The evolution and diversification of oakleaf butterflies.

Oakleaf butterflies in the genus Kallima have a polymorphic wing phenotype, enabling these insects to masquerade as dead leaves. This iconic example of protective resemblance provides an interesting evolutionary paradigm that can be employed to study biodiversity. We integrated multi-omic data analyses and functional validation to infer the evolutionary history of Kallima species and investigate the genetic basis of their variable leaf wing patterns. We find that Kallima butterflies diversified in the eastern Himalayas and dispersed to East and Southeast Asia. Moreover, we find that leaf wing polymorphism is controlled by the wing patterning gene cortex, which has been maintained in Kallima by long-term balancing selection. Our results provide macroevolutionary and microevolutionary insights into a model species originating from a mountain ecosystem.

To and fro in the archipelago: Repeated inter-island dispersal and New Guinea's orogeny affect diversification of Delias, the world's largest butterfly genus.

The world's largest butterfly genus Delias, commonly known as Jezebels, comprises ca. 251 species found throughout Asia, Australia, and Melanesia. Most species are endemic to islands in the Indo-Australian Archipelago or to New Guinea and nearby islands in Melanesia, and many species are restricted to montane habitats over 1200 m. We inferred an extensively sampled and well-supported molecular phylogeny of the group to better understand the spatial and temporal dimensions of its diversification. The remarkable diversity of Delias evolved in just ca. 15-16 Myr (crown age). The most recent common ancestor of a clade with most of the species dispersed out of New Guinea ca. 14 Mya, but at least six subsequently diverging lineages dispersed back to the island. Diversification was associated with frequent dispersal of lineages among the islands of the Indo-Australian Archipelago, and the divergence of sister taxa on a single landmass was rare and occurred only on the largest islands, most notably on New Guinea. We conclude that frequent inter-island dispersal during the Neogene-likely facilitated by frequent sea level change-sparked much diversification during that period. Many extant New Guinea lineages started diversifying 5 Mya, suggesting that orogeny facilitated their diversification. Our results largely agree with the most recently proposed species group classification system, and we use our large taxon sample to extend this system to all described species. Finally, we summarize recent insights to speculate how wing pattern evolution, mimicry, and sexual selection might also contribute to these butterflies' rapid speciation and diversification.

Miocene Climate and Habitat Change Drove Diversification in Bicyclus, Africa's Largest Radiation of Satyrine Butterflies.

Compared to other regions, the drivers of diversification in Africa are poorly understood. We studied a radiation of insects with over 100 species occurring in a wide range of habitats across the Afrotropics to investigate the fundamental evolutionary processes and geological events that generate and maintain patterns of species richness on the continent. By investigating the evolutionary history of Bicyclus butterflies within a phylogenetic framework, we inferred the group's origin at the Oligo-Miocene boundary from ancestors in the Congolian rainforests of central Africa. Abrupt climatic fluctuations during the Miocene (ca. 19-17 Ma) likely fragmented ancestral populations, resulting in at least eight early-divergent lineages. Only one of these lineages appears to have diversified during the drastic climate and biome changes of the early Miocene, radiating into the largest group of extant species. The other seven lineages diversified in forest ecosystems during the late Miocene and Pleistocene when climatic conditions were more favorable-warmer and wetter. Our results suggest changing Neogene climate, uplift of eastern African orogens, and biotic interactions have had different effects on the various subclades of Bicyclus, producing one of the most spectacular butterfly radiations in Africa. [Afrotropics; biodiversity; biome; biotic interactions; Court Jester; extinction; grasslands; paleoclimates; Red Queen; refugia forests; dependent-diversification; speciation.].

Out of Asia: Intercontinental dispersals after the Eocene-Oligocene transition shaped the zoogeography of Limenitidinae butterflies (Lepidoptera: Nymphalidae).

Most members of the nymphalid subfamily Limenitidinae are distributed in tropical regions of Africa, Asia, and the Americas. Previous studies have inferred their higher-level phylogeny and found that Southeast Asia seems to be the center of origin, with numerous dispersal events to other continents. However, the complete biogeographic history of Limenitidinae butterflies is still largely unknown. We sampled 181 taxa from 164 species and used a metagenomic method to obtain 40 genes (mitogenomes and three nuclear ribosomal loci) for inferring the historical biogeography of the group. We find that Limenitidinae originated in eastern Asia during the early Eocene (ca. 52 Ma) and started to diversify and disperse into Africa before the end of Eocene. Intercontinental exchanges between Africa and eastern Asia continued in the early Miocene: Asian Adoliadini and Asian endemic taxa Bhagadatta had African origins in the Oligocene, whereas African Neptini dispersed in the opposite direction from Asia in the early Miocene. In addition, ancestors of the tribes Limenitidini and Adoliadini dispersed into the Neotropics and Australasia multiple times during the early-to-middle Miocene. Eastern Asia is the center of origin of the tribe Limenitidini, with several taxa disjunctly distributed in eastern Asia and the Americas. Our work provides a robust phylogenetic hypothesis of relationships in the tribe Limenitidini and suggests that the alala-species group of Adelpha should be placed in the genus Limenitis. Renamed taxa comb. nov. based on our findings are listed in the text.

The evolution and genetics of sexually dimorphic 'dual' mimicry in the butterfly Elymnias hypermnestra.

Sexual dimorphism is a major component of morphological variation across the tree of life, but the mechanisms underlying phenotypic differences between sexes of a single species are poorly understood. We examined the population genomics and biogeography of the common palmfly Elymnias hypermnestra, a dual mimic in which female wing colour patterns are either dark brown (melanic) or bright orange, mimicking toxic Euploea and Danaus species, respectively. As males always have a melanic wing colour pattern, this makes E. hypermnestra a fascinating model organism in which populations vary in sexual dimorphism. Population structure analysis revealed that there were three genetically distinct E. hypermnestra populations, which we further validated by creating a phylogenomic species tree and inferring historical barriers to gene flow. This species tree demonstrated that multiple lineages with orange females do not form a monophyletic group, and the same is true of clades with melanic females. We identified two single nucleotide polymorphisms (SNPs) near the colour patterning gene WntA that were significantly associated with the female colour pattern polymorphism, suggesting that this gene affects sexual dimorphism. Given WntA's role in colour patterning across Nymphalidae, E. hypermnestra females demonstrate the repeatability of the evolution of sexual dimorphism.

Evolutionary trade-offs between male secondary sexual traits revealed by a phylogeny of the hyperdiverse tribe Eumaeini (Lepidoptera: Lycaenidae).

Male butterflies in the hyperdiverse tribe Eumaeini possess an unusually complex and diverse repertoire of secondary sexual characteristics involved in pheromone production and dissemination. Maintaining multiple sexually selected traits is likely to be metabolically costly, potentially resulting in trade-offs in the evolution of male signals. However, a phylogenetic framework to test hypotheses regarding the evolution and maintenance of male sexual traits in Eumaeini has been lacking. Here, we infer a comprehensive, time-calibrated phylogeny from 379 loci for 187 species representing 91% of the 87 described genera. Eumaeini is a monophyletic group that originated in the late Oligocene and underwent rapid radiation in the Neotropics. We examined specimens of 818 of the 1096 described species (75%) and found that secondary sexual traits are present in males of 91% of the surveyed species. Scent pads and scent patches on the wings and brush organs associated with the genitalia were probably present in the common ancestor of Eumaeini and are widespread throughout the tribe. Brush organs and scent pads are negatively correlated across the phylogeny, exhibiting a trade-off in which lineages with brush organs are unlikely to regain scent pads and vice versa. In contrast, scent patches seem to facilitate the evolution of scent pads, although they are readily lost once scent pads have evolved. Our results illustrate the complex interplay between natural and sexual selection in the origin and maintenance of multiple male secondary sexual characteristics and highlight the potential role of sexual selection spurring diversification in this lineage.

Investigation of an Elevational Gradient Reveals Strong Differences Between Bacterial and Eukaryotic Communities Coinhabiting Nepenthes Phytotelmata.

Elevation is an important determinant of ecological community composition. It integrates several abiotic features and leads to strong, repeatable patterns of community structure, including changes in the abundance and richness of numerous taxa. However, the influence of elevational gradients on microbes is understudied relative to plants and animals. To compare the influence of elevation on multiple taxa simultaneously, we sampled phytotelm communities within a tropical pitcher plant (Nepenthes mindanaoensis) along a gradient from 400 to 1200 m a.s.l. We use a combination of metabarcoding and physical counts to assess diversity and richness of bacteria, micro-eukaryotes, and arthropods, and compare the effect of elevation on community structure to that of regulation by a number of plant factors. Patterns of community structure differed between bacteria and eukaryotes, despite their living together in the same aquatic microhabitats. Elevation influences community composition of eukaryotes to a significantly greater degree than it does bacteria. When examining pitcher characteristics, pitcher dimorphism has an effect on eukaryotes but not bacteria, while variation in pH levels strongly influences both taxa. Consistent with previous ecological studies, arthropod abundance in phytotelmata decreases with elevation, but some patterns of abundance differ between living inquilines and prey.

Anchored phylogenomics illuminates the skipper butterfly tree of life.

BACKGROUND: Butterflies (Papilionoidea) are perhaps the most charismatic insect lineage, yet phylogenetic relationships among them remain incompletely studied and controversial. This is especially true for skippers (Hesperiidae), one of the most species-rich and poorly studied butterfly families. METHODS: To infer a robust phylogenomic hypothesis for Hesperiidae, we sequenced nearly 400 loci using Anchored Hybrid Enrichment and sampled all tribes and more than 120 genera of skippers. Molecular datasets were analyzed using maximum-likelihood, parsimony and coalescent multi-species phylogenetic methods. RESULTS: All analyses converged on a novel, robust phylogenetic hypothesis for skippers. Different optimality criteria and methodologies recovered almost identical phylogenetic trees with strong nodal support at nearly all nodes and all taxonomic levels. Our results support Coeliadinae as the sister group to the remaining skippers, the monotypic Euschemoninae as the sister group to all other subfamilies but Coeliadinae, and the monophyly of Eudaminae plus Pyrginae. Within Pyrginae, Celaenorrhinini and Tagiadini are sister groups, the Neotropical firetips, Pyrrhopygini, are sister to all other tribes but Celaenorrhinini and Tagiadini. Achlyodini is recovered as the sister group to Carcharodini, and Erynnini as sister group to Pyrgini. Within the grass skippers (Hesperiinae), there is strong support for the monophyly of Aeromachini plus remaining Hesperiinae. The giant skippers (Agathymus and Megathymus) once classified as a subfamily, are recovered as monophyletic with strong support, but are deeply nested within Hesperiinae. CONCLUSIONS: Anchored Hybrid Enrichment sequencing resulted in a large amount of data that built the foundation for a new, robust evolutionary tree of skippers. The newly inferred phylogenetic tree resolves long-standing systematic issues and changes our understanding of the skipper tree of life. These resultsenhance understanding of the evolution of one of the most species-rich butterfly families.

Evolution of Hypolimnas butterflies (Nymphalidae): Out-of-Africa origin and Wolbachia-mediated introgression.

Hypolimnas butterflies (Nymphalidae), commonly known as eggflies, are a popular model system for studying a wide range of ecological questions including mimicry, polymorphism, wing pattern evolution, and Wolbachia-host interactions. The lack of a time-calibrated phylogeny for this group has precluded understanding its evolutionary history. We reconstruct a species-level phylogeny using a nine gene dataset and estimate species divergence times. Based on the resulting tree, we investigate the taxon's historical biogeography, examine the evolution of host plant preferences, and test the hypothesis that the endosymbiotic bacterium Wolbachia mediates gene transfer between species. Our analyses indicate that the species are grouped within three strongly supported, deeply divergent clades. However, relationships among these three clades are uncertain. In addition, many Hypolimnas species are not monophyletic or monophyletic with weak support, suggesting widespread incomplete lineage sorting and/or introgression. Biogeographic analysis strongly indicates that the genus diverged from its ancestor in Africa and subsequently dispersed to Asia; the strength of this result is not affected by topological uncertainties. While the larvae of African species feed almost exclusively on Urticaceae, larvae of species found further east often feed on several additional families. Interestingly, we found an identical mitochondrial haplotype in two Hypolimnas species, H. bolina and H. alimena, and a strong association between this mitotype and the Wolbachia strain wBol1a. Future investigations should explore the plausibility of Wolbachia-mediated introgression between species.

Phylogenetics of moth-like butterflies (Papilionoidea: Hedylidae) based on a new 13-locus target capture probe set.

The Neotropical moth-like butterflies (Hedylidae) are perhaps the most unusual butterfly family. In addition to being species-poor, this family is predominantly nocturnal and has anti-bat ultrasound hearing organs. Evolutionary relationships among the 36 described species are largely unexplored. A new, target capture, anchored hybrid enrichment probe set ('BUTTERFLY2.0') was developed to infer relationships of hedylids and some of their butterfly relatives. The probe set includes 13 genes that have historically been used in butterfly phylogenetics. Our dataset comprised of up to 10,898 aligned base pairs from 22 hedylid species and 19 outgroups. Eleven of the thirteen loci were successfully captured from all samples, and the remaining loci were captured from ≥94% of samples. The inferred phylogeny was consistent with recent molecular studies by placing Hedylidae sister to Hesperiidae, and the tree had robust support for 80% of nodes. Our results are also consistent with morphological studies, with Macrosoma tipulata as the sister species to all remaining hedylids, followed by M. semiermis sister to the remaining species in the genus. We tested the hypothesis that nocturnality evolved once from diurnality in Hedylidae, and demonstrate that the ancestral condition was likely diurnal, with a shift to nocturnality early in the diversification of this family. The BUTTERFLY2.0 probe set includes standard butterfly phylogenetics markers, captures sequences from decades-old museum specimens, and is a cost-effective technique to infer phylogenetic relationships of the butterfly tree of life.

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.

Comprehensive phylogeny of Pieridae butterflies reveals strong correlation between diversification and temperature.

Temperature is thought to be a key factor influencing global species richness patterns. We investigate the link between temperature and diversification in the butterfly family Pieridae by combining next generation DNA sequences and published molecular data with fine-grained distribution data. We sampled nearly 600 pierid butterfly species to infer the most comprehensive molecular phylogeny of the family and curated a distribution dataset of more than 800,000 occurrences. We found strong evidence that species in environments with more stable daily temperatures or cooler maximum temperatures in the warm seasons have higher speciation rates. Furthermore, speciation and extinction rates decreased in tandem with global temperatures through geological time, resulting in a constant net diversification.

Global determinants of insect mitochondrial genetic diversity.

Understanding global patterns of genetic diversity is essential for describing, monitoring, and preserving life on Earth. To date, efforts to map macrogenetic patterns have been restricted to vertebrates, which comprise only a small fraction of Earth's biodiversity. Here, we construct a global map of predicted insect mitochondrial genetic diversity from cytochrome c oxidase subunit 1 sequences, derived from open data. We calculate the mitochondrial genetic diversity mean and genetic diversity evenness of insect assemblages across the globe, identify their environmental correlates, and make predictions of mitochondrial genetic diversity levels in unsampled areas based on environmental data. Using a large single-locus genetic dataset of over 2 million globally distributed and georeferenced mtDNA sequences, we find that mitochondrial genetic diversity evenness follows a quadratic latitudinal gradient peaking in the subtropics. Both mitochondrial genetic diversity mean and evenness positively correlate with seasonally hot temperatures, as well as climate stability since the last glacial maximum. Our models explain 27.9% and 24.0% of the observed variation in mitochondrial genetic diversity mean and evenness in insects, respectively, making an important step towards understanding global biodiversity patterns in the most diverse animal taxon.

Bacterial gut symbionts are tightly linked with the evolution of herbivory in ants.

Ants are a dominant feature of terrestrial ecosystems, yet we know little about the forces that drive their evolution. Recent findings illustrate that their diets range from herbivorous to predaceous, with "herbivores" feeding primarily on exudates from plants and sap-feeding insects. Persistence on these nitrogen-poor food sources raises the question of how ants obtain sufficient nutrition. To investigate the potential role of symbiotic microbes, we have surveyed 283 species from 18 of the 21 ant subfamilies using molecular techniques. Our findings uncovered a wealth of bacteria from across the ants. Notable among the surveyed hosts were herbivorous "turtle ants" from the related genera Cephalotes and Procryptocerus (tribe Cephalotini). These commonly harbored bacteria from ant-specific clades within the Burkholderiales, Pseudomonadales, Rhizobiales, Verrucomicrobiales, and Xanthomonadales, and studies of lab-reared Cephalotes varians characterized these microbes as symbiotic residents of ant guts. Although most of these symbionts were confined to turtle ants, bacteria from an ant-specific clade of Rhizobiales were more broadly distributed. Statistical analyses revealed a strong relationship between herbivory and the prevalence of Rhizobiales gut symbionts within ant genera. Furthermore, a consideration of the ant phylogeny identified at least five independent origins of symbioses between herbivorous ants and related Rhizobiales. Combined with previous findings and the potential for symbiotic nitrogen fixation, our results strongly support the hypothesis that bacteria have facilitated convergent evolution of herbivory across the ants, further implicating symbiosis as a major force in ant evolution.

Graphium septentrionicolus Page amp; Treadaway, 2013 (Lepidoptera: Papilionidae) is a distinct species.

After molecular and morphological analyses, the taxon septentrionicolus Page Treadaway, 2013 is shown to be a distinct species, and Graphium adonarensis (Rothschild, 1896) is placed as conspecific with Graphium sarpedon (Linnaeus, 1758). Graphium huangshanensis Wu Ma, 2016 syn. nov. is synonymised with G. septentrionicolus.

Co-circulation of alpha- and beta-coronaviruses in Pteropus vampyrus flying foxes from Indonesia.

Bats are important reservoirs for alpha- and beta-coronaviruses. Coronaviruses (CoV) have been detected in pteropodid bats from several Southeast Asian countries, but little is known about coronaviruses in the Indonesian archipelago in proportion to its mammalian biodiversity. In this study, we screened pooled faecal samples from the Indonesian colonies of Pteropus vampyrus with unbiased next-generation sequencing. Bat CoVs related to Rousettus leschenaultii CoV HKU9 and Eidolon helvum CoV were detected. The 121 faecal samples were further screened using a conventional hemi-nested pan-coronavirus PCR assay. Three positive samples were successfully sequenced, and phylogenetic reconstruction revealed the presence of alpha- and beta-coronaviruses. CoVs belonging to the subgenera Nobecovirus, Decacovirus and Pedacovirus were detected in a single P. vampyrus roost. This study expands current knowledge of coronavirus diversity in Indonesian flying foxes, highlighting the need for longitudinal surveillance of colonies as continuing urbanization and deforestation heighten the risk of spillover events.

LepTraits 1.0 A globally comprehensive dataset of butterfly traits.

Here, we present the largest, global dataset of Lepidopteran traits, focusing initially on butterflies (ca. 12,500 species records). These traits are derived from field guides, taxonomic treatments, and other literature resources. We present traits on wing size, phenology,voltinism, diapause/overwintering stage, hostplant associations, and habitat affinities (canopy, edge, moisture, and disturbance). This dataset will facilitate comparative research on butterfly ecology and evolution and our goal is to inspire future research collaboration and the continued development of this dataset.

SequenceMatrix: concatenation software for the fast assembly of multi-gene datasets with character set and codon information.

We present SequenceMatrix, software that is designed to facilitate the assembly and analysis of multi-gene datasets. Genes are concatenated by dragging and dropping FASTA, NEXUS, or TNT files with aligned sequences into the program window. A multi-gene dataset is concatenated and displayed in a spreadsheet; each sequence is represented by a cell that provides information on sequence length, number of indels, the number of ambiguous bases ("Ns"), and the availability of codon information. Alternatively, GenBank numbers for the sequences can be displayed and exported. Matrices with hundreds of genes and taxa can be concatenated within minutes and exported in TNT, NEXUS, or PHYLIP formats, preserving both character set and codon information for TNT and NEXUS files. SequenceMatrix also creates taxon sets listing taxa with a minimum number of characters or gene fragments, which helps assess preliminary datasets. Entire taxa, whole gene fragments, or individual sequences for a particular gene and species can be excluded from export. Data matrices can be re-split into their component genes and the gene fragments can be exported as individual gene files. SequenceMatrix also includes two tools that help to identify sequences that may have been compromised through laboratory contamination or data management error. One tool lists identical or near-identical sequences within genes, while the other compares the pairwise distance pattern of one gene against the pattern for all remaining genes combined. SequenceMatrix is Java-based and compatible with the Microsoft Windows, Apple MacOS X and Linux operating systems. The software is freely available from http://code.google.com/p/sequencematrix/. © The Willi Hennig Society 2010.

Jamides wananga, a new species from Papua New Guinea and Australia (Lepidoptera: Lycaenidae).

Jamides wananga sp. n. is described and illustrated from Madang Province of Papua New Guinea and Dauan Island in the Torres Strait (Queensland, Australia). The new species is similar in appearance to several other Jamides Hübner species in the bochus-group. DNA sequence data and morphology were used to distinguish the nominotypical subspecies found on the New Guinea mainland from J. w. roxina subsp. n. on Dauan Island. Notes on the habitat and behaviour are also provided.

Detection of Tioman Virus in Pteropus vampyrus Near Flores, Indonesia.

Diverse paramyxoviruses have coevolved with their bat hosts, including fruit bats such as flying foxes (Chiroptera: Pteropodidae). Several of these viruses are zoonotic, but the diversity and distribution of Paramyxoviridae are poorly understood. We screened pooled feces samples from three Pteropus vampyrus colonies and assayed tissues, rectal swabs, and oral swabs from 95 individuals of 23 pteropodid species sampled at 17 sites across the Indonesian archipelago with a conventional paramyxovirus PCR; all tested negative. Samples from 43 individuals were screened with next generation sequencing (NGS), and a single Pteropus vampyrus collected near Flores had Tioman virus sequencing reads. Tioman virus is a bat-borne virus in the genus Pararubulavirus with prior evidence of spillover to humans. This work expands the known range of Tioman virus, and it is likely that this isolated colony likely has sustained intergenerational transmission over a long period.