Morphological Characters Are Compatible with Mitogenomic Data in Resolving the Phylogeny of Nymphalid Butterflies (Lepidoptera: Papilionoidea: Nymphalidae)

Nymphalidae is the largest family of butterflies with their phylogenetic relationships not adequately approached to date. The mitochondrial genomes (mitogenomes) of 11 new nymphalid species were reported and a comparative mitogenomic analysis was conducted together with other 22 available nymphalid mitogenomes. A phylogenetic analysis of the 33 species from all 13 currently recognized nymphalid subfamilies was done based on the mitogenomic data set with three Lycaenidae species as the outgroups. The mitogenome comparison showed that the eleven new mitogenomes were similar with those of other butterflies in gene content and order. The reconstructed phylogenetic trees reveal that the nymphalids are made up of five major clades (the nymphaline, heliconiine, satyrine, danaine and libytheine clades), with sister relationship between subfamilies Cyrestinae and Biblidinae, and most likely between subfamilies Morphinae and Satyrinae. This whole mitogenome-based phylogeny is generally congruent with those of former studies based on nuclear-gene and mitogenomic analyses, but differs considerably from the result of morphological cladistic analysis, such as the basal position of Libytheinae in morpho-phylogeny is not confirmed in molecular studies. However, we found that the mitogenomic phylogeny established herein is compatible with selected morphological characters (including developmental and adult morpho-characters).     

柳紫闪蛱蝶线粒体基因组全序列及与相关蛱蝶类的比较分析(英文)

该文对柳紫闪蛱蝶Apaturailia(鳞翅目:蛱蝶科)的线粒体基因组全序列进行了测定,同时结合其它已知蛱蝶类的相应序列进行了比较分析。结果显示:柳紫闪蛱蝶的线粒体基因组(GenBankaccessionno.:JF437925)是一个15242bp的环状DNA分子,包含13个蛋白质编码基因、2个rRNA基因和22个tRNA基因。13个蛋白编码基因中,除了COI基因的起始密码子是CGA外,其余12个蛋白编码基因都具有标准的ATN起始密码子;柳紫闪蛱蝶与其它已测的10种蛱蝶在基因定位和排列顺序方面几乎相同,只是在非编码序列上存在细微的差异,其核苷酸的构成及密码子使用频率都处于鳞翅目昆虫的范围之内。22个的tRNA基因中,除了tRNASer(AGN)缺少DHU臂,其余的tRNA基因都显示为典型的三叶草结构。基因组共存在9处基因间重叠区(总长度为33bp)以及12个基因间隔区(总长为155bp,最长间隔是49bp,最短的是1bp)。在ND6和Cytb间的间隔区中还发现有(TA)23似微卫星结构。与其他蛱蝶类相似,403bp的AT富集区包含有ATAGA,ATTTA二个保守模块(一个21bp的poly-T,一个10bp的poly-A),以及二个似微卫星的重复结构((TA)10和(TA)7)。     

The complete mitochondrial genome of <Emphasis Type="Italic">Damora sagana</Emphasis> and phylogenetic analyses of the family Nymphalidae

The monotypic genus Damora (Nymphalidae, Heliconiinae) contains a single species, Damora sagana, which is widely distributed across southern China. Herein, its complete mitogenome was sequenced to further understand lepidopteran mitogenome characteristics, reconstruct the nymphalid family phylogeny, and infer the subdivision of Heliconiinae species. The circular mitogenome was 15,151 bp long, abundant in A and T, and comprised of 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and one control region with a gene arrangement typical of lepidopteran mitogenomes. ATN codons initiated all PCGs, except cytochrome c oxidase subunit 1 (COX1), which was initiated by a CGA sequence as has been observed in other lepidopterans. Three PCGs (COX1, COX2 and ND4) employed a single T termination signal, whereas others had the typical complete termination codon (TAA). All tRNA genes were folded into the typical cloverleaf structure except for tRNA-Ser (AGN). The A+T-rich region included the conserved motif ‘ATAGA’ followed by a 17 bp poly-T stretch, which was also observed in tribe Argynnini mitogenomes. A phylogenetic tree was constructed via multiple methods using the 13 PCGs data of D. sagana and other available mitogenomes of nymphalid species. All three phylogenetic trees yielded the same topology. These results were consistent with those from previous studies of most major nymphalid groups, except those regarding tribe subdivision in certain subfamilies such as Argynnini + (Acraeini + Heliconiini) for Heliconiine. Furthermore, our analyses identified that the genus Cethosia was grouped with the genus Acraea composing the tribe Acraeini with strong support.     

Molecular evolution of the wingless gene and its implications for the phylogenetic placement of the butterfly family Riodinidae (Lepidoptera: papilionoidea)

The sequence evolution of the nuclear gene wingless was investigated among 34 representatives of three lepidopteran families (Riodinidae, Lycaenidae, and Nymphalidae) and four outgroups, and its utility for inferring phylogenetic relationships among these taxa was assessed. Parsimony analysis yielded a well-resolved topology supporting the monophyly of the Riodinidae and Lycaenidae, respectively, and indicating that these two groups are sister lineages, with strong nodal support based on bootstrap and decay indices. Although wingless provides robust support for relationships within and between the riodinids and the lycaenids, it is less informative about nymphalid relationships. Wingless does not consistently recover nymphalid monophyly or traditional subfamilial relationships within the nymphalids, and nodal support for all but the most recent branches in this family is low. Much of the phylogenetic information in this data set is derived from first- and second-position substitutions. However, third positions, despite showing uncorrected pairwise divergences up to 78%, also contain consistent signal at deep nodes within the family Riodinidae and at the node defining the sister relationship between the riodinids and lycaenids. Several hypotheses about how third-position signal has been retained in deep nodes are discussed. These include among-site rate variation, identified as a significant factor by maximum likelihood analyses, and nucleotide bias, a prominent feature of third positions in this data set. Understanding the mechanisms which underlie third-position signal is a first step in applying appropriate models to accommodate the specific evolutionary processes involved in each lineage.     

The systematic position of Antirrhea and Caerois, with comments on the classification of the Nymphalidae (Lepidoptera)

Based on a Wagner tree analysis of ninety-two characters (eighty-five larval, one egg, six adult), the nymphalid butterfly genera Antirrhea and Caerois are demonstrated to be the closest relatives of the genus Morpho . Accordingly, Antirrhea and Caerois are formally transferred from the Satyrinae to the Morphinae. Without these two genera, the Morphinae ( sensu Ehrlich) is at best a paraphyletic group. During the study, fourteen nymphalid genera were treated as potential outgroups. The analysis suggests that many currently accepted higher taxa within the Nymphalidae are probably untenable: the Satyrinae and Nymphalinae ( sensu Ehrlich) are both probably polyphyletic; the Biinae ( sensu Miller) must be abandoned, being polyphyletic; the Charaxidae ( sensu Rydon), although probably monophyletic, appear to form a group subordinate to part of the 'Satyrinae'; and Apatura does not cluster with the 'Nymphalinae', but appears to form the sister-group of the 'Satyrinae' (less Antirrhea and Caerois ) plus the Charaxinae. Re-analyses of reduced data sets, in which potentially homoplasious larval head-horn and adult wing venational characters were eliminated, leaves these conclusions essentially unaltered. The authors suggest that a solution to the seemingly intractable problem posed by nymphalid higher classification can be sought by the application of cladistic analysis to a large data set gathered from all developmental stages, with special emphasis on detailed comparative larval morphology.     

Hostplants and classification: a review of nymphalid butterflies

In reviewing the hostplant associations of nymphalid butterflies, particular emphasis is placed on the intractable problem of nymphalid classification. Although offering few certain conclusions, if used in conjunction with more formal morphological characters, the data presented should contribute toward a resolution of the inter-relationships of the many widely recognized groupings within the Nymphalidae, several of which seem to be broadly characterized by typical host families. As a direct result of this analysis, the presumed association between larval hostplants and unpalatability is re-appraised.     

HOST PLANT UTILIZATION,HOST RANGE OSCILLATIONS AND DIVERSIFICATION IN NYMPHALID BUTTERFLIES: A PHYLOGENETIC INVESTIGATION

It has been suggested that phenotypic plasticity is a major factor in the diversification of life, and that variation in host range in phytophagous insects is a good model for investigating this claim. We explore the use of angiosperm plants as hosts for nymphalid butterflies, and in particular the evidence for past oscillations in host range and how they are linked to host shifts and to diversification. At the level of orders of plants, a relatively simple pattern of host use and host shifts emerges, despite the 100 million years of history of the family Nymphalidae. We review the evidence that these host shifts and the accompanying diversifications were associated with transient polyphagous stages, as suggested by the “oscillation hypothesis.” In addition, we investigate all currently polyphagous nymphalid species and demonstrate that the state of polyphagy is rare, has a weak phylogenetic signal, and a very apical distribution in the phylogeny; we argue that these are signs of its transient nature. We contrast our results with data from the bark beetles Dendroctonus, in which a more specialized host use is instead the apical state. We conclude that plasticity in host use is likely to have contributed to diversification in nymphalid butterflies.     

Differential Involvement of Hedgehog Signaling in Butterfly Wing and Eyespot Development

Butterfly eyespots may have evolved from the recruitment of pre-existent gene circuits or regulatory networks into novel locations on the wing. Gene expression data suggests one such circuit, the Hedgehog (Hh) signaling pathway and its target gene engrailed (en), was recruited from a role in patterning the anterior-posterior insect wing axis to a role patterning butterfly eyespots. However, while Junonia coenia expresses hh and en both in the posterior compartment of the wing and in eyespot centers, Bicyclus anynana lacks hh eyespot-specific expression. This suggests that Hh signaling may not be functioning in eyespot development in either species or that it functions in J. coenia but not in B. anynana. In order to test these hypotheses, we performed functional tests of Hh signaling in these species. We investigated the effects of Hh protein sequestration during the larval stage on en expression levels, and on wing size and eyespot size in adults. Hh sequestration led to significantly reduced en expression and to significantly smaller wings and eyespots in both species. But while eyespot size in B. anynana was reduced proportionately to wing size, in J. coenia, eyespots were reduced disproportionately, indicating an independent role of Hh signaling in eyespot development in J. coenia. We conclude that while Hh signaling retains a conserved role in promoting wing growth across nymphalid butterflies, it plays an additional role in eyespot development in some, but not all, lineages of nymphalid butterflies. We discuss our findings in the context of alternative evolutionary scenarios that led to the differential expression of hh and other Hh pathway signaling members across nymphalid species.     

中国蛱蝶科昆虫地理分布与区系分析

分析了中国蛱蝶科已知324种昆虫的分布与区系组成,主要由古北区、东洋区、古北一东洋区共有种类组成,共计309种,占总数的95．37％;其种类组成与澳洲区、新北区有明显差异;特有种类较少,占总数的12．65％;广布种很少,仅占1．74％。同时统计分析了324种蛱蝶在我国各省的贫富指数。     

A single origin for nymphalid butterfly eyespots followed by widespread loss of associated gene expression

Understanding how novel complex traits originate involves investigating the time of origin of the trait, as well as the origin of its underlying gene regulatory network in a broad comparative phylogenetic framework. The eyespot of nymphalid butterflies has served as an example of a novel complex trait, as multiple genes are expressed during eyespot development. Yet the origins of eyespots remain unknown. Using a dataset of more than 400 images of butterflies with a known phylogeny and gene expression data for five eyespot-associated genes from over twenty species, we tested origin hypotheses for both eyespots and eyespot-associated genes. We show that eyespots evolved once within the family Nymphalidae, approximately 90 million years ago, concurrent with expression of at least three genes associated with early eyespot development. We also show multiple losses of expression of most genes from this early three-gene cluster, without corresponding losses of eyespots. We propose that complex traits, such as eyespots, may have originated via co-option of a large pre-existing complex gene regulatory network that was subsequently streamlined of genes not required to fulfill its novel developmental function.     

斐豹蛱蝶线粒体基因组全序列的测定和分析(英文)

该研究对斐豹蛱蝶(Argyreus hyperbius)(鳞翅目:蛱蝶科)线粒体基因组全序列进行了测定和初步分析。结果表明:斐豹蛱蝶线粒体基因全序列全长为15156bp,包含13个蛋白质编码基因、22个tRNA和2个rRNA基因以及1个非编码的A+T富集区,基因排列顺序与其它鳞翅目种类一致;线粒体全序列核苷酸组成和密码子使用显示出明显的A+T偏好(80.8%)和轻微的AT偏移(AT skew,?0.019)。基因组中共存在11个2～52bp不等的基因间隔区,总长96bp;以及14个1～8bp不等的基因重叠区,总长34bp。除COI以CGA作为起始密码子外,13个蛋白质编码基因中的其余12个基因是以ATN作为起始密码子。除COI和COII基因是以单独的一个T为终止密码子,其余11个蛋白质编码基因都是以TAA结尾的。除了缺少DHU臂的tRNASer(AGN),其余的tRNA基因都显示典型的三叶草结构。tRNA(AGN)和ND1之间的基因间隔区包含一个ATACTAA结构域,这个结构域在鳞翅目中是保守的。A+T富集区没有较大的多拷贝重复序列,但是包含一些微小重复结构:ATAGA结构域下游的20bp poly-T结构,ATTTA结构域后的(AT)9重复,以及位于tRNAMet上游的5bp poly-A结构等。这项研究所揭示的斐豹蛱蝶的线粒体基因组特征,不仅为认识蛱蝶科的遗传多样性贡献数据,而且对于该物种的保护生物学、群体遗传学、谱系地理及演化研究等具有重要意义。     

External morphology of adult Libythea celtis (Laicharting [1782]) (Lepidoptera: Nymphalidae)

Libytheinae is a morphologically unusual group of butterflies whose external anatomy has never been described in detail. The subfamily is considered basal within the diverse family Nymphalidae, and libytheines retain many plesiomorphic features, such as a functional female foreleg and smooth larva. In this paper, we describe and illustrate the adult morphology of Libythea celtis Laicharting, make comparisons to other nymphalid subfamilies, and homologize terms whenever possible. We describe major sclerites, sulci, and the presence of cranial and lateral pits on the tibia, the latter which appear to be previously unreported from the lepidopteran literature. When present, sexual dimorphism and geographic variation are noted. It is hoped that the present paper will help stabilize inconsistencies in morphological terms in nymphalid butterflies.     

Functional diversification of lepidopteran opsins following gene duplication.

A comparative approach was taken for identifying amino acid substitutions that may be under positive Darwinian selection and are correlated with spectral shifts among orthologous and paralogous lepidopteran long wavelength-sensitive (LW) opsins. Four novel LW opsin fragments were isolated, cloned, and sequenced from eye-specific cDNAs from two butterflies, Vanessa cardui (Nymphalidae) and Precis coenia (Nymphalidae), and two moths, Spodoptera exigua (Noctuidae) and Galleria mellonella (Pyralidae). These opsins were sampled because they encode visual pigments having a naturally occurring range of lambda(max) values (510-530 nm), which in combination with previously characterized lepidopteran opsins, provide a complete range of known spectral sensitivities (510-575 nm) among lepidopteran LW opsins. Two recent opsin gene duplication events were found within the papilionid but not within the nymphalid butterfly families through neighbor-joining, maximum parsimony, and maximum likelihood phylogenetic analyses of 13 lepidopteran opsin sequences. An elevated rate of evolution was detected in the red-shifted Papilio Rh3 branch following gene duplication, because of an increase in the amino acid substitution rate in the transmembrane domain of the protein, a region that forms the chromophore-binding pocket of the visual pigment. A maximum likelihood approach was used to estimate omega, the ratio of nonsynonymous to synonymous substitutions per site. Branch-specific tests of selection (free-ratio) identified one branch with omega = 2.1044, but the small number of substitutions involved was not significantly different from the expected number of changes under the neutral expectation of omega = 1. Ancestral sequences were reconstructed with a high degree of certainty from these data. Reconstructed ancestral sequences revealed several instances of convergence to the same amino acid between butterfly and vertebrate cone pigments, and between independent branches of the butterfly opsin tree that are correlated with spectral shifts.     

Gene duplication is an evolutionary mechanism for expanding spectral diversity in the long-wavelength photopigments of butterflies

Butterfly long-wavelength (L) photopigments are interesting for comparative studies of adaptive evolution because of the tremendous phenotypic variation that exists in their wavelength of peak absorbance (lambda(max) value). Here we present a comprehensive survey of L photopigment variation by measuring lambda(max) in 12 nymphalid and 1 riodinid species using epi-microspectrophotometry. Together with previous data, we find that L photopigment lambda(max) varies from 510-565 nm in 22 nymphalids, with an even broader 505- to 600-nm range in riodinids. We then surveyed the L opsin genes for which lambda(max) values are available as well as from related taxa and found 2 instances of L opsin gene duplication within nymphalids, in Hermeuptychia hermes and Amathusia phidippus, and 1 instance within riodinids, in the metalmark butterfly Apodemia mormo. Using maximum parsimony and maximum likelihood ancestral state reconstructions to map the evolution of spectral shifts within the L photopigments of nymphalids, we estimate the ancestral pigment had a lambda(max) = 540 nm +/- 10 nm standard error and that blueshifts in wavelength have occurred at least 4 times within the family. We used ancestral state reconstructions to investigate the importance of several amino acid substitutions (Ile17Met, Ala64Ser, Asn70Ser, and Ser137Ala) previously shown to have evolved under positive selection that are correlated with blue spectral shifts. These reconstructions suggest that the Ala64Ser substitution has indeed occurred along the newly identified blueshifted L photopigment lineages. Substitutions at the other 3 sites may also be involved in the functional diversification of L photopigments. Our data strongly suggest that there are limits to the evolution of L photopigment spectral shifts among species with only one L opsin gene and that opsin gene duplication broadens the potential range of lambda(max) values.     

Beyond the colours: discovering hidden diversity in the Nymphalidae of the Yucatan Peninsula in Mexico through DNA barcoding

Background

Recent studies have demonstrated the utility of DNA barcoding in the discovery of overlooked species and in the connection of immature and adult stages. In this study, we use DNA barcoding to examine diversity patterns in 121 species of Nymphalidae from the Yucatan Peninsula in Mexico. Our results suggest the presence of cryptic species in 8 of these 121 taxa. As well, the reference database derived from the analysis of adult specimens allowed the identification of nymphalid caterpillars providing new details on host plant use.

Methodology/Principal Findings

We gathered DNA barcode sequences from 857 adult Nymphalidae representing 121 different species. This total includes four species (Adelpha iphiclus, Adelpha malea, Hamadryas iphtime and Taygetis laches) that were initially overlooked because of their close morphological similarity to other species. The barcode results showed that each of the 121 species possessed a diagnostic array of barcode sequences. In addition, there was evidence of cryptic taxa; seven species included two barcode clusters showing more than 2% sequence divergence while one species included three clusters. All 71 nymphalid caterpillars were identified to a species level by their sequence congruence to adult sequences. These caterpillars represented 16 species, and included Hamadryas julitta, an endemic species from the Yucatan Peninsula whose larval stages and host plant (Dalechampia schottii, also endemic to the Yucatan Peninsula) were previously unknown.

Conclusions/Significance

This investigation has revealed overlooked species in a well-studied museum collection of nymphalid butterflies and suggests that there is a substantial incidence of cryptic species that await full characterization. The utility of barcoding in the rapid identification of caterpillars also promises to accelerate the assembly of information on life histories, a particularly important advance for hyperdiverse tropical insect assemblages.     

Presence of a cerebral factor showing summer-morph-producing hormone activity in the brain of the seasonal non-polyphenic butterflies Vanessa cardui, V. indica and Nymphalis xanthomelas japonica (Lepidoptera: Nymphalidae)

Three species of nymphalid butterflies, Vanessa cardui, V. indica and Nymphalis xanthomelas japonica , do not exhibit seasonal polyphenism in wing coloration. To determine whether seasonal non-polyphenic butterflies possess a cerebral factor affecting wing coloration, we used a Polygonia c-aureum female short-day pupal assay for detection of summer-morph-producing hormone (SMPH) activity in P. c-aureum. When 2% NaCl extracts of 25 brain-equivalents prepared from the pupal brains of V. cardui, V. indica or N. xanthomelas japonica were injected into Polygonia female short-day pupae, all recipients developed into summer-morph adults with dark-yellow wings, and the average grade score (AGS) of summer morphs showing SMPH activity was 3.8, 3.7 and 4.0, respectively. In contrast, when acetone or 80% ethanol extracts prepared from pupal brains were injected into Polygonia pupae, all recipients developed into autumn-morph adults with a dark-brown coloration and each exhibited an AGS of less than 0.5. Our results indicate that a cerebral factor showing SMPH activity is present in the pupal brain of seasonal non-polyphenic nymphalid butterflies, suggesting that a SMPH and cerebral factor showing SMPH activity occur widely among butterfly species. This finding will improve our understanding of the presence of cerebral factors showing interspecific actions of SHPH.     

中国环蛱蝶属的新种,新亚种及新级位：鳞翅目：蛱蝶科

本文描述采自陕西的环蛱蝶属１新种－周环蛱蝶Ｎｅｐｔｉｓｃｈｏｕｉｓｐ．ｎｏｖ．;将黄环蛱蝶伊洛亚种ＮｅｐｔｉｓｔｈｅｍｉｓｉｌｖｓＦｕｈｓｔｏｒｆｅｒ提升为种,即伊洛环蛱蝶ＮｅｐｔｉｓｉｌｖｓＦｒｕｈｓｔｏｒｆｅｒ,认为黄环蛱蝶台湾亚种ＮｅｐｔｉｓｔｈｅｍｉｓｎｉｅｒｉＮｏｍｕｒａ应为伊洛环蛱蝶ＮｅｐｔｉｓｉｌｖｓＦｒｕｈｓｔｏｒｆｅｒ的１个亚种,描述了伊洛环蛱蝶的１新亚种－四川亚种Ｎｅｐｔｉｓｉ     

The Contribution of Tropical Secondary Forest Fragments to the Conservation of Fruit-feeding Butterflies: Effects of Isolation and Age

Concomitant with the rapid loss of tropical mature forests, the relative abundance of secondary forests is increasing steadily and the latter are therefore of growing interest for conservation. We analysed species richness of fruit-feeding nymphalid butterflies in secondary forest fragments of different age and isolation and in mature forest at the eastern margin of the Lore Lindu National Park in Central Sulawesi, Indonesia. From April to August 2001 we collected 2322 individuals of fruit-feeding butterflies, belonging to 33 species. Butterfly species richness increased with succession, but was significantly higher in mature forests than in all types of secondary forest. Isolation of the forest fragments did not have a significant effect on butterfly species richness in the range of distances (up to 1700 m) studied. Rather it appeared to affect only a few species. Species richness of endemic species was higher than of non-endemic species. Although endemic species were most diverse in mature forests, many species captured were restricted to secondary forests. Our results show that mature forest is essential for the conservation of nymphalid butterflies and for the endemic species in this area. However, considering the relatively large number of species found in these rather small habitat islands, secondary forest fragments, especially older successional stages, can be taken into account in conservation efforts and thus contribute to the preservation of tropical biodiversity on a landscape scale.     

Morphology and chaetotaxy of the first instar larvae of six species of the Satyrus (s.l.) series (Lepidoptera: Nymphalidae)

Abstract. The morphology and chaetotaxy of the first instar larvae of six species belonging to the genera Hipparchia, Kanetisa and Chazara are described. Specific characters are stated, drawn mainly from size, setal length and morphology, and the shape of the suranal plate. Several characters, other than chaetotaxy, that are of potential use in nymphalid systematics are discussed. The larval chaetotaxy is briefly compared with that of both heliconiine and danaine first instar larvae.