Jennifer M. Zaspel

Are molecular data supplanting morphological data in modern phylogenetic studies

Morphological data have long served as major sources ofinformation for inferring phylogenetic relationships amongtaxa. With the advent of polymerase chain reaction and modernmolecular approaches to phylogenetics, DNA has become amajor source for phylogenetic inference. Combined analyses ofmolecular and morphological (CAMM) data are not unusual.Studies examining the relative utility of morphological andmolecular data derived from plants and animal groups havebeen performed (e.g. Sanderson & Donoghue, 1989), but theresults of this research were limited by the relative noveltyof molecular data to phylogenetics at the time. Differencesof opinions exist among systematic biologists concerning theutility of morphological data (Scotland

A comparative survey of proboscis morphology and associated structures in fruit-piercing, tear-feeding, and blood-feeding moths in Calpinae (Lepidoptera: Erebidae)

Functional feeding categories for adult species of Calpinae are described. Structures associated with the proboscis were examined using exemplar species in fruit-piercing, blood-feeding, and tear-feeding species using both light microscopy and SEM methods. At least three genera currently placed in Calpini, and several others in related groups lack specialized piercing structures. The proboscis of the tear-feeding species, Hemiceratoides hieroglyphica, is equipped with specialized cuticular hooks not yet observed in other tear-feeding species. Tearing hooks moveable by blood pressure are restricted to Calpini species, and little additional variation within this taxon exists, suggesting proboscis morphology may not be strongly correlated with feeding behavior (e.g., fruit piercing vs. blood feeding). A glossary of terms and character codings for proboscis structures is provided, and morphologies for all included calpine taxa are described. We discuss the taxonomic significance of proboscis morphology in Calpinae and the evolutionary implications of their associated feeding behaviors. This survey indicates morphology provides powerful prediction, but not proof of lepidopteran adult food habits.

Phylogeny and Evolution of Pharmacophagy in Tiger Moths (Lepidoptera: Erebidae: Arctiinae)

The focus of this study was to reconstruct a phylogenetic hypothesis for the moth subfamily Arctiinae (tiger moths, woolly bears) to investigate the evolution of larval and adult pharmacophagy of pyrrolizidine alkaloids (PAs) and the pathway to PA chemical specialization in Arctiinae. Pharmacophagy, collection of chemicals for non-nutritive purposes, is well documented in many species, including the model species Utetheisa ornatrix L. A total of 86 exemplar ingroup species representing tiger moth tribes and subtribes (68 genera) and nine outgroup species were selected. Ingroup species included the most species-rich generic groups to represent the diversity of host-plant associations and pharmacophagous behaviors found throughout Arctiinae. Up to nine genetic markers were sequenced: one mitochondrial (COI barcode region), one nuclear rRNA (D2 region, 28S rRNA), and seven nuclear protein-coding gene fragments: elongation factor 1-α protein, wingless, ribosomal protein subunit S5, carbamoylphosphate synthase domain regions, glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase and cytosolic malate dehydrogenase. A total of 6984 bp was obtained for most species. These data were analyzed using model-based phylogenetic methods: maximum likelihood (ML) and Bayesian inference (BI). Ancestral pharmacophagous behaviors and obligate PA associations were reconstructed using the resulting Bayes topology and Reconstructing Ancestral States in Phylogenies (RASP) software. Our results corroborate earlier studies on the evolution of adult pharmacophagous behaviors, suggesting that this behavior arose multiple times and is concentrated in the phaegopterine-euchromiine-ctenuchine clade (PEC). Our results suggest that PA specialization may have arisen early in the phylogeny of the subfamily and that facultative larval pharmacophagous behaviors are the derived condition.

To be or not to be ... a vampire: a matter of sensillum numbers in Calyptra thalictri?

The mechanisms by which blood feeding in insects has evolved are unclear, primarily because there has been no access to species in which there is a mixture of same-sex blood feeding and non-blood feeding individuals. The discovery of a subset of male Calyptra thalictri (Lepidoptera: Noctuidae: Calpini) that blood feed under constrained experimental conditions, while the majority of these males do not, provides a unique opportunity to investigate members of the same species for potential root mechanisms leading to the ability to blood feed. Previously, C. thalictri populations revealed no morphological differences in the classical structures used for species identification in individuals that took a blood meal compared with those that did not. We report a description of the antennal sensilla and their distribution in male C. thalictri and describe an antennal sensillum distribution dimorphism between individuals that took a blood meal under constrained experimental conditions and those that did not. The number of olfactory sensilla, primarily sensilla coeloconica but also sensilla auricillica, is reduced in C. thalictri males that took a blood meal compared with those that did not. The selectivity of sensilla coeloconica olfactory sensory neurons was investigated. The sensilla coeloconica demonstrated sensitivity to fifteen vertebrate-related volatiles, including ammonia. We propose that the reduction in olfactory sensilla sensitive to vertebrate-related compounds may be correlated to an increase in the likelihood of a male C. thalictri to take a blood meal.

A preliminary molecular phylogenetic assessment of the lichen moths (Lepidoptera: Erebidae: Arctiinae: Lithosiini) with comments on palatability and chemical sequestration

The lichen moth tribe Lithosiini is best known for its hypothesized larval feeding behaviour, lichenivory. The larvae of some species have been found to be capable of sequestering polyphenolics, and the adults of some species are unpalatable to vertebrate predators. However, the chemical basis for the defence is unknown. Here we reconstruct a phylogenetic hypothesis using likelihood methods (maximum likelihood, Bayesian inference) for 65 species of Lithosiini representing 37 genera and seven outgroup species using 2806 bp of sequence data obtained from two mitochondrial gene fragments (COI barcoding region, CytB), and two nuclear gene fragments (RpS5 and ribosomal gene region 28S). Lithosiine species representing four of the seven subtribes and unplaced taxa are included in the analysis. The deeper relationships within the tribe are not strongly supported, and the monophyly of three of the four subtribes is not supported. The placement of the fourth subtribe, Acsalina, is weakly supported. We also conduct a survey of the secondary metabolites present within adults of five species. The species are examined for the presence of lichen polyphenolics and plant secondary metabolites sequestered by arctiines. Seven lichen polyphenolics are identified in the taxa examined, but no plant secondary metabolites are found using either analytical method. The results of this study and prior reports in the literature of chemical sequestration and unpalatability are plotted onto the resulting Bayesian inference phylogeny to examine the evolution of chemical defence within Lithosiini. Species that sequester lichen polyphenolics and unpalatable species occur in each of the major clades recovered in the analysis.

Microbial Diversity Associated with the Fruit-Piercing and Blood-Feeding Moth Calyptra thalictri (Lepidoptera: Noctuidae)

Abstract Previous inventories of the diversity of lepidopteran symbionts have been limited to Eubacteria. We conducted a microbial survey of Calyptra thalictri Borkhausen (Lepidoptera: Noctuidae) by using polymerase chain reaction (PCR) primers for 16S rRNA sequences for Eubacteria, and primers for Archaea, fungi including yeast-like organisms, Microsporidia, and Wolbachia. Heads and abdomens of adult males of this fruit-piercing and blood-feeding moth were assayed separately. High-fidelity PCR and subsequent DNA analyses indicated that at least five microorganisms belonging to the &agr;-, &bgr;-, and &ggr;-Proteobacteria were present. Two eubacterial sequences, related to a Klebsiella sp. and a Sinorhizobium sp., were detected in the abdomens of all nine individuals sampled, and three additional sequences, two related to species in the genus Alcaligenes and one related to a Rhizobium sp., were found in some of the abdominal samples, suggesting all five could be associated with abdominal structures. No Archaea, fungi including yeast-like organisms, Microsporidia, or Wolbachia were detected. These results document the first microbial associates in a fruit-piercing and blood-feeding moth.

LepNet: The Lepidoptera of North America Network

The Lepidoptera of North America Network, or LepNet, is a digitization effort recently launched to mobilize biodiversity data from 3 million specimens of butterflies and moths in United States natural history collections (http://www.lep-net.org/). LepNet was initially conceived as a North American effort but the project seeks collaborations with museums and other organizations worldwide. The overall goal is to transform Lepidoptera specimen data into readily available digital formats to foster global research in taxonomy, ecology and evolutionary biology.

Billbug (Coleoptera: Dryophthoridae: Sphenophorus spp.) Seasonal Biology and DNA-Based Life Stage Association in Indiana Turfgrass

Eleven species of billbugs (Coleoptera: Dryophthoridae: Sphenophorus spp. Schönherr) infest managed turfgrass in North America. However, the regional variation in species composition remains unresolved and the seasonal phenology of several species has not been well documented. The latter gap is largely due to the inability to identify the larval stage to species-a confounding problem with several sympatric insect species. We used field trapping (adults) and soil sampling (larvae and pupae) surveys along with a DNA-based life-stage association to characterize the biology of billbugs associated with turfgrass in the Midwestern United States. Pitfall trapping at four locations in Indiana revealed four billbug species: S. venatus Say, S. parvulus Gyllenhaal, S. minimus Hart, and S. inaequalis Say. Sphenophorus venatus was the most abundant species on warm-season turfgrass while S. parvulus was most abundant on cool-season turfgrass. Investigation of S. venatus seasonal biology revealed two overwintered life stages-larva and adult-which resulted in two overlapping cohorts and two larval generations. Degree-day models describing S. venatus activity were more accurate for first-generation adults and larvae than for overwintering life stages. Maximum-likelihood analyses provided the first molecular species identification of billbug larvae and direct evidence that S. venatus larvae are capable of overwintering above 40°N latitude. Findings clarify the utility of molecular markers (CO1, 18S, and ITS2) for describing billbug larval population dynamics and seasonal phenology in regions where several sympatric billbug species occur. These results support the development of sustainable management strategies based on billbug seasonal phenology in different regions of North America.

HPLC-MS Analysis of Lichen-Derived Metabolites in the Life Stages of Crambidia cephalica (Grote & Robinson)

Tiger moths (Lepidoptera: Erebidae: Arctiinae: Arctiini) are notable for their specialized associations with hosts that produce toxic secondary compounds, and are thus an ideal study system for understanding insect-plant interactions and the evolution of antipredatory defense. Likewise, their sister lineage (Arctiinae: Lithosiini) has been documented feeding on algae and lichens, and is known to sequester lichen-derived secondary compounds from the larval to adult stages. Prevalence of lichenivory in this early radiation (ca. 3000 species) may provide clues to the phylogenetic basis for storied chemical sequestration within all tiger moths. Despite the evolutionary significance of this trait, we lack a basic understanding of the extent of lichenivory among lithosiines, and the distribution of sequestered chemicals among life stages. The dynamics of chemical sequestration throughout the lifecycle for the lichen moth Crambidia cephalica were investigated by testing the hypothesis that lichen-derived metabolites are unequally distributed among life stages, and that laboratory-reared C. cephalica have less metabolite diversity than wild-caught individuals. Crambidia cephalica was reared on Physcia, and analyzed using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Several putative lichen-derived metabolites were detected across three life stages, i.e., larval, pupal, and adult, and differences among life stages and lichen host were observed. These results provide evidence that multiple lichen-derived metabolites are sequestered by C. cephalica; some metabolites are retained through adulthood, and others are lost or modified in earlier life stages. The presence of differing lichen-derived metabolites across life stages may indicate functional properties of the metabolites for C. cephalica with regards to chemical protection from antagonists, and other physiological processes.

Beetles (Insecta: Coleoptera) Associated With the Plains Pocket Gopher, Geomys bursarius (Mammalia: Rodentia: Geomyidae), in Indiana

Abstract The beetle (Insecta: Coleoptera) fauna associated with the underground burrow systems of the plains pocket gopher, Geomys bursarius (Shaw) (Mammalia: Rodentia: Geomyidae), is reported from one of Indianas primary conservation zones, Kankakee Sands, for the first time. Pitfall traps baited with pig dung were placed into active burrow systems and routinely checked from February 2014 to January 2015. A total of 26 species of Coleoptera were found in the burrow systems. We summarize phenological data for the most commonly collected species. Also, a complete checklist of burrow-inhabiting beetles is provided.