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.

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.

The 5S rDNA gene family in mollusks: characterization of transcriptional regulatory regions, prediction of secondary structures, and long-term evolution, with special attention to Mytilidae mussels.

Several reports on the characterization of 5S ribosomal DNA (5S rDNA) in various animal groups have been published to date, but there is a lack of studies analyzing this gene family in a much broader context. Here, we have studied 5S rDNA variation in several molluskan species, including bivalves, gastropods, and cephalopods. The degree of conservation of transcriptional regulatory regions was analyzed in these lineages, revealing a conserved TATA-like box in the upstream region. The evolution of the 120 bp coding region (5S) was also studied, suggesting the occurrence of paralogue groups in razor clams, clams, and cockles. In addition, 5S rDNA sequences from 11 species and 7 genus of Mytilidae Rafinesque, 1815 mussels were sampled and studied in detail. Four different 5S rDNA types, based on the nontranscribed spacer region were identified. The phylogenetic analyses performed within each type showed a between-species gene clustering pattern, suggesting ancestral polymorphism. Moreover, some putative pseudogenized 5S copies were also identified. Our report, together with previous studies that found high degree of intragenomic divergence in bivalve species, suggests that birth-and-death evolution may be the main force driving the evolution of 5S rDNA in these animals, even at the genus level.

Analysis of ITS1 and ITS2 sequences in Ensis razor shells: suitability as molecular markers at the population and species levels, and evolution of these ribosomal DNA spacers.

Internal transcribed spacer 1 and 2 (ITS1 and ITS2) sequences were analysed in Ensis razor shells (Mollusca: Bivalvia: Pharidae). We aimed to (1) test ITS1 and ITS2 as molecular markers at the population level in the successful alien E. directus (Conrad, 1843); (2) test these spacers at the species level in E. directus and three other Ensis species, E. siliqua (L., 1758), E. macha (Molina, 1782), and E. magnus (Schumacher, 1817); and (3) analyse the evolutionary processes that may be shaping Ensis ITS1 and ITS2 extant variation. In E. directus, despite the intragenomic divergence detected, ITS1 and ITS2 were informative in differentiating the geographic areas considered (Denmark and Canada) by means of both the insertion-deletion polymorphism and the nucleotide polymorphism. In this species, the 5.8S ribosomal gene (5.8S) showed scarce polymorphism. At the species level, maximum parsimony and maximum likelihood analyses revealed that ITS1 and ITS2 may be suitable to reconstruct Ensis phylogenetic relationships. Finally, the evolutionary models that best fit the long-term evolution of Ensis ITS1-5.8S-ITS2 are discussed. A mixed process of concerted evolution, birth-and-death evolution, and selection is chosen as an option that may reconcile the long-term evolution of Ensis ITS1-5.8S-ITS2 and 5S ribosomal DNA.

Long-term evolution of 5S ribosomal DNA seems to be driven by birth-and-death processes and selection in Ensis razor shells (Mollusca: Bivalvia).

A study of nucleotide sequence variation of 5S ribosomal DNA from six Ensis species revealed that several 5S ribosomal DNA variants, based on differences in their nontranscribed spacers (NTS), occur in Ensis genomes. The 5S rRNA gene was not very polymorphic, compared with the NTS region. The phylogenetic analyses performed showed a between-species clustering of 5S ribosomal DNA variants. Sequence divergence levels between variants were very large, revealing a lack of sequence homogenization. These results strongly suggest that the long-term evolution of Ensis 5S ribosomal DNA is driven by birth-and-death processes and selection.