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PAX6 is well known as a transcription factor that drives eye development in animals as widely divergent as flies and mammals. In addition to its localization in eyes, PAX6 expression has been reported in the central nervous system, the pancreas, testes, Merkel cells, nasal epithelium, developing cells of the inner ear, and embryonic submandibular salivary gland. Here we show that PAX6 also appears to be present in the mechanosensory neuromasts of the lateral line system in paedomorphic salamanders of the genus Eurycea. Using immunohistochemistry and confocal microscopy to examine a limited number of larvae of two species, listed by the United States of America's federal government as threatened (E. nana) or endangered (E. rathbuni), we found that anti-PAX6 antibody labeled structures that were extranuclear, and labeling was most intense in the apical appendages of the hair cells of the neuromast. This extranuclear localization raises the possibility of an as yet undescribed function for PAX6 as a cytoskeleton-associated protein., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dobbins et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

With limited sampling, geographic variation within a single species can be difficult to distinguish from interspecific variation, confounding our ability to draw accurate species boundaries. We argue that thorough sampling and analysis of contact zones between putative taxa can determine if assortative mating or selection against hybrids exists (supporting the presence of two distinct species), or alternatively if mating is random among genotypes and admixture among adjacent populations is gradual and continuous (supporting geographic variation within a single species). Here, we test two alternative hypotheses for two pairs of named taxa at contact zones within the American milksnake (Lampropeltis triangulum) complex. A prior morphological analysis found areas of gradual intergradation among named taxa, and concluded that the taxa represented geographical races of a single polytypic species. In contrast, a subsequent analysis of gene sequence data, but with limited sampling near the contact zones, hypothesized distinct boundaries between species at the contact zones. At the contact zone between proposed species L. triangulum and Lampropeltis gentilis, we examined a $\sim$700 km-wide transect across the states of Kansas and Missouri, with thorough sampling and reduced-representation genomic-level sequencing, to test the two opposing taxonomic hypotheses. Our transect analyses included examinations of population structure, fixed differences, cline-fitting, and an admixture index analysis. These analyses all supported a gradual and continuous geographic cline across a broad intergrade zone between two geographic forms of L. triangulum, thus providing strong support for a single species in this region (and no support for the recognition of L. gentilis as a distinct species). At a second contact zone between proposed species L. triangulum and Lampropeltis elapsoides (but variously treated as species or subspecies by different researchers) in Kentucky and Tennessee, we re-evaluated morphological data. In this case, the contact zone analysis indicated sympatry and reproductive isolation of the two taxa, and thus strongly supported L. triangulum and L. elapsoides as distinct species. We conclude that detailed studies of contact zones, based on either genetic or morphological data, are essential for distinguishing intraspecific from interspecific variation in the case of widely and continuously distributed taxa. [Contact zones; speciation; species concepts; species delimitation; taxonomy.]., (© The Author(s) 2022. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In most animals, mitochondrial DNA is strictly maternally inherited and non-recombining. One exception to this pattern is called doubly uniparental inheritance (DUI), a phenomenon involving the independent transmission of female and male mitochondrial genomes. DUI is known only from the molluskan class Bivalvia. The phylogenetic distribution of male-transmitted mitochondrial DNA (M mtDNA) in bivalves is consistent with several evolutionary scenarios, including multiple independent gains, losses, and varying degrees of recombination with female-transmitted mitochondrial DNA (F mtDNA). In this study, we use phylogenetic methods to test M mtDNA origination hypotheses and infer the prevalence of mitochondrial recombination in bivalves with DUI. Phylogenetic modeling using site concordance factors supported a single origin of M mtDNA in bivalves coupled with recombination acting over long evolutionary timescales. Ongoing mitochondrial recombination is present in Mytilida and Venerida, which results in a pattern of concerted evolution of F mtDNA and M mtDNA. Mitochondrial recombination could be favored to offset the deleterious effects of asexual inheritance and maintain mitonuclear compatibility across tissues. Cardiida and Unionida have gone without recent recombination, possibly due to an extension of the COX2 gene in male mitochondrial DNA. The loss of recombination could be connected to the role of M mtDNA in sex determination or sexual development. Our results support that recombination events may occur throughout the mitochondrial genomes of DUI species. Future investigations may reveal more complex patterns of inheritance of recombinants, which could explain the retention of signal for a single origination of M mtDNA in protein-coding genes., (© The Author(s) 2023. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Restriction-site-associated DNA sequencing (RADseq) has become an accessible way to obtain genome-wide data in the form of single-nucleotide polymorphisms (SNPs) for phylogenetic inference. Nonetheless, how differences in RADseq methods influence phylogenetic estimation is poorly understood because most comparisons have largely relied on conceptual predictions rather than empirical tests. We examine how differences in ddRAD and 2bRAD data influence phylogenetic estimation in two non-model frog groups. We compare the impact of method choice on phylogenetic information, missing data, and allelic dropout, considering different sequencing depths. Given that researchers must balance input (funding, time) with output (amount and quality of data), we also provide comparisons of laboratory effort, computational time, monetary costs, and the repeatability of library preparation and sequencing. Both 2bRAD and ddRAD methods estimated well-supported trees, even at low sequencing depths, and had comparable amounts of missing data, patterns of allelic dropout, and phylogenetic signal. Compared to ddRAD, 2bRAD produced more repeatable datasets, had simpler laboratory protocols, and had an overall faster bioinformatics assembly. However, many fewer parsimony-informative sites per SNP were obtained from 2bRAD data when using native pipelines, highlighting a need for further investigation into the effects of each pipeline on resulting datasets. Our study underscores the importance of comparing RADseq methods, such as expected results and theoretical performance using empirical datasets, before undertaking costly experiments., Competing Interests: The authors declare that they have no conflicts of interest. This publication is based, in part, on work by DCC while serving at the National Science Foundation. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation or the U.S. government., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Defense against ultraviolet (UV) radiation exposure is essential for survival, especially in high-elevation species. Although some specific genes involved in UV response have been reported, the full view of UV defense mechanisms remains largely unexplored. Herein, we used integrated approaches to analyze UV responses in the highest-elevation frog, Nanorana parkeri . We show less damage and more efficient antioxidant activity in skin of this frog than those of its lower-elevation relatives after UV exposure. We also reveal genes related to UV defense and a corresponding temporal expression pattern in N. parkeri . Genomic and metabolomic analysis along with large-scale transcriptomic profiling revealed a time-dependent coordinated defense mechanism in N. parkeri . We also identified several microRNAs that play important regulatory roles, especially in decreasing the expression levels of cell cycle genes. Moreover, multiple defense genes (i.e., TYR for melanogenesis) exhibit positive selection with function-enhancing substitutions. Thus, both expression shifts and gene mutations contribute to UV adaptation in N. parkeri . Our work demonstrates a genetic framework for evolution of UV defense in a natural environment.

SignificanceTo adapt to arboreal lifestyles, treefrogs have evolved a suite of complex traits that support vertical movement and gliding, thus presenting a unique case for studying the genetic basis for traits causally linked to vertical niche expansion. Here, based on two de novo-assembled Asian treefrog genomes, we determined that genes involved in limb development and keratin cytoskeleton likely played a role in the evolution of their climbing systems. Behavioral and morphological evaluation and time-ordered gene coexpression network analysis revealed the developmental patterns and regulatory pathways of the webbed feet used for gliding in Rhacophorus kio .

As DNA sequencing technologies and methods for delimiting species with genomic data become more accessible and numerous, researchers have more tools than ever to investigate questions in systematics and phylogeography. However, easy access to sophisticated computational tools is not without its drawbacks. Choosing the right approach for one's question can be challenging when presented with multitudinous options, some of which fail to distinguish between species and intraspecific population structure. Here, we employ a methodology that emphasizes intensive geographic sampling, particularly at contact zones between populations, with a focus on differentiating intraspecific genetic clusters from species in the Pantherophis guttatus complex, a group of North American ratsnakes. Using a mitochondrial marker as well as ddRADseq data, we find evidence of mitonuclear discordance which has contributed to historical confusion about the relationships within this group. Additionally, we identify geographically and genetically structured populations within the species Pantherophis emoryi that are congruent with previously described morphological variation. Importantly, we find that these structured populations within P. emoryi are highly admixed throughout the range of the species and show no evidence of any reproductive isolation. Our data support a revision of the taxonomy of this group, and we recognize two species within the complex and three subspecies within P. emoryi. This study illustrates the importance of thorough sampling of contact zones and consideration of gene flow when delimiting species in widespread complexes containing parapatric lineages., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The Himalaya are among the youngest and highest mountains in the world, but the exact timing of their uplift and origins of their biodiversity are still in debate. The Himalayan region is a relatively small area but with exceptional diversity and endemism. One common hypothesis to explain the rich montane diversity is uplift-driven diversification-that orogeny creates conditions favoring rapid in situ speciation of resident lineages. We test this hypothesis in the Himalayan region using amphibians and reptiles, two environmentally sensitive vertebrate groups. In addition, analysis of diversification of the herpetofauna provides an independent source of information to test competing geological hypotheses of Himalayan orogenesis. We conclude that the origins of the Himalayan herpetofauna date to the early Paleocene, but that diversification of most groups was concentrated in the Miocene. There was an increase in both rates and modes of diversification during the early to middle Miocene, together with regional interchange (dispersal) between the Himalaya and adjacent regions. Our analyses support a recently proposed stepwise geological model of Himalayan uplift beginning in the Paleocene, with a subsequent rapid increase of uplifting during the Miocene, finally giving rise to the intensification of the modern South Asian Monsoon., (© The Author(s) 2020. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd.)

Sexually selected traits can be expected to increase in importance when the period of sexual behavior is constrained, such as in seasonally restricted breeders. Anolis lizard male dewlaps are classic examples of multifaceted signaling traits, with demonstrated intraspecific reproductive function reflected in courtship behavior. Fitch and Hillis found a correlation between dewlap size and seasonality in mainland Anolis using traditional statistical methods and suggested that seasonally restricted breeding seasons enhanced the differentiation of this signaling trait. Here, we present two tests of the Fitch-Hillis Hypothesis using new phylogenetic and morphological data sets for 44 species of Mexican Anolis . A significant relationship between dewlap size and seasonality is evident in phylogenetically uncorrected analyses but erodes once phylogeny is accounted for. This loss of strong statistical support for a relationship between a key aspect of dewlap morphology and seasonality also occurs within a species complex ( A. sericeus group) that inhabits seasonal and aseasonal environments. Our results fail to support seasonality as a strong driver of evolution of Anolis dewlap size. We discuss the implications of our results and the difficulty of disentangling the strength of single mechanisms on trait evolution when multiple selection pressures are likely at play., Competing Interests: The authors declare no conflicts of interest., (© 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Many recent species delimitation studies rely exclusively on limited analyses of genetic data analyzed under the multispecies coalescent (MSC) model, and results from these studies often are regarded as conclusive support for taxonomic changes. However, most MSC-based species delimitation methods have well-known and often unmet assumptions. Uncritical application of these genetic-based approaches (without due consideration of sampling design, the effects of a priori group designations, isolation by distance, cytoplasmic-nuclear mismatch, and population structure) can lead to over-splitting of species. Here, we argue that in many common biological scenarios, researchers must be particularly cautious regarding these limitations, especially in cases of well-studied, geographically variable, and parapatrically distributed species complexes. We consider these points with respect to a historically controversial species group, the American milksnakes (Lampropeltis triangulum complex), using genetic data from a recent analysis (Ruane et al. 2014). We show that over-reliance on the program Bayesian Phylogenetics and Phylogeography, without adequate consideration of its assumptions and of sampling limitations, resulted in over-splitting of species in this study. Several of the hypothesized species of milksnakes instead appear to represent arbitrary slices of continuous geographic clines. We conclude that the best available evidence supports three, rather than seven, species within this complex. More generally, we recommend that coalescent-based species delimitation studies incorporate thorough analyses of geographic variation and carefully examine putative contact zones among delimited species before making taxonomic changes., (© The Author(s) 2019. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The exchange of biotas between Eurasia and North America across the Bering land bridge had a major impact on ecosystems of both continents throughout the Cenozoic. This exchange has received particular attention regarding placental mammals dispersing into the Americas, including humans after the last glacial period, and also as an explanation for the disjunct distribution of related seed plants in eastern Asia and eastern North America. Here, we investigate bi-directional dispersal across the Bering land bridge from estimates of dispersal events based on time-calibrated phylogenies of a broad range of plant, fungus and animal taxa. We reveal a long-lasting phase of asymmetrical biotic interchange, with a peak of dispersal from Asia into North America during the late Oligocene warming (26-24 Ma), when dispersal in the opposite direction was greatly decreased. Influx from North America into Asia was lower than in the opposite direction throughout the Cenozoic, but with peak rates of dispersal at the end of the Eocene (40-34 Ma) and again in the early to middle Miocene (16-14 Ma). The strong association between dispersal patterns and environmental changes suggests that plants, fungi and animals have likely dispersed from stable to perturbed environments of North America and Eurasia throughout the Cenozoic., (© The Author(s) 2019. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd.)

Viviparous (live-bearing) vertebrates have evolved repeatedly within otherwise oviparous (egg-laying) clades. Over two-thirds of these changes in vertebrate reproductive parity mode happened in squamate reptiles, where the transition has happened between 98 and 129 times. The transition from oviparity to viviparity requires numerous physiological, morphological, and immunological changes to the female reproductive tract, including eggshell reduction, delayed oviposition, placental development for supply of water and nutrition to the embryo by the mother, enhanced gas exchange, and suppression of maternal immune rejection of the embryo. We performed genomic and transcriptomic analyses of a closely related oviparous-viviparous pair of lizards ( Phrynocephalus przewalskii and Phrynocephalus vlangalii ) to examine these transitions. Expression patterns of maternal oviduct through reproductive development of the egg and embryo differ markedly between the two species. We found changes in expression patterns of appropriate genes that account for each of the major aspects of the oviparity to viviparity transition. In addition, we compared the gene sequences in transcriptomes of four oviparous-viviparous pairs of lizards in different genera ( Phrynocephalus , Eremias , Scincella , and Sphenomorphus ) to look for possible gene convergence at the sequence level. We discovered low levels of convergence in both amino acid replacement and evolutionary rate shift. This suggests that most of the changes that produce the oviparity-viviparity transition are changes in gene expression, so occasional reversals to oviparity from viviparity may not be as difficult to achieve as has been previously suggested., Competing Interests: Conflict of interest statement: D.D.P. and G.Z. are coauthors on a 2014 paper.

Groundwater-dependent species are among the least-known components of global biodiversity, as well as some of the most vulnerable because of rapid groundwater depletion at regional and global scales. The karstic Edwards-Trinity aquifer system of west-central Texas is one of the most species-rich groundwater systems in the world, represented by dozens of endemic groundwater-obligate species with narrow, naturally fragmented distributions. Here, we examine how geomorphological and hydrogeological processes have driven population divergence and speciation in a radiation of salamanders ( Eurycea ) endemic to the Edwards-Trinity system using phylogenetic and population genetic analysis of genome-wide DNA sequence data. Results revealed complex patterns of isolation and reconnection driven by surface and subsurface hydrology, resulting in both adaptive and nonadaptive population divergence and speciation. Our results uncover cryptic species diversity and refine the borders of several threatened and endangered species. The US Endangered Species Act has been used to bring state regulation to unrestricted groundwater withdrawals in the Edwards (Balcones Fault Zone) Aquifer, where listed species are found. However, the Trinity and Edwards-Trinity (Plateau) aquifers harbor additional species with similarly small ranges that currently receive no protection from regulatory programs designed to prevent groundwater depletion. Based on regional climate models that predict increased air temperature, together with hydrologic models that project decreased springflow, we conclude that Edwards-Trinity salamanders and other codistributed groundwater-dependent organisms are highly vulnerable to extinction within the next century., Competing Interests: The authors declare no conflict of interest.

Although many cases of genetic adaptations to high elevations have been reported, the processes driving these modifications and the pace of their evolution remain unclear. Many high-elevation adaptations (HEAs) are thought to have arisen in situ as populations rose with growing mountains. In contrast, most high-elevation lineages of the Qinghai-Tibetan Plateau appear to have colonized from low-elevation areas. These lineages provide an opportunity for studying recent HEAs and comparing them with ancestral low-elevation alternatives. Herein, we compare four frogs (three species of Nanorana and a close lowland relative) and four lizards ( Phrynocephalus ) that inhabit a range of elevations on or along the slopes of the Qinghai-Tibetan Plateau. The sequential cladogenesis of these species across an elevational gradient allows us to examine the gradual accumulation of HEA at increasing elevations. Many adaptations to high elevations appear to arise gradually and evolve continuously with increasing elevational distributions. Numerous related functions, especially DNA repair and energy metabolism pathways, exhibit rapid change and continuous positive selection with increasing elevations. Although the two studied genera are distantly related, they exhibit numerous convergent evolutionary changes, especially at the functional level. This functional convergence appears to be more extensive than convergence at the individual gene level, although we found 32 homologous genes undergoing positive selection for change in both high-elevation groups. We argue that species groups distributed along a broad elevational gradient provide a more powerful system for testing adaptations to high-elevation environments compared with studies that compare only pairs of high-elevation versus low-elevation species., Competing Interests: The authors declare no conflict of interest.

Several previous genomic studies have focused on adaptation to high elevations, but these investigations have been largely limited to endotherms. Snakes of the genus Thermophis are endemic to the Tibetan plateau and therefore present an opportunity to study high-elevation adaptations in ectotherms. Here, we report the de novo assembly of the genome of a Tibetan hot-spring snake ( Thermophis baileyi ) and then compare its genome to the genomes of the other two species of Thermophis , as well as to the genomes of two related species of snakes that occur at lower elevations. We identify 308 putative genes that appear to be under positive selection in Thermophis We also identified genes with shared amino acid replacements in the high-elevation hot-spring snakes compared with snakes and lizards that live at low elevations, including the genes for proteins involved in DNA damage repair ( FEN1 ) and response to hypoxia ( EPAS1 ). Functional assays of the FEN1 alleles reveal that the Thermophis allele is more stable under UV radiation than is the ancestral allele found in low-elevation lizards and snakes. Functional assays of EPAS1 alleles suggest that the Thermophis protein has lower transactivation activity than the low-elevation forms. Our analysis identifies some convergent genetic mechanisms in high-elevation adaptation between endotherms (based on studies of mammals) and ectotherms (based on our studies of Thermophis )., Competing Interests: The authors declare no conflict of interest.

Frogs (Anura) are one of the most diverse groups of vertebrates and comprise nearly 90% of living amphibian species. Their worldwide distribution and diverse biology make them well-suited for assessing fundamental questions in evolution, ecology, and conservation. However, despite their scientific importance, the evolutionary history and tempo of frog diversification remain poorly understood. By using a molecular dataset of unprecedented size, including 88-kb characters from 95 nuclear genes of 156 frog species, in conjunction with 20 fossil-based calibrations, our analyses result in the most strongly supported phylogeny of all major frog lineages and provide a timescale of frog evolution that suggests much younger divergence times than suggested by earlier studies. Unexpectedly, our divergence-time analyses show that three species-rich clades (Hyloidea, Microhylidae, and Natatanura), which together comprise ∼88% of extant anuran species, simultaneously underwent rapid diversification at the Cretaceous-Paleogene (K-Pg) boundary (KPB). Moreover, anuran families and subfamilies containing arboreal species originated near or after the KPB. These results suggest that the K-Pg mass extinction may have triggered explosive radiations of frogs by creating new ecological opportunities. This phylogeny also reveals relationships such as Microhylidae being sister to all other ranoid frogs and African continental lineages of Natatanura forming a clade that is sister to a clade of Eurasian, Indian, Melanesian, and Malagasy lineages. Biogeographical analyses suggest that the ancestral area of modern frogs was Africa, and their current distribution is largely associated with the breakup of Pangaea and subsequent Gondwanan fragmentation., Competing Interests: The authors declare no conflict of interest.

The Rhyparochromidae, the largest family of Lygaeoidea, encompasses more than 1,850 described species, but no mitochondrial genome has been sequenced to date. Here we describe the first mitochondrial genome for Rhyparochromidae: a complete mitochondrial genome of Panaorus albomaculatus (Scott, 1874). This mitochondrial genome is comprised of 16,345 bp, and contains the expected 37 genes and control region. The majority of the control region is made up of a large tandem-repeat region, which has a novel pattern not previously observed in other insects. The tandem-repeats region of P. albomaculatus consists of 53 tandem duplications (including one partial repeat), which is the largest number of tandem repeats among all the known insect mitochondrial genomes. Slipped-strand mispairing during replication is likely to have generated this novel pattern of tandem repeats. Comparative analysis of tRNA gene families in sequenced Pentatomomorpha and Lygaeoidea species shows that the pattern of nucleotide conservation is markedly higher on the J-strand. Phylogenetic reconstruction based on mitochondrial genomes suggests that Rhyparochromidae is not the sister group to all the remaining Lygaeoidea, and supports the monophyly of Lygaeoidea.

True frogs of the genus Rana are widely used as model organisms in studies of development, genetics, physiology, ecology, behavior, and evolution. Comparative studies among the more than 100 species of Rana rely on an understanding of the evolutionary history and patterns of diversification of the group. We estimate a well-resolved, time-calibrated phylogeny from sequences of six nuclear and three mitochondrial loci sampled from most species of Rana, and use that phylogeny to clarify the group's diversification and global biogeography. Our analyses consistently support an "Out of Asia" pattern with two independent dispersals of Rana from East Asia to North America via Beringian land bridges. The more species-rich lineage of New World Rana appears to have experienced a rapid radiation following its colonization of the New World, especially with its expansion into montane and tropical areas of Mexico, Central America, and South America. In contrast, Old World Rana exhibit different trajectories of diversification; diversification in the Old World began very slowly and later underwent a distinct increase in speciation rate around 29-18 Ma. Net diversification is associated with environmental changes and especially intensive tectonic movements along the Asian margin from the Oligocene to early Miocene. Our phylogeny further suggests that previous classifications were misled by morphological homoplasy and plesiomorphic color patterns, as well as a reliance primarily on mitochondrial genes. We provide a phylogenetic taxonomy based on analyses of multiple nuclear and mitochondrial gene loci. [Amphibians; biogeography; diversification rate; Holarctic; transcontinental dispersal., (© The Author(s) 2016. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The Mk model was developed for estimating phylogenetic trees from discrete morphological data, whether for living or fossil taxa. Like any model, the Mk model makes a number of assumptions. One assumption is that transitions between character states are symmetric (i.e., the probability of changing from 0 to 1 is the same as 1 to 0). However, some characters in a data matrix may not satisfy this assumption. Here, we test methods for relaxing this assumption in a Bayesian context. Using empirical data sets, we perform model fitting to illustrate cases in which modeling asymmetric transition rates among characters is preferable to the standard Mk model. We use simulated data sets to demonstrate that choosing the best-fit model of transition-state symmetry can improve model fit and phylogenetic estimation., (© The Author(s) 2015. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

We examine the complex evolution of animal nervous systems and discuss the ramifications of this complexity for inferring the nature of early animals. Although reconstructing the origins of nervous systems remains a central challenge in biology, and the phenotypic complexity of early animals remains controversial, a compelling picture is emerging. We now know that the nervous system and other key animal innovations contain a large degree of homoplasy, at least on the molecular level. Conflicting hypotheses about early nervous system evolution are due primarily to differences in the interpretation of this homoplasy. We highlight the need for explicit discussion of assumptions and discuss the limitations of current approaches for inferring ancient phenotypic states., (Copyright © 2015. Published by Elsevier Ltd.)

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., (© 2015 Wiley Periodicals, Inc.)

Background: The selection of variable sites for inclusion in genomic analyses can influence results, especially when exemplar populations are used to determine polymorphic sites. We tested the impact of ascertainment bias on the inference of population genetic parameters using empirical and simulated data representing the three major continental groups of cattle: European, African, and Indian. We simulated data under three demographic models. Each simulated data set was subjected to three ascertainment schemes: (I) random selection; (II) geographically biased selection; and (III) selection biased toward loci polymorphic in multiple groups. Empirical data comprised samples of 25 individuals representing each continental group. These cattle were genotyped for 47,506 loci from the bovine 50 K SNP panel. We compared the inference of population histories for the empirical and simulated data sets across different ascertainment conditions using F ST and principal components analysis (PCA)., Results: Bias toward shared polymorphism across continental groups is apparent in the empirical SNP data. Bias toward uneven levels of within-group polymorphism decreases estimates of F ST between groups. Subpopulation-biased selection of SNPs changes the weighting of principal component axes and can affect inferences about proportions of admixture and population histories using PCA. PCA-based inferences of population relationships are largely congruent across types of ascertainment bias, even when ascertainment bias is strong., Conclusions: Analyses of ascertainment bias in genomic data have largely been conducted on human data. As genomic analyses are being applied to non-model organisms, and across taxa with deeper divergences, care must be taken to consider the potential for bias in ascertainment of variation to affect inferences. Estimates of F ST , time of separation, and population divergence as estimated by principal components analysis can be misleading if this bias is not taken into account.

The development of efficient sequencing techniques has resulted in large numbers of genomes being available for evolutionary studies. However, only one genome is available for all amphibians, that of Xenopus tropicalis, which is distantly related from the majority of frogs. More than 96% of frogs belong to the Neobatrachia, and no genome exists for this group. This dearth of amphibian genomes greatly restricts genomic studies of amphibians and, more generally, our understanding of tetrapod genome evolution. To fill this gap, we provide the de novo genome of a Tibetan Plateau frog, Nanorana parkeri, and compare it to that of X. tropicalis and other vertebrates. This genome encodes more than 20,000 protein-coding genes, a number similar to that of Xenopus. Although the genome size of Nanorana is considerably larger than that of Xenopus (2.3 vs. 1.5 Gb), most of the difference is due to the respective number of transposable elements in the two genomes. The two frogs exhibit considerable conserved whole-genome synteny despite having diverged approximately 266 Ma, indicating a slow rate of DNA structural evolution in anurans. Multigenome synteny blocks further show that amphibians have fewer interchromosomal rearrangements than mammals but have a comparable rate of intrachromosomal rearrangements. Our analysis also identifies 11 Mb of anuran-specific highly conserved elements that will be useful for comparative genomic analyses of frogs. The Nanorana genome offers an improved understanding of evolution of tetrapod genomes and also provides a genomic reference for other evolutionary studies.

Multicellularity has evolved multiple times, but animals are the only multicellular lineage with nervous systems. This fact implies that the origin of nervous systems was an unlikely event, yet recent comparisons among extant taxa suggest that animal nervous systems may have evolved multiple times independently. Here, we use ancestral gene content reconstruction to track the timing of gene family expansions for the major families of ion-channel proteins that drive nervous system function. We find that animals with nervous systems have broadly similar complements of ion-channel types but that these complements likely evolved independently. We also find that ion-channel gene family evolution has included large loss events, two of which were immediately followed by rounds of duplication. Ctenophores, cnidarians, and bilaterians underwent independent bouts of gene expansion in channel families involved in synaptic transmission and action potential shaping. We suggest that expansions of these family types may represent a genomic signature of expanding nervous system complexity. Ancestral nodes in which nervous systems are currently hypothesized to have originated did not experience large expansions, making it difficult to distinguish among competing hypotheses of nervous system origins and suggesting that the origin of nerves was not attended by an immediate burst of complexity. Rather, the evolution of nervous system complexity appears to resemble a slow fuse in stem animals followed by many independent bouts of gene gain and loss.

Despite the introduction of likelihood-based methods for estimating phylogenetic trees from phenotypic data, parsimony remains the most widely-used optimality criterion for building trees from discrete morphological data. However, it has been known for decades that there are regions of solution space in which parsimony is a poor estimator of tree topology. Numerous software implementations of likelihood-based models for the estimation of phylogeny from discrete morphological data exist, especially for the Mk model of discrete character evolution. Here we explore the efficacy of Bayesian estimation of phylogeny, using the Mk model, under conditions that are commonly encountered in paleontological studies. Using simulated data, we describe the relative performances of parsimony and the Mk model under a range of realistic conditions that include common scenarios of missing data and rate heterogeneity.

Genomic data facilitate opportunities to track complex population histories of divergence and gene flow. We developed a metric, scaled block size (SBS), which uses the nonrecombined block size of introgressed regions of chromosomes to differentiate between recent and ancient types of admixture, and applied it to the reconstruction of admixture in cattle. Cattle are descendants of 2 independently domesticated lineages, taurine and indicine, which diverged more than 200 000 years ago. Several breeds have hybrid ancestry between these divergent lineages. Using 47 506 single-nucleotide polymorphisms, we analyzed the genomic architecture of the ancestry of 1369 individuals. We focused on 4 groups with admixed ancestry, including 2 anciently admixed African breeds (n = 58; n = 43), New World cattle of Spanish origin (n = 51), and known recent hybrids (n = 46). We estimated the ancestry of chromosomal regions for each individual and used the SBS metric to differentiate the timing of admixture among groups and among individuals within groups. By comparing SBS values of test individuals with standards with known recent hybrid ancestry, we were able to differentiate individuals of recent hybrid origin from other admixed cattle. We also estimated ancestry at the chromosomal scale. The X chromosome exhibits reduced indicine ancestry in recent hybrid, New World, and western African cattle, with virtually no evidence of indicine ancestry in New World cattle., (© The American Genetic Association. 2014.)

Background: Most previous studies of morphological and molecular data have consistently supported the monophyly of the true water bugs (Hemiptera: Nepomorpha). An exception is a recent study by Hua et al. (BMC Evol Biol 9: 134, 2009) based on nine nepomorphan mitochondrial genomes. In the analysis of Hua et al. (BMC Evol Biol 9: 134, 2009), the water bugs in the group Pleoidea formed the sister group to a clade that consisted of Nepomorpha (the remaining true water bugs) + Leptopodomorpha (shore bugs) + Cimicomorpha (assassin bugs and relatives) + Pentatomomorpha (stink bugs and relatives), thereby suggesting that fully aquatic hemipterans evolved independently at least twice. Based on these results, Hua et al. (BMC Evol Biol 9: 134, 2009) elevated the Pleoidea to a new infraorder, the Plemorpha., Results: Our reanalysis suggests that the lack of support for the monophyly of the true water bugs (including Pleoidea) by Hua et al. (BMC Evol Biol 9: 134, 2009) likely resulted from inadequate taxon sampling. In particular, long-branch attraction (LBA) between the distant outgroup taxa and Pleoidea, as well as LBA among taxa in the ingroup, made Nepomorpha appear to be polyphyletic. We used three complementary strategies to test and alleviate the effects of LBA: (1) the removal of distant outgroups from the analysis; (2) the addition of closely related outgroups; and (3) the addition of a mitochondrial genome from a second family of Pleoidea. We also performed likelihood-ratio tests to examine the support for monophyly of Nepomorpha with different combinations of taxa included in the analysis. Furthermore, we found that specimens of Helotrephes sp. were misidentified as Paraplea frontalis (Fieber, 1844) by Hua et al. (BMC Evol Biol 9: 134, 2009)., Conclusions: All analyses that included the addition of more taxa significantly and consistently supported the placement of Pleoidea within the Nepomorpha (i.e., supported the monophyly of the traditional true water bugs). Our analyses further support a close relationship between Notonectoidea and Pleoidea within Nepomorpha, and the superfamilies Nepoidea, Ochteroidea, Naucoroidea, and Pleoidea are resolved as monophyletic in all trees with strong support. Our results also confirmed that monophyly of Nepomorpha clearly is not refuted by the mitochondrial genome data.

Ranaviruses have been implicated in recent declines in global amphibian populations. Compared with the family Iridoviridae, to which the genus Ranavirus belongs, ranaviruses have a wide host range in that species/strains are known to infect fish, amphibians and reptiles, presumably due to recent host-switching events. We used eight sequenced ranavirus genomes and two selection-detection methods (site based and branch based) to identify genes that exhibited signatures of positive selection, potentially due to the selective pressures at play during host switching. We found evidence of positive selection acting on four genes via the site-based method, three of which were newly acquired genes unique to ranavirus genomes. Using the branch-based method, we identified eight additional candidate genes that exhibited signatures of dN/dS (non-synonymous/synonymous substitution rate) >1 in the clade where intense host switching had occurred. We found that these branch-specific patterns of elevated dN/dS were enriched in a small group of viral genes that have been acquired most recently in the ranavirus genome, compared with core genes that are shared among all members of the family Iridoviridae. Our results suggest that the group of newly acquired genes in the ranavirus genome may have undergone recent adaptive changes that have facilitated interspecies and interclass host switching.

Anura (frogs and toads) constitute over 88% of living amphibian diversity but many important questions about their phylogeny and evolution remain unresolved. For this study, we developed an efficient method for sequencing anuran mitochondrial DNAs (mtDNAs) by amplifying the mitochondrial genome in 12 overlapping fragments using frog-specific universal primer sets. Based on this method, we generated 47 nearly complete, new anuran mitochondrial genomes and discovered nine novel gene arrangements. By combining the new data and published anuran mitochondrial genomes, we assembled a large mitogenomic data set (11,007 nt) including 90 frog species, representing 39 of 53 recognized anuran families, to investigate their phylogenetic relationships and evolutionary history. The resulting tree strongly supported a paraphyletic arrangement of archaeobatrachian (=nonneobatrachian) frogs, with Leiopelmatoidea branching first, followed by Discoglossoidea, Pipoidea, and Pelobatoidea. Within Neobatrachia, the South African Heleophrynidae is the sister-taxon to all other neobatrachian frogs and the Seychelles-endemic Sooglossidae is recovered as the sister-taxon to Ranoidea. These phylogenetic relationships agree with many nuclear gene studies. The chronogram derived from two Bayesian relaxed clock methods (MultiDivTime and BEAST) suggests that modern frogs (Anura) originated in the early Triassic about 244 Ma and the appearance of Neobatrachia took place in the late Jurassic about 163 Ma. The initial diversifications of two species-rich superfamilies Hyloidea and Ranoidea commenced 110 and 133 Ma, respectively. These times are older than some other estimates by approximately 30-40 My. Compared with nuclear data, mtDNA produces compatible time estimates for deep nodes (>150 Ma), but apparently older estimates for more shallow nodes. Our study shows that, although it evolves relatively rapidly and behaves much as a single locus, mtDNA performs well for both phylogenetic and divergence time inferences and will provide important reference hypotheses for the phylogeny and evolution of frogs.

Estimated phylogenies of evolutionarily diverse taxa will be well supported and more likely to be historically accurate when the analysis contains large amounts of data-many genes sequenced across many taxa. Inferring such phylogenies for non-model organisms is challenging given limited resources for whole-genome sequencing. We take advantage of genomic data from a single species to test the limits of hybridization-based enrichment of hundreds of exons across frog species that diverged up to 250 million years ago. Enrichment success for a given species depends greatly on the divergence time between it and the reference species, and the resulting alignment contains a significant proportion of missing data. However, our alignment generates a well-supported phylogeny of frogs, suggesting that this technique is a practical solution towards resolving relationships across deep evolutionary time.

Previous archeological and genetic research has shown that modern cattle breeds are descended from multiple independent domestication events of the wild aurochs (Bos primigenius) ∼10,000 y ago. Two primary areas of domestication in the Middle East/Europe and the Indian subcontinent resulted in taurine and indicine lines of cattle, respectively. American descendants of cattle brought by European explorers to the New World beginning in 1493 generally have been considered to belong to the taurine lineage. Our analyses of 47,506 single nucleotide polymorphisms show that these New World cattle breeds, as well as many related breeds of cattle in southern Europe, actually exhibit ancestry from both the taurine and indicine lineages. In this study, we show that, although European cattle are largely descended from the taurine lineage, gene flow from African cattle (partially of indicine origin) contributed substantial genomic components to both southern European cattle breeds and their New World descendants. New World cattle breeds, such as Texas Longhorns, provide an opportunity to study global population structure and domestication in cattle. Following their introduction into the Americas in the late 1400s, semiferal herds of cattle underwent between 80 and 200 generations of predominantly natural selection, as opposed to the human-mediated artificial selection of Old World breeding programs. Our analyses of global cattle breed population history show that the hybrid ancestry of New World breeds contributed genetic variation that likely facilitated the adaptation of these breeds to a novel environment.

The accretion of the Indian subcontinent to Eurasia triggered a massive faunal and floral exchange, with Gondwanan taxa entering into Asia and vice versa. The traditional view on the Indian-Asian collision assumes contact of the continental plates during the Early Eocene. Many biogeographic studies rely on this assumption. However, the exact mode and timing of this geological event is still under debate. Here we address, based on an extensive phylogenetic analysis of rhacophorid tree frogs, if there was already a Paleogene biogeographic link between Southeast Asia and India; in which direction faunal exchange occurred between India and Eurasia within the Rhacophoridae; and if the timing of the faunal exchange correlates with one of the recently suggested geological models. Rhacophorid tree frogs showed an early dispersal from India to Asia between 46 and 57 Ma, as reconstructed from the fossil record. During the Middle Eocene, however, faunal exchange ceased, followed by increase of rhacophorid dispersal events between Asia and the Indian subcontinent during the Oligocene that continued until the Middle Miocene. This corroborates recent geological models that argue for a much later final collision between the continental plates. We predict that the Oligocene faunal exchange between the Indian subcontinent and Asia, as shown here for rhacophorid frogs, also applies for other nonvolant organisms with an Indian-Asian distribution, and suggest that previous studies that deal with this faunal interchange should be carefully reinvestigated.

Proteins in the superfamily of voltage-gated ion channels mediate behavior across the tree of life. These proteins regulate the movement of ions across cell membranes by opening and closing a central pore that controls ion flow. The best-known members of this superfamily are the voltage-gated potassium, calcium (Ca(v)), and sodium (Na(v)) channels, which underlie impulse conduction in nerve and muscle. Not all members of this family are opened by changes in voltage, however. NALCN (NA(+) leak channel nonselective) channels, which encode a voltage-insensitive "sodium leak" channel, have garnered a growing interest. This study examines the phylogenetic relationship among Na(v)/Ca(v) voltage-gated and voltage-insensitive channels in the eukaryotic group Opisthokonta, which includes animals, fungi, and their unicellular relatives. We show that NALCN channels diverged from voltage-gated channels before the divergence of fungi and animals and that the closest relatives of NALCN channels are fungal calcium channels, which they functionally resemble.

Accurate delimitation of species is a critical first step in protecting biodiversity. Detection of distinct species is especially important for groups of organisms that inhabit sensitive environments subject to recent degradation, such as creeks, springs, and rivers in arid or semi-desert regions. The genus Dionda currently includes six recognized and described species of minnows that live in clear springs and spring-fed creeks of Texas, New Mexico (USA), and northern Mexico, but the boundaries, delimitation, and characterization of species in this genus have not been examined rigorously. The habitats of some of the species in this genus are rapidly deteriorating, and many local populations of Dionda have been extirpated. Considering the increasing concerns over degradation of their habitat, and pending a more detailed morphological revision of the genus, we undertook a molecular survey based on four DNA regions to examine variation over the range of the genus, test species boundaries, and infer phylogenetic relationships within Dionda. Based on analyses of two mitochondrial (cytb and D-loop) and two nuclear (Rag1 and S7) DNA regions from specimens collected throughout the range of Dionda, we identified 12 distinct species in the genus. Formerly synonymized names are available for two of these species, and four other species remain undescribed. We also redefine the known range of six species. The limited distribution of several of the species, coupled with widespread habitat degradation, suggests that many of the species in this genus should be targets for conservation and recovery efforts., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The long-term persistence of completely asexual species is unexpected. Although asexuality has short-term evolutionary advantages, a lack of genetic recombination leads to the accumulation over time of deleterious mutations. The loss of individual fitness as a result of accumulated deleterious mutations is expected to lead to reduced population fitness and possible lineage extinction. Persistent lineages of asexual, all-female clones (parthenogenetic and gynogenetic species) avoid the negative effects of asexual reproduction through the production of rare males, or otherwise exhibit some degree of genetic recombination. Another form of asexuality, known as androgenesis, results in offspring that are clones of the male parent. Several species of the Asian clam genus Corbicula reproduce via androgenesis. We compared gene trees of mitochondrial and nuclear loci from multiple sexual and androgenetic species across the global distribution of Corbicula to test the hypothesis of long-term clonality of the androgenetic species. Our results indicate that low levels of genetic capture of maternal nuclear DNA from other species occur within otherwise androgenetic lineages of Corbicula. The rare capture of genetic material from other species may allow androgenetic lineages of Corbicula to mitigate the effects of deleterious mutation accumulation and increase potentially adaptive variation. Models comparing the relative advantages and disadvantages of sexual and asexual reproduction should consider the possibility of rare genetic recombination, because such events seem to be nearly ubiquitous among otherwise asexual species.

Voltage-dependent sodium channels are believed to have evolved from calcium channels at the origin of the nervous system. A search of the genome of a single-celled choanoflagellate (the sister group of animals) identified a gene that is homologous to animal sodium channels and has a putative ion selectivity filter intermediate between calcium and sodium channels. Searches of a wide variety of animal genomes, including representatives of each basal lineage, revealed that similar homologs were retained in most lineages. One of these, the Placozoa, does not possess a nervous system. We cloned and sequenced the full choanoflagellate channel and parts of two placozoan channels from mRNA, showing that they are expressed. Phylogenetic analysis clusters the genes for these channels with other known sodium channels. From this phylogeny we infer ancestral states of the ion selectivity filter and show that this state has been retained in the choanoflagellate and placozoan channels. We also identify key gene duplications and losses and show convergent amino acid replacements at important points along the animal lineage.

Phylogenetic analysis has been widely used to test the a priori hypothesis of epidemiological clustering in suspected transmission chains of HIV-1. Among studies showing strong support for relatedness between HIV samples obtained from infected individuals, evidence for the direction of transmission between epidemiologically related pairs has been lacking. During transmission of HIV, a genetic bottleneck occurs, resulting in the paraphyly of source viruses with respect to those of the recipient. This paraphyly establishes the direction of transmission, from which the source can then be inferred. Here, we present methods and results from two criminal cases, State of Washington v Anthony Eugene Whitfield, case number 04-1-0617-5 (Superior Court of the State of Washington, Thurston County, 2004) and State of Texas v Philippe Padieu, case numbers 219-82276-07, 219-82277-07, 219-82278-07, 219-82279-07, 219-82280-07, and 219-82705-07 (219th Judicial District Court, Collin County, TX, 2009), which provided evidence that direction can be established from blinded case samples. The observed paraphyly from each case study led to the identification of an inferred source (i.e., index case), whose identity was revealed at trial to be that of the defendant.

Arabidopsis thaliana knockout lines for the plant-specific eukaryotic translation initiation factors eIFiso4G1 (i4g1) and eIFiso4G2 (i4g2) genes have been obtained. To address the potential for functional redundancy of these genes, homozygous double mutant lines were generated by crossing individual knockout lines. Both single and double mutant plants were analyzed for changes in gross morphology, development, and responses to selected environmental stressors. Single gene knockouts appear to have minimal effect on morphology, germination rate, growth rate, flowering time, or fertility. However, double mutant i4g1/i4g2 knockout plants show reduced germination rates, slow growth rates, moderate chlorosis, impaired fertility and reduced long term seed viability. Double mutant plants also exhibit altered responses to dehydration, salinity, and heat stress. The i4g2 and i4g1/i4g2 double mutant has reduced amounts of chlorophyll a and b suggesting a role in the expression of chloroplast proteins. General protein synthesis did not appear to be affected as the levels of gross protein expression did not appear to change in the mutants. The lack of a phenotype for either of the single mutants suggests there is considerable functional overlap. However, the strong phenotypes observed for the double mutant indicates that the individual gene products may have specialized roles in the expression of proteins involved in plant growth and development.

We present a 6-gene, 420-species maximum-likelihood phylogeny of Ascomycota, the largest phylum of Fungi. This analysis is the most taxonomically complete to date with species sampled from all 15 currently circumscribed classes. A number of superclass-level nodes that have previously evaded resolution and were unnamed in classifications of the Fungi are resolved for the first time. Based on the 6-gene phylogeny we conducted a phylogenetic informativeness analysis of all 6 genes and a series of ancestral character state reconstructions that focused on morphology of sporocarps, ascus dehiscence, and evolution of nutritional modes and ecologies. A gene-by-gene assessment of phylogenetic informativeness yielded higher levels of informativeness for protein genes (RPB1, RPB2, and TEF1) as compared with the ribosomal genes, which have been the standard bearer in fungal systematics. Our reconstruction of sporocarp characters is consistent with 2 origins for multicellular sexual reproductive structures in Ascomycota, once in the common ancestor of Pezizomycotina and once in the common ancestor of Neolectomycetes. This first report of dual origins of ascomycete sporocarps highlights the complicated nature of assessing homology of morphological traits across Fungi. Furthermore, ancestral reconstruction supports an open sporocarp with an exposed hymenium (apothecium) as the primitive morphology for Pezizomycotina with multiple derivations of the partially (perithecia) or completely enclosed (cleistothecia) sporocarps. Ascus dehiscence is most informative at the class level within Pezizomycotina with most superclass nodes reconstructed equivocally. Character-state reconstructions support a terrestrial, saprobic ecology as ancestral. In contrast to previous studies, these analyses support multiple origins of lichenization events with the loss of lichenization as less frequent and limited to terminal, closely related species.

Voltage-dependent sodium channels are critical for electrical excitability. Invertebrates possess a single sodium channel gene; two rounds of genome duplication early in vertebrates increased the number to four. Since the teleost-tetrapod split, independent gene duplications in each lineage have further increased the number of sodium channel genes to 10 in tetrapods and 8 in teleosts. Here we review how the occurrence of multiple sodium channel paralogs has influenced the evolutionary history of three groups of fishes: pufferfish, gymnotiform and mormyriform electric fish. Pufferfish (tetraodontidae) produce a neurotoxin, tetrodotoxin, that binds to and blocks the pore of sodium channels. Pufferfish evolved resistance to their own toxins by amino acid substitutions in the pore of their sodium channels. These substitutions had to occur in parallel across multiple paralogs for organismal resistance to evolve. Gymnotiform and mormyriform fishes independently evolved electric organs to generate electricity for communication and object localization. Two sodium channel genes are expressed in muscle in most fishes. In both groups of weakly electric fishes, one gene lost its expression in muscle and became compartmentalized in the evolutionary novel electric organ, which is a muscle derivative. This gene then evolved at elevated rates, whereas the gene that is still expressed in muscle does not show elevated rates of evolution. In the electric organ-expressing gene, amino acid substitutions occur in parts of the channel involved in determining how long the channel will be open or closed. The enhanced rate of sequence evolution of this gene likely underlies the species-level variations in the electric signal., (© 2009 ISZS, Blackwell Publishing and IOZ/CAS.)

Members of Rhogeessa are hypothesized to have undergone speciation via chromosomal rearrangements in a model termed speciation by monobrachial centric fusions. Recently, mitochondrial cytochrome-b sequence data tentatively supported this hypothesis but could not explicitly test the model's expectations regarding interbreeding among karyotypic forms. These data showed potential evidence for hybridization or incomplete lineage sorting between the karyotypically distinct R. tumida and R. aeneus and identified multiple lineages of karyotypically identical R. tumida. Here, we present a more comprehensive test of speciation by monobrachial centric fusions in Rhogeessa. Our analysis is based on sequence data from two nuclear loci: paternally inherited ZFY and autosomal MPI genes. These data provide results consistent either with incomplete lineage sorting or ancient hybridization to explain alleles shared at low frequency between R. aeneus and R. tumida. Recent and ongoing hybridization between any species can be ruled out. These data confirm the presence of multiple lineages of the 2n=34 karyotypic form ("R. tumida") that are not each other's closest relatives. These results are generally consistent with speciation by monobrachial centric fusions, although additional modes of speciation have also occurred in Rhogeessa. Phylogeographic analyses indicate habitat differences may be responsible for isolation and divergence between different lineages currently referred to as R. tumida.

Animal communication systems are subject to natural selection so the imprint of selection must reside in the genome of each species. Electric fish generate electric organ discharges (EODs) from a muscle-derived electric organ (EO) and use these fields for electrolocation and communication. Weakly electric teleosts have evolved at least twice (mormyriforms, gymnotiforms) allowing a comparison of the workings of evolution in two independently evolved sensory/motor systems. We focused on the genes for two Na(+) channels, Nav1.4a and Nav1.4b, which are orthologs of the mammalian muscle-expressed Na(+) channel gene Nav1.4. Both genes are expressed in muscle in non-electric fish. Nav1.4b is expressed in muscle in electric fish, but Nav1.4a expression has been lost from muscle and gained in the evolutionarily novel EO in both groups. We hypothesized that Nav1.4a might be evolving to optimize the EOD for different sensory environments and the generation of species-specific communication signals. We obtained the sequence for Nav1.4a from non-electric, mormyriform and gymnotiform species, estimated a phylogenetic tree, and determined rates of evolution. We observed elevated rates of evolution in this gene in both groups coincident with the loss of Nav1.4a from muscle and its compartmentalization in EO. We found amino acid substitutions at sites known to be critical for channel inactivation; analyses suggest that these changes are likely to be the result of positive selection. We suggest that the diversity of EOD waveforms in both groups of electric fish is correlated with accelerations in the rate of evolution of the Nav1.4a Na(+) channel gene due to changes in selection pressure on the gene once it was solely expressed in the EO.

Approximately 75% of vertebrate proteins belong to protein families encoded by multiple evolutionarily related genes, a pattern that emerged as a result of gene and genome duplications over the course of vertebrate evolution. In families of genes with similar or related functions, adaptation to a strong selective agent should involve multiple adaptive changes across the entire gene family. However, we know of no evolutionary studies that have explicitly addressed this point. Here, we show how 4 taxonomically diverse species of pufferfishes (Tetraodontidae) each evolved resistance to the guanidinium toxins tetrodotoxin (TTX) and saxitoxin (STX) via parallel amino acid replacements across all 8 sodium channels present in teleost fish genomes. This resulted in diverse suites of coexisting sodium channel types that all confer varying degrees of toxin resistance, yet show remarkable convergence among genes and phylogenetically diverse species. Using site-directed mutagenesis and expression of a vertebrate sodium channel, we also demonstrate that resistance to TTX/STX is enhanced up to 15-fold by single, frequently observed replacements at 2 sites that have not previously been implicated in toxin binding but show similar or identical replacements in pufferfishes and in distantly related vertebrate and nonvertebrate animals. This study presents an example of natural selection acting upon a complete gene family, repeatedly arriving at a diverse but limited number of adaptive changes within the same genome. To be maximally informative, we suggest that future studies of molecular adaptation should consider all functionally similar paralogs of the affected gene family.

Androgenesis is a rare form of asexual male reproduction found in disparate taxa across the Tree of Life. Phylogenetic analyses of mitochondrial genes suggest that androgenesis has arisen repeatedly in the Asian clam genus Corbicula. Two of these androgenetic species have been introduced to North America. Multiple lines of genetic evidence suggest that although nuclear recombination between these two species is rare, mitochondrial genome capture is a frequent consequence of androgenetic parasitism of heterospecific eggs. Egg parasitism may also rarely result in partial nuclear genome capture between closely related species of Corbicula, which provides a mechanism for the otherwise clonal species to avoid the deleterious effects of asexuality. Egg parasitism among congeners may explain why androgenesis has been maintained in Corbicula after fixation and has not yet led to population extinction. This mechanism also provides an explanation for the apparent multiple origins of androgenesis in Corbicula as seen on the mitochondrial DNA phylogeny. We suggest that a single androgenetic lineage may have repeatedly captured mitochondrial genomes (as well as portions of nuclear genomes) from various sexual species, resulting in several distinct androgenetic species with distantly related mtDNA genomes and divergent morphologies.

Evolutionary trajectories of codistributed taxa with comparable ecological preferences and dispersal abilities may be similarly impacted by historical landscape-level processes. Species' responses to changes in a shared biogeographic landscape may be purely concerted, completely independent, or classified as falling within an intermediate part of the continuum bounded by these two extremes. With sufficient molecular data, temporal contrasts of congruence among taxa with respect to these responses can be made. Such contrasts provide insights into the relative influence of ancient versus more recent climatic (and other) impacts on genetic structuring. Using phylogenetic, allele frequency, and genotypic data from two low-mobility, rotting-log-adapted (saproxylic) springtail species (Collembola) from an isolated 100-km-long section of the Great Dividing Range in southeastern Australia, we tested the concerted-response hypothesis over three timescales. Tests of phylogeographic, demographic, and contemporary population-genetic congruence were performed using an integrative approach that draws on both direct (pattern-based) and indirect (scenario-based) analyses. Our data revealed a general pattern of broad-scale similarities in species' responses to the interaction between Pleistocene climatic cycles and landscape setting, overlaid with some species-specific differences on local geographic and more recent temporal scales. This general pattern of phylogeographic congruence was accompanied by evidence for contemporaneous demographic incongruence indicating that, even at relatively small spatial scales, biogeographic context can exert an overarching influence on genetic structuring.