Specimen collection is essential for modern science.

Natural history museums are vital repositories of specimens, samples and data that inform about the natural world; this Formal Comment revisits a Perspective that advocated for the adoption of compassionate collection practices, querying whether it will ever be possible to completely do away with whole animal specimen collection.

Geoclimatic drivers of diversification in the largest arid and semi-arid environment of the Neotropics: Perspectives from phylogeography.

The South American Dry Diagonal, also called the Diagonal of Open Formations, is a large region of seasonally dry vegetation extending from northeastern Brazil to northern Argentina, comprising the Caatinga, Cerrado, and Chaco subregions. A growing body of phylogeography literature has determined that a complex history of climatic changes coupled with more ancient geological events has produced a diverse and endemic-rich Dry Diagonal biota. However, the exact drivers are still under investigation, and their relative strengths and effects are controversial. Pleistocene climatic fluctuations structured lineages via vegetation shifts, refugium formation, and corridors between the Amazon and Atlantic forests. In some taxa, older geological events, such as the reconfiguration of the São Francisco River, uplift of the Central Brazilian Plateau, or the Miocene inundation of the Chaco by marine incursions, were more important. Here, we review the Dry Diagonal phylogeography literature, discussing each hypothesized driver of diversification and assessing degree of support. Few studies statistically test these hypotheses, with most support drawn from associating encountered phylogeographic patterns such as population structure with the timing of ancient geoclimatic events. Across statistical studies, most hypotheses are well supported, with the exception of the Pleistocene Arc Hypothesis. However, taxonomic and regional biases persist, such as a proportional overabundance of herpetofauna studies, and the under-representation of Chaco studies. Overall, both Pleistocene climate change and Neogene geological events shaped the evolution of the Dry Diagonal biota, though the precise effects are regionally and taxonomically varied. We encourage further use of model-based analyses to test evolutionary scenarios, as well as interdisciplinary collaborations to progress the field beyond its current focus on the traditional set of geoclimatic hypotheses.

Speciation Hypotheses from Phylogeographic Delimitation Yield an Integrative Taxonomy for Seal Salamanders (Desmognathus monticola).

Significant advances have been made in species delimitation and numerous methods can test precisely defined models of speciation, though the synthesis of phylogeography and taxonomy is still sometimes incomplete. Emerging consensus treats distinct genealogical clusters in genome-scale data as strong initial evidence of speciation in most cases, a hypothesis that must therefore be falsified under an explicit evolutionary model. We can now test speciation hypotheses linking trait differentiation to specific mechanisms of divergence with increasingly large data sets. Integrative taxonomy can, therefore, reflect an understanding of how each axis of variation relates to underlying speciation processes, with nomenclature for distinct evolutionary lineages. We illustrate this approach here with Seal Salamanders (Desmognathus monticola) and introduce a new unsupervised machine-learning approach for species delimitation. Plethodontid salamanders are renowned for their morphological conservatism despite extensive phylogeographic divergence. We discover 2 geographic genetic clusters, for which demographic and spatial models of ecology and gene flow provide robust support for ecogeographic speciation despite limited phenotypic divergence. These data are integrated under evolutionary mechanisms (e.g., spatially localized gene flow with reduced migration) and reflected in emergent properties expected under models of reinforcement (e.g., ethological isolation and selection against hybrids). Their genetic divergence is prima facie evidence for species-level distinctiveness, supported by speciation models and divergence along axes such as behavior, geography, and climate that suggest an ecological basis with subsequent reinforcement through prezygotic isolation. As data sets grow more comprehensive, species-delimitation models can be tested, rejected, or corroborated as explicit speciation hypotheses, providing for reciprocal illumination of evolutionary processes and integrative taxonomies. [Desmognathus; integrative taxonomy; machine learning; species delimitation.].

The phylogenomic and biogeographic history of the gartersnakes, watersnakes, and allies (Natricidae: Thamnophiini).

North American Thamnophiini (gartersnakes, watersnakes, brownsnakes, and swampsnakes) are an ecologically and phenotypically diverse temperate clade of snakes representing 61 species across 10 genera. In this study, we estimate phylogenetic trees using âˆ¼3,700 ultraconserved elements (UCEs) for 76 specimens representing 75% of all Thamnophiini species. We infer phylogenies using multispecies coalescent methods and time calibrate them using the fossil record. We also conducted ancestral area estimation to identify how major biogeographic boundaries in North America affect broadscale diversification in the group. While most nodes exhibited strong statistical support, analysis of concordant data across gene trees reveals substantial heterogeneity. Ancestral area estimation demonstrated that the genus Thamnophis was the only taxon in this subfamily to cross the Western Continental Divide, even as other taxa dispersed southward toward the tropics. Additionally, levels of gene tree discordance are overall higher in transition zones between bioregions, including the Rocky Mountains. Therefore, the Western Continental Divide may be a significant transition zone structuring the diversification of Thamnophiini during the Neogene and Pleistocene. Here we show that despite high levels of discordance across gene trees, we were able to infer a highly resolved and well-supported phylogeny for Thamnophiini, which allows us to understand broadscale patterns of diversity and biogeography.

Ultraconserved elements-based phylogenomic systematics of the snake superfamily Elapoidea, with the description of a new Afro-Asian family.

The highly diverse snake superfamily Elapoidea is considered to be a classic example of ancient, rapid radiation. Such radiations are challenging to fully resolve phylogenetically, with the highly diverse Elapoidea a case in point. Previous attempts at inferring a phylogeny of elapoids produced highly incongruent estimates of their evolutionary relationships, often with very low statistical support. We sought to resolve this situation by sequencing over 4,500 ultraconserved element loci from multiple representatives of every elapoid family/subfamily level taxon and inferring their phylogenetic relationships with multiple methods. Concatenation and multispecies coalescent based species trees yielded largely congruent and well-supported topologies. Hypotheses of a hard polytomy were not retained for any deep branches. Our phylogenies recovered Cyclocoridae and Elapidae as diverging early within Elapoidea. The Afro-Malagasy radiation of elapoid snakes, classified as multiple subfamilies of an inclusive Lamprophiidae by some earlier authors, was found to be monophyletic in all analyses. The genus Micrelaps was consistently recovered as sister to Lamprophiidae. We establish a new family, Micrelapidae fam. nov., for Micrelaps and assign Brachyophis to this family based on cranial osteological synapomorphy. We estimate that Elapoidea originated in the early Eocene and rapidly diversified into all the major lineages during this epoch. Ecological opportunities presented by the post-Cretaceous-Paleogene mass extinction event may have promoted the explosive radiation of elapoid snakes.

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.].

Complex genetic patterns and distribution limits mediated by native congeners of the worldwide invasive red-eared slider turtle.

Non-native (invasive) species offer a unique opportunity to study the geographical distribution and range limits of species, wherein the evolutionary change driven by interspecific interactions between native and non-native closely related species is a key component. The red-eared slider turtle, Trachemys scripta elegans (TSE), has been introduced and successfully established worldwide. It can coexist with its native congeners T. cataspila, T. venusta and T. taylori in Mexico. We performed comprehensive fieldwork, executed a battery of genetic analyses and applied a novel species distribution modelling approach to evaluate their historical lineage relationships and contemporary population genetic patterns. Our findings support the historical common ancestry between native TSE and non-native (TSEalien ), while also highlighting the genetic differentiation of the exotic lineage. Genetic patterns are associated with their range size/endemism gradient; the microendemic T. taylori showed significant reduced genetic diversity and high differentiation, whereas TSEalien showed the highest diversity and signals of population size expansion. Counter to our expectations, lower naturally occurring distribution overlap and little admixture patterns were found between TSE and its congeners, exhibiting reduced gene flow and clear genetic separation across neighbouring species despite having zones of contact. We demonstrate that these native Trachemys species have distinct climatic niche suitability, probably preventing establishment of and displacement by the TSEalien . Moreover, we found major niche overlap between TSEalien and native species worldwide, supporting our prediction that sites with closer ecological optima to the invasive species have higher establishment risk than those that are closer to the niche-centre of the native species.

Quaternary climatic fluctuations influence the demographic history of two species of sky-island endemic amphibians in the Neotropics.

We evaluated the role of Quaternary climatic fluctuations on the demographic history and population structure of amphibian species endemic to the 'campo rupestre' in the Neotropics, evaluating their distributional shifts, demographic changes, and lineage formation from the end of Pleistocene to present. We chose two anurans endemic to the high-elevation 'campo rupestre' in the Espinhaço Range (ER) in northeastern and southeastern Brazil (Bokermannohyla alvarengai and Bokermannohyla oxente), as models to test the role of Quaternary climatic fluctuations over their distribution range in this region. We collected tissue samples throughout their distribution range and used statistical phylogeography to examine processes of divergence and population demography. We generated spatial-temporal reconstructions using Bayesian inference in a coalescent framework in combination with hind-cast projections of species distribution models (SDMs). We also used the results and literature information to test alternative diversification scenarios via approximate Bayesian computation (ABC). Our results show that Quaternary climatic fluctuations influenced the geographic ranges of both species showing population expansion during the last glacial maximum (LGM) and range contraction during interglacial periods, as inferred from selected ABC models and from past projections of SDMs. We recovered Pleistocene diversification for both species occuring in distinctly unique periods for each taxon. An older and range-restricted lineage was recovered in a geographically isolated geological massif, deserving conservation and further taxonomic study. The diversification and distribution of these amphibian species endemic to the Neotropical 'campo rupestre' were influenced by Quaternary climatic fluctuations. The expansion of cold adapted species restricted to higher elevations during glacial periods and their concomitant retraction during interglacial periods may have been crucial for producing patterns of species richness and endemism along elevation gradients in tropical and subtropical domains. Such processes may influence the evolution of the biota distributed in heterogeneous landscapes with varied topography.

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).

Biogeographic barriers, Pleistocene refugia, and climatic gradients in the southeastern Nearctic drive diversification in cornsnakes (Pantherophis guttatus complex).

The southeastern Nearctic is a biodiversity hotspot that is also rich in cryptic species. Numerous hypotheses (e.g., vicariance, local adaptation, and Pleistocene speciation in glacial refugia) have been tested in an attempt to explain diversification and the observed pattern of extant biodiversity. However, previous phylogeographic studies have both supported and refuted these hypotheses. Therefore, while data support one or more of these diversification hypotheses, it is likely that taxa are forming within this region in species-specific ways. Here, we generate a genomic data set for the cornsnakes (Pantherophis guttatus complex), which are widespread across this region, spanning both biogeographic barriers and climatic gradients. We use phylogeographic model selection combined with hindcast ecological niche models to determine regions of habitat stability through time. This combined approach suggests that numerous drivers of population differentiation explain the current diversity of this group of snakes. The Mississippi River caused initial speciation in this species complex, with more recent divergence events linked to adaptations to ecological heterogeneity and allopatric Pleistocene refugia. Lastly, we discuss the taxonomy of this group and suggest there may be additional cryptic species in need of formal recognition.

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.

Environmental heterogeneity and not vicariant biogeographic barriers generate community-wide population structure in desert-adapted snakes.

Genetic structure can be influenced by local adaptation to environmental heterogeneity and biogeographic barriers, resulting in discrete population clusters. Geographic distance among populations, however, can result in continuous clines of genetic divergence that appear as structured populations. Here, we evaluate the relevant importance of these three factors over a landscape characterized by environmental heterogeneity and the presence of a hypothesized biogeographic barrier in producing population genetic structure within 13 codistributed snake species using a genomic data set. We demonstrate that geographic distance and environmental heterogeneity across western North America contribute to population genomic divergence. Surprisingly, landscape features long thought to contribute to biogeographic barriers play little role in divergence community wide. Our results suggest that isolation by environment is the most important contributor to genomic divergence. Furthermore, we show that models of population clustering that incorporate spatial information consistently outperform nonspatial models, demonstrating the importance of considering geographic distances in population clustering. We argue that environmental and geographic distances as drivers of community-wide divergence should be explored before assuming the role of biogeographic barriers.

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.

The Biogeography of Deep Time Phylogenetic Reticulation.

Most phylogenies are typically represented as purely bifurcating. However, as genomic data have become more common in phylogenetic studies, it is not unusual to find reticulation among terminal lineages or among internal nodes (deep time reticulation; DTR). In these situations, gene flow must have happened in the same or adjacent geographic areas for these DTRs to have occurred and therefore biogeographic reconstruction should provide similar area estimates for parental nodes, provided extinction or dispersal has not eroded these patterns. We examine the phylogeny of the widely distributed New World kingsnakes (Lampropeltis), determine if DTR is present in this group, and estimate the ancestral area for reticulation. Importantly, we develop a new method that uses coalescent simulations in a machine learning framework to show conclusively that this phylogeny is best represented as reticulating at deeper time. Using joint probabilities of ancestral area reconstructions on the bifurcating parental lineages from the reticulating node, we show that this reticulation likely occurred in northwestern Mexico/southwestern US, and subsequently, led to the diversification of the Mexican kingsnakes. This region has been previously identified as an area important for understanding speciation and secondary contact with gene flow in snakes and other squamates. This research shows that phylogenetic reticulation is common, even in well-studied groups, and that the geographic scope of ancient hybridization is recoverable.

Estimating synchronous demographic changes across populations using hABC and its application for a herpetological community from northeastern Brazil.

Many studies propose that Quaternary climatic cycles contracted and/or expanded the ranges of species and biomes. Strong expansion-contraction dynamics of biomes presume concerted demographic changes of associated fauna. The analysis of temporal concordance of demographic changes can be used to test the influence of Quaternary climate on diversification processes. Hierarchical approximate Bayesian computation (hABC) is a powerful and flexible approach that models genetic data from multiple species, and can be used to estimate the temporal concordance of demographic processes. Using available single-locus data, we can now perform large-scale analyses, both in terms of number of species and geographic scope. Here, we first compared the power of four alternative hABC models for a collection of single-locus data. We found that the model incorporating an a priori hypothesis about the timing of simultaneous demographic change had the best performance. Second, we applied the hABC models to a data set of seven squamate and four amphibian species occurring in the Seasonally Dry Tropical Forests (Caatinga) in northeastern Brazil, which, according to paleoclimatic evidence, experienced an increase in aridity during the Pleistocene. If this increase was important for the diversification of associated xeric-adapted species, simultaneous population expansions should be evident at the community level. We found a strong signal of synchronous population expansion in the Late Pleistocene, supporting the increase of the Caatinga during this time. This expansion likely enhanced the formation of communities adapted to high aridity and seasonality and caused regional extirpation of taxa adapted to wet forest.

Using phylogenomics to understand the link between biogeographic origins and regional diversification in ratsnakes.

Globally distributed groups may show regionally distinct rates of diversification, where speciation is elevated given timing and sources of ecological opportunity. However, for most organisms, nearly complete sampling at genomic-data scales to reduce topological error in all regions is unattainable, thus hampering conclusions related to biogeographic origins and rates of diversification. We explore processes leading to the diversity of global ratsnakes and test several important hypotheses related to areas of origin and enhanced diversification upon colonizing new continents. We estimate species trees inferred from phylogenomic scale data (304 loci) while exploring several strategies that consider topological error from each individual gene tree. With a dated species tree, we examine taxonomy and test previous hypotheses that suggest the ratsnakes originated in the Old World (OW) and dispersed to New World (NW). Furthermore, we determine if dispersal to the NW represented a source of ecological opportunity, which should show elevated rates of species diversification. We show that ratsnakes originated in the OW during the mid-Oligocene and subsequently dispersed to the NW by the mid-Miocene; diversification was also elevated in a subclade of NW taxa. Finally, the optimal biogeographic region-dependent speciation model shows that the uptick in ratsnake diversification was associated with colonization of the NW. We consider several alternative explanations that account for regionally distinct diversification rates.

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.

Complex longitudinal diversification across South China and Vietnam in Stejneger's pit viper, Viridovipera stejnegeri (Schmidt, 1925) (Reptilia: Serpentes: Viperidae).

Viridovipera stejnegeri is one of the most common pit vipers in Asia, with a wide distribution in southern China and Vietnam. We investigated historical demography and explored how the environment and climatic factors have shaped genetic diversity and the evolutionary history of this venomous snake. A total of 171 samples from 47 localities were sequenced and analysed for two mitochondrial gene fragments and three nuclear genes. Gene trees reveal the existence of two well-supported clades (Southwest China and Southeast China) with seven distinct and strongly supported, geographically structured subclades within V. stejnegeri. Estimation of divergence time and ancestral area suggests that V. stejnegeri originated at ~6.0 Ma in the late Miocene on the Yunnan-Guizhou Plateau. The estimated date of origin and divergence of the island populations of Taiwan and Hainan closely matches the geological origin of the both islands. The mtDNA gene tree reveals the presence of west-east diversification in V. stejnegeri populations. Complex orogenesis and heterogeneous habitats, as well as climate-mediated habitat differentiation including glacial cycles, all have influenced population structure and the distribution of this taxon. The validity of V. stejnegeri chenbihuii is questionable, and this subspecies most probably represents an invalid taxon.

Phylogeny and tempo of diversification in the superradiation of spiny-rayed fishes.

Spiny-rayed fishes, or acanthomorphs, comprise nearly one-third of all living vertebrates. Despite their dominant role in aquatic ecosystems, the evolutionary history and tempo of acanthomorph diversification is poorly understood. We investigate the pattern of lineage diversification in acanthomorphs by using a well-resolved time-calibrated phylogeny inferred from a nuclear gene supermatrix that includes 520 acanthomorph species and 37 fossil age constraints. This phylogeny provides resolution for what has been classically referred to as the "bush at the top" of the teleost tree, and indicates acanthomorphs originated in the Early Cretaceous. Paleontological evidence suggests acanthomorphs exhibit a pulse of morphological diversification following the end Cretaceous mass extinction; however, the role of this event on the accumulation of living acanthomorph diversity remains unclear. Lineage diversification rates through time exhibit no shifts associated with the end Cretaceous mass extinction, but there is a global decrease in lineage diversification rates 50 Ma that occurs during a period when morphological disparity among fossil acanthomorphs increases sharply. Analysis of clade-specific shifts in diversification rates reveal that the hyperdiversity of living acanthomorphs is highlighted by several rapidly radiating lineages including tunas, gobies, blennies, snailfishes, and Afro-American cichlids. These lineages with high diversification rates are not associated with a single habitat type, such as coral reefs, indicating there is no single explanation for the success of acanthomorphs, as exceptional bouts of diversification have occurred across a wide array of marine and freshwater habitats.

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.