Tod W. Reeder

Evolutionary and ecological causes of the latitudinal diversity gradient in hylid frogs: treefrog trees unearth the roots of high tropical diversity.

Why are there more species in the tropics than in temperate regions? In recent years, this long‐standing question has been addressed primarily by seeking environmental correlates of diversity. But to understand the ultimate causes of diversity patterns, we must also examine the evolutionary and biogeographic processes that directly change species numbers (i.e., speciation, extinction, and dispersal). With this perspective, we dissect the latitudinal diversity gradient in hylid frogs. We reconstruct a phylogeny for 124 hylid species, estimate divergence times and diversification rates for major clades, reconstruct biogeographic changes, and use ecological niche modeling to identify climatic variables that potentially limit dispersal. We find that hylids originated in tropical South America and spread to temperate regions only recently (leaving limited time for speciation). There is a strong relationship between the species richness of each region and when that region was colonized but not between the latitudinal positions of clades and their rates of diversification. Temperature seasonality seemingly limits dispersal of many tropical clades into temperate regions and shows significant phylogenetic conservatism. Overall, our study illustrates how two general principles (niche conservatism and the time‐for‐speciation effect) may help explain the latitudinal diversity gradient as well as many other diversity patterns across taxa and regions.

Hylid Frog Phylogeny and Sampling Strategies for Speciose Clades

How should characters and taxa be sampled to resolve efficiently the phylogeny of ancient and highly speciose groups? We addressed this question empirically in the treefrog family Hylidae, which contains > 800 species and may be nonmonophyletic with respect to other anuran families. We sampled 81 species (54 hylids and 27 outgroups) for two mitochondrial genes (12S, ND1), two nuclear genes (POMC, c-myc), and morphology (144 characters) in an attempt to resolve higher-level relationships. We then added 117 taxa to the combined data set, many of which were sampled for only one gene (12S). Despite the relative incompleteness of the majority of taxa, the resulting trees placed all taxa in the expected higher-level clades with strong support, despite some taxa being > 90% incomplete. Furthermore, we found no relationship between the completeness of a taxon and the support (parsimony bootstrap or Bayesian posterior probabilities) for its localized placement on the tree. Separate analysis of the data set with the most taxa (12S) gives a somewhat problematic estimate of higher-level relationships, suggesting that data sets scored only for some taxa (ND1, nuclear genes, morphology) are important in determining the outcome of the combined analysis. The results show that hemiphractine hylids are not closely related to other hylids and should be recognized as a distinct family. They also show that the speciose genus Hyla is polyphyletic, but that its species can be arranged into three monophyletic genera. A new classification of hylid frogs is proposed. Several potentially misleading signals in the morphological data are discussed.

Why does a trait evolve multiple times within a clade? Repeated evolution of snakelike body form in squamate reptiles

Abstract Why does a trait evolve repeatedly within a clade? When examining the evolution of a trait, evolutionary biologists typically focus on the selective advantages it may confer and the genetic and developmental mechanisms that allow it to vary. Although these factors may be necessary to explain why a trait evolves in a particular instance, they may not be sufficient to explain phylogenetic patterns of repeated evolution or conservatism. Instead, other factors may also be important, such as biogeography and competitive interactions. In squamate reptiles (lizards and snakes) a dramatic transition in body form has occurred repeatedly, from a fully limbed, lizardlike body form to a limb-reduced, elongate, snakelike body form. We analyze this trait in a phylogenetic and biogeographic context to address why this transition occurred so frequently. We included 261 species for which morphometric data and molecular phylogenetic information were available. Among the included species, snakelike body form has evolved about 25 times. Most lineages of snakelike squamates belong to one of two “ecomorphs,” either short-tailed burrowers or long-tailed surface dwellers. The repeated origins of snakelike squamates appear to be associated with the in situ evolution of these two ecomorphs on different continental regions (including multiple origins of the burrowing morph within most continents), with very little dispersal of most limb-reduced lineages between continental regions. Overall, the number of repeated origins of snakelike morphology seems to depend on large-scale biogeographic patterns and community ecology, in addition to more traditional explanations (e.g., selection, development).

Resolving the phylogeny of lizards and snakes (Squamata) with extensive sampling of genes and species

Squamate reptiles (lizards and snakes) are one of the most diverse groups of terrestrial vertebrates. Recent molecular analyses have suggested a very different squamate phylogeny relative to morphological hypotheses, but many aspects remain uncertain from molecular data. Here, we analyse higher-level squamate phylogeny with a molecular dataset of unprecedented size, including 161 squamate species for up to 44 nuclear genes each (33 717 base pairs), using both concatenated and species-tree methods for the first time. Our results strongly resolve most squamate relationships and reveal some surprising results. In contrast to most other recent studies, we find that dibamids and gekkotans are together the sister group to all other squamates. Remarkably, we find that the distinctive scolecophidians (blind snakes) are paraphyletic with respect to other snakes, suggesting that snakes were primitively burrowers and subsequently re-invaded surface habitats. Finally, we find that some clades remain poorly supported, despite our extensive data. Our analyses show that weakly supported clades are associated with relatively short branches for which individual genes often show conflicting relationships. These latter results have important implications for all studies that attempt to resolve phylogenies with large-scale phylogenomic datasets.

Rapid development of multiple nuclear loci for phylogenetic analysis using genomic resources: An example from squamate reptiles

Recently, as genome-scale data have become available for more organisms, the development of phylogenetic markers from nuclear protein-coding loci (NPCL) has become more tractable. However, new methods are needed to efficiently sort the large number of genes from genomic databases into more limited sets appropriate for particular phylogenetic questions, while avoiding introns and paralogs. Here we describe a general methodology for identifying candidate single-copy NPCL from genomic databases. Our method uses information from reference genomes to identify genes with relatively large continuous protein-coding regions (i.e., 700bp). BLAST comparisons are used to help avoid genes with paralogous copies or close relatives (i.e., gene families) that might confound phylogenetic analyses. Exon boundary information is used to identify appropriately spaced potential priming sites. Using this method, we have developed over 25 novel NPCL, which span a variety of desirable evolutionary rates for phylogenetic analyses. Although targeted for higher-level phylogenetics of squamate reptiles, many of these loci appear to be useful across and within other vertebrate clades (e.g., amphibians), and some are relatively rapidly evolving and may be useful for closely-related species (e.g., within genera). This general method can be used whenever large-scale genomic data are available for an appropriate reference species (not necessarily within the focal clade). The method is also well suited for the development of intron regions for lower-level phylogenetic and phylogeographic studies. We provide an online database of alignments and suggested primers for approximately 85 NPCL that should be useful across vertebrates.

Combining Phylogenomics and Fossils in Higher-Level Squamate Reptile Phylogeny: Molecular Data Change the Placement of Fossil Taxa

Molecular data offer great potential to resolve the phylogeny of living taxa but can molecular data improve our understanding of relationships of fossil taxa? Simulations suggest that this is possible, but few empirical examples have demonstrated the ability of molecular data to change the placement of fossil taxa. We offer such an example here. We analyze the placement of snakes among squamate reptiles, combining published morphological data (363 characters) and new DNA sequence data (15,794 characters, 22 nuclear loci) for 45 living and 19 fossil taxa. We find several intriguing results. First, some fossil taxa undergo major changes in their phylogenetic position when molecular data are added. Second, most fossil taxa are placed with strong support in the expected clades by the combined data Bayesian analyses, despite each having >98% missing cells and despite recent suggestions that extensive missing data are problematic for Bayesian phylogenetics. Third, morphological data can change the placement of living taxa in combined analyses, even when there is an overwhelming majority of molecular characters. Finally, we find strong but apparently misleading signal in the morphological data, seemingly associated with a burrowing lifestyle in snakes, amphisbaenians, and dibamids. Overall, our results suggest promise for an integrated and comprehensive Tree of Life by combining molecular and morphological data for living and fossil taxa.

Branch Lengths, Support, and Congruence: Testing the Phylogenomic Approach with 20 Nuclear Loci in Snakes

Many authors have claimed that short branches in the Tree of Life will be very difficult to resolve with strong support, even with the large multilocus data sets now made possible by genomic resources. Short branches may be especially problematic because the underlying gene trees are expected to have discordant phylogenetic histories when the time between branching events is very short. Although there are many examples of short branches that are difficult to resolve, surprisingly, no empirical studies have systematically examined the relationships between branch lengths, branch support, and congruence among genes. Here, we examine these fundamental relationships quantitatively using a data set of 20 nuclear loci for 50 species of snakes (representing most traditionally recognized families). A combined maximum likelihood analysis of the 20 loci gives strong support for 69% of the nodes, but many remain weakly supported, with bootstrap values for 20% ranging from 21% to 66%. For the combined-data tree, we find significant correlations between the length of a branch, levels of bootstrap support, and the proportion of genes that are congruent with that branch in the separate analyses of each gene. We also find that strongly supported conflicts between gene trees over the resolution of individual branches are common (roughly 35% of clades), especially for shorter branches. Overall, our results support the hypothesis that short branches may be very difficult to confidently resolve, even with large, multilocus data sets. Nevertheless, our study provides strong support for many clades, including several that were controversial or poorly resolved in previous studies of snake phylogeny.

Phylogeny of iguanian lizards inferred from 29 nuclear loci, and a comparison of concatenated and species-tree approaches for an ancient, rapid radiation

Iguanian lizards form a diverse clade whose members have been the focus of many comparative studies of ecology, behavior, and evolution. Despite the importance of phylogeny to such studies, interrelationships among many iguanian clades remain uncertain. Within the Old World clade Acrodonta, Agamidae is sometimes found to be paraphyletic with respect to Chamaeleonidae, and recent molecular studies have produced conflicting results for many major clades. Within the largely New World clade Pleurodonta, relationships among the 12 currently recognized major subclades (mostly ranked as families) have been largely unresolved or poorly supported in previous studies. To clarify iguanian evolutionary history, we first infer phylogenies using concatenated maximum-likelihood (ML) and Bayesian analyses of DNA sequence data from 29 nuclear protein-coding genes for 47 iguanian and 29 outgroup taxa. We then estimate a relaxed-clock Bayesian chronogram for iguanians using BEAST. All three methods produce identical topologies. Within Acrodonta, we find strong support for monophyly of Agamidae with respect to Chamaeleonidae, and for almost all relationships within agamids. Within Pleurodonta, we find strong Bayesian support for almost all relationships, and strong ML support for some interfamilial relationships and for monophyly of almost all families (excepting Polychrotidae). Our phylogenetic results suggest a non-traditional biogeographic scenario in which pleurodonts originated in the Northern Hemisphere and subsequently spread southward into South America. The pleurodont portion of the tree is characterized by several very short, deep branches, raising the possibility of deep coalescences that may confound concatenated analyses. We therefore also use 27 of these genes to implement a coalescent-based species-tree approach for pleurodonts. Although this analysis strongly supports monophyly of the pleurodont families, interfamilial relationships are generally different from those in the concatenated tree, and support is uniformly poor. However, a species-tree analysis using only the seven most variable loci yields higher support and more congruence with the concatenated tree. This suggests that low support in the 27-gene species-tree analysis may be an artifact of the many loci that are uninformative for very short branches. This may be a general problem for the application of species-tree methods to rapid radiations, even with phylogenomic data sets. Finally, we correct the non-monophyly of Polychrotidae by recognizing the pleurodont genus Anolis (sensu lato) as a separate family (Dactyloidae), and we correct the non-monophyly of the agamid genus Physignathus by resurrection of the genus Istiurus for the former Physignathus lesueurii.

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.

Evolution of the lizard family Phrynosomatidae as inferred from diverse types of data

The phylogenetic relationships within the iguanian lizard family Phrynosomatidae are inferred from diverse types of data (i.e., mitochondrial rDNA, osteology, coloration, scalation, karyology, and behavior). All 10 currently recognized genera (Callisaurus, Cophosaurus, Holbrookia, Petrosaurus, Phrynosoma, Sator, Sceloporus, Uma, Urosaurus, and Uta) are included in the phylogenetic analyses. The phylogenies inferred from the separate analyses of the DNA sequence data (779 bp; 162 informative characters; 40 species) and non-DNA data (155 informative characters; 59 species) share 26% (10) of their respective clades. Four of the congruent clades (i.e., sand lizards + Phrynosoma, Petrosaurus, Urosaurus, Uta) are strongly supported (-70% bootstrap) in both of the separate analyses while five others are strongly supported in only one, but not both, of the separate analyses. All conflicting hypotheses leading to the taxonomic incongruence (e.g., Sceloporus group interrelationships) are weakly supported (<70% bootstrap) in one or both of the separate analyses. Combining the DNA and non-DNA data for phylogenetic analysis results in a single shortest tree. Overall, the phylogeny from the combined analysis shares more clades in common with the hypotheses inferred from the separate DNA analysis (74%) than with the separate analysis of the non-DNA data (53%). The intergeneric relationships inferred from the combined analysis are more similar to recently published hypotheses based on morphological data, except the Sceloporus group is paraphyletic. Although phrynosomatid intergeneric relationships are well resolved by the combined analysis of the DNA and non-DNA data, the relationships among most genera are nevertheless weakly supported by the separate and combined analyses. This weak support is most likely the result of rapid speciation. The monophyly of the speciose genus Sceloporus (exclusive of Sator) is supported by the separate non-DNA and combined analyses. The inclusion of numerous incomplete taxa (19 species lacking DNA data) in the combined analysis did not decrease resolution among the complete taxa (40 species with DNA and non-DNA data), but the addition of the incomplete taxa did affect the relationships among the complete taxa. Overall, the DNA data are more homoplastic than the non-DNA data, but the degree of character incongruence exhibited within the different partitions and/or sources of the DNA and non-DNA data sets varies greatly.