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Abstract Amphibians represent a diverse group of tetrapods, marked by deep divergence times between their three systematic orders and families. Studying amphibian biology through the genomics lens increases our understanding of the features of this animal class and that of other terrestrial vertebrates. The need for amphibian genomic resources is more urgent than ever due to the increasing threats to this group. Amphibians are one of the most imperiled taxonomic groups, with approximately 41% of species threatened with extinction due to habitat loss, changes in land use patterns, disease, climate change, and their synergistic effects. Amphibian genomic resources have provided a better understanding of ontogenetic diversity, tissue regeneration, diverse life history and reproductive modes, anti-predator strategies, and resilience and adaptive responses. They also serve as essential models for studying broad genomic traits, such as evolutionary genome expansions and contractions, as they exhibit the widest range of genome sizes among all animal taxa and possess multiple mechanisms of genetic sex determination. Despite these features, genome sequencing of amphibians has significantly lagged behind that of other vertebrates, primarily due to the challenges of assembling their large, repeat-rich genomes and the relative lack of societal support. The emergence of long-read sequencing technologies, combined with advanced molecular and computational techniques that improve scaffolding and reduce computational workloads, is now making it possible to address some of these challenges. To promote and accelerate the production and use of amphibian genomics research through international coordination and collaboration, we launched the Amphibian Genomics Consortium (AGC, https://mvs.unimelb.edu.au/amphibian-genomics-consortium ) in early 2023. This burgeoning community already has more than 282 members from 41 countries. The AGC aims to leverage the diverse capabilities of its members to advance genomic resources for amphibians and bridge the implementation gap between biologists, bioinformaticians, and conservation practitioners. Here we evaluate the state of the field of amphibian genomics, highlight previous studies, present challenges to overcome, and call on the research and conservation communities to unite as part of the AGC to enable amphibian genomics research to “leap” to the next level.

Abstract The legume albumin-1 gene family, arising after nodulation, encodes linear a- and b-chain peptides for nutrient storage and defense. Intriguingly, in one prominent legume, Clitoria ternatea, the b-chains are replaced by domains producing ultra-stable cyclic peptides called cyclotides. The mechanism of this gene hijacking is until now unknown. Cyclotides require recruitment of ligase-type asparaginyl endopeptidases (AEPs) for maturation (cyclization), necessitating co-evolution of two gene families. Here we compare a chromosome-level C. ternatea genome with grain legumes to reveal an 8 to 40-fold expansion of the albumin-1 gene family, enabling the additional loci to undergo diversification. Iterative rounds of albumin-1 duplication and diversification create four albumin-1 enriched genomic islands encoding cyclotides, where they are physically grouped by similar pI and net charge values. We identify an ancestral hydrolytic AEP that exhibits neofunctionalization and multiple duplication events to yield two ligase-type AEPs. We propose cyclotides arise by convergence in C. ternatea where their presence enhances defense from biotic attack, thus increasing fitness compared to lineages with linear b-chains and ultimately driving the replacement of b-chains with cyclotides.

Abstract Conservation breeding programs are an effective approach to addressing biodiversity loss. Captive populations are managed to maintain genetic diversity, yet there remains an “implementation gap” in effectively translating molecular genetic data into management. Technological advancements are facilitating rapid generation of genetic data, increasing accessibility for breeding programs. In 2010, Frankham and colleagues proposed a six‐stage process for establishing successful conservation breeding and release programs. Here, we describe the conservation breeding program for the critically endangered Bellinger River turtle (Myuchelys georgesi) and characterize the value of genetic sampling for informing management actions. By generating a chromosome‐level genome and population genetic data, we investigated past and present diversity and assessed relatedness among captive founders. We present a framework modeled on Frankham and colleagues six stages to assist managers in implementing genetic data into actionable conservation strategies. This framework, and worked case study, for managers aims to better guide implementation of genetic approaches into conservation breeding programs.

Abstract* The swift parrot (Lathamus discolor) is a Critically Endangered migratory parrot that breeds in Tasmania and winters on the Australian mainland. Here we provide a reference genome assembly for the swift parrot. We sequence PacBio HiFi reads to create a high-quality reference assembly and identify a complete mitochondrial sequence. We also generate a reference transcriptome from five organs to inform genome annotation. The genome was 1.24 Gb in length and consisted of 847 contigs with a contig N50 of 18.97 Gb and L50 of 20 contigs. This study provides an annotated reference assembly and transcriptomic resources for the swift parrot to assist in future conservation genomic research.

Abstract The number of genome-level resources for non-model species continues to rapidly expand. However, frog species remain underrepresented, with up to 90% of frog genera having no genomic or transcriptomic data. Here, we assemble the first genomic and transcriptomic resources for the recently described southern stuttering frog (Mixophyes australis). The southern stuttering frog is ground-dwelling, inhabiting naturally vegetated riverbanks in south-eastern Australia. Using PacBio HiFi long-read sequencing and Hi-C scaffolding, we generated a high-quality genome assembly, with a scaffold N50 of 369.3 Mb and 95.1% of the genome contained in twelve scaffolds. Using this assembly, we identified the mitochondrial genome, and assembled six tissue-specific transcriptomes. We also bioinformatically characterised novel sequences of two families of antimicrobial peptides (AMPs) in the southern stuttering frog, the cathelicidins and β-defensins. While traditional peptidomic approaches to peptide discovery have typically identified one or two AMPs in a frog species from skin secretions, our bioinformatic approach discovered 12 cathelicidins and two β-defensins that were expressed in a range of tissues. We investigated the novelty of the peptides and found diverse predicted activities. Our bioinformatic approach highlights the benefits of multi-omics resources in peptide discovery and contributes valuable genomic resources in an under-represented taxon.

The Kroombit tinkerfrog (Taudactylus pleione) is a stream-dwelling amphibian of the Myobatrachidae family. It is listed as Critically Endangered and is at high risk of extinction due to chytridiomycosis. Here, we provide the first genome assembly of the evolutionarily distinct Taudactylus genus. We sequenced PacBio HiFi reads to assemble a high-quality long-read genome and identified the mitochondrial genome. We also generated a global transcriptome from a tadpole to improve gene annotation. The genome was 5.52 Gb in length and consisted of 4,196 contigs with a contig N50 of 8.853 Mb and an L50 of 153. This study provides the first genomic resources for the Kroombit tinkerfrog to assist in future phylogenetic, environmental DNA, conservation breeding, and disease susceptibility studies.

The Kroombit tinkerfrog (Taudactylus pleione) is a stream-dwelling amphibian of the Myobatrachidae family. It is listed as Critically Endangered and is at high risk of extinction due to chytridiomycosis. Here, we provide the first genome assembly of the evolutionarily distinct Taudactylus genus. We sequenced PacBio HiFi reads to assemble a high-quality long-read genome and identified the mitochondrial genome. We also generated a global transcriptome from a tadpole to improve gene annotation. The genome was 5.52 Gb in length and consisted of 4,196 contigs with a contig N50 of 8.853 Mb and an L50 of 153. This study provides the first genomic resources for the Kroombit tinkerfrog to assist in future phylogenetic, environmental DNA, conservation breeding, and disease susceptibility studies.

The application of genetic data to conservation management programs can be hindered by the mismatch in timelines for management decisions and the acquisition of genetic data, particularly genomic sequence data that may require outsourcing. While applying genetic principles where data are absent can provide general guidelines for actions, genetic data can often fine-tune actions through adaptive management. We describe the adaptive genetic management of the establishment of a metapopulation of a small arboreal marsupial, the red-tailed phascogale (Phascogale calura). Two captive breeding programs were established as source populations, with genetic principles applied to the establishment of the first program and empirical genetic data used to guide the establishment of the second program. Genetic data from both programs were then used to allocate founders to three new populations to create a metapopulation with diversity both within and among the sites. Building and maintaining the diversity of metapopulations when recovering threatened species will reduce pressure on the original source populations and increase the resilience of the species.

Captive breeding could prevent species extinctions, but selection for captivity may decrease fitness. Here the authors analyse pedigree data on 15 long-running vertebrate breeding programs and find generational fitness changes that processes such as inbreeding depression cannot explain.

Abstract Incorporating mate choice into conservation breeding programs can improve reproduction and the retention of natural behaviors. However, different types of genetic‐based mate choice can have varied consequences for genetic diversity management. As a result, it is important to examine mechanisms of mate choice in captivity to assess its costs and benefits. Most research in this area has focused on experimental pairing trials; however, this resource‐intensive approach is not always feasible in captive settings and can interfere with other management constraints. We used generalized linear mixed models and permutation approaches to investigate overall breeding success in group‐housed Tasmanian devils at three nonmutually exclusive mate choice hypotheses: (a) advantage of heterozygous individuals, (b) advantage of dissimilar mates, and (c) optimum genetic distance, using both 1,948 genome‐wide SNPs and 12 MHC‐linked microsatellites. The managed devil insurance population is the largest such breeding program in Australia and is known to have high variance in reproductive success. We found that nongenetic factors such as age were the best predictors of breeding success in a competitive breeding scenario, with younger females and older males being more successful. We found no evidence of mate choice under the hypotheses tested. Mate choice varies among species and across environments, so we advocate for more studies in realistic captive management contexts as experimental or wild studies may not apply. Conservation managers must weigh up the need to wait for adequate sample sizes to detect mate choice with the risk that genetic changes may occur during this time in captivity. Our study shows that examining and integrating mate choice into the captive management of species housed in realistic, semi‐natural group‐based contexts may be more difficult than previously considered.

The value of genetic management is increasingly recognised in natural resource management particularly in relation to threatened species conservation and translocations. Technical advancements in methodologies available to conservation managers for genetic management present a challenge for long-running programs that monitor genetic diversity over time. Given that microsatellite and single nucleotide polymorphism (SNP) data are not interchangeable, conservation managers are faced with two options: continue using older techniques for consistency (limits genetic knowledge); or discard previous data and move to more precise modern genetic markers (wastes resources and prevents temporal monitoring). To achieve a balance, we should consider whether conservation recommendations made are broadly similar between interpretations of different marker sets, even if estimates are not directly comparable. Here we generated 13,194 SNPs for the woylie (Bettongia penicillata), an Australian marsupial, to provide conservation recommendations across their current range (N = 13 indigenous, reintroduced and haven populations) and provide guidance to conservation managers on how these recommendations align with those previously made using 12 microsatellite markers. Observed heterozygosity was significantly less than expected heterozygosity reflecting the historical bottleneck for the species. Only 4 of the 13 populations showed evidence of inbreeding, and effective population sizes ranged from 9.9 to 268.9. Trends in observed heterozygosity were broadly similar between microsatellite and SNP marker sets. Herein we provide advice for conservation managers undertaking translocations on using genetic data, highlight the benefits of moving to newer SNP based approaches for monitoring conservation actions over time, and show that historical management actions are supported by new technologies.

Abstract Background Recent advances in genomics have greatly increased research opportunities for non-model species. For wildlife, a growing availability of reference genomes means that population genetics is no longer restricted to a small set of anonymous loci. When used in conjunction with a reference genome, reduced-representation sequencing (RRS) provides a cost-effective method for obtaining reliable diversity information for population genetics. Many software tools have been developed to process RRS data, though few studies of non-model species incorporate genome alignment in calling loci. A commonly-used RRS analysis pipeline, Stacks, has this capacity and so it is timely to compare its utility with existing software originally designed for alignment and analysis of whole genome sequencing data. Here we examine population genetic inferences from two species for which reference-aligned reduced-representation data have been collected. Our two study species are a threatened Australian marsupial (Tasmanian devil Sarcophilus harrisii; declining population) and an Arctic-circle migrant bird (pink-footed goose Anser brachyrhynchus; expanding population). Analyses of these data are compared using Stacks versus two widely-used genomics packages, SAMtools and GATK. We also introduce a custom R script to improve the reliability of single nucleotide polymorphism (SNP) calls in all pipelines and conduct population genetic inferences for non-model species with reference genomes. Results Although we identified orders of magnitude fewer SNPs in our devil dataset than for goose, we found remarkable symmetry between the two species in our assessment of software performance. For both datasets, all three methods were able to delineate population structure, even with varying numbers of loci. For both species, population structure inferences were influenced by the percent of missing data. Conclusions For studies of non-model species with a reference genome, we recommend combining Stacks output with further filtering (as included in our R pipeline) for population genetic studies, paying particular attention to potential impact of missing data thresholds. We recognise SAMtools as a viable alternative for researchers more familiar with this software. We caution against the use of GATK in studies with limited computational resources or time.

Aquaculture, conservation, and biological research are reliant on the successful breeding of animals in captivity. Here, Farquharson et al. report that, in captivity, captive-born animals have decreased reproductive success compared to wild-born individuals, across diverse species and contexts.

* The swift parrot ( Lathamus discolor) is a Critically Endangered migratory parrot that breeds in Tasmania and winters on the Australian mainland. Here we provide a reference genome assembly for the swift parrot. We sequence PacBio HiFi reads to create a high-quality reference assembly and identify a complete mitochondrial sequence. We also generate a reference transcriptome from five organs to inform genome annotation. The genome was 1.24 Gb in length and consisted of 847 contigs with a contig N50 of 18.97 Gb and L50 of 20 contigs. This study provides an annotated reference assembly and transcriptomic resources for the swift parrot to assist in future conservation genomic research.

* The brown thornbill ( Acanthiza pusilla) is a songbird endemic to eastern Australia with five recognised subspecies within the brown thornbill. The most notable is the King Island brown thornbill ( Acanthiza pusilla magnirostris) of which there are less than 100 remaining and based on expert elicitation are the most likely Australian bird to become extinct in the next 20 years. We sequenced PacBio HiFi reads of the brown thornbill to generate a high-quality reference genome 1.25Gb in size and contig N50 of 20.1Mb. Additionally, we sequenced mRNA from three tissues to generate a global transcriptome to aid with genome annotation. The generation of a reference genome for the brown thornbill provides an important resource to align additional genomic data which will be produced in the near future.

* The swift parrot ( Lathamus discolor) is a Critically Endangered migratory parrot that breeds in Tasmania and winters on the Australian mainland. Here we provide a reference genome assembly for the swift parrot. We sequence PacBio HiFi reads to create a high-quality reference assembly and identify a complete mitochondrial sequence. We also generate a reference transcriptome from five organs to inform genome annotation. The genome was 1.24 Gb in length and consisted of 847 contigs with a contig N50 of 18.97 Gb and L50 of 20 contigs. This study provides an annotated reference assembly and transcriptomic resources for the swift parrot to assist in future conservation genomic research.

The Kroombit tinkerfrog ( Taudactylus pleione) is a stream-dwelling amphibian of the Myobatrachidae family. It is listed as Critically Endangered and is at high risk of extinction due to chytridiomycosis. Here, we provide the first genome assembly of the evolutionarily distinct Taudactylus genus. We sequenced PacBio HiFi reads to assemble a high-quality long-read genome and identified the mitochondrial genome. We also generated a global transcriptome from a tadpole to improve gene annotation. The genome was 5.52 Gb in length and consisted of 4,196 contigs with a contig N50 of 8.853 Mb and an L50 of 153. This study provides the first genomic resources for the Kroombit tinkerfrog to assist in future phylogenetic, environmental DNA, conservation breeding, and disease susceptibility studies.

The Kroombit tinkerfrog ( Taudactylus pleione) is a stream-dwelling amphibian of the Myobatrachidae family. It is listed as Critically Endangered and is at high risk of extinction due to chytridiomycosis. Here, we provide the first genome assembly of the evolutionarily distinct Taudactylus genus. We sequenced PacBio HiFi reads to assemble a high-quality long-read genome and identified the mitochondrial genome. We also generated a global transcriptome from a tadpole to improve gene annotation. The genome was 5.52 Gb in length and consisted of 4,196 contigs with a contig N50 of 8.853 Mb and an L50 of 153. This study provides the first genomic resources for the Kroombit tinkerfrog to assist in future phylogenetic, environmental DNA, conservation breeding, and disease susceptibility studies.