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Flake and shark samples were purchased from outlets in several coastal Australian regions and genetically barcoded using the cytochrome oxidase subunit 1 (CO1) gene to investigate labelling reliability and species-specific sources of ambiguously labelled fillets. Of the 41 shark fillet samples obtained, 23 yielded high-quality CO1 sequences, out of which 57% (n = 13) were labelled ambiguously (misleading) and 35% (n = 8) incorrectly. In contrast, barramundi fillets, which are widely available and sought after in Australian markets, were shown to be accurately labelled. Species identified from shark samples, including the shortfin mako (n = 3) and the scalloped hammerhead (n = 1), are assessed by the IUCN as endangered and critically endangered, respectively, with several others classified as vulnerable and near threatened.

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Plastic pollution in our oceans is of growing concern particularly due to the presence of toxic additives, such as plasticisers. Therefore, this work aims to develop a comprehensive understanding of the leaching properties of plasticisers from microplastics. This work investigates the leaching of phthalate acid ester (dioctyl terephthalate (DEHT) and diethylhexyl phthalate (DEHP)) and diphenol (bisphenol A (BPA) and bisphenol S (BPS)) plasticisers from polystyrene (PS) microplastics (mean diameter = 136 µm to 1.4 mm) under controlled aqueous conditions (temperature, agitation, pH and salinity). The leaching behaviours of plasticised polymers were quantified using gel permeation chromatography, high performance liquid chromatography and thermal gravimetric analysis, and the particle's plasticisation characterised using differential scanning calorimetry. Leaching rates of phthalate acid ester and diphenol plasticisers were modelled using a diffusion and boundary layer model, whereby these behaviours varied depending on their plasticisation efficiency of PS, the size of the microplastic particle and the surrounding abiotic conditions. Leaching behaviours of DEHT and DEHP were strongly influenced by the microplastic-surface water boundary layer properties, thus wave action (i.e., water agitation) increased the leaching rate of these plasticiser up to 66 % over 21-days, whereas BPA and BPS plasticisers displayed temperature- and size-dependent leaching and were limited by molecular diffusion throughout the bulk polymer (i.e., the microplastic). This information will improve predictions of plasticiser concentration (both that remain in the plastic and released into the surrounding water) at specific time points during the lifetime of a plastic, ultimately ensuring greater accuracy in the assessment of toxicity responses and environmental water quality.

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The El Niño Southern Oscillation (ENSO) is the strongest source of interannual global climate variability, and extreme ENSO events are projected to increase in frequency under climate change. Interannual variability in the Coral Sea circulation has been associated with ENSO, although uncertainty remains regarding ENSO's influence on hydrodynamics and larval dispersal in the adjacent Great Barrier Reef (GBR). We investigated larval connectivity during ENSO events from 2010 to 2017 throughout the GBR, based on biophysical modelling of a widespread predatory reef fish, Lutjanus carponotatus. Our results indicate a well-connected system over the study period with high interannual variability in inter-reef connectivity associated with ENSO. Larval connectivity patterns were highly correlated to variations in the Southern Oscillation Index (SOI). During El Niño conditions and periods of weak SOI, larval dispersal patterns were predominantly poleward in the central and southern regions, reversing to a predominant equatorward flow during very strong SOI and extreme La Niña conditions. These ENSO-linked connectivity patterns were associated with positive connectivity anomalies among reefs. Our findings identify ENSO as an important source of variation in larval dispersal and connectivity patterns in the GBR, which can influence the stability of population dynamics and patterns of biodiversity in the region.

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A criteria-guided workflow was applied to assess the effectiveness of microplastic separation methods on complex marine biological matrices. Efficacy of four methods (nitric acid, HNO3, and potassium hydroxide, KOH, digestions, and sodium chloride, NaCl, and potassium iodide, KI, density flotations) was evaluated on four taxa (hard coral, sponge, sea squirt, sea cucumber) using five microplastics (polyethylene, polystyrene, polyethylene terephthalate, PET, polyvinylchloride, rayon). Matrix clarification was only unacceptably low for KOH. PET discoloured regardless of reagent. Rayon threads unravelled into monofilaments after exposure to all reagents, with discolouration also occurring with HNO3. Recovery rates were overall high, except for dense microplastics treated with NaCl and only KI yielded high rayon recovery efficiency. All polymers were accurately assigned, with subtle spectral changes observed. These results demonstrate specific limitations to separation methods applied to different biological matrices and microplastics and highlight the need to assess their suitability to provide estimates of microplastic contamination.

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Hybridization and introgression are evolutionarily significant phenomena breaking down species boundaries. "Hybrid zones" (regions of species overlap and hybridization) enable quantification of hybridization frequency and examination of mechanisms driving and maintaining gene flow. The hybrid anemonefish Amphiprion leucokranos is found where parent species (A. chrysopterus; A. sandaracinos) distributions overlap. Here, we examine geographic variation in hybridization and introgression, and potential impacts on parent species integrity through assessing relative abundance, social group composition, and genetic structure (mtDNA cytochrome b, 21 microsatellite loci) of taxa at three hybrid zone locations: Kimbe Bay (KB) and Kavieng (KA), Papua New Guinea; the Solomon Islands (SO). Relative abundances of and size disparities between parent species apparently drive hybridization frequency, introgression patterns, and genetic composition of taxa. Conspecific groups are most common in KB (65%) where parent species are similarly abundant. Conversely, mixed species groups dominate SO (82%), where A. chrysopterus is more abundant. Hybrids most commonly cohabit with A. sandaracinos in KB (17%), but with A. chrysopterus in KA (22%) and SO (50%). Genetic differentiation (nDNA) analyses indicate that parent species remain distinct, despite ongoing hybridization and hybrids are genetically similar to A. sandaracinos-resulting from persistent backcrossing with this smallest species. This study shows that hybridization outcomes may depend on the social and ecological context in which taxa hybridize, where relative abundance and disparate size of parent species explain the frequency and patterns of hybridization and introgression in the A. leucokranos hybrid zone, reflecting size-based dominance behaviors of anemonefish social groups.

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BACKGROUND: Understanding the factors shaping population genetic structure is important for evolutionary considerations as well as for management and conservation. While studies have revealed the importance of palaeogeographic changes in shaping phylogeographic patterns in multiple marine fauna, the role of reproductive behaviour is rarely considered in reef fishes. We investigated the population genetics of three commercially important aggregating grouper species in the Indo-West Pacific, namely the camouflage grouper Epinephelus polyphekadion, the squaretail coral grouper Plectropomus areolatus, and the common coral trout P. leopardus, with similar life histories but distinct spatio-temporal characteristics in their patterns of forming spawning aggregations. RESULTS: By examining their mitochondrial control region and 9-11 microsatellite markers, we found an overarching influence of palaeogeographic events in the population structure of all species, with genetic breaks largely coinciding with major biogeographic barriers. The divergence time of major lineages in these species coincide with the Pleistocene glaciations. Higher connectivity is evident in E. polyphekadion and P. areolatus that assemble in larger numbers at fewer spawning aggregations and in distinctive offshore locations than in P. leopardus which has multiple small, shelf platform aggregations. CONCLUSIONS: While palaeogeographic events played an important role in shaping the population structure of the target species, the disparity in population connectivity detected may be partly attributable to differences in their reproductive behaviour, highlighting the need for more investigations on this characteristic and the need to consider reproductive mode in studies of connectivity and population genetics.

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A solid understanding of the spatial ecology of green turtles (Chelonia mydas) is fundamental to their effective conservation. Yet this species, like many marine migratory species, is challenging to monitor and manage because they utilise a variety of habitats that span wide spatio-temporal scales. To further elucidate the connectivity between green turtle rookeries and foraging populations, we sequenced the mtDNA control region of 278 turtles across three foraging sites from the northern Great Barrier Reef (GBR) spanning more than 330 km: Cockle Bay, Green Island and Low Isles. This was performed with a newly developed assay, which targets a longer fragment of mtDNA than previous studies. We used a mixed stock analysis (MSA), which utilises genetic data to estimate the relative proportion of genetically distinct breeding populations found at a given foraging ground. Haplotype and nucleotide diversity was also assessed. A total of 35 haplotypes were identified across all sites, 13 of which had not been found previously in any rookery. The MSA showed that the northern GBR (nGBR), Coral Sea (CS), southern GBR (sGBR) and New Caledonia (NC) stocks supplied the bulk of the turtles at all three sites, with small contributions from other rookeries in the region. Stock contribution shifted gradually from north to south, although sGBR/CS stock dominated at all three sites. The major change in composition occured between Cockle Bay and Low Isles. Our findings, together with other recent studies in this field, show that stock composition shifts with latitude as a natural progression along a coastal gradient. This phenomenon is likely to be the result of ocean currents influencing both post-hatchling dispersal and subsequent juvenile recruitment to diverse coastal foraging sites.

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The application of genome-wide cytonuclear molecular data to identify management and adaptive units at various spatio-temporal levels is particularly important for overharvested large predatory organisms, often characterized by smaller, localized populations. Despite being "near threatened", current understanding of habitat use and population structure of Carcharhinus galapagensis is limited to specific areas within its distribution. We evaluated population structure and connectivity across the Pacific Ocean using genome-wide single-nucleotide polymorphisms (~7200 SNPs) and mitochondrial control region sequences (945 bp) for 229 individuals. Neutral SNPs defined at least two genetically discrete geographic groups: an East Tropical Pacific (Mexico, east and west Galapagos Islands), and another central-west Pacific (Lord Howe Island, Middleton Reef, Norfolk Island, Elizabeth Reef, Kermadec, Hawaii and Southern Africa). More fine-grade population structure was suggested using outlier SNPs: west Pacific, Hawaii, Mexico, and Galapagos. Consistently, mtDNA pairwise ΦST defined three regional stocks: east, central and west Pacific. Compared to neutral SNPs (FST = 0.023-0.035), mtDNA exhibited more divergence (ΦST = 0.258-0.539) and high overall genetic diversity (h = 0.794 ± 0.014; π = 0.004 ± 0.000), consistent with the longstanding eastern Pacific barrier between the east and central-west Pacific. Hawaiian and Southern African populations group within the west Pacific cluster. Effective population sizes were moderate/high for east/west populations (738 and 3421, respectively). Insights into the biology, connectivity, genetic diversity, and population demographics informs for improved conservation of this species, by delineating three to four conservation units across their Pacific distribution. Implementing such conservation management may be challenging, but is necessary to achieve long-term population resilience at basin and regional scales.

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Large amounts of plastic end up in the oceans every year where they fragment into microplastics over time. During this process, microplastics and their associated plasticizers become available for ingestion by different organisms. This study assessed the effects of microplastics (Polyethylene terephthalate; PET) and one plasticizer (Di(2-ethylhexyl)phthalate; DEHP) on mortality, productivity, population sizes and gene expression of the calanoid copepod Parvocalanus crassirostris. Copepods were exposed to DEHP for 48h to assess toxicity. Adults were very healthy following chemical exposure (up to 5120µg L-1), whereas nauplii were severely affected at very low concentrations (48h LC50value of 1.04 ng L-1). Adults exposed to sub-lethal concentrations of DEHP (0.1-0.3µg L-1) or microplastics (10,000-80,000 particles mL-1) exhibited substantial reductions in egg production. Populations were exposed to either microplastics or DEHP for 6 days with 18 days of recovery or for 24 days. Populations exposed to microplastics for 24 days significantly depleted in population size (60±4.1%, p<0.001) relative to controls, whilst populations exposed for only 6 days (with 18 days of recovery) experienced less severe depletions (75±6.0% of control, p<0.05). Populations exposed to DEHP, however, exhibited no recovery and both treatments (6 and 24 days) yielded the same average population size at the termination of the experiment (59±4.9% and 59±3.4% compared to control; p<0.001). These results suggest that DEHP may induce reproductive disorders that can be inherited by subsequent generations. Histone 3 (H3) was significantly (p<0.05) upregulated in both plastic and DEHP treatments after 6 days of exposure, but not after 18 days of recovery. Hsp70-like expression showed to be unresponsive to either DEHP or microplastic exposure. Clearly, microplastics and plasticizers pose a serious threat to zooplankton and potentially to higher trophic levels.

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Hybridisation can produce evolutionary novelty by increasing fitness and adaptive capacity. Heterosis, or hybrid vigour, has been documented in many plant and animal taxa, and is a notable consequence of hybridisation that has been exploited for decades in agriculture and aquaculture. On the contrary, loss of fitness in naturally occurring hybrid taxa has been observed in many cases. This can have negative consequences for the parental species involved (wasted reproductive effort), and has raised concerns for species conservation. This study evaluates the relative fitness of previously documented butterflyfish hybrids of the genus Chaetodon from the Indo-Pacific suture zone at Christmas Island. Histological examination confirmed the reproductive viability of Chaetodon hybrids. Examination of liver lipid content showed that hybrid body condition was not significantly different from parent species body condition. Lastly, size at age data revealed no difference in growth rates and asymptotic length between hybrids and parent species. Based on the traits measured in this study, naturally occurring hybrids of Chaetodon butterflyfishes have similar fitness to their parental species, and are unlikely to supplant parental species under current environmental conditions at the suture zone. However, given sufficient fitness and ongoing genetic exchange between the respective parental species, hybrids are likely to persist within the suture zone.

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Groupers (family Epinephelidae) are a clade of species-rich, biologically diverse reef fishes. Given their ecological variability and widespread distribution across ocean basins, it is important to scrutinize their evolutionary history that underlies present day distributions. This study investigated the patterns and processes by which grouper biodiversity has been generated and what factors have influenced their present day distributions. We reconstructed a robust, time-calibrated molecular phylogeny of Epinephelidae with comprehensive (∼87%) species sampling, whereby diversification rates were estimated and ancestral ranges were reconstructed. Our results indicate that groupers originated in what is now the East Atlantic during the mid-Eocene and diverged successively to form six strongly supported main clades. These clades differ in age (late Oligocene to mid-Miocene), geographic origin (West Atlantic to West Indo-Pacific) and temporal-spatial diversification pattern, ranging from constant rates of diversification to episodes of rapid radiation. Overall, divergence within certain biogeographic regions was most prevalent in groupers, while vicariant divergences were more common in Tropical Atlantic and East Pacific groupers. Our findings reveal that both biological and geographical factors have driven grouper diversification. They also underscore the importance of scrutinizing group-specific patterns to better understand reef fish evolution.

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Christmas Island is located at the overlap of the Indian and Pacific Ocean marine provinces and is a hot spot for marine hybridization. Here, we evaluate the ecological framework and genetic consequences of hybridization between butterflyfishes Chaetodon guttatissimus and Chaetodon punctatofasciatus. Further, we compare our current findings to those from a previous study of hybridization between Chaetodon trifasciatus and Chaetodon lunulatus. For both species groups, habitat and dietary overlap between parental species facilitate frequent heterospecific encounters. Low abundance of potential mates promotes heterospecific pair formation and the breakdown of assortative mating. Despite similarities in ecological frameworks, the population genetic signatures of hybridization differ between the species groups. Mitochondrial and nuclear data from C. guttatissimus × C. punctatofasciatus (1% divergence at cyt b) show bidirectional maternal contributions and relatively high levels of introgression, both inside and outside the Christmas Island hybrid zone. In contrast, C. trifasciatus × C. lunulatus (5% cyt b divergence) exhibit unidirectional mitochondrial inheritance and almost no introgression. Back-crossing of hybrid C. guttatissimus × C. punctatofasciatus and parental genotypes may eventually confound species-specific signals within the hybrid zone. In contrast, hybrids of C. trifasciatus and C. lunulatus may coexist with and remain genetically distinct from the parents. Our results, and comparisons with hybridization studies in other reef fish families, indicate that genetic distance between hybridizing species may be a factor influencing outcomes of hybridization in reef fish, which is consistent with predictions from terrestrially derived hybridization theory.

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The otx2 gene encodes a transcription factor (OTX2) essential in the formation of the brain and sensory systems. Specifically, OTX2-positive cells are associated with axons in the olfactory system of mice and otx2 is upregulated in odour-exposed zebrafish, indicating a possible role in olfactory imprinting. In this study, otx2 was used as a candidate gene to investigate the molecular mechanisms of olfactory imprinting to settlement cues in the coral reef anemonefish, Amphiprion percula. The A. percula otx2 (Ap-otx2) gene was elucidated, validated, and its expression tested in settlement-stage A. percula by exposing them to behaviourally relevant olfactory settlement cues in the first 24 hours post-hatching, or daily throughout the larval phase. In-situ hybridisation revealed expression of Ap-otx2 throughout the olfactory epithelium with increased transcript staining in odour-exposed settlement-stage larval fish compared to no-odour controls, in all scenarios. This suggests that Ap-otx2 may be involved in olfactory imprinting to behaviourally relevant settlement odours in A. percula.

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Extensive ongoing degradation of coral reef habitats worldwide has lead to declines in abundance of coral reef fishes and local extinction of some species. Those most vulnerable are ecological specialists and endemic species. Determining connectivity between locations is vital to understanding recovery and long-term persistence of these species following local extinction. This study explored population connectivity in the ecologically-specialized endemic three-striped butterflyfish (Chaetodon tricinctus) using mt and msatDNA (nuclear microsatellites) to distinguish evolutionary versus contemporary gene flow, estimate self-replenishment and measure genetic diversity among locations at the remote Australian offshore coral reefs of Middleton Reef (MR), Elizabeth Reef (ER), Lord Howe Island (LHI), and Norfolk Island (NI). Mt and msatDNA suggested genetic differentiation of the most peripheral location (NI) from the remaining three locations (MR, ER, LHI). Despite high levels of mtDNA gene flow, there is limited msatDNA gene flow with evidence of high levels of self-replenishment (≥76%) at all four locations. Taken together, this suggests prolonged population recovery times following population declines. The peripheral population (NI) is most vulnerable to local extinction due to its relative isolation, extreme levels of self-replenishment (95%), and low contemporary abundance.

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The rich diversity of coral reef organisms is supported, at least in part, by the diversity of coral reef habitat. Some of the most habitat specialised fishes on coral reefs are obligate coral-dwelling gobies of the genus Gobiodon that inhabit a range of coral species, mostly of the genus Acropora. However, the role of this specialised pattern of habitat use in the evolution of coral-dwelling gobies is not well understood. Diversification of coral-dwelling gobies may be driven by the diversification of their host corals (cospeciation), or alternatively, diversification of these fishes may have occurred independently of the diversification of host corals. The cospeciation hypothesis assumes similar timing in evolution of the gobies and their host corals. We used four genes for each group and the available fossil records to reconstruct and date phylogenies for 20 species of Gobiodon from the Indo-Pacific and the Red Sea, and for 28 species of the coral genus Acropora. Our results indicate that Gobiodon diversified mostly in the last ∼5My, whereas Acropora corals have consistently diversified since the Eocene, making the hypothesis of cospeciation untenable. The fully resolved molecular phylogeny of the genus Gobiodon is in part at odds with previous analyses incorporating morphological data and indicates that some morphological traits form paraphyletic clades within Gobiodon. Our phylogeny supports a hypothesis in which Gobiodon diversified in the Indo-Pacific Ocean and then radiated recently, with multiple new variants found in the Red Sea.

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Much progress has been made toward understanding marine metapopulation dynamics, largely because of multilocus microsatellite surveys able to connect related individuals within the metapopulation. However, most studies are focused on small spatial scales, tens of kilometers, while demographic exchange at larger spatial scales remains poorly documented. Additionally, many small-scale demographic studies conflict with broad-scale phylogeographic patterns concerning levels of marine population connectivity, highlighting a need for data on more intermediate scales. Here, we investigated demographic recruitment processes of a commercially important coral reef fish, the bluespine unicornfish (Naso unicornis) using a suite of mitochondrial DNA (mtDNA) and microsatellite markers. Sampling for this study ranged across the southern Marianas Islands, a linear distance of 250 km and included 386 newly settled postlarval recruits. In contrast with other studies, we report that cohorts of recruits were genetically homogeneous in space and time, with no evidence of temporally stochastic sweepstakes reproduction. The genetic diversity of recruits was high and commensurate with that of the adult population. In addition, there is substantial evidence that 2 recruits, separated by 250 km, were full siblings. This is the largest direct observation of dispersal to date for a coral reef fish. All indications suggest that subpopulations of N. unicornis experience high levels of demographic migrant exchange and metapopulation mixing on a spatial scale of hundreds of kilometers, consistent with high levels of broad-scale genetic connectivity previously reported in this species.

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Marine protected areas (MPAs) are increasingly being advocated and implemented to protect biodiversity on coral reefs. Networks of appropriately sized and spaced reserves can capture a high proportion of species diversity, with gene flow among reserves presumed to promote long term resilience of populations to spatially variable threats. However, numerically rare small range species distributed among isolated locations appear to be at particular risk of extinction and the likely benefits of MPA networks are uncertain. Here we use mitochondrial and microsatellite data to infer evolutionary and contemporary gene flow among isolated locations as well as levels of self-replenishment within locations of the endemic anemonefish Amphiprion mccullochi, restricted to three MPA offshore reefs in subtropical East Australia. We infer high levels of gene flow and genetic diversity among locations over evolutionary time, but limited contemporary gene flow amongst locations and high levels of self-replenishment (68 to 84%) within locations over contemporary time. While long distance dispersal explained the species' integrity in the past, high levels of self-replenishment suggest locations are predominantly maintained by local replenishment. Should local extinction occur, contemporary rescue effects through large scale connectivity are unlikely. For isolated islands with large numbers of endemic species, and high local replenishment, there is a high premium on local species-specific management actions.

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Marine reserves, areas closed to all forms of fishing, continue to be advocated and implemented to supplement fisheries and conserve populations. However, although the reproductive potential of important fishery species can dramatically increase inside reserves, the extent to which larval offspring are exported and the relative contribution of reserves to recruitment in fished and protected populations are unknown. Using genetic parentage analyses, we resolve patterns of larval dispersal for two species of exploited coral reef fish within a network of marine reserves on the Great Barrier Reef. In a 1,000 km(2) study area, populations resident in three reserves exported 83% (coral trout, Plectropomus maculatus) and 55% (stripey snapper, Lutjanus carponotatus) of assigned offspring to fished reefs, with the remainder having recruited to natal reserves or other reserves in the region. We estimate that reserves, which account for just 28% of the local reef area, produced approximately half of all juvenile recruitment to both reserve and fished reefs within 30 km. Our results provide compelling evidence that adequately protected reserve networks can make a significant contribution to the replenishment of populations on both reserve and fished reefs at a scale that benefits local stakeholders.

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Natural hybridization is widespread among coral reef fishes. However, the ecological promoters and evolutionary consequences of reef fish hybridization have not been thoroughly evaluated. Butterflyfishes form a high number of hybrids and represent an appropriate group to investigate hybridization in reef fishes. This study provides a rare test of terrestrially derived hybridization theory in the marine environment by examining hybridization between Chaetodon trifasciatus and C. lunulatus at Christmas Island. Overlapping spatial and dietary ecologies enable heterospecific encounters. Nonassortative mating and local rarity of both parent species appear to permit heterospecific breeding pair formation. Microsatellite loci and mtDNA confirmed the status of hybrids, which displayed the lowest genetic diversity in the sample and used a reduced suite of resources, suggesting decreased adaptability. Maternal contribution to hybridization was unidirectional, and no introgression was detected, suggesting limited, localized evolutionary consequences of hybridization.Comparisons to other reef fish hybridization studies revealed that different evolutionary consequences emerge, despite being promoted by similar factors, possibly due to the magnitude of genetic distance between hybridizing species. This study highlights the need for further enquiry aimed at evaluating the importance and long-term consequences of reef fish hybridization.

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We examined how peripherally isolated endemic species may have contributed to the biodiversity of the Indo-Australian Archipelago biodiversity hotspot by reconstructing the evolutionary history of the wrasse genus Anampses. We identified three alternate models of diversification: the vicariance-based 'successive division' model, and the dispersal-based 'successive colonisation' and 'peripheral budding' models. The genus was well suited for this study given its relatively high proportion (42%) of endemic species, its reasonably low diversity (12 species), which permitted complete taxon sampling, and its widespread tropical Indo-Pacific distribution. Monophyly of the genus was strongly supported by three phylogenetic analyses: maximum parsimony, maximum likelihood, and Bayesian inference based on mitochondrial CO1 and 12S rRNA and nuclear S7 sequences. Estimates of species divergence times from fossil-calibrated Bayesian inference suggest that Anampses arose in the mid-Eocene and subsequently diversified throughout the Miocene. Evolutionary relationships within the genus, combined with limited spatial and temporal concordance among endemics, offer support for all three alternate models of diversification. Our findings emphasise the importance of peripherally isolated locations in creating and maintaining endemic species and their contribution to the biodiversity of the Indo-Australian Archipelago.

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