DNA barcoding of the genus Gampsocleis (Orthoptera, Tettigoniidae) from China.

DNA barcoding is a useful addition to the traditional morphology-based taxonomy. A ca. 650 bp fragment of the 5' end of mitochondrial cytochrome c oxidase subunit I (hereafter COI-5P) DNA barcoding was sued as a practical tool for Gampsocleis species identification. DNA barcodes from 889 specimens belonging to 8 putative Gampsocleis species was analyzed, including 687 newly generated DNA barcodes. These barcode sequences were clustered/grouped into Operational Taxonomic Units (OTUs) using the criteria of five algorithms, namely Barcode Index Number (BIN) System, Assemble Species by Automatic Partitioning (ASAP), a Java program uses an explicit, determinate algorithm to define Molecular Operational Taxonomic Unit (jMOTU), Generalized Mixed Yule Coalescent (GMYC), and Bayesian implementation of the Poisson Tree Processes model (bPTP). The Taxon ID Tree grouped sequences of morphospecies and almost all MOTUs in distinct nonoverlapping clusters. Both long- and short-winged Gampsocleis species are reciprocally monophyletic in the Taxon ID Tree. In BOLD, 889 barcode sequences are assigned to 17 BINs. The algorithms ASAP, jMOTU, bPTP and GMYC clustered the barcode sequences into 6, 13, 10, and 23 MOTUs, respectively. BIN, ASAP, and bPTP algorithm placed three long-winged species, G. sedakovii, G. sinensis and G. ussuriensis within the same MOTU. All species delimitation algorithms split two short-winged species,G. fletcheri and G. gratiosa into at least two MOTUs each, except for ASAP algorithm. More detailed molecular and morphological integrative studies are required to clarify the status of these MOTUs in the future.

New reports on Chinese Anelytra species (Orthoptera, Tettigoniidae: Conocephalinae).

Based on the former studies and new material, Anelytra (Euanelytra) eunigrifrons Ingrisch, 1998 is reported in China for the first time. In addition, the female sex of Anelytra (Euanelytra) spinia Shi & Qiu, 2009 is described. We also confirmed that Anelytra (Euanelytra) spinia Shi & Qiu, 2009 is mainly distributed in Southern China. The examined specimens are deposited in Guangxi Normal University.

Mitochondrial genome of Poecilimon cretensis (Orthoptera: Tettigoniidae: Phaneropterinae): Strong phylogenetic signals in gene overlapping regions.

We report the complete mitochondrial genome of the Cretan bush cricket Poecilimon cretensis. The mitogenome consists of 13 protein-coding regions, 22 tRNAs, two rRNAs, and one control region. The length of mitogenome in P. cretensis varies between15477 and 15631 bp, mainly due to variability in control region. The start and stop codons of protein coding genes exhibit the general pattern in Phaneropterinae. Phylogenetic tree constructed with the mitogenome obtained during this study and 12 mitogenomes of Phaneropterinae downloaded from GenBank, placed P. cretensis in Barbitistini as sister group to Poecilimon luschani. Data indicate that the gene overlapping pattern exhibit strong phylogenetic signals.

A new species of Dasylabris Radoszkowski, 1885 (Hymenoptera: Mutillidae) from India.

We report the complete mitochondrial genome of the Cretan bush cricket Poecilimon cretensis. The mitogenome consists of 13 protein-coding regions, 22 tRNAs, two rRNAs, and one control region. The length of mitogenome in P. cretensis varies between15477 and 15631 bp, mainly due to variability in control region. The start and stop codons of protein coding genes exhibit the general pattern in Phaneropterinae. Phylogenetic tree constructed with the mitogenome obtained during this study and 12 mitogenomes of Phaneropterinae downloaded from GenBank, placed P. cretensis in Barbitistini as sister group to Poecilimon luschani. Data indicate that the gene overlapping pattern exhibit strong phylogenetic signals.

New estimates of genome size in Orthoptera and their evolutionary implications.

Animal genomes vary widely in size, and much of their architecture and content remains poorly understood. Even among related groups, such as orders of insects, genomes may vary in size by orders of magnitude-for reasons unknown. The largest known insect genomes were repeatedly found in Orthoptera, e.g., Podisma pedestris (1C = 16.93 pg), Stethophyma grossum (1C = 18.48 pg) and Bryodemella holdereri (1C = 18.64 pg). While all these species belong to the suborder of Caelifera, the ensiferan Deracantha onos (1C = 19.60 pg) was recently found to have the largest genome. Here, we present new genome size estimates of 50 further species of Ensifera (superfamilies Gryllidea, Tettigoniidea) and Caelifera (Acrididae, Tetrigidae) based on flow cytometric measurements. We found that Bryodemella tuberculata (Caelifera: Acrididae) has the so far largest measured genome of all insects with 1C = 21.96 pg (21.48 gBp). Species of Orthoptera with 2n = 16 and 2n = 22 chromosomes have significantly larger genomes than species with other chromosome counts. Gryllidea genomes vary between 1C = 0.95 and 2.88 pg, and Tetrigidae between 1C = 2.18 and 2.41, while the genomes of all other studied Orthoptera range in size from 1C = 1.37 to 21.96 pg. Reconstructing ancestral genome sizes based on a phylogenetic tree of mitochondrial genomic data, we found genome size values of >15.84 pg only for the nodes of Bryodemella holdereri / B. tuberculata and Chrysochraon dispar / Euthystira brachyptera. The predicted values of ancestral genome sizes are 6.19 pg for Orthoptera, 5.37 pg for Ensifera, and 7.28 pg for Caelifera. The reasons for the large genomes in Orthoptera remain largely unknown, but a duplication or polyploidization seems unlikely as chromosome numbers do not differ much. Sequence-based genomic studies may shed light on the underlying evolutionary mechanisms.

Mitogenome of Xya pfaendleri (Orthoptera: Caelifera): Its comparative description and phylogenetic position within Tridactylidea.

We report the comparative examination of the complete mitochondrial genome of the pygmy mole cricket Xya pfaendleri (Orthoptera: Caelifera: Tridactylidae). The mitogenome consists of 13 protein-coding regions, 22 tRNAs, two rRNAs, and one control region, following the gene order of the ancestral pancrustacean mitogenome. The length of the mitogenome in Xya pfaendleri is 15352 bp. The start and stop codons of the protein-coding genes exhibit the general pattern observed in orthopterans. The data indicate that the pattern of gene overlapping/intergenic sequences exhibits a significant phylogenetic signal. A phylogenetic tree inferred using 12 mitogenomes (seven belonging to Tridactylidea, three to Acrididea, and two to Ensifera) confirms the sister group relationship of Acrididea and Tridactylidea. The relationship among the families of Tridactylidea is Cylindrachetidae + (Ripipterygidae + Tridactylidae). The mitogenome sequences of Xya and Tridactylus constitute a single clade, sharing a last common ancestor 94 million years ago, and rendering the first genus paraphyletic. The present preliminary data suggest that we still have much to learn about the evolution and diversity of Tridactylidea.

Mwhitiwhiti Aotearoa: Phylogeny and synonymy of the silent alpine grasshopper radiation of New Zealand (Orthoptera: Acrididae).

Aotearoa New Zealand has a fauna of endemic alpine grasshoppers, consisting of thirteen species distributed among four genera. The many re-classifications of species within this group and the presence of species complexes highlight the uncertainty that surrounds relationships within and between these genera. High-throughput Next Generation Sequencing was used to assemble the complete mitochondrial genomes, 45S ribosomal cassettes and histone sequences of New Zealands four endemic alpine genera: Alpinacris, Brachaspis, Paprides and Sigaus. Phylogenetic analysis of these molecular datasets, as individual genes, partitions and combinations returned a consistent topology that is incompatible with the current classification. The genera Sigaus, Alpinacris, and Paprides all exhibit paraphyly. A consideration of the pronotum, epiphallus and terminalia of adult specimens reveals species-specific differences, but fails to provide compelling evidence for species groups justifying distinct genera. In combination with phylogenetic, morphological and spatial evidence we propose a simplified taxonomy consisting of a single genus for the mwhitiwhiti Aotearoa species radiation.

Two new species of the genus Diestramima from China (Orthoptera: Rhaphidophoridae: Aemodogryllinae).

The genus Diestramima comprises 41 species from Asia with 31 species distributed in China. In this study, we reconstruct the phylogeny tree of Diestramima species by maximum likelihood and Bayesian inference based on three mitochondrial genes (COI, 12S and 16S). The result indicates that the phylogenetic results are coherent with that based on five molecular markers (COI, 12S, 16S, 18S and 28S). Moreover, two new species, D. pingmengensis sp. nov. He & Zong and D. gulinjingensis. sp. nov. Zong & He are described. Their validities are also supported by morphological features. Furthermore, D. sichuanensis Zhu & Shi, 2022 is treated as a junior synonym of D. guangxiensis Qin, Wang, Liu & Li, 2016 based on both morphological and molecular features.

Evolution of Gene Arrangements in the Mitogenomes of Ensifera and Characterization of the Complete Mitogenome of Schizodactylus jimo.

Gene arrangement (relative location of genes) is another evolutionary marker of the mitogenome that can provide extensive information on the evolutionary mechanism. To explore the evolution of gene arrangements in the mitogenome of diversified Ensifera, we sequenced the mitogenome of the unique dune cricket species found in China and used it for phylogenetic analysis, in combination with 84 known Ensiferan mitogenomes. The mitogenome of Schizodactylus jimo is a 16,428-bp circular molecule that contains 37 genes. We identified eight types of gene arrangement in the 85 ensiferan mitogenomes. The gene location changes (i.e., gene translocation and duplication) were in three gene blocks: I-Q-M-ND2, rrnl-rns-V, and ND3-A-R-N-S-E-F. From the phylogenetic tree, we found that Schizodactylus jimo and most other species share a typical and ancient gene arrangement type (Type I), while Grylloidea has two types (Types II and III), and the other five types are rare and scattered in the phylogenetic tree. We deduced that the tandem replication-random loss model is the evolutionary mechanism of gene arrangements in Ensifera. Selection pressure analysis revealed that purifying selection dominated the evolution of the ensiferan mitochondrial genome. This study suggests that most gene rearrangements in the ensiferan mitogenome are rare accidental events.

A new species of Cephalophlugis Gorochov, 1998 (Orthoptera: Tettigoniidae: Meconematinae: Phlugidini) from Southern Brazil, with bioacoustics and cytogenetics.

Cephalophlugis Gorochov, 1998 is a Neotropical genus of predatory katydids with two valid species, Cephalophlugis cephalotes (Bolvar, 1888) (from So Paulo, Brazil) and Cephalophlugis setosa Gorochov, 2012 (from Sucumbios, Ecuador). Herein, we describe a third species of the genus, Cephalophlugis gaucho sp. nov., from Rio Grande do Sul, Brazil. This species is easily distinguished from the others by the color of the hind wings and the number of spurs on the fore and middle legs. The calling song is composed of a long phrase with a series of syllables that vary in emission rate from beginning to end, with a dominant frequency of 23.43 0.6 (22.1224.37, n=11) kHz. So far, the species sings at the lowest frequency among the Phlugidini. From the chromosomal perspective, C. gaucho sp. nov. presents the karyotype 2n=31, X0, with the complement conserved within Meconematinae.

Study on the Chinese Subfamily Anabropsinae (Orthoptera: Anostostomatidae) VI: One new species of Anabropsis (Pteranabropsis) from Yunnan Province.

This paper is sixth study the Chinese Anabropsinae and reports one new species from Yunnan Province, China, viz. A. (Pteranabropsis) maguanensis sp. nov. (Chinese name: ). Meanwhile, the complete mitochondrical genome of the new species was determined and annotated. The 15, 962 bp circle genome consisted of 13 protein-coding, 22 transfer RNA, 2 ribosomal RNA genes, and an A+T-rich region. It has the typical invertebrate mitochondrial gene arrangement. All protein-coding genes (PCGs) were initiated by typical ATN codon. The nucleotide compositions were significant bias towards AT. All transfer RNA (tRNA) genes had a typical clover-leaf structure, except trnS1 in which the base pairs of the dihydrouridine (DHU) arm was lacking. Overall, six domains and 49 helices were predicted for rrnL, and three structural domains and 27 helices were predicted for rrnS.

Study of the Subfamily Anabropsinae (Orthoptera: Anostostomatidae) in China V: Two new species of Anabropsis (Apteranabropsis) from Guangxi and phylogenetic analysis of the genus Anabropsis.

This paper is the fifth study of the subfamily Anabropsinae in China and contributes two new species from Guangxi, China, viz. Anabropsis (Apteranabropsis) multispinula sp. nov. (Chinese name: ) and Anabropsis (Apteranabropsis) shii sp. nov. (Chinese name: ). The paraproctal processes and male genitalia of Anabropsis (Apteranabropsis) tonkinensis Rehn, 1906 are illustrated. The entire mitochondrial genomes (mitogenomes) of three species of Anabropsis were sequenced. The complete mitochondrial genomes of A. (Apteranabropsis) multispinula sp. nov., A. (Apteranabropsis) shii sp. nov. and A. (Apteranabropsis) guangxiensis were 16023 bp, 16087 bp and 16148 bp in length, respectively. Entire mitogenome and all protein-coding genes (PCGs) displayed high AT-content values. Besides A. (Pteranabropsis) carnarius and A. (Apteranabropsis) guangxiensis, other species of Anabropsis were not found repeated elements in the A+T-rich region. The phylogenetic relationships were constructed among nine samples of eight species of Anabropsis and two outgroups based on 13 PCGs using both Bayesian Inference (BI) and Maximum Likelihood (ML). The phylogenetic analysis did not support for the monophyly of subgenus Apteranabropsis. It's important to obtain more samples to derive accurate phylogenetic relationships.

Contribution to the Chinese subfamily Rhaphidophorinae Walker, 1869 (Orthoptera: Rhaphidophoridae: Rhaphidophorinae) IV: Seven new species of Rhaphidophora and one new mitogenome.

Seven new species and the female sex of Rhaphidophora xishuang Gorochov, 2012 of the rhaphidophorines are described from China. The images illustrating the morphology of the two known species are provided. Meanwhile, the complete mitogenome of Rhaphidophora quadrispina was assembled. The mitogenome was 15892 bp in length and contained the typical gene arrangement. All examined specimens are deposited in the Guangxi Normal University.

The genus Apholidoptera of Pholidopterini (Orthoptera, Tettigoniidae): Molecular phylogeny confirms the generic status.

After a recent revision of the tribe Pholidopterini, the generic status of Apholidoptera becomes questionable and requires to be clarified by determining its phylogenetic position. The present study aims to obtain a phylogenetic tree using published DNA sequence data of Pholidopterini and representative sequences from Apholidoptera kurda, the only species of the genus. Phylogenetic analyses applied to the dataset, including three mitochondrial and one nuclear gene segments, suggested the Apholidoptera kurda within the clade constituting the most basal branch of Pholidopterini, containing five species of Aparapholidoptera. This clade consists of two sister subclades; the first consists of Apholidoptera kurda plus Aparapholidoptera pietschmanni, and the second species in the Aparapholidoptera distincta group. Two subclades share the last common ancestor about 12 million years ago, indicating a deep phylogenetic splitting. Further, both subclades are distinguishable by prominent morphological differences. Considering the ultimate ancestral ages of the con-tribal other genera and phenotypic disparity, we proposed each as a separate genus. This statement confirms the generic status of Apholidoptera with species content of the original description and requires retransferring Aparapholidoptera pietschmanni to Apholidoptera. This nomenclatural change also limits Aparapholidoptera with four species previously given in the Aparapholidoptera distincta group.

Taxonomic revision of the genus Hemigyrus Brunner von Wattenwyl, 1893 (Orthoptera: Tettigoniidae: Pseudophyllinae).

Hemigyrus was established by Brunner von Wattenwyl in 1893. In this study, we collected samples from Chongqing, Guangxi, Hainan, Sichuan, Tibet and Yunnan of China, and reconstructed phylogenetic tree based on COI gene. The results supported the classification of dividing this genus into two subgenera: Tomomima and Hemigyrus. With larger size and evidently branched phylogenetic position, a new subspecies H. (T.) spinosus robustus subsp. nov. Xie, Wang He is described here. H. (H.) acutifolius is firstly reported from China. Males of H. (H.) amplus and H. (H.) acutifolius, females of H. (H.) minor are described for the first time. All materials were deposited in Biological History Museum of East China Normal University (ECNU) and the Shanghai Entomological Museum, Chinese Academy of Sciences (SEM).

Transcriptomic data reveals nuclear-mitochondrial discordance in Gomphocerinae grasshoppers (Insecta: Orthoptera: Acrididae).

The phylogeny of many groups of Orthoptera remains poorly understood. Previous phylogenetic studies largely restricted to few mitochondrial markers found many species in the grasshopper subfamily Gomphocerinae to be para- or polyphyletic, presumably because of incomplete lineage sorting and ongoing hybridization between putatively young lineages. Resolving the phylogeny of the Chorthippus biguttulus species complex is important because many morphologically cryptic species occupy overlapping ranges across Eurasia and serve important ecological functions. We investigated whether multispecies coalescent analysis of 540 genes generated by transcriptome sequencing could resolve the phylogeny of the C. biguttulus complex and related Gomphocerinae species. Our divergence time estimates confirm that Gomphocerinae is a very young radiation, with an age estimated at 1.38 (2.35-0.77) mya for the C. biguttulus complex. Our estimated topology based on complete mitogenomes recovered some species as para- or polyphyletic. In contrast, the multispecies coalescent based on nuclear genes retrieved all species as monophyletic clusters, corroborating most taxonomic hypotheses. Our results underline the importance of using nuclear multispecies coalescent methods for studying young radiations and highlight the need of further taxonomic revision in Gomphocerinae grasshoppers.

Integrative taxonomy base on morphology and molecular phylogeny with description of a new genus, Progoniogryllus gen. nov. and two new species (Orthoptera: Grylloidea: Gryllidae; Gryllinae).

Based on both morphological characters and phylogenetic relationships, one new genus Progoniogryllus gen. nov. and two new species, Progoniogryllus rotundus sp. nov. and Progoniogryllus directus sp. nov., are proposed. The new genus is compared with similar genera of Gryllodes Saussure, 1874, Parasongella Otte, 1987, Callogryllus Sjstedt, 1910 and Goniogryllus Chopard. 1936 by morphological and phylogenetic methods. Based on the knowledge so far, the new genus is very close to Goniogryllus. All the new taxa are described and illustrated, and some information on mating behavior of the new was also provided.

Ptosoproctus gen. nov., a new genus with two new species of Shield-back Katydid, with the first record of genus Eulithoxenus Bey-Bienko, 1951 from China (Orthoptera: Tettigoniidae: Tettigoniinae: Drymadusini).

In this paper, one new genus, Ptosoproctus gen. nov., is established with two new species: P. lanzhouensis sp. nov. and P. baishishanicus sp. nov., described and illustrated based on material collected from northern China. The genus Eulithoxenus Bey-Bienko, 1951 is recorded from China for the first time. E. emeljanovi Mishchenko, 1968 is redescribed. Supplemental description of Uvarovina chinensis Ramme, 1939 is provided. COI and ND2 genes of the three Chinese Drymadusini genera mentioned above were used to reconstruct the phylogenetic tree. Molecular result support the validation of the new genus.

New species of Eugryllacris (Orthoptera: Gryllacrididae) from Yunnan, China.

This paper describes one new species from Yunnan, China and reports the complete mitogenome. The new species differs from other species of the genus in markings on the frons and pronotum with 1 pair of black spots. The complete mitochondrial genome (mitogenome) of this new species was sequenced and analyzed using High-Throughput Sequencing. The mitogenome was 15513bp in length, comprising of 37 genes and a control region, and its gene order and location are identical to the ancestral insect mitogenome. The protein-coding genes (PCGs) had typical ATN initiation codons, and were terminated by typical TAN stop codons except for COII and ND5. Almost all tRNAs could be folded into the typical cloverleaf secondary structure except for tRNASer(AGN). All the type specimens are deposited in the Guangxi Normal University.

Comparative mitogenomes and phylogenetic analysis reveal taxonomic relationship of genera Teredorus and Systolederus (Orthoptera, Tetrigoidea).

The genera Teredorus and Systolederus belong to Tetriginae and Metrodorinae respectively. However, species within these two genera have strikingly similar features, made it difficult to identify clearly by morphological characteristics. In this study, we sequenced the mitochondrial genomes (mitogenomes) of two Teredorus species, and compared them with Systolederus mitochondrial sequences. The sequenced mitogenomes of T. hainanensis and T. bashanensis are 14,946 bp and 14,775 bp in size, respectively. The A+T content of mitogenomes is 76.2% (T. hainanensis) and 74.0% (T. bashanensis). Comparative analysis showed that mitochondrial sequences and structure were similar within these two genera. The results of K2P distances and phylogenetic analysis revealed that Systolederus and Teredorus might be likely considered as one genus of Teredorus. It will provide important resources for further understanding of the taxonomy and phylogenetic relationship of Systolederus and Teredorus.