Erik M. Pilgrim

Molecular phylogenetics of Vespoidea indicate paraphyly of the superfamily and novel relationships of its component families and subfamilies

The 24 000+ described species of Vespoidea include many well‐known stinging wasps, such as paper wasps and hornets (Vespidae), velvet ants (Mutillidae), spider wasps (Pompilidae) and ants (Formicidae). The compelling behaviours of vespoids have been instrumental in developing theories of stepwise evolutionary transitions, which necessarily depend on an understanding of phylogeny, yet, existing morphological phylogenies for Vespoidea conflict. We collected molecular data from four nuclear genes (elongation factor‐1α F2 copy, long‐wavelength rhodopsin, wingless and the D2–D3 regions of 28S ribosomal RNA (2700 bp in total)) to produce the first molecular phylogeny of Vespoidea. We analysed molecular data alone and in combination with published morphological data from Brothers and Carpenter. Parsimony analyses left many deeper nodes unsupported, but suggested paraphyly of three families. Total‐evidence Bayesian inference produced a more resolved tree, in which the monophyly of Vespoidea was nevertheless ambiguous. Bayesian inference of molecular data alone returned a well‐resolved consensus with posterior probabilities of over 95% for most nodes. We used this topology as the best estimate of phylogeny at the family and subfamily levels. Notable departures from previous estimates include: (i) paraphyly of Vespoidea resulting from the nesting of Apoidea within a lineage comprising Formicidae, Scoliidae and two subfamilies of Bradynobaenidae; (ii) paraphyly of Bradynobaenidae, Mutillidae and Tiphiidae; (iii) a sister relationship between Rhopalosomatidae and Vespidae; and (iv) Rhopalosomatidae + Vespidae as sister to all other vespoids/apoids. We discuss character evidence in light of the new phylogeny, and propose a new classification of Aculeata that recognizes eight superfamilies: Apoidea, Chrysidoidea, Formicoidea, Pompiloidea, Scolioidea, Tiphioidea, Thynnoidea and Vespoidea.

Incorporation of DNA barcoding into a large-scale biomonitoring program: opportunities and pitfalls

Abstract Taxonomic identification of benthic macroinvertebrates is critical to protocols used to assess the biological integrity of aquatic ecosystems. The time, expense, and inherent error rate of species-level morphological identifications has necessitated use of genus- or family-level identifications in most large, statewide bioassessment programs. Use of coarse-scale taxonomy can obscure signal about biological condition, particularly if the range of species tolerances is large within genera or families. We hypothesized that integration of deoxyribonucleic acid (DNA) barcodes (partial cytochrome c oxidase subunit I sequences) into bioassessment protocols would provide greater discriminatory ability than genus-level identifications and that this increased specificity could lead to more sensitive assessments of water quality and habitat. Analysis of DNA barcodes from larval specimens of Ephemeroptera, Plecoptera, and Trichoptera (EPT) taxa collected as part of Marylands Biological Stream Survey (MBSS) revealed ∼2 to 3× as many DNA-barcode groups or molecular operational taxonomic units (mOTUs) as morphologically identified genera. As expected, geographic distributions for several mOTUs were tighter than for the parent genus, but few mOTUs showed closer associations with water-quality variables or physical-habitat features than did the genus in which they belonged. The need for improved protocols for the consistent generation of DNA barcodes is discussed.

A Molecular Method for Associating the Dimorphic Sexes of Velvet Ants (Hymenoptera: Mutillidae)

Abstract For several groups of insects, including the velvet ants, male–female associations are difficult because of extreme dimorphism between the sexes. Many species, or even genera, are known from only a single sex. In this study, we investigate the use of DNA sequences of the internal transcribed spacer regions (ITS1 & ITS2) as a means of associating males and females in velvet ants. We compare ITS sequences between sexes in taxa where the associations are well documented and uncontested. We compare these sequences to other closely related species. The DNA sequence data show little or no variability between sexes, while much greater differences exist between species. This method should prove a valuable technique for making associations between previously unmatched males and females in velvet ants, and likely other hymenopteran taxa.

Cryptic Biodiversity in Streams: A Comparison of Macroinvertebrate Communities Based on Morphological and DNA Barcode Identifications

Abstract: Species-level identifications are difficult or impossible for many larval aquatic macroinvertebrates. We described the taxonomic composition of macroinvertebrate communities from 5 coastal streams in 3 neighboring catchments in southern California. We compared taxonomic identifications based on deoxyribonucleic acid (DNA) barcoding (cytochrome c oxidase subunit I [COI]) with morphological identifications of the same specimens. We examined 5870 individuals, and barcodes with sequence lengths >350 base pairs (bp) for 91% of those specimens. We used the naturally occurring gaps in divergence frequencies for each order (usually 2% level of genetic divergence) to delimit putative species for all taxonomic groups except Simulium (3%) and Baetis (1%). We identified 200 species across these 5 streams. We identified 104 more species via barcodes than via morphology (200 vs 96, a 108% increase). Richness increases were greatest for Chironomidae (60 more species), Ephemeroptera (10 species), Acari (10 species), and Trichoptera (6 species). Forty-five percent of the genera/species identified morphologically represented >2 species. Many (86) species identified with barcodes were represented by only 1 or 2 specimens and were found at only 1 stream. Thus, species rarity (either spatially or numerically) appears to be a common characteristic of these streams. Barcoding increased total richness at each site by 12 to 40 taxa over morphology alone, and increased the difference between reference and impact sites in terms of lost taxa. These results suggest that macroinvertebrate biodiversity in streams has been underestimated substantially in the past, as has the biodiversity lost in response to environmental stress. The potential of DNA barcoding will not be fully realized until we can assign traits, such as habitat preference, ecological function, and pollution tolerance, at the species level.

Phylogeny of the Sympetrinae (Odonata: Libellulidae): further evidence of the homoplasious nature of wing venation

Abstract Sympetrinae is the largest subfamily of the diverse dragonfly family Libellulidae. This subfamily, like most libellulid subfamilies, is defined currently by a few wing venation characters, none of which are synapomorphies for the taxon. In this study, we used DNA sequence data from the nuclear locus elongation factor‐1α and the mitochondrial loci 16S and 12S rRNA, together with 38 wing venation characters, to test the monophyly of the Sympetrinae and several other libellulid subfamilies. No analysis recovered Sympetrinae as monophyletic, partly because of the position of Leucorrhinia (of the subfamily Leucorrhininae) as a strongly supported sister to Sympetrum (of Sympetrinae) in all analyses. The subfamilies Brachydiplactinae, Leucorrhininae, Trameinae and Trithemistinae were also found not to be monophyletic. Libellulinae was the only subfamily supported strongly as monophyletic. Consistency indices and retention indices of wing venation characters used to define various subfamilies were closer to zero than unity, showing that many of these characters were homoplasious, and therefore not useful for a classification scheme within Libellulidae.

Does DNA barcoding improve performance of traditional stream bioassessment metrics

Abstract: Benthic macroinvertebrate community composition is used to assess wetland and stream condition and to help differentiate the effects of stressors among sites. Deoxyribonucleic acid (DNA) barcoding has been promoted as a way to increase taxonomic resolution and, thereby, to increase the sensitivity of bioassessment metrics. We compared the ability of several commonly used bioassessment metrics calculated with data derived from morphology and from DNA barcoding to detect differences in stream condition of 6 paired sites in southern California with relatively subtle impacts to habitat. At each site, we sampled an upstream (reference) reach and a downstream (impact) reach with armored stream banks. We counted and identified ∼600 organisms/ sample based on morphology (generally to species, but to genus for midges). We then extracted mitochondrial (mt)DNA from each individual and sequenced the ∼658-base pair (bp) barcoding region of the cytochrome c oxidase subunit I (COI) gene. Most (91%) organisms yielded sequences >350 bp in length, but high failure rates for all taxa collected from 1 stream required that we exclude it from analysis. Sixteen metrics calculated with morphological data showed subtle but not significant differences in community composition between armored and unarmored reaches. The statistical power of 10 of the 16 metrics was substantially higher when calculated with DNA than with morphological data, and we were able to discern differences between armored and unarmored reaches with the DNA data. These differences were associated with increased taxonomic richness detected for midges, mayflies, noninsects, caddisflies, and black flies when DNA data were used. Our results suggest that identifications based on DNA barcoding have the potential to improve power to detect small changes in stream condition.

Evaluating Ethanol-based Sample Preservation to Facilitate Use of DNA Barcoding in Routine Freshwater Biomonitoring Programs Using Benthic Macroinvertebrates

Molecular methods, such as DNA barcoding, have the potential to enhance biomonitoring programs worldwide. Altering routinely used sample preservation methods to protect DNA from degradation may pose a potential impediment to application of DNA barcoding and metagenomics for biomonitoring using benthic macroinvertebrates. Using higher volumes or concentrations of ethanol, requirements for shorter holding times, or the need to include additional filtering may increase cost and logistical constraints to existing biomonitoring programs. To address this issue we evaluated the efficacy of various ethanol-based sample preservation methods at maintaining DNA integrity. We evaluated a series of methods that were minimally modified from typical field protocols in order to identify an approach that can be readily incorporated into existing monitoring programs. Benthic macroinvertebrates were collected from a minimally disturbed stream in southern California, USA and subjected to one of six preservation treatments. Ten individuals from five taxa were selected from each treatment and processed to produce DNA barcodes from the mitochondrial gene cytochrome c oxidase I (COI). On average, we obtained successful COI sequences (i.e. either full or partial barcodes) for between 93–99% of all specimens across all six treatments. As long as samples were initially preserved in 95% ethanol, successful sequencing of COI barcodes was not affected by a low dilution ratio of 2∶1, transfer to 70% ethanol, presence of abundant organic matter, or holding times of up to six months. Barcoding success varied by taxa, with Leptohyphidae (Ephemeroptera) producing the lowest barcode success rate, most likely due to poor PCR primer efficiency. Differential barcoding success rates have the potential to introduce spurious results. However, routine preservation methods can largely be used without adverse effects on DNA integrity.

Potential for DNA-based identification of Great Lakes fauna: match and mismatch between taxa inventories and DNA barcode libraries

DNA-based identification of mixed-organism samples offers the potential to greatly reduce the need for resource-intensive morphological identification, which would be of value both to bioassessment and non-native species monitoring. The ability to assign species identities to DNA sequences found depends on the availability of comprehensive DNA reference libraries. Here, we compile inventories for aquatic metazoans extant in or threatening to invade the Laurentian Great Lakes and examine the availability of reference mitochondrial COI DNA sequences (barcodes) in the Barcode of Life Data System for them. We found barcode libraries largely complete for extant and threatening-to-invade vertebrates (100% of reptile, 99% of fish, and 92% of amphibian species had barcodes). In contrast, barcode libraries remain poorly developed for precisely those organisms where morphological identification is most challenging; 46% of extant invertebrates lacked reference barcodes with rates especially high among rotifers, oligochaetes, and mites. Lack of species-level identification for many aquatic invertebrates also is a barrier to matching DNA sequences with physical specimens. Attaining the potential for DNA-based identification of mixed-organism samples covering the breadth of aquatic fauna requires a concerted effort to build supporting barcode libraries and voucher collections.

Molecular and Morphological Study of Species-Level Questions within the Dragonfly Genus Sympetrum (Odonata: Libellulidae)

Abstract This study combines morphological and molecular data to address several questions of species validity within the dragonfly genus Sympetrum. We compared morphological characters (genitalia and other putatively diagnostic characters) and DNA sequences from mitochondrial cytochrome oxidase I (COI) and nuclear internal transcribed spacer (ITS) regions between these disputed taxa and their close relatives. Specimens of Sympetrum nigrescens Lucas shared COI haplotypes with Sympetrum striolatum (Charpentier), and no morphological characters consistently diagnosed S. nigrescens, which therefore becomes a junior synonym of S. striolatum. Similarly, Sympetrum occidentale Bartenev shared identical COI and ITS sequences with Sympetrum semicinctum (Say), and the supposed diagnostic morphological characters overlapped with the intraspecific variation within S. semicinctum. Sympetrum occidentale becomes a junior synonym of S. semicinctum. In a third case, the genetic distance between Sympetrum signiferum Cannings & Garrison and Sympetrum vicinum (Hagen) was lower than that found between most undisputed species. However, the morphological characters that distinguish S. signiferum from S. vicinum were distinct and consistent, and they supported the retention of S. signiferum as a valid species. In the fourth case, neither morphological nor genetic data were able to distinguish Sympetrum janeae Carle consistently from Sympetrum internum Montgomery, or Sympetrum rubicundulum (Say); in addition, genetic distances between individuals of S. internum and S. rubicundulum were small or nonexistent. Further studies are necessary to test the species status of S. janeae and its close relatives.

Comparison of four species-delimitation methods applied to a DNA barcode data set of insect larvae for use in routine bioassessment

Abstract: Species delimitation (grouping individuals into distinct taxonomic groups) is an essential part of evolutionary, conservation, and molecular ecology. Deoxyribonucleic acid (DNA) barcodes, short fragments of the cytochrome c oxidase subunit I (COI) gene, are being used in environmental bioassessments to assign specimens to putative species, but no method for delimiting DNA barcodes into species-level entities is universally accepted. We investigated the effect of delimitation methods on outcomes of bioassessments based on DNA barcodes. We used 2 tree-construction methods (neighbor joining [NJ], maximum likelihood [ML]) and 4 classes of species-delimitation criteria (distance-based, bootstrap support, reciprocal monophyly, and coalescentbased) with a DNA barcode data set consisting of 3 genera and 2202 COI sequences. We compared species delimitations for Baetis (Ephemeroptera:Baetidae), Eukiefferiella (Diptera:Chironomidae), and Simulium (Diptera: Simuliidae) from different streams. We assessed congruence among trees and compared species abundances and estimated species richness among methods. NJ followed by use of a standard barcoding distance cutoff (2%) yielded the greatest number of putative species. All other delimitation methods yielded similar, but lower, richness. Differences in species delimitations produced by various methods might have been caused by confounding factors, such as possible parthenogenesis in Baetis and rare haplotypes in abundant species of Baetis and Simulium. Eukiefferiella presented the fewest discrepancies among delimitations. Each method can be regarded as producing a separate line of evidence contributing to the delimitation of separately evolving lineages. The increased resolution offered by DNA barcoding can yield important insights into the natural history of organisms, but the power of these observations is limited without the use of multigene and multilocus data sets.