Climate Change Alters Sexual Signaling in a Desert-Adapted Frog.

AbstractClimate change is altering species' habitats, phenology, and behavior. Although sexual behaviors impact population persistence and fitness, climate change's effects on sexual signals are understudied. Climate change can directly alter temperature-dependent sexual signals, cause changes in body size or condition that affect signal production, or alter the selective landscape of sexual signals. We tested whether temperature-dependent mating calls of Mexican spadefoot toads (Spea multiplicata) had changed in concert with climate in the southwestern United States across 22 years. We document increasing air temperatures, decreasing rainfall, and changing seasonal patterns of temperature and rainfall in the spadefoots' habitat. Despite increasing air temperatures, spadefoots' ephemeral breeding ponds have been getting colder at most elevations, and male calls have been slowing as a result. However, temperature-standardized call characters have become faster, and male condition has increased, possibly due to changes in the selective environment. Thus, climate change might generate rapid, complex changes in sexual signals with important evolutionary consequences.

Disentangling the developmental origins of a novel phenotype: enhancement versus reversal of environmentally induced gene expression.

Increasing evidence suggests that many novel traits might have originated via plasticity-led evolution (PLE). Yet, little is known of the developmental processes that underpin PLE, especially in its early stages. One such process is 'phenotypic accommodation', which occurs when, in response to a change in the environment, an organism experiences adjustments across variable parts of its phenotype that improve its fitness. Here, we asked if environmentally induced changes in gene expression are enhanced or reversed during phenotypic accommodation of a novel, complex phenotype in spadefoot toad tadpoles (Spea multiplicata). More genes than expected were affected by both the environment and phenotypic accommodation in the liver and brain. However, although phenotypic accommodation primarily reversed environmentally induced changes in gene expression in liver tissue, it enhanced these changes in brain tissue. Thus, depending on the tissue, phenotypic accommodation may either minimize functional disruption via reversal of gene expression patterns or promote novelty via enhancement of existing expression patterns. Our study thereby provides insights into the developmental origins of a novel phenotype and the incipient stages of PLE.

Transcriptomic bases of a polyphenism.

Polyphenism-in which multiple distinct phenotypes are produced from a single genotype owing to differing environmental conditions-is commonplace, but its molecular bases are poorly understood. Here, we examine the transcriptomic bases of a polyphenism in Mexican spadefoot toads (Spea multiplicata). Depending on their environment, their tadpoles develop into either a default "omnivore" morph or a novel "carnivore" morph. We compared patterns of gene expression among sibships that exhibited high versus low production of carnivores when reared in conditions that induce the carnivore morph versus those that do not. We found that production of the novel carnivore morph actually involved changes in fewer genes than did the maintenance of the default omnivore morph in the inducing environment. However, only body samples showed this pattern; head samples showed the opposite pattern. We also found that changes to lipid metabolism (especially cholesterol biosynthesis) and peroxisome contents and function might be crucial for establishing and maintaining differences between the morphs. Thus, our findings suggest that carnivore phenotype might have originally evolved following the breakdown of robustness mechanisms that maintain the default omnivore phenotype, and that the carnivore morph is developmentally regulated by lipid metabolism and peroxisomal form, function, and/or signaling. This study also serves as a springboard for further exploration into the nature and causes of plasticity in an emerging model system.

Genetic accommodation in the wild: evolution of gene expression plasticity during character displacement.

Ecological character displacement is considered crucial in promoting diversification, yet relatively little is known of its underlying mechanisms. We examined whether evolutionary shifts in gene expression plasticity ('genetic accommodation') mediate character displacement in spadefoot toads. Where Spea bombifrons and S. multiplicata occur separately in allopatry (the ancestral condition), each produces alternative, diet-induced, larval ecomorphs: omnivores, which eat detritus, and carnivores, which specialize on shrimp. By contrast, where these two species occur together in sympatry (the derived condition), selection to minimize competition for detritus has caused S. bombifrons to become nearly fixed for producing only carnivores, suggesting that character displacement might have arisen through an extreme form of genetic accommodation ('genetic assimilation') in which plasticity is lost. Here, we asked whether we could infer a signature of this process in regulatory changes of specific genes. In particular, we investigated whether genes that are normally expressed more highly in one morph ('biased' genes) have evolved reduced plasticity in expression levels among S. bombifrons from sympatry compared to S. bombifrons from allopatry. We reared individuals from sympatry vs. allopatry on detritus or shrimp and measured the reaction norms of nine biased genes. Although different genes displayed different patterns of gene regulatory evolution, the combined gene expression profiles revealed that sympatric individuals had indeed lost the diet-induced gene expression plasticity present in allopatric individuals. Our data therefore provide one of the few examples from natural populations in which genetic accommodation/assimilation can be traced to regulatory changes of specific genes. Such genetic accommodation might mediate character displacement in many systems.

Genetic Variants Underlying Plasticity in Natural Populations of Spadefoot Toads: Environmental Assessment versus Phenotypic Response.

Many organisms facultatively produce different phenotypes depending on their environment, yet relatively little is known about the genetic bases of such plasticity in natural populations. In this study, we describe the genetic variation underlying an extreme form of plasticity--resource polyphenism--in Mexican spadefoot toad tadpoles, Spea multiplicata. Depending on their environment, these tadpoles develop into one of two drastically different forms: a carnivore morph or an omnivore morph. We collected both morphs from two ponds that differed in which morph had an adaptive advantage and performed genome-wide association studies of phenotype (carnivore vs. omnivore) and adaptive plasticity (adaptive vs. maladaptive environmental assessment). We identified four quantitative trait loci associated with phenotype and nine with adaptive plasticity, two of which exhibited signatures of minor allele dominance and two of which (one phenotype locus and one adaptive plasticity locus) did not occur as minor allele homozygotes. Investigations into the genetics of plastic traits in natural populations promise to provide novel insights into how such complex, adaptive traits arise and evolve.

Comparative transcriptomics reveals that a novel form of phenotypic plasticity evolved via lineage-specific changes in gene expression.

Novel forms of phenotypic plasticity may evolve by lineage-specific changes or by co-opting mechanisms from more general forms of plasticity. Here, we evaluated whether a novel resource polyphenism in New World spadefoot toads (genus Spea) evolved by co-opting mechanisms from an ancestral form of plasticity common in anurans-accelerating larval development rate in response to pond drying. We compared overlap in differentially expressed genes between alternative trophic morphs constituting the polyphenism in Spea versus those found between tadpoles of Old World spadefoot toads (genus Pelobates) when experiencing different pond-drying regimes. Specifically, we (1) generated a de novo transcriptome and conducted differential gene expression analysis in Spea multiplicata, (2) utilized existing gene expression data and a recently published transcriptome for Pelobates cultripes when exposed to different drying regimes, and (3) identified unique and overlapping differentially expressed transcripts. We found thousands of differentially expressed genes between S. multiplicata morphs that were involved in major developmental reorganization, but the vast majority of these were not differentially expressed in P. cultripes. Thus, S. multiplicata's novel polyphenism appears to have arisen primarily through lineage-specific changes in gene expression and not by co-opting existing patterns of gene expression involved in pond-drying plasticity. Therefore, although ancestral stress responses might jump-start evolutionary innovation, substantial lineage-specific modification might be needed to refine these responses into more complex forms of plasticity.

Identification of candidate loci for adaptive phenotypic plasticity in natural populations of spadefoot toads.

Phenotypic plasticity allows organisms to alter their phenotype in direct response to changes in the environment. Despite growing recognition of plasticity's role in ecology and evolution, few studies have probed plasticity's molecular bases-especially using natural populations. We investigated the genetic basis of phenotypic plasticity in natural populations of spadefoot toads (Spea multiplicata). Spea tadpoles normally develop into an "omnivore" morph that is favored in long-lasting, low-density ponds. However, if tadpoles consume freshwater shrimp or other tadpoles, they can alternatively develop (via plasticity) into a "carnivore" morph that is favored in ephemeral, high-density ponds. By combining natural variation in pond ecology and morph production with population genetic approaches, we identified candidate loci associated with each morph (carnivores vs. omnivores) and loci associated with adaptive phenotypic plasticity (adaptive vs. maladaptive morph choice). Our candidate morph loci mapped to two genes, whereas our candidate plasticity loci mapped to 14 genes. In both cases, the identified genes tended to have functions related to their putative role in spadefoot tadpole biology. Our results thereby form the basis for future studies into the molecular mechanisms that mediate plasticity in spadefoots. More generally, these results illustrate how diverse loci might mediate adaptive plasticity.

Male sexual signal predicts phenotypic plasticity in offspring: implications for the evolution of plasticity and local adaptation.

In a rapidly changing world, understanding the processes that influence a population's ability to respond to natural selection is critical for identifying how to preserve biodiversity. Two such processes are phenotypic plasticity and sexual selection. Whereas plasticity can facilitate local adaptation, sexual selection potentially impedes local adaptation, especially in rapidly changing or variable environments. Here we hypothesize that, when females preferentially choose males that sire plastic offspring, sexual selection can actually facilitate local adaptation to variable or novel environments by promoting the evolution of adaptive plasticity. We tested this hypothesis by evaluating whether male sexual signals could indicate plasticity in their offspring and, concomitantly, their offspring's ability to produce locally adapted phenotypes. Using spadefoot toads ( Spea multiplicata) as our experimental system, we show that a male sexual signal predicts plasticity in his offspring's resource-use morphology. Specifically, faster-calling males (which are preferred by females) produce more plastic offspring; such plasticity, in turn, enables these males' offspring to respond adaptively to the spadefoots' highly variable environment. The association between a preferred male signal and adaptive plasticity in his offspring suggests that female mate choice can favour the evolution and maintenance of phenotypic plasticity and thereby foster adaptation to a variable environment. This article is part of the theme issue 'The role of plasticity in phenotypic adaptation to rapid environmental change'.

Genome of Spea multiplicata, a Rapidly Developing, Phenotypically Plastic, and Desert-Adapted Spadefoot Toad.

Frogs and toads (anurans) are widely used to study many biological processes. Yet, few anuran genomes have been sequenced, limiting research on these organisms. Here, we produce a draft genome for the Mexican spadefoot toad, Spea multiplicata, which is a member of an unsequenced anuran clade. Atypically for amphibians, spadefoots inhabit deserts. Consequently, they possess many unique adaptations, including rapid growth and development, prolonged dormancy, phenotypic (developmental) plasticity, and adaptive, interspecies hybridization. We assembled and annotated a 1.07 Gb Sp. multiplicata genome containing 19,639 genes. By comparing this sequence to other available anuran genomes, we found gene amplifications in the gene families of nodal, hyas3, and zp3 in spadefoots, and obtained evidence that anuran genome size differences are partially driven by variability in intergenic DNA content. We also used the genome to identify genes experiencing positive selection and to study gene expression levels in spadefoot hybrids relative to their pure-species parents. Completion of the Sp. multiplicata genome advances efforts to determine the genetic bases of spadefoots' unique adaptations and enhances comparative genomic research in anurans.

Call a spade a spade: taxonomy and distribution of Pelobates, with description of a new Balkan endemic.

The genomic era contributes to update the taxonomy of many debated terrestrial vertebrates. In an accompanying work, we provided a comprehensive molecular assessment of spadefoot toads (Pelobates) using genomic data. Our results call for taxonomic updates in this group. First, nuclear phylogenomics confirmed the species-level divergence between the Iberian P.cultripes and its Moroccan relative P.varaldii. Second, we inferred that P.fuscus and P.vespertinus, considered subspecies until recently, feature partial reproductive isolation and thus deserve a specific level. Third, we evidenced cryptic speciation and diversification among deeply diverged lineages collectively known as Pelobatessyriacus. Populations from the Near East correspond to the Eastern spadefoot toad P.syriacus sensu stricto, which is represented by two subspecies, one in the Levant (P.s.syriacus) and the other in the rest of the range (P.s.boettgeri). Populations from southeastern Europe correspond to the Balkan spadefoot toad, P.balcanicus. Based on genetic evidence, this species is also polytypic: the nominal P.b.balcanicus inhabits the Balkan Peninsula; a new subspecies P.b.chloeae ssp. nov. appears endemic to the Peloponnese. In this paper, we provide an updated overview of the taxonomy and distribution of all extant Pelobates taxa and describe P.b.chloeae ssp. nov.

A new species of Leptobrachium (Anura, Megophryidae) from western Thailand.

We describe a new species of the genus Leptobrachium from the Khao Laem Mountain, Suan Phung District, Ratchaburi Province, Tenasserim Region, western Thailand, based on molecular and morphological evidences. The new species, Leptobrachium tenasserimense sp. nov., can be distinguished from all other congeners by the following combination of characters: (1) adult SVL of 41.4-58.8 mm in males and 54.7-58.6 mm in females; (2) rounded finger and toe tips; (3) relative finger lengths: II

Effects of chlorothalonil on development and growth of amphibian embryos and larvae.

Chlorothalonil is a broad spectrum fungicide widely used in agricultural and urban environments, yet little is known regarding its effects on amphibians. We examined effects of chlorothalonil on growth, malformations, and mortality in embryos and larvae of Xenopus laevis and Spea multiplicata, and assessed variation in sensitivity among aquatic organisms using a species sensitivity distribution (SSD). Chlorothalonil induced gut malformations in X. laevis embryos and inhibited growth. Tail degeneration was observed in larvae of both species and reduced tail length to total length ratios occurred at environmentally relevant concentrations (5.9 and 11.0 µg/L). The mechanism of tail degeneration is unclear, but alteration in the expression of genes involved in tail resorption is a hypothesized mechanism. Larval amphibians were more sensitive than invertebrates and fish. Based on our results and the range of reported environmental concentrations, chlorothalonil may pose a risk to larval amphibians in certain habitats and scenarios.