The accuracy of the DNA barcoding tool depends on the existence of a comprehensive archived library of sequences reliably determined at species level by expert taxonomists. However, misidentifications are not infrequent, especially following large-scale DNA barcoding campaigns on diverse and taxonomically complex groups. In this study we used the species-rich flea beetle genus Longitarsus, that requires a high level of expertise for morphological species identification, as a case study to assess the accuracy of the DNA barcoding tool following several optimization procedures. We built a cox1 reference database of 1502 sequences representing 78 Longitarsus species, among which 117 sequences (32 species) were newly generated using a non-invasive DNA extraction method that allows keeping reference voucher specimens. Within this dataset we identified 69 taxonomic inconsistencies using barcoding gap analysis and tree topology methods. Threshold optimisation and a posteriori taxonomic revision based on newly generated reference sequences and metadata allowed resolving 44 sequences with ambiguous and incorrect identification and provided a significant improvement of the DNA barcoding accuracy and identification efficacy. Unresolved taxonomic uncertainties, due to overlapping intra-and inter-specific levels of divergences, mainly regards the Longitarsus pratensis species complex and polyphyletic groups L. melanocephalus, L. nigrofasciatus and L. erro. Such type of errors indicates either poorly established taxonomy or any biological processes that make mtDNA groups poorly predictive of species boundaries (e.g. recent speciation or interspecific hybridisation), thus providing directions for further integrative taxonomic and evolutionary studies. Overall, this study underlines the importance of reference vouchers and high-quality metadata associated to sequences in reference databases and corroborates, once again, the key role of taxonomists in any step of the DNA barcoding pipeline in order to generate and maintain a correct and functional reference library.

No abstract

The recently published mitochondrial genome of the fingerprint oyster Alectryonella plicatula (Gmelin, 1791) with GenBank accession number MW143047 was resolved in an unexpected phylogenetic position, as sister to the Pacific cupped oyster Magallana gigas (Thunberg, 1793) and share with this species three typical gene duplications that represent robust synapomorphies of the Magallana clade. In this study, we verified the identity of MW143047 using direct comparisons of single gene sequences, DNA barcoding and phylogenetic analyses. BLAST searches using as query each of the 12 protein coding genes (PCGs) and rRNA genes extracted from MW143047 retrieved M. gigas as best hit with 100% sequence identity for all genes. MW143047 is nested within the clade formed by M. gigas sequences, with virtually zero-length terminal branch, both in the cox1 gene tree (based on 3639 sequences) and in the 16S gene tree (based on 1839 sequences), as well as in the Maximum Likelihood mitogenomic tree based on concatenated sequence of 12 PCGs. Our findings suggest that the original specimen used for mitogenome sequencing was misidentified and represents an individual of M. gigas. This study reinforces the notion that morphological shell analysis alone is not sufficient for oyster identification, not even at high taxonomic ranks such as subfamilies. While it is well established that morphological identification of oysters should be validated by molecular data, this study emphasizes that also molecular data should be taxonomically verified by means of DNA barcoding and phylogenetic analyses. The implications of the publication of taxonomically misidentified sequences and mitogenomes are discussed.

In the current global warming phase, relict glacial areas are one of the most threatened ecosystems in the world. They are “cold-spots” of biodiversity and of great interest both from the ecological and conservation point of view. We investigated the biological communities (plants and arthropods) hosted by one of the southernmost European glaciers: Calderone Glacier, the last preserved glacier of the Apennines mountain chain (Italy). We analyzed supraglacial debris and the nearby moraine and we found a rather diverse and peculiar biodiversity, which includes also new species. Some arthropods, such as the springtail Desoria calderonis, are particularly sensitive to the presence of ice at microtopographic level. Among plants, only Arabis alpina caucasica is able to grow on the supraglacial debris, perhaps because of factors related to the seed germination and seedling survival on this habitat type. Calderone glacier, with its particular biogeographic location, is probably currently acting as one of the last refugia for rare and endemic cryophilic species within the Mediterranean Region during the ongoing warm period.

The recently published mitochondrial genome of the fingerprint oyster Alectryonella plicatula (Gmelin, 1791) with GenBank accession number MW143047 was resolved in an unexpected phylogenetic position, as sister to the Pacific cupped oyster Magallana gigas (Thunberg, 1793) and share with this species three typical gene duplications that represent robust synapomorphies of the Magallana clade. In this study, we verified the identity of MW143047 using direct comparisons of single gene sequences, DNA barcoding and phylogenetic analyses. BLAST searches using each of the 12 protein coding genes and rRNA genes extracted from MW143047 as query retrieved M. gigas as best hit with 100% sequence identity. MW143047 is nested within the clade formed by M. gigas sequences, with virtually no difference between their terminal branch lengths, both in the cox1 gene tree (based on 3639 sequences) and in the 16S gene tree (based on 1839 sequences), as well as in the Maximum Likelihood mitogenomic tree based on concatenated sequence of 12 PCGs. Our findings suggest that the original specimen used for mitogenome sequencing was misidentified and represents an individual of M. gigas. This study reinforces the notion that morphological shell analysis alone is not sufficient for oyster identification, not even at high taxonomic ranks such as subfamilies. While it is well established that morphological identification of oysters should be validated by molecular data, this study emphasizes that also molecular data should be taxonomically validated by means of DNA barcoding and phylogenetic analyses. The implications of the publication of taxonomically misidentified sequences and mitogenomes are discussed