Soowon Cho

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.

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.

Molecular phylogenetics of heliothine moths (Lepidoptera: Noctuidae: Heliothinae), with comments on the evolution of host range and pest status

Abstract The Heliothinae are a cosmopolitan subfamily of about 365 species that include some of the world’s most injurious crop pests. This study re‐assesses evolutionary relationships within heliothines, providing an improved phylogeny and classification to support ongoing intensive research on heliothine genomics, systematics, and biology. Our phylogeny estimate is based on two nuclear gene regions, namely elongation factor‐1α (EF‐1α; 1240 bp) and dopa decarboxylase (DDC; 687 bp), and on the barcoding region of mitochondrial cytochrome oxidase I (COI; 708 bp), providing a total of 2635 bp. These were sequenced for 71 heliothines, representing all major genera and nearly all recognized subgenera and species groups, and for 16 outgroups representing all major lineages of trifine Noctuidae. Analysis of the combined data by maximum likelihood, unweighted parsimony and Bayesian methods gave nearly identical topologies, and the individual gene trees showed only one case of potentially strong conflict. Relationships among genera and subgenera are resolved with strong bootstrap support. The earliest‐diverging lineages (c. 200 species in total) consist almost entirely of host specialists, reflecting the inferred ancestral heliothine host range under parsimony. The remaining species form a clade – the Heliothis group – that includes most of the polyphages (30% of heliothines) and all of the major pests. Many other species in the Heliothis group, however, are host specialists. Our results extend previous efforts to subdivide this large clade, and show the most notorious pest groups, the corn earworm complex (Helicoverpa) and the tobacco budworm (Heliothis virescens) group, to be closely related, joining with a small oligophagous genus in what we term the major‐pest lineage. Thus, genomic/experimental results from one model pest may extrapolate well to other pest species. The frequency of evolutionary expansion and contraction in host range appears to increase dramatically at the base of the Heliothis group, in contrast to the case for earlier‐diverging lineages. We ascribe this difference provisionally to differential evolutionary constraints arising from contrasting life‐history syndromes. Host‐specific behaviour and crypsis, coupled with low fecundity and vagility, may discourage host‐range expansion in earlier‐diverging lineages. By contrast, in the Heliothis group, the absence of host‐specific traits, coupled with high vagility and fecundity, may more readily permit expansion or contraction of the host range in response to varying ecological pressures such as host species abundance or differential competition and predation.

A molecular phylogeny for the leaf-roller moths (Lepidoptera: Tortricidae) and its implications for classification and life history evolution.

Background Tortricidae, one of the largest families of microlepidopterans, comprise about 10,000 described species worldwide, including important pests, biological control agents and experimental models. Understanding of tortricid phylogeny, the basis for a predictive classification, is currently provisional. We present the first detailed molecular estimate of relationships across the tribes and subfamilies of Tortricidae, assess its concordance with previous morphological evidence, and re-examine postulated evolutionary trends in host plant use and biogeography. Methodology/Principal Findings We sequenced up to five nuclear genes (6,633 bp) in each of 52 tortricids spanning all three subfamilies and 19 of the 22 tribes, plus up to 14 additional genes, for a total of 14,826 bp, in 29 of those taxa plus all 14 outgroup taxa. Maximum likelihood analyses yield trees that, within Tortricidae, differ little among data sets and character treatments and are nearly always strongly supported at all levels of divergence. Support for several nodes was greatly increased by the additional 14 genes sequenced in just 29 of 52 tortricids, with no evidence of phylogenetic artifacts from deliberately incomplete gene sampling. There is strong support for the monophyly of Tortricinae and of Olethreutinae, and for grouping of these to the exclusion of Chlidanotinae. Relationships among tribes are robustly resolved in Tortricinae and mostly so in Olethreutinae. Feeding habit (internal versus external) is strongly conserved on the phylogeny. Within Tortricinae, a clade characterized by eggs being deposited in large clusters, in contrast to singly or in small batches, has markedly elevated incidence of polyphagous species. The five earliest-branching tortricid lineages are all species-poor tribes with mainly southern/tropical distributions, consistent with a hypothesized Gondwanan origin for the family. Conclusions/Significance We present the first robustly supported phylogeny for Tortricidae, and a revised classification in which all of the sampled tribes are now monophyletic.

Lycorma delicatula (Hemiptera: Auchenorrhyncha: Fulgoridae: Aphaeninae) finally, but suddenly arrived in Korea

A history of name changes in two fulgorid species –Lycorma delicatula and Limois emelianovi– is reviewed. Lycorma delicatula was once mistakenly reported to occur in Korea. Now, it has suddenly become common in western Korea, creating the suspicion that it has recently arrived from China and settled in Korea. A brief morphological and biological description of L. delicatula is provided, and its original Korean name, “ggot‐mae‐mi”, is revalidated. Limois emelianovi, sometimes considered a synonym of emeljanovi, is the correct name for this species, as emeljanovi is simply another transliteration of the personal name Emelianov, Emeljanov or Emel’yanov. The name emelianovi stands correct based on the International Code of Zoological Nomenclature code 32.5.1, because there is no internal evidence of an inadvertent error, and an incorrect transliteration is not considered an inadvertent error. The cytochrome oxidase I (COI) barcoding regions of both species were sequenced and compared for future reference.

Phylogeny and feeding trait evolution of the mega-diverse Gelechioidea (Lepidoptera: Obtectomera): new insight from 19 nuclear genes

The Gelechioidea (>18 000 species), one of the largest superfamilies of Lepidoptera, are a major element of terrestrial ecosystems and include important pests and biological model species. Despite much recent progress, our understanding of the classification, phylogeny and evolution of Gelechioidea remains limited. Building on recent molecular studies of this superfamily and a recently revised family/subfamily classification, we provide an independent estimate of among‐family relationships, with little overlap in gene sample. We analysed up to five nuclear genes, totalling 6633 bp, for each of 77 gelechioids, plus up to 14 additional genes, for a total of 14 826 bp, in 45 of those taxa and all 19 outgroup taxa. Our maximum‐likelihood (ML) analyses, like those of previous authors, strongly support monophyly for most multiply‐sampled families and subfamilies, but very weakly support most relationships above the family level. Our tree looks superficially divergent from that of the most recent molecular study of gelechioids, but when the previous tree is re‐rooted to accord maximally with ours, the two phylogenies agree entirely on the deepest‐level divergences in Gelechioidea, and strongly though incompletely on among‐family relationships within the major groups. This concordance between independent studies is evidence that the groupings (or at least the unrooted branching order) are probably accurate, despite the low bootstrap values. After re‐rooting, both trees divide the families into three monophyletic groups: a ‘Gelechiid Assemblage,’ consisting of Gelechiidae and Cosmopterigidae; a ‘Scythridid Assemblage,’ consisting of Stathmopodidae, Scythrididae, Blastobasidae, Elachistidae, Momphidae, Coleophoridae and Batrachedridae; and a ‘Depressariid Assemblage,’ consisting of Autostichidae, Xyloryctidae, Lecithoceridae, Oecophoridae, Depressariidae and Lypusidae. Within the largest family, Gelechiidae, our results strongly support the pairing of Anomologinae with Gelechiinae, in accordance with a recent study of this family. Relationships among the other subfamilies, however, conflict moderately to strongly between studies, leaving the intrafamily phylogeny unsettled. Within the ‘Scythridid Assemblage,’ both trees support an ‘SSB clade’ consisting of Blastobasidae + (Scythrididae + Stathmopodidae), strongly resolved only in our results. Coleophoridae + Batrachedridae is supported, albeit weakly, in both trees, and only Momphidae differ in position between studies. Within the ‘Depressariid Assemblage,’ both trees support an ‘AXLO’ clade consisting of Autostichidae, Xyloryctidae, Lecithoceridae and Oecophoridae. The monophyly of this clade and relationships therein are supported weakly in previous results but strongly in ours. The recently re‐defined family Depressariidae is paraphyletic in our tree, but the evidence against depressariid monophyly is very weak. There is moderate support for a core group of Depressariidae consisting, among the seven subfamilies we sampled, of Depressariinae, Aeolanthinae and Hypertrophinae. We show that gelechioids have a higher total number and percentage of species that are saprophagous as larvae than any other apoditrysian superfamily, that saprophagy is concentrated primarily in the ‘AXLO clade,’ and that the ancestral gelechioid condition was probably feeding on live plants. Among the living‐plant feeders, concealed external feeding was probably the ancestral state. The multiple origins of internal feeding of various kinds, including leaf mining (otherwise almost unknown in Apoditrysia), are restricted mostly to the Scythridid and Gelechiid Assemblages. The traits that predispose or permit lineages to adopt these unusual life histories are worthy of study.

Phylogeny of Rhigonematomorpha based on the complete mitochondrial genome of Rhigonema thysanophora (Nematoda: Chromadorea)

We determined the complete mitochondrial genome sequence of Rhigonema thysanophora, the first representative of Rhigonematomorpha, and used this sequence along with 57 other nematode species for phylogenetic analyses. The R. thysanophora mtDNA is 15 015 bp and identical to all other chromadorean nematode mtDNAs published to date in that it contains 36 genes (lacking atp8) encoded in the same direction. Phylogenetic analyses of nucleotide and amino acid sequence data for the 12 protein‐coding genes recovered Rhigonematomorpha as the sister group to the heterakoid species, Ascaridia columbae (Ascaridomorpha). The organization of R. thysanophora mtDNA resembles the most common pattern for the Rhabditomorpha+Ascaridomorpha+Diplogasteromorpha clade in gene order, but with some substantial gene rearrangements. This similarity in gene order is in agreement with the sequence‐based analyses that indicate a close relationship between Rhigonematomorpha and Rhabditomorpha+Ascaridomorpha+Diplogasteromorpha. These results are consistent with certain analyses of nuclear SSU rDNA for R. thysanophora and some earlier classification systems that asserted phylogenetic affinity between Rhigonematomorpha and Ascaridomorpha, but inconsistent with morphology‐based phylogenetic hypotheses that suggested a close (taxonomic) relationship between rhigonematomorphs and oxyuridomorphs (pinworms). These observations must be tempered by noting that few rhigonematomorph species have been sequenced and included in phylogenetic analyses, and preliminary studies based on SSU rDNA suggest the group is not monophyletic. Additional mitochondrial genome sequences of rhigonematids are needed to characterize their phylogenetic relationships within Chromadorea, and to increase understanding of mitochondrial genome evolution.

Preserving and vouchering butterflies and moths for large-scale museum-based molecular research

Butterflies and moths (Lepidoptera) comprise significant portions of the world’s natural history collections, but a standardized tissue preservation protocol for molecular research is largely lacking. Lepidoptera have traditionally been spread on mounting boards to display wing patterns and colors, which are often important for species identification. Many molecular phylogenetic studies have used legs from pinned specimens as the primary source for DNA in order to preserve a morphological voucher, but the amount of available tissue is often limited. Preserving an entire specimen in a cryogenic freezer is ideal for DNA preservation, but without an easily accessible voucher it can make specimen identification, verification, and morphological work difficult. Here we present a procedure that creates accessible and easily visualized “wing vouchers” of individual Lepidoptera specimens, and preserves the remainder of the insect in a cryogenic freezer for molecular research. Wings are preserved in protective holders so that both dorsal and ventral patterns and colors can be easily viewed without further damage. Our wing vouchering system has been implemented at the University of Maryland (AToL Lep Collection) and the University of Florida (Florida Museum of Natural History, McGuire Center of Lepidoptera and Biodiversity), which are among two of the largest Lepidoptera molecular collections in the world.

A review of the genus Adoxophyes (Lepidoptera Tortricidae) in Korea, with description of A. paraorana sp. nov.

Abstract We reviewed the genus Adoxophyes (Lepidoptera: Tortricidae) from Korea and recognized three species of the genus. Among them, A. paraorana sp. nov., formerly misidentified as A. orana in Korea, is described as new to science. Adoxophyes orana, a notorious pest known in most Eurasian countries for a long time, turns out to be only recently introduced or found in Korea. Photographs of the adults and genitalia of the species are provided. Specific distinction was supported by the COI barcode study.

Korea Barcode of Life Database System (KBOL)

A major concern regarding the collection and storage of biodiversity information is the inefficiency of conventional taxonomic approaches in dealing with a large number of species. This inefficiency has increased the demand for automated, rapid, and reliable molecular identification systems and large-scale biological databases. DNA-based taxonomic approaches are now arguably a necessity in biodiversity studies. In particular, DNA barcoding using short DNA sequences provides an effective molecular tool for species identification. We constructed a large-scale database system that holds a collection of 5531 barcode sequences from 2429 Korean species. The Korea Barcode of Life database (KBOL, http://koreabarcode.org) is a web-based database system that is used for compiling a high volume of DNA barcode data and identifying unknown biological specimens. With the KBOL system, users can not only link DNA barcodes and biological information but can also undertake conservation activities, including environmental management, monitoring, and detecting significant organisms.