Akito Y. Kawahara

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.

Phylogenomics provides strong evidence for relationships of butterflies and moths

Butterflies and moths constitute some of the most popular and charismatic insects. Lepidoptera include approximately 160 000 described species, many of which are important model organisms. Previous studies on the evolution of Lepidoptera did not confidently place butterflies, and many relationships among superfamilies in the megadiverse clade Ditrysia remain largely uncertain. We generated a molecular dataset with 46 taxa, combining 33 new transcriptomes with 13 available genomes, transcriptomes and expressed sequence tags (ESTs). Using HaMStR with a Lepidoptera-specific core-orthologue set of single copy loci, we identified 2696 genes for inclusion into the phylogenomic analysis. Nucleotides and amino acids of the all-gene, all-taxon dataset yielded nearly identical, well-supported trees. Monophyly of butterflies (Papilionoidea) was strongly supported, and the group included skippers (Hesperiidae) and the enigmatic butterfly–moths (Hedylidae). Butterflies were placed sister to the remaining obtectomeran Lepidoptera, and the latter was grouped with greater than or equal to 87% bootstrap support. Establishing confident relationships among the four most diverse macroheteroceran superfamilies was previously challenging, but we recovered 100% bootstrap support for the following relationships: ((Geometroidea, Noctuoidea), (Bombycoidea, Lasiocampoidea)). We present the first robust, transcriptome-based tree of Lepidoptera that strongly contradicts historical placement of butterflies, and provide an evolutionary framework for genomic, developmental and ecological studies on this diverse insect order.

A framework to assess evolutionary responses to anthropogenic light and sound

Human activities have caused a near-ubiquitous and evolutionarily-unprecedented increase in environmental sound levels and artificial night lighting. These stimuli reorganize communities by interfering with species-specific perception of time-cues, habitat features, and auditory and visual signals. Rapid evolutionary changes could occur in response to light and noise, given their magnitude, geographical extent, and degree to which they represent unprecedented environmental conditions. We present a framework for investigating anthropogenic light and noise as agents of selection, and as drivers of other evolutionary processes, to influence a range of behavioral and physiological traits such as phenological characters and sensory and signaling systems. In this context, opportunities abound for understanding contemporary and rapid evolution in response to human-caused environmental change.

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.

Phylogeny and Biogeography of Hawkmoths (Lepidoptera: Sphingidae): Evidence from Five Nuclear Genes

Background The 1400 species of hawkmoths (Lepidoptera: Sphingidae) comprise one of most conspicuous and well-studied groups of insects, and provide model systems for diverse biological disciplines. However, a robust phylogenetic framework for the family is currently lacking. Morphology is unable to confidently determine relationships among most groups. As a major step toward understanding relationships of this model group, we have undertaken the first large-scale molecular phylogenetic analysis of hawkmoths representing all subfamilies, tribes and subtribes. Methodology/Principal Findings The data set consisted of 131 sphingid species and 6793 bp of sequence from five protein-coding nuclear genes. Maximum likelihood and parsimony analyses provided strong support for more than two-thirds of all nodes, including strong signal for or against nearly all of the fifteen current subfamily, tribal and sub-tribal groupings. Monophyly was strongly supported for some of these, including Macroglossinae, Sphinginae, Acherontiini, Ambulycini, Philampelini, Choerocampina, and Hemarina. Other groupings proved para- or polyphyletic, and will need significant redefinition; these include Smerinthinae, Smerinthini, Sphingini, Sphingulini, Dilophonotini, Dilophonotina, Macroglossini, and Macroglossina. The basal divergence, strongly supported, is between Macroglossinae and Smerinthinae+Sphinginae. All genes contribute significantly to the signal from the combined data set, and there is little conflict between genes. Ancestral state reconstruction reveals multiple separate origins of New World and Old World radiations. Conclusions/Significance Our study provides the first comprehensive phylogeny of one of the most conspicuous and well-studied insects. The molecular phylogeny challenges current concepts of Sphingidae based on morphology, and provides a foundation for a new classification. While there are multiple independent origins of New World and Old World radiations, we conclude that broad-scale geographic distribution in hawkmoths is more phylogenetically conserved than previously postulated.

Phylotranscriptomics: Saturated third codon positions radically influence the estimation of trees based on next-gen data.

Recent advancements in molecular sequencing techniques have led to a surge in the number of phylogenetic studies that incorporate large amounts of genetic data. We test the assumption that analyzing large number of genes will lead to improvements in tree resolution and branch support using moths in the superfamily Bombycoidea, a group with some interfamilial relationships that have been difficult to resolve. Specifically, we use a next-gen data set that included 19 taxa and 938 genes (∼1.2M bp) to examine how codon position and saturation might influence resolution and node support among three key families. Maximum likelihood, parsimony, and species tree analysis using gene tree parsimony, on different nucleotide and amino acid data sets, resulted in largely congruent topologies with high bootstrap support compared with prior studies that included fewer loci. However, for a few shallow nodes, nucleotide and amino acid data provided high support for conflicting relationships. The third codon position was saturated and phylogenetic analysis of this position alone supported a completely different, potentially misleading sister group relationship. We used the program RADICAL to assess the number of genes needed to fix some of these difficult nodes. One such node originally needed a total of 850 genes but only required 250 when synonymous signal was removed. Our study shows that, in order to effectively use next-gen data to correctly resolve difficult phylogenetic relationships, it is necessary to assess the effects of synonymous substitutions and third codon positions.

Increased gene sampling strengthens support for higher-level groups within leaf-mining moths and relatives (Lepidoptera: Gracillariidae)

BackgroundResearchers conducting molecular phylogenetic studies are frequently faced with the decision of what to do when weak branch support is obtained for key nodes of importance. As one solution, the researcher may choose to sequence additional orthologous genes of appropriate evolutionary rate for the taxa in the study. However, generating large, complete data matrices can become increasingly difficult as the number of characters increases. A few empirical studies have shown that augmenting genes even for a subset of taxa can improve branch support. However, because each study differs in the number of characters and taxa, there is still a need for additional studies that examine whether incomplete sampling designs are likely to aid at increasing deep node resolution. We target Gracillariidae, a Cretaceous-age (~100 Ma) group of leaf-mining moths to test whether the strategy of adding genes for a subset of taxa can improve branch support for deep nodes. We initially sequenced ten genes (8,418 bp) for 57 taxa that represent the major lineages of Gracillariidae plus outgroups. After finding that many deep divergences remained weakly supported, we sequenced eleven additional genes (6,375 bp) for a 27-taxon subset. We then compared results from different data sets to assess whether one sampling design can be favored over another. The concatenated data set comprising all genes and all taxa and three other data sets of different taxon and gene sub-sampling design were analyzed with maximum likelihood. Each data set was subject to five different models and partitioning schemes of non-synonymous and synonymous changes. Statistical significance of non-monophyly was examined with the Approximately Unbiased (AU) test.ResultsPartial augmentation of genes led to high support for deep divergences, especially when non-synonymous changes were analyzed alone. Increasing the number of taxa without an increase in number of characters led to lower bootstrap support; increasing the number of characters without increasing the number of taxa generally increased bootstrap support. More than three-quarters of nodes were supported with bootstrap values greater than 80% when all taxa and genes were combined. Gracillariidae, Lithocolletinae + Leucanthiza, and Acrocercops and Parectopa groups were strongly supported in nearly every analysis. Gracillaria group was well supported in some analyses, but less so in others. We find strong evidence for the exclusion of Douglasiidae from Gracillarioidea sensu Davis and Robinson (1998). Our results strongly support the monophyly of a G.B.R.Y. clade, a group comprised of Gracillariidae + Bucculatricidae + Roeslerstammiidae + Yponomeutidae, when analyzed with non-synonymous changes only, but this group was frequently split when synonymous and non-synonymous substitutions were analyzed together.Conclusions1) Partially or fully augmenting a data set with more characters increased bootstrap support for particular deep nodes, and this increase was dramatic when non-synonymous changes were analyzed alone. Thus, the addition of sites that have low levels of saturation and compositional heterogeneity can greatly improve results. 2) Gracillarioidea, as defined by Davis and Robinson (1998), clearly do not include Douglasiidae, and changes to current classification will be required. 3) Gracillariidae were monophyletic in all analyses conducted, and nearly all species can be placed into one of six strongly supported clades though relationships among these remain unclear. 4) The difficulty in determining the phylogenetic placement of Bucculatricidae is probably attributable to compositional heterogeneity at the third codon position. From our tests for compositional heterogeneity and strong bootstrap values obtained when synonymous changes are excluded, we tentatively conclude that Bucculatricidae is closely related to Gracillariidae + Roeslerstammiidae + Yponomeutidae.

A molecular phylogeny for the oldest (nonditrysian) lineages of extant Lepidoptera, with implications for classification, comparative morphology and life-history evolution

Within the insect order Lepidoptera (moths and butterflies), the so‐called nonditrysian superfamilies are mostly species‐poor but highly divergent, offering numerous synapomorphies and strong morphological evidence for deep divergences. Uncertainties remain, however, and tests of the widely accepted morphological framework using other evidence are desirable. The goal of this paper is to test previous hypotheses of nonditrysian phylogeny against a data set consisting of 61 nonditrysian species plus 20 representative Ditrysia and eight outgroups (Trichoptera), nearly all sequenced for 19 nuclear genes (up to 14 700 bp total). We compare our results in detail with those from previous studies of nonditrysians, and review the morphological evidence for and against each grouping The major conclusions are as follows. (i) There is very strong support for Lepidoptera minus Micropterigidae and Agathiphagidae, here termed Angiospermivora, but no definitive resolution of the position of Agathiphagidae, although support is strongest for alliance with Micropterigidae, consistent with another recent molecular study. (ii) There is very strong support for Glossata, which excludes Heterobathmiidae, but weak support for relationships among major homoneurous clades. Eriocraniidae diverge first, corroborating the morphological clade Coelolepida, but the morphological clades Myoglossata and Neolepidoptera are never monophyletic in the molecular trees; both are contradicted by strong support for Lophocoronoidea + Hepialoidea, the latter here including Mnesarchaeoidea syn.n. (iii) The surprising grouping of Acanthopteroctetidae + Neopseustidae, although weakly supported here, is consistent with another recent molecular study. (iv) Heteroneura is very strongly supported, as is a basal split of this clade into Nepticuloidea + Eulepidoptera. Relationships within Nepticuloidea accord closely with recent studies based on fewer genes but many more taxa. (v) Eulepidoptera are split into a very strongly supported clade consisting of Tischeriidae + Palaephatidae + Ditrysia, here termed Euheteroneura, and a moderately supported clade uniting Andesianidae with Adeloidea. (vi) Relationships within Adeloidea are strongly resolved and Tridentaformidae fam.n. is described for the heretofore problematic genus Tridentaforma Davis, which is strongly supported in an isolated position within the clade. (vii) Within Euheteroneura, the molecular evidence is conflicting with respect to the sister group to Ditrysia, but strongly supports paraphyly of Palaephatidae. We decline to change the classification, however, because of strong morphological evidence supporting palaephatid monophyly. (viii) We review the life histories and larval feeding habits of all nonditrysian families and assess the implications of our results for hypotheses about early lepidopteran phytophagy. The first host record for Neopseustidae, which needs confirmation, suggests that larvae of this family may be parasitoids.

The evolutionary origins of ritualized acoustic signals in caterpillars

Animal communication signals can be highly elaborate, and researchers have long sought explanations for their evolutionary origins. For example, how did signals such as the tail-fan display of a peacock, a firefly flash or a wolf howl evolve? Animal communication theory holds that many signals evolved from non-signalling behaviours through the process of ritualization. Empirical evidence for ritualization is limited, as it is necessary to examine living relatives with varying degrees of signal evolution within a phylogenetic framework. We examine the origins of vibratory territorial signals in caterpillars using comparative and molecular phylogenetic methods. We show that a highly ritualized vibratory signal—anal scraping—originated from a locomotory behaviour—walking. Furthermore, comparative behavioural analysis supports the hypothesis that ritualized vibratory signals derive from physical fighting behaviours. Thus, contestants signal their opponents to avoid the cost of fighting. Our study provides experimental evidence for the origins of a complex communication signal, through the process of ritualization.

DNA barcoding reveals a largely unknown fauna of Gracillariidae leaf-mining moths in the Neotropics.

Higher taxa often show increasing species richness towards tropical low latitudes, a pattern known as the latitudinal biodiversity gradient (LBG). A rare reverse LBG (with greater richness towards temperate high latitudes) is exhibited by Gracillariidae leaf‐mining moths, in which most described species occur in northern temperate areas. We carried out the first assessment of gracillariid species diversity in two Neotropical regions to test whether the relatively low tropical species diversity of this family is genuine or caused by insufficient sampling and a strong taxonomic impediment. Field surveys in six French Guianan and one Ecuadorian site produced 516 gracillariid specimens that were DNA barcoded to facilitate identification and to match larvae inside leaf mines with adults. Species delineation from sequence data was approximated using Automatic Barcode Gap Discovery and Refined Single Linkage Analysis through the Barcode Index Number system, and the proportion of described/undescribed species was estimated after comparison with types of 83% of described species. Locally, alpha‐diversity far exceeds that of any known temperate fauna, with as many as 108 candidate species (59.3% as singletons) collected at one site, and with an estimated species richness lower bound of 240 species. Strikingly, at least 85% of the species collected as adults were found to be undescribed. Our sampling represents the most thorough survey of gracillariid species diversity in the Neotropics to date and the results from both our molecular and morphological analyses indicate that the current reverse LBG seen in this group is an artefact of insufficient sampling and a strong description deficit in the Neotropics.