Jesse W. Breinholt

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.

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.

Using phylogenetically-informed annotation (PIA) to search for light-interacting genes in transcriptomes from non-model organisms

BackgroundTools for high throughput sequencing and de novo assembly make the analysis of transcriptomes (i.e. the suite of genes expressed in a tissue) feasible for almost any organism. Yet a challenge for biologists is that it can be difficult to assign identities to gene sequences, especially from non-model organisms. Phylogenetic analyses are one useful method for assigning identities to these sequences, but such methods tend to be time-consuming because of the need to re-calculate trees for every gene of interest and each time a new data set is analyzed. In response, we employed existing tools for phylogenetic analysis to produce a computationally efficient, tree-based approach for annotating transcriptomes or new genomes that we term Phylogenetically-Informed Annotation (PIA), which places uncharacterized genes into pre-calculated phylogenies of gene families.ResultsWe generated maximum likelihood trees for 109 genes from a Light Interaction Toolkit (LIT), a collection of genes that underlie the function or development of light-interacting structures in metazoans. To do so, we searched protein sequences predicted from 29 fully-sequenced genomes and built trees using tools for phylogenetic analysis in the Osiris package of Galaxy (an open-source workflow management system). Next, to rapidly annotate transcriptomes from organisms that lack sequenced genomes, we repurposed a maximum likelihood-based Evolutionary Placement Algorithm (implemented in RAxML) to place sequences of potential LIT genes on to our pre-calculated gene trees. Finally, we implemented PIA in Galaxy and used it to search for LIT genes in 28 newly-sequenced transcriptomes from the light-interacting tissues of a range of cephalopod mollusks, arthropods, and cubozoan cnidarians. Our new trees for LIT genes are available on the Bitbucket public repository (http://bitbucket.org/osiris_phylogenetics/pia/) and we demonstrate PIA on a publicly-accessible web server (http://galaxy-dev.cnsi.ucsb.edu/pia/).ConclusionsOur new trees for LIT genes will be a valuable resource for researchers studying the evolution of eyes or other light-interacting structures. We also introduce PIA, a high throughput method for using phylogenetic relationships to identify LIT genes in transcriptomes from non-model organisms. With simple modifications, our methods may be used to search for different sets of genes or to annotate data sets from taxa outside of Metazoa.

Moth tails divert bat attack: Evolution of acoustic deflection

Significance Bats and moths have been engaged in acoustic warfare for more than 60 million y. Yet almost half of moth species lack bat-detecting ears and still face intense bat predation. We hypothesized that the long tails of one group of seemingly defenseless moths, saturniids, are an anti-bat strategy designed to divert bat attacks. Using high-speed infrared videography, we show that the spinning hindwing tails of luna moths lure echolocating bat attacks to these nonessential appendages in over half of bat–moth interactions. Further we show that long hindwing tails have independently evolved multiple times in saturniid moths. This finding expands our knowledge of antipredator deflection strategies, the limitations of bat sonar, and the extent of a long-standing evolutionary arms race. Adaptations to divert the attacks of visually guided predators have evolved repeatedly in animals. Using high-speed infrared videography, we show that luna moths (Actias luna) generate an acoustic diversion with spinning hindwing tails to deflect echolocating bat attacks away from their body and toward these nonessential appendages. We pit luna moths against big brown bats (Eptesicus fuscus) and demonstrate a survival advantage of ∼47% for moths with tails versus those that had their tails removed. The benefit of hindwing tails is equivalent to the advantage conferred to moths by bat-detecting ears. Moth tails lured bat attacks to these wing regions during 55% of interactions between bats and intact luna moths. We analyzed flight kinematics of moths with and without hindwing tails and suggest that tails have a minimal role in flight performance. Using a robust phylogeny, we find that long spatulate tails have independently evolved four times in saturniid moths, further supporting the selective advantage of this anti-bat strategy. Diversionary tactics are perhaps more common than appreciated in predator–prey interactions. Our finding suggests that focusing on the sensory ecologies of key predators will reveal such countermeasures in prey.

Underground evolution: New roots for the old tree of lumbricid earthworms

Earthworms belonging to the family Lumbricidae are extremely abundant in terrestrial temperate regions. They affect soil properties and nutrient cycling, thus shaping plant community composition and aboveground food webs. Some lumbricids are also model organisms in ecology and toxicology. Despite the intense research efforts dedicated to lumbricids over the last 130years, the evolutionary relationships and taxonomic classification of these organisms are still subject to great debate. Resolution of their systematics is hampered by the structural simplicity of the earthworm body plan and the existence of cryptic species. We sampled 160 earthworm specimens belonging to 84 lumbricid species (28 genera) and 22 Lumbricoidea outgroups, sequenced two nuclear genes, four mitochondrial genes and seven mitochondrial tRNAs and examined 22 morphological characters. We then applied a combination of phylogenetic methods to generate the first robust genus-level phylogeny of the Lumbricidae. Our results show that the current Lumbricidae classification and the underlying hypotheses of character evolution must be revised. Our chronogram suggests that lumbricids emerged in the Lower Cretaceous in the holarctic region and that their diversification has been driven by tectonic processes (e.g. Laurasia split) and geographical isolation. Our chronogram and character reconstruction analysis reveal that spermathecae number does not follow a gradual pattern of reduction and that parthenogenesis arose from sexual relatives multiple times in the group; the same analysis also indicates that both epigeic and anecic earthworms evolved from endogeic ancestors. These findings emphasize the strong and multiple changes to which morphological and ecological characters are subjected, challenging the hypothesis of character stasis in Lumbricidae.

Resolving relationships among the megadiverse butterflies and moths with a novel pipeline for anchored phylogenomics

Abstract. The advent of next‐generation sequencing technology has allowed for thecollection of large portions of the genome for phylogenetic analysis. Hybrid enrichment and transcriptomics are two techniques that leverage next‐generation sequencing and have shown much promise. However, methods for processing hybrid enrichment data are still limited. We developed a pipeline for anchored hybrid enrichment (AHE) read assembly, orthology determination, contamination screening, and data processing for sequences flanking the target “probe” region. We apply this approach to study the phylogeny of butterflies and moths (Lepidoptera), a megadiverse group of more than 157,000 described species with poorly understood deep‐level phylogenetic relationships. We introduce a new, 855 locus AHE kit for Lepidoptera phylogenetics and compare resulting trees to those from transcriptomes. The enrichment kit was designed from existing genomes, transcriptomes, and expressed sequence tags and was used to capture sequence data from 54 species from 23 lepidopteran families. Phylogenies estimated from AHE data were largely congruent with trees generated from transcriptomes, with strong support for relationships at all but the deepest taxonomic levels. We combine AHE and transcriptomic data to generate a new Lepidoptera phylogeny, representing 76 exemplar species in 42 families. The tree provides robust support for many relationships, including those among the seven butterfly families. The addition of AHE data to an existing transcriptomic dataset lowers node support along the Lepidoptera backbone, but firmly places taxa with AHE data on the phylogeny. Combining taxa sequenced for AHE with existing transcriptomes and genomes resulted in a tree with strong support for (Calliduloidea + Gelechioidea + Thyridoidea) + (Papilionoidea + Pyraloidea + Macroheterocera). To examine the efficacy of AHE at a shallow taxonomic level, phylogenetic analyses were also conducted on a sister group representing a more recent divergence, the Saturniidae and Sphingidae. These analyses utilized sequences from the probe region and data flanking it, nearly doubled the size of the dataset; resulting trees supported new phylogenetics relationships, especially within the Saturniidae and Sphingidae (e.g., Hemarina derived in the latter). We hope that our data processing pipeline, hybrid enrichment gene set, and approach of combining AHE data with transcriptomes will be useful for the broader systematics community.

A molecular phylogeny and revised higher-level classification for the leaf-mining moth family Gracillariidae and its implications for larval host-use evolution

Gracillariidae are one of the most diverse families of internally feeding insects, and many species are economically important. Study of this family has been hampered by lack of a robust and comprehensive phylogeny. In the present paper, we sequenced up to 22 genes in 96 gracillariid species, representing all previously recognized subfamilies and genus groups, plus 20 outgroups representing other families and superfamilies. Following objective identification and removal of two rogue taxa, two datasets were constructed: dataset 1, which included 12 loci totalling 9927 bp for 94 taxa, and dataset 2, which supplemented dataset 1 with 10 additional loci for 10 taxa, for a total of 22 loci and 16 167 bp. Maximum likelihood analyses strongly supported the monophyly of Gracillariidae and most previously recognized subfamilies and genus groups. On this basis, we propose a new classification consisting of eight subfamilies, four of which are newly recognized or resurrected: Acrocercopinae Kawahara & Ohshima subfam. n.; Gracillariinae Stainton; Lithocolletinae Stainton; Marmarinae Kawahara & Ohshima subfam. n.; Oecophyllembiinae Réal & Balachowsky; Parornichinae Kawahara & Ohshima subfam. n.; Ornixolinae Kuznetzov & Baryshnikova stat. rev.; and Phyllocnistinae Zeller. The subfamily Gracillariinae is restricted to the monophyletic group comprising Gracillaria Haworth and closely related genera. We also formally transfer Acrocercops scriptulata Meyrick to Ornixolinae and use the name Diphtheroptila Vári, creating Diphtheroptila scriptulata comb. n. An exploratory mapping of larval host‐use traits on the phylogeny shows strong conservation of modes of leaf mining but much higher lability of associations with host plant orders and families, suggesting that host shifts could play a significant role in gracillariid diversification.

Body size affects the evolution of eyespots in caterpillars

Significance Eyespots are a widespread form of antipredator defense that have long captured the imagination of evolutionary biologists, geneticists, psychologists, and artists. These markings are particularly common within Lepidoptera, and eyespots on caterpillars have been shown to deter avian predators; however, why eyespots have evolved in particular caterpillar species, and why they are not even more widespread, remain unclear. Here we answer this question using a powerful three-pronged approach. Our phylogenetically controlled analysis of hawkmoths demonstrates that eyespots are typically restricted to large caterpillars, and our field and laboratory experiments provide an explanation for this. Eyespots are costly to small caterpillars because they enhance detectability without providing a protective advantage, but they are beneficial to large caterpillars because they deter predators. Many caterpillars have conspicuous eye-like markings, called eyespots. Despite recent work demonstrating the efficacy of eyespots in deterring predator attack, a fundamental question remains: Given their protective benefits, why have eyespots not evolved in more caterpillars? Using a phylogenetically controlled analysis of hawkmoth caterpillars, we show that eyespots are associated with large body size. This relationship could arise because (i) large prey are innately conspicuous; (ii) large prey are more profitable, and thus face stronger selection to evolve such defenses; and/or (iii) eyespots are more effective on large-bodied prey. To evaluate these hypotheses, we exposed small and large caterpillar models with and without eyespots in a 2 × 2 factorial design to avian predators in the field. Overall, eyespots increased prey mortality, but the effect was particularly marked in small prey, and eyespots decreased mortality of large prey in some microhabitats. We then exposed artificial prey to naïve domestic chicks in a laboratory setting following a 2 × 3 design (small or large size × no, small, or large eyespots). Predators attacked small prey with eyespots more quickly, but were more wary of large caterpillars with large eyespots than those without eyespots or with small eyespots. Taken together, these data suggest that eyespots are effective deterrents only when both prey and eyespots are large, and that innate aversion toward eyespots is conditional. We conclude that the distribution of eyespots in nature likely results from selection against eyespots in small caterpillars and selection for eyespots in large caterpillars (at least in some microhabitats).

Evolution of Manduca sexta hornworms and relatives: Biogeographical analysis reveals an ancestral diversification in Central America

The hawkmoth genus Manduca is a diverse group of very large, conspicuous moths that has served as an important model across many biological disciplines. Two species in particular, the tobacco hornworm (Manduca sexta) and the tomato hornworm (Manduca quinquemaculatus) have been researched extensively. Studies across biological fields have referred to these two species as being closely related or even sister species, but the extent to which these two model organisms are related remains largely unknown. We conducted a comprehensive multi-gene phylogenetic analysis of Manduca, based on both an ML and Bayesian framework, which resulted in a monophyletic Manduca but only when two other genera, Dolba and Euryglottis are included. We tentatively conclude that the sister group to Manduca sexta comprises the Caribbean M. afflicta and M. johanni, and the sister lineage to this clade includes M. quinquemaculatus and the Hawaiian M. blackburni. Thus, M. sexta and M. quinquemaculatus are closely related, but are not sister species. Biogeographical analyses reveal an ancestral center of diversification in Central America, and Manduca appears to have subsequently colonized North and South America. Our phylogeny provides an important foundation for comparative studies of two model organisms and their relatives.

Molecular phylogenetics of the burrowing crayfish genus Fallicambarus (Decapoda: Cambaridae)

Ainscough, B.J., Breinholt, J.W., Robison, H.W. & Crandall, K.A. (2013). Molecular phylogenetics of the burrowing crayfish genus Fallicambarus (Decapoda: Cambaridae). —Zoologica Scripta, 42, 306–316.