The mitogenome of a Malagasy butterfly Malaza fastuosus (Mabille, 1884) recovered from the holotype collected over 140 years ago adds support for a new subfamily of Hesperiidae (Lepidoptera).

Malaza fastuosus is a lavishly patterned skipper butterfly from a genus that has three described species, all endemic to the mainland of Madagascar. To our knowledge, M. fastuosus has not been collected for nearly 50 years. To evaluate the power of our techniques to recover DNA, we used a single foreleg of an at least 140-year-old holotype specimen from the collection of the Natural History Museum London with no destruction of external morphology to extract DNA and assemble a complete mitogenome from next generation sequencing reads. The resulting 15 540 bp mitogenome contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and an A+T rich region, similarly to other Lepidoptera mitogenomes. Here we provide the first mitogenome also for Trapezitinae (Rachelia extrusus). Phylogenetic analysis of available skipper mitogenomes places Malaza outside of Trapezitinae and Barcinae + Hesperiinae, with a possible sister relationship to Heteropterinae. Of these, at least Heteropterinae, Trapezitinae, and almost all Hesperiinae have monocot-feeding caterpillars. Malaza appears to be an evolutionarily highly distinct ancient lineage, morphologically with several unusual hesperiid features. The monotypic subfamily Malazinae Lees & Grishin subfam. nov. (type genus Malaza) is proposed to reflect this morphological and molecular evidence.

Unravelling relationships among the shared stripes of sailors: Mitogenomic phylogeny of Limenitidini butterflies (Lepidoptera, Nymphalidae, Limenitidinae), focusing on the genera Athyma and Limenitis.

The phylogenetic relationships of the nymphalid butterfly tribe Limenitidini are best known for the genera Limenitis and Adelpha, model taxa for evolutionary processes such as Batesian mimicry and rapid adaptive radiations. Whereas these American limenitidines have received the most attention, phylogenetic relationships of their Asian relatives are still controversial and largely unexplored. Even one of the largest genera in Asia, Athyma, is polyphyletic. To clarify the phylogenetic relationships of these Asian Limenitidini, a total of 53 representatives were sampled; 37 have their mitogenomes sequenced for the first time. Our phylogenetic results confirm that mitogenomic data provides well-resolved relationships at most major levels of the phylogeny, even using different partition schemes or different inference methods. Interestingly, our results show that some Athyma taxa are embedded within the genus Limenitis, whereas the genus Tacola, previously considered to be a synonym of Athyma, needs to be recognized as a valid clade. Additionally, the other Limenitidini genera in Asia (namely Tarattia, Litinga, Sumalia, Pandita and Patsuia) are now grouped either within Athyma or Limenitis, so these genera need to be sunk. Importantly, we also show that the mainly Old World Limenitis and entirely New World Adelpha are sister groups, confirming the relevance of Asian lineages to global studies of Limenitis evolution.

DNA barcodes reveal deeply neglected diversity and numerous invasions of micromoths in Madagascar 1.

Madagascar is a prime evolutionary hotspot globally, but its unique biodiversity is under threat, essentially from anthropogenic disturbance. There is a race against time to describe and protect the Madagascan endangered biota. Here we present a first molecular characterization of the micromoth fauna of Madagascar. We collected 1572 micromoths mainly using light traps in both natural and anthropogenically disturbed habitats in 24 localities across eastern and northwest Madagascar. We also collected 1384 specimens using a Malaise trap in a primary rain forest at Andasibe, eastern Madagascar. In total, we DNA barcoded 2956 specimens belonging to 1537 Barcode Index Numbers (BINs), 88.4% of which are new to BOLD. Only 1.7% of new BINs were assigned to species. Of 47 different families found, Dryadaulidae, Bucculatricidae, Bedelliidae, Batrachedridae, and Blastobasidae are newly reported for Madagascar and the recently recognized Tonzidae is confirmed. For test faunas of Canada and Australia, 98.9%-99.4% of Macroheterocera BINs exhibited the molecular synapomorphy of a phenylalanine in the 177th complete DNA barcode codon. Non-macroheteroceran BINs could thus be sifted out efficiently in the Malaise sample. The Madagascar micromoth fauna shows highest affinity with the Afrotropics (146 BINs also occur in the African continent). We found 22 recognised pests or invasive species, mostly occurring in disturbed habitats. Malaise trap samples show high temporal turnover and alpha diversity with as many as 507 BINs collected; of these, astonishingly, 499 (98.4%) were novel to BOLD and 292 (57.6%) were singletons. Our results provide a baseline for future surveys across the island.

Systematics and historical biogeography of the old world butterfly subtribe Mycalesina (Lepidoptera: Nymphalidae: Satyrinae).

BACKGROUND: Butterflies of the subtribe Mycalesina have radiated successfully in almost all habitat types in Africa, Madagascar, the Indian subcontinent, Indo-China and Australasia. Studies aimed at understanding the reasons behind the evolutionary success of this spectacular Old World butterfly radiation have been hampered by the lack of a stable phylogeny for the group. Here, we have reconstructed a robust phylogenetic framework for the subtribe using 10 genes from 195 exemplar taxa. RESULTS: We recovered seven well supported clades within the subtribe corresponding to the five traditional genera (Lohora, Heteropsis, Hallelesis, Bicyclus, Mycalesis), one as recently revised (Mydosama) and one newly revised genus (Culapa). The phylogenetic relationships of these mycalesine genera have been robustly established for the first time. Within the proposed phylogenetic framework, we estimated the crown age of the subtribe to be 40 Million years ago (Mya) and inferred its ultimate origin to be in Asia. Our results reveal both vicariance and dispersal as factors responsible for the current widespread distribution of the group in the Old World tropics. We inferred that the African continent has been colonized at least twice by Asian mycalesines within the last 26 and 23 Mya. In one possible scenario, an Asian ancestor gave rise to Heteropsis on continental Africa, which later dispersed into Madagascar and most likely back colonised Asia. The second colonization of Africa by Asian ancestors resulted in Hallelesis and Bicyclus on continental Africa, the descendants of which did not colonise other regions but rather diversified only in continental Africa. The genera Lohora and Mydosama are derivatives of ancestors from continental Asia. CONCLUSION: Our proposed time-calibrated phylogeny now provides a solid framework within which we can implement mechanistic studies aimed at unravelling the ecological and evolutionary processes that culminated in the spectacular radiation of mycalesines in the Old World tropics.

A mitochondrial genome phylogeny of owlet moths (Lepidoptera: Noctuoidea), and examination of the utility of mitochondrial genomes for lepidopteran phylogenetics.

A phylogenetic hypothesis for the lepidopteran superfamily Noctuoidea was inferred based on the complete mitochondrial (mt) genomes of 12 species (six newly sequenced). The monophyly of each noctuoid family in the latest classification was well supported. Novel and robust relationships were recovered at the family level, in contrast to previous analyses using nuclear genes. Erebidae was recovered as sister to (Nolidae+(Euteliidae+Noctuidae)), while Notodontidae was sister to all these taxa (the putatively basalmost lineage Oenosandridae was not included). In order to improve phylogenetic resolution using mt genomes, various analytical approaches were tested: Bayesian inference (BI) vs. maximum likelihood (ML), excluding vs. including RNA genes (rRNA or tRNA), and Gblocks treatment. The evolutionary signal within mt genomes had low sensitivity to analytical changes. Inference methods had the most significant influence. Inclusion of tRNAs positively increased the congruence of topologies, while inclusion of rRNAs resulted in a range of phylogenetic relationships varying depending on other analytical factors. The two Gblocks parameter settings had opposite effects on nodal support between the two inference methods. The relaxed parameter (GBRA) resulted in higher support values in BI analyses, while the strict parameter (GBDH) resulted in higher support values in ML analyses.

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).

BACKGROUND: Mitogenomic 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. RESULTS: We 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. CONCLUSIONS: Thirty 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.

Towards a mitogenomic phylogeny of Lepidoptera.

The backbone phylogeny of Lepidoptera remains unresolved, despite strenuous recent morphological and molecular efforts. Molecular studies have focused on nuclear protein coding genes, sometimes adding a single mitochondrial gene. Recent advances in sequencing technology have, however, made acquisition of entire mitochondrial genomes both practical and economically viable. Prior phylogenetic studies utilised just eight of 43 currently recognised lepidopteran superfamilies. Here, we add 23 full and six partial mitochondrial genomes (comprising 22 superfamilies of which 16 are newly represented) to those publically available for a total of 24 superfamilies and ask whether such a sample can resolve deeper lepidopteran phylogeny. Using recoded datasets we obtain topologies that are highly congruent with prior nuclear and/or morphological studies. Our study shows support for an expanded Obtectomera including Gelechioidea, Thyridoidea, plume moths (Alucitoidea and Pterophoroidea; possibly along with Epermenioidea), Papilionoidea, Pyraloidea, Mimallonoidea and Macroheterocera. Regarding other controversially positioned higher taxa, Doidae is supported within the new concept of Drepanoidea and Mimallonidae sister to (or part of) Macroheterocera, while among Nymphalidae butterflies, Danainae and not Libytheinae are sister to the remainder of the family. At the deepest level, we suggest that a tRNA rearrangement occurred at a node between Adeloidea and Ditrysia+Palaephatidae+Tischeriidae.

The genome sequence of the Hoary Footman, Eilema caniola (Hübner, 1808).

We present a genome assembly from one female Eilema caniola (the Hoary Footman; Arthropoda; Insecta; Lepidoptera; Erebidae). The genome sequence is 781.7 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the W and Z sex chromosomes. The mitochondrial genome has also been assembled and is 15.42 kilobases in length. Gene annotation of this assembly on Ensembl identified 22,953 protein coding genes.

The genome sequence of the Peppered Grey, Eudonia truncicolella (Stainton, 1849).

We present a genome assembly from an individual male Eudonia truncicolella (the Peppered Grey; Arthropoda; Insecta; Lepidoptera; Crambidae). The genome sequence is 499.1 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.38 kilobases in length.

The genome sequence of the Chequered Fruit-tree Tortrix, Pandemis corylana (Fabricius, 1794).

We present a genome assembly from an individual male Pandemis corylana (the Chequered Fruit-tree Tortrix; Arthropoda; Insecta; Lepidoptera; Tortricidae). The genome sequence is 441.6 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.53 kilobases in length. Gene annotation of this assembly on Ensembl identified 19,608 protein coding genes.

Global arthropod beta-diversity is spatially and temporally structured by latitude.

Global biodiversity gradients are generally expected to reflect greater species replacement closer to the equator. However, empirical validation of global biodiversity gradients largely relies on vertebrates, plants, and other less diverse taxa. Here we assess the temporal and spatial dynamics of global arthropod biodiversity dynamics using a beta-diversity framework. Sampling includes 129 sampling sites whereby malaise traps are deployed to monitor temporal changes in arthropod communities. Overall, we encountered more than 150,000 unique barcode index numbers (BINs) (i.e. species proxies). We assess between site differences in community diversity using beta-diversity and the partitioned components of species replacement and richness difference. Global total beta-diversity (dissimilarity) increases with decreasing latitude, greater spatial distance and greater temporal distance. Species replacement and richness difference patterns vary across biogeographic regions. Our findings support long-standing, general expectations of global biodiversity patterns. However, we also show that the underlying processes driving patterns may be regionally linked.

The genome sequence of the Ochreous Pearl, Anania crocealis (Hübner, 1796).

We present a genome assembly from an individual male Anania crocealis (the Ochreous Pearl; Arthropoda; Insecta; Lepidoptera; Crambidae). The genome sequence is 624.0 megabases in span. Most of the assembly is scaffolded into 29 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.33 kilobases in length. Gene annotation of this assembly on Ensembl identified 20,293 protein coding genes.

The genome sequence of the Chestnut, Conistra vaccinii (Linnaeus, 1761).

We present a genome assembly from an individual male Conistra vaccinii (the Chestnut; Arthropoda; None; Lepidoptera; Noctuidae). The genome sequence is 720.8 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.44 kilobases in length. Gene annotation of this assembly on Ensembl identified 13,109 protein coding genes.

The genome sequence of the Autumnal Rustic, Eugnorisma glareosa (Esper, 1788).

We present a genome assembly from an individual male Eugnorisma glareosa (the Autumnal Rustic; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 631.0 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.39 kilobases in length. Gene annotation of this assembly on Ensembl identified 19,768 protein coding genes.

The genome sequence of the Grey Pine Carpet, Thera obeliscata (Hübner, 1787).

We present a genome assembly from an individual male Thera obeliscata (the Grey Pine Carpet; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 404.7 megabases in span. Most of the assembly is scaffolded into 18 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 17.5 kilobases in length.

The genome sequence of the Chevron, Eulithis testata (Linnaeus, 1761).

We present a genome assembly from an individual male Eulithis testata (the Chevron; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 308.1 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.9 kilobases in length. Gene annotation of this assembly on Ensembl identified 16,167 protein coding genes.

The genome sequence of the Brown-spot Pinion, Agrochola litura (Linnaeus, 1761).

We present a genome assembly from an individual female Agrochola litura (the Brown-spot Pinion; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 772.2 megabases in span. Most of the assembly is scaffolded into 32 chromosomal pseudomolecules, including the W and Z sex chromosomes. The mitochondrial genome has also been assembled and is 15.55 kilobases in length. Gene annotation of this assembly on Ensembl identified 19,500 protein coding genes.

The genome sequence of the Ashy Button, Acleris sparsana (Denis & Schiffermüller, 1775).

We present a genome assembly from an individual male Acleris sparsana (the Ashy Button; Arthropoda; Insecta; Lepidoptera; Tortricidae). The genome sequence is 589.5 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 16.4 kilobases in length. Gene annotation of this assembly on Ensembl identified 22,123 protein coding genes.

The identity of Argyrialacteella (Fabricius, 1794) (Lepidoptera, Pyraloidea, Crambinae), synonyms, and related species revealed by morphology and DNA capture in type specimens.

In this study the aim was to resolve the taxonomy of several species of Argyria Hübner (Pyraloidea, Crambinae) with previously unrecognised morphological variation. By analysing the DNA barcode (COI-5P) in numerous specimens, the aim was to reconstruct phylogenetic relationships between species, to provide better evidence for synonymies, and to circumscribe their geographical distribution. Using an innovative DNA hybridisation capture protocol, the DNA barcode of the lectotype of Argyrialacteella (Fabricius, 1794) was partially recovered for comparison with the 229 DNA barcode sequences of Argyria specimens available in the Barcode of Life Datasystems, and this firmly establishes the identity of the species. The same protocol was used for the following type specimens: the Argyriaabronalis (Walker, 1859) holotype, thus confirming the synonymy of this name with A.lacteella, the holotype of A.lusella (Zeller, 1863), syn. rev., the holotype of A.multifacta Dyar, 1914, syn. nov. newly synonymised with A.lacteella, and a specimen of Argyriadiplomochalis Dyar, 1913, collected in 1992. In addition, nine specimens of A.lacteella, A.diplomochalis, A.centrifugens Dyar, 1914 and A.gonogramma Dyar, 1915, from North to South America were sampled using classical COI amplification and Sanger sequencing. Argyriagonogramma Dyar, described from Bermuda, is the name to be applied to the more widespread North American species formerly identified as A.lacteella. Following morphological study of its holotype, Argyriavestalis Butler, 1878, syn. nov. is also synonymised with A.lacteella. The name A.pusillalis Hübner, 1818, is considered a nomen dubium associated with A.gonogramma. The adult morphology is diagnosed and illustrated, and distributions are plotted for A.lacteella, A.diplomochalis, A.centrifugens, and A.gonogramma based on slightly more than 800 specimens. For the first time, DNA barcode sequences are provided for the Antillean A.diplomochalis. This work provides a modified, improved protocol for the efficient hybrid capture enrichment of DNA barcodes from 18th and 19th century type specimens in order to solve taxonomic issues in Lepidoptera.