Estimating belowground plant abundance with DNA metabarcoding.

Most work on plant community ecology has been performed above ground, neglecting the processes that occur in the soil. DNA metabarcoding, in which multiple species are computationally identified in bulk samples, can help to overcome the logistical limitations involved in sampling plant communities belowground. However, a major limitation of this methodology is the quantification of species' abundances based on the percentage of sequences assigned to each taxon. Using root tissues of five dominant species in a semi-arid Mediterranean shrubland (Bupleurum fruticescens, Helianthemum cinereum, Linum suffruticosum, Stipa pennata and Thymus vulgaris), we built pairwise mixtures of relative abundance (20%, 50% and 80% biomass), and implemented two methods (linear model fits and correction indices) to improve estimates of root biomass. We validated both methods with multispecies mixtures that simulate field-collected samples. For all species, we found a positive and highly significant relationship between the percentage of sequences and biomass in the mixtures (R2  = .44-.66), but the equations for each species (slope and intercept) differed among them, and two species were consistently over- and under-estimated. The correction indices greatly improved the estimates of biomass percentage for all five species in the multispecies mixtures, and reduced the overall error from 17% to 6%. Our results show that, through the use of post-sequencing quantification methods on mock communities, DNA metabarcoding can be effectively used to determine not only species' presence but also their relative abundance in field samples of root mixtures. Importantly, knowledge of these aspects will allow us to study key, yet poorly understood, belowground processes.

Enabling large-scale feather mite studies: an Illumina DNA metabarcoding pipeline.

Feather mites are among the most common and diverse ectosymbionts of birds, yet basic questions such as the nature of their relationship remain largely unanswered. One reason for feather mites being understudied is that their morphological identification is often virtually impossible when using female or young individuals. Even for adult male specimens this task is tedious and requires advanced taxonomic expertise, thus hampering large-scale studies. In addition, molecular-based methods are challenging because the low DNA amounts usually obtained from these tiny mites do not reach the levels required for high-throughput sequencing. This work aims to overcome these issues by using a DNA metabarcoding approach to accurately identify and quantify the feather mite species present in a sample. DNA metabarcoding is a widely used molecular technique that takes advantage of high-throughput sequencing methodologies to assign the taxonomic identity to all the organisms present in a complex sample (i.e., a sample made up of multiple specimens that are hard or impossible to individualise). We present a high-throughput method for feather mite identification using a fragment of the COI gene as marker and Illumina Miseq technology. We tested this method by performing two experiments plus a field test over a total of 11,861 individual mites (5360 of which were also morphologically identified). In the first experiment, we tested the probability of detecting a single feather mite in a heterogeneous pool of non-conspecific individuals. In the second experiment, we made 2 × 2 combinations of species and studied the relationship between the proportion of individuals of a given species in a sample and the proportion of sequences retrieved to test whether DNA metabarcoding can reliably quantify the relative abundance of mites in a sample. Here we also tested the efficacy of degenerate primers (i.e., a mixture of similar primers that differ in one or several bases that are designed to increase the chance of annealing) and investigated the relationship between the number of mismatches and PCR success. Finally, we applied our DNA metabarcoding pipeline to a total of 6501 unidentified and unsorted feather mite individuals sampled from 380 European passerine birds belonging to 10 bird species (field test). Our results show that this proposed pipeline is suitable for correct identification and quantitative estimation of the relative abundance of feather mite species in complex samples, especially when dealing with a moderate number (> 30) of individuals per sample.

A new species of Enchytraeus (Enchytraeidae, Oligochaeta) from the profundal of Lake Van, the world's largest soda Lake (Turkey, East Anatolia).

Enchytraeus polatdemiri sp. nov. (Enchytaeidae, Oligochaeta) was discovered in the framework of a sampling campaign of the benthic invertebrate fauna of the hyperalkaline Lake Van in Eastern Anatolia, Turkey, the third-largest closed lake and the largest soda lake on Earth. It was the only oligochaete species found in all samples. DNA sequencing included a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene, and a fragment of the nuclear histone 3 (H3) gene. For comparison, specimens from laboratory cultures of E. albidus Henle, 1837, a widespread and morphologically similar species, were sequenced as well. The new species differs from E. albidus in comparatively small body size, 2 or 3 chaetae per bundle, saddle-shaped clitellum, absence of a copulatory field between the male pores and vasa deferentia usually not extending beyond the clitellum. The individual gene trees of COI and H3, as well as the combined phylogenetic analysis of both trees, recovered Enchytraeus polatdemiri sp. nov. as a monophyletic group within the genus Enchytraeus, closely related to E. albidus, but with an average p-distance for COI of 14.5 %. E. polatdemiri sp. nov. may have evolved from a local population of Enchytraeus albidus, a species well-adapted to changing salinity conditions, or from a common ancestor into an extremophile species that dwells and reproduces in the profundal of a strongly alkaline soda lake.

The complete mitochondrial genome of the feather mite Trouessartia rubecula Jablonska, 1968 (Astigmata: Analgoidea: Trouessartiidae).

We assembled and annotated the complete mitochondrial genome of Trouessartia rubecula, the first feather mite complete mitochondrial genome from the largest feather mite superfamily Analgoidea (ca. 1150 spp). The mitogenome was composed of 13 protein, 17 tRNA, and 2 rRNA-coding genes and was 14,125 bp in length.