Correction: Integrated Taxonomy Reveals Hidden Diversity in Northern Australian Fishes: A New Species of Seamoth (Genus Pegasus).

[This corrects the article DOI: 10.1371/journal.pone.0149415.].

Assessing DNA for fish identifications from reference collections: the good, bad and ugly shed light on formalin fixation and sequencing approaches.

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present. Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.

-Redescription and distributional range extension of the Speckled Siphonfish, Siphamia guttulata (Pisces: Apogonidae).

During seabed biodiversity surveys between 2003 and 2005 from the Torres Strait (Papua New Guinea) to the southern Great Barrier Reef (Queensland), hundreds of Siphamia specimens were collected. After Gon Allen's (2012) revision allowed greater interrogation of the Siphamia species present, a re-examination of preserved and frozen Siphamia specimens at the CSIRO Australian National Fish Collection (ANFC) was warranted. The material was re-identified as four commonly collected species (S. cuneiceps, S. roseigaster, S. tubifer, and S. tubulata) and a fifth unidentified species that appeared to key to S. guttulata, previously known only from the type locality. Further detailed investigations including an analysis of meristic, morphometric and COI barcoding data confirmed the identity of S. guttulata from almost the entire length of the Great Barrier Reef, Queensland, from the Torres Strait in the north to the Northumberland Islands Group in the south. This study provides a redescription of Siphamia guttulata and highlights the importance of re-assessing the taxonomic status of museum material after revisionary studies.

Aphyonid-clade species of Australia (Teleostei, Bythitidae) with four species new to Australian waters and a new species of Barathronus.

During voyages in 2017 off southern and southeastern Australia, the Australian Research Vessel Investigator deployed a series of demersal beam trawls to depths of around 5000 metres. Nineteen specimens of the rarely caught aphyonid-clade of the ophidiiform family Bythitidae, representing five species, were caught. Four of these are new to Australian waters:  Barathronus pacificus Nielsen and Eagle, 1974 known from the northeastern and southwestern Pacific Ocean, Paraphyonus bolini (Nielsen, 1974) known from the western Indian and western Pacific Oceans, Paraphyonus rassi (Nielsen, 1975) known from the Atlantic Ocean and Sciadonus pedicellaris Garman, 1899, known from the northeastern Atlantic and northeastern and southwestern Pacific Oceans. Also included are Aphyonus gelatinosus Günther, 1878 known from all oceans including ten specimens from Australian waters, Barathronus maculatus Shcherbachev, 1976 known from South Africa to the westernmost Pacific including 13 specimens from Australian waters, Sciadonus longiventralis Nielsen, 2018 known from the holotype collected off New South Wales and finally Barathronus algrahami n. sp. known from the holotype caught off South Australia and four paratypes from off Taiwan and northern Philippines. Close examination of specimens collected during recent voyages combined with recent and ongoing studies by the first author and DNA COI barcoding analysis enabled an assessment of the aphyonid-clade species hitherto recorded from Australian waters. An identification key to the eight aphyonid clade species known from Australian waters is provided.

Effects of Including Misidentified Sharks in Life History Analyses: A Case Study on the Grey Reef Shark Carcharhinus amblyrhynchos from Papua New Guinea.

Fisheries observer programs are used around the world to collect crucial information and samples that inform fisheries management. However, observer error may misidentify similar-looking shark species. This raises questions about the level of error that species misidentifications could introduce to estimates of species' life history parameters. This study addressed these questions using the Grey Reef Shark Carcharhinus amblyrhynchos as a case study. Observer misidentification rates were quantified by validating species identifications using diagnostic photographs taken on board supplemented with DNA barcoding. Length-at-age and maturity ogive analyses were then estimated and compared with and without the misidentified individuals. Vertebrae were retained from a total of 155 sharks identified by observers as C. amblyrhynchos. However, 22 (14%) of these were sharks were misidentified by the observers and were subsequently re-identified based on photographs and/or DNA barcoding. Of the 22 individuals misidentified as C. amblyrhynchos, 16 (73%) were detected using photographs and a further 6 via genetic validation. If misidentified individuals had been included, substantial error would have been introduced to both the length-at-age and the maturity estimates. Thus validating the species identification, increased the accuracy of estimated life history parameters for C. amblyrhynchos. From the corrected sample a multi-model inference approach was used to estimate growth for C. amblyrhynchos using three candidate models. The model averaged length-at-age parameters for C. amblyrhynchos with the sexes combined were L∞ = 159 cm TL and L0 = 72 cm TL. Females mature at a greater length (l50 = 136 cm TL) and older age (A50 = 9.1 years) than males (l50 = 123 cm TL; A50 = 5.9 years). The inclusion of techniques to reduce misidentification in observer programs will improve the results of life history studies and ultimately improve management through the use of more accurate data for assessments.

Integrated Taxonomy Reveals Hidden Diversity in Northern Australian Fishes: A New Species of Seamoth (Genus Pegasus).

Fishes are one of the most intensively studied marine taxonomic groups yet cryptic species are still being discovered. An integrated taxonomic approach is used herein to delineate and describe a new cryptic seamoth (genus Pegasus) from what was previously a wide-ranging species. Preliminary mitochondrial DNA barcoding indicated possible speciation in Pegasus volitans specimens collected in surveys of the Torres Strait and Great Barrier Reef off Queensland in Australia. Morphological and meristic investigations found key differences in a number of characters between P. volitans and the new species, P. tetrabelos. Further mt DNA barcoding of both the COI and the slower mutating 16S genes of additional specimens provided strong support for two separate species. Pegasus tetrabelos and P. volitans are sympatric in northern Australia and were frequently caught together in trawls at the same depths.

Rediscovery of the Threatened River Sharks, Glyphis garricki and G. glyphis, in Papua New Guinea.

Recent surveys of the shark and ray catches of artisanal fishers in the Western Province of Papua New Guinea (PNG) resulted in the rediscovery of the threatened river sharks, Glyphis garricki and Glyphis glyphis. These represent the first records of both species in PNG since the 1960s and 1970s and highlight the lack of studies of shark biodiversity in PNG. Two individuals of G. garricki and three individuals of G. glyphis were recorded from coastal marine waters of the Daru region of PNG in October and November 2014. The two G. garricki specimens were small individuals estimated to be 100-105 cm and ~113 cm total length (TL). The three G. glyphis specimens were all mature, one a pregnant female and two adult males. These are the first adults of G. glyphis recorded to date providing a more accurate maximum size for this species, i.e. ~260 cm TL. A single pup which was released from the pregnant female G. glyphis, was estimated to be ~65 cm TL. Anecdotal information from the fishers of pregnant females of G. glyphis containing 6 or 7 pups provides the first estimate of litter size for this species. The jaws of the pregnant female G. glyphis were retained and a detailed description of the dentition is provided, since adult dentition has not been previously documented for this species. Genetic analyses confirmed the two species cluster well within samples from these species collected in northern Australia.

Establishing the evolutionary compatibility of potential sources of colonizers for overfished stocks: a population genomics approach.

Identifying fish stock structure is fundamental to pinpoint stocks that might contribute colonizers to overfished stocks. However, a stock's potential to contribute to rebuilding hinges on demographic connectivity, a challenging parameter to measure. With genomics as a new tool, fisheries managers can detect signatures of natural selection and thus identify fishing areas likely to contribute evolutionarily compatible colonizers to an overfished area (i.e. colonizers that are not at a fitness disadvantage in the overfished area and able to reproduce at optimal rates). Identifying evolutionarily compatible stocks would help narrow the focus on establishing demographic connectivity where it matters. Here, we genotype 4723 SNPs in 616 orange roughy (Hoplostethus atlanticus) across five fishing areas off the Tasmanian coast in Australia. We ask whether these areas form a single genetic unit, and test for signatures of local adaptation. Results from amova, structure, discriminant analysis of principal components, BAYESASS and isolation by distance suggest that sampled locations are subjected to geneflow amounts that are above what is needed to establish 'drift connectivity'. However, it remains unclear whether there is a single panmictic population or several highly connected populations. Most importantly, we did not find any evidence of local adaptation, suggesting that the examined orange roughy stocks are evolutionarily compatible. The data have helped test an assumption of the orange roughy management programme and to formulate hypotheses regarding stock demographic connectivity. Overall, our results demonstrate the potential of genomics to inform fisheries management, even when evidence for stock structure is sparse.

Cryptic diversity in flathead fishes (Scorpaeniformes: Platycephalidae) across the Indo-West Pacific uncovered by DNA barcoding.

Identification of taxonomical units underpins most biological endeavours ranging from accurate biodiversity estimates to the effective management of sustainably harvested, protected or endangered species. Successful species identification is now frequently based on a combination of approaches including morphometrics and DNA markers. Sequencing of the mitochondrial COI gene is an established methodology with an international campaign directed at barcoding all fishes. We employed COI sequencing alongside traditional taxonomic identification methods and uncovered instances of deep intraspecific genetic divergences among flathead species. Sixty-five operational taxonomic units (OTUs) were observed across the Indo-West Pacific from just 48 currently recognized species. The most comprehensively sampled taxon, Platycephalus indicus, exhibited the highest levels of genetic diversity with eight lineages separated by up to 16.37% genetic distance. Our results clearly indicate a thorough reappraisal of the current taxonomy of P. indicus (and its three junior synonyms) is warranted in conjunction with detailed taxonomic work on the other additional Platycephalidae OTUs detected by DNA barcoding.