Anna E. Savage

The external appearance of frog skin varies among species and across body regions. Although this variation has long been recognized, it remains an understudied aspect of frog diversity. Further, the evolutionary processes driving this variation are unclear because previous work has been largely qualitative. Here we quantify the skin texture of 187 species spanning 45 of the 57 frog families using standardized gel-based profilometry. Using phylogenetic comparative models we explore the extent to which skin texture differs among body regions, how these differences have evolved across major frog clades, and whether microhabitat, climate, and body size help explain texture patterns. We find that the ventral posterior region, which functions in water uptake and rehydration, tends to be rougher than other body regions, yet skin texture evolves at similar rates across the body. Microhabitat, particularly arboreality, is linked to greater skin texture variation among body regions. Among species occupying more terrestrial microhabitats (e.g., arboreal, burrowing, leaflitter), we find that body size and local climate has little effect on skin texture. By comparing skin texture across a wide range of species and environments worldwide, our study tests hypotheses about external skin diversity in frogs that have previously received limited comparative evaluation and highlights texture as an informative component of amphibian skin biology.

Major histocompatibility complex (MHC) immune loci illustrate how natural selection shapes functional genetic diversity in wild populations. Balancing selection favors high MHC diversity within individuals and populations that persists beyond speciation, leading to shared allelic lineages among taxa. However, some vertebrates show markedly lower allelic diversity and even the loss of entire MHC gene classes. Variation in life history and disease prevalence makes sea turtles an important group for studying interspecific MHC diversity, but this has been minimally explored. We sequenced class I and class II MHC genes in over 300 individuals from loggerhead (Caretta caretta), green (Chelonia mydas), leatherback (Dermochelys coriacea), and Kemp's ridley (Lepidochelys kempii) sea turtles. We recovered 162 class I and 308 class II functionally distinct alleles, many of which were shared among species. Codon usage analyses suggest that the shared alleles have been maintained through speciation. High allele counts and evidence of diversifying and positive selection suggest balancing selection maintains considerable MHC diversity in sea turtles. However, we found two notable exceptions: (i) D. coriacea had extremely low nucleotide and allelic diversity across all MHC loci and (ii) one MHC class II gene copy on a different chromosome from the core MHC genomic region showed little evidence of positive selection and almost no genetic variability among all four species, suggesting an atypical and potentially functionally distinct gene. Our study finds notably high allelic and nucleotide diversity in sea turtle MHC promoted by balancing selection, yet evolutionary pressures vary considerably between species and gene copies.

New insights into the diversity of lymphocyte functions challenges previous dogma about the rigid divide between innate and adaptive immunity. While T cells with canonically diversified receptors are crucial for recognizing novel antigens, other T cell lineages express innate-like receptors that recognize conserved molecular patterns. The relative frequency of innate-like to diversifying T cell receptors (iTCRs: dTCRs) varies greatly across vertebrate species and across ontogeny within species. These within-species dynamics can potentially be explained by developmental constraints on immunity, pathogen diversity and exposure, or by trade-offs associated with specificity. To better understand how these factors shape T cell repertoires, we constructed an agent-based model of TCR evolution inspired by the diversity of ontogenic life histories in amphibians but applicable to an array of vertebrate species. Our model features two life stages with distinct parasite populations and life history costs. The model predicts that changes in ontogeny (stage duration, T cell maturation time) and environmental factors (parasite diversity, parasite complexity) exert drastic effects on the stage structure of T cell investment strategies. A better understanding of the evolutionary pressures that shape TCR diversity will provide new insights into lymphocyte evolution and immune investment across organismal development.

The genus Pristimantis diversified in the tropical Andes mountains and is the most speciose genus of terrestrial vertebrates. Pristimantis are notable among frogs in that they thrive at high elevations (>2,000 m) and are direct developers without a tadpole stage. Despite their ecological significance, little is known about the genetic and physiological traits enabling their success. We conducted transcriptomic analysis on seven Pristimantis species sampled across elevations in the Ecuadorean Andes to explore three hypotheses for their success: (i) unique genes are under selection relative to all other frogs, (ii) common selection occurs across all direct developers, or (iii) common selection occurs across all high-elevation frog clades. Comparative analysis with 34 frog species revealed unique positive selection in Pristimantis genes related to aerobic respiration, hemostasis, signaling, cellular transportation of proteins and ions, and immunity. Additionally, we detected positive selection across all direct developers for genes associated with oxygenase activity and metal ion binding. While many genes under selection in Pristimantis were not positively selected in other high-elevation frog species, we identified some shared genes and pathways linked to lipid metabolism, innate immunity, and cellular redox processes. We observed more positive selection in duplicated- versus single-copy genes, while relaxed purifying selection was prevalent in single-copy genes. Notably, copy number of an innate immunity complement gene was positively correlated with Pristimantis species elevation. Our findings contribute novel insights into the genetic basis of adaptation in Pristimantis and provide a foundation for future studies on the evolutionary mechanisms leading to direct development and coping with high elevations.

Driven by co-evolution with pathogens, host immunity continuously adapts to optimize defence against pathogens within a given environment. Recent advances in genetics, genomics and transcriptomics have enabled a more detailed investigation into how immunogenetic variation shapes the diversity of immune responses seen across domestic and wild animal species. However, a deeper understanding of the diverse molecular mechanisms that shape immunity within and among species is still needed to gain insight into—and generate evolutionary hypotheses on—the ultimate drivers of immunological differences. Here, we discuss current advances in our understanding of molecular evolution underpinning jawed vertebrate immunity. First, we introduce the immunome concept, a framework for characterizing genes involved in immune defence from a comparative perspective, then we outline how immune genes of interest can be identified. Second, we focus on how different selection modes are observed acting across groups of immune genes and propose hypotheses to explain these differences. We then provide an overview of the approaches used so far to study the evolutionary heterogeneity of immune genes on macro and microevolutionary scales. Finally, we discuss some of the current evidence as to how specific pathogens affect the evolution of different groups of immune genes. This review results from the collective discussion on the current key challenges in evolutionary immunology conducted at the ESEB 2021 Online Satellite Symposium: Molecular evolution of the vertebrate immune system, from the lab to natural populations. Abstract Reviewing current advances in our understanding of molecular evolution underpinning vertebrate immunity, we propose hypotheses to explain differences in selection modes across immune genes and discuss supporting evidence.

Anthropogenic habitat disturbance is fundamentally altering patterns of disease transmission and immunity across the vertebrate tree of life. Most studies linking anthropogenic habitat change and disease focus on habitat loss and fragmentation, but these processes often lead to a third process that is equally important: habitat split. Defined as spatial separation between the multiple classes of natural habitat that many vertebrate species require to complete their life cycles, habitat split has been linked to population declines in vertebrates, e.g. amphibians breeding in lowland aquatic habitats and overwintering in fragments of upland terrestrial vegetation. Here, we link habitat split to enhanced disease risk in amphibians (i) by reviewing the biotic and abiotic forces shaping elements of immunity and (ii) through a spatially oriented field study focused on tropical frogs. We propose a framework to investigate mechanisms by which habitat split influences disease risk in amphibians, focusing on three broad host factors linked to immunity: (i) composition of symbiotic microbial communities, (ii) immunogenetic variation, and (iii) stress hormone levels. Our review highlights the potential for habitat split to contribute to host‐associated microbiome dysbiosis, reductions in immunogenetic repertoire, and chronic stress, that often facilitate pathogenic infections and disease in amphibians and other classes of vertebrates. We highlight that targeted habitat‐restoration strategies aiming to connect multiple classes of natural habitats (e.g. terrestrial–freshwater, terrestrial–marine, marine–freshwater) could enhance priming of the vertebrate immune system through repeated low‐load exposure to enzootic pathogens and reduced stress‐induced immunosuppression.

Regional genetic differentiation of mitochondrial lineages occurs in migratory species with natal philopatry such as sea turtles. However, early juvenile dispersal represents a key opportunity for gene flow and colonization of new regions through founder events, making it an important yet under‐studied life stage. To assess connectivity among sea turtle life stages and ocean basins, we sequenced mitochondrial DNA (mtDNA) fragments from 35 juveniles sampled in the Gulf of Mexico from the rarely observed dispersal stage across three species: green turtles (Chelonia mydas; n = 30), hawksbills (Eretmochelys imbricata; n = 3), and loggerheads (Caretta caretta; n = 2). We estimated green turtle rookery contributions using a many‐to‐many Bayesian mixed stock analysis that incorporated dispersal probabilities based on rookery size and transport via ocean currents. We assembled a gene tree including 709 distinct mtDNA control region haplotypes from the literature for all seven extant sea turtle species to assess gaps in life‐stage data across ocean basins, as well as contextualize the lineages we sampled from dispersing juveniles. Our results indicate a high likelihood that green turtles sampled in the Gulf of Mexico originated from rookeries along the coast of Mexico, with smaller contributions from Costa Rica and Suriname. The gene tree analysis yielded species‐level relationships consistent with those presented previously, while intra‐species relationships between lineages and ocean basins differed, particularly within loggerhead and green turtle clades. Our results highlight the lack of genetic data from juvenile sea turtles, especially the early dispersal stage, and the potential for these data to answer broader questions of connectivity and diversification across species and lineages.

Fibropapillomatosis (FP) is a sea turtle disease characterized by benign tumor development on the skin, eyes, and/or internal organs. It primarily affects juvenile green turtles (Chelonia mydas) in coastal foraging sites. The Indian River Lagoon (IRL), Florida, USA, is a coastal green turtle foraging site where the observed FP annual rate averaged 49% between 1983 and 2018. While FP is no longer considered a major cause of sea turtle mortality and most individuals fully recover, the overall dynamics of this disease are poorly understood because prior disease history is unknown for individuals without FP at capture time, and future disease outcome is unknown for individuals with FP at capture time. To better evaluate FP dynamics for green turtles in the IRL, we developed a hierarchical model for predicting disease state change. We used data from 4149 captures of 3700 individual green turtles captured in the IRL. The hierarchical disease state model contained two levels: Level 1 modeled whether an individual would develop FP, and Level 2 modeled disease state progression, including states for pre‐FP affliction, active FP affliction, and full recovery from FP. From the hierarchical model, we estimated 99.8% (95% credibility intervals 99.1%–100%) of juvenile green turtles in the IRL developed FP, indicating that nearly every individual in the IRL is affected by this disease. The model also suggested that turtles quickly developed FP upon recruitment to the IRL and then recovered at different rates, with most completely recovering before emigrating from the IRL as they mature. This is the first analysis of long‐term sea turtle data suggesting nearly every turtle in an aggregation both develops and recovers from FP.

Characterizing polymorphism at the major histocompatibility complex (MHC) genes is key to understanding the vertebrate immune response to disease. Despite being globally afflicted by the infectious tumour disease fibropapillomatosis (FP), immunogenetic variation in sea turtles is minimally explored. We sequenced theα₁peptide-binding region of MHC class I genes (162 bp) from 268 juvenile green (Chelonia mydas) and 88 loggerhead (Caretta caretta) sea turtles in Florida, USA. We recovered extensive variation (116 alleles) and trans-species polymorphism. Supertyping analysis uncovered three functional MHC supertypes corresponding to the three well-supported clades in the phylogeny. We found significant evidence of positive selection at seven amino acid sites in the class I exon. Random forest modelling and risk ratio analysis ofCh. mydasalleles uncovered one allele weakly associated with smooth FP tumour texture, which may be associated with disease outcome. Our study represents the first characterization of MHC class I diversity inCh. mydasand the largest sample of sea turtles used to date in any study of adaptive genetic variation, revealing tremendous genetic variation and high adaptive potential to viral pathogen threats. The novel associations we identified between MHC diversity and FP outcomes in sea turtles further highlight the importance of evaluating genetic predictors of disease, including MHC and other functional markers.