Immediate genetic augmentation and enhanced habitat connectivity are required to secure the future of an iconic endangered freshwater fish population

Genetic diversity is rapidly lost from small, isolated populations by genetic drift. Measuring the level of genetic drift using effective population size (Ne) is highly useful for management. Single-cohort genetic Ne estimators approximate the number of breeders in one season (Nb): a value <100 signals likely inbreeding depression. Per-generation Ne <1000 estimated from multiple cohorts signals reduced adaptive potential. Natural populations rarely meet assumptions of Ne-estimation, so interpreting estimates is challenging. Macquarie perch is an endangered Australian freshwater fish threatened by severely reduced range, habitat loss and fragmentation. To counteract low Ne, augmented gene flow is being implemented in several populations. In the Murrumbidgee River, unknown effects of water management on among-site connectivity impede the design of effective interventions.

Using DArT SNPs for 328 Murrumbidgee individuals sampled across several sites and years with different flow conditions, we assessed population structure, site isolation, heterozygosity, inbreeding, and Ne. We tested for inbreeding depression, assessed genetic diversity and dispersal and evaluated whether individuals translocated from Cataract Reservoir to the Murrumbidgee River bred, and interbred with local fish.

We found strong genetic structure, indicating complete or partial isolation of river fragments. This structure violates assumptions of Ne estimation, resulting in strongly downwardly biased Nb estimates unless assessed per-site, highlighting the necessity to account for population structure while estimating Ne. Inbreeding depression was not detected, but with low Nb at each site, inbreeding and inbreeding depression are likely. These results flagged the necessity to address within-river population connectivity through flow-management and genetic mixing through translocations among sites and from other populations. Three detected genetically diverse offspring of a translocated Cataract fish and a local parent indicated that genetic mixing is in progress. Including admixed individuals in estimates yielded lower Ne but higher heterozygosity, suggesting heterozygosity is a preferable indicator of genetic augmentation.

Methods
This repository contains genetic and phenotypic data for southeatern Australian freshwater fish, Macquarie perch, analyzed in Pavlova A, Pearce L, Sturgiss F, Lake E, Sunnucks P, & Lintermans M. (2024). Immediate genetic augmentation and enhanced habitat connectivity are required to secure the future of an iconic endangered freshwater fish population. Evolutionary Applications, doi: 10.1111/eva.70019/

Genetic data- SNP genotypes from reduced representation sequencing data- were obtained by Diversity Arrays Technology (DArT). File Report_DMacq23-8576_6_moreOrders_SNP_mapping_2.csv is original DArT genotype data file for Macquarie perch (Macquaria australasica), an output of DS14 pipeline by Diversity Arrays Technology. Genotypes are for individuals from Murrumbidgee River and Cataract Reservoir populations (the only ones used during SNP genotyping). SNP genotypes are obtained from reduced-representation sequencing data.

This data was filtered and analyzed in R using script 2.Analyses_of_DArT_genotypes.R. This script is run to filter genotypes to remove loci with mean read depth <6 and >50, loci with reproducibility <95%, loci missing in >10% of individuals, individuals with >20% of missing data, loci with heterozygosity significantly higher than 0.5 in either Murrumbidgee (analysed as a whole) or Cataract Reservoir. One random SNP per DArT-tag was retained, to reduce loci that are highly non-independent through very close physical linkage. The final complete dataset of 3,447 biallelic SNPs scored for 375 individuals (328 Murrumbidgee, 47 Cataract Reservoir) and its subsets were used for assessment of genetic diversity, population structure, effective population sizes. A subset of data comprising adult individuals was analyzed for population structure, FST and isolation-by-distance, after retaining a single SNP locus within a 50-Kb fragment.

Apart from the genotype file, three additional files used as input by this script are provided: Murrumbidgee_CataractDam_covariate.age.cohort.growth.csv - a covariate file with additional information for each genotyped individual; column descriptions are given in the R script; Dgeo.csv- a matrix of river distances (in km) for isolation-by-distance analysis; and Dgen.csv- a matrix of FST-values for isolation-by-distance analysis, produced by sambar during analyses of adults. 

All results of genetic and phenotypic analyses for each individual are summarized in MurrCat_data_results.csv.