Integrative taxonomy, phylogenetics and historical biogeography of subgenus Aeschyntelus Stål, 1872 (Hemiptera: Heteroptera: Rhopalidae).

The subgenus Aeschyntelus includes six species that show variations in body color and shape, thus making it difficult to identify them based on morphological identification alone. To date, no genetic study has evaluated species within this genus. Herein, we collected 171 individuals from 90 localities of Rhopalus and employed an integrative taxonomic approach that incorporated morphological data, mitochondrial genomic data (COI, whole mitochondrial data) and nuclear genomic data (18S + 28S rRNAs, nuclear genome-wide SNPs) to delineate species boundaries. Our analyses confirmed the status of nine described species of Rhopalus and proposed the recognition of one new species known as Rhopalus qinlinganus sp. nov., which is classified within the subgenus Aeschyntelus. Discrepancies arising from nuclear and mitochondrial data suggest the presence of mito-nuclear discordance. Specifically, mitochondrial data indicated admixture within Clade A, comprising R. kerzhneri and R. latus, whereas genome-wide SNPs unambiguously identified two separate species, aligning with morphological classification. Conversely, mitochondrial data clearly distinguished Clade B- consisting of R. sapporensis into two lineages, whereas genome-wide SNPs unequivocally identified a single species. Our study also provides insights into the evolutionary history of Aeschyntelus, thus indicating that it likely originated in East Asia during the middle Miocene. The development of Aeschyntelus biodiversity in the southwestern mountains of China occurred via an uplift-driven diversification process. Our findings highlight the necessity of integrating both morphological and multiple molecular datasets for precise species identification, particularly when delineating closely related species. Additionally, it reveals the important role of mountain orogenesis on speciation within the southwestern mountains of China.

Identifying a potentially invasive population in the native range of a species: The enlightenment from the phylogeography of the yellow spotted stink bug, Erthesina fullo (Hemiptera: Pentatomidae).

The yellow spotted stink bug (YSSB), Erthesina fullo (Thunberg, 1783) is an important Asian pest that has recently successfully invaded Europe and an excellent material for research on the initial stage of biological invasion. Here, we reported the native evolutionary history, recent invasion history, and potential invasion threats of YSSB for the first time based on population genetic methods [using double digest restriction-site associated DNA (ddRAD) data and mitochondrial COI and CYTB] and ecological niche modelling. The results showed that four lineages (east, west, southwest, and Hainan Island) were established in the native range with a strong east-west differentiation phylogeographical structure, and the violent climate fluctuation might cause population divergence during the Middle and Upper Pleistocene. In addition, land bridges and monsoon promote dispersal and directional genetic exchanging between island populations and neighboring continental populations. The east lineage (EA) was identified as the source of invasion in Albania. EA had the widest geographical distribution among all other lineages, with a star-like haplotype network with the main haplotype as the core. It also had a rapid population expansion history, indicating that the source lineage might have stronger diffusion ability and adaptability. Our findings provided a significant biological basis for fine tracking of invasive source at the lineage or population level and promote early invasion warning of potential invasive species on a much subtler lineage level.

Fabrication and application of carrier-free and carrier-based nanopesticides in pest management.

Pesticides are widely used for pest control to promote sustained and stable growth of agricultural production. However, indiscriminate pesticide usage poses a great threat to environmental and human health. In recent years, nanotechnology has shown the ability to increase the performance of conventional pesticides and has great potential for improving adhesion to crop foliage, solubility, stability, targeted delivery, and so forth. This review discusses two types of nanopesticides, namely, carrier-free nanopesticides and carrier-based nanopesticides, that can precisely release necessary and sufficient amounts of active ingredients. At first, the basic characterization and preparation methods of these two distinct types of nanopesticides are briefly summarized. Subsequently, current applications and future perspectives on scientific examples and strategies for promoting the usage efficacy and reducing the environmental risks of these nanopesticides were also described. Overall, nanopesticides can promote higher crop yields and lay the foundation for sustainable agriculture and global food security.

Identification and expression patterns of somatic piRNAs and PIWI genes in Riptortus pedestris (Hemiptera: Alydidae).

RNA interference (RNAi)-based gene silencing is a feasible and sustainable technology for the management of hemipteran pests by double-stranded RNA involvement, including small-interfering RNA, microRNA, and Piwi-interacting RNA (piRNA) pathways, that may help to decrease the usage of chemical insecticides. However, only a few data are available on the somatic piRNAs and their biogenesis genes in Riptortus pedestris, which serves as a significant pest of soybean (Glycine max). In this study, two family members of the PIWI gene were identified and characterized in R. pedestris, containing Argonaute3 (RpAgo3) and Aubergine (RpAub) genes with conserved protein domains, and their clusters were validated by phylogenetic analysis. In addition, they were widely expressed in all developmental stages of the whole body of R. pedestris and had lower expression levels in R. pedestris guts under different rearing conditions based on previous transcriptome sequencing. Furthermore, abundant clean reads were filtered to a total number of 45,998 piRNAs with uridine bias at the first nucleotide (nt) position and 26-32 nt in length by mapping onto the reference genome of R. pedestris according to our previous whole-transcriptome sequencing. Finally, our data revealed that gut bacterial changes were significantly positively or negatively associated with differentially expressed piRNAs among the five comparison groups with Pearson correlation analysis. In conclusion, these findings paved new avenues for the application of RNAi-based biopesticides for broad-spectrum hemipteran pest control.

Mitochondrial introgression and mito-nuclear discordance obscured the closely related species boundaries in Cletus Stål from China (Heteroptera: Coreidae).

Accurate taxonomy and delimitation are of great importance for pest control strategies and management programs. Here, we focus on Cletus (Insecta: Hemiptera: Coreidae), which includes many crop pests. The species boundaries still conflict and only cytochrome c oxidase subunit I (COI) barcoding has been previously used for molecular studies. We generated new mitochondrial genome and nuclear genome-wide SNPs to explore the species boundaries of 46 Cletus samples from China using multiple species delimitation approaches. All results recovered a monophyly with high support, except for two closely related species in clade I - C. punctiger and C. graminis. Mitochondrial data demonstrated admixture in clade I, while genome-wide SNPs unambiguously identified two separate species, which were confirmed by morphological classification. Inconsistent nuclear and mitochondrial data indicated mito-nuclear discordance. Mitochondrial introgression is the most likely explanation, and more extensive sampling and more comprehensive data are needed to ascertain a pattern. Accurate species delimitation will shed light on species status; thus, an accurate taxonomy is of particular concern, as there is a pressing need to implement precise control of agricultural pests and to perform further research on diversification.

Parallel metatranscriptome analysis reveals degradation of plant secondary metabolites by beetles and their gut symbionts.

Switching to a new host plant is a driving force for divergence and speciation in herbivorous insects. This process of incorporating a novel host plant into the diet may require a number of adaptations in the insect herbivores that allow them to consume host plant tissue that may contain toxic secondary chemicals. As a result, herbivorous insects are predicted to have evolved efficient ways to detoxify major plant defences and increase fitness by either relying on their own genomes or by recruiting other organisms such as microbial gut symbionts. In the present study we used parallel metatranscriptomic analyses of Altica flea beetles and their gut symbionts to explore the contributions of beetle detoxification mechanisms versus detoxification by their gut consortium. We compared the gut meta-transcriptomes of two sympatric Altica species that feed exclusively on different host plant species as well as their F1 hybrids that were fed one of the two host plant species. These comparisons revealed that gene expression patterns of Altica are dependent on both beetle species identity and diet. The community structure of gut symbionts was also dependent on the identity of the beetle species, and the gene expression patterns of the gut symbionts were significantly correlated with beetle species and plant diet. Some of the enriched genes identified in the beetles and gut symbionts are involved in the degradation of secondary metabolites produced by plants, suggesting that Altica flea beetles may use their gut microbiota to help them feed on and adapt to their host plants.

Two Apriona Species Sharing a Host Niche Have Different Gut Microbiome Diversity.

The adaptability of herbivorous insects to toxic plant defense compounds is partly related to the structure of the gut microbiome. To overcome plant resistance, the insect gut microbiome should respond to a wide range of allelochemicals derived from dietary niches. Nevertheless, for sibling herbivorous insect species, whether the gut microbiome contributes to success in food niche competition is unclear. Based on 16S rDNA high-throughput sequencing, the gut microbiomes of two Apriona species that share the same food niche were investigated in this study to determine whether the gut microbiome contributes to insect success in food-niche competition. Our observations indicated that the gut microbiome tended to play a part in host niche competition between the two Apriona species. The gut microbiome of Apriona swainsoni had many enriched pathways that can help degrade plant toxic secondary compounds, including xenobiotic biodegradation and metabolism, terpenoid and polyketide metabolism, and secondary metabolite biosynthesis. Meanwhile, A. swainsoni hosted a much greater variety of microorganisms and had more viable bacteria than A. germari. We conclude that gut microbes may influence the coevolution of herbivores and host plants. Gut bacteria may not only serve to boost nutritional relationships, but may also play an important role in insect food niche competition.

Phylogeny of Coreoidea based on mitochondrial genomes show the paraphyly of Coreidae and Alydidae.

Coreoidea (Insecta: Hemiptera: Heteroptera) is a widely distributed and agriculturally important bugs. However, the phylogeny of Coreoidea lacked consensus on higher-level relationships and several studies by comparative morphological characters and molecular data suggested the non-monophyly of two families: Coreidae and Alydidae. The mitochondrial genome (mitogenome) has long been thought to be a significant marker to understand phylogenetic relationships, but the mitogenome in Alydidae is scarce to date. In the present study, we gathered the mitogenomes of 28 species from four families of Coreoidea excluding Hyocephalidae (Alydidae, Coreidae, Rhopalidae, and Stenocephalidae), including four newly sequenced mitogenomes of Alydidae, and conducted mitogenomic organization and phylogenetic studies. We used maximum likelihood and Bayesian inference methods to infer the higher-level phylogeny from the perspective of mitogenomes, primarily to investigate the phylogenetic relationship betweeen Coreidae and Alydidae. We add evidence that neither Alydidae nor Coreidae are monophyletic based on mitogenomes. Newly sequenced mitogenomes of Alydidae have traditional gene structure and gene rearrangement was not found. Alydinae was always recovered as closely related to Pseudophloeinae of the coreid subfamily with high support. The placement of the coreid subfamily Hydarinae and alydid subfamily Micrelytrinae are unstable depending on approach used. In terms of the length and nucleotide composition of the protein coding genes in mitogenomes, Pseudophloeinae and Hydarinae of coreid were more similar to Alydidae. The unsettled classification issues of Coreidae and Alydidae by mitogenomes were demonstrated in this work, indicating that further study is needed.

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.

Gut bacterial communities and their contribution to performance of specialist Altica flea beetles.

Host plant shifts are a common mode of speciation in herbivorous insects. Although insects can evolve adaptations to successfully incorporate a new host plant, it is becoming increasingly recognized that the gut bacterial community may play a significant role in allowing insects to detoxify novel plant chemical defenses. Here, we examined differences in gut bacterial communities between Altica flea beetle species that feed on phylogenetically unrelated host plants in sympatry. We surveyed the gut bacterial communities of three closely related flea beetles from multiple locations using 16S rRNA amplicon sequencing. The results showed that the beetle species shared a high proportion (80.7%) of operational taxonomic units. Alpha-diversity indicators suggested that gut bacterial diversity did not differ among host species, whereas geography had a significant effect on bacterial diversity. In contrast, analyses of beta-diversity showed significant differences in gut bacterial composition among beetle species when we used species composition and relative abundance metrics, but there was no difference in composition when species presence/absence and phylogenetic distance indices were used. Within host beetle species, gut bacterial composition varied significantly among sites. A metagenomic functionality analysis predicted that the gut microbes had functions involved in xenobiotic biodegradation and metabolism as well as metabolism of terpenoids and polyketides. These predictions, however, did not differ among beetle host species. Antibiotic curing experiments showed that development time was significantly prolonged, and there was a significant decline in body weight of newly emerged adults in beetles lacking gut bacteria, suggesting the beetles may receive a potential benefit from the gut microbe-insect interaction. On the whole, our results suggest that although the gut bacterial community did not show clear host-specific patterns among Altica species, spatiotemporal variability is an important determinant of gut bacterial communities. Furthermore, the similarity of communities among these beetle species suggests that microbial facilitation may not be a determinant of host plant shifts in Altica.

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.

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.

Endogenous cellulolytic enzyme systems in the longhorn beetle Mesosa myops (Insecta: Coleoptera) studied by transcriptomic analysis.

The Cerambycidae (longhorn beetle) is a large family of Coleoptera with xylophagous feeding habits. Cellulose digestion plays an important role in these wood-feeding insects. In this study, transcriptomic technology was used to obtain one glycoside hydrolase family 45 (GH45) cellulase and seven GH5 cellulases from Mesosa myops, a typical longhorn beetle. Analyses of expression dynamics and evolutionary relationships provided a complete description of the cellulolytic system. The expression dynamics related to individual development indicated that endogenous GH45 and GH5 cellulases dominate cellulose digestion in M. myops. Evolutionary analyses suggested that GH45 cellulase gene is a general gene in the Coleoptera Suborder Polyphaga. Evolutionary analyses also indicated that the GH5 cellulase group in Lamiinae longhorn beetles is closely associated with wood feeding. This study demonstrated that there is a complex endogenous cellulolytic system in M. myops that is dominated by cellulases belonging to two glycoside hydrolase families.

The ncRNA-mediated regulatory networks of defensins and lysozymes in Riptortus pedestris: involvement in response to gut bacterial disturbances.

Insects depend on humoral immunity against intruders through the secretion of antimicrobial peptides (AMPs) and immune effectors via NF-κB transcription factors, and their fitness is improved by gut bacterial microbiota. Although there are growing numbers of reports on noncoding RNAs (ncRNAs) involving in immune responses against pathogens, comprehensive studies of ncRNA-AMP regulatory networks in Riptortus pedestris, which is one of the widely distributed pests in East Asia, are still not well understood under feeding environmental changes. The objective of this study employed the whole-transcriptome sequencing (WTS) to systematically identify the lncRNAs (long noncoding RNA) and circRNAs (circular RNA) and to obtain their differential expression from the R. pedestris gut under different feeding conditions. Functional annotation indicated that they were mainly enriched in various biological processes with the GO and KEGG databases, especially in immune signaling pathways. Five defensin (four novel members) and eleven lysozyme (nine novel members) family genes were identified and characterized from WTS data, and meanwhile, phylogenetic analysis confirmed their classification. Subsequently, the miRNA-mRNA interaction network of above two AMPs and lncRNA-involved ceRNA (competing endogenous RNA) regulatory network of one lysozyme were predicted and built based on bioinformatic prediction and calculation, and the expression patterns of differentially expressed (DE) defensins, and DE lysozymes and related DE ncRNAs were estimated and selected among all the comparison groups. Finally, to integrate the analyses of WTS and previous 16S rRNA amplicon sequencing, we conducted the Pearson correlation analysis to reveal the significantly positive or negative correlation between above DE AMPs and ncRNAs, as well as most changes in the gut bacterial microbiota at the genus level of R. pedestris. Taken together, the present observations provide great insights into the ncRNA regulatory networks of AMPs in response to rearing environmental changes in insects and uncover new potential strategies for pest control in the future.

Changes in the gut bacterial community affect miRNA profiles in Riptortus pedestris under different rearing conditions.

Insects possess complex and dynamic gut microbial system, which contributes to host nutrient absorption, reproduction, energy metabolism, and protection against stress. However, there are limited data on interactions of host-gut bacterial microbiota through miRNA (microRNA) regulation in a significant pest, Riptortus pedestris. Here, we performed the 16S rRNA amplicon sequencing and small RNA sequencing from the R. pedestris gut under three environmental conditions and antibiotic treatment, suggesting that we obtained a large amount of reads by assembly, filtration and quality control. The 16S rRNA amplicon sequencing results showed that the abundance and diversity of gut bacterial microbiota were significantly changed between antibiotic treatment and other groups, and they are involved in metabolism and biosynthesis-related function based on functional prediction. Furthermore, we identified different numbers of differentially expressed unigenes (DEGs) and differentially expressed miRNAs (DEMs) based on high-quality mappable reads, which were enriched in various immune-related pathways, including Toll-like receptor, RIG-I-like receptor, NOD-like receptor, JAK/STAT, PI3K/Akt, NF-κB, MAPK signaling pathways, and so forth, using GO and KEGG enrichment analysis. Later on, the identified miRNAs and their target genes in the R. pedestris gut were predicted and randomly selected to construct an interaction network. Finally, our study indicated that alterations in the gut bacterial microbiota are significantly positively or negatively associated with DEMs of the Toll/Imd signaling pathway with Pearson correlation analysis. Taken together, the results of our study lay the foundation for further deeply understanding the interactions between the gut microbiota and immune responses in R. pedestris through miRNA regulation, and provide the new basis for pest management in hemipteran pests.

Integrated analysis of miRNA profiles and gut bacterial changes in Altica viridicyanea following antibiotic treatment.

The gut bacteria involves in insect homeostasis by playing essential roles in host physiology, metabolism, innate immunity, and so forth. microRNAs (miRNAs) are endogenous small noncoding RNAs that posttranscriptionally regulate gene expression to affect immune or metabolic processes in insects. For several non-model insects, the available knowledge on the relationship between changes in the gut bacteria and miRNA profiles is limited. In this study, we investigated the gut bacterial diversity, composition, and function from Altica viridicyanea feeding on normal- and antibiotic-treated host plants using 16S rRNA amplicon sequencing; antibiotics have been shown to affect the body weight and development time in A. viridicyanea, suggesting that the gut bacteria of the normal sample were more diverse and abundant than those of the antibiotic-fed group, and most of them were involved in various physical functions by enrichment analysis. Furthermore, we executed small RNA transcriptome sequencing using the two experimental groups to obtain numerous sRNAs, such as piRNAs, siRNAs, and known and novel miRNAs, by data mapping and quality control, and furthermore, a total of 224 miRNAs were identified as significantly differentially expressed miRNAs, of which some DEMs and their target genes participated in immune- and metabolism-related pathways based on GO and KEGG annotation. Besides, regarding the regulatory roles of miRNA and target genes, a interaction network of DEM-target gene pairs from eight immune- or metabolism-related signaling pathways were constructed. Finally, we discovered that DEMs from above pathways were significantly positively or negatively correlated with gut bacterial alterations following antibiotic treatment. Collectively, the observations of this study expand our understanding of how the disturbance of gut bacteria affects miRNA profiles in A. viridicyanea and provide new valuable resources from extreme ranges for future studies on the adaptive evolution in insects.

The lncRNA-mediated ceRNA network of Altica viridicyanea is involved in the regulation of the Toll/Imd signaling pathway under antibiotic treatment.

Long noncoding RNAs (lncRNAs) play significant roles in the regulation of mRNA expression or in shaping the competing endogenous RNA (ceRNA) network by targeting miRNA. The insect gut is one of the most important tissues due to direct contact with external pathogens and functions in the immune defense against pathogen infection through the innate immune system and symbionts, but there are limited observations on the role of the lncRNA-involved ceRNA network of the Toll/Imd pathway and correlation analysis between this network and bacterial microbiota in the Altica viridicyanea gut. In this research, we constructed and sequenced six RNA sequencing libraries using normal and antibiotic-reared samples, generating a total of 17,193 lncRNAs and 26,361 mRNAs from massive clean data by quality control and bioinformatic analysis. Furthermore, a set of 8,539 differentially expressed lncRNAs (DELs) and 13,263 differentially expressed mRNAs (DEMs), of which related to various immune signaling pathways, such as the Toll/Imd, JAK/STAT, NF-κB, and PI3K-Akt signaling pathways, were obtained between the two experimental groups in A. viridicyanea. In addition, numerous GO and KEGG enrichment analyses were used to annotate the DELs and their target genes. Moreover, six Toll family members and nineteen signal genes from the Toll/Imd signaling pathway were identified and characterized using online tools, and phylogenetic analyses of the above genes proved their classification. Next, a lncRNA-miRNA-mRNA network of the Toll/Imd pathway was built, and it contained different numbers of DEMs in this pathway and related DELs based on prediction and annotation. In addition, qRT-PCR validation and sequencing data were conducted to show the expression patterns of the above DELs and DEMs related to the Toll/Imd signaling pathway. Finally, the correlated investigations between DELs or DEMs of the Toll/Imd signaling pathway and most changes in the gut bacterial microbiota revealed significantly positive or negative relationships between them. The present findings provide essential evidence for innate immune ceRNAs in the beetle gut and uncover new potential relationships between innate immune pathways and the gut bacterial microbiota in insects.

The anthropogenic effect of land use on population genetics of Malcus inconspicuus.

Since the beginning of the Holocene era, human activities have seriously impacted animal habitats and vegetative environments. Species that are dependent on natural habitats or with narrow niches might be more severely affected by habitat changes. Malcus inconspicuus is distributed in subtropical China and highly dependent on the mountain environment. Our study investigated the role of the mountainous landscape in the historical evolution of M. inconspicuus and the impact of Holocene human activities on it. A phylogeographical approach was implemented with integrative datasets including double-digest restriction site-associated DNA (ddRAD), mitochondrial data, and distribution data. Three obvious clades and an east-west phylogeographical pattern were found in subtropical China. Mountainous landscape has "multifaceted" effects on the evolutionary history of M. inconspicuus, it has contributed to population differentiation, provided glacial refuges, and provided population expansion corridors during the postglacial period. The effective population size (Ne) of M. inconspicuus showed a sharp decline during the Holocene era, which revealed a significantly negative correlation with the development of cropland in a hilly area at the same time and space. It supported that the species which are highly dependent on natural habitats might undergo greater impact when the habitat was damaged by agricultural activities and we should pay more attention to them, especially in the land development of their distribution areas.