First Report of Tomato spotted wilt virus infecting Helichrysum bracteatum in China.

Strawflower (Helichrysum bracteatum, Asteraceae) , an annual or biennial herb, is one of the most popular flowers in the world because of the colorful flowers and the long flowering period. However, the ornamental plants belonging to Asteraceae are susceptible to numerous viruses such as cucumber mosaic virus (CMV) (Cucumovirus, Bromoviridae) , potato virus Y (Potyvirus, Potyviridae), tomato mosaic virus (ToMV) (Tobamovirus, Virgaviridae), tobacco mosaic virus (TMV) (Tobamovirus, Virgaviridae), chrysanthemum virus B (CVB) (Carlavirus, Betaflexiviridae), tomato aspermy virus (TAV) (Cucumovirus, Bromoviridae), tomato spotted wilt virus (TSWV) (Orthotospovirus tomatomaculae, Tospoviridae), and impatiens necrotic spot virus (INSV) (Orthotospovirus impatiensnecromaculae, Orthotospovirus) resulting in severe yield loss (Verma et al. 2003; Raj et al. 2007; Kondo et al. 2011; Liu et al. 2014; Marys et al. 2014; Min et al. 2020; Gautam et al. 2021; Read et al. 2022; Supakitthanakorn et al. 2022). Among these viruses, the TSWV, a thrips-transmitted negative-stranded RNA virus, is well known to cause viral disease in several plant species while is less reported in Helichrysum, especially in China. In April 2021, viral attack symptoms were detected on the leaves of H. bracteatum during our routine checks in the greenhouse located at Shunyi District, Beijing, China, such as wilting, shrinking, chlorotic blotches, chlorotic ring spots. To investigate the virus infecting H. bracteatum, in total of 25 symptomatic and 5 asymptomatic leaves were sampled and tested by the effective double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using antisera against CMV, PVY/PVX, ToMV, TMV, CVB, TAV, TSWV, INSV, separately (Agdia, USA). Only the TSWV showed positive in symptomatic samples, and asymptomatic samples were all negative, which implied TSWV infection. To further confirm the virus type of TSWV isolated from H. bracteatum samples, the genomic RNA of the virus was isolated using reverse transcription and polymerase chain reaction (RT-PCR), and then was cloned, sequenced and analyzed. Total RNA of five symptomatic leaves (ELISA-positive) were extracted using the FastPure Plant Total RNA Isolation Kit (Vazyme, China), and then were reverse transcribed by HiScript II Reverse Transcriptase (Vazyme, China). Each genome segments were amplified using Phanta Max Super-Fidelity DNA Polymerase (Vazyme, China) with TSWV-specific primers newly designed and listed in Table S1. The PCR setup was as follow: 95°C for 30 s, followed by 35 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 1.5 min, with a final extension at 72°C for 10 min. All PCR products were cloned into the TA/Blunt-Zero vector (Vazyme, China) and sequenced (GENEWIZ, Inc.). We assembled and then analyzed the evolutionary relationship of three genomic fragments, that is, TSWV-BJFC-Hb S (2923 bp), M (4785 bp) and L (8971 bp) using the BLAST tools. The results showed high similarity with TSWV-henan isolated from pepper in China (99.6% to TSWV-S (MT799179.1), 99.8 % to TSWV-M (MT799178.1) and 99.8 % TSWV-L (MT799177.1)). These sequences have been submitted to the GenBank (OM982910, OM982911 and OM937131). Taking all of these evidences together, the viral disease observed in H. bracteatum was closely associated with TSWV. TSWV is currently widespread in China, infecting Nasturtium, Chrysanthemum and cowpea (Xiao et al. 2015; Hu et al. 2018; Yu et al. 2021). Epidemics of TSWV have also been reported in several other countries such as Korea, North Carolina, Turkey and India (Renukadevi et al. 2015; Koehler et al. 2016; Kwak et al. 2021; Erilmez, S. 2022). This is the first report of TSWV infection on H. bracteatum in China. Due to the fast spread and serious economic losses of TSWV, the rapid detection may be the essential way to prevent this viral disease among crops (Macharia et al. 2014).

CRISPR/Cas9-Mediated genomic knock out of tyrosine hydroxylase and yellow genes in cricket Gryllus bimaculatus.

Gryllus bimaculatus is an emerging model organism in various fields of biology such as behavior, neurology, physiology and genetics. Recently, application of reverse genetics provides an opportunity of understanding the functional genomics and manipulating gene regulation networks with specific physiological response in G. bimaculatus. By using CRISPR/Cas9 system in G. bimaculatus, we present an efficient knockdown of Tyrosine hydroxylase (TH) and yellow-y, which are involved in insect melanin and catecholamine-biosynthesis pathway. As an enzyme catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine, TH confines the first step reaction in the pathway. Yellow protein (dopachrome conversion enzyme, DCE) is also involved in the melanin biosynthetic pathway. The regulation system and molecular mechanism of melanin biogenesis in the pigmentation and their physiological functions in G. bimaculatus hasn't been well defined by far for lacking of in vivo models. Deletion and insertion of nucleotides in target sites of both TH and Yellow are detected in both F0 individuals and the inheritable F1 progenies. We confirm that TH and yellow-y are down-regulated in mutants by quantitative real-time PCR analysis. Compared with the control group, mutations of TH and yellow-y genes result in defects in pigmentation. Most F0 nymphs with mutations of TH gene die by the first instar, and the only adult had significant defects in the wings and legs. However, we could not get any homozygotes of TH mutants for all the F2 die by the first instar. Therefore, TH gene is very important for the growth and development of G. bimaculatus. When the yellow-y gene is knocked out, 71.43% of G. bimaculatus are light brown, with a slight mosaic on the abdomen. The yellow-y gene can be inherited stably through hybridization experiment with no obvious phenotype except lighter cuticular color. The present loss of function study indicates the essential roles of TH and yellow in pigmentation, and TH possesses profound and extensive effects of dopamine synthesis in embryonic development in G. bimaculatus.

Clarifying Recent Adaptive Diversification of the Chrysanthemum-Group on the Basis of an Updated Multilocus Phylogeny of Subtribe Artemisiinae (Asteraceae: Anthemideae).

Understanding the roles played by geography and ecology in driving species diversification and in the maintenance of species cohesion is the central objective of evolutionary and ecological studies. The multi-phased orogenesis of Qinghai-Tibetan Plateau (QTP) and global climate changes over late-Miocene has profoundly influenced the environments and evolution of organisms in this region and the vast areas of Asia. In this study, we investigate the lineage diversification of Chrysanthemum-group in subtribe Artemisiinae (tribe Anthemideae, Asteraceae) likely under the effects of climate changes during this period. Using DNA sequences of seven low-copy nuclear loci and nrITS and the coalescent analytical methods, a time-calibrated phylogeny of subtribe Artemisiinae was reconstructed with emphasis on Chrysanthemum-group. The monophyletic Chrysanthemum-group was well resolved into two major clades corresponding to Chrysanthemum and Ajania, two genera which can be well identified by capitulum morphology but have been intermingled in previous plastid and ITS trees. Within Chrysanthemum, a later divergence between Ch. indicum-complex and Ch. zawadskii-complex can be recognized. The time frames of these sequential divergences coincide with the late Cenozoic uplift of the Northern QTP and the concomitant climatic heterogeneity between eastern and inland Asia. Reconstruction of historical biogeography suggested the origin of Chrysanthemum-group in Central Asia, followed by eastward migration of Chrysanthemum and in situ diversification of Ajania. Within Chrysanthemum, Ch. indicum-complex and Ch. zawadskii-complex exhibited contemporary distributional division, the former in more southern and the latter in more northern regions. The geographic structure of the three lineages in Chrysanthemum-group have been associated with the niche differentiation, and environmental heterogenization in Asia interior.

A new species of Parapteronemobius from Zhejiang, China (Orthoptera: Trigonidiidae: Nemobiinae).

Parapteronemobius contains 7 species or subspecies distributed in East Asia with only one species recorded from China. In this study, we reported the second species, P. chenggong He sp. nov., from Zhejiang. The new species differs from P. dibrachiatus in body size, male genitalia and COI gene. The phylogeny of this genus in subfamily Nemobiinae are discussed based on COI, 18S and 28S genes. The type specimens are deposited in the Museum of Biology, East China Normal University (ECNU).

A new species of genus Hexacentrus Serville, 1831 from Taiwan (Orthoptera: Tettigoniidae: Hexacentrinae).

There are 29 species or subspecies in genus Hexacentrus occurring in Asia, Africa and Australia. Because of its similar appearance, it is not easy to distinguish them by traditional methods. In this study, we collected samples and sequenced COI genes from wide range. By reconstructing the gene tree, we found one new species, H. formosanus Chen et He sp. nov., from Taiwan. The new species is similar to H. expansus or H. inflatissimus, but differs from the former in male Cu2 vein of left tegmina curved and slender, and spectrum of male left tegmina slender and subsquare; differs from the later by body size smaller and female tegmina narrow and short. The type specimens are deposited in National Museum of Natural Science, Taichung, Taiwan (NMNS). H. japonicus hareyamai is treated as species level, H. hareyamai stat. nov.

A new species of genus Homoeoxipha Saussure, 1874 from China (Orthoptera: Trigonidiidae: Trigonidiinae).

The genus Homoeoxipha contains 11 species worldwide with 4 recorded from China. In this study, we reconstructed the gene tree based on COI gene, and reported one new species, H. oscillantenna He sp. nov. The new species is similar to H. eurylobus Ma, Liu Zhang, but differs in the color of fore femur and the structure of male genitalia. Calling songs and nymph morphology are also provided. The type specimens are deposited in the Museum of Biology, East China Normal University (ECNU).

Dysfunction of CYC2g is responsible for the evolutionary shift from radiate to disciform flowerheads in the Chrysanthemum group (Asteraceae: Anthemideae).

Evolutionary shifts among radiate, disciform and discoid flowerheads have occurred repeatedly in a number of major lineages across the Asteraceae phylogeny; such transitions may also appear within evolutionarily young groups. Although several studies have demonstrated that CYC2 genes partake in regulating floral morphogenesis in Asteraceae, the evolution of capitulum forms within a recently diverging lineage has remained poorly understood. Here, we study the molecular regulation of the shift from a radiate to a disciform capitulum within the Chrysanthemum group. This is a recently radiating group mainly comprising two genera, Chrysanthemum and Ajania, that are phylogenetically intermingled but distinct in flowerhead morphology: Chrysanthemum spp. with radiate capitula and Ajania spp. with disciform capitula. We found that the morphogenesis of zygomorphy in the marginal floret in Ajania was disrupted soon after floral primordium emergence; CYC2g, one of the CYC2 copies that was expressed prominently in the ray floret of Chrysanthemum was not expressed in flowerheads of Ajania. Weakening the expression of ClCYC2g in Chrysanthemum lavandulifolium led to the gradual transition of a ray flower toward the disc-like form. Molecular evolutionary analyses indicated that the disciform capitulum might have evolved only once, approximately 8 Mya, arising from dysfunction of the CYC2g orthologs. A 20-nt deletion, including a putative TATA-box of the Ajania-type CYC2g promoter, appeared to inhibit the expression of the gene. Considering the divergent habitats of Chrysanthemum and Ajania, we propose that the shift from radiate to disciform capitulum must have been related to changes in pollination strategies under selective pressure.

The first phylogenetic study of sword-tail crickets from China inferred from COI, 18S and 28S genes, with the establishment of two new genera and description of one new species (Orthoptera: Grylloidea: Trigonidiidae).

The phylogenetic tree of Chinese Trigonidiinae species was reconstructed based on one mitochondrial fragment (COI) and two nuclear ribosomal loci (18S and 28S). According to the concatenated trees, we define four genus groups with high nodal supports. The subgenus Paratrigonidium should be upgraded to genus status. A new genus Abstrigonidium He gen. nov. is established and Paratrigonidium chloropodum is selected as type species (A. chloropodum comb. nov.). Amusurgus (Paranaxipha) fujianensis is moved to genus Sectus as S. fujianensis comb. nov. A new species named as Emerasoma curvicerca He gen. et sp. nov. is reported. It differs from other genera by asymmetric genitalia, and lacking stridulatory files and mirror. The type specimens are deposited in Museum of Biology, East China Normal University (ECNU).

Report of a new genus Mirigryllus belonging to tribe Modicogryllini, with a new species M. nigrus from Zhejiang, China (Orthoptera: Gryllidae: Gryllinae: Modicogryllini).

A new genus (Mirigryllus He gen. nov.) with a new species (M. nigrus He sp. nov.) is reported from Zhejiang, China. The morphology and calling songs of the new species are provided. The features that different from tribe Modicogryllini spp. include long and thin posterior epiphallic lobes, ectoparameron transparent. Two genes, COI and 18S, of 11 Modicogryllini species were sequenced, and phylogenetic tree was reconstructed. The results support the validation of the new species. The type specimens are deposited in Museum of Biology, East China Normal University (ECNU).

A new species of Goniurosaurus from Hainan Island, China based on molecular and morphological data (Squamata: Sauria: Eublepharidae).

Three species of geckos in the genus Goniurosaurus have been recorded from Hainan Island in China. We describe a new species, Goniurosaurus kwanghua sp. nov. Zhu He, bringing the number of this genus in Hainan Island to four. We conducted phylogenetic analyses with two mitochondrial genes (16S Cytb) and two nuclear genes (RAG1 C-MOS) to validate this new species. The new species is similar to G. hainanensis, but differs in the following characters: (1) middle section of the nuchal loop wide and posteriorly protracted, (2) wider body bands with dark markings, (3) yellow stripes on posterior side of humerus and femur, linked to the first and third body bands, respectively. The type specimens are deposited in the Museum of Biology, East China Normal University (ECNU).

First record of Lissotrachelus ferrugineonotatus cricket producing ultrasound (Orthoptera: Grylloidea).

The range of hearing for human is about 50 to 20,000 Hz, but some animals can produce ultrasounds over 20000 Hz. These include bats, cetaceans, rodents and even frogs (Feng et al., 2006). Katydids and crickets are famous singing insects belonging to order Orthoptera. Many katydids can produce ultrasounds even over 100 kHz (Montealegre-Z et al., 2006). However, the dominant frequency of most crickets ranges from 2 to 8 kHz (Walker Carlysle, 1975; Robillard Desutter-Grandcolas, 2004). In previous studies, only Eneopterinae crickets were reported to produce high-frequency songs (Robillard et al. 2007; Robillard Tan 2013). We report that Lissotrachelus ferrugineonotatus crickets produce ultrasounds with peak at 26.25 kHz.

A pilot phylogeny study of Nemobiinae inferred from 18S, 28s and COI genes, with descriptions of two new genera and a new species from Hainan, China (Orthoptera: Grylloidea: Trigonidiidae).

The phylogenetic tree of Chinese Nemobiinae species was reconstructed based on one mitochondrial COI fragment and two nuclear ribosomal loci (18S and 28S). According to both of concatenated and coalescent trees, Dianemobius, Polionemobius and Pteronemobius (except P. jianfenglingensis Liu Shi) were shown to be monophyletic with highly nodal supports. As to P. jianfenglingensis, it should be moved into a newly established genus, Giganemobius Shen He gen. nov. In addition, a new mute species named as Qionemobius hsterus Shen He gen. nov. sp. nov. was reported. It differs from other genera by having six apical spurs on hind tibia, but lacking forewings. The type specimens are deposited in Museum of Biology, East China Normal University (ECNU).

A new genus with a new species of Shield-back Katydid, with comments on the phylogeny and diagnosis of the genus Kansua Uvarov and the tribe Drymadusini (Orthoptera: Tettigoniidae: Tettigoniinae) from China.

In this paper, a new species, Sichuana cryptospina sp. nov., is described from Sichuan, China. COI genes were used to analyze the phylogenetic relationships with the species belonging to Drymadusini from China. Phylogenetic reconstruction supports the monophyly of Drymadusini while the relationships among genera are still unclear. The new species should be placed in the new genus named Sichuana gen. nov. Additionally, Kansua Uvarov should be moved into this tribe, with supplementary expression of the tribe diagnosis. Types of the new species in this paper are preserved in the Museum of Biology, East China Normal University (ECNU).

Cacoplistes (Laminogryllus) brevisparamerus Wang, Zhang, Wei amp; Liu, 2017: A junior synonym of Cacoplistes (Laminogryllus) choui Liu amp; Shi, 2012 (Orthoptera: Grylloidea: Phalangopsidae: Cachoplistinae).

Cacoplistes (Laminogryllus) spp. from Guangxi were compared by morphology and molecular studies. Morphology results showed that differences of genitalia were caused by movable epiphallus. Molecular result showed that only 0.3% substitution sites of COI gene were detected. Thus, C. (L.) brevisparamerus syn. is the junior synonym of C. (L.) choui.

An integrative taxonomy of Vescelia pieli pieli species complex based on morphology, genes and songs from China (Orthoptera: Grylloidea: Phalangopsidae: Phaloriinae).

There are nine species or subspecies belonging to genus Vescelia Stål, 1877 in the world, with two species distributed in China, V. pieli pieli and V. liangi. During 2018-2019, Vescelia spp. from Hainan, Guangdong and Fujian were systematically investigated. Here, we described two new species, Vescelia dulcis He sp. nov. and V. pieli monotonia He subsp. nov. The new species are similar to V. pieli pieli in morphology except for minute differences in the genitalia. But phylogenetic reconstruction and differences in songs support the validity of the new species. The type specimens are deposited in Museum of Biology, East China Normal University (ECNU).

A new genus of cricket with one new species from western Yunnan, China (Orthoptera: Gryllidae: Gryllinae).

Most male crickets can produce songs mostly for attracting females. Songs are specific among species,. this depending on physiology and the structural characteristics of the forewings. Nevertheless, some species belonging to subfamily Gryllinae have lost their singing abilities (mute species). Chinese examples include Agryllus spp., Conoblemmus spp., Goniogryllus spp. and Callogryllus yunnanus. Males of these genera are either wingless or have forwings similar to those of females. These crickets have been poorly studied in China. In this study, we describe one new genus Asonicogryllus He gen. nov. with a new species Asonicogryllus kwanghua sp. nov. from western Yunnan. The type specimens are deposited in Museum of Biology, East China Normal University (ECNU).

New and little-known crickets from Southern Guangxi, China (Orthoptera: Grylloidea: Gryllidae: Phalangopsidae; Trigonidiidae).

The crickets from Southern Guangxi, China have been investigated from 2017-2019. Teleogryllus (Brachyteleogryllus) commodus and Lissotrachelus ferrugineonotatus are first reported from Guangxi. Two new species, Polionemobius marblus He sp. nov. and Ornebius panda He sp. nov., are described along with their calling songs. The COI genes of above species and Cacoplistes brevisparamerus, Gorochovius trinervus are provided. The type specimens are deposited in Museum of Biology, East China Normal University (ECNU).

Three new species of genus Mecopoda Serville, 1831 from China (Orthoptera: Tettigoniidae: Mecopodinae).

Only six species or subspecies of genus Mecopoda have been recorded with two species recorded from China prior to this study. We describe 3 new species, M. hainanensis He sp. nov., M. fallax He sp. nov. and M. marmorata He sp. nov. from Yunnan and Hainan. Although M. hainanensis is similar to M. niponensis, M. fallax is similar to M. elongata elongata, their songs can be used for identification. The result of molecular study also supports the validity of three new species. The type specimens are deposited in Museum of Biology, East China Normal University (ECNU).

A new species of the subgenus Duolandrevus (Eulandrevus) Kirby, 1906 from Guangxi, China (Orthoptera: Gryllidae: Landrevinae).

Seven species of the subgenus Duolandrevus (Eulandrevus) Kirby, 1906 have been recorded from China prior to this study. Their morphology is similar, and genitalia can be used for distinguishing different species in earlier research. We describe one new species, D. (E.) obsidianus He sp. nov. from Guangxi. The new species is similar to related species in gross morphology, but showed differences in genitalia with epiphallic lateral lobes bifurcated apically in right angle. The songs and COI genes are first compared in this study, and the results support the validity of the new species. The type specimens are deposited in Museum of Biology, East China Normal University (ECNU).

Patterning the Asteraceae Capitulum: Duplications and Differential Expression of the Flower Symmetry CYC2-Like Genes.

There are several types of capitulum in the Asteraceae due to different combinations of florets varying in corolla shape and stamen development. Previous studies have shown that the formation of ray florets on a radiate capitulum may be related to the parallel co-option of CYC2-like genes among independent Asteraceae lineages. The present work tests that hypothesis and attempts to shed light on the pattern of evolution of the Asteraceae capitulum and floral heteromorphism under the regulation of CYC2-like genes. In this study, the evolutionary history of CYC2-like genes in the Asterales was reconstructed and their expression patterns were examined in species representing different capitulum types and several major Asteraceae lineages. To clarify the role of CYC2d clade genes in morphogenesis of ray flowers, overexpression of ClCYC2d was conducted in Chrysanthemum lavandulifolium. Our results show that there are six CYC2-like members in the Asteraceae; they are results of five duplication events starting from a single-copy gene in the common ancestor of the Goodeniaceae-Calyceraceae-Asteraceae group and completing before the divergence of the subfamily Carduoideae of Asteraceae. Spatial expression pattern of each of the Asteraceae CYC2-like members is conserved across the family. All the six members contribute to the development of the complexity of a capitulum: To form a ray floret, either CYC2c or CYC2g plays an essential role, while CYC2d represses the development of dorsal corolla lobes and stamens of the floret. In sum, the developmental program of making a ray flower is conserved involving functionally divergent CYC2-like genes. Based on extensive species sampling, this study provides an overview of the mode of regulation of CYC2-like genes that patterns the capitulum architectures and their transitions.