Towards a better future for DNA barcoding: Evaluating monophyly- and distance-based species identification using COI gene fragments of Dacini fruit flies.

The utility of a universal DNA 'barcode' fragment (658 base pairs of the Cytochrome C Oxidase I [COI] gene) has been established as a useful tool for species identification, and widely criticized as one for understanding the evolutionary history of a group. Large amounts of COI sequence data have been produced that hold promise for rapid species identification, for example, for biosecurity. The fruit fly tribe Dacini holds about a thousand species, of which 80 are pests of economic concern. We generated a COI reference library for 265 species of Dacini containing 5601 sequences that span most of the COI gene using circular consensus sequencing. We compared distance metrics versus monophyly assessments for species identification and although we found a 'soft' barcode gap around 2% pairwise distance, the exceptions to this rule dictate that a monophyly assessment is the only reliable method for species identification. We found that all fragments regularly used for Dacini fruit fly identification >450 base pairs long provide similar resolution. 11.3% of the species in our dataset were non-monophyletic in a COI tree, which is mostly due to species complexes. We conclude with recommendations for the future generation and use of COI libraries. We revise the generic assignment of Dacus transversus stat. rev. Hardy 1982, and Dacus perpusillus stat. rev. Drew 1971 and we establish Dacus maculipterus White 1998 syn. nov. as a junior synonym of Dacus satanas Liang et al. 1993.

Revision of the Genus Rhagastis Rothschild & Jordan, 1903 (Lepidoptera: Sphingidae) from China, Based on Morphological and Phylogenetic Analyses.

Here, the taxonomy of the genus Rhagastis Rothschild & Jordan, 1903 (Lepidoptera, Sphingidae, Macroglossinae, Macroglossini) from China is revised based on differences in wing morphology, male and female genitalia, and the phylogenetic relationship of the DNA barcodes. Subspecies of Rhagastis albomarginatus (Rothschild, 1894) and R. castor (Walker, 1856) are treated as "good" species, namely Rhagastis dichroae Mell, 1922 stat. nov.; R. everetti Rothschild & Jordan, 1903 stat. nov.; R. aurifera (Butler, 1875) stat. rev.; R. chinensis Mell, 1922 stat. nov.; R. formosana Clark, 1925 stat. nov.; and R. jordani Oberthür, 1904 stat. rev. The distribution maps, biological notes, and ecological records of the genus Rhagastis Rothschild & Jordan, 1903 from China are given, and a species inventory of genus Rhagastis in the world is also included.

Local adaptation and migratory habits balance spatial-genetic structure between continental and insular chestnut tiger butterflies in East Asia.

Geographic and climatic differences between islands and continents may affect the evolution of their biota, and promote divergent selection in species distributed in both landscapes. To assess spatial-genetic structure, we genotyped 18 expressed sequence tag-simple sequence repeat (EST-SSR) loci and sequenced two mtDNA markers (ND5 and COI) and one nuclear marker (EF1α) in two subspecies of the butterfly Parantica sita. Compared with nuclear markers, mtDNA had a stronger signal of population structure. Approximate Bayesian computation (ABC) suggested that a continuous-gene-flow model best described the data. According to this model, the two subspecies diverged approximately 23.1 kya, with 10 times more introgression from the continental (ssp. sita) to the insular subspecies (ssp. niphonica) than vice versa. Ecological niche modelling was performed to predict the paleo- and current potential distributions and elucidate the geohistorical process, which revealed a northeastern, insular origin. Winter precipitation and annual temperature range were the main determinants of the subspecies distributions. Maximum-likelihood population-effects models showed that the population differentiation of the insular and continental subspecies was primarily affected by environmental resistance and local climate. Sex-biased migration capacity and long-term precipitation-driven divergence between the continental and insular lineages shaped the current genetic structure of P. sita. Evidence from the nuclear markers confirmed intersubspecific gene flow despite adaptive divergence between the subspecies. These results imply that the continental subspecies is still capable of returning to the island and introgressing with the insular subspecies.

Immature stages of Neptis reducta Fruhstorfer, 1908 (Lepidoptera, Nymphalidae, Neptini): morphology, ecology and food specialty.

Neptis reducta Fruhsorfer, 1908 is currently recognized as a nymphalid butterfly found only in Taiwan and Thailand, with previously recorded larval hostplant and immature biology involving mis-identifications. The present study recognizes Aphananthe aspera (Cannabaceae) as the sole larval hostplant for N. reducta, and the nymphalid butterfly is the first known lepidopterous insect specialized on Aphananthe.

The Aboveground Vegetation Type and Underground Soil Property Mediate the Divergence of Soil Microbiomes and the Biological Interactions.

The composition of the soil microbiome is influenced by environmental (abiotic) variables and biological interactions (biotic factors). To determine whether the aboveground vegetation and soil physicochemical properties were the main determinant of beta-diversity and biological interaction of soil microbial community, we sampled soils from the temperate coniferous forest and grassland. Clustering of operational taxonomic units was conducted using 16S rRNA gene. We found that the microbial composition of the rhizospheres, in which root exudates influence the microbial environment, show lower alpha-diversity than that of nonroot soils. The nonsignificant rhizosphere effect suggested other undetermined factors or stochastic processes accounted for microbial diversity in the rhizosphere. More significant microbe-microbe interactions were observed in forest and rhizosphere soils relative to the grassland soils. The elevated number of positive correlations for relative abundances in forest soil implied beneficial associations being common among bacteria, in particular within the rhizosphere environment. The particular soil properties generated by root exudates also alter the physicochemical properties of soil such as K and pH value, and might in turn favor the adoption of teamwork-cooperation strategies for microbe-microbe interactions, represented as large clusters of positive associations among bacterial taxa. Specific biological interactions differentiated the microbiomes within forest soils. Thus, the environmental selection pressure of aboveground vegetation accounts for differences between soil microbiomes while biotic factors are responsible for fine-scale differences of the microbial community in forest soils.

DNA barcoding and morphological data reveal a new Hyposoter (Hymenoptera: Ichneumonidae: Porizontinae) reared from a rare zygaenid moth Artona flavipuncta Hampson, 1900 in Taiwan.

A new species of Hyposoter Foerster, 1869 is discovered based on morphology of adults and cocoons, biology, and DNA barcoding evidence. It is herein described as Hyposoter distriangulum Chen, Huang & Hsu sp. nov. , which is a common parasitoid of a rare zygaenid moth Artona flavipuncta Hampson, 1900. The differences between similar Hyposoter species in Japan and Taiwan are also presented.

Continuation of the genetic divergence of ecological speciation by spatial environmental heterogeneity in island endemic plants.

Divergent selection plays a critical role not only as a speciation driver but also in maintaining post-speciation divergence. In the absence of direct evidence, ancestral interspecific gene flow between incipient species can reflect ancient selective pressure for ecological speciation. In the present study, two late-Pleistocene diverged species endemic to Taiwan, Scutellaria playfairii and S. tashiroi, were spatially and ecologically partitioned with partial overlap. Multilocus genome-scan analyses and in silico evaluation revealed ancestral interspecific gene flow but distinct genetic compositions, implying that adaptive divergence contributed to their speciation. Ecological niche modeling and principal component analysis suggested incomplete divergent niches between the two species; the species distribution is therefore consistent with Hutchinson's metaphor of multidimensional hypervolume niches rather than attributable to a single factor. Constraint ordination analysis supported this inference of a combination of variables explaining the genetic structure. The rare occurrence of hybrids in the sympatric population suggested hybrid breakdown, providing further evidence of divergent selection blocking gene flow. The correlation of environmental variables with integrated genetic components demonstrated that environmental heterogeneity maintains the species and population differentiation. This study highlights the importance of environmental heterogeneity and divergent selection for the rapid speciation and recent diversification of island plants.

Imbalanced positive selection maintains the functional divergence of duplicated DIHYDROKAEMPFEROL 4-REDUCTASE genes.

Gene duplication could be beneficial by functional division but might increase the risk of genetic load. The dynamics of duplicated paralogs number could involve recombination, positive selection, and functional divergence. Duplication of DIHYDROFLAVONOL 4-REDUCTASE (DFR) has been reported in several organisms and may have been retained by escape from adaptive conflict (EAC). In this study, we screened the angiosperm DFR gene focusing on a diversified genus Scutellaria to investigate how these duplicated genes are retained. We deduced that gene duplication involved multiple independent events in angiosperms, but the duplication of DFR was before the divergence of Scutellaria. Asymmetric positive selective pressures resulted in different evolutionary rates between the duplicates. Different numbers of regulatory elements, differential codon usages, radical amino acid changes, and differential gene expressions provide evidences of functional divergence between the two DFR duplicates in Scutellaria, implying adaptive subfunctionalization between duplicates. The discovery of pseudogenes accompanying a reduced replacement rate in one DFR paralogous gene suggested possibly leading to "loss of function" due to dosage imbalance after the transient adaptive subfunctionalization in the early stage of duplication. Notwithstanding, episodic gene duplication and functional divergence may be relevant to the diversification of ecological function of DFR gene in Scutellaria.

Diversifying selection of the anthocyanin biosynthetic downstream gene UFGT accelerates floral diversity of island Scutellaria species.

BACKGROUND: Adaptive divergence, which usually explains rapid diversification within island species, might involve the positive selection of genes. Anthocyanin biosynthetic pathway (ABP) genes are important for floral diversity, and are related to stress resistance and pollination, which could be responsible for species diversification. Previous studies have shown that upstream genes of ABP are subject to selective constraints and have a slow evolutionary rate, while the constraints on downstream genes are lower. RESULTS: In this study, we confirmed these earlier observations of heterogeneous evolutionary rate in upstream gene CHS and the downstream gene UFGT, both of which were expressed in Scutellaria sp. inflorescence buds. We found a higher evolutionary rate and positive selection for UFGT. The codons under positive selection corresponded to the diversified regions, and the presence or absence of an α-helix might produce conformation changes in the Rossmann-like fold structure. The significantly high evolutionary rates for UFGT genes in Taiwanese lineages suggested rapid accumulation of amino acid mutations in island species. The results showed positive selection in closely related species and explained the high diversification of floral patterns in these recently diverged species. In contrast, non-synonymous mutation rate decreases in long-term divergent species could reduce mutational load and prevent the accumulation of deleterious mutations. CONCLUSIONS: Together with the positive selection of transcription factors for ABP genes described in a previous study, these results confirmed that positive selection takes place at a translational level and suggested that the high divergence of ABP genes is related to the floral diversity in island Scutellaria species.

Immature biology and morphology of an obligate myrmecophilous butterfly Catapaecilma major moltrechti (Wileman) (Lepidoptera: Lycaenidae) from Taiwa.

The immature biology and morphology of Catapaecilma major moltrechti (Wileman) from Taiwan are reported. The larva of this taxon is proven to be largely predacious on a few scale insect species attended by ant Crematogester rogenhoferi in satellite nests constructed by the ants, but it also consumes plant material within or near ants' satellite nests in elder instars. The discovery of the food habit of this taxon is interesting as the larva of C. major is known to be phytophagous in India and Sri Lanka.

Descriptions of two new notodontid species from the relic Fagus forests in northeastern Taiwan (Lepidoptera, Notodontidae).

Two new Fagus-feeding notodontids, i.e. Syntypistis taipingshanensis Wu & Hsu sp. n. and Pheosiopsis seni Wu & Hsu sp. n. are recently discovered from the relic Fagus forests in northeastern Taiwan. Based on the genitalia structures, the closely relatives of two new species are S. melana Wu & Fang, 2003 and P. albalienata Kishida & Kobayashi, 2005, respectively, both occurring in the Fagus forests of Southern China.

Changes of diet and dominant intestinal microbes in farmland frogs.

BACKGROUND: Agricultural activities inevitably result in anthropogenic interference with natural habitats. The diet and the gut microbiota of farmland wildlife can be altered due to the changes in food webs within agricultural ecosystems. In this work, we compared the diet and intestinal microbiota of the frog Fejervarya limnocharis in natural and farmland habitats in order to understand how custom farming affects the health of in vivo microbial ecosystems. RESULTS: The occurrence, abundance, and the numbers of prey categories of stomach content were significantly different between the frogs inhabiting natural and farmland habitats. In addition, differences in the abundance, species richness, and alpha-diversity of intestinal microbial communities were also statistically significant. The microbial composition, and particularly the composition of dominant microbes living in intestines, indicated that the land use practices might be one of factors affecting the gut microbial community composition. Although the first three dominant microbial phyla Bacteroidetes, Firmicutes, and Proteobacteria found in the intestines of frogs were classified as generalists among habitats, the most dominant gut bacterial phylum Bacteroidetes in natural environments was replaced by the microbial phylum Firmicutes in farmland frogs. Increased intestinal microbial richness of the farmland frogs, which is mostly contributed by numerous microbial species of Proteobacteria, Actinobacteria, Acidobacteria, and Planctomycetes, not only reflects the possible shifts in microbial community composition through the alteration of external ecosystem, but also indicates the higher risk of invasion by disease-related microbes. CONCLUSIONS: This study indicates that anthropogenic activities, such as the custom farming, have not only affected the food resources of frogs, but also influenced the health and in vivo microbial ecosystem of wildlife.