chemical phenotype with a previously described species, and provide additional support for undescribed taxa of Reticulitermes.     

Phylogeny and taxonomy of endangered South and South-east Asian freshwater turtles elucidated by mtDNA sequence variation (Testudines: Geoemydidae: Batagur, Callagur, Hardella, Kachuga, Pangshura)

Using DNA sequences of the mitochondrial cytochrome b gene, we investigated phylogeny and taxonomy of South and South-east Asian turtles of all species and subspecies of the genera Batagur, Callagur, Hardella, Kachuga and Pangshura. We found three major clades: (i) a moderately to well-supported clade containing all large riverine species assigned so far to Batagur, Callagur and Kachuga ; (ii) a well-supported monophylum comprising the four Pangshura species; and (iii) Hardella that could constitute either the sister-taxon of Pangshura or of a clade comprising Batagur, Callagur, Kachuga and Pangshura. The genus Kachuga is clearly polyphyletic. Therefore, we recommend placing all Batagur, Callagur and Kachuga species in one genus. According to the International Code of Zoological Nomenclature Batagur Gray, 1856, being originally erected at higher rank, takes precedence over the simultaneously published name Kachuga Gray, 1856, and the younger name Callagur Gray, 1870, resulting in an expanded genus Batagur. Indonesian and Malaysian Batagur baska proved to be highly distinct from our sequences of this species from the Sundarbans (Bangladesh, adjacent India), suggesting that a previously unidentified species is involved. This finding is of high conservation relevance in the critically endangered B. baska. The currently recognized subspecies within Hardella thurjii , Pangshura smithii and P. tentoria do not correspond well with mtDNA clades. Considering that the two subspecies of H. thurjii are likely to be based only on individual ontogenetic differences, we propose abandoning the usage of subspecies within H. thurjii . In the Ghaghra River, Uttar Pradesh (India) we detected shared haplotypes in P. smithii and P. tentoria, implying that the unusual morphological characters of the Ghaghra River population of P. tentoria could be the result of interspecific hybridization.     

Geographical divergence in the Japanese land snail Euhadra herklotsi inferred from its molecular phylogeny and genital characters

We studied genetic variation within the Japanese land snail Euhadra herklotsi, which occurs on Kyushu and the surrounding islands, using partial sequences of the mitochondrial COI gene and nuclear ITS2 genes. The phylogenetic analysis revealed the existence of two major clades: clade N in the north and clade S in the south. These clades were parapatric and overlapped in southern Kyushu. Genetic divergence was high in clade N, whereas it was much lower in clade S. In addition, isolation-by-distance within each clade was implied. Since no current geographical barriers separate these clades, the genetic structure of clade S might have been influenced by historical events, such as volcanic activity, and a resulting population bottleneck followed by range expansion. The genital characteristics of clade-S snails were distinct from those of clade-N snails, and snails in both clades were sympatric at one locality. The shells of clade-N snails were generally larger than those of clade-S snails, but the shell-size variation within each clade could not be explained simply by environmental variables. Our study suggests that E. herklotsi likely consists of two sibling species. The taxonomic status of the previously proposed subspecies of E. herklotsi and related species requires reassessment.     

An ITS-based phylogenetic framework for the genus Vorticella: finding the molecular and morphological gaps in a taxonomically difficult group

Vorticella includes more than 100 currently recognized species and represents one of the most taxonomically challenging genera of ciliates. Molecular phylogenetic analysis of Vorticella has been performed so far with only sequences coding for small subunit ribosomal RNA (SSU rRNA); only a few of its species have been investigated using other genetic markers owing to a lack of similar sequences for comparison. Consequently, phylogenetic relationships within the genus remain unclear, and molecular discrimination between morphospecies is often difficult because most regions of the SSU rRNA gene are too highly conserved to be helpful. In this paper, we move molecular systematics for this group of ciliates to the infrageneric level by sequencing additional molecular markers—fast-evolving internal transcribed spacer (ITS) regions—in a broad sample of 66 individual samples of 28 morphospecies of Vorticella collected from Asia, North America and Europe. Our phylogenies all featured two strongly supported, highly divergent, paraphyletic clades (I, II) comprising the morphologically defined genus Vorticella. Three major lineages made up clade I, with a relatively well-resolved branching order in each one. The marked divergence of clade II from clade I confirms that the former should be recognized as a separate taxonomic unit as indicated by SSU rRNA phylogenies. We made the first attempt to elucidate relationships between species in clade II using both morphological and multi-gene approaches, and our data supported a close relationship between some morphospecies of Vorticella and Opisthonecta, indicating that relationships between species in the clade are far more complex than would be expected from their morphology. Different patterns of helix III of ITS2 secondary structure were clearly specific to clades and subclades of Vorticella and, therefore, may prove useful for resolving phylogenetic relationships in other groups of ciliates.     

A phylogenetic analysis of the boreal lichen Mycoblastus sanguinarius (Mycoblastaceae, lichenized Ascomycota) reveals cryptic clades correlated with fatty acid profiles

Lichens are a prominent feature of northern conifer forests and a large number of species are thought to be circumboreal. Whether or not circumboreal lichen species really constitute monophyletic groups has seldom been tested. We investigated molecular phylogenetic patterns in the mycobiont of Mycoblastus sanguinarius, a well known epiphytic lichen species of the boreal forest, based on material collected from across the high latitude northern hemisphere. A three-locus dataset of internal transcribed spacer rDNA, translation elongation factor 1-α and replication licensing factor Mcm7 DNA sequences revealed that material treated until now as belonging to M. sanguinarius does indeed form a monophyletic group within the genus and is distinct from a strongly supported Mycoblastus affinis. The M. sanguinarius complex appears closely related to the rare Mycoblastus glabrescens, which is currently known only from the Pacific Northwest and was rediscovered during the present study. However, within M. sanguinarius s.lat. in the northern hemisphere, two deeply divergent and morphologically coherent species can be recovered, one of which matches the southern hemisphere species Mycoblastus sanguinarioides and turns out to be widespread in North America and Asia, and one of which corresponds to M. sanguinarius s.str. Both M. sanguinarius and M. sanguinarioides exhibit additional low-level genetic differentiation into geographically structured clades, the most prominent of which are distributed in East Asia/eastern North America and western North America/Europe, respectively. Individuals from these lowest-level clades are morphologically indistinguishable but chemical analyses by thin layer chromatography revealed that each clade possesses its own fatty acid profile, suggesting that chemical differentiation precedes morphological differentiation and may be a precursor to speciation.     

Molecular systematics of new world gopher, bull, and pinesnakes (Pituophis: Colubridae), a transcontinental species complex

Pituophis melanoleucus (gopher, bull, and pinesnakes) is among the most widely distributed polytypic species complexes in North America, with most authors recognizing from a single transcontinental species (the melanoleucus complex, composed of 15 subspecies) to four (monotypic and polytypic) species. We used mitochondrial gene sequences from the two middle American species, P. deppei and P. lineaticollis, and from 13 subspecies from most of the range of the melanoleucus complex to test various phylogenetic hypotheses for Pituophis. Maximum parsimony and maximum likelihood methods identified the same major clades within Pituophis and indicated that two segments of the melanoleucus complex, the lodingi-melanoleucus-mugitus eastern pinesnake clade and the affinis-annectens-bimaris-catenifer-deserticola- sayi-ruthveni-vertebr alis clade from central and western United States and northern Mexico, represent divergent, allopatric lineages with no known intergradation zone. We recognize each of these two groupings as a different species. Our data also indicate that some ruthveni are more closely related to sayi than to other ruthveni. Nonetheless, ruthveni is an allopatric taxon diagnosable from its closest relatives by a combination of morphometric characters, and because it is likely that at least some of these traits are independent and genetically inherited, we interpret this as evidence that ruthveni has attained the status of independent evolutionary lineage, despite the fact that it retains strong genetic affinities with sayi. The endemic Baja Californian gopher snakes (bimaris and vertebralis) are considered by some taxonomists as a different species, P. vertebralis, but we discovered that these serpents belong to two different clades and hence we do not agree with the recognition of P. vertebralis as presently defined. In summary, we believe that three distinct species are included in the melanoleucus complex, Pituophis melanoleucus (sensu stricto), P. catenifer, and P. ruthveni, and that their recognition better represents the evolutionary diversity within this species complex.