Yu Feng Hsu

The evolution of alternative parasitic life histories in large blue butterflies

Large blue (Maculinea) butterflies are highly endangered throughout the Palaearctic region, and have been the focus of intense conservation research. In addition, their extraordinary parasitic lifestyles make them ideal for studies of life history evolution. Early instars consume flower buds of specific host plants, but later instars live in ant nests where they either devour the brood (predators), or are fed mouth-to-mouth by the adult ants (cuckoos). Here we present the phylogeny for the group, which shows that it is a monophyletic clade nested within Phengaris, a rare Oriental genus whose species have similar life histories. Cuckoo species are likely to have evolved from predatory ancestors. As early as five million years ago, two Maculinea clades diverged, leading to the different parasitic strategies seen in the genus today. Contrary to current belief, the two recognized cuckoo species show little genetic divergence and are probably a single ecologically differentiated species. On the other hand, some of the predatory morphospecies exhibit considerable genetic divergence and may contain cryptic species. These findings have important implications for conservation and reintroduction efforts.

The Complete Mitochondrial Genome of the Near-Threatened Swallowtail, Agehana maraho (Lepidoptera: Papilionidae): Evaluating Sequence Variability and Suitable Markers for Conservation Genetic Studies

ABSTRACT: Agehana maraho (Shiraki and Sonan, 1934) is a near-threatened swallowtail butterfly endemic to Taiwan. As a first step in evaluating the most variable molecular markers for further population genetic and conservation studies of this and other insects, the entire mitochondrial genome (mitogenome) was sequenced (16,094bp). The most distinctive structure of the Agehana mitogenome is the control region (CR; 1,270bp). This is the longest CR found so far in any lepidopteran, and it also represents the first known case of two units of macro repeats within a tandem region. In a comparison with another 12 lepidopteran mitogenomes, the genes atp8, atp6, and nad6 were found to be more variable than cox1, suggesting an undue focus on cox1 (COI) in identification and phylogeographic studies. A combination of these first three genes plus the CR, comprising micro as well as macro repeats, may thus provide more suitable markers for conservation genetic studies, not only of this near-threatened species, but also of many other insects.

Ancient Neotropical origin and recent recolonisation: Phylogeny, biogeography and diversification of the Riodinidae (Lepidoptera: Papilionoidea)

We present the first dated higher-level phylogenetic and biogeographic analysis of the butterfly family Riodinidae. This family is distributed worldwide, but more than 90% of the c. 1500 species are found in the Neotropics, while the c. 120 Old World species are concentrated in the Southeast Asian tropics, with minor Afrotropical and Australasian tropical radiations, and few temperate species. Morphologically based higher classification is partly unresolved, with genera incompletely assigned to tribes. Using 3666bp from one mitochondrial and four nuclear markers for each of 23 outgroups and 178 riodinid taxa representing all subfamilies, tribes and subtribes, and 98 out of 145 described genera of riodinids, we estimate that Riodinidae split from Lycaenidae about 96Mya in the mid-Cretaceous and started to diversify about 81Mya. The Riodinidae are monophyletic and originated in the Neotropics, most likely in lowland proto-Amazonia. Neither the subfamily Euselasiinae nor the Nemeobiinae are monophyletic as currently constituted. The enigmatic, monotypic Neotropical genera Styx and Corrachia (most recently treated in Euselasiinae: Corrachiini) are highly supported as derived taxa in the Old World Nemeobiinae, with dispersal most likely occurring across the Beringia land bridge during the Oligocene. Styx and Corrachia, together with all other nemeobiines, are the only exclusively Primulaceae-feeding riodinids. The steadily increasing proliferation of the Neotropical Riodininae subfamily contrasts with the decrease in diversification in the Old World, and may provide insights into factors influencing the diversification rate of this relatively ancient clade of Neotropical insects.

Mitogenomic sequences effectively recover relationships within brush-footed butterflies (Lepidoptera: Nymphalidae).

BackgroundMitogenomic phylogenies have revealed well-supported relationships for many eukaryote groups. In the order Lepidoptera, 113 species mitogenomes had been sequenced (May 14, 2014). However, these data are restricted to ten of the forty-three recognised superfamilies, while it has been challenging to recover large numbers of mitogenomes due to the time and cost required for primer design and sequencing. Nuclear rather than mitochondrial genes have been preferred to reconstruct deep-level lepidopteran phylogenies, without seriously evaluating the potential of entire mitogenomes. Next-generation sequencing methods remove these limitations by providing efficiently massive amounts of sequence data. In the present study, we simultaneously obtained a large number of nymphalid butterfly mitogenomes to evaluate the utility of mitogenomic phylogenies by comparing reconstructions to the now quite well established phylogeny of Nymphalidae.ResultsWe newly obtained 30 nymphalid mitogenomes via pyrosequencing on the Roche 454 GS Junior system, and combined these sequences with publicly accessible data to provide a 70-taxa dataset covering 37 genes for a 15,495 bp alignment. Polymorphic sites were not homogeneously distributed across the gene. Two gene regions, nad6 and 3’ end of nad5, were most variable, whereas the cox1 and 5’ ends of rrnL were most conserved. Phylogenetic relationships inferred by two likelihood methods were congruent and strongly supported (>0.95 posterior probability; ML bootstrap >85%), across the majority of nodes for multiple partitioning strategies and substitution models. Bayes factor results showed that the most highly partitioned dataset is the preferred strategy among different partitioning schemes. The most striking phylogenetic findings were that the subfamily Danainae not Libytheinae was sister of the remaining brush-footed butterflies and that, within Limenitidini, the genus Athyma was clearly polyphyletic. None of the single-gene phylogenies recovered the highly supported topologies generated on the basis of the whole mitogenomic data.ConclusionsThirty mitogenomes were assembled with 89% completeness from the contigs of pyrosequencing-derived reads. Entire mitogenomes or higher-quality sequences could be obtained by increasing pyrosequencing read coverage or by additional Sanger sequencing. Our mitogenomic phylogenies provide robust nodal support at a range of levels, demonstrating that mitogenomes are both accurate and efficient molecular markers for inferring butterfly phylogeny.

Female sex pheromone and male behavioral responses of the bombycid moth Trilocha varians: comparison with those of the domesticated silkmoth Bombyx mori

Analysis of female sex pheromone components and subsequent field trap experiments demonstrated that the bombycid moth Trilocha varians uses a mixture of (E,Z)-10,12-hexadecadienal (bombykal) and (E,Z)-10,12-hexadecadienyl acetate (bombykyl acetate) as a sex pheromone. Both of these components are derivatives of (E,Z)-10,12-hexadecadienol (bombykol), the sex pheromone of the domesticated silkmoth Bombyx mori. This finding prompted us to compare the antennal and behavioral responses of T. varians and B. mori to bombykol, bombykal, and bombykyl acetate in detail. The antennae of T. varians males responded to bombykal and bombykyl acetate but not to bombykol, and males were attracted only when lures contained both bombykal and bombykyl acetate. In contrast, the antennae of B. mori males responded to all the three components. Behavioral analysis showed that B. mori males responded to neither bombykal nor bombykyl acetate. Meanwhile, the wing fluttering response of B. mori males to bombykol was strongly inhibited by bombykal and bombykyl acetate, thereby indicating that bombykal and bombykyl acetate act as behavioral antagonists for B. mori males. T. varians would serve as a reference species for B. mori in future investigations into the molecular mechanisms underlying the evolution of sex pheromone communication systems in bombycid moths.

Phylogeny and Historical Biogeography of Asian Pterourus Butterflies (Lepidoptera: Papilionidae): A Case of Intercontinental Dispersal from North America to East Asia

The phylogenetic status of the well-known Asian butterflies often known as Agehana (a species group, often treated as a genus or a subgenus, within Papilio sensu lato) has long remained unresolved. Only two species are included, and one of them especially, Papilio maraho, is not only rare but near-threatened, being monophagous on its vulnerable hostplant, Sassafras randaiense (Lauraceae). Although the natural history and population conservation of “Agehana” has received much attention, the biogeographic origin of this group still remains enigmatic. To clarify these two questions, a total of 86 species representatives within Papilionidae were sampled, and four genes (concatenated length 3842 bp) were used to reconstruct their phylogenetic relationships and historical scenarios. Surprisingly, “Agehana” fell within the American Papilio subgenus Pterourus and not as previously suggested, phylogenetically close to the Asian Papilio subgenus Chilasa. We therefore formally synonymize Agehana with Pterourus. Dating and biogeographic analysis allow us to infer an intercontinental dispersal of an American ancestor of Asian Pterourus in the early Miocene, which was coincident with historical paleo-land bridge connections, resulting in the present “East Asia-America” disjunction distribution. We emphasize that species exchange between East Asia and America seems to be a quite frequent occurrence in butterflies during the Oligocene to Miocene climatic optima.

Molecular Phylogeny, Laboratory Rearing, and Karyotype of the Bombycid Moth, Trilocha varians

Abstract This study describes the molecular phylogeny, laboratory rearing, and karyotype of a bombycid moth, Trilocha varians (F. Walker) (Lepidoptera: Bombycidae), which feeds on leaves of Ficus spp. (Rosales: Moraceae). The larvae of this species were collected in Taipei city, Taiwan, and the Ryukyu Archipelago (Ishigaki and Okinawa Islands, Japan). Molecular phylogenetic analyses revealed that T. varians belongs to the subfamily Bombycinae, thus showing a close relationship to the domesticated silkworm Bombyx mori (L.), a lepidopteran model insect. A laboratory method was developed for rearing T. varians and the time required for development from the embryo to adult was determined. From oviposition to adult emergence, the developmental zero was 10.47 °C and total effective temperature was 531.2 day—degrees, i.e., approximately 30 days for one generation when reared at 28 °C. The haploid of T. varians consisted of n = 26 chromosomes. In highly polyploid somatic nuclei, females showed a large heterochromatin body, indicating that the sex chromosome system in T. varians is WZ/ZZ (female/male). The results of the present study should facilitate the utilization of T. varians as a reference species for B. mori, thereby leading to a greater understanding of the ecology and evolution of bombycid moths.

A new lycaenid butterfly exclusively associated with the subalpine sclerophyllous oak forests in Taiwan (Lepidoptera, Lycaenidae, Theclinae)

Subalpine sclerophyllous oak forest grows at high elevation in Taiwan, distantly separated from similar forest communities found in western China, Tibet and India. An investigation on bud‐breaking phenology of oaks and associated phytophagous insects in this community revealed an undescribed species of Teratozephyrus lycaenid. This and other oak‐associated insects suggest that the presence of this oak community in Taiwan resulted from a more widely distributed community. The newly discovered Teratozephyrus turned out to be most closely related to T. nuwaii, described from western China, and is described below as T. elatus sp. nov. Two other species of Teratozephyrus also inhabit Taiwan, but both are associated with broad‐leaved oak forests. Of them, T. arisanus is widespread both in China and Taiwan, is morphologically distinct from the other Teratozephyrus species and is exclusively associated with broad‐leaved oaks. The other, T. yugaii, probably gained its broad‐leaved oak association secondarily as no similar species is found in the broad‐leaved oak communities on continental Asia, and it favours cool, higher‐elevation environments even though its host plant ranges into far lower elevations.

A new species of Potanthus from montane forests in Taiwan providing additional evidence of biotic relationship between the Philippines and the Asian continent (Lepidoptera, Hesperiidae; Hesperiinae)

A previously undescribed, montane forest-dwelling Potanthus is reported as a species endemic to Taiwan. Its closest relatives are suggested to be P. flavus, distributed from the continental Asia to Japan, and P. niobe from the Philippines, based upon two synapomorphies in the genital structures of both sexes. This new species inhabits altitudes higher than all the congeneric members previously known from Taiwan. Its presence fills the gap in the P. flavus + P. niobe distribution between the Philippines and the Asian continent, demonstrating a biotic connection between these regions.

Systematic Problems Surrounding Howarthia melli (Forster) (Lepidoptera: Lycaenidae: Theclinae), with Description of a New Species and a Review of Rhododendron-Association in Lycaenid Butterflies

Abstract Within the genus Howarthia, Howarthia melli (Forster) is the most frequently reported species across the southern provinces of China. An examination of the type series of H. melli, in combination with results of rearing experiments, reveals that three species have been frequently confused under the name H. melli. The three are superficially similar but not closely related to each other within the genus. H. melli is much more restricted in distribution than previously thought, with most literature records referring to the other two species, Howarthia cheni Chou & Wang and an undescribed species, Howarthia cyanea sp. n., described herein. We provide a discussion of the relationships of these species and a review of Rhododendron-association in Lycaenidae.