A New Species of Scymnus (Coleoptera, Coccinellidae) from Pakistan with Mitochondrial Genome and Its Phylogenetic Implications.

In this study, a new species of the subgenus Pullus belonging to the Scymnus genus from Pakistan, Scymnus (Pullus) cardi sp. nov., was described and illustrated, with information on its distribution, host plants, and prey. Additionally, the completed mitochondrial genome (mitogenome) of the new species using high-throughput sequencing technology was obtained. The genome contains the typical 37 genes (13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs) and a non-coding control region, and is arranged in the same order as that of the putative ancestor of beetles. The AT content of the mitogenome is approximately 85.1%, with AT skew and GC skew of 0.05 and -0.43, respectively. The calculated values of relative synonymous codon usage (RSCU) determine that the codon UUA (L) has the highest frequency. Furthermore, we explored the phylogenetic relationship among 59 representatives of the Coccinellidae using Bayesian inference and maximum likelihood methods, the results of which strongly support the monophyly of Coccinellinae. The phylogenetic results positioned Scymnus (Pullus) cardi in a well-supported clade with Scymnus (Pullus) loewii and Scymnus (Pullus) rubricaudus within the genus Scymnus and the tribe Scymnini. The mitochondrial sequence of S. (P.) cardi will contribute to the mitochondrial genome database and provide helpful information for the identification and phylogeny of Coccinellidae.

Host genetics and larval host plant modulate microbiome structure and evolution underlying the intimate insect-microbe-plant interactions in Parnassius species on the Qinghai-Tibet Plateau.

Insects harbor a remarkable diversity of gut microbiomes critical for host survival, health, and fitness, but the mechanism of this structured symbiotic community remains poorly known, especially for the insect group consisting of many closely related species that inhabit the Qinghai-Tibet Plateau. Here, we firstly analyzed population-level 16S rRNA microbial dataset, comprising 11 Parnassius species covering 5 subgenera, from 14 populations mostly sampled in mountainous regions across northwestern-to-southeastern China, and meanwhile clarified the relative importance of multiple factors on gut microbial community structure and evolution. Our findings indicated that both host genetics and larval host plant modulated gut microbial diversity and community structure. Moreover, the effect analysis of host genetics and larval diet on gut microbiomes showed that host genetics played a critical role in governing the gut microbial beta diversity and the symbiotic community structure, while larval host plant remarkably influenced the functional evolution of gut microbiomes. These findings of the intimate insect-microbe-plant interactions jointly provide some new insights into the correlation among the host genetic background, larval host plant, the structure and evolution of gut microbiome, as well as the mechanisms of high-altitude adaptation in closely related species of this alpine butterfly group.

Characterization of Four Complete Mitogenomes of Monolepta Species and Their Related Phylogenetic Implications.

Monolepta is one of the diverse genera in the subfamily Galerucinae, including 708 species and 6 sub-species worldwide. To explore the information on the mitogenome characteristics and phylogeny of the section "Monoleptites", especially the genus Monolepta, we obtained the newly completed mitochondrial genomes (mitogenomes) of four Monolepta species using high-throughput sequencing technology. The lengths of these four new mitochondrial genomes are 16,672 bp, 16,965 bp, 16,012 bp, and 15,866 bp in size, respectively. All four mitochondrial genomes include 22 transfer RNA genes (tRNAs), 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), and one control region, which is consistent with other Coleoptera. The results of the nonsynonymous with synonymous substitution rates showed that ND6 had the highest evolution rate, while COI displayed the lowest evolution rate. The substitution saturation of three datasets (13 PCGs_codon1, 13 PCGs_codon2, 13 PCGs_codon3) showed that there was no saturation across all datasets. Phylogenetic analyses based on three datasets (ND1, 15 genes of mitogenomes, and 13 PCGs_AA) were carried out using maximum likelihood (ML) and Bayesian inference (BI) methods. The results showed that mitogenomes had a greater capacity to resolve the main clades than the ND1 gene at the suprageneric and species levels. The section "Monoleptites" was proven to be a monophyletic group, while Monolepta was a non-monophyletic group. Based on ND1 data, the newly sequenced species whose antennal segment 2 was shorter than 3 were split into several clades, while, based on the mitogenomic dataset, the four newly sequenced species had close relationships with Paleosepharia. The species whose antennal segment 2 was as long as 3 were split into two clades, which indicated that the characteristic of "antennal segment 2 as long as 3" of the true "Monolepta" evolved multiple times in several subgroups. Therefore, to explore the relationships among the true Monolepta, the most important thing is to perform a thorough revision of Monolepta and related genera in the future.

Dispersal from the Qinghai-Tibet plateau by a high-altitude butterfly is associated with rapid expansion and reorganization of its genome.

Parnassius glacialis is a typical "Out of the QTP" alpine butterfly that originated on the Qinghai-Tibet Plateau (QTP) and dispersed into relatively low-altitude mountainous. Here we assemble a chromosome-level genome of P. glacialis and resequence 9 populations in order to explore the genome evolution and local adaptation of this species. These results indicated that the rapid accumulation and slow unequal recombination of transposable elements (TEs) contributed to the formation of its large genome. Several ribosomal gene families showed extensive expansion and selective evolution through transposon-mediated processed pseudogenes. Additionally, massive structural variations (SVs) of TEs affected the genetic differentiation of low-altitude populations. These low-altitude populations might have experienced a genetic bottleneck in the past and harbor genes with selective signatures which may be responsible for the potential adaptation to low-altitude environments. These results provide a foundation for understanding genome evolution and local adaptation for "Out of the QTP" of P. glacialis.

Diapause-Linked Gene Expression Pattern and Related Candidate Duplicated Genes of the Mountain Butterfly Parnassius glacialis (Lepidoptera: Papilionidae) Revealed by Comprehensive Transcriptome Profiling.

The mountain butterfly Parnassius glacialis is a representative species of the genus Parnassius, which probably originated in the high-altitude Qinhai-Tibet Plateau in the Miocene and later dispersed eastward into relatively low-altitude regions of central to eastern China. However, little is known about the molecular mechanisms underlying the long-term evolutionary adaptation to heterogeneous environmental conditions of this butterfly species. In this study, we obtained the high-throughput RNA-Seq data from twenty-four adult individuals in eight localities, covering nearly all known distributional areas in China, and firstly identified the diapause-linked gene expression pattern that is likely to correlate with local adaptation in adult P. glacialis populations. Secondly, we found a series of pathways responsible for hormone biosynthesis, energy metabolism and immune defense that also exhibited unique enrichment patterns in each group that are probably related to habitat-specific adaptability. Furthermore, we also identified a suite of duplicated genes (including two transposable elements) that are mostly co-expressed to promote the plastic responses to different environmental conditions. Together, these findings can help us to better understand this species' successful colonization to distinct geographic areas from the western to eastern areas of China, and also provide us with some insights into the evolution of diapause in mountain Parnassius butterfly species.

Complete mitochondrial genome of Distenia punctulatoides (Coleoptera: Chrysomeloidea: Disteniinae) and its phylogenetic implications.

The family Disteniidae is a moderately large and widely distributed lineage. Distenia punctulatoides belongs to the family Disteniidae from the cerambycoid assemblage. Here, we report the complete mitogenome of D. punctulatoides, which is 15,675 bp in length. It contains 37 genes and a noncoding control region, which are arranged in the same order as that of the putative ancestor of beetles. The total base composition of the new mitogenome is 40.2% for A, 17.1% for C, 10.0% for G, and 32.7% for T. The new mitogenomic organization, nucleotide composition, and codon usage do not differ significantly from other beetles. Using available complete mitogenomes, the high-level phylogeny of the family Disteniidae was explored. The phylogenetic analyses showed that Disteniidae were monophyletic, and the genus Distenia grouped with the genus Clytomelegena as sister groups. Combining the morphological and molecular data, Typodryas Thomson, 1864 is suggested to be a junior synonym of Distenia Lepeletier and Audinet-Serville, 1828.

Seven new mitochondrial genomes of phytophagous scarab beetles (Coleoptera: Scarabaeidae) and phylogenetic implications.

Among Scarabaeidae, the phytophagous scarab lineage including Melolonthinae, Cetoniinae, Dynastinae, and Rutelinae is considered important due to its members roles as agricultural pests or pollinators. In this study, the near-complete mitochondrial genomes of seven species from six genera in the phytophagous scarab lineage were newly sequenced: Anomala russiventris (Fairmaire, 1893); Apogonia cf. basalis (Moser, 1915); Apogonia splendida (Boheman, 1858); Coenochilus striatus (Westwood, 1874); Trichogomphus mongol (Arrow, 1908); Sophrops subrugatus (Moser, 1921) and Tetraserica leishanica (Liu, Bai, Yang Ahrens, 2014). The complete mitochondrial genomes from the 6 species include 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and 1 control region, which have a highly conserved gene arrangement, except for Tr. mongol with the rearrangement of 2 tRNA genes (tRNA-Ile and tRNA-Gln), which is a potential identified subfamily-level character of Dynastinae. In order to test whether the mitogenomic data are suited for high-level phylogenetic inferences, the substitution saturation and heterogeneity were analyzed. The results showed no sign that the phylogenetic inferences were biased by substitution saturation or the low heterogeneity of the sequence composition for most pairwise comparisons between the sequences for the entire dataset (13 PCGs) and the amino acids dataset (13 PCGs_AA). Based on the combined data of 13 PCGs and 13 PCGs_AA from the mitogenomes of 37 taxa, the phylogeny of the phytophagous lineage was explored using RAxML and Bayesian methods. The results confirmed that Cetoniinae, Rutelinae, and Dynastinae are monophyletic, and that the latter two are sister groups. Melolonthinae is a paraphyletic group, and its tribes, Diplotaxini, Euchirini, Melolonthini, Rhizotrogini, and Sericini, are a monophyletic group. The subfamily rank of Dynastinae and the tribe rank of Anomalini and Adoretini are supported.

Notes on spotted-elytron species of Gallerucida Motschulsky with the description of six new species from China (Coleoptera, Chrysomelidae, Galerucinae).

In this study, fifteen species of Gallerucida Motschulsky, 1860 (Coleoptera: Chrysomelidae: Galerucinae), with spotted elytra, from China are reviewed, including one new record: G.balyi (Duvivier, 1885), six new species: G.fortispina Xu & Yang, sp. nov., G.levifasciata Xu & Nie, sp. nov., G.nigrovittata Xu & Yang, sp. nov., G.octodecimpunctata Xu & Yang, sp. nov., G.piceusfasciata Xu & Yang, sp. nov., G.rufipectoralis Xu & Nie, sp. nov., and Aplosonyxgansuica (Chen, 1942), comb. nov. is removed from genus Gallerucida. A key to the spotted-elytron species of Gallerucida from China is given as well as habitus photographs of the related species and Aplosonyxgansuica comb. nov. and photographs of the aedeagus of each new species.

Five new species of the leaf-beetle genus Monolepta Chevrolat (Coleoptera, Chrysomelidae, Galerucinae) from China.

In this study, five new species of the leaf-beetle genus Monolepta Chevrolat, 1836 (Coleoptera, Chrysomelidae, Galerucinae) are described from China: M.albipunctata sp. nov., M.alticola sp. nov., M.bivittata sp. nov., M.mengsongensis sp. nov., and M.rubripennis sp. nov. A key and catalogue to the 68 Chinese species of Monolepta with the second and third antennomeres of equal length are given as well as photographs of the habitus and aedeagus of the new species and type habitus images of 37 known species.

The draft genome of the specialist flea beetle Altica viridicyanea (Coleoptera: Chrysomelidae).

BACKGROUND: Altica (Coleoptera: Chrysomelidae) is a highly diverse and taxonomically challenging flea beetle genus that has been used to address questions related to host plant specialization, reproductive isolation, and ecological speciation. To further evolutionary studies in this interesting group, here we present a draft genome of a representative specialist, Altica viridicyanea, the first Alticinae genome reported thus far. RESULTS: The genome is 864.8 Mb and consists of 4490 scaffolds with a N50 size of 557 kb, which covered 98.6% complete and 0.4% partial insect Benchmarking Universal Single-Copy Orthologs. Repetitive sequences accounted for 62.9% of the assembly, and a total of 17,730 protein-coding gene models and 2462 non-coding RNA models were predicted. To provide insight into host plant specialization of this monophagous species, we examined the key gene families involved in chemosensation, detoxification of plant secondary chemistry, and plant cell wall-degradation. CONCLUSIONS: The genome assembled in this work provides an important resource for further studies on host plant adaptation and functionally affiliated genes. Moreover, this work also opens the way for comparative genomics studies among closely related Altica species, which may provide insight into the molecular evolutionary processes that occur during ecological speciation.

The complete mitochondrial genome of the seed-borer weevil, Bruchidius uberatus (Coleoptera: Chrysomelidae: Bruchinae).

In this study, the complete 15,892 bp mitochondrial genome of Bruchidius uberatus (Fåhraeus) was sequenced using Illumina NovaSeq6000 platform. The mitogenome is a double-stranded circular molecule of 15,892 bp in length with 22 transfer RNA genes, 13 protein-coding genes and two ribosomal RNA genes as in other insects. Twenty-five species from 8 subfamilies of Chrysomelidae were selected as ingroups and 3 species of Lamiinae as outgroups for phylogenetic analysis based on mitogenome. The results showed that the subfamily Bruchinae was monophyly. Genus Bruchidius had more closed relationship with Acanthoscelides than Callosobruchus in Bruchinae with high support values.

The contribution of mitochondrial metagenomics to large-scale data mining and phylogenetic analysis of Coleoptera.

A phylogenetic tree at the species level is still far off for highly diverse insect orders, including the Coleoptera, but the taxonomic breadth of public sequence databases is growing. In addition, new types of data may contribute to increasing taxon coverage, such as metagenomic shotgun sequencing for assembly of mitogenomes from bulk specimen samples. The current study explores the application of these techniques for large-scale efforts to build the tree of Coleoptera. We used shotgun data from 17 different ecological and taxonomic datasets (5 unpublished) to assemble a total of 1942 mitogenome contigs of >3000 bp. These sequences were combined into a single dataset together with all mitochondrial data available at GenBank, in addition to nuclear markers widely used in molecular phylogenetics. The resulting matrix of nearly 16,000 species with two or more loci produced trees (RAxML) showing overall congruence with the Linnaean taxonomy at hierarchical levels from suborders to genera. We tested the role of full-length mitogenomes in stabilizing the tree from GenBank data, as mitogenomes might link terminals with non-overlapping gene representation. However, the mitogenome data were only partly useful in this respect, presumably because of the purely automated approach to assembly and gene delimitation, but improvements in future may be possible by using multiple assemblers and manual curation. In conclusion, the combination of data mining and metagenomic sequencing of bulk samples provided the largest phylogenetic tree of Coleoptera to date, which represents a summary of existing phylogenetic knowledge and a defensible tree of great utility, in particular for studies at the intra-familial level, despite some shortcomings for resolving basal nodes.

The complete mitochondrial genome of the cowpea weevil, Callosobruchus maculates (Coleoptera: Chrysomelidae: Bruchinae) and a related phylogenetic analysis of Chrysomelidae.

In this study, the complete 17,809 bp mitochondrial genome of Callosobruchus maculates (F.) (Coleoptera: Chrysomelidae: Bruchinae) was sequenced using Illumina's HiSeq2000 platform. The mitogenome is a double-stranded circular molecule of 17,809 bp in length with 21 transfer RNA genes, 13 protein-coding genes, and two ribosomal RNA genes as in other insects. Specially, there is a 2008 bp-inserted segment between ND2 and tRNA-Trp from 1180 to 3187, which cannot be aligned to any known gene of mitogenomes. To estimate the taxonomic status of Bruchinae, total 17 species from eight subfamilies of Chrysomelidae were selected as ingroups and three species of Lamiinae as outgroups for phylogenetic analysis based on mitogenome. The results showed that three major lineages were formed, including a basal 'Eumolpine' clade (Cassidinae, Eumolpinae, Cryptocephalinae, Clytrinae), ''Criocerine' clade (Criocerinae, Bruchinae) and 'Chrysomeline' clade (Chrysomelinae, Galerucinae s. l.). Bruchinae showed more closed relationship with Criocerinae than other subfamilies. More thorough taxon sampling will be needed to well understand the relationship in Chrysomelidae.

The phylogeny of Galerucinae (Coleoptera: Chrysomelidae) and the performance of mitochondrial genomes in phylogenetic inference compared to nuclear rRNA genes.

With efficient sequencing techniques, full mitochondrial genomes are rapidly replacing other widely used markers, such as the nuclear rRNA genes, for phylogenetic analysis but their power to resolve deep levels of the tree remains controversial. We studied phylogenetic relationships of leaf beetles (Chrysomelidae) in the tribes Galerucini and Alticini (root worms and flea beetles) based on full mitochondrial genomes (103 newly sequenced), and compared their performance to the widely sequenced nuclear rRNA genes (full 18S, partial 28S). Our results show that: (i) the mitogenome is phylogenetically informative from subtribe to family level, and the per-nucleotide contribution to nodal support is higher than that of rRNA genes, (ii) the Galerucini and Alticini are reciprocally monophyletic sister groups, if the classification is adjusted to accommodate several 'problematic genera' that do not fit the dichotomy of lineages based on the presence (Alticini) or absence (Galerucini) of the jumping apparatus, and (iii) the phylogenetic results suggest a new classification system of Galerucini with eight subtribes: Oidina, Galerucina, Hylaspina, Metacyclina, Luperina, Aulacophorina, Diabroticina and Monoleptina.

How many genera and species of Galerucinaes. str. do we know? Updated statistics (Coleoptera, Chrysomelidae).

Galerucinae s. str. is a rich group of leaf beetles. A new, up-to date checklist of Galerucinae genera in the world is provided, including the number of valid species of each genus. Genera and species were counted in literature published before the end of 2016. In summary, 7145 species (7132 recent, 13 fossils) and 192 subspecies from 543 genera (542 recent, 1 fossil) were quantified in Galerucinae s. str. In comparison with the previous catalogue of worldwide Galerucinae (Wilcox 1971-1973), an additional 91 valid genera, 1341 valid species (1337 recent, 4 fossils) and 38 subspecies have been published; 43 genera were synonymized, four genera were transferred into Alticini, two subgenera were elevated to genus rank, and one genus was downgraded to subgenus rank. The updated list of references to taxonomic publications on Galerucinae s. str. from the period 1971-2016 is provided.

Complete mitochondrial genome of Spiniphilus spinicornis (Coleoptera: Vesperidae: Philinae) and phylogenetic analysis among Cerambycoidea.

Spiniphilus spinicornis belongs to subfamily Philinae of family Vesperidae from cerambycoid assemblage. The first complete mitogenome of Spiniphilus spinicornis was reported. The genome is 15 707 bp in length and contains the typical 37 genes that are arranged in the same order as that of the putative ancestor of beetles. The total base composition of the mitogenome is 30.9% for A, 11.1% for C, 19.3% for G, and 38.6% for T. The genome organization, nucleotide composition, and codon usage do not differ significantly from other martens. The percentage of A + T is 69.5%. The first complete mitogenome of subfamily Philinae could be used in studies of molecular systematics, phylogenetic, and conservation genetics. Phylogenetic analysis showed that Cerambycoidea was monophyly with high support value. Lamiinae, Cerambycinae, and Philinae were monophyly too, respectively.

Comparative transcriptome analysis of chemosensory genes in two sister leaf beetles provides insights into chemosensory speciation.

Divergence in chemosensory traits has been posited as an important component of chemosensory speciation in insects. In particular, chemosensory genes expressed in the peripheral sensory neurons are likely to influence insect behaviors such as preference for food, oviposition sites, and mates. Despite their key role in insect behavior and potentially speciation, the underlying genetic basis for divergence in chemosensory traits remains largely unexplored. One way to ascertain the role of chemosensory genes in speciation is to make comparisons of these genes across closely related species to detect the genetic signatures of divergence. Here, we used high throughput transcriptome analysis to compare chemosensory genes of the sister leaf beetles species Pyrrhalta maculicollis and P. aenescens, whose sexual isolation and host plant preference are mediated by divergent chemical signals. Although there was low overall divergence between transcriptome profiles, there were a number of genes that were differentially expressed between the species. Furthermore, we also detected two chemosensory genes under positive selection, one of which that was also differentially expressed between the species, suggesting a possible role for these genes in chemical-based premating reproductive isolation and host use. Combined with the available chemical and ecological work in this system, further studies of the divergent chemosensory genes presented here will provide insight into the process of chemosensory speciation among Pyrrhalta beetles.

Adaptation to different host plant ages facilitates insect divergence without a host shift.

Host shifts and subsequent adaption to novel host plants are important drivers of speciation among phytophagous insects. However, there is considerably less evidence for host plant-mediated speciation in the absence of a host shift. Here, we investigated divergence of two sympatric sister elm leaf beetles, Pyrrhalta maculicollis and P. aenescens, which feed on different age classes of the elm Ulmus pumila L. (seedling versus adult trees). Using a field survey coupled with preference and performance trials, we show that these beetle species are highly divergent in both feeding and oviposition preference and specialize on either seedling or adult stages of their host plant. An experiment using artificial leaf discs painted with leaf surface wax extracts showed that host plant chemistry is a critical element that shapes preference. Specialization appears to be driven by adaptive divergence as there was also evidence of divergent selection; beetles had significantly higher survival and fecundity when reared on their natal host plant age class. Together, the results identify the first probable example of divergence induced by host plant age, thus extending how phytophagous insects might diversify in the absence of host shifts.

Male mate recognition via cuticular hydrocarbons facilitates sexual isolation between sympatric leaf beetle sister species.

Chemical signals in insects have been documented to play an important role in mate recognition, and divergence in chemical signals can often cause sexual isolation between closely related species or populations within species. We investigated the role of cuticular hydrocarbons (CHCs), short distance chemical signals, in male mate recognition between the two sympatric elm leaf beetles, Pyrrhalta maculicollis and Pyrrhaltaaenescens. Mating experiments demonstrated that strong sexual isolation between the two species was driven by CHCs divergence. Males preferred to mate with conspecific females with intact conspecific CHCs or conspecific CHCs reapplied after removal. Males also preferred heterospecific females that were treated with conspecific CHCs. Chemical analysis showed that the CHC profiles differ significantly between species. In P. maculicollis dimethyl-branched alkanes between C29 and C35 account for the majority of the saturated alkanes while the CHC profile of P. aenescens mostly consisted of monomethyl-branched alkanes between C22 and C29. Additionally, some compounds, such as 12,18-diMeC32, 12,18-diMeC34, are unique to P. maculicollis.