Performance of DNA metabarcoding, standard barcoding, and morphological approach in the identification of host–parasitoid interactions

<div><p>Understanding interactions between herbivores and parasitoids is essential for successful biodiversity protection and monitoring and for biological pest control. Morphological identifications employ insect rearing and are complicated by insects’ high diversity and crypsis. DNA barcoding has been successfully used in studies of host–parasitoid interactions as it can substantially increase the recovered real host–parasitoid diversity distorted by overlooked species complexes, or by species with slight morphological differences. However, this approach does not allow the simultaneous detection and identification of host(s) and parasitoid(s). Recently, high-throughput sequencing has shown high potential for surveying ecological communities and trophic interactions. Using mock samples comprising insect larvae and their parasitoids, we tested the potential of DNA metabarcoding for identifying individuals involved in host–parasitoid interactions to different taxonomic levels, and compared it to standard DNA barcoding and morphological approaches. For DNA metabarcoding, we targeted the standard barcoding marker cytochrome oxidase subunit I using highly degenerate primers, 2*300 bp sequencing on a MiSeq platform, and RTAX classification using paired-end reads. Additionally, using a large host–parasitoid dataset from a Central European floodplain forest, we assess the completeness and usability of a local reference library by confronting the number of Barcoding Index Numbers obtained by standard barcoding with the number of morphotypes. Overall, metabarcoding recovery was high, identifying 92.8% of the taxa present in mock samples, and identification success within individual taxonomic levels did not significantly differ among metabarcoding, standard barcoding, and morphology. Based on the current local reference library, 39.4% parasitoid and 90.7% host taxa were identified to the species level. DNA barcoding estimated higher parasitoid diversity than morphotyping, especially in groups with high level of crypsis. This study suggests the potential of metabarcoding for effectively recovering host–parasitoid diversity, together with more accurate identifications obtained from building reliable and comprehensive reference libraries, especially for parasitoids.</p></div

Detailed composition of the individual mock samples (S1–S5) recovered by DNA metabarcoding (Illumina MiSeq).

<p>The taxonomic assignment of recovered host and parasitoid taxa is emphasized together with the proportion of putative symbionts. The width of each sector corresponds to the relative proportion of its reads (n_r = total number of reads). Taxonomic levels are displayed hierarchically from order (the innermost layer) to species level (the outermost layer). The inset table shows organisms (H = host, P = parasitoid) put in the mock samples, and their identification to the lowest possible taxonomic level based on consensus of the three methods (morphological identification, standard barcoding and metabarcoding). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187803#pone.0187803.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187803#pone.0187803.s003" target="_blank">S3</a> Tables for details.</p

Dynamic Formation of Asexual Diploid and Polyploid Lineages: Multilocus Analysis of <em>Cobitis</em> Reveals the Mechanisms Maintaining the Diversity of Clones

<div><p>Given the hybrid genomic constitutions and increased ploidy of many asexual animals, the identification of processes governing the origin and maintenance of clonal diversity provides useful information about the evolutionary consequences of interspecific hybridization, asexuality and polyploidy. In order to understand the processes driving observed diversity of biotypes and clones in the <em>Cobitis taenia</em> hybrid complex, we performed fine-scale genetic analysis of Central European hybrid zone between two sexual species using microsatellite genotyping and mtDNA sequencing. We found that the hybrid zone is populated by an assemblage of clonally (gynogenetically) reproducing di-, tri- and tetraploid hybrid lineages and that successful clones, which are able of spatial expansion, recruit from two ploidy levels, i.e. diploid and triploid. We further compared the distribution of observed estimates of clonal ages to theoretical distributions simulated under various assumptions and showed that new clones are most likely continuously recruited from ancestral populations. This suggests that the clonal diversity is maintained by dynamic equilibrium between origination and extinction of clonal lineages. On the other hand, an interclonal selection is implied by nonrandom spatial distribution of individual clones with respect to the coexisting sexual species. Importantly, there was no evidence for sexually reproducing hybrids or clonally reproducing non-hybrid forms. Together with previous successful laboratory synthesis of clonal <em>Cobitis</em> hybrids, our data thus provide the most compelling evidence that 1) the origin of asexuality is causally linked to interspecific hybridization; 2) successful establishment of clones is not restricted to one specific ploidy level and 3) the initiation of clonality and polyploidy may be dynamic and continuous in asexual complexes.</p> </div

Europe-wide distribution, reproductive pathways and contact zone in Odra R. basin of studied species.

<p>(a) Distribution area of <i>C. tanaitica</i> (blue circles), <i>C. taenia</i> (red area), and <i>C. elongatoides</i> (yellow area), with the directions of postglacial colonization of Europe by <i>C. taenia</i> (red arrows) and <i>C. elongatoides</i> (yellow arrow). Secondary contact zones are indicated by zigzag symbols, and the dispersal of clonal lineages is indicated by dotted arrows (modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045384#pone.0045384-Janko2" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045384#pone.0045384-Culling1" target="_blank">[46]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045384#pone.0045384-Janko7" target="_blank">[64]</a>); (b) inferred reproductive pathways in <i>Cobitis</i> complex. Sperm at straight arrow indicates true fertilization, whereas sperm at round arrow indicates a triggering the egǵs development without paternal genetic contribution to the offspring; (c) Sampling sites in the Odra R. hybrid zone. For each locality, the presence of <i>C. taenia</i> and <i>C. elongatoides</i> is indicated by red and yellow dots, respectively and we indicate co-occurring hybrid biotypes, and their number in parentheses, if more than one. For each biotype on a given sample site, we list the presence of clones (MLL) and their absolute frequencies in parentheses, if more than one.</p

Histograms of the frequency distribution of pairwise distances in bp among genotyped individuals.

<p>(a–b) Data from <i>C. elongatoides</i> and <i>C. taenia</i>; (c–d) Distributions from observed (natural) and simulated diploid hybrids; (e–f) distributions from the observed (natural) and simulated triploid hybrids.</p

Observed and simulated distribution of clonal age estimators.

<p>(a) Correlation between the observed values of <i>dist.bp</i> and Tomiuk and Loeschcke’s <i>I</i>; (b, d) Distributions of Hartigan’s <i>D</i> values calculated from simulated <i>dist.bp</i> histograms at every 200^th generation of the simulation. Each value was calculated from a 50∶50 mixture of sympatric and allopatric clones. Arrows indicate the observed value; frequency distributions of the observed values of <i>dist.bp,</i> and <i>I</i>, respectively (c, e).</p

Unrooted statistical parsimony networks of haplotypes belonging to <i>C. taenia</i>-like (T) (upper panel) and <i>C. elongatoides</i>-like (E) (lower panel) clades (sensu [<b>13</b>]).

<p>White circles denote haplotypes found in sexual individuals only, dark grey circles denote those found in hybrids only, and light grey circles denote haplotypes shared by both hybrid and sexual individuals. The sizes of haplotypes are proportional to their frequency. Small blank circles represent missing (unobserved) haplotypes. Newly sequenced haplotypes are in bold. Rectangles delimit the hybrid clades I and II.</p

The plots of <i>dist.mut</i> and <i>dist.bp</i> values as a function of true age.

<p>Each gray line tracks the evolution of one clonal lineage over the time. Simulations are shown for the 2 values of mutation rate (µ) under SSM mutation model. At selected times, we represent the boxplots showing the median of simulated values of <i>dist.mut</i> and <i>dist.bp</i>. as well as first and third quartile; whiskers represent 1.5 times the interquartile range.</p

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