Transcript- and annotation-guided genome assembly of the European starling.

The European starling, Sturnus vulgaris, is an ecologically significant, globally invasive avian species that is also suffering from a major decline in its native range. Here, we present the genome assembly and long-read transcriptome of an Australian-sourced European starling (S. vulgaris vAU), and a second, North American, short-read genome assembly (S. vulgaris vNA), as complementary reference genomes for population genetic and evolutionary characterization. S. vulgaris vAU combined 10× genomics linked-reads, low-coverage Nanopore sequencing, and PacBio Iso-Seq full-length transcript scaffolding to generate a 1050 Mb assembly on 6222 scaffolds (7.6 Mb scaffold N50, 94.6% busco completeness). Further scaffolding against the high-quality zebra finch (Taeniopygia guttata) genome assigned 98.6% of the assembly to 32 putative nuclear chromosome scaffolds. Species-specific transcript mapping and gene annotation revealed good gene-level assembly and high functional completeness. Using S. vulgaris vAU, we demonstrate how the multifunctional use of PacBio Iso-Seq transcript data and complementary homology-based annotation of sequential assembly steps (assessed using a new tool, saaga) can be used to assess, inform, and validate assembly workflow decisions. We also highlight some counterintuitive behaviour in traditional busco metrics, and present buscomp, a complementary tool for assembly comparison designed to be robust to differences in assembly size and base-calling quality. This work expands our knowledge of avian genomes and the available toolkit for assessing and improving genome quality. The new genomic resources presented will facilitate further global genomic and transcriptomic analysis on this ecologically important species.

Historical museum samples enable the examination of divergent and parallel evolution during invasion.

During the Anthropocene, Earth has experienced unprecedented habitat loss, native species decline and global climate change. Concurrently, greater globalization is facilitating species movement, increasing the likelihood of alien species establishment and propagation. There is a great need to understand what influences a species' ability to persist or perish within a new or changing environment. Examining genes that may be associated with a species' invasion success or persistence informs invasive species management, assists with native species preservation and sheds light on important evolutionary mechanisms that occur in novel environments. This approach can be aided by coupling spatial and temporal investigations of evolutionary processes. Here we use the common starling, Sturnus vulgaris, to identify parallel and divergent evolutionary change between contemporary native and invasive range samples and their common ancestral population. To do this, we use reduced-representation sequencing of native samples collected recently in northwestern Europe and invasive samples from Australia, together with museum specimens sampled in the UK during the mid-19th century. We found evidence of parallel selection on both continents, possibly resulting from common global selective forces such as exposure to pollutants. We also identified divergent selection in these populations, which might be related to adaptive changes in response to the novel environment encountered in the introduced Australian range. Interestingly, signatures of selection are equally as common within both invasive and native range contemporary samples. Our results demonstrate the value of including historical samples in genetic studies of invasion and highlight the ongoing and occasionally parallel role of adaptation in both native and invasive ranges.

Reevaluating claims of ecological speciation in Halichoeres bivittatus.

Allopatry has traditionally been viewed as the primary driver of speciation in marine taxa, but the geography of the marine environment and the larval dispersal capabilities of many marine organisms render this view somewhat questionable. In marine fishes, one of the earliest and most highly cited empirical examples of ecological speciation with gene flow is the slippery dick wrasse, Halichoeres bivittatus. Evidence for this cryptic or incipient speciation event was primarily in the form of a deep divergence in a single mitochondrial locus between the northern and southern Gulf of Mexico, combined with a finding that these two haplotypes were associated with different habitat types ("tropical" vs. "subtropical") in the Florida Keys and Bermuda, where they overlap. Here, we examine habitat assortment in the Florida Keys using a broader sampling of populations and habitat types than were available for the original study. We find no evidence to support the claim that haplotype frequencies differ between habitat types, and little evidence to support any differences between populations in the Keys. These results undermine claims of ecological speciation with gene flow in Halichoeres bivittatus. Future claims of this type should be supported by multiple lines of evidence that illuminate potential mechanisms and allow researchers to rule out alternative explanations for spatial patterns of genetic differences.

Comparing the dietary niche overlap and ecomorphological differences between invasive Hemidactylus mabouia geckos and a native gecko competitor.

Hemidactylus mabouia is one of the most successful, widespread invasive reptile species and has become ubiquitous across tropical urban settings in the Western Hemisphere. Its ability to thrive in close proximity to humans has been linked to the rapid disappearance of native geckos. However, aspects of Hemidactylus mabouia natural history and ecomorphology, often assumed to be linked with this effect on native populations, remain understudied or untested. Here, we combine data from ∂15N and ∂13C stable isotopes, stomach contents, and morphometric analyses of traits associated with feeding and locomotion to test alternate hypotheses of displacement between H. mabouia and a native gecko, Phyllodactylus martini, on the island of Curaçao. We demonstrate substantial overlap of invertebrate prey resources between the species, with H. mabouia stomachs containing larger arthropod prey as well as vertebrate prey. We additionally show that H. mabouia possesses several morphological advantages, including larger sizes in feeding-associated traits and limb proportions that could offer a propulsive locomotor advantage on vertical surfaces. Together, these findings provide the first support for the hypotheses that invasive H. mabouia and native P. martini overlap in prey resources and that H. mabouia possess ecomorphological advantages over P. martini. This work provides critical context for follow-up studies of H. mabouia and P. martini natural history and direct behavioral experiments that may ultimately illuminate the mechanisms underlying displacement on this island and act as a potential model for other systems with Hemidactylus mabouia invasions.

Emergence of an evolutionary innovation: Gene expression differences associated with the transition between oviparity and viviparity.

Our understanding of the evolution of complex biological traits is greatly advanced by examining taxa with intermediate phenotypes. The transition from oviparity (egg-laying) to viviparity (live-bearing) has occurred independently in many animal lineages, but there are few phenotypic intermediates. The lizard Saiphos equalis exhibits bimodal reproduction, with some viviparous populations, and other oviparous populations with long egg-retention, a rare trait where most of embryonic development occurs inside the mother prior to late ovipositioning. We posit that oviparous S. equalis represent an intermediate form between "true" oviparity and viviparity. We used transcriptomics to compare uterine gene expression in these two phenotypes, and provide a molecular model for the genetic control and evolution of reproductive mode. Many genes are differentially expressed throughout the reproductive cycle of both phenotypes, which have clearly different gene expression profiles overall. The differentially expressed genes within oviparous and viviparous individuals have broadly similar biological functions putatively important for sustaining embryos, including uterine remodelling, respiratory gas and water exchange, and immune regulation. These functional similarities indicate either that long egg-retention is an exaptation for viviparity, or might reflect parallel evolution of similar gravidity-related changes in gene expression in long egg-retention oviparity. In contrast, gene expression changes across the reproductive cycle of long egg-retaining oviparous S. equalis are dramatically different from those of "true" oviparous skinks (such as Lampropholis guichenoti), supporting our assertion that oviparous S. equalis exhibit an intermediate phenotype between "true" oviparity and viviparity.

The Regulation of Uterine Proinflammatory Gene Expression during Pregnancy in the Live-Bearing Lizard, Pseudemoia entrecasteauxii.

The evolutionary transition from egg-laying to live-bearing in amniote vertebrates (reptiles and mammals) requires the development of a closer association between the maternal and embryonic tissue to facilitate gas and nutrient exchange with the embryo. Because the embryo is an allograft to the father and mother, it could be considered foreign by the maternal immune system and thus be immunologically rejected during pregnancy. In eutherian ("placental") mammals, the proinflammatory genes interleukin 1B (IL1B), tumor necrosis factor (TNF) and tumor necrosis factor receptor superfamily 1A (TNFRSF1A) are tightly regulated in the pregnant uterus to prevent embryonic rejection. We tested whether inflammation is similarly regulated in pregnant viviparous reptiles by comparing the expression of IL1B, TNF, and TNFRSF1A in the pregnant and nonpregnant uterus of the viviparous lizard, Pseudemoia entrecasteauxii. We found statistically significant support for the downregulation of pregnant uterine TNF mRNA expression in P. entrecasteauxii, but no statistically significant changes in mRNA expression of TNFRSF1A or IL1B between pregnant and nonpregnant uteri. Although these genes are apparently not regulated at the transcriptional level, our immunofluorescence microscopy analyses nonetheless demonstrate that the IL1B proteins are stored intracellularly during pregnancy, possibly resulting in inhibition of inflammatory response. We therefore conclude that processes of both transcriptional (TNF) and posttranslational (IL1B) gene regulation may reduce inflammation in the pregnant uterus of this viviparous reptile. Our study is important because it demonstrates that regulating the maternal immune system to prevent embryonic rejection may be important in reptilian pregnancy as it is in mammalian pregnancy.

Live birth in an archosauromorph reptile.

Live birth has evolved many times independently in vertebrates, such as mammals and diverse groups of lizards and snakes. However, live birth is unknown in the major clade Archosauromorpha, a group that first evolved some 260 million years ago and is represented today by birds and crocodilians. Here we report the discovery of a pregnant long-necked marine reptile (Dinocephalosaurus) from the Middle Triassic (∼245 million years ago) of southwest China showing live birth in archosauromorphs. Our discovery pushes back evidence of reproductive biology in the clade by roughly 50 million years, and shows that there is no fundamental reason that archosauromorphs could not achieve live birth. Our phylogenetic models indicate that Dinocephalosaurus determined the sex of their offspring by sex chromosomes rather than by environmental temperature like crocodilians. Our results provide crucial evidence for genotypic sex determination facilitating land-water transitions in amniotes.

Comparative genomics of hormonal signaling in the chorioallantoic membrane of oviparous and viviparous amniotes.

In oviparous amniotes (reptiles, birds, and mammals) the chorioallantoic membrane (CAM) lines the inside of the egg and acts as the living point of contact between the embryo and the outside world. In livebearing (viviparous) amniotes, communication during embryonic development occurs across placental tissues, which form between the uterine tissue of the mother and the CAM of the embryo. In both oviparous and viviparous taxa, the CAM is at the interface of the embryo and the external environment and can transfer signals from there to the embryo proper. To understand the evolution of placental hormone production in amniotes, we examined the expression of genes involved in hormone synthesis, metabolism, and hormone receptivity in the CAM of species across the amniote phylogeny. We collected transcriptome data for the chorioallantoic membranes of the chicken (oviparous), the lizards Lerista bougainvillii (both oviparous and viviparous populations) and Pseudemoia entrecasteauxii (viviparous), and the horse Equus caballus (viviparous). The viviparous taxa differ in their mechanisms of nutrient provisioning: L. bougainvillii is lecithotrophic (embryonic nourishment is provided via the yolk only), but P. entrecasteauxii and the horse are placentotrophic (embryos are nourished via placental transport). Of the 423 hormone-related genes that we examined, 91 genes are expressed in all studied species, suggesting that the chorioallantoic membrane ancestrally had an endocrine function. Therefore, the chorioallantoic membrane appears to be a highly hormonally active organ in all amniotes. No genes are expressed only in viviparous species, suggesting that the evolution of viviparity has not required the recruitment of any specific hormone-related genes. Our data suggest that the endocrine function of the CAM as a placental tissue evolved in part through co-option of ancestral gene expression patterns.

Reptile Pregnancy Is Underpinned by Complex Changes in Uterine Gene Expression: A Comparative Analysis of the Uterine Transcriptome in Viviparous and Oviparous Lizards.

The evolution of new organs is difficult to study because most vertebrate organs evolved only once, more than 500 million years ago. An ideal model for understanding complex organ evolution is the placenta, a structure that is present in live bearing reptiles and mammals (amniotes), which has evolved independently more than 115 times. Using transcriptomics, we characterized the uterine gene expression patterns through the reproductive cycle of a viviparous skink lizard, Pseudemoia entrecasteauxii Then we compare these patterns with the patterns of gene expression from two oviparous skinks Lampropholis guichenoti and Lerista bougainvillii While thousands of genes are differentially expressed between pregnant and non-pregnant uterine tissue in the viviparous skink, few differentially expressed genes were identified between gravid and non-gravid oviparous skinks. This finding suggests that in P. entrecasteauxii, a pregnant-specific gene expression profile has evolved, allowing for the evolution of pregnancy-specific innovations in the uterus. We find substantial gene expression differences between the uterus of the chorioallantoic and the yolk sac placenta in P. entrecasteauxii, suggesting these placental regions are specialized for different placental functions. In particular, the chorioallantoic placenta is likely a major site of nutrient transport by membrane-bound transport proteins, while the yolk sac placenta also likely transports nutrients but via apocrine secretions. We discuss how the evolution of transcription factor networks is likely to underpin the evolution of the new transcriptional states in the uterine tissue of viviparous reptiles.

Basking behavior predicts the evolution of heat tolerance in Australian rainforest lizards.

There is pressing urgency to understand how tropical ectotherms can behaviorally and physiologically respond to climate warming. We examine how basking behavior and thermal environment interact to influence evolutionary variation in thermal physiology of multiple species of lygosomine rainforest skinks from the Wet Tropics of northeastern Queensland, Australia (AWT). These tropical lizards are behaviorally specialized to exploit canopy or sun, and are distributed across marked thermal clines in the AWT. Using phylogenetic analyses, we demonstrate that physiological parameters are either associated with changes in local thermal habitat or to basking behavior, but not both. Cold tolerance, the optimal sprint speed, and performance breadth are primarily influenced by local thermal environment. Specifically, montane lizards are more cool tolerant, have broader performance breadths, and higher optimum sprinting temperatures than their lowland counterparts. Heat tolerance, in contrast, is strongly affected by basking behavior: there are two evolutionary optima, with basking species having considerably higher heat tolerance than shade skinks, with no effect of elevation. These distinct responses among traits indicate the multiple selective pressures and constraints that shape the evolution of thermal performance. We discuss how behavior and physiology interact to shape organisms' vulnerability and potential resilience to climate change.

Allelic expression of mammalian imprinted genes in a matrotrophic lizard, Pseudemoia entrecasteauxii.

Genomic imprinting is a process that results in the differential expression of genes depending on their parent of origin. It occurs in both plants and live-bearing mammals, with imprinted genes typically regulating the ability of an embryo to manipulate the maternal provision of nutrients. Genomic imprinting increases the potential for selection to act separately on paternally and maternally expressed genes, which increases the number of opportunities that selection can facilitate embryonic control over maternal nutrient provision. By looking for imprinting in an independent matrotrophic lineage, the viviparous lizard Pseudemoia entrecasteauxii (Scincidae), we test the hypothesis that genomic imprinting facilitates the evolution of substantial placental nutrient transport to embryos (matrotrophy). We sequenced transcriptomes from the embryonic component of lizard placentae to determine whether there are parent-of-origin differences in expression of genes that are imprinted in mammals. Of these genes, 19 had sufficiently high expression in the lizard to identify polymorphisms in transcribed sequences. We identified bi-allelic expression in 17 genes (including insulin-like growth factor 2), indicating that neither allele was imprinted. These data suggest that either genomic imprinting has not evolved in this matrotrophic skink or, if it has, it has evolved in different genes to mammals. We outline how these hypotheses can be tested. This study highlights important differences between mammalian and reptile pregnancy and the absence of any shared imprinting genes reflects fundamental differences in the way that pregnancy has evolved in these two lineages.

Which came first: The lizard or the egg? Robustness in phylogenetic reconstruction of ancestral states.

Changes in parity mode between egg-laying (oviparity) and live-bearing (viviparity) have occurred repeatedly throughout vertebrate evolution. Oviparity is the ancestral amniote state, and viviparity has evolved many times independently within amniotes (especially in lizards and snakes), with possibly a few reversions to oviparity. In amniotes, the shelled egg is considered a complex structure that is unlikely to re-evolve if lost (i.e., it is an example of Dollo's Principle). However, a recent ancestral state reconstruction analysis concluded that viviparity was the ancestral state of squamate reptiles (lizards and snakes), and that oviparity re-evolved from viviparity many times throughout the evolutionary history of squamates. Here, we re-evaluate support for this provocative conclusion by testing the sensitivity of the analysis to model assumptions and estimates of squamate phylogeny. We found that the models and methods used for parity mode reconstruction are highly sensitive to the specific estimate of phylogeny used, and that the point estimate of phylogeny used to suggest that viviparity is the root state of the squamate tree is far from an optimal phylogenetic solution. The ancestral state reconstructions are also highly sensitive to model choice and specific values of model parameters. A method that is designed to account for biases in taxon sampling actually accentuates, rather than lessens, those biases with respect to ancestral state reconstructions. In contrast to recent conclusions from the same data set, we find that ancestral state reconstruction analyses provide highly equivocal support for the number and direction of transitions between oviparity and viviparity in squamates. Moreover, the reconstructions of ancestral parity state are highly dependent on the assumptions of each model. We conclude that the common ancestor of squamates was oviparous, and subsequent evolutionary transitions to viviparity were common, but reversals to oviparity were rare. The three putative reversals to oviparity with the strongest phylogenetic support occurred in the snakes Eryx jayakari and Lachesis, and the lizard, Liolaemus calchaqui. Our results emphasize that because the conclusions of ancestral state reconstruction studies are often highly sensitive to the methods and assumptions of analysis, researchers should carefully consider this sensitivity when evaluating alternative hypotheses of character-state evolution.

Evaluating the performance of anchored hybrid enrichment at the tips of the tree of life: a phylogenetic analysis of Australian Eugongylus group scincid lizards.

BACKGROUND: High-throughput sequencing using targeted enrichment and transcriptomic methods enables rapid construction of phylogenomic data sets incorporating hundreds to thousands of loci. These advances have enabled access to an unprecedented amount of nucleotide sequence data, but they also pose new questions. Given that the loci targeted for enrichment are often highly conserved, how informative are they at different taxonomic scales, especially at the intraspecific/phylogeographic scale? We investigate this question using Australian scincid lizards in the Eugongylus group (Squamata: Scincidae). We sequenced 415 anchored hybrid enriched (AHE) loci for 43 individuals and mined 1650 exons (1648 loci) from transcriptomes (transcriptome mining) from 11 individuals, including multiple phylogeographic lineages within several species of Carlia, Lampropholis, and Saproscincus skinks. We assessed the phylogenetic information content of these loci at the intergeneric, interspecific, and phylogeographic scales. As a further test of the utility at the phylogeographic scale, we used the anchor hybrid enriched loci to infer lineage divergence parameters using coalescent models of isolation with migration. RESULTS: Phylogenetic analyses of both data sets inferred very strongly supported trees at all taxonomic levels. Further, AHE loci yielded estimates of divergence times between closely related lineages that were broadly consistent with previous population-level analyses. CONCLUSIONS: Anchored-enriched loci are useful at the deep phylogeny and phylogeographic scales. Although overall phylogenetic support was high throughout the Australian Eugongylus group phylogeny, there were nonetheless some conflicting or unresolved relationships, especially regarding the placement of Pseudemoia, Cryptoblepharus, and the relationships amongst closely-related species of Tasmanian Niveoscincus skinks.

Ancestral state reconstructions require biological evidence to test evolutionary hypotheses: A case study examining the evolution of reproductive mode in squamate reptiles.

To understand evolutionary transformations it is necessary to identify the character states of extinct ancestors. Ancestral character state reconstruction is inherently difficult because it requires an accurate phylogeny, character state data, and a statistical model of transition rates and is fundamentally constrained by missing data such as extinct taxa. We argue that model based ancestral character state reconstruction should be used to generate hypotheses but should not be considered an analytical endpoint. Using the evolution of viviparity and reversals to oviparity in squamates as a case study, we show how anatomical, physiological, and ecological data can be used to evaluate hypotheses about evolutionary transitions. The evolution of squamate viviparity requires changes to the timing of reproductive events and the successive loss of features responsible for building an eggshell. A reversal to oviparity requires that those lost traits re-evolve. We argue that the re-evolution of oviparity is inherently more difficult than the reverse. We outline how the inviability of intermediate phenotypes might present physiological barriers to reversals from viviparity to oviparity. Finally, we show that ecological data supports an oviparous ancestral state for squamates and multiple transitions to viviparity. In summary, we conclude that the first squamates were oviparous, that frequent transitions to viviparity have occurred, and that reversals to oviparity in viviparous lineages either have not occurred or are exceedingly rare. As this evidence supports conclusions that differ from previous ancestral state reconstructions, our paper highlights the importance of incorporating biological evidence to evaluate model-generated hypotheses.

Snake and bird predation drive the repeated convergent evolution of correlated life history traits and phenotype in the Izu Island Scincid lizard (Plestiodon latiscutatus).

Predation may create strong natural selection pressure on the phenotype and life history characteristics of prey species. The Izu scincid lizards (Plestiodon latiscutatus) that inhabit the four Japanese Izu Islands with only bird predators are drab brown, mature later, lay small clutches of large eggs, and hatch large neonates. In contrast, skinks on seven islands with both snake and bird predators are conspicuously colored, mature early, lay large clutches of small eggs, and hatch small neonates. We test the hypothesis that these suites of traits have evolved independently on each island via natural selection pressures from one of two predator regimes--birds-only and birds + snakes. Using two mtDNA genes and a nuclear locus, we infer a time-calibrated phylogeny of P. latiscutatus that reveals a basal split between Mikura and all islands south, and Miyake, all islands north, and the Izu Peninsula. Populations inhabiting Miyake, Niijima, Shikine, and Toshima are not monophyletic, suggesting either multiple colonizations or an artifact of incomplete lineage sorting (ILS). We therefore developed novel phylogenetic comparative analyses that assume either a multiple colonization or more restrictive single colonization ILS scenario and found 1) statistically significant support for the of different suites of phenotypic and life history characteristics with the presence of bird-only or bird + snake predator assemblages, and 2) strong phylogenetic support for at least two independent derivations of either the "bird-only" or "snakes + birds" phenotypes regardless of colonization scenario. Finally, our time-calibrated phylogeographic analysis supports the conclusion that the ancestor to modern Izu Island P. latiscutatus dispersed from the mainland to the Izu proto-islands between 3-7.6 million years ago (Ma). These lineages remained present in the area during successive formation of the islands, with one lineage re-colonizing the mainland 0.24-0.7 Ma.

Boom and bust: ancient and recent diversification in bichirs (Polypteridae: Actinopterygii), a relictual lineage of ray-finned fishes.

Understanding the history that underlies patterns of species richness across the Tree of Life requires an investigation of the mechanisms that not only generate young species-rich clades, but also those that maintain species-poor lineages over long stretches of evolutionary time. However, diversification dynamics that underlie ancient species-poor lineages are often hidden due to a lack of fossil evidence. Using information from the fossil record and time calibrated molecular phylogenies, we investigate the history of lineage diversification in Polypteridae, which is the sister lineage of all other ray-finned fishes (Actinopterygii). Despite originating at least 390 million years (Myr) ago, molecular timetrees support a Neogene origin for the living polypterid species. Our analyses demonstrate polypterids are exceptionally species depauperate with a stem lineage duration that exceeds 380 million years (Ma) and is significantly longer than the stem lineage durations observed in other ray-finned fish lineages. Analyses of the fossil record show an early Late Cretaceous (100.5-83.6 Ma) peak in polypterid genus richness, followed by 60 Ma of low richness. The Neogene species radiation and evidence for high-diversity intervals in the geological past suggest a "boom and bust" pattern of diversification that contrasts with common perceptions of relative evolutionary stasis in so-called "living fossils."

The evolution of viviparity: molecular and genomic data from squamate reptiles advance understanding of live birth in amniotes.

Squamate reptiles (lizards and snakes) are an ideal model system for testing hypotheses regarding the evolution of viviparity (live birth) in amniote vertebrates. Viviparity has evolved over 100 times in squamates, resulting in major changes in reproductive physiology. At a minimum, all viviparous squamates exhibit placentae formed by the appositions of maternal and embryonic tissues, which are homologous in origin with the tissues that form the placenta in therian mammals. These placentae facilitate adhesion of the conceptus to the uterus as well as exchange of oxygen, carbon dioxide, water, sodium, and calcium. However, most viviparous squamates continue to rely on yolk for nearly all of their organic nutrition. In contrast, some species, which rely on the placenta for at least a portion of organic nutrition, exhibit complex placental specializations associated with the transport of amino acids and fatty acids. Some viviparous squamates also exhibit reduced immunocompetence during pregnancy, which could be the result of immunosuppression to protect developing embryos. Recent molecular studies using both candidate-gene and next-generation sequencing approaches have suggested that at least some of the genes and gene families underlying these phenomena play similar roles in the uterus and placenta of viviparous mammals and squamates. Therefore, studies of the evolution of viviparity in squamates should inform hypotheses of the evolution of viviparity in all amniotes, including mammals.

Developmental sequences of squamate reptiles are taxon specific.

Recent studies in comparative vertebrate embryology have focused on two related questions. One concerns the existence of a phylotypic period, or indeed any period, during development in which sequence variation among taxa is constrained. The second question concerns the degree to which developmental characters exhibit a phylogenetic signal. These questions are important because they underpin attempts to understand the evolution of developmental characters and their links to adult morphology. To address these questions, we compared the sequence of developmental events spanning the so-called phylotypic period of vertebrate development in squamate reptiles (lizards and snakes), from the formation of the primary optic placode to the first appearance of scale anlagen. We used Bayesian phylogenetic ancestral state reconstruction analyses and estimates of Bayesian posterior probabilities of the rank order of developmental events to determine the level of support for phylogenetically associated variation in development. We assessed the amount of variation in event sequences by plotting the proportions of reconstructed ranks (excluding unlikely events, PP < 0.05) associated with each event. Sequence variability was the lowest towards the middle of the phylotypic period and involved three events (allantois contacts chorion, maximum number of pharyngeal slits, and appearance of the apical epidermal ridge [AER]); these events each had only two reconstructed ranks. Squamate clades also differed in the rank order of developmental events. Of the 20 events in our analyses, 12 had strongly supported (PP ≥ 0.95) sequence ranks that differed at two or more internal nodes of the tree. For example, gekkotans are distinguished by the late appearance of the allantois bud compared to all other squamates (ranks 7 and 8 vs. rank 3, respectively) and Serpentes are distinguished by the earlier completion of torsion (rank 3) compared to acrodonts and pleurodonts (ranks 7 and 5, respectively). Clade specific sequences of developmental events mean that investigators should not extend observations on the development on particular squamate species to distantly related taxa for use in comparative studies.

Evidence for determinism in species diversification and contingency in phenotypic evolution during adaptive radiation.

Adaptive radiation (AR) theory predicts that groups sharing the same source of ecological opportunity (EO) will experience deterministic species diversification and morphological evolution. Thus, deterministic ecological and morphological evolution should be correlated with deterministic patterns in the tempo and mode of speciation for groups in similar habitats and time periods. We test this hypothesis using well-sampled phylogenies of four squamate groups that colonized the New World (NW) in the Late Oligocene. We use both standard and coalescent models to assess species diversification, as well as likelihood models to examine morphological evolution. All squamate groups show similar early pulses of speciation, as well as diversity-dependent ecological limits on clade size at a continental scale. In contrast, processes of morphological evolution are not easily predictable and do not show similar pulses of early and rapid change. Patterns of morphological and species diversification thus appear uncoupled across these groups. This indicates that the processes that drive diversification and disparification are not mechanistically linked, even among similar groups of taxa experiencing the same sources of EO. It also suggests that processes of phenotypic diversification cannot be predicted solely from the existence of an AR or knowledge of the process of diversification.

Molecular phylogenetics of squirrelfishes and soldierfishes (Teleostei: Beryciformes: Holocentridae): reconciling more than 100 years of taxonomic confusion.

Squirrelfishes and soldierfishes (Holocentridae) are among the most conspicuous species in the nocturnal reef fish community. However, there is no clear consensus regarding their evolutionary relationships, which is reflected in a complicated taxonomic history. We collected DNA sequence data from multiple single copy nuclear genes and one mitochondrial gene sampled from over fifty percent of the recognized holocentrid species and infer the first species-level phylogeny of the Holocentridae. Our results strongly support the monophyly of the clades Myripristinae (soldierfishes) and Holocentrinae (squirrelfishes). The molecular phylogenies differ with regard to previous hypotheses of relationships within the Myriprisitinae, resolving a clade of cryptic reef associated and deep water non-reef dwelling lineages (Corniger+Plectrypops+Ostichthys) that is the sister lineage to a monophyletic Myripristis. Within Holocentrinae, Neoniphon and Sargocentron are strongly supported as paraphyletic, while Holocentrus is nested within Sargocentron. Using Bayesian ancestral state reconstruction methods, we demonstrate the taxonomically diagnostic characters for Neoniphon and Sargocentron likely represent character states with a complex evolutionary history that is not reflective of shared common ancestry. We propose a new classification for Holocentrinae, recognizing four lineages that are treated as genera: Sargocentron Fowler, 1904, Holocentrus Scopoli, 1777, Flameo Jordan and Evermann, 1898, and Neoniphon Castelnau, 1875.