Susan J. Weller

Integration of DNA barcoding into an ongoing inventory of complex tropical biodiversity

Inventory of the caterpillars, their food plants and parasitoids began in 1978 for todays Area de Conservacion Guanacaste (ACG), in northwestern Costa Rica. This complex mosaic of 120 000 ha of conserved and regenerating dry, cloud and rain forest over 0–2000 m elevation contains at least 10 000 species of non‐leaf‐mining caterpillars used by more than 5000 species of parasitoids. Several hundred thousand specimens of ACG‐reared adult Lepidoptera and parasitoids have been intensively and extensively studied morphologically by many taxonomists, including most of the co‐authors. DNA barcoding — the use of a standardized short mitochondrial DNA sequence to identify specimens and flush out undisclosed species — was added to the taxonomic identification process in 2003. Barcoding has been found to be extremely accurate during the identification of about 100 000 specimens of about 3500 morphologically defined species of adult moths, butterflies, tachinid flies, and parasitoid wasps. Less than 1% of the species have such similar barcodes that a molecularly based taxonomic identification is impossible. No specimen with a full barcode was misidentified when its barcode was compared with the barcode library. Also as expected from early trials, barcoding a series from all morphologically defined species, and correlating the morphological, ecological and barcode traits, has revealed many hundreds of overlooked presumptive species. Many but not all of these cryptic species can now be distinguished by subtle morphological and/or ecological traits previously ascribed to ‘variation’ or thought to be insignificant for species‐level recognition. Adding DNA barcoding to the inventory has substantially improved the quality and depth of the inventory, and greatly multiplied the number of situations requiring further taxonomic work for resolution.

A Large-Scale, Higher-Level, Molecular Phylogenetic Study of the Insect Order Lepidoptera (Moths and Butterflies)

Background Higher-level relationships within the Lepidoptera, and particularly within the species-rich subclade Ditrysia, are generally not well understood, although recent studies have yielded progress. We present the most comprehensive molecular analysis of lepidopteran phylogeny to date, focusing on relationships among superfamilies. Methodology / Principal Findings 483 taxa spanning 115 of 124 families were sampled for 19 protein-coding nuclear genes, from which maximum likelihood tree estimates and bootstrap percentages were obtained using GARLI. Assessment of heuristic search effectiveness showed that better trees and higher bootstrap percentages probably remain to be discovered even after 1000 or more search replicates, but further search proved impractical even with grid computing. Other analyses explored the effects of sampling nonsynonymous change only versus partitioned and unpartitioned total nucleotide change; deletion of rogue taxa; and compositional heterogeneity. Relationships among the non-ditrysian lineages previously inferred from morphology were largely confirmed, plus some new ones, with strong support. Robust support was also found for divergences among non-apoditrysian lineages of Ditrysia, but only rarely so within Apoditrysia. Paraphyly for Tineoidea is strongly supported by analysis of nonsynonymous-only signal; conflicting, strong support for tineoid monophyly when synonymous signal was added back is shown to result from compositional heterogeneity. Conclusions / Significance Support for among-superfamily relationships outside the Apoditrysia is now generally strong. Comparable support is mostly lacking within Apoditrysia, but dramatically increased bootstrap percentages for some nodes after rogue taxon removal, and concordance with other evidence, strongly suggest that our picture of apoditrysian phylogeny is approximately correct. This study highlights the challenge of finding optimal topologies when analyzing hundreds of taxa. It also shows that some nodes get strong support only when analysis is restricted to nonsynonymous change, while total change is necessary for strong support of others. Thus, multiple types of analyses will be necessary to fully resolve lepidopteran phylogeny.

Evolutionary character analysis tracing character change on a cladogram

Abstract— There has been little formal discussion concerning character analysis in cladistics, even though characters and their character state trees are central to phylogenetic analyses. We refer to this field as Evolutionary Character Analysis. This paper defines the components of evolutionary character analysis: character state trees, transmodal characters, cladogram characters, attribute and character phylogenies; and the use of these components in phylogenetic inference and evolutionary studies.

Can Deliberately Incomplete Gene Sample Augmentation Improve a Phylogeny Estimate for the Advanced Moths and Butterflies (Hexapoda: Lepidoptera)?

Abstract This paper addresses the question of whether one can economically improve the robustness of a molecular phylogeny estimate by increasing gene sampling in only a subset of taxa, without having the analysis invalidated by artifacts arising from large blocks of missing data. Our case study stems from an ongoing effort to resolve poorly understood deeper relationships in the large clade Ditrysia ( > 150,000 species) of the insect order Lepidoptera (butterflies and moths). Seeking to remedy the overall weak support for deeper divergences in an initial study based on five nuclear genes (6.6 kb) in 123 exemplars, we nearly tripled the total gene sample (to 26 genes, 18.4 kb) but only in a third (41) of the taxa. The resulting partially augmented data matrix (45% intentionally missing data) consistently increased bootstrap support for groupings previously identified in the five-gene (nearly) complete matrix, while introducing no contradictory groupings of the kind that missing data have been predicted to produce. Our results add to growing evidence that data sets differing substantially in gene and taxon sampling can often be safely and profitably combined. The strongest overall support for nodes above the family level came from including all nucleotide changes, while partitioning sites into sets undergoing mostly nonsynonymous versus mostly synonymous change. In contrast, support for the deepest node for which any persuasive molecular evidence has yet emerged (78–85% bootstrap) was weak or nonexistent unless synonymous change was entirely excluded, a result plausibly attributed to compositional heterogeneity. This node (Gelechioidea + Apoditrysia), tentatively proposed by previous authors on the basis of four morphological synapomorphies, is the first major subset of ditrysian superfamilies to receive strong statistical support in any phylogenetic study. A “more-genes-only” data set (41 taxa×26 genes) also gave strong signal for a second deep grouping (Macrolepidoptera) that was obscured, but not strongly contradicted, in more taxon-rich analyses.

Phylogenetic Studies of ribosomal RNA variation in higher moths and butterflies (Lepidoptera: Ditrysia)

The selection of exemplars has been shown both theoretically and empirically to affect tree topology, but the importance of the number and nature of taxa used to represent higher taxonomic lineages in molecular studies is rarely stressed. In our rRNA study of higher moths and butterflies (Lepidoptera: Ditrysia), the selection of different exemplars and outgroups caused major tree rearrangements. We also examined the effectiveness with which conserved rRNA regions track the diversification of Lepidoptera. Homoplasy is as prevalent at the few variable sites of conserved regions (18E, 18J, 28F) as at the many variable sites of a more rapidly evolving region (28B). Finally, 28B sequence variation differs qualitatively among lepidopteran superfamilies of presumed comparable age, the Papilionoidea (true butterflies) and Noctuoidea (cutworm moths and relatives).

Advances in Insect Chemical Ecology: A quest for alkaloids: the curious relationship between tiger moths and plants containing pyrrolizidine alkaloids

Introduction A curious relationship exists between a group of plants, the pyrrolizidine alkaloids they contain, and tiger moths of the family Arctiidae. Tiger moths possess an impressive array of chemicals, either produced de novo or sequestered from plants, that protect them to a greater or lesser degree from predators and parasites. These chemicals include cyossin (Teas et al. , 1966), biogenic amines (Bisset et al. , 1959, 1960; Rothschild and Aplin, 1971), pyrazines (Rothschild et al. , 1984), polyphenolics (Hesbacher et al. , 1995), iridoid glycosides (Bowers and Stamp, 1997), and cardenolides (Rothschild et al. , 1970, 1973; Wink and von Nickisch-Rosenegk, 1997); however, no group of compounds, it seems, has influenced the natural history and behavior of tiger moths as the pyrrolizidine alkaloids (PAs) have done (Weller et al. , 2000a). Several excellent reviews have been written about these compounds from the perspective of their chemistry, the plants that produce them (Bull et al. ; 1968; Mattocks, 1986; Hartmann and Witte, 1995), and the insects that utilize them (Schneider, 1986; Boppre, 1990; Hartmann and Ober, 2000), but none has focussed exclusively, on their intimate relationships with tiger moths. The members of the family Arctiidae, which numbers over 11 000 species, are often brilliantly colored (Watson and Goodger, 1986; Holloway, 1988; Weller et al. , 2000a). In addition to standard aposematic red, yellow, or black patterns, adults and larvae may have iridescent blue and green, or even pearly white, coloration. White can be considered aposematic when individuals rest conspicuously on green vegetation.

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.

Abdominal Modifications Occurring in Wasp Mimics of the Ctenuchine-Euchromiine Clade (Lepidoptera: Arctiidae)

Abstract Members of the arctiine moth tribes Ctenuchini and Euchromiini exhibit extreme morphologies including the evolution of convincing wasp mimicry. The lepidopteran abdomen is constricted to produce the visual effect of a hymenopteran petiole (petiole mimic). Many of these species also possess an abdominal “ventral valve” that has been ascribed both a defensive and a courtship function. We examined 85 species (45 genera) to determine the structural variation and distribution of these unique morphologies. Width-length ratios of modified and unmodified segments were generated from 52 species to describe the abdominal constriction in petiole mimics and unmodified abdomen. Here, we provide the first detailed descriptions and illustrations of the morphologies of petiole mimics and ventral valves. Two independent derivations of petiole mimics occur in this clade. Similarly, not all ventral valves are homologous. We propose the phrase “subabdominal pouch” to replace “ventral valve” because it more accurately describes these morphological structures. Petiole mimics may involve modification of either sternite II (SII) or III, but only SIII petiole mimics lack subabdominal pouches. This result suggests that the presence of one morphological innovation (petiole mimic) limits the possession of the other (androconial type).