First records raise questions: DNA barcoding of Odonata in the middle of the Mediterranean.

We present the results of the first-ever DNA barcoding study of odonates from the Maltese Islands. In total, 10 morphologically identified species were collected during a two-week long expedition in 2018. Eighty cytochrome c oxidase subunit I (COI) barcodes were obtained from the collected specimens. Intra- and interspecific distances ranged from 0.00% to 2.24% and 0.48% to 17.62%, respectively. Successful species identification based on ascribing a single morphological species to a single Barcode Index Number (BIN) was achieved for eight species (80%). In the case of two species, Ischnura genei and Anax parthenope, BINs were shared with other closely related species. The taxonomic status of I. genei is questionable and the phylogenetic relationship between A. imperator/parthenope is not clear. Further studies involving a series of adult specimens collected in a wide spatial range and nuclear markers are necessary to resolve these cases. Therefore, this dataset serves as an initial DNA barcode reference library for Maltese odonates, within a larger project: Aquatic Macroinvertebrates DNA Barcode Library of Malta.

Contrasting morphological and DNA barcode-suggested species boundaries among shallow-water amphipod fauna from the southern European Atlantic coast.

In this study we compared DNA barcode-suggested species boundaries with morphology-based species identifications in the amphipod fauna of the southern European Atlantic coast. DNA sequences of the cytochrome c oxidase subunit I barcode region (COI-5P) were generated for 43 morphospecies (178 specimens) collected along the Portuguese coast which, together with publicly available COI-5P sequences, produced a final dataset comprising 68 morphospecies and 295 sequences. Seventy-five BINs (Barcode Index Numbers) were assigned to these morphospecies, of which 48 were concordant (i.e., 1 BIN = 1 species), 8 were taxonomically discordant, and 19 were singletons. Twelve species had matching sequences (<2% distance) with conspecifics from distant locations (e.g., North Sea). Seven morphospecies were assigned to multiple, and highly divergent, BINs, including specimens of Corophium multisetosum (18% divergence) and Dexamine spiniventris (16% divergence), which originated from sampling locations on the west coast of Portugal (only about 36 and 250 km apart, respectively). We also found deep divergence (4%-22%) among specimens of seven species from Portugal compared to those from the North Sea and Italy. The detection of evolutionarily meaningful divergence among populations of several amphipod species from southern Europe reinforces the need for a comprehensive re-assessment of the diversity of this faunal group.

Principles for Constructing DNA Barcode Reference Libraries.

All DNA barcode methods rely on reference sequences linked to well-curated voucher specimens. Definitions for and locations of DNA barcode reference libraries are not standardized, and vary throughout the literature. Standardizing, and centralizing reference specimens would provide an unambiguous source, analogous to reference genomes, to reproduce identifications and improve a library. This chapter proposes a working definition of a DNA barcode reference library, consistent with DNA barcode data standards, along with principles and methods to consider when producing or using such a library. These methods allow explicit traceback to sequence-sources which elevate the value of voucher specimens, and create a potential for community curation.

Fish DNA barcoding around large marine infrastructure for improved biodiversity assessment and monitoring.

Accurate species-level identification is pivotal for environmental assessments and monitoring. The PERU LNG terminal is composed of large marine infrastructure located on the central coast of Peru. Since construction, taxonomically challenging species such as drum fishes (Sciaenidae) have been attracted to the new hard-bottom habitat. We conducted a DNA barcoding study to investigate fish diversity and constructed a DNA barcode reference library. We examined 56 vouchered specimens and identified 24 unique species. Intra- and interspecific divergence estimates ranged between 0 and 0.64% and 11 and 35.5%, respectively. We assessed the efficiency of the reference library to identify 29 non-vouchered specimens. We had 82.5% efficiency by using both our reference library (n = 17) and GenBank (n = 24). We highlight the importance of implementing molecular barcoding for complementing biodiversity assessments in marine environments. This study represents a first step towards generating a comprehensive DNA barcode reference library for marine fishes in Peru.

Using herbarium-derived DNAs to assemble a large-scale DNA barcode library for the vascular plants of Canada.

PREMISE OF THE STUDY: Constructing complete, accurate plant DNA barcode reference libraries can be logistically challenging for large-scale floras. Here we demonstrate the promise and challenges of using herbarium collections for building a DNA barcode reference library for the vascular plant flora of Canada. METHODS: Our study examined 20,816 specimens representing 5076 of 5190 vascular plant species in Canada (98%). For 98% of the specimens, at least one of the DNA barcode regions was recovered from the plastid loci rbcL and matK and from the nuclear ITS2 region. We used beta regression to quantify the effects of age, type of preservation, and taxonomic affiliation (family) on DNA sequence recovery. RESULTS: Specimen age and method of preservation had significant effects on sequence recovery for all markers, but influenced some families more (e.g., Boraginaceae) than others (e.g., Asteraceae). DISCUSSION: Our DNA barcode library represents an unparalleled resource for metagenomic and ecological genetic research working on temperate and arctic biomes. An observed decline in sequence recovery with specimen age may be associated with poor primer matches, intragenomic variation (for ITS2), or inhibitory secondary compounds in some taxa.