Identifying populations at highest risk from climate change is a critical component of conservation efforts. However, vulnerability assessments are usually applied at the species level, even though intraspecific variation in exposure, sensitivity, and adaptive capacity play a crucial role in determining vulnerability. Genomic data can inform intraspecific vulnerability by identifying signatures of local adaptation that reflect population-level variation in sensitivity and adaptive capacity. Here, we address the question of local adaptation to temperature and the genetic basis of thermal tolerance in two stream frogs (Ascaphus truei and A. montanus). Building on previous physiological and temperature data, we used whole genome resequencing of tadpoles from four sites spanning temperature gradients in each species to test for signatures of local adaptation. To support these analyses, we developed the first annotated reference genome for A. truei. We then expanded the geographic scope of our analysis using targeted capture at an additional 11 sites per species. We found evidence of local adaptation to temperature based on physiological and genomic data in A. montanus and genomic data in A. truei, suggesting similar levels of sensitivity (i.e., susceptibility) among populations regardless of stream temperature. However, invariant thermal tolerances across temperatures in A. truei suggests that populations occupying warmer streams may be most sensitive. We identified high levels of evolutionary potential in both species based on genomic and physiological data. While further integration of these data is needed to comprehensively evaluate spatial variation in vulnerability, this work illustrates the value of genomics in identifying spatial patterns of climate change vulnerability.

Although biologists have described biofluorescence in a diversity of taxa, there have been few systematic efforts to document the extent of biofluorescence within a taxonomic group or investigate its general significance. Through a field survey across South America, we discover and document patterns of biofluorescence in tropical amphibians. We more than triple the number of anuran species that have been tested for this trait. We find evidence for ecological tuning (i.e., the specific adaptation of a signal to the environment in which it is received) of the biofluorescent signals. For 56.58% of species tested, the fluorescence excitation peak matches the wavelengths most abundant at twilight, the light environment in which most frogs are active. Additionally, biofluorescence emission spans both wavelengths of low availability in twilight and the peak sensitivity of green-sensitive rods in the anuran eye, likely increasing contrast of this signal for a conspecific receiver. We propose an expanded key for testing the ecological significance of biofluorescence in future studies, providing potential explanations for the other half of fluorescent signals not originally meeting formerly proposed criteria. With evidence of tuning to the ecology and sensory systems of frogs, our results suggest frog biofluorescence is likely functioning in anuran communication.

The recurrent evolution of resistance to cardiotonic steroids (CTS) across diverse animals most frequently involves convergent amino-acid substitutions in the H1-H2 extracellular loop of Na+, K+-ATPase (NKA). Previous work revealed that hystricognath rodents (e.g. chinchilla) and pterocliform birds (sandgrouse) have convergently evolved amino-acid insertions in the H1-H2 loop, but their functional significance was not known. Using protein engineering, we show that these insertions have distinct effects on CTS resistance in homologs of each of the two species that strongly depend on intramolecular interactions with other residues. Removing the insertion in the chinchilla NKA unexpectedly increases CTS resistance and decreases NKA activity. In the sandgrouse NKA, the amino acid insertion and substitution Q111R both contribute to an augmented CTS resistance without compromising ATPase activity levels. Molecular docking simulations provide additional insight into the biophysical mechanisms responsible for the context-specific mutational effects on CTS insensitivity of the enzyme. Our results highlight the diversity of genetic substrates that underlie CTS insensitivity in vertebrate NKA and reveal how amino-acid insertions can alter the phenotypic effects of point mutations at key sites in the same protein domain.

Studies in vertebrate genomics require sampling from a broad range of tissue types, taxa, and localities. Recent advancements in long-read and long-range genome sequencing have made it possible to produce high-quality chromosome-level genome assemblies for almost any organism. However, adequate tissue preservation for the requisite ultra-high molecular weight DNA (uHMW DNA) remains a major challenge. Here we present a comparative study of preservation methods for field and laboratory tissue sampling, across vertebrate classes and different tissue types. We find that no single method is best for all cases. Instead, the optimal storage and extraction methods vary by taxa, by tissue, and by down-stream application. Therefore, we provide sample preservation guidelines that ensure sufficient DNA integrity and amount required for use with long-read and long-range sequencing technologies across vertebrates. Our best practices generate the uHMW DNA needed for the high-quality reference genomes for Phase 1 of the Vertebrate Genomes Project (VGP), whose ultimate mission is to generate chromosome-level reference genome assemblies of all ~70,000 extant vertebrate species.

This presents a de novo genome assembly of Leptodactylus fuscus. High molecular weight DNA was extracted from a L. fuscus embryo which had been preserved in ethanol upon collection in Garzón, Huila, Colombia. The library was prepared and sequenced with 10X Genomics Chromium. Linked reads were processed by Long Ranger basic v2.2.2 and assembled with Supernova v2.1.1. The assembled genome is 2.42 Gb with 16,530 scaffolds >=10 kb, and scaffold N50 = 363 kb. The assembly size of contigs larger than 10 kb (1.26 Gb) is about half of the estimated genome size (2.4 Gb). 72.6% of the BUSCO Tetrapoda gene annotations (version odb10) were identified.

Gene duplication is an important source of evolutionary innovation, but the functional distinction between duplicates can be opposed by ongoing gene conversion between them. Here we document a tandem duplication of Na+,K+-ATPase subunit α1 (ATP1A1) sharedby frogs in the genus Leptodactylus,a group of species that feeds on toxic toads. One ATP1A1 paralog evolved resistance to toad toxins while the other paralog retained ancestral susceptibility. Frequent non-allelic gene conversion homogenized most of the paralog sequences,yet the two Leptodactylusparalogs are distinguished by 12 amino acid substitutions that were maintained by strong selection that counteracted gene conversion. Protein-engineering experiments show that two substitutions substantially increase toxin resistance, whereas the 10 additional substitutions mitigate deleterious pleiotropic effects on enzyme function. Our results highlight how neofunctionalized gene duplicates can help pinpoint functional substitutions and their interactions with the genetic backgrounds on which they arise.

Isthmian Central America (ICA) is one of the most biodiverse regions in the world, hosting an exceptionally high number of species per unit area. ICA was formed < 25 million years ago and, consequently, its biotic assemblage is relatively young and derived from both colonization and in situ diversification. Despite intensive taxonomic work on the local fauna, the potential forces driving genetic divergences and ultimately speciation in ICA remain poorly studied. Here, we used a landscape genetics approach to test whether isolation by distance, topography, habitat suitability, or environment drive the genetic diversity of the regional frog assemblage. To this end, we combined data on landscape features and mitochondrial DNA sequence variation for nine co-distributed amphibian species with disparate life histories. In five species, we found that at least one of the factors tested explained patterns of genetic divergence. However, rather than finding a general pattern, our results revealed idiosyncratic responses to historical and ecological processes, indicating that intrinsic life-history characteristics may determine the effect of different drivers of isolation on genetic divergence in ICA. Our work also suggests that the convergence of several factors promoting isolation among populations over a heterogeneous landscape might maximize genetic differentiation despite short geographical distances. In conclusion, abiotic factors and geographical features have differentially affected the genetic diversity across the regional frog assemblage. Much more complex models (i.e. considering multiple drivers), beyond simple vicariance of Caribbean and Pacific lineages are needed to better understand the evolutionary history of ICA’s diverse biotas.