Biodiversity of Philippine marine fishes: A DNA barcode reference library based on voucher specimens.

Accurate identification of fishes is essential for understanding their biology and to ensure food safety for consumers. DNA barcoding is an important tool because it can verify identifications of both whole and processed fishes that have had key morphological characters removed (e.g., filets, fish meal); however, DNA reference libraries are incomplete, and public repositories for sequence data contain incorrectly identified sequences. During a nine-year sampling program in the Philippines, a global biodiversity hotspot for marine fishes, we developed a verified reference library of cytochrome c oxidase subunit I (COI) sequences for 2,525 specimens representing 984 species. Specimens were primarily purchased from markets, with additional diversity collected using rotenone or fishing gear. Species identifications were verified based on taxonomic, phenotypic, and genotypic data, and sequences are associated with voucher specimens, live-color photographs, and genetic samples catalogued at Smithsonian Institution, National Museum of Natural History. The Biodiversity of Philippine Marine Fishes dataset is released herein to increase knowledge of species diversity and distributions and to facilitate accurate identification of market fishes.

Sampling multiple life stages significantly increases estimates of marine biodiversity.

Biodiversity assessments are critical for setting conservation priorities, understanding ecosystem function and establishing a baseline to monitor change. Surveys of marine biodiversity that rely almost entirely on sampling adult organisms underestimate diversity because they tend to be limited to habitat types and individuals that can be easily surveyed. Many marine animals have planktonic larvae that can be sampled from the water column at shallow depths. This life stage often is overlooked in surveys but can be used to relatively rapidly document diversity, especially for the many species that are rare or live cryptically as adults. Using DNA barcode data from samples of nemertean worms collected in three biogeographical regions-Northeastern Pacific, the Caribbean Sea and Eastern Tropical Pacific-we found that most species were collected as either benthic adults or planktonic larvae but seldom in both stages. Randomization tests show that this deficit of operational taxonomic units collected as both adults and larvae is extremely unlikely if larvae and adults were drawn from the same pool of species. This effect persists even in well-studied faunas. These results suggest that sampling planktonic larvae offers access to a different subset of species and thus significantly increases estimates of biodiversity compared to sampling adults alone. Spanish abstract is available in the electronic supplementary material.

Evolution and phylogeny of the deep-sea isopod families Desmosomatidae Sars, 1897 and Nannoniscidae Hansen, 1916 (Isopoda: Asellota).

In the deep sea, the phylogeny and biogeography of only a few taxa have been well studied. Although more than 200 species in 32 genera have been described for the asellote isopod families Desmosomatidae Sars, 1897 and Nannoniscidae Hansen, 1916 from all ocean basins, their phylogenetic relationships are not completely understood. There is little doubt about the close relationship of these families, but the taxonomic position of a number of genera is so far unknown. Based on a combined morphological phylogeny using the Hennigian method with a dataset of 107 described species and a molecular phylogeny based on three markers (COI, 16S, and 18S) with 75 species (most new to science), we could separate Desmosomatidae and Nannoniscidae as separate families. However, we could not support the concept of the subfamilies Eugerdellatinae Hessler, 1970 and Desmosomatinae Hessler, 1970. Most genera of both families were well supported, but several genera appear as para- or even polyphyletic. Within both families, convergent evolution and analogies caused difficulty in defining apomorphies for phylogenetic reconstructions and this is reflected in the results of the concatenated molecular tree. There is no biogeographic pattern in the distribution as the genera occur over the entire Atlantic and Pacific Ocean, showing no specific phylogeographical pattern. Poor resolution at deep desmosomatid nodes may reflect the long evolutionary history of the family and rapid evolutionary radiations. Supplementary Information: The online version contains supplementary material available at 10.1007/s13127-021-00509-9.

DROP: Molecular voucher database for identification of Drosophila parasitoids.

Molecular identification is increasingly used to speed up biodiversity surveys and laboratory experiments. However, many groups of organisms cannot be reliably identified using standard databases such as GenBank or BOLD due to lack of sequenced voucher specimens identified by experts. Sometimes a large number of sequences are available, but with too many errors to allow identification. Here, we address this problem for parasitoids of Drosophila by introducing a curated open-access molecular reference database, DROP (Drosophila parasitoids). Identifying Drosophila parasitoids is challenging and poses a major impediment to realize the full potential of this model system in studies ranging from molecular mechanisms to food webs, and in biological control of Drosophila suzukii. In DROP, genetic data are linked to voucher specimens and, where possible, the voucher specimens are identified by taxonomists and vetted through direct comparison with primary type material. To initiate DROP, we curated 154 laboratory strains, 856 vouchers, 554 DNA sequences, 16 genomes, 14 transcriptomes, and six proteomes drawn from a total of 183 operational taxonomic units (OTUs): 114 described Drosophila parasitoid species and 69 provisional species. We found species richness of Drosophila parasitoids to be heavily underestimated and provide an updated taxonomic catalogue for the community. DROP offers accurate molecular identification and improves cross-referencing between individual studies that we hope will catalyse research on this diverse and fascinating model system. Our effort should also serve as an example for researchers facing similar molecular identification problems in other groups of organisms.

Misidentification of Bellator gymnostethus (Gilbert, 1892) as Prionotus ruscarius Gilbert amp; Starks, 1904 (Scorpaeniformes: Triglidae).

The checklist by Robertson et al. (2017) of fishes from the tropical eastern Pacific included information on three members of the family Triglidae: Bellator loxias (Jordan, 1897), Prionotus ruscarius and P. stephanophrys Lockington, 1881. Unfortunately, the identification of four specimens as P. ruscarius is incorrect, as they are Bellator gymnostethus. We thank Benjamin Victor for bringing these misidentifications to our notice through his work with mtDNA sequence data from the Barcode of Life Database (BOLD: http://www.boldsystems.org). The photographs of the four specimens on the BOLD website clearly depict a Bellator species rather than a Prionotus. However, the photograph in the 2017 paper (Figure 75, page 78), is correctly identified and labelled as P. ruscarius, and this species was collected on the cruise of the Miguel Oliver discussed in Robertson et al. (2017), see Benavides Moreno et al. (2019). This correction brings the number of triglids collected on that cruise to four species.

Documenting decapod biodiversity in the Caribbean from DNA barcodes generated during field training in taxonomy.

DNA barcoding is a useful tool to identify the components of mixed or bulk samples, as well as to determine individuals that lack morphologically diagnostic features. However, the reference database of DNA barcode sequences is particularly sparsely populated for marine invertebrates and for tropical taxa. We used samples collected as part of two field courses, focused on graduate training in taxonomy and systematics, to generate DNA sequences of the barcode fragments of cytochrome c oxidase subunit I (COI) and mitochondrial ribosomal 16S genes for 447 individuals, representing at least 129 morphospecies of decapod crustaceans. COI sequences for 36% (51/140) of the species and 16S sequences for 26% (37/140) of the species were new to GenBank. Automatic Barcode Gap Discovery identified 140 operational taxonomic units (OTUs) which largely coincided with the morphospecies delimitations. Barcode identifications (i.e. matches to identified sequences) were especially useful for OTUs within Synalpheus, a group that is notoriously difficult to identify and rife with cryptic species, a number of which we could not identify to species, based on morphology. Non-concordance between morphospecies and barcode OTUs also occurred in a few cases of suspected cryptic species. As mitochondrial pseudogenes are particularly common in decapods, we investigate the potential for this dataset to include pseudogenes and discuss the utility of these sequences as species identifiers (i.e. barcodes). These results demonstrate that material collected and identified during training activities can provide useful incidental barcode reference samples for under-studied taxa.

A DNA barcode reference library of French Polynesian shore fishes.

The emergence of DNA barcoding and metabarcoding opened new ways to study biological diversity, however, the completion of DNA barcode libraries is fundamental for such approaches to succeed. This dataset is a DNA barcode reference library (fragment of Cytochrome Oxydase I gene) for 2,190 specimens representing at least 540 species of shore fishes collected over 10 years at 154 sites across the four volcanic archipelagos of French Polynesia; the Austral, Gambier, Marquesas and Society Islands, a 5,000,000 km2 area. At present, 65% of the known shore fish species of these archipelagoes possess a DNA barcode associated with preserved, photographed, tissue sampled and cataloged specimens, and extensive collection locality data. This dataset represents one of the most comprehensive DNA barcoding efforts for a vertebrate fauna to date. Considering the challenges associated with the conservation of coral reef fishes and the difficulties of accurately identifying species using morphological characters, this publicly available library is expected to be helpful for both authorities and academics in various fields.

Reference DNA barcodes and other mitochondrial markers for identifying Caribbean Octocorals.

DNA barcoding is a useful tool for documenting the diversity of metazoans. The most commonly used barcode markers, 16S and COI, are not considered suitable for species identification within some "basal" phyla of metazoans. Nevertheless metabarcoding studies of bulk mixed samples commonly use these markers and may obtain sequences for "basal" phyla. We sequenced mitochondrial DNA fragments of cytochrome oxidase c subunit I (COI), 16S ribosomal RNA (16S), NADH dehydrogenase subunits 2 (16S-ND2), 6 (ND6-ND3) and 4L (ND4L-MSH) for 27 species of Caribbean octocorals to create a reference barcode dataset and to compare the utility of COI and 16S to other markers more typically used for octocorals. The most common genera (Erythropodium, Ellisella, Briareum, Plexaurella, Muriceopsis and Pterogorgia) were effectively distinguished by small differences (5 or more substitutions or indels) in COI and 16S sequences. Gorgonia and Antillogorgia were effectively distinguished from each other by unique haplotypes, but the small genetic differences make distance approaches ineffective for these taxa. Plexaura, Pseudoplexaura and Eunicea were indistinguishable from each other but were generally effectively distinguished from other genera, further supporting the idea that these genera have undergone a rapid endemic radiation in the Caribbean.

Nuclear introns help unravel the diversification history of the Australo-Pacific Petroica robins.

Australo-Pacific Petroica robins are known for their striking variability in sexual plumage coloration. Molecular studies in recent years have revised the taxonomy of species and subspecies boundaries across the southwest Pacific and New Guinea. However, these studies have not been able to resolve phylogenetic relationships within Petroica owing to limited sampling of the nuclear genome. Here, we sequence five nuclear introns across all species for which fresh tissue was available. Nuclear loci offer support for major geographic lineages that were first inferred from mtDNA. We find almost no shared nuclear alleles between currently recognized species within the New Zealand and Australian lineages, whereas the Pacific robin radiation has many shared alleles. Multilocus coalescent species trees based on nuclear loci support a sister relationship between the Australian lineage and the Pacific robin radiation-a node that is poorly supported by mtDNA. We also find discordance in support for a sister relationship between the similarly plumaged Rose Robin (P. rosea) and Pink Robin (P. rodinogaster). Our nuclear data complement previous mtDNA studies in suggesting that the phenotypically cryptic eastern and western populations of Australia's Scarlet Robin (P. boodang) are genetically distinct lineages at the early stages of divergence and speciation.

A genetic fingerprint of Amphipoda from Icelandic waters - the baseline for further biodiversity and biogeography studies.

Amphipods constitute an abundant part of Icelandic deep-sea zoobenthos yet knowledge of the diversity of this fauna, particularly at the molecular level, is scarce. The present work aims to use molecular methods to investigate genetic variation of the Amphipoda sampled during two IceAGE collecting expeditions. The mitochondrial cytochrome oxidase subunit 1 (COI) of 167 individuals originally assigned to 75 morphospecies was analysed. These targeted morhospecies were readily identifiable by experts using light microscopy and representative of families where there is current ongoing taxonomic research. The study resulted in 81 Barcode Identity Numbers (BINs) (of which >90% were published for the first time), while Automatic Barcode Gap Discovery revealed the existence of 78 to 83 Molecular Operational Taxonomic Units (MOTUs). Six nominal species (Rhachotropis helleri, Arrhis phyllonyx, Deflexilodes tenuirostratus, Paroediceros propinquus, Metopa boeckii, Astyra abyssi) appeared to have a molecular variation higher than the 0.03 threshold of both p-distance and K2P usually used for amphipod species delineation. Conversely, two Oedicerotidae regarded as separate morphospecies clustered together with divergences in the order of intraspecific variation. The incongruence between the BINs associated with presently identified species and the publicly available data of the same taxa was observed in case of Paramphithoe hystrix and Amphilochus manudens. The findings from this research project highlight the necessity of supporting molecular studies with thorough morphology species analyses.

Rhachotropis (Eusiroidea, Amphipoda) from the North East Atlantic.

The genus Rhachotropis has the widest geographic and bathymetric distribution of all amphipod genera worldwide. Molecular and morphological investigations of specimens sampled around Iceland and off the Norwegian coast allow the first insights into the relationships of North East Atlantic Rhachotropis. The 31 cytochrome oxidase subunit I (COI) sequences generated for this study were assigned 13 Barcode Index Numbers (BINs) in the Barcode of Life database (BOLD), of which 12 are new to the database. Molecular analyses of COI and 16S sequences could not confirm a theory that depth has a greater influence on the phylogeny of Rhachotropis than geographic distance. Although the North East Atlantic is a well-studied area, our molecular investigations revealed the genus Rhachotropis may contain cryptic species, which indicates a higher biodiversity than currently known. For example, the specimens which key to Rhachotropis helleri is a complex of three COI clades, two of which cannot be identified with morphological traits. One specimen of each of the clades in the cladogram was documented by high definition photographs. A special focus was on the visual morphology of the eyes, as this character shows interspecific differences within the genus Rhachotropis in response to fixation in ethanol. Detailed morphological investigation showed that some clades thought to be indistinguishable can be separated by minute but consistent morphological characters. Datamining Genbank to examine all registered COI-sequences of R. aculeata, the only previously known Rhachotropis BIN in the North Atlantic and sub-Arctic, showed R. aculeata to be subdivided by an Arctic and a North Atlantic population.

Deep-water bony fishes collected by the B/O Miguel Oliver on the shelf edge of Pacific Central America: an annotated, illustrated and DNA-barcoded checklist.

An annotated and photographically illustrated checklist with DNA barcodes of the species of bony fishes collected during a month-long research cruise of the Spanish Research vessel B/O Miguel Oliver is presented. The vessel made trawls on the continental shelf of the Pacific coast of Central America, in November-December 2010, at depths of 108-1625 m. This list, based on 707 specimens (of a total of 876 specimens collected during the whole expedition), includes 129 species belonging to 15 orders, 61 families, and 97 genera. New information is presented on the geographical distributions of more than a third of those species, with 29 species (22.4%) representing new records from Central American waters and 17 species (13.2%) having expanded latitudinal ranges. Data on capture depths demonstrate increased depth ranges due to new minimum and/or maximum known depths for 31 species, i.e. 24% of those captured. Tissue samples from frozen specimens were used to obtain DNA barcodes of 682 (96.5%) individuals belonging to 118 species (91.4% of those recorded here), which have been made publically available in Genbank. Those data include barcodes for 84 species (65.1% of the total collected, and 77.1% of those for which barcodes were obtained) and 30 genera (30.9% of those collected) for which no species barcodes have been previously published. Barcodes of 54 species represent the first genetic sequences of any type published for those species. The abundance of new data indicate that there is still much to learn about the composition and geographical and depth distributions of the fish fauna of the shelf edge and continental slope of this region.

DNA barcode data accurately assign higher spider taxa.

The use of unique DNA sequences as a method for taxonomic identification is no longer fundamentally controversial, even though debate continues on the best markers, methods, and technology to use. Although both existing databanks such as GenBank and BOLD, as well as reference taxonomies, are imperfect, in best case scenarios "barcodes" (whether single or multiple, organelle or nuclear, loci) clearly are an increasingly fast and inexpensive method of identification, especially as compared to manual identification of unknowns by increasingly rare expert taxonomists. Because most species on Earth are undescribed, a complete reference database at the species level is impractical in the near term. The question therefore arises whether unidentified species can, using DNA barcodes, be accurately assigned to more inclusive groups such as genera and families-taxonomic ranks of putatively monophyletic groups for which the global inventory is more complete and stable. We used a carefully chosen test library of CO1 sequences from 49 families, 313 genera, and 816 species of spiders to assess the accuracy of genus and family-level assignment. We used BLAST queries of each sequence against the entire library and got the top ten hits. The percent sequence identity was reported from these hits (PIdent, range 75-100%). Accurate assignment of higher taxa (PIdent above which errors totaled less than 5%) occurred for genera at PIdent values >95 and families at PIdent values ≥ 91, suggesting these as heuristic thresholds for accurate generic and familial identifications in spiders. Accuracy of identification increases with numbers of species/genus and genera/family in the library; above five genera per family and fifteen species per genus all higher taxon assignments were correct. We propose that using percent sequence identity between conventional barcode sequences may be a feasible and reasonably accurate method to identify animals to family/genus. However, the quality of the underlying database impacts accuracy of results; many outliers in our dataset could be attributed to taxonomic and/or sequencing errors in BOLD and GenBank. It seems that an accurate and complete reference library of families and genera of life could provide accurate higher level taxonomic identifications cheaply and accessibly, within years rather than decades.

Recurrent hybridization and recent origin obscure phylogenetic relationships within the 'white-headed' gull (Larus sp.) complex.

Species complexes that have undergone recent radiations are often characterized by extensive allele sharing due to recent ancestry and (or) introgressive hybridization. This can result in discordant evolutionary histories of genes and heterogeneous genomes, making delineating species limits difficult. Here we examine the phylogenetic relationships among a complex group of birds, the white-headed gulls (Aves: Laridae), which offer a unique window into the speciation process due to their recent evolutionary history and propensity to hybridize. Relationships were examined among 17 species (61 populations) using a multilocus approach, including mitochondrial and nuclear intron DNA sequences and microsatellite genotype information. Analyses of microsatellite and intron data resulted in some species-based groupings, although most species were not represented by a single cluster. Considerable allele and haplotype sharing among white-headed gull species was observed; no locus contained a species-specific clade. Despite this, our multilocus approach provided better resolution among some species than previous studies. Interestingly, most clades appear to correspond to geographic locality: our BEAST analysis recovered strong support for a northern European/Icelandic clade, a southern European/Russian clade, and a western North American/canus clade, with weak evidence for a high latitude clade spanning North America and northwestern Europe. This geographical structuring is concordant with behavioral observations of pervasive hybridization in areas of secondary contact. The extent of allele and haplotype sharing indicates that ecological and sexual selection are likely not strong enough to complete reproductive isolation within several species in the white-headed gull complex. This suggests that just a few genes are driving the speciation process.

DNA barcoding applied to ex situ tropical amphibian conservation programme reveals cryptic diversity in captive populations.

Amphibians constitute a diverse yet still incompletely characterized clade of vertebrates, in which new species are still being discovered and described at a high rate. Amphibians are also increasingly endangered, due in part to disease-driven threats of extinctions. As an emergency response, conservationists have begun ex situ assurance colonies for priority species. The abundance of cryptic amphibian diversity, however, may cause problems for ex situ conservation. In this study we used a DNA barcoding approach to survey mitochondrial DNA (mtDNA) variation in captive populations of 10 species of Neotropical amphibians maintained in an ex situ assurance programme at El Valle Amphibian Conservation Center (EVACC) in the Republic of Panama. We combined these mtDNA sequences with genetic data from presumably conspecific wild populations sampled from across Panama, and applied genetic distance-based and character-based analyses to identify cryptic lineages. We found that three of ten species harboured substantial cryptic genetic diversity within EVACC, and an additional three species harboured cryptic diversity among wild populations, but not in captivity. Ex situ conservation efforts focused on amphibians are therefore vulnerable to an incomplete taxonomy leading to misidentification among cryptic species. DNA barcoding may therefore provide a simple, standardized protocol to identify cryptic diversity readily applicable to any amphibian community.

Using DNA barcoding to assess Caribbean reef fish biodiversity: expanding taxonomic and geographic coverage.

This paper represents a DNA barcode data release for 3,400 specimens representing 521 species of fishes from 6 areas across the Caribbean and western central Atlantic regions (FAO Region 31). Merged with our prior published data, the combined efforts result in 3,964 specimens representing 572 species of marine fishes and constitute one of the most comprehensive DNA barcoding "coverages" for a region reported to date. The barcode data are providing new insights into Caribbean shorefish diversity, allowing for more and more accurate DNA-based identifications of larvae, juveniles, and unknown specimens. Examples are given correcting previous work that was erroneous due to database incompleteness.

Construction and analysis of phylogenetic trees using DNA barcode data.

The assembly of sequence data obtained from DNA barcodes into phylogenies or NJ trees has proven highly useful in estimating relatedness among species as well as providing a framework in which hypotheses regarding the evolution of traits or species distributions may be investigated. In this chapter, we outline the process by which DNA sequence data is assembled into a phylogenetically informative matrix, and then provide details on the methods to reconstruct NJ or phylogenetic trees that employ DNA barcode data, using only barcode data or combining barcodes with other data.

Introduction to animal DNA barcoding protocols.

Procedures and protocols common to many DNA barcoding projects are summarized. Planning for any project should emphasize front-end procedures, especially the "genetic lockdown" of collected materials for downstream genetic procedures. Steps further into the DNA barcoding process chain, such as sequencing, data processing, and other back-end functions vary slightly, if at all, among projects and are presented elsewhere in the volume. Point-of-collection sample and tissue handling and data/metadata handling are stressed. Specific predictions of the future workflows and mechanics of DNA barcoding are difficult, so focus is on that which most or all future methods and technologies will surely share.

DNA barcoding fishes.

This chapter is an overview of the techniques for DNA barcoding of fishes from field collection to DNA sequence analysis. Recommendations for modifications of field protocols and best tissue sampling practices are made. A variety of DNA extraction protocols is provided, including high-throughput robot-assisted methods. A pair of well-tested forward and reverse primers for PCR amplification and sequencing are presented. These primers have been successfully used for DNA barcode on a wide array of marine fish taxa and also work well in most freshwater and cartilaginous fishes. Recipes and cycling protocols for both PCR amplification and sequencing and cleanup methods for the reaction products are provided. A method for the consistent production of high-quality DNA barcodes from DNA sequence data is given and stringent guidelines for judging the quality of raw sequence data are laid out.

Multiple shifts between violet and ultraviolet vision in a family of passerine birds with associated changes in plumage coloration.

Colour vision in diurnal birds falls into two discrete classes, signified by the spectral sensitivity of the violet- (VS) or ultraviolet-sensitive (UVS) short wavelength-sensitive type 1 (SWS1) single cone. Shifts between sensitivity classes are rare; three or four are believed to have happened in the course of avian evolution, one forming UVS higher passerines. Such shifts probably affect the expression of shortwave-dominated plumage signals. We have used genomic DNA sequencing to determine VS or UVS affinity in fairy-wrens and allies, Maluridae, a large passerine family basal to the known UVS taxa. We have also spectrophotometrically analysed male plumage coloration as perceived by the VS and UVS vision systems. Contrary to any other investigated avian genus, Malurus (fairy-wrens) contains species with amino acid residues typical of either VS or UVS cone opsins. Three bowerbird species (Ptilonorhynchidae) sequenced for outgroup comparison carry VS opsin genes. Phylogenetic reconstructions render one UVS gain followed by one or more losses as the most plausible evolutionary scenario. The evolution of avian ultraviolet sensitivity is hence more complex, as a single shift no longer explains its distribution in Passeriformes. Character correlation analysis proposes that UVS vision is associated with shortwave-reflecting plumage, which is widespread in Maluridae.