A Phylogenetic Analysis of Armored Scale Insects, 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. Also, in some groups, the adult female never sheds the second instars cuticle, and remains within its confines, a habit referred to as the pupillarial habit. Here we expand upon recent phylogenetic work (Morse and Normark 2006) by analyzing a partitioned dataset including armored scale and endoysmbiont DNA from one hundred and twenty three species of armored scales, represented by two hundred and fifty-four samples. Included were fragments of the nuclear protein-coding gene Elongation Factor 1α (EF1α), the D2 and D3 expansion segments of the large subunit ribosomal RNA gene 28S, and a region of mitochondrial DNA encompassing the 3\u27 portion of cytochrome oxidase I (COI), and the 5\u27 portion of cytochrome oxidase II (COII). Ribosomal 16S from the primary bacterial endosymbiont Uzinura diaspidicola was amplified as well. Two versions of our dataset were analyzed due to concerns over the possible effects of missing data. The first version (the full dataset) contained all 254 taxa, with every taxon having at least both the 28S and EF1α fragments. The second version (the core dataset) had only the 113 taxa for which all four fragments were available. Maximum parsimony, maximum likelihood, and Bayesian analyses were run on both versions of the dataset, as well as individually for each fragment. We find that our results were consistent across methods, and between the two versions of the dataset. It appears that including missing data had little effect on topology. Our results mirror that of the classic taxonomy, however we reconstruct a general lack of monophyly at the subfamily, tribal, and subtribal levels. Within the two major subfamilies, we reconstruct that the same developmental pathway has evolved independently. We reconstruct independent replacements of the pupillarial habit with the scale cover, followed by independent origins of early PGE. In each case there appears to be increased diversity in clades associated with the scale cover and early PGE. In light of this apparent increase we propose a new adaptive scenario under which early PGE may have evolved – the removal of male-killing paternal chromosomes. We also reconstruct the ancestor to the armored scales to Australasian in origins, and to have an ancestral diet breath that includes members of the Rosids and/or Monocot plant groups

Pest control services on farms vary among bird species on diversified, low-intensity farms

Avian species provide pest control services in some agricultural systems, which may incentivize farmers to conserve natural habitats for native biodiversity. A critical component of this equation, however, is verifying that avian species are consuming potential pest species in the agricultural ecosystems. We used a DNA metabarcoding approach to determine the frequency of pest presence in songbird fecal samples collected from birds caught on diversified, low-intensity farms in New England, USA, during the bird breeding season. Twelve species of insect pest were identified in fecal samples, and across all songbird species 12.6% of samples included DNA from at least one pest. Frequency of pest presence depended on songbird species, with Common Yellowthroats and Gray Catbirds eating pests more frequently than Song Sparrows. Pests were also more frequently found in fecal samples collected from hatch-year birds and birds caught later in the year. Although we observed a lower frequency of pest consumption than observed in previous comparable research, growers can likely improve pest control by songbirds by promoting the woody, non-crop habitat types preferred by insectivorous species, in our system specifically Gray Catbirds and Common Yellowthroats

Parasite Prevalence May Drive the Biotic Impoverishment of New England (USA) Bumble Bee Communities

Numerous studies have reported a diversity of stressors that may explain continental-scale declines in populations of native pollinators, particularly those in the genus Bombus. However, there has been little focus on the identification of the local-scale dynamics that may structure currently impoverished Bombus communities. For example, the historically diverse coastal-zone communities of New England (USA) now comprise only a few species and are primarily dominated by a single species, B. impatiens. To better understand the local-scale factors that might be influencing this change in community structure, we examined differences in the presence of parasites in different species of Bombus collected in coastal-zone communities. Our results indicate that Bombus species that are in decline in this region were more likely to harbor parasites than are B. impatiens populations, which were more likely to be parasite-free and to harbor fewer intense infections or co-infections. The contrasting parasite burden between co-occurring winners and losers in this community may impact the endgame of asymmetric contests among species competing for dwindling resources. We suggest that under changing climate and landscape conditions, increasing domination of communities by healthy, synanthropic Bombus species (such as B. impatiens) may be another factor hastening the further erosion of bumble bee diversity

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

Analysis of time-to-event for observational studies: Guidance to the use of intensity models

This paper provides guidance for researchers with some mathematical background on the conduct of time-to-event analysis in observational studies based on intensity (hazard) models. Discussions of basic concepts like time axis, event definition and censoring are given. Hazard models are introduced, with special emphasis on the Cox proportional hazards regression model. We provide check lists that may be useful both when fitting the model and assessing its goodness of fit and when interpreting the results. Special attention is paid to how to avoid problems with immortal time bias by introducing time-dependent covariates. We discuss prediction based on hazard models and difficulties when attempting to draw proper causal conclusions from such models. Finally, we present a series of examples where the methods and check lists are exemplified. Computational details and implementation using the freely available R software are documented in Supplementary Material. The paper was prepared as part of the STRATOS initiative.Comment: 28 pages, 12 figures. For associated Supplementary material, see http://publicifsv.sund.ku.dk/~pka/STRATOSTG8

Categorization of species as native or nonnative using DNA sequence signatures without a complete reference library.

New genetic diagnostic approaches have greatly aided efforts to document global biodiversity and improve biosecurity. This is especially true for organismal groups in which species diversity has been underestimated historically due to difficulties associated with sampling, the lack of clear morphological characteristics, and/or limited availability of taxonomic expertise. Among these methods, DNA sequence barcoding (also known as "DNA barcoding") and by extension, meta-barcoding for biological communities, has emerged as one of the most frequently utilized methods for DNA-based species identifications. Unfortunately, the use of DNA barcoding is limited by the availability of complete reference libraries (i.e., a collection of DNA sequences from morphologically identified species), and by the fact that the vast majority of species do not have sequences present in reference databases. Such conditions are critical especially in tropical locations that are simultaneously biodiversity rich and suffer from a lack of exploration and DNA characterization by trained taxonomic specialists. To facilitate efforts to document biodiversity in regions lacking complete reference libraries, we developed a novel statistical approach that categorizes unidentified species as being either likely native or likely nonnative based solely on measures of nucleotide diversity. We demonstrate the utility of this approach by categorizing a large sample of specimens of terrestrial insects and spiders (collected as part of the Moorea BioCode project) using a generalized linear mixed model (GLMM). Using a training data set of known endemic (n = 45) and known introduced species (n = 102), we then estimated the likely native/nonnative status for 4,663 specimens representing an estimated 1,288 species (412 identified species), including both those specimens that were either unidentified or whose endemic/introduced status was uncertain. Using this approach, we were able to increase the number of categorized specimens by a factor of 4.4 (from 794 to 3,497), and the number of categorized species by a factor of 4.8 from (147 to 707) at a rate much greater than chance (77.6% accuracy). The study identifies phylogenetic signatures of both native and nonnative species and suggests several practical applications for this approach including monitoring biodiversity and facilitating biosecurity

Validating Morphometrics with DNA Barcoding to Reliably Separate Three Cryptic Species of Bombus Cresson (Hymenoptera: Apidae)

Despite their large size and striking markings, the identification of bumble bees (Bombus spp.) is surprisingly difficult. This is particularly true for three North American sympatric species in the subgenus Pyrobombus that are often misidentified: B. sandersoni Franklin, B. vagans Smith B. perplexus Cresson. Traditionally, the identification of these cryptic species was based on observations of differences in hair coloration and pattern and qualitative comparisons of morphological characters including malar length. Unfortunately, these characteristics do not reliably separate these species. We present quantitative morphometric methods to separate these species based on the malar length to width ratio (MRL) and the ratios of the malar length to flagellar segments 1 (MR1) and 3 (MR3) for queens and workers, and validated our determinations based on DNA barcoding. All three measurements discriminated queens of B. sandersoni and B. vagans with 100% accuracy. For workers, we achieved 99% accuracy by combining both MR1 and MR3 measurements, and 100% accuracy differentiating workers using MRL. Moreover, measurements were highly repeatable within and among both experienced and inexperienced observers. Our results, validated by genetic evidence, demonstrate that malar measurements provide accurate identifications of B. vagans and B. sandersoni. There was considerable overlap in the measurements between B. perplexus and B. sandersoni. However, these species can usually be reliably separated by combining malar ratio measurements with other morphological features like hair color. The ability to identify bumble bees is key to monitoring the status and trends of their populations, and the methods we present here advance these efforts

Rapid and cost-effective generation of single specimen multilocus barcoding data from whole arthropod communities by multiple levels of multiplexing

In light of the current biodiversity crisis, molecular barcoding has developed into an irreplaceable tool. Barcoding has been considerably simplified by developments in high throughput sequencing technology, but still can be prohibitively expensive and laborious when community samples of thousands of specimens need to be processed. Here, we outline an Illumina amplicon sequencing approach to generate multilocus data from large collections of arthropods. We reduce cost and effort up to 50-fold, by combining multiplex PCRs and DNA extractions from pools of presorted and morphotyped specimens and using two levels of sample indexing. We test our protocol by generating a comprehensive, community wide dataset of barcode sequences for several thousand Hawaiian arthropods from 14 orders, which were collected across the archipelago using various trapping methods. We explore patterns of diversity across the Archipelago and compare the utility of different arthropod trapping methods for biodiversity explorations on Hawaii, highlighting undergrowth beating as highly efficient method. Moreover, we show the effects of barcode marker, taxonomy and relative biomass of the targeted specimens and sequencing coverage on taxon recovery. Our protocol enables rapid and inexpensive explorations of diversity patterns and the generation of multilocus barcode reference libraries across whole ecosystems

Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111

For highly pathogenic viruses, reporter assays that can be rapidly performed are critically needed to identify potentially functional mutations for further study under maximal containment (e.g., biosafety level 4 [BSL-4]). The Ebola virus nucleoprotein (NP) plays multiple essential roles during the viral life cycle, yet few tools exist to study the protein under BSL-2 or equivalent containment. Therefore, we adapted reporter assays to measure NP oligomerization and virion-like particle (VLP) production in live cells and further measured transcription and replication using established minigenome assays. As a proof-of-concept, we examined the NP-R111C substitution, which emerged during the 20132016 Western African Ebola virus disease epidemic and rose to high frequency. NP-R111C slightly increased NP oligomerization and VLP budding but slightly decreased transcription and replication. By contrast, a synthetic charge-reversal mutant, NP-R111E, greatly increased oligomerization but abrogated transcription and replication. These results are intriguing in light of recent structures of NP oligomers, which reveal that the neighboring residue, K110, forms a salt bridge with E349 on adjacent NP molecules. By developing and utilizing multiple reporter assays, we find that the NP-111 position mediates a complex interplay between NP\u27s roles in protein structure, virion budding, and transcription and replication