Whole Snake Genomes from Eighteen Families of Snakes (Serpentes: Caenophidia) and Their Applications to Systematics.

We present genome assemblies for 18 snake species representing 18 families (Serpentes: Caenophidia): Acrochordus granulatus, Aparallactus werneri, Boaedon fuliginosus, Calamaria suluensis, Cerberus rynchops, Grayia smithii, Imantodes cenchoa, Mimophis mahfalensis, Oxyrhabdium leporinum, Pareas carinatus, Psammodynastes pulverulentus, Pseudoxenodon macrops, Pseudoxyrhopus heterurus, Sibynophis collaris, Stegonotus admiraltiensis, Toxicocalamus goodenoughensis, Trimeresurus albolabris, and Tropidonophis doriae. From these new genome assemblies, we extracted thousands of loci commonly used in systematic and phylogenomic studies on snakes, including target-capture datasets composed of UCEs and AHEs, as well as traditional Sanger loci. Phylogenies inferred from the two target-capture loci datasets were identical with each other, and strongly congruent with previously published snake phylogenies. To show additional utility of these non-model genomes for investigative evolutionary research, we mined the genome assemblies of two New Guinea island endemics in our dataset (Stegonotus admiraltiensis and Tropidonophis doriae) for the ATP1a3 gene, a thoroughly researched indicator of resistance to toad toxin ingestion by squamates. We find that both these snakes possess the genotype for toad toxin resistance despite their endemism to New Guinea, a region absent of any toads until the human-mediated introduction of Cane Toads in the 1930s. These species possess identical substitutions that suggest the same bufotoxin resistance as their Australian congenerics (Stegonotus cucullatus and Tropidonophis mairii) which forage on invasive Cane Toads. Herein, we show the utility of short-read high coverage genomes, as well as improving the deficit of available squamate genomes with associated voucher specimens.

Phylogenomics of Psammodynastes and Buhoma (Elapoidea: Serpentes), with the description of a new Asian snake family.

Asian mock vipers of the genus Psammodynastes and African forest snakes of the genus Buhoma are two genera belonging to the snake superfamily Elapoidea. The phylogenetic placements of Psammodynastes and Buhoma within Elapoidea has been extremely unstable which has resulted in their uncertain and debated taxonomy. We used ultraconserved elements and traditional nuclear and mitochondrial markers to infer the phylogenetic relationships of these two genera with other elapoids. Psammodynastes, for which a reference genome has been sequenced, were found, with strong branch support, to be a relatively early diverging split within Elapoidea that is sister to a clade consisting of Elapidae, Micrelapidae and Lamprophiidae. Hence, we allocate Psammodynastes to its own family, Psammodynastidae new family. However, the phylogenetic position of Buhoma could not be resolved with a high degree of confidence. Attempts to identify the possible sources of conflict in the rapid radiation of elapoid snakes suggest that both hybridisation/introgression during the rapid diversification, including possible ghost introgression, as well as incomplete lineage sorting likely have had a confounding role. The usual practice of combining mitochondrial loci with nuclear genomic data appears to mislead phylogeny reconstructions in rapid radiation scenarios, especially in the absence of genome scale data.

Integrative methods reveal multiple drivers of diversification in rice paddy snakes.

Divergence dating analyses in systematics provide a framework to develop and test biogeographic hypotheses regarding speciation. However, as molecular datasets grow from multilocus to genomic, sample sizes decrease due to computational burdens, and the testing of fine-scale biogeographic hypotheses becomes difficult. In this study, we use coalescent demographic models to investigate the diversification of poorly known rice paddy snakes from Southeast Asia (Homalopsidae: Hypsiscopus), which have conflicting dates of origin based on previous studies. We use coalescent modeling to test the hypothesis that Hypsiscopus diversified 2.5 mya during the Khorat Plateau uplift in Thailand. Additionally, we use ecological niche analyses to identify potential differences in the niche space of the two most widely distributed species in the past and present. Our results suggest Hypsiscopus diversified ~ 2.4 mya, supporting that the Khorat Plateau may have initiated the diversification of rice paddy snakes. We also find significant niche differentiation and shifts between species of Hypsiscopus, indicating that environmental differences may have sustained differentiation of this genus after the Khorat Plateau uplift. Our study expands on the diversification history of snakes in Southeast Asia, and highlights how results from smaller multilocus datasets can be useful in developing and testing biogeographic hypotheses alongside genomic datasets.

Speciation rates are unrelated to the formation of population structure in Malagasy gemsnakes.

Speciation rates vary substantially across the tree of life. These rates should be linked to the rate at which population structure forms if a continuum between micro and macroevolutionary patterns exists. Previous studies examining the link between speciation rates and the degree of population formation in clades have been shown to be either correlated or uncorrelated depending on the group, but no study has yet examined the relationship between speciation rates and population structure in a young group that is constrained spatially to a single-island system. We examine this correlation in 109 gemsnakes (Pseudoxyrhophiidae) endemic to Madagascar and originating in the early Miocene, which helps control for extinction variation across time and space. We find no relationship between rates of speciation and the formation rates of population structure over space in 33 species of gemsnakes. Rates of speciation show low variation, yet population structure varies widely across species, indicating that speciation rates and population structure are disconnected. We suspect this is largely due to the persistence of some lineages not susceptible to extinction. Importantly, we discuss how delimiting populations versus species may contribute to problems understanding the continuum between shallow and deep evolutionary processes.

Effects of the egg incubation environment on turtle carapace development.

Developing organisms are often exposed to fluctuating environments that destabilize tissue-scale processes and induce abnormal phenotypes. This might be common in species that lay eggs in the external environment and with little parental care, such as many reptiles. In turtles, morphological development has provided striking examples of abnormal phenotypic patterns, though the influence of the environment remains unclear. To this end, we compared fluctuating asymmetry, as a proxy for developmental instability, in turtle hatchlings incubated in controlled laboratory and unstable natural conditions. Wild and laboratory hatchlings featured similar proportions of supernumerary scales (scutes) on the dorsal shell (carapace). Such abnormal scutes likely elevated shape asymmetry, which was highest in natural nests. Moreover, we tested the hypothesis that hot and dry environments cause abnormal scute formation by subjecting eggs to a range of hydric and thermal laboratory incubation regimes. Shape asymmetry was similar in hatchlings incubated at five constant temperatures (26-30°C). A hot (30°C) and severely Dry substrate yielded smaller hatchlings but scutes were not overtly affected. Our study suggests that changing nest environments contribute to fluctuating asymmetry in egg-laying reptiles, while clarifying the conditions at which turtle shell development remains buffered from the external environment.

Empirical and philosophical problems with the subspecies rank.

Species-level taxonomy derives from empirical sources (data and techniques) that assess the existence of spatiotemporal evolutionary lineages via various species "concepts." These concepts determine if observed lineages are independent given a particular methodology and ontology, which relates the metaphysical species concept to what "kind" of thing a species is in reality. Often, species concepts fail to link epistemology back to ontology. This lack of coherence is in part responsible for the persistence of the subspecies rank, which in modern usage often functions as a placeholder between the evolutionary events of divergence or collapse of incipient species. Thus, prospective events like lineages merging or diverging require information from unknowable future information. This is also conditioned on evidence that the lineage already has a detectably distinct evolutionary history. Ranking these lineages as subspecies can seem attractive given that many lineages do not exhibit intrinsic reproductive isolation. We argue that using subspecies is indefensible on philosophical and empirical grounds. Ontologically, the rank of subspecies is either identical to that of species or undefined in the context of evolutionary lineages representing spatiotemporally defined individuals. Some species concepts more inclined to consider subspecies, like the Biological Species Concept, are disconnected from evolutionary ontology and do not consider genealogy. Even if ontology is ignored, methods addressing reproductive isolation are often indirect and fail to capture the range of scenarios linking gene flow to species identity over space and time. The use of subspecies and reliance on reproductive isolation as a basis for an operational species concept can also conflict with ethical issues governing the protection of species. We provide a way forward for recognizing and naming species that links theoretical and operational species concepts regardless of the magnitude of reproductive isolation.

Ecological Divergence and the History of Gene Flow in the Nearctic Milksnakes (Lampropeltis triangulum Complex).

Many phylogeographic studies on species with large ranges have found genetic-geographic structure associated with changes in habitat and physical barriers preventing or reducing gene flow. These interactions with geographic space, contemporary and historical climate, and biogeographic barriers have complex effects on contemporary population genetic structure and processes of speciation. While allopatric speciation at biogeographic barriers is considered the primary mechanism for generating species, more recently it has been shown that parapatric modes of divergence may be equally or even more common. With genomic data and better modeling capabilities, we can more clearly define causes of speciation in relation to biogeography and migration between lineages, the location of hybrid zones with respect to the ecology of parental lineages, and differential introgression of genes between taxa. Here, we examine the origins of three Nearctic milksnakes (Lampropeltis elapsoides, Lampropeltis triangulum and Lampropeltis gentilis) using genome-scale data to better understand species diversification. Results from artificial neural networks show that a mix of a strong biogeographic barrier, environmental changes, and physical space has affected genetic structure in these taxa. These results underscore conspicuous environmental changes that occur as the sister taxa L. triangulum and L. gentilis diverged near the Great Plains into the forested regions of the Eastern Nearctic. This area has been recognized as a region for turnover for many vertebrate species, but as we show here the contemporary boundary does not isolate these sister species. These two species likely formed in the mid-Pleistocene and have remained partially reproductively isolated over much of this time, showing differential introgression of loci. We also demonstrate that when L. triangulum and L. gentilis are each in contact with the much older L. elapsoides, some limited gene flow has occurred. Given the strong agreement between nuclear and mtDNA genomes, along with estimates of ecological niche, we suggest that all three lineages should continue to be recognized as unique species. Furthermore, this work emphasizes the importance of considering complex modes of divergence and differential allelic introgression over a complex landscape when testing mechanisms of speciation. [Cline; delimitation; Eastern Nearctic; Great Plains; hybrids; introgression; speciation.].

Citizen science and online data: Opportunities and challenges for snake ecology and action against snakebite.

The secretive behavior and life history of snakes makes studying their biology, distribution, and the epidemiology of venomous snakebite challenging. One of the most useful, most versatile, and easiest to collect types of biological data are photographs, particularly those that are connected with geographic location and date-time metadata. Photos verify occurrence records, provide data on phenotypes and ecology, and are often used to illustrate new species descriptions, field guides and identification keys, as well as in training humans and computer vision algorithms to identify snakes. We scoured eleven online and two offline sources of snake photos in an attempt to collect as many photos of as many snake species as possible, and attempt to explain some of the inter-species variation in photograph quantity among global regions and taxonomic groups, and with regard to medical importance, human population density, and range size. We collected a total of 725,565 photos-between 1 and 48,696 photos of 3098 of the world's 3879 snake species (79.9%), leaving 781 "most wanted" species with no photos (20.1% of all currently-described species as of the December 2020 release of The Reptile Database). We provide a list of most wanted species sortable by family, continent, authority, and medical importance, and encourage snake photographers worldwide to submit photos and associated metadata, particularly of "missing" species, to the most permanent and useful online archives: The Reptile Database, iNaturalist, and HerpMapper.

Phylogenetics of mud snakes (Squamata: Serpentes: Homalopsidae): A paradox of both undescribed diversity and taxonomic inflation.

Mud snakes (Serpentes: Homalopsidae) are a family of 55 described, mainly aquatic, species primarily distributed throughout mainland Southeast Asia and the Indo-Australian Archipelago. Although they have been the focus of prior research, the basic relationships amongst genera and species remain poorly known. We used a combined mitochondrial and nuclear gene dataset to infer their phylogenetic relationships, using the highest levels of taxon and geographic sampling for any homalopsid phylogeny to date (62% generic and 62% species coverage; 140 individuals). Our results recover two reciprocally monophyletic groups: the fangless Brachyorrhos and its sister clade comprised of all rear-fanged homalopsids. Most genera and interspecific relationships were monophyletic and strongly supported, but intergeneric relationships and intraspecific population structure lack support. We find evidence of both undescribed diversity as well as cases of taxonomic inflation within several species. Tree-based species delimitation approaches (mPTP) support potential new candidate species as distinct from their conspecifics and also suggest that many named taxa may not be distinct species. Divergence date estimation and lineage-through-time analyses indicate lower levels of speciation in the Eocene, with a subsequent burst in diversification in the Miocene. Homalopsids may have diversified most rapidly during the Pliocene and Pleistocene, possibly in relation to tectonic shifts and sea-level fluctuations that took place in Sundaland and the Sahul Shelf. Our analyses provide new insights on homalopsid taxonomy, a baseline phylogeny for the family, and further biogeographic implications demonstrating how dynamic tectonics and Quaternary sea level changes may have shaped a widespread, diverse family of snakes.

Citizen science and online data: opportunities and challenges for snake ecology and action against snakebite

The secretive behavior and life history of snakes makes studying their biology, distribution, and the epidemiology of venomous snakebite challenging. One of the most useful, most versatile, and easiest to collect types of biological data are photographs, particularly those that are connected with geographic location and date-time metadata. Photos verify occurrence records, provide data on phenotypes and ecology, and are often used to illustrate new species descriptions, field guides and identification keys, as well as in training humans and computer vision algorithms to identify snakes. We scoured eleven online and two offline sources of snake photos in an attempt to collect as many photos of as many snake species as possible, and attempt to explain some of the inter-species variation in photograph quantity among global regions and taxonomic groups, and with regard to medical importance, human population density, and range size. We collected a total of 725,565 photos—between 1 and 48,696 photos of 3098 of the world's 3879 snake species (79.9%), leaving 781 “most wanted” species with no photos (20.1% of all currently-described species as of the December 2020 release of The Reptile Database). We provide a list of most wanted species sortable by family, continent, authority, and medical importance, and encourage snake photographers worldwide to submit photos and associated metadata, particularly of “missing” species, to the most permanent and useful online archives: The Reptile Database, iNaturalist, and HerpMapper.

Interrogating Genomic-Scale Data for Squamata (Lizards, Snakes, and Amphisbaenians) Shows no Support for Key Traditional Morphological Relationships.

Genomics is narrowing uncertainty in the phylogenetic structure for many amniote groups. For one of the most diverse and species-rich groups, the squamate reptiles (lizards, snakes, and amphisbaenians), an inverse correlation between the number of taxa and loci sampled still persists across all publications using DNA sequence data and reaching a consensus on the relationships among them has been highly problematic. In this study, we use high-throughput sequence data from 289 samples covering 75 families of squamates to address phylogenetic affinities, estimate divergence times, and characterize residual topological uncertainty in the presence of genome-scale data. Importantly, we address genomic support for the traditional taxonomic groupings Scleroglossa and Macrostomata using novel machine-learning techniques. We interrogate genes using various metrics inherent to these loci, including parsimony-informative sites (PIS), phylogenetic informativeness, length, gaps, number of substitutions, and site concordance to understand why certain loci fail to find previously well-supported molecular clades and how they fail to support species-tree estimates. We show that both incomplete lineage sorting and poor gene-tree estimation (due to a few undesirable gene properties, such as an insufficient number of PIS), may account for most gene and species-tree discordance. We find overwhelming signal for Toxicofera, and also show that none of the loci included in this study supports Scleroglossa or Macrostomata. We comment on the origins and diversification of Squamata throughout the Mesozoic and underscore remaining uncertainties that persist in both deeper parts of the tree (e.g., relationships between Dibamia, Gekkota, and remaining squamates; among the three toxicoferan clades Iguania, Serpentes, and Anguiformes) and within specific clades (e.g., affinities among gekkotan, pleurodont iguanians, and colubroid families).

Interrogating genomic-scale data for squamata (lizards, snakes, and amphisbaenians) shows no support for key traditional morphological relationships

Genomics is narrowing uncertainty in the phylogenetic structure for many amniote groups. For one of the most diverse and species-rich groups, the squamate reptiles (lizards, snakes, and amphisbaenians), an inverse correlation between the number of taxa and loci sampled still persists across all publications using DNA sequence data and reaching a consensus on the relationships among them has been highly problematic. In this study, we use high-throughput sequence data from 289 samples covering 75 families of squamates to address phylogenetic affinities, estimate divergence times, and characterize residual topological uncertainty in the presence of genome-scale data. Importantly, we address genomic support for the traditional taxonomic groupings Scleroglossa and Macrostomata using novel machine-learning techniques. We interrogate genes using various metrics inherent to these loci, including parsimony-informative sites (PIS), phylogenetic informativeness, length, gaps, number of substitutions, and site concordance to understand why certain loci fail to find previously well-supported molecular clades and how they fail to support species-tree estimates. We show that both incomplete lineage sorting and poor gene-tree estimation (due to a few undesirable gene properties, such as an insufficient number of PIS), may account for most gene and species-tree discordance. We find overwhelming signal for Toxicofera, and also show that none of the loci included in this study supports Scleroglossa or Macrostomata. We comment on the origins and diversification of Squamata throughout the Mesozoic and underscore remaining uncertainties that persist in both deeper parts of the tree (e.g., relationships between Dibamia, Gekkota, and remaining squamates; among the three toxicoferan clades Iguania, Serpentes, and Anguiformes) and within specific clades (e.g., affinities among gekkotan, pleurodont iguanians, and colubroid families)

Interrogating genomic-scale data for squamata (lizards, snakes, and amphisbaenians) shows no support for key traditional morphological relationships

Genomics is narrowing uncertainty in the phylogenetic structure for many amniote groups. For one of the most diverse and species-rich groups, the squamate reptiles (lizards, snakes, and amphisbaenians), an inverse correlation between the number of taxa and loci sampled still persists across all publications using DNA sequence data and reaching a consensus on the relationships among them has been highly problematic. In this study, we use high-throughput sequence data from 289 samples covering 75 families of squamates to address phylogenetic affinities, estimate divergence times, and characterize residual topological uncertainty in the presence of genome-scale data. Importantly, we address genomic support for the traditional taxonomic groupings Scleroglossa and Macrostomata using novel machine-learning techniques. We interrogate genes using various metrics inherent to these loci, including parsimony-informative sites (PIS), phylogenetic informativeness, length, gaps, number of substitutions, and site concordance to understand why certain loci fail to find previously well-supported molecular clades and how they fail to support species-tree estimates. We show that both incomplete lineage sorting and poor gene-tree estimation (due to a few undesirable gene properties, such as an insufficient number of PIS), may account for most gene and species-tree discordance. We find overwhelming signal for Toxicofera, and also show that none of the loci included in this study supports Scleroglossa or Macrostomata. We comment on the origins and diversification of Squamata throughout the Mesozoic and underscore remaining uncertainties that persist in both deeper parts of the tree (e.g., relationships between Dibamia, Gekkota, and remaining squamates; among the three toxicoferan clades Iguania, Serpentes, and Anguiformes) and within specific clades (e.g., affinities among gekkotan, pleurodont iguanians, and colubroid families)

The Origins and Diversification of the Exceptionally Rich Gemsnakes (Colubroidea: Lamprophiidae: Pseudoxyrhophiinae) in Madagascar.

Processes leading to spectacular diversity of both form and species on islands have been well-documented under island biogeography theory, where distance from source and island size are key factors determining immigration and extinction resistance. But far less understood are the processes governing in situ diversification on the world's mega islands, where large and isolated land masses produced morphologically distinct radiations from related taxa on continental regions. Madagascar has long been recognized as a natural laboratory due to its isolation, lack of influence from adjacent continents, and diversification of spectacular vertebrate radiations. However, only a handful of studies have examined rate shifts of in situ diversification for this island. Here, we examine rates of diversification in the Malagasy snakes of the family Pseudoxyrhophiinae (gemsnakes) to understand if rates of speciation were initially high, enhanced by diversification into distinct biomes, and associated with key dentition traits. Using a genomic sequence-capture data set for 366 samples, we determine that all previously described and newly discovered species are delimitable and therefore useful candidates for understanding diversification trajectories through time. Our analysis detected no shifts in diversification rate between clades or changes in biome or dentition type. Remarkably, we demonstrate that rates of diversification of the gemsnake radiation, which originated in Madagascar during the early Miocene, remained steady throughout the Neogene. However, we do detect a significant slowdown in diversification during the Pleistocene. We also comment on the apparent paradox where most living species originated in the Pleistocene, despite diversification rates being substantially higher during the earlier 15 myr.

Phylogenomics using formalin-fixed and 100+ year-old intractable natural history specimens.

Museum specimens provide a wealth of information to biologists, but obtaining genetic data from formalin-fixed and fluid-preserved specimens remains challenging. While DNA sequences have been recovered from such specimens, most approaches are time-consuming and produce low data quality and quantity. Here, we use a modified DNA extraction protocol combined with high-throughput sequencing to recover DNA from formalin-fixed and fluid-preserved snakes that were collected over a century ago and for which little or no modern genetic materials exist in public collections. We successfully extracted DNA and sequenced ultraconserved elements (x¯ = 2318 loci) from 10 fluid-preserved snakes and included them in a phylogeny with modern samples. This phylogeny demonstrates the general use of such specimens in phylogenomic studies and provides evidence for the placement of enigmatic snakes, such as the rare and never-before sequenced Indian Xylophis stenorhynchus. Our study emphasizes the relevance of museum collections in modern research and simultaneously provides a protocol that may prove useful for specimens that have been previously intractable for DNA sequencing.

Asynchronous demographic responses to Pleistocene climate change in Eastern Nearctic vertebrates.

Pleistocene climatic cycles altered species distributions in the Eastern Nearctic of North America, yet the degree of congruent demographic response to the Pleistocene among codistributed taxa remains unknown. We use a hierarchical approximate Bayesian computational approach to test if population sizes across lineages of snakes, lizards, turtles, mammals, birds, salamanders and frogs in this region expanded synchronously to Late Pleistocene climate changes. Expansion occurred in 75% of 74 lineages, and of these, population size trajectories across the community were partially synchronous, with coexpansion found in at least 50% of lineages in each taxonomic group. For those taxa expanding outside of these synchronous pulses, factors related to when they entered the community, ecological thresholds or biotic interactions likely condition their timing of response to Pleistocene climate change. Unified timing of population size change across communities in response to Pleistocene climate cycles is likely rare in North America.

Comparing species tree estimation with large anchored phylogenomic and small Sanger-sequenced molecular datasets: an empirical study on Malagasy pseudoxyrhophiine snakes.

BACKGROUND: Using molecular data generated by high throughput next generation sequencing (NGS) platforms to infer phylogeny is becoming common as costs go down and the ability to capture loci from across the genome goes up. While there is a general consensus that greater numbers of independent loci should result in more robust phylogenetic estimates, few studies have compared phylogenies resulting from smaller datasets for commonly used genetic markers with the large datasets captured using NGS. Here, we determine how a 5-locus Sanger dataset compares with a 377-locus anchored genomics dataset for understanding the evolutionary history of the pseudoxyrhophiine snake radiation centered in Madagascar. The Pseudoxyrhophiinae comprise ~86 % of Madagascar's serpent diversity, yet they are poorly known with respect to ecology, behavior, and systematics. Using the 377-locus NGS dataset and the summary statistics species-tree methods STAR and MP-EST, we estimated a well-supported species tree that provides new insights concerning intergeneric relationships for the pseudoxyrhophiines. We also compared how these and other methods performed with respect to estimating tree topology using datasets with varying numbers of loci. METHODS: Using Sanger sequencing and an anchored phylogenomics approach, we sequenced datasets comprised of 5 and 377 loci, respectively, for 23 pseudoxyrhophiine taxa. For each dataset, we estimated phylogenies using both gene-tree (concatenation) and species-tree (STAR, MP-EST) approaches. We determined the similarity of resulting tree topologies from the different datasets using Robinson-Foulds distances. In addition, we examined how subsets of these data performed compared to the complete Sanger and anchored datasets for phylogenetic accuracy using the same tree inference methodologies, as well as the program *BEAST to determine if a full coalescent model for species tree estimation could generate robust results with fewer loci compared to the summary statistics species tree approaches. We also examined the individual gene trees in comparison to the 377-locus species tree using the program MetaTree. RESULTS: Using the full anchored dataset under a variety of methods gave us the same, well-supported phylogeny for pseudoxyrhophiines. The African pseudoxyrhophiine Duberria is the sister taxon to the Malagasy pseudoxyrhophiines genera, providing evidence for a monophyletic radiation in Madagascar. In addition, within Madagascar, the two major clades inferred correspond largely to the aglyphous and opisthoglyphous genera, suggesting that feeding specializations associated with tooth venom delivery may have played a major role in the early diversification of this radiation. The comparison of tree topologies from the concatenated and species-tree methods using different datasets indicated the 5-locus dataset cannot beused to infer a correct phylogeny for the pseudoxyrhophiines under any method tested here and that summary statistics methods require 50 or more loci to consistently recover the species-tree inferred using the complete anchored dataset. However, as few as 15 loci may infer the correct topology when using the full coalescent species tree method *BEAST. MetaTree analyses of each gene tree from the Sanger and anchored datasets found that none of the individual gene trees matched the 377-locus species tree, and that no gene trees were identical with respect to topology. CONCLUSIONS: Our results suggest that ≥50 loci may be necessary to confidently infer phylogenies when using summaryspecies-tree methods, but that the coalescent-based method *BEAST consistently recovers the same topology using only 15 loci. These results reinforce that datasets with small numbers of markers may result in misleading topologies, and further, that the method of inference used to generate a phylogeny also has a major influence on the number of loci necessary to infer robust species trees.

Independent Demographic Responses to Climate Change among Temperate and Tropical Milksnakes (Colubridae: Genus Lampropeltis).

The effects of Late Quaternary climate change have been examined for many temperate New World taxa, but the impact of Pleistocene glacial cycles on Neotropical taxa is less well understood, specifically with respect to changes in population demography. Here, we examine historical demographic trends for six species of milksnake with representatives in both the temperate and tropical Americas to determine if species share responses to climate change as a taxon or by area (i.e., temperate versus tropical environments). Using a multilocus dataset, we test for the demographic signature of population expansion and decline using non-genealogical summary statistics, as well as coalescent-based methods. In addition, we determine whether range sizes are correlated with effective population sizes for milksnakes. Results indicate that there are no identifiable trends with respect to demographic response based on location, and that species responded to changing climates independently, with tropical taxa showing greater instability. There is also no correlation between range size and effective population size, with the largest population size belonging to the species with the smallest geographic distribution. Our study highlights the importance of not generalizing the demographic histories of taxa by region and further illustrates that the New World tropics may not have been a stable refuge during the Pleistocene.

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.

Speciation at the Mogollon Rim in the Arizona Mountain Kingsnake (Lampropeltis pyromelana).

Studies of speciation and taxon delimitation are usually decoupled. Combining these methods provides a stronger theoretical ground for recognizing new taxa and understanding processes of speciation. Using coalescent methods, we examine speciation, post-speciation population demographics, and taxon delimitation in the Arizona Mountain Kingsnake (Lampropeltis pyromelana), a species restricted to high elevations in southwestern United States and northern Mexico (SW). These methods provide a solid foundation for understanding how biogeographic barriers operate at the regional scale in the SW. Bayesian species delimitation methods, using three loci from samples of L. pyromelana taken throughout their range, show strong support for the existence of two species that are separated by low elevation habitats found between the Colorado Plateau/ Mogollon Rim and the Sierra Madre Occidental. Our results suggest an allopatric mode of speciation given the near absence of gene flow over time, which resulted in two lineages of unequal population sizes. Speciation likely occurred prior to the Pleistocene, during the aridification of the SW and/or the uplift of the Colorado Plateau, and while these species occupy similar high-elevation niches, they are isolated by xeric conditions found in the intervening low deserts. Furthermore, post-speciation demographics suggest that populations of both lineages were not negatively impacted by climate change throughout the Pleistocene. Finally, our results suggest that at least for this group, where divergence is old and gene flow is low, Bayesian species delimitation performs well.