Characterization of microsatellite loci in neotropical Heliconius butterflies

The Heliconius butterflies offer exceptional opportunities for the study of the ecology and evolution of mimicry. Despite previous reports of difficulties in the development of microsatellite loci in Lepidoptera, we characterize 15 polymorphic loci in H. erato that show promise for genetic mapping and population studies in this and other species. Levels of variation were high, in both numbers and size ranges of alleles. The loci showed broad amplification success across the genus and in two other genera. All loci that amplified in a population of H. melpomene were polymorphic.     

蛱蝶翅鳞片的超微结构观察

对我国东北地区典型常见蛱蝶科15属20种蝴蝶翅鳞片的超微结构进行了扫描电镜观察。结果显示：蛱蝶翅鳞片形态上可分为窄叶形、阔叶形和圆叶形3种,鳞片长65～135 μm,宽35～85 μm,间距48～112 μm。蛱蝶翅鳞片的超微结构可分为拱桥形、棋盘形和筛孔形3 种。拱桥形结构和棋盘形结构比较接近,二者与筛孔形结构差异较明显。在已观察的种类中,线蛱蝶属红线蛱蝶翅鳞片上的纵肋突起最小（200 nm×300 nm）,闪蛱蝶属柳紫闪蛱蝶翅鳞片上的纵肋突起最大（590 nm×560 nm）。鳞片具有相似的形状、结构和排列,尤其是同属蝴蝶翅鳞片超微结构的形状和尺寸差异较小,表明它们之间的亲缘关系接近。     

Molecular systematics of the butterfly genus Ithomia (Lepidoptera: Ithomiinae): a composite phylogenetic hypothesis based on seven genes

Butterflies in the nymphalid subfamily Ithomiinae are brightly colored and involved in mimicry. Here we present a phylogenetic hypothesis for 23 of the 24 species in the genus Ithomia, based on seven different gene regions, representing 5 linkage groups and 4469 bp. We sequenced varying length regions of the following genes: (1) elongation factor 1alpha (Ef1alpha; 1028 bp); (2) tektin (tektin; 715 bp); (3) wingless (wg; 405 bp); (4) ribosomal protein L5 (RpL5; 722 bp, exons 1, 2, 3, and introns 1 and 2); and (5) mitochondrial cytochrome oxidase I, II (Co1 and Co2 and intervening leucine tRNA; 1599 bp). The results show incongruence between some genetic loci, although when alternate topologies are compared statistically it was generally true that one topology was supported by a majority of loci sampled. This highlights the need to sample widely across the genome in order to obtain a well-supported phylogenetic hypothesis. A combined evidence topology is presented based on a Bayesian analysis of all the gene regions, except the fast-evolving RpL5. The resulting hypothesis is concordant with the most probable relationships determined from our topological comparisons, although in some parts of the tree relationships remain weakly supported. The tree suggests diversification has largely occurred within biogeographic regions such as Central America, the Amazon, the southern and northern Andes, with only occasional dispersal (or vicariance) between such regions. This phylogenetic hypothesis can now be used to investigate patterns of diversification across the genus, such as the potential role of color pattern changes in speciation.     

Complex dynamics underlie the evolution of imperfect wing pattern convergence in butterflies

Adaptive radiation is characterized by rapid diversification that is strongly associated with ecological specialization. However, understanding the evolutionary mechanisms fueling adaptive diversification requires a detailed knowledge of how natural selection acts at multiple life‐history stages. Butterflies within the genus Adelpha represent one of the largest and most diverse butterfly lineages in the Neotropics. Although Adelpha species feed on an extraordinary diversity of larval hosts, convergent evolution is widespread in this group, suggesting that selection for mimicry may contribute to adaptive divergence among species. To investigate this hypothesis, we conducted predation studies in Costa Rica using artificial butterfly facsimiles. Specifically, we predicted that nontoxic, palatable Adelpha species that do not feed on host plants in the family Rubiaceae would benefit from sharing a locally convergent wing pattern with the presumably toxic Rubiaceae‐feeding species via reduced predation. Contrary to expectations, we found that the presumed mimic was attacked significantly more than its locally convergent model at a frequency paralleling attack rates on both novel and palatable prey. Although these data reveal the first evidence for protection from avian predators by the supposed toxic, Rubiaceae‐feeding Adelpha species, we conclude that imprecise mimetic patterns have high costs for Batesian mimics in the tropics.     

The phylogenetic relationships of Chalcosiinae (Lepidoptera, Zygaenoidea, Zygaenidae)

The chalcosiine zygaenid moths constitute one of the most striking groups within the lower-ditrysian Lepidoptera, with highly diverse mimetic patterns, chemical defence systems, scent organs, copulatory mechanisms, hostplant utilization and diapause biology, plus a very disjunctive biogeographical pattern. In this paper we focus on the genus-level phylogenetics of this subfamily. A cladistic study was performed using 414 morphological and biochemical characters obtained from 411 species belonging to 186 species-groups of 73 genera plus 21 outgroups. Phylogenetic analysis using maximum parsimony leads to the following conclusions: (1) neither the current concept of Zygaenidae nor that of Chalcosiinae is monophyletic; (2) the previously proposed sister-group relationship of Zygaeninae + Chalcosiinae is rejected in favour of the relationship (Zygaeninae + ((Callizygaeninae + Cleoda ) + ( Heteropan + Chalcosiinae))); (3) except for the monobasic Aglaopini, none of the tribes sensu Alberti (1954 ) is monophyletic; (4) chalcosiine synapomorphies include structures of the chemical defence system, scent organs of adults and of the apodemal system of the male genitalia. A paired metathoracic androconial organ and a series of abdominal tergal corematal organs have been discovered, both being new to Lepidoptera. Due to highly homoplastic patterns in copulatory structures and wings that demonstrate significant sexual dimorphism, polymorphism and mimicry, 17 of the 69 'true' chalcosiine genera ( c . 25%) are shown to be either paraphyletic or polyphyletic. The present classification is therefore very misleading. Reductions of various parts of the male genitalia in some groups are accompanied by morphological and functional replacement involving the 8th abdominal segment. A prominent but convergent lock and key mechanism is revealed.  © 2005 The Linnean Society of London, Zoological Journal of the Linnean Society , 2005, 143 , 161–341.     

The phylogenetics and biochemistry of host-plant specialization in Melitaeine butterflies (Lepidoptera: Nymphalidae)

Butterflies in the tribe Melitaeini (Lepidoptera: Nymphalidae) are known to utilize host plants belonging to 16 families, although most host-plant records are from four families. Of the 16 host-plant families, 12 produce secondary plant metabolites called iridoids. Earlier studies have shown that larvae of several melitaeine species use iridoids as feeding stimulants and sequester these compounds for larval defense. I investigate the evolutionary history of host-plant use in the tribe Melitaeini by testing a recent phylogenetic hypothesis of 65 species representing the four major species groups of the tribe. By simple character optimization of host-plant families and presence/absence of iridoids in the host plants, I find that plant chemistry is a more conservative trait than plant taxonomy. The ancestral host plant(s) of the entire tribe most likely contained iridoids and were likely to be in the plant family Plantaginaceae. A major host shift from plants containing iridoids to plants not containing iridoids has happened three times independently. The results show that the evolution of host-plant use in melitaeines has been (and still is) a dynamic process when considering plant taxonomy, but is relatively stable when considering host-plant chemistry.     

The first vespiform tineid moth (Lepidoptera: Tineidae)

Abstract Vespitinea gurkharum gen. et sp.n. is described from specimens reared from a bracket-fungus ( Ganoderma - Polyporaceae) from lowland rain-forest in Brunei. This striking wasp-like moth is suggested to be a Batesian mimic of species of Vespidae and Pompilidae (Hymenoptera) and the first example of this mimicry type recorded from the Tineoidea. Abdominal, eye and wing-coupling modifications suggest that it is diurnal and a behavioural mimic. The systematic position of Vespitinea is discussed with reference to biology and adult and larval morphology.     

The evolution of ecologically important characters in Heliconius butterflies (Lepidoptera: Nymphalidae): a cladistic review

The biology of Heliconius butterflies has provided a rich source of data to test theories of ecological genetics, coevolution and community ecology. Many putatively adaptive characters have been discussed with reference to a phylogenetic hypothesis based on a variety of morphological and life-history traits interpreted from an evolutionary taxonomic perspective. Here, alternate interpretations of characters on the traditional tree and a more recent mitochondrial DNA cladogram with a substantially different topology are compared and contrasted. It is shown that many characters ostensibly providing support for the traditional phylogenetic hypothesis are almost equally parsimoniously distributed and in some cases more parsimoniously distributed on the mtDNA tree than on the tree inferred from those characters. Discussion of alternate evolutionary scenarios based on the mDNA-based topology is presented for pupal mating, pollen feeding, foodplant coevolution, and other ecologically significant features.     

The roles of wing color pattern and geography in the evolution of Neotropical Preponini butterflies

Diversification rates and evolutionary trajectories are known to be influenced by phenotypic traits and the geographic history of the landscapes that organisms inhabit. One of the most conspicuous traits in butterflies is their wing color pattern, which has been shown to be important in speciation. The evolution of many taxa in the Neotropics has also been influenced by major geological events. Using a dated, species‐level molecular phylogenetic hypothesis for Preponini, a colorful Neotropical butterfly tribe, we evaluated whether diversification rates were constant or varied through time, and how they were influenced by color pattern evolution and biogeographical events. We found that Preponini originated approximately 28 million years ago and that diversification has increased through time consistent with major periods of Andean uplift. Even though some clades show evolutionarily rapid transitions in coloration, contrary to our expectations, these shifts were not correlated with shifts in diversification. Involvement in mimicry with other butterfly groups might explain the rapid changes in dorsal color patterns in this tribe, but such changes have not increased species diversification in this group. However, we found evidence for an influence of major Miocene and Pliocene geological events on the tribe''s evolution. Preponini apparently originated within South America, and range evolution has since been dynamic, congruent with Andean geologic activity, closure of the Panama Isthmus, and Miocene climate variability.     

Phylogeography and demography of Guianan harlequin toads (Atelopus): diversification within a refuge

We investigated the genetic structure of populations of Guianan harlequin toads (genus Atelopus) and their evolutionary affinities to extra-Guianan congeners. Phylogenetic analysis of mitochondrial cytochrome b (cyt b) and NADH dehydrogenase subunit 2 (ND2) gene sequences produced well-supported clades largely corresponding to the four recognized taxa in the Guianas (Atelopus spumarius hoogmoedi, Atelopus spumarius barbotini, Atelopus franciscus, and Atelopus flavescens). Our findings suggest that the Guianan A. spumarius represent distinct evolutionary lineages that merit distinction from Amazonian conspecifics, and that the status of A. flavescens and A. franciscus is somewhat less clear. Approximately 69% of the observed genetic variation is accounted for by differences between these four recognized taxa. Coalescent-based estimates of gene flow between taxa suggest that these lineages are largely isolated from one another. Negligible rates of migration between populations and significant divergence within such close proximity suggests that although the region inhabited by these taxa is almost entirely undisturbed, significant habitat heterogeneity exists as to have produced a remarkable diversification of Atelopus within the eastern Guiana Shield. These results contradict the commonly held view of the Guiana Shield as a 'refuge' whose stability during late Tertiary and Quaternary climatic fluctuations served as a biotic reservoir. Instead, we provide evidence that climatic fluctuations during this time had a diversifying effect within the Guianan region.     

Karyotypic diversity and speciation in Agrodiaetus butterflies

That chromosomal rearrangements may play an important role in maintaining postzygotic isolation between well-established species is part of the standard theory of speciation. However, little evidence exists on the role of karyotypic change in speciation itself--in the establishment of reproductive barriers between previously interbreeding populations. The large genus Agrodiaetus (Lepidoptera: Lycaenidae) provides a model system to study this question. Agrodiaetus butterflies exhibit unusual interspecific diversity in chromosome number, from n= 10 to n= 134; in contrast, the majority of lycaenid butterflies have n= 23/24. We analyzed the evolution of karyotypic diversity by mapping chromosome numbers on a thoroughly sampled mitochondrial phylogeny of the genus. Karyotypic differences accumulate gradually between allopatric sister taxa, but more rapidly between sympatric sister taxa. Overall, sympatric sister taxa have a higher average karyotypic diversity than allopatric sister taxa. Differential fusion of diverged populations may account for this pattern because the degree of karyotypic difference acquired between allopatric populations may determine whether they will persist as nascent biological species in secondary sympatry. This study therefore finds evidence of a direct role for chromosomal rearrangements in the final stages of animal speciation. Rapid karyotypic diversification is likely to have contributed to the explosive speciation rate observed in Agrodiaetus, 1.6 species per million years.     

Thermoregulatory significance of wing melanization in Pieris butterflies (Lepidoptera: Pieridae): physics,posture, and pattern

Summary Pieris butterflies use a novel behavioral posture for thermoregulation called reflectance basking, in which the wings are used as solar reflectors to reflect radiation to the body. As a means of exploring the thermoregulatory significance of wing melanization patterns, I examine the relation of basking posture and wing color pattern to body temperature. A mathematical model of the reflectance process predicts certain combinations of dorsal wing melanization pattern and basking posture that maximize body temperature. Laboratory experiments and field observations show that this model correctly predicts qualitative differences in the relation of body temperature to basking posture based on differences in the extent of dorsal melanization on the wing margins, both between species and between sexes within species of Pieris. This is the first demonstration in insects that coloration of the entire wing surface can affect thermoregulation. Model and experimental results suggest that, in certain wing regions, increased melanization can reduce body temperature in Pieris; this effect of melanization is exactly the opposite of that found in other Pierid butterflies that use their wings as solar absorbers. These results are discussed in terms of the evolution of wing melanization pattern and thermoregulatory behavior in butterflies.     

Hybridization and transgressive exploration of colour pattern and wing morphology in Heliconius butterflies

Hybridization can generate novel phenotypes distinct from those of parental lineages, a phenomenon known as transgressive trait variation. Transgressive phenotypes might negatively or positively affect hybrid fitness, and increase available variation. Closely related species of Heliconius butterflies regularly produce hybrids in nature, and hybridization is thought to play a role in the diversification of novel wing colour patterns despite strong stabilizing selection due to interspecific mimicry. Here, we studied wing phenotypes in first‐ and second‐generation hybrids produced by controlled crosses between either two co‐mimetic species of Heliconius or between two nonmimetic species. We quantified wing size, shape and colour pattern variation and asked whether hybrids displayed transgressive wing phenotypes. Discrete traits underlain by major‐effect loci, such as the presence or absence of colour patches, generate novel phenotypes. For quantitative traits, such as wing shape or subtle colour pattern characters, hybrids only exceed the parental range in specific dimensions of the morphological space. Overall, our study addresses some of the challenges in defining and measuring phenotypic transgression for multivariate traits and our data suggest that the extent to which transgressive trait variation in hybrids contributes to phenotypic diversity depends on the complexity and the genetic architecture of the traits.     

Shifting distributions and speciation: species divergence during rapid climate change

Questions about how shifting distributions contribute to species diversification remain virtually without answer, even though rapid climate change during the Pleistocene clearly impacted genetic variation within many species. One factor that has prevented this question from being adequately addressed is the lack of precision associated with estimates of species divergence made from a single genetic locus and without incorporating processes that are biologically important as populations diverge. Analysis of DNA sequences from multiple variable loci in a coalescent framework that (i) corrects for gene divergence pre-dating speciation, and (ii) derives divergence-time estimates without making a priori assumptions about the processes underlying patterns of incomplete lineage sorting between species (i.e. allows for the possibility of gene flow during speciation), is critical to overcoming the inherent logistical and analytical difficulties of inferring the timing and mode of speciation during the dynamic Pleistocene. Estimates of species divergence that ignore these processes, use single locus data, or do both can dramatically overestimate species divergence. For example, using a coalescent approach with data from six loci, the divergence between two species of montane Melanoplus grasshoppers is estimated at between 200,000 and 300,000 years before present, far more recently than divergence estimates made using single-locus data or without the incorporation of population-level processes. Melanoplus grasshoppers radiated in the sky islands of the Rocky Mountains, and the analysis of divergence between these species suggests that the isolation of populations in multiple glacial refugia was an important factor in promoting speciation. Furthermore, the low estimates of gene flow between the species indicate that reproductive isolation must have evolved rapidly for the incipient species boundaries to be maintained through the subsequent glacial periods and shifts in species distributions.     

Mimicry on the QT(L): genetics of speciation in Mimulus

Ecological studies suggest that hummingbird-pollinated plants in North America mimic each other to increase visitation by birds. Published quantitative trait locus (QTL) data for two Mimulus species indicate that floral traits associated with hummingbird versus bee pollination results from a few loci with major effects on morphology, as predicted by classical models for the evolution of mimicry. Thus, the architecture of genetic divergence associated with speciation may depend on the ecological context.     

Effects of changing climate on species diversification in tropical forest butterflies of the genus Cymothoe (Lepidoptera: Nymphalidae)

Extant clades may differ greatly in their species richness, suggesting differential rates of species diversification. Based on phylogenetic trees, it is possible to identify potential correlates of such differences. Here, we examine species diversification in a clade of 82 tropical African forest butterfly species (Cymothoe), together with its monotypic sister genus Harma. Our aim was to test whether the diversification of the Harma–Cymothoe clade correlates with end‐Miocene global cooling and desiccation, or with Pleistocene habitat range oscillations, both postulated to have led to habitat fragmentation. We first generated a species‐level phylogenetic tree for Harma and Cymothoe, calibrated within an absolute time scale, and then identified temporal and phylogenetic shifts in species diversification. Finally, we assessed correlations between species diversification and reconstructed global temperatures. Results show that, after the divergence of Harma and Cymothoe in the Miocene (15 Mya), net species diversification was low during the first 7 Myr. Coinciding with the onset of diversification of Cymothoe around 7.5 Mya, there was a sharp and significant increase in diversification rate, suggesting a rapid radiation, and correlating with a reconstructed period of global cooling and desiccation in the late Miocene, rather than with Pleistocene oscillations. Our estimated age of 4 Myr for a clade of montane species corresponds well with the uplift of the Eastern Arc Mountains where they occur. We conclude that forest fragmentation caused by changing climate in the late Miocene as well as the Eastern Arc Mountain uplift are both likely to have promoted species diversification in the Harma–Cymothoe clade. Cymothoe colonized Madagascar much later than most other insect lineages and, consequently, had less time available for diversification on the island. We consider the diversification of Cymothoe to be a special case compared with other butterfly clades studied so far, both in terms of its abrupt diversification rate increase and its recent occurrence (7 Myr). It is clear that larval host plant shift(s) cannot explain the difference in diversification between Cymothoe and Harma; however, such a shift(s) may have triggered differential diversification rates within Cymothoe. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, ●● , ●●–●●.     

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.     

The geography of speciation in narrow‐range endemics of the ‘Haenydra’ lineage (Coleoptera,Hydraenidae, Hydraena)

Aim We test whether species of western Mediterranean aquatic Coleoptera of the ‘Haenydra’ lineage (Hydraenidae, Hydraena) originated through: (1) successive periods of dispersal and speciation, (2) range fragmentation by random vicariance, or (3) range fragmentation by geographic isolation owing to a general reduction of population density. Location Europe. Methods To discriminate between scenarios we use contrasting predictions of the relationship between phylogenetic and geographic distance. The phylogeny was based on 3 kb of four mitochondrial and two nuclear gene fragments of about half of the known species of ‘Haenydra’, including most western Mediterranean taxa. Divergences were estimated using a molecular clock. The relationship between phylogenetic and geographic distance was tested using bivariate plots, Mantel tests and comparison of the observed phylogeny with the one minimizing geographic distances between species, as measured using Euclidean minimum spanning trees (EMSTs). Results The monophyly of ‘Haenydra’ was strongly supported, although its phylogenetic placement was not resolved. ‘Haenydra’ was estimated to be of late Miocene age, with most species originating during the Pleistocene. In two clades (Hydraena tatii and Hydraena emarginata clades) there was a significant association between geographic and phylogenetic distance, and the reconstructed phylogeny was identical to that obtained through the EMST, demonstrating a strong non‐randomness of the geographic distribution of the species. In two other clades (Hydraena iberica and Hydraena bitruncata clades) there was no association between geographic and phylogenetic distance, and the observed phylogeny was not the one minimizing geographic distances. In one of the clades this seems to be due to a secondary, recent range expansion of one species (H. iberica), which erased the geographic signal of their distributions. Main conclusions We show that it is possible to obtain strong evidence of stasis of the geographic ranges of narrow‐range endemic species through the study of their phylogenetic relationships and current distributions. In at least two of the studied clades, current species seem to have originated through the fragmentation of a more widely distributed species, without further range movements. A process of range expansion and fragmentation may have occurred repeatedly within the ‘Haenydra’ lineage, contributing to the accumulation of narrow‐range endemics in Mediterranean Pleistocene refugia.     

Independent inheritance of preference and performance in hybrids between host races of Mitoura butterflies (Lepidoptera: Lycaenidae)

Divergent natural selection contributes to reproductive isolation among populations adapting to different habitats or resources if hybrids between populations are intermediate in phenotype and suffer an associated, environmentally dependent reduction in fitness. This prediction was tested using two host races of Mitoura butterflies. Thirty-five F1 hybrid and parental lines were created, larvae were raised on the two host plants, and oviposition preferences were assayed in choice arenas. Larvae from both reciprocal hybrid crosses suffered a host-specific reduction in performance: when reared on incense cedar, hybrid survival was approximately 30% less than the survival of pure lines of the cedar-associated host race. The performance of hybrid larvae reared on the other host, MacNab cypress, was not reduced relative to parental genotypes. Females from both reciprocal hybrid crosses preferred to oviposit on incense cedar, the same host that resulted in the reduced survival of hybrid larvae. Thus, dominance is implicated in the inheritance of traits involved in both preference and performance, which do not appear to be genetically linked in Mitoura butterflies. Gene flow between host races may be reduced because the correlation between preference and performance that was previously described in parental populations is essentially broken by hybridization.