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

Environmental conditions experienced during the larval dispersal of marine organisms can determine the 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 the genetic structure of recently settled recruits on the east coast of Tasmania over 3 months of peak settlement during 2012 (August-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 the dispersal of J. edwardsii could be a possible driver of the resulting phenotype of settlers.

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.

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.

Efficiency of ddRAD target enriched sequencing across spiny rock lobster species (Palinuridae: Jasus).

Double digest restriction site-associated DNA sequencing (ddRADseq) and target capture sequencing methods are used to explore population and phylogenetic questions in non-model organisms. ddRADseq offers a simple and reliable protocol for population genomic studies, however it can result in a large amount of missing data due to allelic dropout. Target capture sequencing offers an opportunity to increase sequencing coverage with little missing data and consistent orthologous loci across samples, although this approach has generally been applied to conserved markers for deeper evolutionary questions. Here, we combine both methods to generate high quality sequencing data for population genomic studies of all marine lobster species from the genus Jasus. We designed probes based on ddRADseq libraries of two lobster species (Jasus edwardsii and Sagmariasus verreauxi) and evaluated the captured sequencing data in five other Jasus species. We validated 4,465 polymorphic loci amongst these species using a cost effective sequencing protocol, of which 1,730 were recovered from all species, and 4,026 were present in at least three species. The method was also successfully applied to DNA samples obtained from museum specimens. This data will be further used to assess spatial-temporal genetic variation in Jasus species found in the Southern Hemisphere.

Seagrass proliferation precedes mortality during hypo-salinity events: a stress-induced morphometric response.

Halophytes, such as seagrasses, predominantly form habitats in coastal and estuarine areas. These habitats can be seasonally exposed to hypo-salinity events during watershed runoff exposing them to dramatic salinity shifts and osmotic shock. The manifestation of this osmotic shock on seagrass morphology and phenology was tested in three Indo-Pacific seagrass species, Halophila ovalis, Halodule uninervis and Zostera muelleri, to hypo-salinity ranging from 3 to 36 PSU at 3 PSU increments for 10 weeks. All three species had broad salinity tolerance but demonstrated a moderate hypo-salinity stress response--analogous to a stress induced morphometric response (SIMR). Shoot proliferation occurred at salinities <30 PSU, with the largest increases, up to 400% increase in shoot density, occurring at the sub-lethal salinities <15 PSU, with the specific salinity associated with peak shoot density being variable among species. Resources were not diverted away from leaf growth or shoot development to support the new shoot production. However, at sub-lethal salinities where shoots proliferated, flowering was severely reduced for H. ovalis, the only species to flower during this experiment, demonstrating a diversion of resources away from sexual reproduction to support the investment in new shoots. This SIMR response preceded mortality, which occurred at 3 PSU for H. ovalis and 6 PSU for H. uninervis, while complete mortality was not reached for Z. muelleri. This is the first study to identify a SIMR in seagrasses, being detectable due to the fine resolution of salinity treatments tested. The detection of SIMR demonstrates the need for caution in interpreting in-situ changes in shoot density as shoot proliferation could be interpreted as a healthy or positive plant response to environmental conditions, when in fact it could signal pre-mortality stress.