Toward reconstructing the evolution of advanced moths and butterflies (Lepidoptera: Ditrysia): an initial molecular study

Background

In the mega-diverse insect order Lepidoptera (butterflies and moths; 165,000 described species), deeper relationships are little understood within the clade Ditrysia, to which 98% of the species belong. To begin addressing this problem, we tested the ability of five protein-coding nuclear genes (6.7 kb total), and character subsets therein, to resolve relationships among 123 species representing 27 (of 33) superfamilies and 55 (of 100) families of Ditrysia under maximum likelihood analysis.

Results

Our trees show broad concordance with previous morphological hypotheses of ditrysian phylogeny, although most relationships among superfamilies are weakly supported. There are also notable surprises, such as a consistently closer relationship of Pyraloidea than of butterflies to most Macrolepidoptera. Monophyly is significantly rejected by one or more character sets for the putative clades Macrolepidoptera as currently defined (P < 0.05) and Macrolepidoptera excluding Noctuoidea and Bombycoidea sensu lato (P ≤ 0.005), and nearly so for the superfamily Drepanoidea as currently defined (P < 0.08). Superfamilies are typically recovered or nearly so, but usually without strong support. Relationships within superfamilies and families, however, are often robustly resolved. We provide some of the first strong molecular evidence on deeper splits within Pyraloidea, Tortricoidea, Geometroidea, Noctuoidea and others. Separate analyses of mostly synonymous versus non-synonymous character sets revealed notable differences (though not strong conflict), including a marked influence of compositional heterogeneity on apparent signal in the third codon position (nt3). As available model partitioning methods cannot correct for this variation, we assessed overall phylogeny resolution through separate examination of trees from each character set. Exploration of "tree space" with GARLI, using grid computing, showed that hundreds of searches are typically needed to find the best-feasible phylogeny estimate for these data.

Conclusion

Our results (a) corroborate the broad outlines of the current working phylogenetic hypothesis for Ditrysia, (b) demonstrate that some prominent features of that hypothesis, including the position of the butterflies, need revision, and (c) resolve the majority of family and subfamily relationships within superfamilies as thus far sampled. Much further gene and taxon sampling will be needed, however, to strongly resolve individual deeper nodes.     

Evolutionary dynamics of rDNA clusters on chromosomes of moths and butterflies (Lepidoptera)

We examined chromosomal distribution of major ribosomal DNAs (rDNAs), clustered in the nucleolar organizer regions (NORs), in 18 species of moths and butterflies using fluorescence in situ hybridization with a codling moth (Cydia pomonella) 18S rDNA probe. Most species showed one or two rDNA clusters in their haploid karyotype but exceptions with 4–11 clusters also occurred. Our results in a compilation with previous data revealed dynamic evolution of rDNA distribution in Lepidoptera except Noctuoidea, which showed a highly uniform rDNA pattern. In karyotypes with one NOR, interstitial location of rDNA prevailed, whereas two-NOR karyotypes showed mostly terminally located rDNA clusters. A possible origin of the single interstitial NOR by fusion between two NOR-chromosomes with terminal rDNA clusters lacks support in available data. In some species, spreading of rDNA to new, mostly terminal chromosome regions was found. The multiplication of rDNA clusters without alteration of chromosome numbers rules out chromosome fissions as a major mechanism of rDNA expansion. Based on rDNA dynamics in Lepidoptera and considering the role of ordered nuclear architecture in karyotype evolution, we propose ectopic recombination, i.e., homologous recombination between repetitive sequences of non-homologous chromosomes, as a primary motive force in rDNA repatterning.     

Phylogenetic study of heliconiine butterflies based on morphology and restriction analysis of ribosomal RNA genes

Relationships of ten heliconiine butterflies (genera Dryas and Heliconius , family Nymphalidae) were elucidated by phylogenetic analysis of characters based on ribosomal DNA restriction site variation and morphology. Agraulis vanillae , also a heliconiine, was used as the outgroup species. Although neither the morphological nor the molecular data unambiguously resolve relationships among the heliconiines, a combined analysis of both data sets results in a tree that is similar to traditional systematic arrangements and previous views of radiation in the group. Both pupal-mating and nonpupal-mating species group as clades in the combined analysis. However, the restriction site data alone do not support the monophyly of the pupal-mating clade, and the morphological data alone do not support the monophyly of the non-pupal-mating clade. Furthermore, relationships of H. melpomene, H. cydno and the silvaniform species depart from traditional arrangements based on morphology and reproductive compatibility experiments. All trees support the independent evolution of similar wing patterns of species previously suggested to be members of mimicry complexes. Several mimicry complexes appear to have a member in each of the two major monophyletic groups (pupal-mating and non-pupal-mating clades).     

Interspecific variation in mitochondrial serine transfer RNA (UCN) in Euptychiina butterflies (Lepidoptera: Satyrinae): structure and alignment

The nucleotide variation and structural patterns of mitochondrial RNA molecule have been proposed as useful tools in molecular systematics; however, their usefulness is always subject to a proper assessment of homology in the sequence alignment. The present study describes the secondary structure of mitochondrial tRNA for the amino acid serine (UCN) on 13 Euptychiina species and the evaluation of its potential use for evolutionary studies in this group of butterflies. The secondary structure of tRNAs showed variation among the included species except between Hermeuptychia sp1 and sp2. Variation was concentrated in the ribotimidina-pseudouridine-cystosine (TψC), dihydrouridine (DHU) and variable loops and in the DHU and TψC arms. These results suggest this region as a potential marker useful for taxonomic differentiation of species in this group and also confirm the importance of including information from the secondary structure of tRNA to optimize the alignments.     

Phylogenetic relationships in brown argus butterflies (Lepidoptera: Lycaenidae: Aricia) from northwestern Europe

Lycaenid butterflies of the Aricia agestisartaxerxes complex pose an unresolved taxonomic and conservation problem in northwestern Europe. Two key issues require resolution: (i) how many species of Aricia occur in northwestern Europe and what are their distributions?; (ii) how is the morphological variation observed in northwestern Europe best explained? We investigated phylogenetic relationships and phylogeographic patterns in this species group using mitochondrial and nuclear markers in comparison with morphological variation. A 325 bp fragment of the mitochondrial cytochromeb gene was sequenced from 179 individuals representing 18 populations from the UK and Scandinavia. Seventeen enzymecoding loci were analysed from 538 individuals from the same populations. Highly congruent phylogenies between mitochondrial and allozyme markers demonstrate that the sample is composed of two closely related species, A. agestis and A. artaxerxes. Both marker types also suggest that Scottish and northern Scandinavian A. artaxerxes populations are conspecific, and consequently do not support the endemic status of A. artaxerxes in the UK. The subspecies division of British populations of A. artaxerxes is also not supported by phylogenetic analyses. Allozyme and mitochondrial analyses cluster two populations from the Peak District, UK, differently. The former suggests that they are A. artaxerxes whilst the latter suggests they are A. agestis. Further research is required to find the reason for this disagreement, which could be associated with the different dynamics of nuclear and mitochondrial genes across a hybrid zone between the two species. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75 , 27–37.     

Intraspecific variation in wing and pupal melanization in copper butterflies (Lepidoptera: Lycaenidae)

Melanin is a widespread pigment causing variation in skin darkness, with darker phenotypes typically reaching higher equilibrium temperatures than lighter ones. Therefore, selection is expected to favour darker phenotypes in colder environments. In the present study, we show intraspecific variation in pupal (and wing) melanization along an altitudinal gradient in two species of copper butterflies. Both, pupal and wing melanization increased with increasing altitude. Consistent with the thermal melanism hypothesis, darker (high-altitude) pupae reached higher equilibrium temperatures than paler (low-altitude) ones. However, as temperature differences were rather small despite pronounced differences in melanization, we cannot rule out that factors (e.g. ultraviolet protection, disease resistance) other than temperature comprise the principal selective agents. Mechanistically, variation in melanization might be related to variation in hormone titres, as demonstrated by low-altitude pupae showing higher ecdysteroid and juvenile hormone titres compared to high-altitude ones. Furthermore, we report sex differences in wing melanization, with males being darker than females, which is potentially related to a higher flight activity of males.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 301–312.     

Chromosome number variation within Philaethria butterflies (Lepidoptera: Nymphalidae,Heliconiini)

The haploid chromosome number of the South American butterfly Philaethria dido varies from 12 to 88. Eight different numbers have been found in this species complex. The related Ph. pygmalion and Ph. wernickei usually show only n=29, a very frequent number in the Lepidoptera; numbers of n=15 and n=21 for these species need confirmation. The most common chromosome number for Ph. dido is also the highest, n=88, and is found in many parts of northern and central Brazil on the Amazon river and its tributaries, as well as adjacent parts of other countries. The other numbers were observed mainly in northern South America and along the east coast. Two very different numbers were found together in four localities. We did not find specimens with meiotic features suggesting hybridization between individuals with different chromosome numbers. The diverse numbers in Ph. dido may belong to good sibling species, distinguishable externally by very minor characters. Since Ph. dido is a very primitive species in the tribe Heliconiini, dating probably from the early Tertiary, it probably has had many opportunities to undergo divergent chromosome evolution in isolation. Its strong, high flight and broad ecological valence would then permit rapid spreading out and coexistence of different chromosome forms, which in some cases have been noted to show diverse behaviour in the field.Dedicated to Professor Hans Bauer on the occasion of his eightieth birthday     

Phylogenetic utility of the nuclear gene dopa decarboxylase in noctuoid moths (Insecta: Lepidoptera: noctuoidea)

Phylogenetic utility for the nuclear gene encoding dopa decarboxylase (DDC), little used in systematics, was recently demonstrated within the noctuid moth subfamily Heliothinae. Here we extend the test of the utility of a 709-bp DDC fragment to deeper levels, analyzing 49 species representing major groups across the superfamily Noctuoidea. Parsimony, distance, and maximum-likelihood analyses recover all or nearly all of a set of "test clades" supported by clear morphological synapomorphies, spanning a wide range of taxonomic levels. DDC also upholds a recent proposal that the Noctuidae are paraphyletic. Nt3 contributes a majority of the signal and recovers the basal split between Notodontidae and all other noctuoids, despite a plateau of nt3 divergence at this level. However, nonsynonymous changes also support groups at all levels, and in contrast to nt3, amino acid divergence shows no plateau. The utility of DDC promises to extend back to the early Tertiary and Cretaceous, a time span for which few suitable genes have been identified.     

Widespread decoupling of mtDNA variation and species integrity in Grammia tiger moths (Lepidoptera: Noctuidae)

Abstract. We investigate the diversity of the North American tiger moth genus Grammia Rambur (Lepidoptera: Noctuidae) by comparing mitochondrial DNA (mtDNA) ‘barcode’ fragments of cytochrome oxidase I with non‐molecular characters such as morphology, ecology, behaviour and distribution. Mitochondrial DNA genealogy is strikingly at odds with morpho‐species taxonomy for most of the 28 sampled species, as haplotypic polyphyly not only is taxonomically widespread, but involves multiple shared haplotypes among two to four species. Morpho‐ecological traits show that those species sharing haplotypes are often not closely related. Furthermore, high mtDNA divergences occur within species. Haplotypic variation is highly discordant with species taxonomy, but variation at a continental scale reveals significant geographic structuring of haplogroups, transcending morpho‐species boundaries. A nested clade analysis and comparison of non‐molecular with mtDNA data indicate that most discordance between mtDNA and taxonomy in Grammia is explained best by taxonomically and geographically widespread ongoing hybridization events resulting in mtDNA introgression. We hypothesize that broad areas of sympatry, interspecifically compatible genitalic structure, and species overlap in pheromone components facilitate hybridization, with disparate interspecies abundances promoting mitochondrial introgression. The molecular evolution of Grammia challenges the view that interspecific gene exchange occurs rarely and is restricted to recently diverged species. These results show the value of mtDNA in detecting cryptic hybridization, while highlighting the inherent dangers of drawing taxonomic conclusions based solely on mtDNA.     

基于线粒体ND1和16S rRNA基因序列探讨中国蝴蝶12科的系统发育关系(鳞翅目: 双孔次亚目: 蝶类)

对中国12科共32种代表蝶类的ND1基因和16S rRNA 基因进行了序列测定（包括新测30种ND1基因和9种16S rRNA基因）和比较分析, 同时采用邻接法、最大似然法和贝叶斯法构建了12科蝶类的系统发育树, 探讨了其高级分类群的系统发育关系。序列分析的结果显示: 经比对处理后的两个基因总长度为869 bp, 其中保守位点373个, 可变位点496个, 简约信息位点375个; A+T的平均含量为80.2％, 明显高于C+G的平均含量19.8％。分子系统树表明: 蛱蝶科不是单系群; 珍蝶类、斑蝶类和喙蝶类位于蛱蝶科内; 粉蝶科和凤蝶科具有共同祖先。据此建议: 绢蝶科应归入凤蝶科; 蚬蝶科归入灰蝶科; 珍蝶类、斑蝶类和喙蝶类作为蛱蝶科中的亚科, 眼蝶类从蛱蝶科中分离出来独立成科。另外, 环蝶类的系统分类地位还有待于进一步研究。     

Phylogeny of the superfamily Gelechioidea (Lepidoptera: Ditrysia): an exemplar approach

Phylogenetic relationships within the megadiverse lepidopteran superfamily Gelechioidea have been poorly understood and consequently the family level classification has been problematic. An analysis of phylogeny using 193 characters, including 241 informative character states, derived from larval, pupal and adult morphology and larval ecology, was performed to resolve the phylogeny of the Gelechioidea. 143 species representing the diversity of the putative Gelechioidea were included, supplemented with 13 species representing 11 other Ditrysian families. The monophyly of the Gelechioidea was supported, although only with homoplastic characters. The putative position of the Gelechioidea as the sister group of the Apoditrysia was not supported, since the Gelechioidea was nested within this clade. The Gelechioidea was divided into two main lineages: (1) the gelechiid lineage constituting Deoclonidae, Syringopainae, a re‐composed Coleophoridae (including Coelopoetinae and Batrachedrinae as paraphyletic with Stathmopodinae, and Coleophorinae nested within it), Momphidae, Pterolonchidae, Scythrididae, Cosmopterigidae, and Gelechiidae, and (2) the oecophorid lineage constituting the “autostichid” family assemblage (including taxa formerly assigned to Autostichinae, Holcopogoninae, Symmocinae, Glyphidoceridae and Lecithoceridae), Xyloryctidae s.l. (including a paraphyletic Xyloryctidae of authors, some oecophorids of authors, Deuterogoniinae and Blastobasinae), Oecophoridae s.s., Amphisbatidae s.s., Carcinidae, Stenomati[n/d]ae, Chimabachidae and Elachistidae (including Depressariinae s.s., Telechrysis, Ethmiinae, Hypertrophinae s.l., miscellaneous “amphisbatids”sensu authors, Aeolanthinae, Parametriotinae, Agonoxeninae and Elachistinae). Detritivory/fungivory may have evolved only twice within Gelechioidea, though the evolution of larval food substrate use frequently reverses. To avoid an unnecessary further proliferation of names, it is recommended that no further family group names are introduced within the Gelechioidea, unless based on a rigorous analysis of inter‐relationships.     

Revised systematics and higher classification of pierid butterflies (Lepidoptera: Pieridae) based on molecular data

The butterfly family Pieridae comprises approximately 1000 described species placed in 85 genera, but the higher classification has not yet been settled. We used molecular data from eight gene regions (one mitochondrial and seven nuclear protein‐coding genes) comprising a total of ~6700 bp from 96 taxa to infer a well‐supported phylogenetic hypothesis for the family. Based on this hypothesis, we revise the higher classification for all pierid genera. We resurrect the tribe Teracolini stat. rev. in the subfamily Pierinae to include the genera Teracolus, Pinacopteryx, Gideona, Ixias, Eronia, Colotis and most likely Calopieris. We transfer Hebomoia to the tribe Anthocharidini and assign the previously unplaced genera Belenois and Dixeia to the subtribe Aporiina. Three lineages near the base of Pierinae (Leptosia, Elodina and Nepheronia + Pareronia) remain unplaced. For each of these, we describe and delineate new tribes: Elodinini Braby tribus nova, Leptosiaini Braby tribus nova and Nepheroniini Braby tribus nova. The proposed higher classification is based on well‐supported monophyletic groups and is likely to remain stable even with the addition of more data.     

Linnaeus's butterflies (Lepidoptera: Papilionoidea and Hesperioidea)

The names of all the species of butterflies described by Linnaeus under "Papilio" are researched. Of the 305 names treated, 243 (c. 80%) are currently valid as specific (241) or subspecific (2), 29 are junior synonyms, 14 are invalid (one of these applying to a fake), and for 13 the identity is unknown or uncertain. Six species of moths misidentified by Linnaeus as butterflies are cited in the study, but details are not included. One hundred and fifty-two lectotypes have been designated, representing about 56% of the sum of the valid names and junior synonyms. Of these, 99 were selected from specimens in the Linnean Society of London, 52 from Queen Ludovica Ulrica's collection, Uppsala, and one lectotype is a Petiver specimen from the collection of Sir Hans Sloane. Linnaeus described at least five species, possibly eight, from the literature alone. All Linnaean material examined is documented, as are 'subsequent' specimens that are associated with Linnaean material. Synonymy and homonymy are discussed and presented, as are the identities of type localities. Care has been taken to achieve a practical balance between Linnaean and current species identities. Linnaean material studied included specimens from The Linnean Society of London, Museum Ludovicae Ulricae (Uppsala University), the Clerck and De Geer collections in the Naturhistoriska Riksmuseet, Stockholm, and the collection of James Petiver, now part of the collection of Sir Hans Sloane housed in The Natural History Museum, London.     

The fossil record and taphonomy of butterflies and moths (Insecta,Lepidoptera): implications for evolutionary diversity and divergence-time estimates

Background

It is conventionally accepted that the lepidopteran fossil record is significantly incomplete when compared to the fossil records of other, very diverse, extant insect orders. Such an assumption, however, has been based on cumulative diversity data rather than using alternative statistical approaches from actual specimen counts.

Results

We reviewed documented specimens of the lepidopteran fossil record, currently consisting of 4,593 known specimens that are comprised of 4,262 body fossils and 331 trace fossils. The temporal distribution of the lepidopteran fossil record shows significant bias towards the late Paleocene to middle Eocene time interval. Lepidopteran fossils also record major shifts in preservational style and number of represented localities at the Mesozoic stage and Cenozoic epoch level of temporal resolution. Only 985 of the total known fossil specimens (21.4%) were assigned to 23 of the 40 extant lepidopteran superfamilies. Absolute numbers and proportions of preservation types for identified fossils varied significantly across superfamilies. The secular increase of lepidopteran family-level diversity through geologic time significantly deviates from the general pattern of other hyperdiverse, ordinal-level lineages.

Conclusion

Our statistical analyses of the lepidopteran fossil record show extreme biases in preservation type, age, and taxonomic composition. We highlight the scarcity of identified lepidopteran fossils and provide a correspondence between the latest lepidopteran divergence-time estimates and relevant fossil occurrences at the superfamily level. These findings provide caution in interpreting the lepidopteran fossil record through the modeling of evolutionary diversification and in determination of divergence time estimates.

Electronic supplementary material

The online version of this article (doi:10.1186/s12862-015-0290-8) contains supplementary material, which is available to authorized users.     

Phylogenetic relationships among genera of danaine butterflies (Lepidoptera: Nymphalidae) as implied by morphology and DNA sequences

Cladistic relationships among genera and subtribes of Danaini (the milkweed butterflies) were inferred by analysis of data combined from five sources: morphology of adults and immature stages, and DNA sequences from three gene regions. The results corroborate and greatly increase support for prior hypotheses based on morphology alone. A new index summarizing incongruence among data partitions, the Partition Congruence Proportion (PCP), is introduced. The significance of the inferred pattern of phylogenetic relationships for comparative chemical ecology of milkweed butterflies is briefly discussed.     

Molecular phylogeny of swallowtail butterflies of the tribe Papilionini (Papilionidae, Lepidoptera).

Swallowtail butterflies of the tribe Papilionini number about 225 species and are currently used as model organisms in several research areas, including genetics, chemical ecology and phylogenetics of host plant utilization and mimicry, mechanisms of speciation, and conservation. We have inferred phylogenetic relationships for a sample of 18 species of the genus Papilio (sensu lato) and five outgroup taxa by sequencing two stretches of mitochondrial DNA that correspond to segments 12886-13370 and 12083-12545 of Drosophila melanogaster mitochondrial DNA and consist of sections of the genes for the large ribosomal RNA and subunit 1 of NADH-dehydrogenase. Our data support the monophyly of Papilio and, within it, of several traditionally recognized subgroups. Species belonging to groups that utilize primarily Rutaceae as larval foodplants form two clusters, corresponding to Old World and American taxa, respectively, while two previously recognized clades-of American and South Asian-Austronesian origin-whose members were known to feed mostly on Lauraceae and Magnoliaceae, are observed to form a clade. The sister group of Papilio is found to be the South Asian genus Meandrusa, which also happens to feed on Lauraceae. The latter plant family is therefore the probable larval host of the ancestor Papilio and the shift to Rutaceae (which four-fifths of extant Papilio species use as foodplants) is more likely to have occurred only after the initial diversification of the genus.     

Phylogenetic utility and comparative morphology of the composite scale brushes in male phycitine moths (Lepidoptera, Pyralidae)

Male pyralid moths in the subfamily of Phycitinae are known to possess composite scale brush structures associated with the 8th abdominal sternite, but the histology and the structural morphology of these organs have not been adequately explored. As such, the phylogenetic utility of these structures is unknown. We examine the pre-genitalic abdominal histology of male Dioryctria reniculelloides (Pyralidae: Phycitinae) associated with the composite scale brushes, as well as structural morphology within the genus Dioryctria and two closely related genera. The composite scale brushes are composed of fused scales. The musculature associated with the base of sternum 8 shows considerable modification compared to previously described Lepidoptera. Complex glandular tissue was also found associated with the scale brush structures, suggesting secretory function. Phylogenetic utility of ultrastructure and gross morphology was examined for major Dioryctria species groups. Many characters were homoplasious, but several supported the monophyly of the genus, as well as some internal relationships. In conclusion, the combination of ultrastructural, gross morphological and histological characters can be a rich source of information for elucidating a range of evolutionary relationships within the subfamily.     

Skipping flights in Ypthima butterflies (Lepidoptera: Nymphalidae)

The skipping flight patterns of three species of Ypthima (Lepidoptera: Nymphalidae) were analyzed using high‐speed video recordings to clarify how wings move and how driving forces are produced. All three species showed a flight pattern that includes a pause that accounts for about 50% of a flap cycle when their wings completely close after each upstroke. The observed pause causes the “skipping” flight trajectory based on the clap–fling mechanism. Pause duration was correlated with upstroke wing motion, suggesting the contribution of the latter to a long pause duration. This is also supported by the temporal relationship between the wing and body motions. The aerodynamic power necessary for the pause flight was calculated for the three species.     

Genomic organization of ribosomal RNA genes in Bromus (Poaceae).

Restriction site maps of the rDNA genes of nine Bromus species are described. The rDNA repeat units ranged from 8.2 to 11.1 kbp in length. Intraspecific length variation was observed in the BamHI digestions in three of the nine species. Restriction site variation was observed mainly in the intergenic spacer (IGS) but was also detected in the coding region. A unique KpnI site was present in the IGS of Bromus tectorum and Bromus sericeus (subgenus Stenobromus); in addition, B. sericeus contained an extra EcoRI site. An additional DraI site was observed in the IGS of Bromus trinii (subgenus Neobromus). A BstEII site in the IGS, common to seven of the species, was absent in B. tectorum and B. sericeus. In the coding region, a 2.1-kbp BstEII fragment was present in four subgenera represented by Bromus inermis and Bromus erectus (subgenus Festucaria), Bromus marginatus and Bromus carinatus (subgenus Ceratochloa), B. tectorum and B. sericeus (subgenus Stenobromus), and B. trinii (subgenus Neobromus); a similar fragment of only 1.1 kbp was present in Bromus mollis and Bromus arvensis (subgenus Bromus). An additional BamHI site was present in the coding region of B. erectus. Ribosomal DNA data suggested that B. mollis and B. arvensis (subgenus Bromus) are genetically isolated from the other subgenera, which showed a derived relationship. Restriction site mapping of the rDNA genes could provide useful molecular data for species identification and population and evolutionary studies in Bromus. Key words : Bromus, ribosomal DNA, restriction maps, evolutionary relationships.