Environmental DNA survey in areas of the Kimberley region at risk of invasion by cane toads

No cane toad eDNA was detected at any of the sites. However, at Lake Gregory, Paruku Rangers observed cane toads near the sampling site during field work. We attribute the false negative detections to the small number of sites covered at this large site, as well as the small volume of water sampled. This highlights the importance of using multiple detection methods when carrying out cane toad monitoring. In the future, large water samples should be collected to avoid false negative detections. Continuous sample collection during rangers’ programmed activities will complement monitoring programs and target early incursions of cane toads in the area

Standard operating procedure for environmental DNA field sample collection

The environmental DNA (eDNA) technique represents a sensitive tool that allows for cost-effective monitoring of plant and animal species across large geographical areas. This standard operating procedure provides a step-by-step guide to a simple eDNA water sample collection method developed for use by non-scientists

New Australian frontier in freshwater fish invasion via Torres Strait Islands

All continents, excluding Antarctica and the Artic, have been affected by incursion from alien freshwater fish species. Australia has not been spared. Four hundred and fifty species have now been declared on the ornamental importation list, making management a real challenge. With approximately 25 non-native species documented, Papua New Guinea (PNG) has likely some problems with invasive freshwater fish. Many of these species have been intentionally introduced to increase access to food as a protein source for remote communities or have spread naturally from western parts of Java and Indonesia, and now constitute a large biomass on some floodplain areas in PNG. The Torres Strait is located between PNG and northern Queensland and was previously a land bridge, though now under higher sea levels the region exists as a series of approximately 300 islands. The threat of further range extension of freshwater fish from PNG into northern Queensland via the Torres Strait Islands is significant, with two invasive fish species already recorded on northern islands of the Torres Strait (climbing perch, Anabas testudineus which has been continually recorded for the past decade; and recently the GIFT tilapia, Oreochromis niloticus). Here we present a case to control further spread of invasive freshwater fish species towards Australia, using a Land and Sea Ranger program, where Rangers are trained to be confident in the identification of pest fish species and to implement strategies to protect their borders from potential future incursions. The success of this program relies on Rangers to continue partaking in surveillance monitoring of coastal waters, checking and controlling for any new invasive species moving from PNG into Australian waters. We outline the biosecurity obligation under Article 14 of the Treaty between the two nations, which identifies the importance of conservation and protection of coastal floodplains from invasive species, and the spread between both nations

Environmental DNA analysis confirms extant populations of the cryptic Irwin’s turtle within its historical range

Background Approximately 50% of freshwater turtles worldwide are currently threatened by habitat loss, rural development and altered stream flows. Paradoxically, reptiles are understudied organisms, with many species lacking basic geographic distribution and abundance data. The iconic Irwin’s turtle, Elseya irwini, belongs to a unique group of Australian endemic freshwater turtles capable of cloacal respiration. Water resource development, increased presence of saltwater crocodiles and its cryptic behaviour, have made sampling for Irwin’s turtle in parts of its range problematic, resulting in no confirmed detections across much of its known range for > 25 years. Here, we used environmental DNA (eDNA) analysis for E. irwini detection along its historical and contemporary distribution in the Burdekin, Bowen and Broken River catchments and tributaries. Five replicate water samples were collected at 37 sites across those three river catchments. Environmental DNA was extracted using a glycogen-aided precipitation method and screened for the presence of E. irwini through an eDNA assay targeting a 127 base pair-long fragment of the NADH dehydrogenase 4 (ND4) mitochondrial gene. Results Elseya irwini eDNA was detected at sites within its historic distribution in the lower Burdekin River, where the species had not been formally recorded for > 25 years, indicating the species still inhabits the lower Burdekin area. We also found higher levels of E. iriwni eDNA within its contemporary distribution in the Bowen and Broken Rivers, matching the prevailing scientific view that these areas host larger populations of E. irwini. Conclusions This study constitutes the first scientific evidence of E. irwini presence in the lower Burdekin since the original type specimens were collected as part of its formal description, shortly after the construction of the Burdekin Falls Dam. From the higher percentage of positive detections in the upper reaches of the Broken River (Urannah Creek), we conclude that this area constitutes the core habitat area for the species. Our field protocol comprises a user-friendly, time-effective sampling method. Finally, due to safety risks associated with traditional turtle sampling methods in the Burdekin River (e.g., estuarine crocodiles) we propose eDNA sampling as the most pragmatic detection method available for E. irwini

Long distance (>20 km) downstream detection of endangered stream frogs suggests an important role for eDNA in surveying for remnant amphibian populations

Background Globally, amphibian species have suffered drastic population declines over the past 40 years. Hundreds of species are now listed as Critically Endangered, with many of these considered “possibly extinct”. Most of these species are stream-dwelling frogs inhabiting remote, montane areas, where remnant populations are hard to find using traditional surveys. Environmental DNA (eDNA) could revolutionize surveys for ‘missing’ and endangered amphibian populations by screening water samples from downstream sections to assess presence in the upstream catchments. However, the utility of this survey technique is dependent on quantifying downstream detection probability and distances. Methods Here we tested downstream detection distances in two endangered stream frogs (Litoria lorica and L. nannotis) that co-occur in a remote stream catchment in north-east Australia, and for which we know precise downstream distributional limits from traditional surveys. Importantly, the two last populations of L. lorica persist in this catchment: one small (~1,000 frogs) and one very small (~100 frogs). We conducted eDNA screening at a series of sites kilometers downstream from the populations using precipitation from two fixed water volumes (15 and 100 mL) and via water filtering (mean 1,480 L). Results We detected L. nannotis and the small L. lorica population (~1,000 frogs) at most sampling sites, including 22.8 km downstream. The filtration method was highly effective for far-downstream detection, as was precipitation from 100 mL water samples, which also resulted in consistent detections at the far-downstream sites (including to 22.8 km). In contrast, we had limited downstream detection success for the very small L. lorica population (~100 frogs). Discussion The ecological aspects of our study system, coupled with thorough traditional surveys, enabled us to measure downstream eDNA detection distances with accuracy. We demonstrate that eDNA from a small population of approximately 1,000 frogs can be detected as far as 22.8 km downstream from the population. Water filtration is considered best for eDNA detection of rare aquatic species—indeed it was effective in this study—but we also achieved far-downstream detections when precipitating eDNA from 100 mL water samples. Collecting small water volumes for subsequent precipitation in the lab is more practical than filtration when surveying remote areas. Our downstream detection distances (>20 km) suggest eDNA is a valuable tool for detecting rare stream amphibians. We provide recommendations on optimal survey methods

Telomere dynamics in the Pacific crown-of-thorns seastar (Acanthaster cf. solaris): effect of age, diet, and tissue type

Population irruptions of crown-of-thorns seastar (CoTS, Acanthaster spp.) represent a perennial threat to Indo-Pacific coral reefs. Age determination of CoTS is challenging, thereby hindering understanding and management of this nuisance species. Telomeres, which are protective DNA structure found at the ends of eukaryotic chromosomes that shorten at each cell division, have been used to estimate age in wild animals. To investigate the use of telomeres in CoTS, we optimized a quantitative PCR protocol to measure relative telomere length (rTL) in CoTS for the first time. Comparing rTL among four age groups (4, 7, 16, > 24 months post-settlement), we found that adult CoTS generally exhibit shorter rTL than juveniles, which is the first evidence of age-related telomere attrition in CoTS. However, there was large within-age class variation, and no significant relationships were found between adult CoTS rTL and potential age-indicating external features. Furthermore, we found accelerated telomere attrition under sub-optimal diet, where individuals that were fed crustose coralline algae for 16 months exhibited shorter rTL than their counterparts fed on coral. A positive correlation was found between rTL of tube feet and pyloric caeca, suggesting synchronization of telomere dynamics across somatic tissues in CoTS. Overall, our results suggest that rTL could be used to classify CoTS into broad age groups, though individual variation constrains the ability to resolve specific cohorts. The present study contributes to the understanding of telomere dynamics in marine invertebrates, while laying the groundwork for future research into rTL as biomarker for age and potentially stress for CoTS

Can environmental DNA be used to detect first arrivals of the cane toad, Rhinella marina, into novel locations?

Eradicating invasive species is difficult, but success is more likely when populations are small after arrival. The cane toad, Rhinella marina, is an invasive pest species that threatens native fauna worldwide. Increasingly, environmental DNA (eDNA) is used as a technique to monitor the presence of invasive species given its power to detect low numbers of individuals. We aimed to investigate eDNA persistence in freshwater at three different temperatures (25, 30 and 35°C) and eDNA detection thresholds for R. marina using controlled experiments in aquaria. For the latter, two water volumes and two cane toad exposure times were used (800 or 200 L volume with 5 or 30 min exposure). A 15‐ml water sample was collected from each replicated aquaria and preserved with 5 ml Longmire's buffer. Environmental DNA was extracted and four technical quantitative PCR replicates were analyzed targeting the cane toad 16S rDNA mitochondrial gene. Environmental DNA decayed rapidly in water and was reliably detected for up to 3 days after cane toad removal, regardless of the temperature treatment. Also, cane toad eDNA was detected in the water after a 5‐min initial exposure of a single individual in 800 L of water. Under the physical parameters tested here, a positive detection means that a cane toad has been in contact with the water body between 1 and 3 days prior to the sampling event. The results of the present study show the importance of eDNA for determining the presence of a species that occurs at low abundance in a small water body, such as at the onset of a cane toad invasion

Disentangling genetic from environmental effects on phenotypic variability of Southern rock lobster (Jasus edwardsii) postlarvae

Environmental conditions experienced during larval dispersal of marine organisms can determine size-at-settlement of recruits. It is, therefore, not uncommon that larvae undergoing different dispersal histories would exhibit phenotypic variability at recruitment. Here we investigated morphological differences in recently settled southern rock lobster (Jasus edwardsii) recruits, known as pueruli, along a latitudinal and temporal gradient on the east coast of Tasmania, Australia. We further explored whether natural selection could be driving morphological variation. We used double digest restriction-site associated DNA sequencing (ddRADseq) to assess differences in genetic structure of recently settled recruits on the east coast of Tasmania over three months of peak settlement during 2012 (August, September and October). Phenotypic differences in pueruli between sites and months of settlement were observed, with significantly smaller individuals found at the northernmost site. Also, there was a lack of overall genetic divergence; however, significant differences in pairwise FST values between settlement months were observed at the southernmost study site, located at an area of confluence of ocean currents. Specifically, individuals settling into the southernmost earlier in the season were genetically different from those settling later. The lack of overall genetic divergence in the presence of phenotypic variation indicates that larval environmental history during dispersal of J. edwardsii could be a possible driver of the resulting phenotype of settlers

Temporal genetic patterns of diversity and structure evidence chaotic genetic patchiness in a spiny lobster

Population structure of many marine organisms is spatially patchy and varies within and between years, a phenomenon defined as chaotic genetic patchiness. This results from the combination of planktonic larval dispersal and environmental stochasticity. Additionally, in species with bi-partite life, postsettlement selection can magnify these genetic differences. The high fecundity (up to 500,000 eggs annually) and protracted larval duration (12–24 months) and dispersal of the southern rock lobster, Jasus edwardsii, make it a good test species for chaotic genetic patchiness and selection during early benthic life. Here, we used double digest restriction site-associated DNA sequencing (ddRADseq) to investigate chaotic genetic patchiness and postsettlement selection in this species. We assessed differences in genetic structure and diversity of recently settled pueruli across four settlement years and between two sites in southeast Australia separated by approximately 1,000 km. Postsettlement selection was investigated by identifying loci under putative positive selection between recently settled pueruli and postpueruli and quantifying differences in the magnitude and strength of the selection at each year and site. Genetic differences within and among sites through time in neutral SNP markers indicated chaotic genetic patchiness. Recently settled puerulus at the southernmost site exhibited lower genetic diversity during years of low puerulus catches, further supporting this hypothesis. Finally, analyses of outlier SNPs detected fluctuations in the magnitude and strength of the markers putatively under positive selection over space and time. One locus under putative positive selection was consistent at both locations during the same years, suggesting the existence of weak postsettlement selection

Practical eDNA sampling methods inferred from particle size distribution and comparison of capture techniques for a Critically Endangered elasmobranch

Environmental DNA (eDNA) methods are increasingly applied in the marine environment to identify species and community structure. To establish widely applicable eDNA techniques for elasmobranchs, we used the Critically Endangered largetooth sawfish (Pristis pristis Linnaeus, 1758) as a model species for: (1) assessing eDNA particle size distribution; (2) assessing the efficiency of long-term preservation of water samples; and (3) comparing the efficiency and detection sensitivity of filtration and precipitation methods. Water samples (1 L) collected from a tank containing one largetooth sawfish specimen were sequentially filtered through five filter membranes of decreasing pore size (20, 10, 5, 1.2, and 0.45 μm). The proportion of sawfish eDNA retained within each size class was determined through quantitative real-time PCR (qPCR) using a species-specific TaqMan probe assay. A linear mixed-effects model (lme) showed that the 1.2 and 20 μm filters captured most of the eDNA particles present in the sampled water. Additionally, whole water samples (0.375 L) were preserved in Longmire's buffer, stored at tropical ambient temperatures (26.3°C ± 3.0 SD) and extracted at five time points: immediately, one, two, and three months after collection, as well as frozen and extracted three months later, to assess the preservation efficiency of Longmire's buffer via qPCR analysis. A linear mixed-effects model showed that samples maintained maximal eDNA yield for at least three months after collection at ambient storage. Lastly, when comparing the filtration and precipitation methods, filtration using 0.45 μm pore size was more sensitive to capture of large-tooth sawfish eDNA than filtration with 20 μm filter or water precipitation. However, water precipitation was more efficient when accounting for volume of water processed. These results provide options for best capture and preservation of elasmobranch eDNA