We tracked temporal changes in protist diversity at the Long Term Ecological Research (LTER) station MareChiara in the Gulf of Naples (Mediterranean Sea) on eight dates in 2011 using a metabarcoding approach. Illumina analysis of the V4 and V9 fragments of the 18S rDNA produced 869 522 and 1 410 071 sequences resulting in 6517 and 6519 OTUs, respectively. Marked compositional variations were recorded across the year, with less than 2% of OTUs shared among all samples and similar patterns for the two marker tags. Alveolata, Stramenopiles and Rhizaria were the most represented groups. A comparison with light microscopy data indicated an over-representation of Dinophyta in the sequence dataset, whereas Bacillariophyta showed comparable taxonomic patterns between sequence and light microscopy data. Shannon diversity values were stable from February to September, increasing thereafter with a peak in December. Community variance was mainly explained by seasonality (as temperature), trophic status (as chlorophyll a), and influence of coastal waters (as salinity). Overall, the background knowledge of the system provided a sound context for the result interpretation, showing that LTER sites provide an ideal setting for high-throughput sequencing (HTS) metabarcoding characterisation of protist assemblages and their relationships with environmental variations.

HmtDB (http://www.hmtdb.uniba.it:8080/hmdb) is a open resource created to support population genetics and mitochondrial disease studies. The database hosts human mitochondrial genome sequences annotated with population and variability data, the latter being estimated through the application of the SiteVar software based on site-specific nucleotide and amino acid variability calculations. The annotations are manually curated thus adding value to the quality of the information provided to the end-user. Classifier tools implemented in HmtDB allow the prediction of the haplogroup for any human mitochondrial genome currently stored in HmtDB or externally submitted de novo by an end-user. Haplogroup definition is based on the Phylotree system. End-users accessing HmtDB are hence allowed to (i) browse the database through the use of a multi-criterion ‘query’ system; (ii) analyze their own human mitochondrial sequences via the ‘classify’ tool (for complete genomes) or by downloading the ‘fragment-classifier’ tool (for partial sequences); (iii) download multi-alignments with reference genomes as well as variability data.

Nucleotide sequences from the plastome are currently the main source for assessing taxonomic and phylogenetic relationships in flowering plants and their historical biogeography at all hierarchical levels. One major exception is the large and economically important genus Quercus (oaks). Whereas differentiation patterns of the nuclear genome are in agreement with morphology and the fossil record, diversity patterns in the plastome are at odds with established taxonomic and phylogenetic relationships. However, the extent and evolutionary implications of this incongruence has yet to be fully uncovered. The DNA sequence divergence of four Euro-Mediterranean Group Ilex oak species (Quercus ilex L., Q. coccifera L., Q. aucheri Jaub. & Spach., Q. alnifolia Poech.) was explored at three chloroplast markers (rbcL, trnK/matK, trnH-psbA). Phylogenetic relationships were reconstructed including worldwide members of additional 55 species representing all Quercus subgeneric groups. Family and order sequence data were harvested from gene banks to better frame the observed divergence in larger taxonomic contexts. We found a strong geographic sorting in the focal group and the genus in general that is entirely decoupled from species boundaries. High plastid divergence in members of Quercus Group Ilex, including haplotypes shared with related, but long isolated oak lineages, point towards multiple geographic origins of this group of oaks. The results suggest that incomplete lineage sorting and repeated phases of asymmetrical introgression among ancestral lineages of Group Ilex and two other main Groups of Eurasian oaks (Cyclobalanopsis and Cerris) caused this complex pattern. Comparison with the current phylogenetic synthesis also suggests an initial high- versus mid-latitude biogeographic split within Quercus. High plastome plasticity of Group Ilex reflects geographic area disruptions, possibly linked with high tectonic activity of past and modern distribution ranges, that did not leave imprints in the nuclear genome of modern species and infrageneric lineages.

DNA barcoding may be particularly important in influencing ecology, economic issues, and the fundamental crisis facing biodiversity as a standardized, species-level identification tool for taxonomy assessment. Trees play important roles in the conservation of many land ecosystems, the wood trade, and the definition of biogeographical processes; nevertheless, peculiar biological, evolutionary and taxonomical features will probably constitute an intriguing challenge to barcoders. We examined whether four marker regions (trnh-psba, rbcL, rpoc1, matK) proposed by the Consortium for the Barcode of Life (CBOL) matched species taxonomy in a preliminary tree biodiversity survey of Italian forested land. Our objective was to provide a test of future in situ applications of DNA barcodes by evaluating the efficacy of species discrimination under the criteria of uniformity of methods and natural co-occurrence of the species in the main forest ecosystems. Fifty-two species were included in a floristic study. We obtained 73% total discrimination success, with trnH-psbA as the best performing marker and oaks as the least responsive plants to the markers used. A further taxon-based study of Quercus (thirty specimens, 12 species) revealed that this genus is refractory to barcoding (0% discrimination success), a probable consequence of low variation rate at the plastid genome level, hybridization, and the incidence of biogeography. We conclude that some species-rich tree genera in small geographical regions may prove exceptionally difficult to barcode. Until more efficient markers are developed, we recommend that improved and diversified sampling (multiple locations of sympatric and co-occurring congenerics) be embraced as a timely and important goal for the precise assessment of haplotype specificity to facilitate the productive application of barcoding in practice.