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Integrating genomics and evolution into the conservation and breeding management of a Threatened Australian frog

This study integrates genome-wide SNP data and evolutionary methods to characterize population structure, genetic diversity, and founder relatedness in the threatened Southern Stuttering Frog (*Mixophyes australis*), providing a genomic framework to optimize its conservation breeding program and guide future reintroduction efforts.

Original authors: Kamryn Carter, Robyn E. Shaw, Garry Daly, Rachel Melrose, Amber R. Harrison, Jarrad Prangell, Aimee J. Silla, Phillip G. Byrne, Damien Esquerré

Published 2026-09-29

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CC BY 4.0

Original authors: Kamryn Carter, Robyn E. Shaw, Garry Daly, Rachel Melrose, Amber R. Harrison, Jarrad Prangell, Aimee J. Silla, Phillip G. Byrne, Damien Esquerré

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

In the quiet corners of temperate forests along Australia's eastern coast, a large, ground-dwelling frog known as the Southern Stuttering Frog faces a precarious future. This species, native to New South Wales, has suffered a dramatic decline over the last few decades, largely due to a devastating fungal disease that has wiped out populations across much of its range. When wild numbers drop too low, conservationists often turn to breeding programs, capturing animals to raise them in safety before releasing them back into the wild. However, keeping a species alive in captivity is not just about providing food and shelter; it is about preserving the invisible genetic blueprint that allows the species to adapt and survive. If a captive group is too closely related, the offspring may suffer from inbreeding, losing the genetic variety needed to fight disease or handle environmental changes. To manage this, scientists are increasingly turning to genomics, a field that reads the DNA of organisms to understand their family trees, diversity, and health without needing to rely on written records that often do not exist for wild animals.

A team of researchers has now applied these powerful genetic tools to the Southern Stuttering Frog, creating a roadmap for how to manage its recovery. The study focuses on a new conservation breeding program established at Symbio Wildlife Park, which began in 2022 with frogs collected from two separate creek systems in the wild. The scientists wanted to know if the frogs brought into captivity were genetically similar or different, how they compared to the remaining wild populations, and how best to pair them for breeding to keep the species healthy. By analyzing thousands of tiny variations in the frogs' DNA, the team uncovered a clear split in the species' history: the frogs living in the north are genetically distinct from those in the south, separated by a natural barrier that has stopped them from mixing for a long time.

The researchers found that the frogs currently held in the breeding program, sourced from the northern part of the species' range, are genetically healthy and diverse. They are not overly related to one another, which is a promising start for a captive population. However, the study also highlighted a troubling reality for the southern populations. The few remaining wild frogs in the south, and the few that survived in captivity before the current program began, show signs of severe genetic erosion. Their genetic diversity is low, and they carry high levels of inbreeding, a condition that makes them more vulnerable to extinction. The data suggests that the southern group has been cut off from the northern group for a long time, likely by the Hunter River region, creating two distinct genetic lineages within the same species.

This discovery has immediate implications for how conservationists should manage the breeding program. While the study suggests that mixing the northern and southern groups is unlikely to cause outbreeding depression based on predictive models, the researchers explicitly recommend conducting experimental tests first to confirm this. Strategically designed mating pairs crossing individuals both within and between the northern populations, as well as newly sourced captive groups, are now planned to quantify the outcome of population mixing on offspring viability. Within the northern group, the scientists used their genetic data to identify family relationships that were previously unknown. They found that while most frogs in the program are unrelated, a few are close relatives, such as parents and offspring or siblings. By knowing exactly who is related to whom, managers can now pair frogs that are not closely related, ensuring that the next generation retains as much genetic variety as possible.

The study also tested different ways of calculating these family relationships using DNA, comparing three common methods to see which worked best for this specific type of data. They found that while the exact numbers varied slightly between the methods, they all agreed on the ranking of who was most related to whom. This gives managers confidence that they can use these tools to make informed decisions even without a written family tree. The researchers suggest that the best strategy moving forward is to treat the captive population as a collection of smaller, semi-isolated groups that occasionally exchange individuals, mimicking the natural movement of frogs in the wild. This approach would prevent the entire group from becoming too similar while still allowing new genetic material to enter the mix.

Looking ahead, the team emphasizes the urgent need to find and study the last remaining wild frogs in the south, particularly a small group in the Watagans National Park. Without more samples from these southern survivors, it is impossible to know exactly how different they are from the northern frogs or whether they can ever be safely mixed. The researchers also call for continued monitoring of the breeding program and any future reintroduced populations to ensure they remain genetically healthy. By combining the science of evolution with the practical needs of conservation, this work provides a clear, data-driven path forward for saving the Southern Stuttering Frog, turning a collection of individual animals into a resilient population capable of surviving in a changing world.

Technical Summary: Integrating Genomics and Evolution into the Conservation and Breeding Management of a Threatened Australian Frog

Problem Statement
Conservation Breeding Programs (CBPs) are critical for safeguarding endangered species, yet they often lack the genomic data necessary to manage genetic diversity effectively, particularly when pedigree records are incomplete or absent. The Southern Stuttering Frog (Mixophyes australis), a large terrestrial amphibian endemic to temperate forests in eastern New South Wales (NSW), Australia, faces severe threats from habitat loss and Chytridiomycosis (caused by Batrachochytrium dendrobatidis). The species has experienced a significant range contraction, with populations south of Sydney largely extirpated. A new CBP was established in 2022 to create an insurance population and facilitate reintroduction. However, without a clear understanding of the population structure, gene flow patterns, and the genetic relatedness of founders, there is a risk of inbreeding depression, loss of adaptive potential, or outbreeding depression if distinct evolutionary lineages are mixed. The study addresses the challenge of integrating evolutionary biology and genomics to inform management decisions for this newly described threatened species.

Methodology
The study utilized genome-wide Single Nucleotide Polymorphism (SNP) data generated via Diversity Arrays Technology (DArTseq) to analyze two distinct datasets:

Conservation Dataset: 73 samples from the CBP founders at Symbio Wildlife Park (sourced from two wild creek systems, populations A and B) and five samples from a reintroduced population (R1).
Wild Dataset: 59 samples sourced from Mahony et al. (2023), comprising 55 Mixophyes australis samples from populations S1–S4 across the extant range (between Port Macquarie and Sydney) and four samples from the extinct range (between Sydney and Nowra).

Data processing involved rigorous filtering in RStudio using the dartRverse package to remove low-reproducibility loci, adjust for read depth, and filter for minor allele frequency. To address ascertainment bias between datasets generated at different times, analyses were often performed independently, with metrics standardized where cross-comparisons were necessary.

Key analytical approaches included:

Population Structure: Principal Coordinates Analysis (PCoA), pairwise fixation index ( $F_{ST}$ ) calculations, Isolation by Distance (IBD) testing via Mantel tests, and clustering analyses using Bayesian (STRUCTURE) and sparse non-negative matrix factorization (sNMF) methods.
Genetic Diversity: Calculation of observed ( $H_O$ ) and expected ( $H_E$ ) heterozygosity, inbreeding coefficients ( $F_{IS}$ ), allelic richness (AR), private alleles (PA), and effective population size ( $N_e$ ).
Relatedness Estimation: Comparison of three distinct estimators—Genomic Relationship Matrix (GRM), EMIBD9 (a likelihood-based expectation-maximization method), and TrioML (implemented in COANCESTRY)—to determine kinship coefficients ( $\Phi$ ) among founders. This included visualizing relatedness networks to identify close kin.

Key Results

Population Structure: Wild populations exhibit significant genetic structuring. There is a clear divergence between northern populations (S1–S3) and the southern population (S4), separated by the Hunter River region. The southern population (S4) is genetically distinct and shows very high differentiation ( $F_{ST} > 0.25$ ) from northern groups. Within the northern range, isolation by distance is evident, though S1 and S2 are more closely clustered despite geographic separation. In contrast, the CBP founders (Populations A and B) and the reintroduced population (R1) show minimal genetic differentiation ( $F_{ST} < 0.1$ ), indicating they originate from a genetically similar source pool.
Genetic Diversity: The southern population (S4) displays severely eroded genetic diversity, characterized by low heterozygosity, high inbreeding ( $F_{IS} = 0.599$ ), low allelic richness, and a critically low effective population size ( $N_e \approx 1.7$ ). Northern populations show moderate heterozygosity but varying levels of inbreeding; S3 appears the healthiest demographically, while S1 and S2 possess higher unique diversity (private alleles). The conservation population (A+B) exhibits moderate heterozygosity and low inbreeding, comparable to healthy wild populations, suggesting the founders captured a representative sample of genetic variation.
Relatedness and Methodology: All three relatedness estimators (GRM, EMIBD9, TrioML) produced consistent rankings of pairwise relatedness, though absolute values varied. GRM and TrioML estimated a low background kinship ( $\Phi \approx 0.007–0.015$ ) for the breeding population, equivalent to third cousins or unrelated individuals. EMIBD9 yielded higher estimates ( $\Phi \approx 0.09–0.10$ ), likely due to its design for highly inbred, small populations. Within the subset of 26 active breeders, GRM and TrioML identified approximately 8 parent-offspring/sibling relationships, one half-sibling, and two first cousins, while EMIBD9 identified fewer close relationships.

Significance and Claims
The paper claims to provide a foundational framework for the long-term genetic management of M. australis by demonstrating how genomic data can directly inform conservation breeding decisions in the absence of pedigree records. The study highlights the following contributions:

Management Unit Definition: It identifies a significant genetic break between northern and southern populations, suggesting that the southern lineage (S4) requires distinct management and that mixing northern and southern founders should be approached with caution pending further sampling of the remnant southern population.
Founder Assessment: It confirms that the current CBP founders are genetically diverse and not significantly structured, allowing for flexible mate-pairing strategies within the program without immediate risk of outbreeding depression.
Methodological Guidance: By comparing relatedness estimators, the authors provide a practical framework for managers, recommending a focus on the relative ranking of relatedness rather than absolute values when selecting breeding pairs. They suggest GRM for routine speed and TrioML for robustness, while cautioning against the use of EMIBD9 for larger, less inbred datasets.
Conservation Strategy: The findings support the strategy of maintaining multiple semi-isolated subpopulations with controlled exchange to preserve local adaptation while minimizing inbreeding. The study emphasizes the need for continued genetic monitoring, further sampling of the remnant southern population, and experimental crossing trials to validate the viability of mixing populations before reintroduction.

Ultimately, the paper asserts that integrating evolutionary processes and genomics into conservation assessments is vital for maintaining the adaptive potential of threatened species, particularly in the face of emerging diseases like Chytridiomycosis.

Technical Summary: Integrating Genomics and Evolution into the Conservation and Breeding Management of a Threatened Australian Frog

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