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.

First Account of Phylogeographic Variation, Larval Characters, and Laboratory Rearing of the Endangered Cobblestone Tiger Beetle Cicindelidia marginipennis, Dejean, 1831 with Observations of Their Natural History.

The cobblestone tiger beetle, Cicindelidia marginipennis (Dejean, 1831) is a North American species specializing in riparian habitats from New Brunswick, Canada, to Alabama in the United States. In the United States, this species is state-listed as threatened or endangered range-wide and periodically receives consideration for federal listing, mostly due to habitat decline. Despite its conservation status, intraspecific genetic diversity for this species has not been explored and little is known about its natural history. To support further inquiry into the biology of C. marginipennis, this study provides the first look at range-wide genetic diversity using mitochondrial DNA (mtDNA), describes all three larval instars, and describes natural history characteristics from captive rearing and field observation. Based on mtDNA analyses, our results suggest that geographically based population structure may exist throughout the range, with individuals from Alabama possessing haplotypes not found elsewhere in our sampling. Further genetic analyses, particularly multi-locus analyses, are needed to determine whether the Alabama population represents a separate cryptic species. Our morphological analysis and descriptions of larval instars reveal a combination of characteristics that can be used to differentiate C. marginipennis from closely related and co-occurring species. Based on our field observations, we find that the larval "throw pile" of soil excavated from burrows is a key search image for locating larvae, and we provide descriptions and detailed photographs to aid surveys. Lastly, we find that this species can be successfully reared in captivity and provide guidelines to aid future recovery efforts.

Towards a catalogue of biodiversity databases: An ontological case study.

Biodiversity informatics depends on digital access to credible information about species. Many online resources host species' data, but the lack of categorisation for these resources inhibits the growth of this entire field. To explore possible solutions, we examined the (now retired) Biodiversity Information Projects of the World (BIPW) dataset created by the Biodiversity Information Standards (TDWG); this project, which ran from 2007-2015 (officially removed from the TDWG website in 2018) was an attempt at organising the Web's biodiversity databases into an indexed list. To do this, we applied a simple classification scheme to score databases within BIPW based on nine data categories, to characterise trends and current compositions of this biodiversity e-infrastructure. Primarily, we found that of 600 databases investigated from BIPW, only 315 (~53%) were accessible at the time of this writing, underscoring the precarious nature of the biodiversity information landscape. Many of these databases are still available, but suffer accessibility issues such as link rot, thus putting the information they contain in danger of being lost. We propose that a community-driven database of biodiversity databases with an accompanying ontology could facilitate efficient discovery of relevant biodiversity databases and support smaller databases - which have the greatest risk of being lost.

Geographic Life History Differences Predict Genomic Divergence Better than Mitochondrial Barcodes or Phenotype.

Species diversity can be inferred using multiple data types, however, results based on genetic data can be at odds with patterns of phenotypic variation. Tiger beetles of the Cicindelidiapolitula (LeConte, 1875) species complex have been taxonomically problematic due to extreme phenotypic variation within and between populations. To better understand the biology and taxonomy of this group, we used mtDNA genealogies and multilocus nuclear analyses of 34,921 SNPs to elucidate its evolutionary history and evaluate the validity of phenotypically circumscribed species and subspecies. Genetic analyses recovered two divergent species that are also ecologically distinct, based on adult life history. These patterns are incongruous with the phenotypic variation that informed prior taxonomy, and most subspecies were not supported as distinct evolutionary lineages. One of the nominal subspecies was found to be a cryptic species; consequently, we elevate C. p.laetipennis (Horn, 1913) to a full species. Although nuclear and mtDNA datasets recovered broadly similar evolutionary units, mito-nuclear discordance was more common than expected, being observed between nearly all geographically overlapping taxonomic pairs. Additionally, a pattern of 'mitochondrial displacement' was observed, where mitochondria from one species unidirectionally displace others. Overall, we found that geographically associated life history factors better predict genomic divergence than phenotype and mitochondrial genealogies, and consequently taxon identifications based on mtDNA (e.g., DNA barcodes) may be misleading.

Mitogenome metadata: current trends and proposed standards.

Mitogenome metadata are descriptive terms about the sequence, and its specimen description that allow both to be digitally discoverable and interoperable. Here, we review a sampling of mitogenome metadata published in the journal Mitochondrial DNA between 2005 and 2014. Specifically, we have focused on a subset of metadata fields that are available for GenBank records, and specified by the Genomics Standards Consortium (GSC) and other biodiversity metadata standards; and we assessed their presence across three main categories: collection, biological and taxonomic information. To do this we reviewed 146 mitogenome manuscripts, and their associated GenBank records, and scored them for 13 metadata fields. We also explored the potential for mitogenome misidentification using their sequence diversity, and taxonomic metadata on the Barcode of Life Datasystems (BOLD). For this, we focused on all Lepidoptera and Perciformes mitogenomes included in the review, along with additional mitogenome sequence data mined from Genbank. Overall, we found that none of 146 mitogenome projects provided all the metadata we looked for; and only 17 projects provided at least one category of metadata across the three main categories. Comparisons using mtDNA sequences from BOLD, suggest that some mitogenomes may be misidentified. Lastly, we appreciate the research potential of mitogenomes announced through this journal; and we conclude with a suggestion of 13 metadata fields, available on GenBank, that if provided in a mitogenomes's GenBank record, would increase their research value.

Fast fish face fewer mitochondrial mutations: Patterns of dN/dS across fish mitogenomes.

Mitochondrial DNA is routinely used to answer a variety of biological questions; and there is growing evidence suggesting that its accumulation of mutations is influenced by life history, effective population size and cellular energy requirements. This study examines the influence of phylogenetic patterns of metabolic activity on the evolution of mitochondrial DNA in fishes, given energy requirements associated with high performance versus sedentary life histories. It was determined that all 13 protein coding genes of the mitogenome experience a relaxation of purifying selection in sedentary fishes. This phenomenon was not detected in nuclear housekeeping genes, suggesting that it can be explained by the energy requirements of these groups, and possibly their effective population sizes. This study also examined the subunit binding sites of two subunits of cytochrome c oxidase (COXI and COXIII), and did not detect any differences in selection between these groups of fishes. These cytochrome c oxidase subunits interact with subunits that are encoded by the nuclear genome and it has been suggested that a unique form of coevolution occurs between these genomes in order to maintain function, and may have implications for speciation. Although this was not a main focus of this study, our preliminary results suggest that substitutions in subunit binding site regions are rare. The results from this study add to the growing literature on the complex relationship between mitochondrial DNA and the evolution of life histories across the tree of life.

The hemiptera (insecta) of Canada: constructing a reference library of DNA barcodes.

DNA barcode reference libraries linked to voucher specimens create new opportunities for high-throughput identification and taxonomic re-evaluations. This study provides a DNA barcode library for about 45% of the recognized species of Canadian Hemiptera, and the publically available R workflow used for its generation. The current library is based on the analysis of 20,851 specimens including 1849 species belonging to 628 genera and 64 families. These individuals were assigned to 1867 Barcode Index Numbers (BINs), sequence clusters that often coincide with species recognized through prior taxonomy. Museum collections were a key source for identified specimens, but we also employed high-throughput collection methods that generated large numbers of unidentified specimens. Many of these specimens represented novel BINs that were subsequently identified by taxonomists, adding barcode coverage for additional species. Our analyses based on both approaches includes 94 species not listed in the most recent Canadian checklist, representing a potential 3% increase in the fauna. We discuss the development of our workflow in the context of prior DNA barcode library construction projects, emphasizing the importance of delineating a set of reference specimens to aid investigations in cases of nomenclatural and DNA barcode discordance. The identification for each specimen in the reference set can be annotated on the Barcode of Life Data System (BOLD), allowing experts to highlight questionable identifications; annotations can be added by any registered user of BOLD, and instructions for this are provided.

Corroborating molecular species discovery: Four new pine-feeding species of Chionaspis (Hemiptera, Diaspididae).

The genus Chionaspis (Hemiptera, Diaspididae) includes two North American species of armored scale insects feeding on Pinaceae: Chionaspis heterophyllae Cooley, and Chionaspis pinifoliae (Fitch). Despite the economic impact of conifer-feeding Chionaspis on horticulture, the species diversity in this group has only recently been systematically investigated using samples from across the group's geographic and host range. This paper provides morphological recognition characters for four new species that were recently hypothesized to exist on the basis of molecular evidence. The new species, here described, are Chionaspis brachycephalon Vea sp. n., Chionaspis caudata Vea sp. n., Chionaspis sonorae Vea sp. n. and Chionaspis torreyanae Vea sp. n.  One of the new species, Chionaspis caudata Vea, has a gland spine at the apex of the pygidium, between the median lobes, unlike any other species of Chionaspis. An identification key to the species of Chionaspis feeding on pine in North America is provided.

A phylogenetic analysis of armored scale insects (Hemiptera: Diaspididae), based upon nuclear, mitochondrial, and endosymbiont gene sequences.

Armored scale insects (Hemiptera: Diaspididae) are among the most invasive insects in the world. They have unusual genetic systems, including diverse types of paternal genome elimination (PGE) and parthenogenesis. Intimate relationships with their host plants and bacterial endosymbionts make them potentially important subjects for the study of co-evolution. Here, we expand upon recent phylogenetic work (Morse and Normark, 2006) by analyzing armored scale and endosymbiont DNA sequences from 125 species of armored scale insect, represented by 253 samples and eight outgroup species. We used fragments of four different gene regions: the nuclear protein-coding gene Elongation Factor 1α (EF1α), the large ribosomal subunit (28S) rDNA, a mitochondrial region spanning parts of cytochrome oxidase I (COI) and cytochrome oxidase II (COII), and the small ribosomal subunit (16S) rDNA from the primary bacterial endosymbiont Uzinura diaspidicola. Maximum likelihood, and Bayesian analyses were performed producing highly congruent topological results. A comparison of two datasets, one with and one without missing data, found that missing data had little effect on topology. Our results broadly corroborate several major features of the existing classification, although we do not find any of the subfamilies, tribes or subtribes to be monophyletic as currently constituted. Using ancestral state reconstruction we estimate that the ancestral armored scale had the late PGE sex system, and it may as well have been pupillarial, though results differed between reconstruction methods. These results highlight the need for a complete revision of this family, and provide the groundwork for future taxonomic work in armored scale insects.