Genetic differentiation and phylogeography of Erythroneurini (Hemiptera, Cicadellidae, Typhlocybinae) in the southwestern karst area of China.

Erythroneurini is the largest tribe of the microleafhopper subfamily Typhlocybinae. Most prior research on this tribe has focused on traditional classification, phylogeny, and control of agricultural pests, and the phylogeography of the group remains poorly understood. In this study, the mitochondrial genomes of 10 erythroneurine species were sequenced, and sequences of four genes were obtained for 12 geographical populations of Seriana bacilla. The new sequence data were combined with previously available mitochondrial DNA sequence data and analyzed using Bayesian and Maximum-Likelihood-based phylogenetic methods to elucidate relationships among genera and species and estimate divergence times. Seriana was shown to be derived from within Empoascanara. Phylogeographic and population genetic analysis of the endemic Chinese species Seriana bacilla suggest that the species diverged about 54.85 Mya (95% HPD: 20.76-66.23 million years) in the Paleogene period and that population divergence occurred within the last 14 million years. Ancestral area reconstruction indicates that Seriana bacilla may have originated in the central region of Guizhou, and geographical barriers are the main factors affecting gene flow among populations. Ecological niche modeling using the MaxEnt model suggests that the distribution of the species was more restricted in the past but is likely to expand in the future years 2050 and 2070.

Phylogenomics of the leafhopper genus Neoaliturus Distant, 1918 (Hemiptera: Cicadellidae: Deltocephalinae) reveals genetically divergent lineages in the invasive beet leafhopper.

Phylogenomic analysis based on nucleotide sequences of 398 nuclear gene loci for 67 representatives of the leafhopper genus Neoaliturus yielded well-resolved estimates of relationships among species of the genus. Subgenus Neoaliturus (Neoaliturus) is consistently paraphyletic with respect to Neoaliturus (Circulifer). The analysis revealed the presence of at least ten genetically divergent clades among specimens consistent with the previous morphology-based definition of the leafhopper genus "Circulifer" which includes three previously recognized "species complexes." Specimens of the American beet leafhopper, N. tenellus (Baker), collected from the southwestern USA consistently group with one of these clades, comprising specimens from the eastern Mediterranean. Some of the remaining lineages are consistent with ecological differences previously observed among eastern Mediterranean populations and suggest that N. tenellus, as previously defined, comprises multiple monophyletic species, distinguishable by slight morphological differences.

Mitogenomic Analysis and Phylogenetic Implications for the Deltocephaline Tribe Chiasmini (Hemiptera: Cicadellidae: Deltocephalinae).

The grassland leafhopper tribe Chiasmini (Cicadellidae: Deltocephalinae) presently comprises 324 described species worldwide, with the highest species diversity occurring in the Nearctic region but a greater diversity of genera occurring in the Old World. In China, this tribe comprises 39 described species in 11 genera, but the fauna remains understudied. The complete mitogenomes of three species of this tribe have been sequenced previously. In order to better understand the phylogenetic position of Chiasmini within the subfamily Deltocephalinae and to investigate relationships among Chiasmini genera and species, we sequenced and analyzed the complete mitogenomes of 13 species belonging to seven genera from China. Comparison of the newly sequenced mitogenomes reveals a closed circular double-stranded structure containing 37 genes with a total length of 14,805 to 16,269 bp and a variable number of non-coding A + T-rich regions. The gene size, gene order, gene arrangement, base composition, codon usage, and secondary structure of tRNAs of the newly sequenced mitogenomes of these 13 species are highly conserved in Chiasmini. The ATN codon is commonly used as the start codon in protein-coding genes (PCGs), except for ND5 in Doratura sp. and ATP6 in Nephotettix nigropictus, which use the rare GTG start codon. Most protein-coding genes have TAA or TAG as the stop codon, but some genes have an incomplete T stop codon. Except for the tRNA for serine (trnS1(AGN)), the secondary structure of the other 21 tRNAs is a typical cloverleaf structure. In addition to the primary type of G-U mismatch, five other types of tRNA mismatches were observed: A-A, A-C, A-G, U-C, and U-U. Chiasmini mitochondrial genomes exhibit gene overlaps with three relatively stable regions: the overlapping sequence between trnW and trnC is AAGTCTTA, the overlapping sequence between ATP8 and ATP6 is generally ATGATTA, and the overlapping sequence between ND4 and ND4L is generally TTATCAT. The largest non-coding region is the control region, which exhibits significant length and compositional variation among species. Some Chiasmini have tandem repeat structures within their control regions. Unlike some other deltocephaline leafhoppers, the sequenced Chiasmini lack mitochondrial gene rearrangements. Phylogenetic analyses of different combinations of protein-coding and ribosomal genes using maximum likelihood and Bayesian methods under different models, using either amino acid or nucleotide sequences, are generally consistent and also agree with results of prior analyses of nuclear and partial mitochondrial gene sequence data, indicating that complete mitochondrial genomes are phylogenetically informative at different levels of divergence within Chiasmini and among leafhoppers in general. Apart from Athysanini and Opsiini, most of the deltocephaline tribes are recovered as monophyletic. The results of ML and BI analyses show that Chiasmini is a monophyletic group with seven monophyletic genera arranged as follows: ((Zahniserius + (Gurawa + (Doratura + Aconurella))) + (Leofa + (Exitianus + Nephotettix))).

Climate change may drive the distribution of tribe Zyginelline pests in China and the Indo-China Peninsula to shift towards higher latitude river-mountain systems.

BACKGROUND: Tribe Zyginelline leafhoppers can transmit plant viruses and are important pests that affect agriculture, forestry, and animal husbandry, causing serious economic losses. The potential distribution patterns of Zyginellini will change under climate change. Therefore, the best-performing random forest and maximum entropy models among 12 commonly used ecological niche models, alongside an ensemble model, were selected to predict the changes in habitat suitability distribution of Zyginellini under current and future climate scenarios [represented by two shared socio-economic pathways (SSPs), namely SSP126 and SSP585, for three periods (2050s, 2070s, and 2090s)] in China and the Indo-China Peninsula for the first time. RESULTS: The results revealed that the distribution of Zyginellini was mainly dominated by minimum temperature of coldest month. Under current and future climate scenarios, Zyginellini was mostly distributed southeast of the 400 mm equivalent precipitation line in China, and Vietnam. Under the future SSP126 scenario, the alert areas will mainly be concentrated in Hunan, Jiangxi, Zhejiang, Anhui, and Hebei in China, alongside Myanmar and Thailand in the Indo-China Peninsula. Meanwhile, in the SSP585 scenario, the alert areas in China will increase, whereas there will be little change in the Indo-China Peninsula. Interestingly, from the current to the future, the cores of Zyginelline distribution occurred around rivers and mountains, and shifted from Guizhou along the Yuanjiang River system to higher latitudes in Hunan. CONCLUSION: Zyginellini prefers higher latitude river-mountain systems under climate change. Our results will contribute to effective pest control strategies and biogeographical research for Zyginellini alongside other Cicadellidae insects. © 2023 Society of Chemical Industry.

Investigating Monophyly of Typhlocybini Based on Complete Mitochondrial Genomes with Characterization and Comparative Analysis of 19 Species (Hemiptera: Cicadellidae: Typhlocybinae).

Tribes of the leafhopper subfamily Typhlocybinae have traditionally been defined based on differences in forewing and hindwing venation. Except for Typhlocybini (sensu lato), the classification of tribes is relatively stable. The monophyly of Typhlocybini needs to be examined, and the relationships among genera within Typhlocybini have not been resolved. Few mitogenome sequences representative of major lineages of Typhlocybini have been available to facilitate a comprehensive phylogenetic analysis of the tribe. In this study, the complete mitogenomes of 19 species of Typhlocybini were sequenced. The gene arrangements of the 19 new mitogenomes are consistent with ancestral insect mitogenomes. Phylogenetic analyses by both maximum-likelihood and Bayesian methods of 67 species of Typhlocybinae suggest that Zyginellini is paraphyletic with respect to Typhlocybini. The phylogenetic relationships within Typhlocybini are discussed, and the major results show that the Farynala and Linnavuoriana complexes previously recognized based on morphological characters correspond to monophyletic lineages.

Morphological and ultrastructural studies of the internal reproductive systems of two deltocephaline leafhoppers, Nephotettixcincticeps and Deltocephalusvulgaris (Hemiptera, Cicadellidae, Deltocephalinae).

Insects have highly variable reproductive systems, reflecting a diversity of reproductive strategies and adaptations. Such variation has been widely used to classify and estimate phylogenetic relationships. Here, the morphology and ultrastructure of the internal reproductive systems of two leafhopper species are described and illustrated, using both light and transmission electron microscopy, and representing two tribes of Deltocephalinae: in Chiasmini, Nephotettixcincticeps (Uhler, 1896), and in Deltocephalini, Deltocephalusvulgaris (Dash & Viraktamath, 1998). Tables comparing the morphology of male and female internal reproductive structures of these studied species are provided and indicate that the main differences are in the relative shapes, sizes, and colors of these structures. The overall structure and organization, including details of the ultrastructure, of these two leafhopper species' male and female internal reproductive systems are very similar to those of previously studied leafhoppers. The main differences observed among species include the number of testicular follicles, the relative position of seminal vesicles and the degree of development of the accessory glands in the male, the number of ovaries, and the shape and color of the vagina and spermatheca in the female.

Structural Characteristics of Mitochondrial Genomes of Eight Treehoppers (Hemiptera: Membracidae: Centrotinae) and Their Phylogenetic Implications.

Complete mitochondrial genomes were newly sequenced for eight species of the treehopper subfamily Centrotinae (Hemiptera: Membracidae), four of which represent genera for which mitogenomes were not previously available. The new mitogenomes are generally similar in overall structure, gene order, base composition, and nucleotide content to those of previously sequenced species of the subfamily. Phylogenetic analyses were conducted using both maximum likelihood and Bayesian inference methods based on three separate nucleotide sequence datasets in which RNA gene sequences and/or third codon positions were either included or excluded from the concatenated protein-coding gene alignments. The results are consistent with previous phylogenies based on morphology and partial nuclear genome data, except for the lack of support for the monophyly of Leptocentrini. These results show that mitogenome sequences are informative of both ancient and recent divergence patterns within Centrotinae.

A new genus of Empoascini (Hemiptera, Cicadellidae, Typhlocybinae) from Papua New Guinea.

Ivmaka longicaudatus gen. et sp. nov. is described based on specimens from Papua New Guinea. Habitus photos and illustrations of the male genitalia of this new species are given and differences between the new genus with closely related genera are discussed.

A new species of Kana Distant representing the first record of Evacanthinae (Hemiptera: Cicadellidae) from Madagascar.

Kana ranomafanensis sp. n. is described and illustrated based on specimens from montane rainforests in eastern Madagascar. This is the first record of the diverse and widely distributed leafhopper subfamily Evacanthinae from Madagascar. A revised diagnosis of Kana Distant is provided and the new species is compared to species from the Indomalayan and Afrotropical regions. Evidence from previous molecular divergence time analyses suggests that ancestors of the new species arrived in Madagascar via transoceanic dispersal. The ovipositor of the genus and the male genitalia of Kana decora (Melichar), previously known only from the female holotype from Sri Lanka, are illustrated for the first time.

A key to genera of Dikraneurini from China, with description of a new species of Cornicola Ohara & Hayashi (Hemiptera, Cicadellidae, Typhlocybinae).

The leafhopper genus Cornicola Ohara & Hayashi, previously recorded from Japan, is recorded from China for the first time and a new species, C.maculatus Xu, Dietrich & Qin, sp. nov., is described and illustrated, including its color polymorphism. This genus has male genitalia and hind wing venation similar to those found in Empoascini but it is more appropriately placed in Dikraneurini. A key to species of Cornicola is given together with a key to the genera of Dikraneurini from China.

Functional and evolutionary implications of protein and metal content of leafhopper brochosomes.

Brochosomes derived from the specialized glandular segments of the Malpighian tubules (MTs) form superhydrophobic coatings for insects of Membracoidea, and have multiple hypothetical functions. However, the constituents, biosynthesis and evolutionary origin of brochosomes remain poorly understood. We investigated general chemical and physical characteristics of the integumental brochosomes (IBs) of the leafhopper Psammotettix striatus, determined the constituents of IBs, identified the unigenes involved in brochosomal protein synthesis, and investigated the potential associations among brochosomal protein synthesis, amino acid composition of food source, and the possible roles of endosymbionts in brochosome production. The results show that IBs are mainly composed of glycine- and tyrosine-rich proteins and some metal elements, which contain both essential and non-essential amino acids (EAAs and NEAAs) for insects, including EAAs deficient in the sole food source. All 12 unigenes involved in synthesizing the 12 brochosomal proteins (BPs) with high confidence are exclusively highly expressed in the glandular segment of MTs, confirming that brochosomes are synthesized by this segment. The synthesis of BPs is one of the key synapomorphies of Membracoidea but may be lost secondarily in a few lineages. The synthesis of BPs might be related to the symbiosis of leafhoppers/treehoppers with endosymbionts that provide these insects with EAAs, including those are deficient in the sole diet (i.e., plant sap) and could only be made available by the symbionts. We hypothesize that the functional modification of MTs have combined with the application of BPs enabling Membracoidea to colonize and adapt to novel ecological niches, and evolve to the dramatic diversification of this hemipteran group (in particular the family Cicadellidae). This study highlights the importance of evolutionary plasticity and multiple functions of MTs in driving the adaptations and evolution of sap-sucking insects of Hemiptera.

Phylogenetic Analysis of the Genus Planaphrodes Hamilton (Hemiptera, Cicadellidae, Aphrodinae) Based on Morphological Characteristics, with Revision of Species from China, Korea and Japan.

A morphology-based phylogeny of the Holarctic leafhopper genus Planaphrodes Hamilton is reconstructed for the first time based on 39 discrete male adult morphological characters. The results support the monophyly of Planaphrodes, with the included species forming two monophyletic lineages defined mainly by the number and location of aedeagus processes. The position of Planaphrodes in the Aphrodini was resolved as follows: (Stroggylocephalus + (Anoscopus + (Planaphrodes + Aphrodes))). The fauna of Planaphrodes from China, Japan and Korea are reviewed and six species are recognized, including two new species: P. bifasciatus (Linnaeus), P. sahlbergii (Signoret), P. nigricans (Matsumura), P. laevus (Rey), P. baoxingensis sp. nov. (China: Sichuan) and P. faciems sp. nov. (China: Hubei). Acocephalus alboguttatus Kato, 1933 syn. nov. and Aphrodes daiwenicus Kuoh, 1981 syn. nov. are considered junior synonyms of Planaphrodes sahlbergii (Signoret, 1879). Planaphrodes bella Choe, 1981 is a junior synonym of Planaphrodes nigricans (Matsumura, 1912). A checklist and key to species of Planaphrodes are provided.

Guilt by Association: DNA Barcoding-Based Identification of Potential Plant Hosts of Phytoplasmas from Their Insect Carriers.

Phytoplasmas are small phloem-restricted and insect-transmissible bacteria that infect many plant species, including important crops and ornamental plants, causing severe economic losses. Our previous studies screened phytoplasmas in hundreds of leafhoppers collected from natural habitats worldwide and identified multiple genetically different phytoplasmas in seven leafhopper species (potential insect vectors). As an initial step toward determining the impact of these phytoplasmas on the ecosystem, ribulose 1,5-biphosphate carboxylase large subunit (rbcL), a commonly used plant DNA barcoding marker, was employed to identify the plant species that the phytoplasma-harboring leafhoppers feed on. The DNA of 17 individual leafhoppers was PCR amplified using universal rbcL primers. PCR products were cloned, and five clones per amplicon were randomly chosen for Sanger sequencing. Moreover, Illumina high-throughput sequencing on selected PCR products was conducted and confirmed no missing targets in Sanger sequencing. The nucleotide BLAST results revealed 14 plant species, including six well-known plant hosts of phytoplasmas such as tomato, alfalfa, and maize. The remaining species have not been documented as phytoplasma hosts, expanding our knowledge of potential plant hosts. Notably, the DNA of tomato and maize (apparently cultivated in well-managed croplands) was detected in some phytoplasma-harboring leafhopper species sampled in non-crop lands, suggesting the spillover/spillback risk of phytoplasma strains between crop and non-crop areas. Furthermore, our results indicate that barcoding (or metabarcoding) is a valuable tool to study the three-way interactions among phytoplasmas, plant hosts, and vectors. The findings contribute to a better understanding of phytoplasma host range, host shift, and disease epidemiology.

Unraveling the hierarchical genetic structure of tea green leafhopper, Matsumurasca onukii, in East Asia based on SSRs and SNPs.

Matsumurasca onukii (Matsuda, R. (1952). Oyo-Kontyu Tokyo, 8(1): 19-21), one of the dominant pests in major tea production areas in Asia, currently is known to occur in Japan, Vietnam, and China, and severely threatens tea production, quality, and international trade. To elucidate the population genetic structure of this species, 1633 single nucleotide polymorphisms (SNPs) and 18 microsatellite markers (SSRs) were used to genotype samples from 27 sites representing 18 geographical populations distributed throughout the known range of the species in East Asia. Analyses of both SNPs and SSRs showed that M. onukii populations in Yunnan exhibit high-genetic differentiation and structure compared with the other populations. The Kagoshima (JJ) and Shizuoka (JS) populations from Japan were separated from populations from China by SNPs, but clustered with populations from Jinhua (JH), Yingde (YD), Guilin (GL), Fuzhou (FZ), Hainan (HQ), Leshan (CT), Chongqing (CY), and Zunyi (ZY) tea plantations in China and the Vietnamese Vinh Phuc (VN) population based on the SSR data. In contrast, CT, CY, ZY, and Shaanxi (SX) populations clustered together based on SNPs, but were separated by SSRs. Both marker datasets identified significant geographic differentiation among the 18 populations. Various environmental and anthropogenic factors, including geographical barriers to migration, human transport of hosts (Camellia sinesis [L.] O. Kuntze) and adaptation of M. onukii to various local climatic zones possibly account for the rapid spread of this pest in Asia. The results demonstrate that SNPs from high-throughput genotyping data can be used to reveal subtle genetic substructure at broad scales in r-strategist insects.

Description of a new species of the leafhopper genus Tambocerus (Hemiptera: Cicadellidae: Deltocephalinae) from Guizhou, China.

A new leafhopper species, Tambocerus fanjingensis sp. nov., is described and illustrated from Guizhou Province, China. A checklist of the Chinese species of Tambocerus is provided and a key based on the male genitalia is given to distinguish the Chinese species of the genus. The type specimens of the new species are deposited in the Institute of Entomology, Guizhou University, Guiyang, China (GUGC).

Comparison of Traditional and Next-Generation Approaches for Uncovering Phytoplasma Diversity, with Discovery of New Groups, Subgroups and Potential Vectors.

Despite several decades' effort to detect and identify phytoplasmas (Mollicutes) using PCR and Sanger sequencing focusing on diseased plants, knowledge of phytoplasma biodiversity and vector associations remains highly incomplete. To improve protocols for documenting phytoplasma diversity and ecology, we used DNA extracted from phloem-feeding insects and compared traditional Sanger sequencing with a next-generation sequencing method, Anchored Hybrid Enrichment (AHE) for detecting and characterizing phytoplasmas. Among 22 of 180 leafhopper samples that initially tested positive for phytoplasmas using qPCR, AHE yielded phytoplasma 16Sr sequences for 20 (19 complete and 1 partial sequence) while Sanger sequencing yielded sequences for 16 (11 complete and 5 partial). AHE yielded phytoplasma sequences for an additional 7 samples (3 complete and 4 partial) that did not meet the qPCR threshold for phytoplasma positivity or yielded non-phytoplasma sequences using Sanger sequencing. This suggests that AHE is more efficient for obtaining phytoplasma sequences. Twenty-three samples with sufficient data were classified into eight 16Sr subgroups (16SrI-B, I-F, I-AO, III-U, V-C, IX-J, XI-C, XXXVII-A), three new subgroups (designated as 16SrVI-L, XV-D, XI-G) and three possible new groups. Our results suggest that screening phloem-feeding insects using qPCR and AHE sequencing may be the most efficient method for discovering new phytoplasmas.

A New genus of flightless leafhoppers (Hemiptera: Cicadellidae: Megophthalminae) from the Pramo of Northern South America.

Diablophthalmus, gen. nov., is described and illustrated based on two new species, D. wygodzinskyorum sp. n., and D. marmoratus sp. n. from Pramo habitats in Colombia. These insects are the first recorded members of tribe Megophthalmini from South America. Morphologically convergent features shared with other soil-dwelling Membracoidea are discussed.

Mitogenomic phylogeny of Typhlocybinae (Hemiptera: Cicadellidae) reveals homoplasy in tribal diagnostic morphological traits.

The subfamily Typhlocybinae is a ubiquitous, highly diverse group of mostly tiny, delicate leafhoppers. The tribal classification has long been controversial and phylogenetic methods have only recently begun to test the phylogenetic status and relationships of tribes. To shed light on the evolution of Typhlocybinae, we performed phylogenetic analyses based on 28 newly sequenced and 19 previously sequenced mitochondrial genomes representing all currently recognized tribes. The results support the monophyly of the subfamily and its sister-group relationship to Mileewinae. The tribe Zyginellini is polyphyletic with some included genera derived independently within Typhlocybini. Ancestral character state reconstruction suggests that some morphological characters traditionally considered important for diagnosing tribes (presence/absence of ocelli, development of hind wing submarginal vein) are homoplastic. Divergence time estimates indicate that the subfamily arose during the Middle Cretaceous and that the extant tribes arose during the Late Cretaceous. Phylogenetic results support establishment of a new genus, Subtilissimia Yan & Yang gen. nov., with two new species, Subtilissimia fulva Yan & Yang sp. nov. and Subtilissimia pellicula Yan & Yang sp. nov.; but indicate that two previously recognized species of Farynala distinguished only by the direction of curvature of the processes of the aedeagus are synonyms, that is, Farynala dextra Yan & Yang, 2017 equals Farynala sinistra Yan & Yang, 2017 syn. nov. A key to tribes of Typhlocybinae is provided.

Revision of the genus Homa Distant (Hemiptera: Cicadellidae: Typhlocybinae).

The leafhopper genus Homa Distant is revised. Four new species, H. osificata Xu, Dietrich Qin sp. nov., H. oretinia Xu, Dietrich Qin sp. nov., H. asilata Xu, Dietrich Qin sp. nov., and H. algulata Xu, Dietrich Qin sp. nov., are described from Thailand. H. haematoptilus (Kirkaldy) is redescribed based on specimens from the Oriental Region. All included species are illustrated and a key is provided to separate species for which males are known.

Characterization, Comparison of Four New Mitogenomes of Centrotinae (Hemiptera: Membracidae) and Phylogenetic Implications Supports New Synonymy.

To explore the phylogenetic relationships of the subfamily Centrotinae from the mitochondrial genome data, four complete mitogenomes (Anchon lineatus, Anchon yunnanensis, Gargara genistae and Tricentrus longivalvulatus) were sequenced and analyzed. All the newly sequenced mitogenomes contain 37 genes. Among the 13 protein-coding genes (PCGs) of the Centrotinae mitogenomes, a sliding window analysis and the ratio of Ka/Ks suggest that atp8 is a relatively fast evolving gene, while cox1 is the slowest. All PCGs start with ATN, except for nad5 (start with TTG), and stop with TAA or the incomplete stop codon T, except for nad2 and cytb (terminate with TAG). All tRNAs can fold into the typical cloverleaf secondary structure, except for trnS1, which lacks the dihydrouridine (DHU) arm. The BI and ML phylogenetic analyses of concatenated alignments of 13 mitochondrial PCGs among the major lineages produce a well-resolved framework. Phylogenetic analyses show that Membracoidea, Smiliinae and Centrotinae, together with tribes Centrotypini and Leptobelini are recovered as well-supported monophyletic groups. The tribe Gargarini (sensu Wallace et al.) and its monophyly are supported.