Planning and implementing genetic rescue of an endangered freshwater fish population in a regulated river, where low flow reduces breeding opportunities and may trigger inbreeding depression.

Augmenting depleted genetic diversity can improve the fitness and evolutionary potential of wildlife populations, but developing effective management approaches requires genetically monitored test cases. One such case is the small, isolated and inbred Cotter River population of an endangered Australian freshwater fish, the Macquarie perch Macquaria australasica, which over 3 years (2017-2019) received 71 translocated migrants from a closely related, genetically more diverse population. We used genetic monitoring to test whether immigrants bred, interbred with local fish and augmented population genetic diversity. We also investigated whether levels of river flow affected recruitment, inbreeding depression and juvenile dispersal. Fish length was used to estimate the age, birth year cohort and growth of 524 individuals born between 2016 and 2020 under variable flow conditions. DArT genome-wide genotypes were used to assess individual ancestry, heterozygosity, short-term effective population size and identify parent-offspring and full-sibling families. Of 442 individuals born after translocations commenced, only two (0.45%) were of mixed ancestry; these were half-sibs with one translocated parent in common. Numbers of breeders and genetic diversity for five birth year cohorts of the Cotter River fish were low, especially in low-flow years. Additionally, individuals born in the year of lowest flow evidently suffered from inbreeding depression for juvenile growth. The year of highest flow was associated with the largest number of breeders, lowest inbreeding in the offspring and greatest juvenile dispersal distances. Genetic diversity decreased in the upstream direction, flagging restricted access of breeders to the most upstream breeding sites, exacerbated by low river flow. Our results suggest that the effectiveness of translocations could be increased by focussing on upstream sites and moving more individuals per year; using riverine sources should be considered. Our results indicate that river flow sufficient to facilitate fish movement through the system would increase the number of breeders, promote individuals' growth, reduce inbreeding depression and promote genetic rescue.

Fish and macroinvertebrate assemblages reveal extensive degradation of the world's rivers.

Rivers suffer from multiple stressors acting simultaneously on their biota, but the consequences are poorly quantified at the global scale. We evaluated the biological condition of rivers globally, including the largest proportion of countries from the Global South published to date. We gathered macroinvertebrate- and fish-based assessments from 72,275 and 37,676 sites, respectively, from 64 study regions across six continents and 45 nations. Because assessments were based on differing methods, different systems were consolidated into a 3-class system: Good, Impaired, or Severely Impaired, following common guidelines. The proportion of sites in each class by study area was calculated and each region was assigned a Köppen-Geiger climate type, Human Footprint score (addressing landscape alterations), Human Development Index (HDI) score (addressing social welfare), % rivers with good ambient water quality, % protected freshwater key biodiversity areas; and % of forest area net change rate. We found that 50% of macroinvertebrate sites and 42% of fish sites were in Good condition, whereas 21% and 29% were Severely Impaired, respectively. The poorest biological conditions occurred in Arid and Equatorial climates and the best conditions occurred in Snow climates. Severely Impaired conditions were associated (Pearson correlation coefficient) with higher HDI scores, poorer physico-chemical water quality, and lower proportions of protected freshwater areas. Good biological conditions were associated with good water quality and increased forested areas. It is essential to implement statutory bioassessment programs in Asian, African, and South American countries, and continue them in Oceania, Europe, and North America. There is a need to invest in assessments based on fish, as there is less information globally and fish were strong indicators of degradation. Our study highlights a need to increase the extent and number of protected river catchments, preserve and restore natural forested areas in the catchments, treat wastewater discharges, and improve river connectivity.

A national-scale dataset for threats impacting Australia's imperiled flora and fauna.

Australia is in the midst of an extinction crisis, having already lost 10% of terrestrial mammal fauna since European settlement and with hundreds of other species at high risk of extinction. The decline of the nation's biota is a result of an array of threatening processes; however, a comprehensive taxon-specific understanding of threats and their relative impacts remains undocumented nationally. Using expert consultation, we compile the first complete, validated, and consistent taxon-specific threat and impact dataset for all nationally listed threatened taxa in Australia. We confined our analysis to 1,795 terrestrial and aquatic taxa listed as threatened (Vulnerable, Endangered, or Critically Endangered) under Australian Commonwealth law. We engaged taxonomic experts to generate taxon-specific threat and threat impact information to consistently apply the IUCN Threat Classification Scheme and Threat Impact Scoring System, as well as eight broad-level threats and 51 subcategory threats, for all 1,795 threatened terrestrial and aquatic threatened taxa. This compilation produced 4,877 unique taxon-threat-impact combinations with the most frequently listed threats being Habitat loss, fragmentation, and degradation (n = 1,210 taxa), and Invasive species and disease (n = 966 taxa). Yet when only high-impact threats or medium-impact threats are considered, Invasive species and disease become the most prevalent threats. This dataset provides critical information for conservation action planning, national legislation and policy, and prioritizing investments in threatened species management and recovery.

Reproductive ecology of a critically endangered alpine galaxiid.

Stocky galaxias Galaxias tantangara is a newly described freshwater fish restricted to a single population, occupying a 3 km reach of a small headwater stream in the upper Murrumbidgee River catchment of south-eastern Australia. This species is listed as critically endangered under IUCN Red List criteria, and knowledge of the species' ecology is critical for future conservation efforts to establish additional populations by translocation and captive breeding. This study details the first account of spawning and reproductive ecology of G. tantangara, including reproductive development, timing of spawning and a description of one spawning site. Peak gonadosomatic index was observed in March/April in males and in October in females. Absolute fecundity ranged from 211 oocytes for a 76 mm length to caudal fork (LCF) fish to 810 oocytes for a 100 mm LCF fish. The observation of spent females in mid-November 2017 and discovery of an egg mass 8 days later suggest that spawning had occurred, and over a relatively short period. Larvae were subsequently detected in monthly electrofishing surveys in December 2017. Findings from this study provide new understanding of existing and future threats to G. tantangara and have important implications for conservation management of not only this species but also other closely related threatened Galaxias species.

Multi-year pair-bonding in Murray cod (Maccullochella peelii).

Mating strategies in fishes are known to include polygyny, polyandry and monogamy and provide valuable insights regarding powerful evolutionary forces such as sexual selection. Monogamy is a complex of mating systems that has been relatively neglected. Previous work on mating strategies in fishes has often been based on observation and focused on marine species rather than freshwater fishes. SNPs are increasingly being used as a molecular ecology tool in non-model organisms, and methods of probabilistic genetic analysis of such datasets are becoming available for use in the absence of parental genotypes. This approach can be used to infer mating strategies. The long-term pair bonding seen in mammals, reptiles and birds has not been recorded in freshwater fishes-in every other respect an extremely diverse group. This study shows that multi-year pair bonding occurs in an Australian Percichthyid fish that exhibits paternal care of eggs and larvae. Using SNPs, full sibling pairs of larvae were found over multiple years in a three-year study. Stable isotope signatures of the larvae support the genetic inference that full sibling pairs shared a common mother, the ultimate source of that isotopic signature during oogenesis. Spatial and temporal clustering also suggests that the full sibling larvae are unlikely to be false positive identifications of the probabilistic identification of siblings. For the first time, we show multi-year pair bonding in a wild freshwater fish. This will have important conservation and management implications for the species. This approach could provide insights into many behavioural, ecological and evolutionary questions, particularly if this is not a unique case. Our findings are likely to initiate interest in seeking more examples of monogamy and alternative mating strategies in freshwater fishes, particularly if others improve methods of analysis of SNP data for identification of siblings in the absence of parental genotypes.

Impact of 2019-2020 mega-fires on Australian fauna habitat.

Australia's 2019-2020 mega-fires were exacerbated by drought, anthropogenic climate change and existing land-use management. Here, using a combination of remotely sensed data and species distribution models, we found these fires burnt ~97,000 km2 of vegetation across southern and eastern Australia, which is considered habitat for 832 species of native vertebrate fauna. Seventy taxa had a substantial proportion (>30%) of habitat impacted; 21 of these were already listed as threatened with extinction. To avoid further species declines, Australia must urgently reassess the extinction vulnerability of fire-impacted species and assist the recovery of populations in both burnt and unburnt areas. Population recovery requires multipronged strategies aimed at ameliorating current and fire-induced threats, including proactively protecting unburnt habitats.

Signatures of polygenic adaptation associated with climate across the range of a threatened fish species with high genetic connectivity.

Adaptive differences across species' ranges can have important implications for population persistence and conservation management decisions. Despite advances in genomic technologies, detecting adaptive variation in natural populations remains challenging. Key challenges in gene-environment association studies involve distinguishing the effects of drift from those of selection and identifying subtle signatures of polygenic adaptation. We used paired-end restriction site-associated DNA sequencing data (6,605 biallelic single nucleotide polymorphisms; SNPs) to examine population structure and test for signatures of adaptation across the geographic range of an iconic Australian endemic freshwater fish species, the Murray cod Maccullochella peelii. Two univariate gene-association methods identified 61 genomic regions associated with climate variation. We also tested for subtle signatures of polygenic adaptation using a multivariate method (redundancy analysis; RDA). The RDA analysis suggested that climate (temperature- and precipitation-related variables) and geography had similar magnitudes of effect in shaping the distribution of SNP genotypes across the sampled range of Murray cod. Although there was poor agreement among the candidate SNPs identified by the univariate methods, the top 5% of SNPs contributing to significant RDA axes included 67% of the SNPs identified by univariate methods. We discuss the potential implications of our findings for the management of Murray cod and other species generally, particularly in relation to informing conservation actions such as translocations to improve evolutionary resilience of natural populations. Our results highlight the value of using a combination of different approaches, including polygenic methods, when testing for signatures of adaptation in landscape genomic studies.

Severe consequences of habitat fragmentation on genetic diversity of an endangered Australian freshwater fish: A call for assisted gene flow.

Genetic diversity underpins the ability of populations to persist and adapt to environmental changes. Substantial empirical data show that genetic diversity rapidly deteriorates in small and isolated populations due to genetic drift, leading to reduction in adaptive potential and fitness and increase in inbreeding. Assisted gene flow (e.g. via translocations) can reverse these trends, but lack of data on fitness loss and fear of impairing population "uniqueness" often prevents managers from acting. Here, we use population genetic and riverscape genetic analyses and simulations to explore the consequences of extensive habitat loss and fragmentation on population genetic diversity and future population trajectories of an endangered Australian freshwater fish, Macquarie perch Macquaria australasica. Using guidelines to assess the risk of outbreeding depression under admixture, we develop recommendations for population management, identify populations requiring genetic rescue and/or genetic restoration and potential donor sources. We found that most remaining populations of Macquarie perch have low genetic diversity, and effective population sizes below the threshold required to retain adaptive potential. Our simulations showed that under management inaction, smaller populations of Macquarie perch will face inbreeding depression within a few decades, but regular small-scale translocations will rapidly rescue populations from inbreeding depression and increase adaptive potential through genetic restoration. Despite the lack of data on fitness loss, based on our genetic data for Macquarie perch populations, simulations and empirical results from other systems, we recommend regular and frequent translocations among remnant populations within catchments. These translocations will emulate the effect of historical gene flow and improve population persistence through decrease in demographic and genetic stochasticity. Increasing population genetic connectivity within each catchment will help to maintain large effective population sizes and maximize species adaptive potential. The approach proposed here could be readily applicable to genetic management of other threatened species to improve their adaptive potential.

Methods to maximise recovery of environmental DNA from water samples.

The environmental DNA (eDNA) method is a detection technique that is rapidly gaining credibility as a sensitive tool useful in the surveillance and monitoring of invasive and threatened species. Because eDNA analysis often deals with small quantities of short and degraded DNA fragments, methods that maximize eDNA recovery are required to increase detectability. In this study, we performed experiments at different stages of the eDNA analysis to show which combinations of methods give the best recovery rate for eDNA. Using Oriental weatherloach (Misgurnus anguillicaudatus) as a study species, we show that various combinations of DNA capture, preservation and extraction methods can significantly affect DNA yield. Filtration using cellulose nitrate filter paper preserved in ethanol or stored in a -20°C freezer and extracted with the Qiagen DNeasy kit outperformed other combinations in terms of cost and efficiency of DNA recovery. Our results support the recommendation to filter water samples within 24hours but if this is not possible, our results suggest that refrigeration may be a better option than freezing for short-term storage (i.e., 3-5 days). This information is useful in designing eDNA detection of low-density invasive or threatened species, where small variations in DNA recovery can signify the difference between detection success or failure.

Exposure of the freshwater bivalve Hyridella australis to metal contaminated sediments in the field and laboratory microcosms: metal uptake and effects.

Metal uptake and induced toxic effects on Hyridella australis were investigated by establishing 28 day exposure-dose-response relationships (EDR) of transplanted H. australis at four sites along a sediment metal contamination gradient in the mine affected Molonglo River, NSW. Laboratory exposure of this organism to the same sediments, collected from in situ sites, was run concurrently. Metal concentrations in whole organisms, individual tissues and sub-cellular tissue fractions were measured as organism metal dose. Total antioxidant capacity (TAOC), lipid peroxidation (MDA) and lysosomal membrane destabilisation (LMS) were measured as biological responses. H. australis accumulated significantly higher tissue zinc concentrations compared to the other metals. In situ organisms at the mine affected sites accumulated more metals than organisms in laboratory microcosms. Accumulated zinc, cadmium and the total metal concentrations in whole organism tissues reflected exposure-dose relationships. Sub-cellular analysis showed that most of the accumulated metals, both in the field and laboratory exposed organisms, were detoxified over 28 days exposure. Clear exposure and dose dependent responses of decreased TAOC and measurable increases in MDA and LMS with increased metal exposure and dose were evident in H. australis caged in the river. In contrast, a dose-response relationship was only evident for cadmium in laboratory exposed organisms. Organisms caged at mine affected sites showed stronger EDR relationships than those exposed in laboratory microcosms as they were exposed to additional sources of dissolved zinc and cadmium. Exposure in laboratory microcosms underestimated metal uptake and effects, thus assessment of metal contaminated sediments should be undertaken "in situ".

Who's your mama? Riverine hybridisation of threatened freshwater Trout Cod and Murray Cod.

Rates of hybridization and introgression are increasing dramatically worldwide because of translocations, restocking of organisms and habitat modifications; thus, determining whether hybridization is occuring after reintroducing extirpated congeneric species is commensurately important for conservation. Restocking programs are sometimes criticized because of the genetic consequences of hatchery-bred fish breeding with wild populations. These concerns are important to conservation restocking programs, including those from the Australian freshwater fish family, Percichthyidae. Two of the better known Australian Percichthyidae are the Murray Cod, Maccullochella peelii and Trout Cod, Maccullochella macquariensis which were formerly widespread over the Murray Darling Basin. In much of the Murrumbidgee River, Trout Cod and Murray Cod were sympatric until the late 1970s when Trout Cod were extirpated. Here we use genetic single nucleotide polymorphism (SNP) data together with mitochondrial sequences to examine hybridization and introgression between Murray Cod and Trout Cod in the upper Murrumbidgee River and consider implications for restocking programs. We have confirmed restocked riverine Trout Cod reproducing, but only as inter-specific matings, in the wild. We detected hybrid Trout Cod-Murray Cod in the Upper Murrumbidgee, recording the first hybrid larvae in the wild. Although hybrid larvae, juveniles and adults have been recorded in hatcheries and impoundments, and hybrid adults have been recorded in rivers previously, this is the first time fertile F1 have been recorded in a wild riverine population. The F1 backcrosses with Murray cod have also been found to be fertile. All backcrosses noted were with pure Murray Cod. Such introgression has not been recorded previously in these two species, and the imbalance in hybridization direction may have important implications for restocking programs.

Detection of dwarf gourami iridovirus (Infectious spleen and kidney necrosis virus) in populations of ornamental fish prior to and after importation into Australia, with the first evidence of infection in domestically farmed Platy (Xiphophorus maculatus).

The movement of ornamental fish through international trade is a major factor for the transboundary spread of pathogens. In Australia, ornamental fish which may carry dwarf gourami iridovirus (DGIV), a strain of Infectious spleen and kidney necrosis virus (ISKNV), have been identified as a biosecurity risk despite relatively stringent import quarantine measures being applied. In order to gain knowledge of the potential for DGIV to enter Australia, imported ornamental fish were sampled prior to entering quarantine, during quarantine, and post quarantine from wholesalers and aquatic retail outlets in Australia. Samples were tested by quantitative polymerase chain reaction (qPCR) for the presence of megalocytivirus. Farmed and wild ornamental fish were also tested. Megalocytivirus was detected in ten of fourteen species or varieties of ornamental fish. Out of the 2086 imported gourami tested prior to entering quarantine, megalocytivirus was detected in 18.7% of fish and out of the 51 moribund/dead ornamental fish tested during the quarantine period, 68.6% were positive for megalocytivirus. Of fish from Australian wholesalers and aquatic retail outlets 14.5% and 21.9%, respectively, were positive. Out of 365 farmed ornamental fish, ISKNV-like megalocytivirus was detected in 1.1%; these were Platy (Xiphophorus maculatus). Megalocytivirus was not detected in free-living breeding populations of Blue gourami (Trichopodus trichopterus) caught in Queensland. This study showed that imported ornamental fish are vectors for DGIV and it was used to support an import risk analysis completed by the Australian Department of Agriculture. Subsequently, the national biosecurity policy was revised and from 1 March 2016, a health certification is required for susceptible families of fish to be free of this virus prior to importation.