Multi-scale dissection of wing transparency in the clearwing butterfly Phanus vitreus.

Optical transparency is rare in terrestrial organisms, and often originates through loss of pigmentation and reduction in scattering. The coloured wings of some butterflies and moths have repeatedly evolved transparency, offering examples of how they function optically and biologically. Because pigments are primarily localized in the scales that cover a colourless wing membrane, transparency has often evolved through the complete loss of scales or radical modification of their shape. Whereas bristle-like scales have been well documented in glasswing butterflies, other scale modifications resulting in transparency remain understudied. The butterfly Phanus vitreus achieves transparency while retaining its scales and exhibiting blue/cyan transparent zones. Here, we investigate the mechanism of wing transparency in P. vitreus by light microscopy, focused ion beam milling, microspectrophotometry and optical modelling. We show that transparency is achieved via loss of pigments and vertical orientation in normal paddle-like scales. These alterations are combined with an anti-reflective nipple array on portions of the wing membrane being more exposed to light. The blueish coloration of the P. vitreus transparent regions is due to the properties of the wing membrane, and local scale nanostructures. We show that scale retention in the transparent patches might be explained by these perpendicular scales having hydrophobic properties.

Ultrastructure of androconia and surrounding scales of nine species of Hesperiidae (Lepidoptera).

The ultrastructure of androconia and their surrounding scales of nine species in nine genera across four subfamilies of Hesperiidae is studied. This provides a basis for the classification and identification of some genera and species. The wing surface of the scent glands patches was cut with scissors, observed and photographed under an S-4800 scanning electron microscope (at 10.0 kV accelerated pressure). There were significant differences in the types of scent glands patches across subfamilies. The scent glands patches of Pyrginae and Dudaminae are mainly in the costal fold of the forewing, while those of Coeliadinae and Hesperiinae are mainly in the line or circular stigma on the wing surface. The length, breadth and aperture of the androconia were further measured and the data are analysed by variance and multiple comparisons. There are significant differences amongst the subfamilies, except for Dudaminae and Pyrginae. In Hesperiinae, Telicotacolon (Fabricius, 1775) and Ampittiavirgata (Leech, 1890) have no significant difference in the aperture of the androconia, but are significantly different from Thymelicusleoninus (Butler, 1878). There are significant differences in the aperture between Pyrgusalveus's (Hübner, 1803) androconium and the second androconium of Loboclabifasciata (Bremer & Grey, 1853), but not with the first androconium of Loboclabifasciata. The morphology of androconia in the scent glands patches is very similar in Hesperiinae; all are rod-shaped and paddle-like. The scale types around the scent glands patches are different, but there are one or two similar types. To a certain extent, the aperture of the androconia reflects the genetic relationships between subfamilies and species. The differences in scale type and structure of scent glands patches can be used as a reference for the classification of subfamilies and genera in Hesperiidae.

Complete mitochondrial genome sequence of Abraximorpha davidii (Lepidoptera: Hesperiidae: Tagiadinae).

Taxonomic status and phylogenetic position of some skippers within Hesperiidae remains a controversial issue, here, we sequenced and analyzed the complete mitochondrial genome (mitogenome) of Abraximorpha davidii, one of species in Hesperiidae. This mitogenome is 15,469 bp long and encodes 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), and two ribosomal RNA unit genes (rRNAs). The overall base composition of the mitogenome is A 40.2%, T 41.4%, C 11.2%, and G 7.2%, with a high A + T content of 81.6%. Except for cox1 starting with CGA, all other PCGs start with the standard ATN codons (seven ATG and five ATT). Most of the PCGs terminate with the stop codon TAA, whereas cox1, cox2, nad5, and nad4 end with the incomplete codon T-. Phylogenetic analysis showed that A. davidii is closely related to Daimio tethys and Tagiades vajuna, then this clade clusters Ctenoptilum vasava and Celaenorrhinus maculosa.

Cryptic biodiversity of tropical hesperiid caterpillar-attacking parasitoid wasps: three new species of Creagrura Townes (Hymenoptera, Ichneumonidae, Cremastinae) from Costa Rica and Perú.

Background: We describe three new species of the previously monotypic genus Creagrura Townes from Central and South America: C.alejandromasisi sp. n. and C.rogerblancoi sp. n. from Costa Rica and C.allpahuaya sp. n. from Peru, all of which emphasise the unknown parasitoid insect diversity yet to be revealed in the tropics. New information: Host relationships of the two Costa Rican species are described in detail. In addition, it is inferred that the Creagrura wasps find and oviposit in the caterpillar when it is exposed at night, rather than when it is concealed during daylight hours.

An updated list of butterflies (Lepidoptera, Papilionoidea) of two Guatemalan seasonally dry forests.

Guatemala has a great diversity of butterflies, although there have been few intensive surveys on Lepidoptera in the country so far. We present an updated list of 218 species in 149 genera, 19 subfamilies, and six families of butterflies sampled at two seasonally dry forests in the Salamá and Motagua valleys in central and eastern Guatemala, by integrating new data from field surveys conducted in 2014-2021 into our previously published data (Yoshimoto et al. 2018, 2019), with Amblyscirteselissaelissa Godman, 1900, Repensflorus (Godman, 1900), and Niconiadesnikko Hayward, 1948 (Hesperiidae: Hesperiinae) as new country records. We collected a hairstreak species, Chalybshassan (Stoll, 1790) (Lycaenidae: Theclinae), at the Motagua Valley site, representing the second record for Guatemala since the early 20th century, after we rediscovered it at the Salamá Valley site in 2011 and 2012 (Yoshimoto and Salinas-Gutiérrez 2015). Nymphalidae and Hesperiidae had larger numbers of species than the other four families at both sites. In Pieridae and Nymphalidae, species composition was similar between the sites, whereas in Lycaenidae, Riodinidae, and Papilionidae it differed more greatly between the sites. These results confirm the relatively high lepidopteran diversity of Guatemalan dry forests, noteworthy for the small areas that comprise the study sites, and represent marked similarities and differences in butterfly fauna and phenology within these forests.

It's a moth! It's a butterfly! It's the complete mitochondrial genome of the American moth-butterfly Macrosoma conifera (Warren, 1897) (Insecta: Lepidoptera: Hedylidae)!

The taxonomic placement of the moth-butterfly, Macrosoma conifera (Warren 1897) (Lepidoptera: Hedylidae), has been controversial. The 15,344 bp complete M. conifera circular mitogenome, assembled by genome skimming, consists of 81.7% AT nucleotides, 22 tRNAs, 13 protein-coding genes, 2 rRNAs and a control region in the typical butterfly gene order. Macrosoma conifera COX1 features an atypical CGA start codon while ATP6, COX1, COX2, and ND5 exhibit incomplete stop codons completed by the post-transcriptional addition of 3' A residues. Phylogenetic reconstruction places M. conifera as sister to the skippers (Hesperiidae), which is consistent with several recent phylogenetic analyses.

Hasora mavis Evans, 1934 syn. n. is confirmed to be the female of H. leucospila leucospila (Mabille, 1891) (Hesperiidae, Coeliadinae) by DNA barcoding.

Based upon the comparison of the 649 bp COI gene sequences, Hasora mavis Evans, 1934 is proved to be the female of a sexually dimorphic species, H. leucospila leucospila (Mabille, 1891), and thus treated as a junior subjective synonym of the latter.

Resurrection of Caltoris ranrunna (Sonan) from synonymy as a skipper endemic to Taiwan based on COI barcode and morphology.

Caltoris ranrunna (Sonan, 1936) is resurrected from synonymy of Caltoris cahira austeni (Moore, 1883) to represent a Caltoris species endemic to Taiwan based upon COI barcode divergence and morphological diagnosis in genitalia of both sexes. This species distributes allopatrically from C. cahira of continental Asia and Andamans.

Complete mitochondrial genome of the silver stripped skipper, Leptalina unicolor (Lepidoptera: Hesperiidae).

The silver stripped skipper, Leptalina unicolor Bremer and Grey, 1853 (Lepidoptera: Hesperiidae), is listed as endangered insect in South Korea. We sequenced the whole genome (15,854 bp) of L. unicolor species using Next-Generation Sequencing method and the subsequent gap-filling method. This genome included a set of typical genes and one major non-coding A + T-rich region, with an arrangement identical to that observed in most lepidopteran genomes. Twelve protein-coding genes (PCGs) had the typical ATN start codon, whereas COI had the atypical CGA codon that is frequently found in the start region of the lepidopteran COI. The 757-bp long A + T-rich region was the second largest among completely sequenced Hesperiidae, which ranged from 234 to 793 bp. Phylogenetic reconstruction was performed by maximum-likelihood method using the concatenated sequences of 13 PCGs and two rRNAs of available species of Hesperiidae, including that of L. unicolor (a total of 28 species). The resulting phylogeny provided strong support for monophyletic Heteropterinae in which L. unicolor belongs, with the highest nodal support and a sister relationship between current L. unicolor and co-subfamilial species Carterocephalus silvicola with a bootstrap value of 91%.

Rediscovery of an elusive species, Halpe sikkima Moore, 1882 (Hesperiidae, Hesperiinae) from Hainan, China.

Almost 70 years after its first record from Hainan by Evans in 1949, the second specimen of Halpe sikkima Moore, 1882 was found on the island. The male genitalia of this species are illustrated and re-described. The COI sequence is published for the first time.

Aggregated occurrence records of the federally endangered Poweshiek skipperling (Oarisma poweshiek).

BACKGROUND: Primary biodiversity data records that are open access and available in a standardised format are essential for conservation planning and research on policy-relevant time-scales. We created a dataset to document all known occurrence data for the Federally Endangered Poweshiek skipperling butterfly [Oarisma poweshiek (Parker, 1870; Lepidoptera: Hesperiidae)]. The Poweshiek skipperling was a historically common species in prairie systems across the upper Midwest, United States and Manitoba, Canada. Rapid declines have reduced the number of verified extant sites to six. Aggregating and curating Poweshiek skipperling occurrence records documents and preserves all known distributional data, which can be used to address questions related to Poweshiek skipperling conservation, ecology and biogeography. Over 3500 occurrence records were aggregated over a temporal coverage from 1872 to present. Occurrence records were obtained from 37 data providers in the conservation and natural history collection community using both "HumanObservation" and "PreservedSpecimen" as an acceptable basisOfRecord. Data were obtained in different formats and with differing degrees of quality control. During the data aggregation and cleaning process, we transcribed specimen label data, georeferenced occurrences, adopted a controlled vocabulary, removed duplicates and standardised formatting. We examined the dataset for inconsistencies with known Poweshiek skipperling biogeography and phenology and we verified or removed inconsistencies by working with the original data providers. In total, 12 occurrence records were removed because we identified them to be the western congener Oarisma garita (Reakirt, 1866). This resulting dataset enhances the permanency of Poweshiek skipperling occurrence data in a standardised format. NEW INFORMATION: This is a validated and comprehensive dataset of occurrence records for the Poweshiek skipperling (Oarisma poweshiek) utilising both observation and specimen-based records. Occurrence data are preserved and available for continued research and conservation projects using standardised Darwin Core formatting where possible. Prior to this project, much of these occurrence records were not mobilised and were being stored in individual institutional databases, researcher datasets and personal records. This dataset aggregates presence data from state conservation agencies, natural heritage programmes, natural history collections, citizen scientists, researchers and the U.S. Fish & Wildlife Service. The data include opportunistic observations and collections, research vouchers, observations collected for population monitoring and observations collected using standardised research methodologies. The aggregated occurrence records underwent cleaning efforts that improved data interoperablitity, removed transcription errors and verified or removed uncertain data. This dataset enhances available information on the spatiotemporal distribution of this Federally Endangered species. As part of this aggregation process, we discovered and verified Poweshiek skipperling occurrence records from two previously unknown states, Nebraska and Ohio.

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.

Revalidation of an endemic Cuban Skipper, Chiomara gundlachi (Lepidoptera: Hesperiidae).

In the present work we reassess the taxonomic status of one species of Hesperiidae, subfamily Pyrginae, originally described from Cuba. As part of a larger project on the Greater Antillean butterflies, we obtained COI barcode sequences for Cuban specimens of Chiomara mithrax (Möschler, 1879). The comparison of these sequences revealed deep divergences from others belonging to continental specimens. Inspired by these results we performed morphological comparisons including genitalia structures, which revealed correlating differences between specimens from Cuba and the continent. Therefore, we propose the necessary changes in its taxonomy.

Mitogenomes of Giant-Skipper Butterflies reveal an ancient split between deep and shallow root feeders.

Background: Giant-Skipper butterflies from the genus Megathymus are North American endemics. These large and thick-bodied Skippers resemble moths and are unique in their life cycles. Grub-like at the later stages of development, caterpillars of these species feed and live inside yucca roots. Adults do not feed and are mostly local, not straying far from the patches of yucca plants. Methods: Pieces of muscle were dissected from the thorax of specimens and genomic DNA was extracted (also from the abdomen of a specimen collected nearly 60 years ago). Paired-end libraries were prepared and sequenced for 150bp from both ends. The mitogenomes were assembled from the reads followed by a manual gap-closing procedure and a phylogenetic tree was constructed using a maximum likelihood method from an alignment of the mitogenomes. Results: We determined mitogenome sequences of nominal subspecies of all five known species of Megathymus and Agathymus mariae to confidently root the phylogenetic tree. Pairwise sequence identity indicates the high similarity, ranging from 88-96% among coding regions for 13 proteins, 22 tRNAs and 2 rRNA, with a gene order typical for mitogenomes of Lepidoptera. Phylogenetic analysis confirms that Giant-Skippers (Megathymini) originate within the subfamily Hesperiinae and do not warrant a subfamily rank. Genus Megathymus is monophyletic and splits into two species groups. M. streckeri and M. cofaqui caterpillars feed deep in the main root system of yucca plants and deposit frass underground. M. ursus, M. beulahae and M. yuccae feed in the yucca caudex and roots near the ground, and deposit frass outside through a "tent" (a silk tube projecting from the center of yucca plant). M. yuccae and M. beulahae are sister species consistently with morphological similarities between them. Conclusions: We constructed the first DNA-based phylogeny of the genus Megathymus from their mitogenomes. The phylogeny agrees with morphological considerations.

Ten genes and two topologies: an exploration of higher relationships in skipper butterflies (Hesperiidae).

Despite multiple attempts to infer the higher-level phylogenetic relationships of skipper butterflies (Family Hesperiidae), uncertainties in the deep clade relationships persist. The most recent phylogenetic analysis included fewer than 30% of known genera and data from three gene markers. Here we reconstruct the higher-level relationships with a rich sampling of ten nuclear and mitochondrial markers (7,726 bp) from 270 genera and find two distinct but equally plausible topologies among subfamilies at the base of the tree. In one set of analyses, the nuclear markers suggest two contrasting topologies, one of which is supported by the mitochondrial dataset. However, another set of analyses suggests mito-nuclear conflict as the reason for topological incongruence. Neither topology is strongly supported, and we conclude that there is insufficient phylogenetic evidence in the molecular dataset to resolve these relationships. Nevertheless, taking morphological characters into consideration, we suggest that one of the topologies is more likely.

The complete mitochondrial genome of the butterfly Hasora anura (Lepidoptera: Hesperiidae).

The complete mitochondrial genome (mitogenome) of Hasora anura was sequenced and analyzed in the study. The mitogenome is a typical circular DNA molecule of 15 290 bp, including 37 genes and a putative control region. All protein-coding genes, except for COI begins with the CGA codon as observed in other lepidopterans, start with a typical ATN initiation codon. Eleven genes terminate with TAA or TAG codons, and two genes use a single T residue as the termination codon. All tRNAs have the typical clover-leaf structure, except that the dihydrouridine (DHU) arm of tRNASer(AGN) forms a simple loop. In the sampled species of Hesperiidae, Lobocla bifasciatus (Celaenorrhinus maculosus (Daimio Tethys, Ctenoptilum vasava)), Erynnis montanus, Carterocephalus silvicola, Ampittia dioscorides (Potanthus flavus (Ochlodes venata, Polytremis nascens)), Choaspes benjaminii (Hasora vitta, Hasora anura), are recovered in phylogenetic analyses with high supports.

The complete mitochondrial genome of the Daimio tethys (Lepidoptera: Hesperoidea: Hesperiidae).

The complete mitochondrial genome of Daimio tethys (Hesperoidea: Hesperiidae) is a circular molecule of 15,341 bp in length, containing 37 typical animal mitochondrial genes: 13 protein coding genes (ATP6, ATP8, COI-III, ND1-6, ND4L, Cytb), 2 rRNA genes, 22 tRNA genes and a non-coding AT-rich region. Its gene order and content are identical to all other available butterfly mitogenomes. All PCGs initiate with typical ATN codons, except for COI, which is initiated by the CGA codon as observed in other butterfly species. Ten PCGs use complete termination codons TAA or TAG, whereas the COI, COII and ND5 genes end with a single T. A total of 179 bp of intergenic spacers are interspersed in 11 regions, ranging in size from 1 to 82 bp. The AT-rich region is 415 bp long and contains some conserved structures characteristic of the lepidopteran mitogenomes, including the motif ATAGA followed by a 17 bp poly-T stretch and a microsatellite-like (AT)8 element preceded by the ATTA motif.

Complete mitochondrial genome of the Chinese skipper, Polytremis jigongi (Lepidoptera: Hesperiidae).

The sequence of the mitochondrial genome (mitogenome) of Polytremis jigongi (Lepidoptera: Hesperiidae) has been presented in this article. It is 15,353 bp in length, with an A + T content of 80.9% containing 13 protein-coding genes, 22 tRNAs, 2 rRNAs, and a noncoding control region (D-loop). All of the 37 typical animal mitochondrial genes were found. All protein-coding genes started with ATN as a start codon except for the gene COX1 that uses CGA as in other lepidopteran species. Five protein-coding genes use incomplete stop codon TA or T, while the others use TAA as stop codons. Most of the tRNA genes can be folded into a typical cloverleaf structure. Nucleotide composition is similar to other insects, showing a high bias toward A + T. Phylogenetic analysis showed that the genome sequence of P. jigongi is close to Hesperiidae.

The complete mitochondrial genome of Hasora vitta (Butler, 1870) (Lepidoptera: Hesperiidae).

The complete mitochondrial genome (mitogenome) of Hasora vitta was sequenced and analyzed in the study. The mitogenome is 15 282 bp in size, including 37 genes and a putative control region. Thirteen protein-coding genes all start with a typical ATN codon, expect that COI gene uses CGA as its initial codon. Majority of the 13 PCGs have a complete termination codon (TAA or TAG) except for COII and ND4 have a single T residue. All tRNAs have typical clover-leaf secondary structure, except for tRNASer((AGN)), in which the dihydrouridine (DHU) arm is a simple loop. In the sampled species of Hesperiidae, Hasora vitta, Choaspes benjaminii, Ampittia dioscorides, the remaining sampled species, Celaenorrhinus maculosus (Daimio tethys, Ctenoptilum vasava), Potanthus flavus (Polytremis nascens, Ochlodes venata), are recovered in phylogenetic analyses with high supports.

Complete mitochondrial DNA genome of Polytremis nascens (Lepidoptera: Hesperiidae).

In this study, the complete mitochondrial DNA (mtDNA) sequence of Polytremis nascens (Lepidoptera: Hesperiidae) was determined. The 15,392 bp mitogenome with GenBank accession number KM981865 contained 13 protein genes, 22 tRNAs, 2 rRNAs, and a non-coding control region (D-loop). All the 37 typical animal mitochondrial genes were found. The overall base composition was 39.7% A, 40.7% T, 7.7% G and 11.9% C, with a high A + T content (80.4%). This complete mitogenome of P. nascens provides a basic data for studies on species identification, molecular systematics and conservation genetics.