Meta-Fish-Lib: A generalised, dynamic DNA reference library pipeline for metabarcoding of fishes.

The accuracy and reliability of DNA metabarcoding analyses depend on the breadth and quality of the reference libraries that underpin them. However, there are limited options available to obtain and curate the huge volumes of sequence data that are available on public repositories such as NCBI and BOLD. Here, we provide a pipeline to download, clean and annotate mitochondrial DNA sequence data for a given list of fish species. Features of this pipeline include (a) support for multiple metabarcode markers; (b) searches on species synonyms and taxonomic name validation; (c) phylogeny assisted quality control for identification and removal of misannotated sequences; (d) automatically generated coverage reports for each new GenBank release update; and (e) citable, versioned DOIs. As an example we provide a ready-to-use curated reference library for the marine and freshwater fishes of the U.K. To augment this reference library for environmental DNA metabarcoding specifically, we generated 241 new MiFish-12S sequences for 88 U.K. marine species, and make available new primer sets useful for sequencing these. This brings the coverage of common U.K. species for the MiFish-12S fragment to 93%, opening new avenues for scaling up fish metabarcoding across wide spatial gradients. The Meta-Fish-Lib reference library and pipeline is hosted at https://github.com/genner-lab/meta-fish-lib.

An In Silico Comparison of Protocols for Dated Phylogenomics.

In the age of genome-scale DNA sequencing, choice of molecular marker arguably remains an important decision in planning a phylogenetic study. Using published genomes from 23 primate species, we make a standardized comparison of four of the most frequently used protocols in phylogenomics, viz., targeted sequence-enrichment using ultraconserved element and exon-capture probes, and restriction-site-associated DNA sequencing (RADseq and ddRADseq). Here, we present a procedure to perform in silico extractions from genomes and create directly comparable data sets for each class of marker. We then compare these data sets in terms of both phylogenetic resolution and ability to consistently and precisely estimate clade ages using fossil-calibrated molecular-clock models. Furthermore, we were also able to directly compare these results to previously published data sets from Sanger-sequenced nuclear exons and mitochondrial genomes under the same analytical conditions. Our results show-although with the exception of the mitochondrial genome data set and the smallest ddRADseq data set-that for uncontroversial nodes all data classes performed equally well, that is they recovered the same well supported topology. However, for one difficult-to-resolve node comprising a rapid diversification, we report well supported but conflicting topologies among the marker classes consistent with the mismodeling of gene tree heterogeneity as demonstrated by species tree analyses of single nucleotide polymorphisms. Likewise, clade age estimates showed consistent discrepancies between data sets under strict and relaxed clock models; for recent nodes, clade ages estimated by nuclear exon data sets were younger than those of the UCE, RADseq and mitochondrial data, but vice versa for the deepest nodes in the primate phylogeny. This observation is explained by temporal differences in phylogenetic informativeness (PI), with the data sets with strong PI peaks toward the present underestimating the deepest node ages. Finally, we conclude by emphasizing that while huge numbers of loci are probably not required for uncontroversial phylogenetic questions-for which practical considerations such as ease of data generation, sharing, and aggregating, therefore become increasingly important-accurately modeling heterogeneous data remains as relevant as ever for the more recalcitrant problems.

Nuclear environmental DNA resolves fine-scale population genetic structure in an aquatic habitat.

There is considerable potential for nuclear genomic material in environmental DNA (eDNA) to inform us of population genetic structure within aquatic species. We tested if nuclear allelic composition data sourced from eDNA can resolve fine scale spatial genetic structure of the cichlid fish Astatotilapia calliptera in Lake Masoko, Tanzania. In this ∼35 m deep crater lake the species is diverging into two genetically distinguishable ecomorphs, separated by a thermo-oxycline at ∼15 m that divides biologically distinct water masses. We quantified population genetic structure along a depth transect using single nucleotide polymorphisms (SNPs) derived from genome sequencing of 530 individuals. This population genetic structure was reflected in a focal set of SNPs that were also reliably amplified from eDNA - with allele frequencies derived from eDNA reflecting those of fish within each depth zone. Thus, by targeting known genetic variation between populations within aquatic eDNA, we measured genetic structure within the focal species.

Invalid lectotypification for Synodontisvictoriae Boulenger, 1906 (Siluriformes, Mochokidae) by Poll (1971), and the designation of a new lectotype.

The lectotype and paralectotype of Synodontisvictoriae Boulenger, 1906, designated by Poll (1971), were examined. Inconsistencies between data presented for the designated lectotype and the illustrated individual raise the question of whether lectotypification by Poll is valid. This case is not formally regulated by the International Code of Zoological Nomenclature, but based on Article 74.5, the lectotypification for S.victoriae should be considered invalid because it cannot unambiguously indicate a single name-bearing specimen. Thus, we designate a new lectotype for S.victoriae (BMNH 1906.5.30.191, Entebbe, standard length 188.2 mm) out of two syntypes and provide illustrations and new morphometric and meristic data for both specimens.

Correction: Schistosoma species detection by environmental DNA assays in African freshwaters.

[This corrects the article DOI: 10.1371/journal.pntd.0008129.].

Schistosoma species detection by environmental DNA assays in African freshwaters.

BACKGROUND: Schistosomiasis is a neglected tropical parasitic disease associated with severe pathology, mortality and economic loss worldwide. Programs for disease control may benefit from specific and sensitive diagnostic methods to detect Schistosoma trematodes in aquatic environments. Here we report the development of novel environmental DNA (eDNA) qPCR assays for the presence of the human-infecting species Schistosoma mansoni, S. haematobium and S. japonicum. METHODOLOGY/PRINCIPAL FINDINGS: We first tested the specificity of the assays across the three species using genomic DNA preparations which showed successful amplification of target sequences with no cross amplification between the three focal species. In addition, we evaluated the specificity of the assays using synthetic DNA of multiple Schistosoma species, and demonstrated a high overall specificity; however, S. japonicum and S. haematobium assays showed cross-species amplification with very closely-related species. We next tested the effectiveness of the S. mansoni assay using eDNA samples from aquaria containing infected host gastropods, with the target species revealed as present in all infected aquaria. Finally, we evaluated the effectiveness of the S. mansoni and S. haematobium assays using eDNA samples from eight discrete natural freshwater sites in Tanzania, and demonstrated strong correspondence between infection status established using eDNA and conventional assays of parasite prevalence in host snails. CONCLUSIONS/SIGNIFICANCE: Collectively, our results suggest that eDNA monitoring is able to detect schistosomes in freshwater bodies, but refinement of the field sampling, storage and assay methods are likely to optimise its performance. We anticipate that environmental DNA-based approaches will help to inform epidemiological studies and contribute to efforts to control and eliminate schistosomiasis in endemic areas.

Climate Change Drives Poleward Increases and Equatorward Declines in Marine Species.

Marine environments have increased in temperature by an average of 1°C since pre-industrial (1850) times [1]. Given that species ranges are closely allied to physiological thermal tolerances in marine organisms [2], it may therefore be expected that ocean warming would lead to abundance increases at poleward side of ranges and abundance declines toward the equator [3]. Here, we report a global analysis of abundance trends of 304 widely distributed marine species over the last century, across a range of taxonomic groups from phytoplankton to fish and marine mammals. Specifically, using a literature database, we investigate the extent that the direction and strength of long-term species abundance changes depend on the sampled location within the latitudinal range of species. Our results show that abundance increases have been most prominent where sampling has taken place at the poleward side of species ranges, and abundance declines have been most prominent where sampling has taken place at the equatorward side of species ranges. These data provide evidence of omnipresent large-scale changes in abundance of marine species consistent with warming over the last century and suggest that adaptation has not provided a buffer against the negative effects of warmer conditions at the equatorward extent of species ranges. On the basis of these results, we suggest that projected sea temperature increases of up to 1.5°C over pre-industrial levels by 2050 [4] will continue to drive latitudinal abundance shifts in marine species, including those of importance for coastal livelihoods.

Environmental DNA-based xenomonitoring for determining Schistosoma presence in tropical freshwaters.

BACKGROUND: Schistosomiasis is a neglected tropical disease that infects over 200 million people worldwide. Control measures can benefit from improved surveillance methods in freshwaters, with environmental DNA (eDNA) surveys having the potential to offer effective and rapid detection of schistosomes. However, sampling eDNA directly from natural water bodies can lead to inaccurate estimation of infection risk if schistosome eDNA is rare in the environment. Here we report a xenomonitoring method that allows schistosome infections of host snail species to be determined from eDNA in water used to house those snails. METHODS: Host snail species were collected and placed in containers of water and allowed to shed cercariae, and then water samples were filtered and tested using qPCR assays specific to the African species Schistosoma mansoni and Schistosoma haematobium. We evaluated this "eDNA-based xenomonitoring" approach by experimentally comparing the results to those obtained from direct qPCR screening of tissue sourced from the snails in the experiment. RESULTS: We found that our method accurately diagnosed the presence of S. mansoni-infected snails in all tests, and S. haematobium-infected snails in 92% of tests. Moreover, we found that the abundance of Schistosoma eDNA in experiments was directly dependent on the number and biomass of infected snails. CONCLUSIONS: These results provide a strong indication that this surveillance method combining the utility of eDNA-based monitoring with the reliability of traditional xenomonitoring approaches could be used to accurately assay the presence of Schistosoma species in natural habitats. This approach may be well-suited for epidemiological studies and monitoring in endemic areas, where it can assist schistosomiasis control by indicating infection risk from freshwaters and guiding necessary interventions to eliminate the disease.

Persistence of environmental DNA in marine systems.

As environmental DNA (eDNA) becomes an increasingly valuable resource for marine ecosystem monitoring, understanding variation in its persistence across contrasting environments is critical. Here, we quantify the breakdown of macrobial eDNA over a spatio-temporal axis of locally extreme conditions, varying from ocean-influenced offshore to urban-inshore, and between winter and summer. We report that eDNA degrades 1.6 times faster in the inshore environment than the offshore environment, but contrary to expectation we find no difference over season. Analysis of environmental covariables show a spatial gradient of salinity and a temporal gradient of pH, with salinity-or the biotic correlates thereof-most important. Based on our estimated inshore eDNA half-life and naturally occurring eDNA concentrations, we estimate that eDNA may be detected for around 48 h, offering potential to collect ecological community data of high local fidelity. We conclude by placing these results in the context of previously published eDNA decay rates.

One thousand DNA barcodes of piranhas and pacus reveal geographic structure and unrecognised diversity in the Amazon.

Piranhas and pacus (Characiformes: Serrasalmidae) are a charismatic but understudied family of Neotropical fishes. Here, we analyse a DNA barcode dataset comprising 1,122 specimens, 69 species, 16 genera, 208 localities, and 34 major river drainages in order to make an inventory of diversity and to highlight taxa and biogeographic areas worthy of further sampling effort and conservation protection. Using four methods of species discovery-incorporating both tree and distance based techniques-we report between 76 and 99 species-like clusters, i.e. between 20% and 33% of a priori identified taxonomic species were represented by more than one mtDNA lineage. There was a high degree of congruence between clusters, with 60% supported by three or four methods. Pacus of the genus Myloplus exhibited the most intraspecific variation, with six of the 13 species sampled found to have multiple lineages. Conversely, piranhas of the Serrasalmus rhombeus group proved difficult to delimit with these methods due to genetic similarity and polyphyly. Overall, our results recognise substantially underestimated diversity in the serrasalmids, and emphasise the Guiana and Brazilian Shield rivers as biogeographically important areas with multiple cases of across-shield and within-shield diversifications. We additionally highlight the distinctiveness and complex phylogeographic history of rheophilic taxa in particular, and suggest multiple colonisations of these habitats by different serrasalmid lineages.

A preliminary inventory of the catfishes of the lower Rio Nhamundá, Brazil (Ostariophysi, Siluriformes).

The Rio Nhamundá is a poorly-known clearwater river draining the southern Guiana Shield of Brazil. In this study we report the findings of a preliminary ichthyological survey, focusing on catfishes (Siluriformes). We identify a total of 36 species (31 genera, seven families) from the Nhamundá, including 11 species already recorded from the river. Overall, our survey results show that even rapid surveys can provide important information on Amazon fish biodiversity, suggesting potential new species, providing range extensions for nominal species, and additionally highlighting taxa in need of taxonomic revision and genetic study. As well as the traditional forms of data collected on biodiversity surveys (i.e. preserved specimen vouchers), our study also provides "new" types of data in the form of DNA barcodes and images of fishes exhibiting colouration in life, information that will be invaluable in future work addressing difficult groups. O Rio Nhamundá é um rio de água clara, pouco conhecido, que drena parte do Escudo das Guianas em território brasileiro. Nesse estudo, nós reportamos os resultados de um levantamento ictiofaunístico preliminar dessa área, tendo como foco os bagres (Siluriformes). Nós identificamos um total de 36 espécies (31 gêneros, sete famílias) provenientes de nossa coleta, e adicionamos 11 espécies já conhecidas para o rio. De maneira geral, os resultados de nossa pesquisa mostram que mesmo levantamentos rápidos podem gerar informações importantes sobre a biodiversidade de peixes amazônicos, sugerindo potenciais espécies novas, ampliando a área de distribuição de espécies, além de apontar a necessidade de revisões taxonômicas e estudos genéticos para alguns taxa. Para além das formas tradicionais de dados coletados em pesquisas de biodiversidade (i.e. espécimes preservados), nosso estudo fornece "novas" formas de dados, como DNA barcodes e imagens com o padrão de coloração dos espécimes vivos, informações essas que serão de valor inestimável para futuros estudos que abordem grupos taxonômicos difíceis.

Integrative taxonomy reveals a new species of pacu (Characiformes: Serrasalmidae: Myloplus) from the Brazilian Amazon

Pacus of the genus Myloplus represent a formidable taxonomic challenge, and particularly so for the case of M. asterias and M. rubripinnis, two widespread and common species that harbor considerable morphological diversity. Here we apply DNA barcoding and multiple species discovery methods to find candidate species in this complex group. We report on one well-supported lineage that is also morphologically and ecologically distinct. This lineage represents a new species that can be distinguished from congeners by the presence of dark chromatophores on lateral-line scales, which gives the appearance of a black lateral line. It can be further diagnosed by having 25-29 branched dorsal-fin rays (vs. 18-24), 89-114 perforated scales from the supracleithrum to the end of hypural plate (vs. 56-89), and 98-120 total lateral line scales (vs. 59-97). The new species is widely distributed in the Amazon basin, but seems to have a preference for black- and clearwater habitats. This ecological preference and black lateral line color pattern bears a striking similarity to the recently described silver dollar Metynnis melanogrammus.(AU)

Pacus do gênero Myloplus representam um desafio taxonômico formidável, e particularmente o caso de M. asterias e M. rubripinnis, duas espécies amplamente distribuídas e comuns que abrigam uma considerável diversidade morfológica. Aplicamos aqui a tecnologia do DNA barcoding e múltiplos métodos de descoberta de espécies para encontrar possíveis espécies novas nesse grupo complexo. Registramos uma linhagem bem suportada que também é distinta morfológica e ecologicamente. Essa linhagem representa uma nova espécie que pode ser distinguida das demais congêneres por apresentar cromatóforos escuros nas escamas da linha lateral que conferem uma aparência de linha lateral preta. Ela pode ser adicionalmente diagnosticada por ter 25-29 raios ramificados na nadadeira dorsal (vs. 18-24), 89-114 escamas perfuradas do supracleitro até o final da placa hipural (vs. 56-89) e 98-120 escamas totais na linha lateral (vs. 59-97). A nova espécie é amplamente distribuída na bacia Amazônica, mas aparentemente possui preferência por habitats de água preta e clara. A preferência ecológica e o padrão de colorido escuro da linha lateral consistem em semelhanças impressionantes com o silver dólar recém descrito Metynnis melanogrammus.(AU)

Barcoding and border biosecurity: identifying cyprinid fishes in the aquarium trade.

BACKGROUND: Poorly regulated international trade in ornamental fishes poses risks to both biodiversity and economic activity via invasive alien species and exotic pathogens. Border security officials need robust tools to confirm identifications, often requiring hard-to-obtain taxonomic literature and expertise. DNA barcoding offers a potentially attractive tool for quarantine inspection, but has yet to be scrutinised for aquarium fishes. Here, we present a barcoding approach for ornamental cyprinid fishes by: (1) expanding current barcode reference libraries; (2) assessing barcode congruence with morphological identifications under numerous scenarios (e.g. inclusion of GenBank data, presence of singleton species, choice of analytical method); and (3) providing supplementary information to identify difficult species. METHODOLOGY/PRINCIPAL FINDINGS: We sampled 172 ornamental cyprinid fish species from the international trade, and provide data for 91 species currently unrepresented in reference libraries (GenBank/Bold). DNA barcodes were found to be highly congruent with our morphological assignments, achieving success rates of 90-99%, depending on the method used (neighbour-joining monophyly, bootstrap, nearest neighbour, GMYC, percent threshold). Inclusion of data from GenBank (additional 157 spp.) resulted in a more comprehensive library, but at a cost to success rate due to the increased number of singleton species. In addition to DNA barcodes, our study also provides supporting data in the form of specimen images, morphological characters, taxonomic bibliography, preserved vouchers, and nuclear rhodopsin sequences. Using this nuclear rhodopsin data we also uncovered evidence of interspecific hybridisation, and highlighted unrecognised diversity within popular aquarium species, including the endangered Indian barb Puntius denisonii. CONCLUSIONS/SIGNIFICANCE: We demonstrate that DNA barcoding provides a highly effective biosecurity tool for rapidly identifying ornamental fishes. In cases where DNA barcodes are unable to offer an identification, we improve on previous studies by consolidating supplementary information from multiple data sources, and empower biosecurity agencies to confidently identify high-risk fishes in the aquarium trade.

Spider: an R package for the analysis of species identity and evolution, with particular reference to DNA barcoding.

Spider: SPecies IDentity and Evolution in R is a new R package implementing a number of useful analyses for DNA barcoding studies and associated research into species delimitation and speciation. Included are functions essential for generating important summary statistics from DNA barcode data, assessing specimen identification efficacy, and for testing and optimizing divergence threshold limits. In terms of investigating evolutionary and taxonomic questions, techniques for assessing diagnostic nucleotides and probability of reciprocal monophyly are also provided. Additionally, a sliding window function offers opportunities to analyse information across a gene, essential for marker design in degraded DNA studies. Spider capitalizes on R's extensible ethos and offers an integrated platform ideal for the analysis of both nucleotide and morphological data. The program can be obtained from the comprehensive R archive network (CRAN, http://cran.r-project.org) and from the R-Forge package development site (http://spider.r-forge.r-project.org/).

Sliding window analyses for optimal selection of mini-barcodes, and application to 454-pyrosequencing for specimen identification from degraded DNA.

DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI) is over 600 base pairs (bp), amplification fails when the DNA molecule is degraded into smaller fragments. However, relevant information for specimen identification may not be evenly distributed along the barcoding region, and a shorter target can be sufficient for identification purposes. This study proposes a new, widely applicable, method to compare the performance of all potential 'mini-barcodes' for a given molecular marker and to objectively select the shortest and most informative one. Our method is based on a sliding window analysis implemented in the new R package SPIDER (Species IDentity and Evolution in R). This method is applicable to any taxon and any molecular marker. Here, it was tested on earthworm DNA that had been degraded through digestion by carnivorous landsnails. A 100 bp region of 16 S rDNA was selected as the shortest informative fragment (mini-barcode) required for accurate specimen identification. Corresponding primers were designed and used to amplify degraded earthworm (prey) DNA from 46 landsnail (predator) faeces using 454-pyrosequencing. This led to the detection of 18 earthworm species in the diet of the snail. We encourage molecular ecologists to use this method to objectively select the most informative region of the gene they aim to amplify from degraded DNA. The method and tools provided here, can be particularly useful (1) when dealing with degraded DNA for which only small fragments can be amplified, (2) for cases where no consensus has yet been reached on the appropriate barcode gene, or (3) to allow direct analysis of short reads derived from massively parallel sequencing without the need for bioinformatic consolidation.