Dense Geographic and Genomic Sampling Reveals Paraphyly and a Cryptic Lineage in a Classic Sibling Species Complex.

Incomplete or geographically biased sampling poses significant problems for research in phylogeography, population genetics, phylogenetics, and species delimitation. Despite the power of using genome-wide genetic markers in systematics and related fields, approaches such as the multispecies coalescent remain unable to easily account for unsampled lineages. The Empidonax difficilis/Empidonax occidentalis complex of small tyrannid flycatchers (Aves: Tyrannidae) is a classic example of widely distributed species with limited phenotypic geographic variation that was broken into two largely cryptic (or "sibling") lineages following extensive study. Though the group is well-characterized north of the US Mexico border, the evolutionary distinctiveness and phylogenetic relationships of southern populations remain obscure. In this article, we use dense genomic and geographic sampling across the majority of the range of the E. difficilis/E. occidentalis complex to assess whether current taxonomy and species limits reflect underlying evolutionary patterns, or whether they are an artifact of historically biased or incomplete sampling. We find that additional samples from Mexico render the widely recognized species-level lineage E. occidentalis paraphyletic, though it retains support in the best-fit species delimitation model from clustering analyses. We further identify a highly divergent unrecognized lineage in a previously unsampled portion of the group's range, which a cline analysis suggests is more reproductively isolated than the currently recognized species E. difficilis and E. occidentalis. Our phylogeny supports a southern origin of these taxa. Our results highlight the pervasive impacts of biased geographic sampling, even in well-studied vertebrate groups like birds, and illustrate what is a common problem when attempting to define species in the face of recent divergence and reticulate evolution.

Exploring Chihuahuan Desert diversification in the gray-banded kingsnake, Lampropeltis alterna (Serpentes: Colubridae).

Within many biomes, the cause of phylogeographic structure remains unknown even across regions throughout North America, including within the biodiverse Chihuahuan Desert. For example, little is known about population structure or the timing of diversification of Chihuahuan endemics. This is due largely to the lack of population genomic studies within this region. We generated ultra-conserved element data for the gray-banded kingsnake (Lampropeltis alterna) to investigate lineage divergence and historical demography across the Chihuahuan Desert. We found three unique lineages corresponding to the Trans-Pecos and Mapimian biogeographic regions of the Chihuahuan Desert, and a distinct population in the Sierra Madre Occidental. Using several mutation rates to calibrate the timing of divergence among these lineages, we show that lineage divergence likely occurred during the Pleistocene, which indicates that careful consideration needs to be used when applying mutation rates to ultra-conserved elements. We suggest that biogeographic provinces within the Chihuahuan Desert may have served as allopatric refugia during climatic fluctuations of the Quaternary. This work serves as an important template for further testing biogeographic hypotheses within the region.

Species delimitation and biogeography of the gnatcatchers and gnatwrens (Aves: Polioptilidae).

The New World avian family Polioptilidae (gnatcatchers and gnatwrens) is distributed from Argentina to Canada and includes 15 species and more than 60 subspecies. No study to date has evaluated phylogenetic relationships within this family and the historical pattern of diversification within the group remains unknown. Moreover, species limits, particularly in widespread taxa that show geographic variation, remain unclear. In this study, we delimited species and estimated phylogenetic relationships using multilocus data for the entire family. We then used the inferred diversity along with alternative taxonomic classification schemes to evaluate how lumping and splitting of both taxa and geographical areas influenced biogeographic inference. Species-tree analyses grouped Polioptilidae into four main clades: Microbates, Ramphocaenus, a Polioptila guianensis complex, and the remaining members of Polioptila. Ramphocaenus melanurus was sister to the clade containing M. cinereiventris and M. collaris, which formed a clade sister to all species within Polioptila. Polioptila was composed of two clades, the first of which included the P. guianensis complex; the other contained all remaining species in the genus. Using multispecies coalescent modeling, we inferred a more than 3-fold increase in species diversity, of which 87% represent currently recognized species or subspecies. Much of this diversity corresponded to subspecies that occur in the Neotropics. We identified three polyphyletic species, and delimited 4-6 previously undescribed candidate taxa. Probabilistic modeling of geographic ranges on the species tree indicated that the family likely had an ancestral origin in South America, with all three genera independently colonizing North America. Support for this hypothesis, however, was sensitive to the taxonomic classification scheme used and the number of geographical areas allowed. Our study proposes the first phylogenetic hypothesis for Polioptilidae and provides genealogical support for the reclassification of species limits. Species limits and the resolution of geographical areas that taxa inhabit influence the inferred spatial diversification history.

Phylogenomic insights into the diversification of salamanders in the Isthmura bellii group across the Mexican highlands.

Mountain formation in Mexico has played an important role in the diversification of many Mexican taxa. The Trans-Mexican Volcanic Belt in particular has served as both a cradle of diversification and conduit for dispersal. We investigated the evolutionary history of the Isthmura bellii group of salamanders, a widespread amphibian across the Mexican highlands, using sequence capture of ultraconserved elements. Results suggest that the I. bellii group probably originated in southeastern Mexico in the late Miocene and later dispersed across the Trans-Mexican Volcanic Belt and into the Sierra Madre Occidental. Pre-Pleistocene uplift of the Trans-Volcanic Belt likely promoted early diversification by serving as a mesic land-bridge across central Mexico. These findings highlight the importance of the Trans-Volcanic Belt in generating Mexico's rich biodiversity.

Integrative species delimitation in practice: Revealing cryptic lineages within the short-nosed skink Plestiodon brevirostris (Squamata: Scincidae).

Integrative taxonomy has been generally considered as a goal in systematics for more than a decade. Here, we employed environmental, molecular, and morphological data to evaluate the species boundaries within the short-nosed skink Plestiodon brevirostris from south-central Mexico, one member of the morphologically conservative P. brevirostris group. Our molecular dataset includes one mitochondrial and two nuclear loci. The mitochondrial fragment includes the full length of the gene coding for the NADH dehydrogenase subunit 1 protein, a segment of the gene coding for 16S ribosomal RNA, and flanking tRNAs. The nuclear dataset includes fragments of the genes coding for the megakaryoblastic leukemia 1 and RNA fingerprint 35 proteins. We employed phylogenetic reconstruction, analyses of population structure and morphological variation, and species delimitation methods (including the integration of the three kinds of data in a unified probabilistic framework) to evaluate species limits. Our results suggest that P. brevirostris represents four distinct species. The information provided by each kind of data allowed us to discern between alternative explanations for the observed patterns of geographic structure. Two of the newly recognized lineages are poorly differentiated morphologically but apparently differ in environmental preferences and are allopatric. Additionally, one lineage is microendemic and parapatric with respect to another one. Moreover, our phylogenetic analyses suggest that other taxa within the P. brevirostris group may represent species complexes. We discuss our results in the context of integrative species delimitation.

Cryptic genetic diversity, population structure, and gene flow in the Mojave rattlesnake (Crotalus scutulatus).

The Mojave rattlesnake (Crotalus scutulatus) inhabits deserts and arid grasslands of the western United States and Mexico. Despite considerable interest in its highly toxic venom and the recognition of two subspecies, no molecular studies have characterized range-wide genetic diversity and population structure or tested species limits within C. scutulatus. We used mitochondrial DNA and thousands of nuclear loci from double-digest restriction site associated DNA sequencing to infer population genetic structure throughout the range of C. scutulatus, and to evaluate divergence times and gene flow between populations. We find strong support for several divergent mitochondrial and nuclear clades of C. scutulatus, including splits coincident with two major phylogeographic barriers: the Continental Divide and the elevational increase associated with the Central Mexican Plateau. We apply Bayesian clustering, phylogenetic inference, and coalescent-based species delimitation to our nuclear genetic data to test hypotheses of population structure. We also performed demographic analyses to test hypotheses relating to population divergence and gene flow. Collectively, our results support the existence of four distinct lineages within C. scutulatus, and genetically defined populations do not correspond with currently recognized subspecies ranges. Finally, we use approximate Bayesian computation to test hypotheses of divergence among multiple rattlesnake species groups distributed across the Continental Divide, and find evidence for co-divergence at this boundary during the mid-Pleistocene.

Genomic data reveal ancient microendemism in forest scorpions across the California Floristic Province.

The California Floristic Province (CFP) in western North America is a globally significant biodiversity hotspot. Elucidating patterns of endemism and the historical drivers of this diversity has been an important challenge of comparative phylogeography for over two decades. We generated phylogenomic data using ddRADseq to examine genetic structure in Uroctonus forest scorpions, an ecologically restricted and dispersal-limited organism widely distributed across the CFP north to the Columbia River. We coupled our genetic data with species distribution models (SDMs) to determine climatically suitable areas for Uroctonus both now and during the Last Glacial Maximum. Based on our analyses, Uroctonus is composed of two major genetic groups that likely diverged over 2 million years ago. Each of these groups itself contains numerous genetic groups that reveal a pattern of vicariance and microendemism across the CFP. Migration rates among these populations are low. SDMs suggest forest scorpion habitat has remained relatively stable over the last 21 000 years, consistent with the genetic data. Our results suggest tectonic plate rafting, mountain uplift, river drainage formation and climate-induced habitat fragmentation have all likely played a role in the diversification of Uroctonus. The intricate pattern of genetic fragmentation revealed across a temporal continuum highlights the potential of low-dispersing species to shed light on small-scale patterns of biodiversity and the underlying processes that have generated this diversity in biodiversity hotspots.

Hidden histories of gene flow in highland birds revealed with genomic markers.

Genomic studies are revealing that divergence and speciation are marked by gene flow, but it is not clear whether gene flow has played a prominent role during the generation of biodiversity in species-rich regions of the world where vicariance is assumed to be the principal mode by which new species form. We revisit a well-studied organismal system in the Mexican Highlands, Aphelocoma jays, to test for gene flow among Mexican sierras. Prior results from mitochondrial DNA (mtDNA) largely conformed to the standard model of allopatric divergence, although there was also evidence for more obscure histories of gene flow in a small sample of nuclear markers. We tested for these 'hidden histories' using genomic markers known as ultraconserved elements (UCEs) in concert with phylogenies, clustering algorithms and newer introgression tests specifically designed to detect ancient gene flow (e.g. ABBA/BABA tests). Results based on 4303 UCE loci and 2500 informative SNPs are consistent with varying degrees of gene flow among highland areas. In some cases, gene flow has been extensive and recent (although perhaps not ongoing today), whereas in other cases there is only a trace signature of ancient gene flow among species that diverged as long as 5 million years ago. These results show how a species complex thought to be a model for vicariance can reveal a more reticulate history when a broader portion of the genome is queried. As more organisms are studied with genomic data, we predict that speciation-with-bouts-of-gene-flow will turn out to be a common mode of speciation.

Species delimitation using Bayes factors: simulations and application to the Sceloporus scalaris species group (Squamata: Phrynosomatidae).

Current molecular methods of species delimitation are limited by the types of species delimitation models and scenarios that can be tested. Bayes factors allow for more flexibility in testing non-nested species delimitation models and hypotheses of individual assignment to alternative lineages. Here, we examined the efficacy of Bayes factors in delimiting species through simulations and empirical data from the Sceloporus scalaris species group. Marginal-likelihood scores of competing species delimitation models, from which Bayes factor values were compared, were estimated with four different methods: harmonic mean estimation (HME), smoothed harmonic mean estimation (sHME), path-sampling/thermodynamic integration (PS), and stepping-stone (SS) analysis. We also performed model selection using a posterior simulation-based analog of the Akaike information criterion through Markov chain Monte Carlo analysis (AICM). Bayes factor species delimitation results from the empirical data were then compared with results from the reversible-jump MCMC (rjMCMC) coalescent-based species delimitation method Bayesian Phylogenetics and Phylogeography (BP&P). Simulation results show that HME and sHME perform poorly compared with PS and SS marginal-likelihood estimators when identifying the true species delimitation model. Furthermore, Bayes factor delimitation (BFD) of species showed improved performance when species limits are tested by reassigning individuals between species, as opposed to either lumping or splitting lineages. In the empirical data, BFD through PS and SS analyses, as well as the rjMCMC method, each provide support for the recognition of all scalaris group taxa as independent evolutionary lineages. Bayes factor species delimitation and BP&P also support the recognition of three previously undescribed lineages. In both simulated and empirical data sets, harmonic and smoothed harmonic mean marginal-likelihood estimators provided much higher marginal-likelihood estimates than PS and SS estimators. The AICM displayed poor repeatability in both simulated and empirical data sets, and produced inconsistent model rankings across replicate runs with the empirical data. Our results suggest that species delimitation through the use of Bayes factors with marginal-likelihood estimates via PS or SS analyses provide a useful and complementary alternative to existing species delimitation methods.

Coalescent species delimitation in milksnakes (genus Lampropeltis) and impacts on phylogenetic comparative analyses.

Both gene-tree discordance and unrecognized diversity are sources of error for accurate estimation of species trees, and can affect downstream diversification analyses by obscuring the correct number of nodes, their density, and the lengths of the branches subtending them. Although the theoretical impact of gene-tree discordance on evolutionary analyses has been examined previously, the effect of unsampled and cryptic diversity has not. Here, we examine how delimitation of previously unrecognized diversity in the milksnake (Lampropeltis triangulum) and use of a species-tree approach affects both estimation of the Lampropeltis phylogeny and comparative analyses with respect to the timing of diversification. Coalescent species delimitation indicates that L. triangulum is not monophyletic and that there are multiple species of milksnake, which increases the known species diversity in the genus Lampropeltis by 40%. Both genealogical and temporal discordance occurs between gene trees and the species tree, with evidence that mitochondrial DNA (mtDNA) introgression is a main factor. This discordance is further manifested in the preferred models of diversification, where the concatenated gene tree strongly supports an early burst of speciation during the Miocene, in contrast to species-tree estimates where diversification follows a birth-death model and speciation occurs mostly in the Pliocene and Pleistocene. This study highlights the crucial interaction among coalescent-based phylogeography and species delimitation, systematics, and species diversification analyses.

Caves as microrefugia: Pleistocene phylogeography of the troglophilic North American scorpion Pseudouroctonus reddelli.

BACKGROUND: Survival in microrefugia represents an important paradigm in phylogeography for explaining rapid postglacial re-colonization by species in temperate regions. Microrefugia may allow populations to persist in areas where the climatic conditions on the surface have become unfavourable. Caves generally contain stable microclimates and may represent microrefugia for species capable of exploiting both cave and surface habitats (troglophiles). We examine the phylogeography of the troglophilic North American vaejovid scorpion Pseudouroctonus reddelli using 1,993 base pairs of mitochondrial and nuclear DNA sequence data generated from 12 populations. We use (i) descriptive measures of genetic diversity and population genetics statistics, (ii) reconstructions of phylogeographical structure, spatial diffusion during diversification, and population sizes through time, and (iii) species distribution modelling to test predictions of the hypothesis that caves serve as microrefugia. We compare phylogeographical patterns in P. reddelli with other troglophilic species across the Edwards Plateau karst region of Texas. RESULTS: Results revealed high haplotype and nucleotide diversity and substantial phylogeographical structure, probably generated during the Pleistocene. Spatial diffusion occurred along the southern edge of the Edwards Plateau from multiple refugia along the Balcones Escarpment. There was little evidence for population and geographical expansion. Species distribution models predicted substantial reductions in suitable epigean habitat for P. reddelli at the Last Glacial Maximum (LGM). CONCLUSIONS: High genetic diversity, strong phylogeographical structure, diffusion from multiple refugia, and unfavourable climatic conditions at the LGM collectively support the hypothesis that caves served as microrefugia for P. reddelli. Similar patterns of genetic structure in P. reddelli and other troglophilic species across the Edwards Plateau karst region of Texas suggest that caves serving as microrefugia are important for the formation, maintenance, and future survival of troglophilic species in temperate karst regions.

A multilocus phylogeny of a major New World avian radiation: the Vireonidae.

The family Vireonidae represents one of the most widespread and well-known New World avian radiations, but a robust species-level phylogeny of the group is lacking. Here, we infer a phylogeny of Vireonidae using multilocus data obtained from 221 individuals from 46 of 52 vireonid species (representing all four genera) and five "core Corvoidea" outgroups. Our results show Vireonidae to be monophyletic, consistent with a single colonization of the New World by an Asian ancestor. Cyclarhis and Vireolanius are monophyletic genera that diverged early from the rest of Vireonidae. Hylophilus is polyphyletic, represented by three distinct clades concordant with differences in morphology, habitat, and voice. The poorly known South American species Hylophilus sclateri is embedded within the genus Vireo. Vireo, in turn, consists of several well-supported intrageneric clades. Overall, tropical vireonid species show much higher levels of intraspecific genetic structure than temperate species and several currently recognized species are probably comprised of multiple cryptic species.

A comprehensive multilocus assessment of sparrow (Aves: Passerellidae) relationships.

The New World sparrows (Emberizidae) are among the best known of songbird groups and have long-been recognized as one of the prominent components of the New World nine-primaried oscine assemblage. Despite receiving much attention from taxonomists over the years, and only recently using molecular methods, was a "core" sparrow clade established allowing the reconstruction of a phylogenetic hypothesis that includes the full sampling of sparrow species diversity. In this paper, we use mitochondrial DNA gene sequences from all 129 putative species of sparrow and four additional (nuclear) loci for a subset of these taxa to resolve both generic and species level relationships. Hypotheses derived from our mitochondrial (2184 base pairs) and nuclear (5705 base pairs) DNA data sets were generally in agreement with respect to clade constituency but differed somewhat with respect to among-clade relationships. Sparrow diversity is defined predominantly by eight well-supported clades that indicate a lack of monophyly for at least three currently recognized genera. Ammodramus is polyphyletic and requires the naming of two additional genera. Spizella is also polyphyletic with Tree Sparrow (Spizella arborea) as a taxonomic "outlier". Pselliophorus is embedded within a larger Atlapetes assemblage and should be merged with that group. This new hypothesis of sparrow relationships will form the basis for future comparative analyses of variation within songbirds.

A new species of bunchgrass lizard (Squamata: Phrynosomatidae) from the southern sky islands of the Sierra Madre Occidental, Mexico.

A new species of bunchgrass lizard in the Sceloporus scalaris group is described from the southern portion of the Sierra Madre Occidental in Mexico. The new species, Sceloporus aurantius sp. nov., was previously confused with S. brownorum but differs from this and all but one species within the S. scalaris group by a lack of blue belly patches in males. It shares with S. chaneyi an absence of blue belly patches, but differs from this species in size, number of dorsal scales, number of scales around midbody, and presence of an un-patterned morph. The new species further differs from S. chaneyi, and all other species in the S. scalaris species group, by unique phylogenetic position revealed through species delimitation based on multi-locus nuclear DNA. Principal component analyses of 24 traditional morphological characters used to describe previous S. scalaris group taxa indicate that these characters may be of limited use to delineate species in this species group. However, male lateral and ventral coloration may still be an important character for diagnosing species.

Speciational history of North American Haemorhous finches (Aves: Fringillidae) inferred from multilocus data.

We investigated species relationships and timing of speciation in North American Haemorhous finches by using a mitochondrial phylogeographic approach combined with a multilocus species tree reconstruction. Haemorhous purpureus and H. cassinii were strongly supported as sister taxa, and H. mexicanus was sister to H. purpureus+H. cassinii. Our divergence times indicated that diversification within Haemorhous occurred progressively from the Late Miocene into the Pleistocene. Our inferred pattern of speciation demonstrates the complexity of the origins of North American birds, and provides additional evidence that a single cause for speciation in closely related North American birds, such as Late Pleistocene glacial-interglacial cycles, is unlikely.

Multilocus phylogeny and biogeography of the New World Pheucticus grosbeaks (Aves: Cardinalidae).

Using a multilocus approach, we investigated the tempo and pattern of diversification in a widely distributed New World songbird, the cardinalid genus Pheucticus. Each of the three geographic groups recovered (North American, Middle American, and South American) was comprised of a pair of currently recognized species, and four, three, and three geographically and genetically distinct phylogeographic lineages respectively. Diversification within Pheucticus appears to have occurred at a relatively constant pace throughout the Pleistocene and evenly across a broad latitudinal distribution. The Isthmus of Panama completion and Pleistocene glacial cycles both appear to have played prominent roles in the diversification of this group.

An asymmetry in niche conservatism contributes to the latitudinal species diversity gradient in New World vertebrates.

The Tropical Niche Conservatism hypothesis is a leading explanation for why biodiversity increases towards the equator. The model suggests that most lineages have tropical origins, with few dispersing into temperate regions. However, biotas are comprised of lineages with differing geographical origins, thus it is unclear whether lineages that originated on different continents will exhibit similar patterns of niche conservatism. Here, we summarised biogeographical patterns of New World vertebrates and compared species diversity patterns between families that originated in North and South America. Overall, families with southern origins exhibit niche conservatism with many lineages restricted to the Neotropics, whereas many northern-origin families are distributed across the Neotropics and the Nearctic. Consequently, northern lineages have contributed to high tropical biodiversity, but southern lineages have contributed relatively little to temperate biodiversity in North America. The asymmetry in niche conservatism between northern and southern lineages is an important contributor to the biodiversity gradient.

Diversification in the Mexican horned lizard Phrynosoma orbiculare across a dynamic landscape.

The widespread montane Mexican horned lizard Phrynosoma orbiculare (Squamata: Phrynosomatidae) represents an ideal species to investigate the relative impacts of Neogene vicariance and Quaternary climate change on lineage diversification across the Mexican highlands. We used mitochondrial DNA to examine the maternal history of P. orbiculare and estimate the timing and tempo of lineage diversification. Based on our results, we inferred 11 geographically structured, well supported mitochondrial lineages within this species, suggesting P. orbiculare represents a species complex. Six divergences between lineages likely occurred during the Late Miocene and Pliocene, and four splits probably happened during the Pleistocene. Diversification rate appeared relatively constant through time. Spatial and temporal divergences between lineages of P. orbiculare and co-distributed taxa suggest that a distinct period of uplifting of the Transvolcanic Belt around 7.5-3 million years ago broadly impacted diversification in taxa associated with this mountain range. To the north, several river drainages acting as filter barriers differentially subdivided co-distributed highland taxa through time. Diversification patterns observed in P. orbiculare provide additional insight into the mechanisms that impacted differentiation of highland taxa across the complex Mexican highlands.

Relative roles of Neogene vicariance and Quaternary climate change on the historical diversification of bunchgrass lizards (Sceloporus scalaris group) in Mexico.

Neogene vicariance during the Miocene and Pliocene and Quaternary climate change have synergistically driven diversification in Mexican highland taxa. We investigated the impacts of these processes on genetic diversification in the widely distributed bunchgrass lizards in the Sceloporus scalaris group. We searched for correlations between timing in diversification and timing of (1) a period of marked volcanism across the Trans-Mexican Volcanic Belt in central Mexico 3-7.5million years ago (Ma) and (2) a transition to larger glacial-interglacial cycles during the mid-Pleistocene. From our phylogenetic analyses of mitochondrial DNA we identified two major clades that contained 13 strongly supported lineages. One clade contained lineages from the two northern sierras of Mexico, and the other clade included lineages associated with the Trans-Mexican Volcanic Belt and Central Mexican Plateau. Results provided support for Neogene divergences within the S. scalaris group in response to uplift of the Trans-Mexican Volcanic Belt, a pattern observed in several co-distributed taxa, and suggested that Quaternary climate change likely had little effect on diversification between lineages. Uplift of the Trans-Mexican Volcanic Belt during specific time periods appears to have strongly impacted diversification in Mexican highland taxa.

A multilocus perspective on the speciation history of a North American aridland toad (Anaxyrus punctatus).

Interpretations of phylogeographic patterns can change when analyses shift from single gene-tree to multilocus coalescent analyses. Using multilocus coalescent approaches, a species tree and divergence times can be estimated from a set of gene trees while accounting for gene-tree stochasticity. We utilized the conceptual strengths of a multilocus coalescent approach coupled with complete range-wide sampling to examine the speciation history of a broadly distributed, North American warm-desert toad, Anaxyrus punctatus. Phylogenetic analyses provided strong support for three major lineages within A. punctatus. Each lineage broadly corresponded to one of three desert regions. Early speciation in A. punctatus appeared linked to late Miocene-Pliocene development of the Baja California peninsula. This event was likely followed by a Pleistocene divergence associated with the separation of the Chihuahuan and Sonoran Deserts. Our multilocus coalescent-based reconstruction provides an informative contrast to previous single gene-tree estimates of the evolutionary history of A. punctatus.