DNA barcoding of fresh seafood in Australian markets reveals misleading labelling and sale of endangered species

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

Comparative characterization of a temperature responsive gene (lactate dehydrogenase-B, ldh-b) in two congeneric tropical fish, Lates calcarifer and Lates niloticus

The characterization of candidate loci is a critical step in obtaining insight into adaptation and acclimation of organisms. In this study of two non-model tropical (to sub-tropical) congeneric perciformes (Lates calcarifer and Lates niloticus) we characterized both coding and non-coding regions of lactate dehydrogenase-B (ldh-b), a locus which exhibits temperature-adaptive differences among temperate and sub-tropical populations of the North American killifish Fundulus heteroclitus. Ldh-b was 5,004 and 3,527 bp in length in L. calcarifer and L. niloticus, respectively, with coding regions comprising 1,005 bp in both species. A high level of sequence homology existed between species for both coding and non-coding regions of ldh-b (> 97% homology), corresponding to a 98.5% amino acid sequence homology. All six known functional sites within the encoded protein sequence (LDH-B) were conserved between the two Lates species. Ten simple sequence repeat (SSR) motifs (mono-, di-, tri- and tetranucleotide) and thirty putative microRNA elements (miRNAs) were identified within introns 1, 2, 5 and 6 of both Lates species. Five single nucleotide polymorphisms (SNPs) were also identified within miRNA containing intron regions. Such SNPs are implicated in several complex human conditions and/or diseases (as demonstrated by extensive genome-wide association studies). This novel characterization serves as a platform to further examine how non-model species may respond to changes in their native temperatures, which are expected to increase by up to 6°C over the next century

Ingestion and depuration of microplastics by a planktivorous coral reef fish, Pomacentrus amboinensis

Microplastics are ubiquitous contaminants in marine environments and organisms. Concerns about potential impacts on marine organisms are usually associated with uptake of microplastics, especially via ingestion. This study used environmentally relevant exposure conditions to investigate microplastic ingestion and depuration kinetics of the planktivorous damselfish, Pomacentrus amboinensis. Irregular shaped blue polypropylene (PP) particles (longest length 125–250 μm), and regular shaped blue polyester (PET) fibers (length 600–700 μm) were selected based on physical and chemical characteristics of microplastics commonly reported in the marine environment, including in coral reef ecosystems. Individual adult damselfish were exposed to a single dose of PP particles and PET fibers at concentrations reported for waters of the Great Barrier Reef (i.e., environmentally relevant concentrations, ERC), or future projected higher concentrations (10x ERC, 100x ERC). Measured microplastic concentrations were similar to their nominal values, confirming that PP particles and PET fibers were present at the desired concentrations and available for ingestion by individual damselfish. Throughout the 128-h depuration period, the 88 experimental fish were sampled 2, 4, 8, 16, 32, 64, and 128-h post microplastic exposure and their gastrointestinal tracts (GIT) analyzed for ingested microplastics. While damselfish ingested both experimental microplastics at all concentrations, body burden, and depuration rates of PET fibers were significantly larger and longer, respectively, compared to PP particles. For both microplastic types, exposure to higher concentrations led to an increase in body burden and lower depuration rates. These findings confirm ingestion of PP particles and PET fibers by P. amboinensis and demonstrate for the first time the influence of microplastic characteristics and concentrations on body burden and depuration rates. Finally, despite measures put in place to prevent contamination, extraneous microplastics were recovered from experimental fish, highlighting the challenge to completely eliminate contamination in microplastic exposure studies. These results are critical to inform and continuously improve protocols for future microplastics research, and to elucidate patterns of microplastic contamination and associated risks in marine organisms

The El Niño Southern Oscillation drives multidirectional inter-reef larval connectivity in the Great Barrier Reef

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

Closing the gap: mixed stock analysis of three foraging populations of green turtles (Chelonia mydas) on the Great Barrier Reef

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.Weused 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

The application of genetics to marine management and conservation: examples from the Indo-Pacific

Molecular tools and analyses have played pivotal roles in uncovering the processes and patterns of biodiversity in the Indian and Pacific oceans. However, integrating genetic results into management and conservation objectives has been challenging, with few examples that show practical applicability. This review aims to address some of the perceived barriers to an enhanced approach that integrates molecular data into management and conservation goals, by reviewing papers relevant to both conservation and fisheries management in the Indo-Pacific region, particularly with respect to phylogeography, connectivity, and species identification, as well as stock delineation, restoration of depleted wild stocks, mislabeled marine resources and "molecular forensics." We also highlight case studies from each of these areas that illustrate how molecular analyses are relevant to conservation and management in the Indo- Pacific, spanning a variety of vertebrate and invertebrate species. We discuss the application of genetic data to the design and evaluation of the effectiveness of marine protected area networks, stock delineation, and restoration and the usage of exclusion tests and parentage analyses for fisheries management. We conclude that there is a distinct need for increasing public awareness and ownership of genetically unique lineages and, ultimately, the increased inclusion of genetic research into management policy and conservation. Finally, we make a case for the importance of clear and effective communication for promoting public awareness, public ownership, and for achieving conservation goals within the region

Phylogeography of the reef fish Cephalopholis argus (Epinephelidae) indicates Pleistocene isolation across the indo-pacific barrier with contemporary overlap in the coral triangle

Background: \ud The Coral Triangle (CT), bounded by the Philippines, the Malay Peninsula, and New Guinea, is the epicenter of marine biodiversity. Hypotheses that explain the source of this rich biodiversity include 1) the center of origin, 2) the center of accumulation, and 3) the region of overlap. Here we contribute to the debate with a phylogeographic survey of a widely distributed reef fish, the Peacock Grouper (Cephalopholis argus; Epinephelidae) at 21 locations (N = 550) using DNA sequence data from mtDNA cytochrome b and two nuclear introns (gonadotropin-releasing hormone and S7 ribosomal protein).\ud \ud Results: \ud Population structure was significant (ΦST = 0.297, P < 0.001; FST = 0.078, P < 0.001; FST = 0.099, P < 0.001 for the three loci, respectively) among five regions: French Polynesia, the central-west Pacific (Line Islands to northeastern Australia), Indo-Pacific boundary (Bali and Rowley Shoals), eastern Indian Ocean (Cocos/Keeling and Christmas Island), and western Indian Ocean (Diego Garcia, Oman, and Seychelles). A strong signal of isolation by distance was detected in both mtDNA (r = 0.749, P = 0.001) and the combined nuclear loci (r = 0.715, P < 0.001). We detected evidence of population expansion with migration toward the CT. Two clusters of haplotypes were detected in the mtDNA data (d = 0.008), corresponding to the Pacific and Indian Oceans, with a low level of introgression observed outside a mixing zone at the Pacific-Indian boundary.\ud \ud Conclusions: \ud We conclude that the Indo-Pacific Barrier, operating during low sea level associated with glaciation, defines the primary phylogeographic pattern in this species. These data support a scenario of isolation on the scale of 105 year glacial cycles, followed by population expansion toward the CT, and overlap of divergent lineages at the Pacific-Indian boundary. This pattern of isolation, divergence, and subsequent overlap likely contributes to species richness at the adjacent CT and is consistent with the region of overlap hypothesis

Chimerism in Wild Adult Populations of the Broadcast Spawning Coral Acropora millepora on the Great Barrier Reef

Chimeras are organisms containing tissues or cells of two or more genetically distinct individuals, and are known to exist in at least nine phyla of protists, plants, and animals. Although widespread and common in marine invertebrates, the extent of chimerism in wild populations of reef corals is unknown.The extent of chimerism was explored within two populations of a common coral, Acropora millepora, on the Great Barrier Reef, Australia, by using up to 12 polymorphic DNA microsatellite loci. At least 2% and 5% of Magnetic Island and Pelorus Island populations of A. millepora, respectively, were found to be chimeras (3% overall), based on conservative estimates. A slightly less conservative estimate indicated that 5% of colonies in each population were chimeras. These values are likely to be vast underestimates of the true extent of chimerism, as our sampling protocol was restricted to a maximum of eight branches per colony, while most colonies consist of hundreds of branches. Genotypes within chimeric corals showed high relatedness, indicating that genetic similarity is a prerequisite for long-term acceptance of non-self genotypes within coral colonies.While some brooding corals have been shown to form genetic chimeras in their early life history stages under experimental conditions, this study provides the first genetic evidence of the occurrence of coral chimeras in the wild and of chimerism in a broadcast spawning species. We hypothesize that chimerism is more widespread in corals than previously thought, and suggest that this has important implications for their resilience, potentially enhancing their capacity to compete for space and respond to stressors such as pathogen infection

Naturally occurring hybrids of coral reef butterflyfishes have similar fitness compared to parental species.

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

The trematode genera Paragonimus and Schistosoma in East Asia: molecular evolution, phylogeny and biogeography

Trematode infections affect one fifth of the worlds' population, with more than 200 million Chinese alone being at risk (World Health Organisation 1997, Hotez et at 1997). Of particular interest to this study are the Asian species of the trematode genera Paragonimus and Schistosoma, which infect many mammals, including humans. It is becoming clear that within both genera species complexes exist that require further characterisation. Molecular markers have given some insight into the nature of these complexes. In this thesis additional molecular markers are investigated as sources of information about the nature and distribution of the S. japonicum, P. westermani and P. ohirai species complexes. These two genera are of additional interest because they are thought to have shared a similar biogeographic history in Asia, which should be reflected in their molecular evolution. I have obtained DNA sequences from one nuclear (ITS1) and one mitochondrial (ND1) marker of five Paragonimus species (P. westermani, P. miyazakii, P. macrorchis, P. ohirai and P. iloktsuenensis) present in East Asia, including numerous geographic isolates of the P. westermani and P. ohirai species complexes. In addition, I have obtained ITS1 sequences of three schistosome species (S. japonicum, S malayensis and S. mekongi) present in East Asia, which comprise the S. japonicum species complex and an African schistosome species (S. mansoni) for comparative purposes. I could differentiate all species using ITS1 and ND1 sequence data. ITS1 is contained within the ribosomal DNA gene cluster, which is repeated in tandem many times. ITS1 does not experience the constraints of coding regions and theoretically undergoes sequence homogenisation within individuals and species. Despite this, intra-individual sequence variation was sometimes greater than inter-species variation, particularly among northeast Asian P. westermani isolates. Such variation confounded phylogenetic inferences for this group. There are some interesting aspects relating to processes of concerted evolution generally, when considering the differences within and between the two trematode genera, Paragonimus and Schistosoma, in this study. Additionally, a transcriptional enhancer motif (TATAAT) is embedded within the repeats of the ITS1 of the Asian schistosomes. The repeats are the cause of size variation and given the improbability of such motifs occurring by chance, I propose that the abundant, large variants containing multiple copies of repeats may have a role in the stage- or tissue-specific regulation of transcription of the ribosomal genes. In contrast, the ND1 gene codes for a mitochondrial protein and is therefore functionally constrained. Phylogenetic inferences could be made from ND1 sequence data obtained from Paragonimus. However, many clones had to be sequenced per individual to achieve this, as multiple ND1 lineages occurred within individuals of all Paragonimus species and strains investigated. Presumed pseudogenes were identified, which may be present in either the nuclear genome or in different types of mitochondria, as Paragonimus species have two types of mitochondria which differ structurally. It seems likely that both nuclear pseudogenes and heteroplasmic mitochondrial genes occur, though this hypothesis remains to be tested. The Paragonimus westermani species complex has been further studied, to determine the origins of parthenogenetic triploid forms. Ribosomal DNA-ITS restriction fragments, ND1 sequences and simple sequence repeat (SSRs) "fingerprints" of triploids from China, Korea and Japan all indicate that the triploids are genetically different from one another. I conclude from this that triploids may have arisen more than once independently, possibly by relatively rare coatings between diploid and tetraploid P. westermani individuals, which occur in sympatry with the triploids in NE China. Alternatively, triploid lineages may have arisen once and diverged subsequently by mutation alone. Despite their suggested shared biogeographic history, members of the two genera exhibit rather different properties with respect to their ND1 and ITS1 genes. This is likely to be a reflection of their different phylogenetic histories. Molecular techniques have been used successfully to infer phylogenies which could be used to evaluate a previously published biogeographic hypothesis, but only to a limited extent, using the markers. It is clear that the situation for P. westermani in particular is complex and requires further investigation. Molecular evolution of these markers has proved to be an interesting component of the study that has brought to light some novel ideas and applications of these markers, not as phylogenetic tools, but as tools to study processes of concerted evolution in the nuclear genome and mitochondrial evolution