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

Cryptic diversity in the North American Dromochorus tiger beetles (Coleoptera: Carabidae: Cicindelinae): a congruence-based method for species discovery

A fundamental problem in biodiversity science is determining the number of species in any taxon, and there is a growing awareness that cryptic diversity contributes to this problem – even in well-studied groups. Discovering cryptic species requires several lines of evidence to elucidate congruent patterns across data-types, and distinguish unrecognized species. Tiger beetles are among the most well-studied insect groups; yet few new North American species have been described since the mid-20th century, suggesting that that the number of morphologically distinct species is reaching an asymptote. We explore the possibility that more species exist in the fauna as cryptic species, by analysing a broad geographic sample of all species in the genus Dromochorus. We employ a ‘taxonomic congruence’ approach, where we first generate species hypotheses from patterns of reciprocal monophyly across the mitochondrial and nuclear datasets, and test these hypotheses through congruence with population structure, morphological measures and ecological divergence. We find broad congruence that supports eight species of Dromochorus, more than doubling the known diversity. We also validate a previously ambiguous taxon, and re-describe previously named species. Lastly, we identify new diagnostic morphological characters, include an updated dichotomous key and provide updated natural history/ecological characteristics for the genus and individual species

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 Cicindelidia politula (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

Discovery of cryptic species diversity in North American pine-feeding chionaspis scale insects (Hemiptera: Diaspididae)

The pine-needle scale insects; Chionaspis pinifoliae and C. heterophyllae are armored scale insects (Hemiptera: Diaspididae) with extensive native ranges throughout North America. In particular, C. pinifoliae is found on almost every species in the genus Pinus. Both species are economically important pests on pines, and over a century of scientific literature has considered these as only two morphological species. The life history of scale insects suggests they may form strongly structured metapopulations, resulting in high rates of host race formation and possibly speciation. Such newly originated species are likely to be morphologically similar. The geographic distribution and host use of these two species suggests they may represent a group of species that have gone unsampled due to their broad distribution or unrecognized due to their similar morphology. To explore the potential for species diversity in pine-needle scale insects I collected 366 individual insects from 320 localities across North America, representing 51 host species within the Pinaceae. I estimated species diversity by inferring species boundaries using genealogical concordance across allele genealogies of two nuclear loci and one mitochondrial locus. Using Maximum Likelihood allele genealogies in a majority-rule consensus to assess congruence, I conservatively detect 10 species in this group. However some of these 10 species contain morphological subgroups that conventional taxonomy would recognize as their own species. I explored alternate species delimitations using a range of species delimitation schemes based on genealogical concordance and mitochondrial divergence. I analyzed these delimitation schemes as species in a Bayesian species tree analysis, and infer that a delimitation of 26 species is the optimum scheme. This 26-species scheme also recognizes most of the aforementioned unique morphological subgroups, as species. Additionally, the genetic analyses for the above work occasionally recovered parasitoid sequences, from the 28S D2 and D3 subunits of ribosomal RNA. I included these sequences in a phylogeny of over 500 specimens from 18 of 19 families within the Chalcidoidea. I compared the phylogenetic results against a comprehensive list of parasitoids recorded from C. pinifoliae and C. heterophyllae, and find the 28s sequences indicate the parasitoid diversity utilizing these pine-feeding scale insects is much higher than previously thought

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

The Hemiptera (Insecta) of Canada: Constructing a Reference Library of DNA Barcodes

<div><p>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.</p></div

A three dimensional visualization of taxonomic congruence between BINs and species identifications, based on morphology, for three families of Canadian Hemiptera (Aphididae, Cicadellidae, and Miridae).

<p>The taxonomists who identified these specimens are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125635#pone.0125635.s012" target="_blank">S7 Table</a>. A robustly concordant BIN (one species per BIN, with many specimens in that BIN) occupies a forward position in the plane along the left wall, whereas discordant BINs (those with many specimens, and several species names) appear towards the upper right quadrant. The colors correspond to the concordance status of the same BIN on BOLD. In many cases, BINs that are concordant in this curated dataset are discordant on BOLD. This disparity highlights the utility of defining a reference set of specimens, as library specimens will be grouped by BOLD in BINs with misidentified specimens.</p