Aquatic suspended particulate matter as source of eDNA for fish metabarcoding.

The use of environmental DNA (eDNA) for monitoring aquatic macrofauna allows the non-invasive species determination and measurement of their DNA abundance and typically involves the analysis of eDNA captured from water samples. In this proof-of-concept study, we focused on the novel use of eDNA extracted from archived suspended particulate matter (SPM) for identifying fish species using metabarcoding, which benefits from the prospect of retrospective monitoring and also analysis of fish communities through time. We used archived SPM samples of the German Environmental Specimen Bank (ESB), which were collected using sedimentation traps from different riverine points in Germany. Environmental DNA was extracted from nine SPM samples differing in location, organic content, and porosity (among other factors) using four different methods for the isolation of high-quality DNA. Application of the PowerSoil DNA Isolation Kit with an overnight incubation in lysis buffer, resulted in DNA extraction with the highest purity and eDNA metabarcoding of these eDNA fragments was used to detect a total of 29 fish taxa among the analyzed samples. Here we demonstrated for the first time that SPM is a promising source of eDNA for metabarcoding analysis, which could provide valuable retrospective information (when using archived SPM) for fish monitoring, complementing the currently used approaches.

A validated workflow for rapid taxonomic assignment and monitoring of a national fauna of bees (Apiformes) using high throughput DNA barcoding.

Improved taxonomic methods are needed to quantify declining populations of insect pollinators. This study devises a high-throughput DNA barcoding protocol for a regional fauna (United Kingdom) of bees (Apiformes), consisting of reference library construction, a proof-of-concept monitoring scheme, and the deep barcoding of individuals to assess potential artefacts and organismal associations. A reference database of cytochrome oxidase c subunit 1 (cox1) sequences including 92.4% of 278 bee species known from the UK showed high congruence with morphological taxon concepts, but molecular species delimitations resulted in numerous split and (fewer) lumped entities within the Linnaean species. Double tagging permitted deep Illumina sequencing of 762 separate individuals of bees from a UK-wide survey. Extracting the target barcode from the amplicon mix required a new protocol employing read abundance and phylogenetic position, which revealed 180 molecular entities of Apiformes identifiable to species. An additional 72 entities were ascribed to nuclear pseudogenes based on patterns of read abundance and phylogenetic relatedness to the reference set. Clustering of reads revealed a range of secondary operational taxonomic units (OTUs) in almost all samples, resulting from traces of insect species caught in the same traps, organisms associated with the insects including a known mite parasite of bees, and the common detection of human DNA, besides evidence for low-level cross-contamination in pan traps and laboratory procedures. Custom scripts were generated to conduct critical steps of the bioinformatics protocol. The resources built here will greatly aid DNA-based monitoring to inform management and conservation policies for the protection of pollinators.

Genetic Exchange among Bdelloid Rotifers Is More Likely Due to Horizontal Gene Transfer Than to Meiotic Sex.

Although strict asexuality is supposed to be an evolutionary dead end, morphological, cytogenetic, and genomic data suggest that bdelloid rotifers, a clade of microscopic animals, have persisted and diversified for more than 60 Myr in an ameiotic fashion. Moreover, the genome of bdelloids of the genus Adineta comprises 8%-10% of genes of putative non-metazoan origin, indicating that horizontal gene transfers are frequent within this group and suggesting that this mechanism may also promote genetic exchanges among bdelloids as well. To test this hypothesis, we used five independent sequence markers to study the genetic diversity of 576 Adineta vaga individuals from a park in Belgium. Haplowebs and GMYC analyses revealed the existence of six species among our sampled A. vaga individuals, with strong evidence of both intra- and interspecific recombination. Comparison of genomic regions of three allele-sharing individuals further revealed signatures of genetic exchanges scattered among regions evolving asexually. Our findings suggest that bdelloids evolve asexually but exchange DNA horizontally both within and between species.

Sexual species are separated by larger genetic gaps than asexual species in rotifers.

Why organisms diversify into discrete species instead of showing a continuum of genotypic and phenotypic forms is an important yet rarely studied question in speciation biology. Does species discreteness come from adaptation to fill discrete niches or from interspecific gaps generated by reproductive isolation? We investigate the importance of reproductive isolation by comparing genetic discreteness, in terms of intra- and interspecific variation, between facultatively sexual monogonont rotifers and obligately asexual bdelloid rotifers. We calculated the age (phylogenetic distance) and average pairwise genetic distance (raw distance) within and among evolutionarily significant units of diversity in six bdelloid clades and seven monogonont clades sampled for 4211 individuals in total. We find that monogonont species are more discrete than bdelloid species with respect to divergence between species but exhibit similar levels of intraspecific variation (species cohesiveness). This pattern arises because bdelloids have diversified into discrete genetic clusters at a faster net rate than monogononts. Although sampling biases or differences in ecology that are independent of sexuality might also affect these patterns, the results are consistent with the hypothesis that bdelloids diversified at a faster rate into less discrete species because their diversification does not depend on the evolution of reproductive isolation.

Effects of phylogenetic reconstruction method on the robustness of species delimitation using single-locus data.

Coalescent-based species delimitation methods combine population genetic and phylogenetic theory to provide an objective means for delineating evolutionarily significant units of diversity. The generalised mixed Yule coalescent (GMYC) and the Poisson tree process (PTP) are methods that use ultrametric (GMYC or PTP) or non-ultrametric (PTP) gene trees as input, intended for use mostly with single-locus data such as DNA barcodes. Here, we assess how robust the GMYC and PTP are to different phylogenetic reconstruction and branch smoothing methods. We reconstruct over 400 ultrametric trees using up to 30 different combinations of phylogenetic and smoothing methods and perform over 2000 separate species delimitation analyses across 16 empirical data sets. We then assess how variable diversity estimates are, in terms of richness and identity, with respect to species delimitation, phylogenetic and smoothing methods. The PTP method generally generates diversity estimates that are more robust to different phylogenetic methods. The GMYC is more sensitive, but provides consistent estimates for BEAST trees. The lower consistency of GMYC estimates is likely a result of differences among gene trees introduced by the smoothing step. Unresolved nodes (real anomalies or methodological artefacts) affect both GMYC and PTP estimates, but have a greater effect on GMYC estimates. Branch smoothing is a difficult step and perhaps an underappreciated source of bias that may be widespread among studies of diversity and diversification. Nevertheless, careful choice of phylogenetic method does produce equivalent PTP and GMYC diversity estimates. We recommend simultaneous use of the PTP model with any model-based gene tree (e.g. RAxML) and GMYC approaches with BEAST trees for obtaining species hypotheses.

The widely used small subunit 18S rDNA molecule greatly underestimates true diversity in biodiversity surveys of the meiofauna.

Molecular tools have revolutionized the exploration of biodiversity, especially in organisms for which traditional taxonomy is difficult, such as for microscopic animals (meiofauna). Environmental (eDNA) metabarcode surveys of DNA extracted from sediment samples are increasingly popular for surveying biodiversity. Most eDNA surveys use the nuclear gene-encoding small-subunit rDNA gene (18S) as a marker; however, different markers and metrics used for delimiting species have not yet been evaluated against each other or against morphologically defined species (morphospecies). We assessed more than 12,000 meiofaunal sequences of 18S and of the main alternatively used marker [Cytochrome c oxidase subunit I (COI) mtDNA] belonging to 55 datasets covering three taxonomic ranks. Our results show that 18S reduced diversity estimates by a factor of 0.4 relative to morphospecies, whereas COI increased diversity estimates by a factor of 7.6. Moreover, estimates of species richness using COI were robust among three of four commonly used delimitation metrics, whereas estimates using 18S varied widely with the different metrics. We show that meiofaunal diversity has been greatly underestimated by 18S eDNA surveys and that the use of COI provides a better estimate of diversity. The suitability of COI is supported by cross-mating experiments in the literature and evolutionary analyses of discreteness in patterns of genetic variation. Furthermore its splitting of morphospecies is expected from documented levels of cryptic taxa in exemplar meiofauna. We recommend against using 18S as a marker for biodiversity surveys and suggest that use of COI for eDNA surveys could provide more accurate estimates of species richness in the future.