A Chromosome-Level Genome Assembly of the Non-Hematophagous Leech Whitmania pigra (Whitman 1884): Identification and Expression Analysis of Antithrombotic Genes.

Despite being a non-hematophagous leech, Whitmania pigra is widely used in traditional Chinese medicine for the treatment of antithrombotic diseases. In this study, we provide a high quality genome of W. pigra and based on which, we performed a systematic identification of the potential antithrombotic genes and their corresponding proteins. We identified twenty antithrombotic gene families including thirteen coagulation inhibitors, three platelet aggregation inhibitors, three fibrinolysis enhancers, and one tissue penetration enhancer. Unexpectedly, a total of 79 antithrombotic genes were identified, more than a typical blood-feeding Hirudinaria manillensis, which had only 72 antithrombotic genes. In addition, combining with the RNA-seq data of W. pigra and H. manillensis, we calculated the expression levels of antithrombotic genes of the two species. Five and four gene families had significantly higher and lower expression levels in W. pigra than in H. manillensis, respectively. These results showed that the number and expression level of antithrombotic genes of a non-hematophagous leech are not always less than those of a hematophagous leech. Our study provides the most comprehensive collection of antithrombotic biomacromolecules from a non-hematophagous leech to date and will significantly enhance the investigation and utilization of leech derivatives in thrombosis therapy research and pharmaceutical applications.

Revisiting the Asian Buffalo Leech (Hirudinaria manillensis) Genome: Focus on Antithrombotic Genes and Their Corresponding Proteins.

Leeches are well-known annelids due to their obligate blood-feeding habits. Some leech species secrete various biologically active substances which have important medical and pharmaceutical value in antithrombotic treatments. In this study, we provided a high-quality genome of the Asian buffalo leech (Hirudinaria manillensis), based on which we performed a systematic identification of potential antithrombotic genes and their corresponding proteins. Combining automatic and manual prediction, we identified 21 antithrombotic gene families including fourteen coagulation inhibitors, three platelet aggregation inhibitors, three fibrinolysis enhancers, and one tissue penetration enhancer. A total of 72 antithrombotic genes, including two pseudogenes, were identified, including most of their corresponding proteins forming three or more disulfide bonds. Three protein families (LDTI, antistasin, and granulin) had internal tandem repeats containing 6, 10, and 12 conserved cysteines, respectively. We also measured the anticoagulant activities of the five identified hirudins (hirudin_Hman1 ~ hirudin_Hman5). The results showed that three (hirudin_Hman1, hirudin_Hman2, and hirudin_Hman5), but not the remaining two, exhibited anticoagulant activities. Our study provides the most comprehensive collection of antithrombotic biomacromolecules from a leech to date. These results will greatly facilitate the research and application of leech derivatives for medical and pharmaceutical purposes in the treatment of thrombotic diseases.

Impacts of future climate on the distribution of 4 bumblebee species in the Qinghai-Tibet Plateau.

Climate warming threatens dozens of bumblebee species across the world, including alpine areas. In plateaus with vast and continuous high-altitude areas, bumblebees' response to climate warming may be relatively optimistic. To study the species' responses to future climate in the Qinghai-Tibet Plateau, we quantified the suitable areas for 4 local bumblebee indicator species under current and future climate scenarios (Shared Socio-economic Pathway 126, 245, 370, and 585 in 2,100) using MaxEnt models. Suitable areas of indicator species were stacked to obtain the species richness layer. According to the acreage and connectivity of suitable areas and the acreage of the high richness area, a warmed climate will be more suitable for bumblebees' distribution compared to the current climate. The SSP 126 and SSP 245 scenario will be the 2 most suitable. Meanwhile, with climate warming, suitable areas and the high richness areas will move to high altitudes and their altitude range will decrease. The greater suitability in warmed climates may be caused by the topography of plateaus, which provides an opportunity for bumblebees to migrate to cooler areas. However, mitigation of warming is still necessary because an excessively warm climate will decrease bumblebees' habitat suitability. In plateaus, species in lower altitudes will migrate to higher altitudes, conservation in high altitudes should pay attention to not only original species, but also immigrated species. In lower altitudes, many species will migrate to higher altitudes, then local bumblebee diversity will decrease. Local conservation should be focused on these lower altitude areas.

Evolutionary Adaptation of Genes Involved in Galactose Derivatives Metabolism in Oil-Tea Specialized Andrena Species.

Oil-tea (Camellia oleifera) is a woody oil crop whose nectar includes galactose derivatives that are toxic to honey bees. Interestingly, some mining bees of the genus Andrena can entirely live on the nectar (and pollen) of oil-tea and are able to metabolize these galactose derivatives. We present the first next-generation genomes for five and one Andrena species that are, respectively, specialized and non-specialized oil-tea pollinators and, combining these with the published genomes of six other Andrena species which did not visit oil-tea, we performed molecular evolution analyses on the genes involved in the metabolizing of galactose derivatives. The six genes (NAGA, NAGA-like, galM, galK, galT, and galE) involved in galactose derivatives metabolism were identified in the five oil-tea specialized species, but only five (with the exception of NAGA-like) were discovered in the other Andrena species. Molecular evolution analyses revealed that NAGA-like, galK, and galT in oil-tea specialized species appeared under positive selection. RNASeq analyses showed that NAGA-like, galK, and galT were significantly up-regulated in the specialized pollinator Andrena camellia compared to the non-specialized pollinator Andrena chekiangensis. Our study demonstrated that the genes NAGA-like, galK, and galT have played an important role in the evolutionary adaptation of the oil-tea specialized Andrena species.

Digestive Tract Morphology and Gut Microbiota Jointly Determine an Efficient Digestive Strategy in Subterranean Rodents: Plateau Zokor.

Rodents' lifestyles vary in different environments, and to adapt to various lifestyles specific digestion strategies have been developed. Among these strategies, the morphology of the digestive tracts and the gut microbiota are considered to play the most important roles in such adaptations. However, how subterranean rodents adapt to extreme environments through regulating gut microbial diversity and morphology of the digestive tract has yet to be fully studied. Here, we conducted the comparisons of the gastrointestinal morphology, food intake, food assimilation, food digestibility and gut microbiota of plateau zokor Eospalax baileyi in Qinghai-Tibet Plateau and laboratory rats Rattus norvegicus to further understand the survival strategy in a typical subterranean rodent species endemic to the Qinghai-Tibet Plateau. Our results revealed that plateau zokor evolved an efficient foraging strategy with low food intake, high food digestibility, and ultimately achieved a similar amount of food assimilation to laboratory rats. The length and weight of the digestive tract of the plateau zokor was significantly higher than the laboratory rat. Particularly, the weight and length of the large intestine and cecum in plateau zokor is three times greater than that of the laboratory rat. Microbiome analysis showed that genus (i.e., Prevotella, Oscillospira, CF231, Ruminococcus and Bacteroides), which are usually associated with cellulose degradation, were significantly enriched in laboratory rats, compared to plateau zokor. However, prediction of metagenomic function revealed that both plateau zokor and laboratory rats shared the same functions in carbohydrate metabolism and energy metabolism. The higher digestibility of crude fiber in plateau zokor was mainly driven by the sizes of cecum and cecum tract, as well as those gut microbiota which associated with cellulose degradation. Altogether, our results highlight that both gut microbiota and the morphology of the digestive tract are vital to the digestion in wild rodents.

Population genomics and phylogeography of Colletes gigas, a wild bee specialized on winter flowering plants.

Diet specialization may affect the population genetic structure of pollinators by reducing gene flow and driving genetic differentiation, especially in pollen-specialist bees. Colletes gigas is a pollen-specialist pollinator of Camellia oleifera, one of the most important staple oil crops in China. Ca. oleifera blooms in cold climates and contains special compounds that make it an unusable pollen source to other pollinators. Thus, C. gigas undoubtedly plays a key role as the main pollinator of Ca. oleifera, with biological and economic significance. Here, we use a population genomic approach to analyze the roles of geography and climate on the genetic structure, genetic diversity, and demographic history of C. gigas. A total of 1,035,407 SNPs were identified from a 582.77 Gb dataset. Clustering and phylogenetic analyses revealed a marked genetic structure, with individuals grouped into nine local clusters. A significant isolation by distance was detected by both the Mantel test (R = .866, p = .008) and linear regression (R 2 = .616, p < .001). Precipitation and sunshine duration were positively and significantly (R ≥ .765, p ≤ .016) correlated with observed heterozygosity (H o) and expected heterozygosity (H e). These results showed that C. gigas populations had a distinct phylogeographic pattern determined by geographical distance and environmental factors (precipitation and sunshine duration). In addition, an analysis of paleogeographic dynamics indicated that C. gigas populations exhibited patterns of glacial expansion and interglacial contraction, likely resulting from post-glacial habitat contraction and fragmentation. Our results indicated that the peculiar phylogeographic patterns in C. gigas populations may be related to their specialization under long-term adaptation to host plants. This work improves our understanding of the population genetics in pollen-specialist bees. The distinct genetic clusters identified in this study should be taken into consideration for the protection and utilization of this specialized crop pollinator.

The Potential Roles of Unique Leaf Structure for the Adaptation of Rheum tanguticum Maxim. ex Balf. in Qinghai-Tibetan Plateau.

Leaves are essential plant organs with numerous variations in shape and size. The leaf size is generally smaller in plants that thrive in areas of higher elevation and lower annual mean temperature. The Qinghai-Tibetan Plateau is situated at an altitude of >4000 m with relatively low annual average temperatures. Most plant species found on the Qinghai-Tibetan Plateau have small leaves, with Rheum tanguticum Maxim. ex Balf. being an exception. Here, we show that the large leaves of R. tanguticum with a unique three-dimensional (3D) shape are potentially an ideal solution for thermoregulation with little energy consumption. With the increase in age, the shape of R. tanguticum leaves changed from a small oval plane to a large palmatipartite 3D shape. Therefore, R. tanguticum is a highly heteroblastic species. The leaf shape change during the transition from the juvenile to the adult phase of the development in R. tanguticum is a striking example of the manifestation of plant phenotypic plasticity. The temperature variation in different parts of the leaf was a distinct character of leaves of over-5-year-old plants. The temperature of single-plane leaves under strong solar radiation could accumulate heat rapidly and resulted in temperatures much higher than the ambient temperature. However, leaves of over-5-year-old plants could lower leaf temperature by avoiding direct exposure to solar radiation and promoting local airflow to prevent serious tissue damage by sunburn. Furthermore, the net photosynthesis rate was correlated with the heterogeneity of the leaf surface temperature. Our results demonstrate that the robust 3D shape of the leaf is a strategy that R. tanguticum has developed evolutionarily to adapt to the strong solar radiation and low temperature on the Qinghai-Tibetan Plateau.

Molecular evolution of bumble bee vitellogenin and vitellogenin-like genes.

Vitellogenin (Vg), a storage protein, has been significantly studied for its egg yolk precursor role in oviparous animals. Recent studies found that vitellogenin and its Vg-like homologs were fundamentally involved in many other biological processes in social insects such as female caste differences and oxidative stress resilience. In this study, we conducted the first large-scale molecular evolutionary analyses of vitellogenin coding genes (Vg) and Vg-like genes of bumble bees, a primitively eusocial insect belonging to the genus Bombus. We obtained sequences for each of the four genes (Vg, Vg-like-A, Vg-like-B, and Vg-like-C) from 27 bumble bee genomes (nine were newly sequenced in this study), and sequences from the two closest clades of Bombus, including five Apis species and five Tetragonula species. Our molecular evolutionary analyses show that in bumble bee, the conventional Vg experienced strong positive selection, while the Vg-like genes showed overall relaxation of purifying selection. In Apis and Tetragonula; however, all four genes were found under purifying selection. Furthermore, the conventional Vg showed signs of strong positive selection in most subgenera in Bombus, apart from the obligate parasitic subgenus Psithyrus which has no caste differentiation. Together, these results indicate that the conventional Vg, a key pleiotropic gene in social insects, is the most rapidly evolving copy, potentially due to its multiple known social functions for both worker and queen castes. This study shows that concerted evolution and purifying selection shaped the evolution of the Vg gene family following their ancient gene duplication and may be the leading forces behind the evolution of new potential protein function enabling functional social pleiotropy.

Complete mitochondrial genome of Actias dubernardi (Lepidoptera: Saturniidae).

The complete mitochondrial genome of Actias dubernardi (Lepidoptera: Saturniidae) is 15,270 bp in length, containing 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and a putative control region. All of the protein-coding genes (PCGs) use the standard start codon ATN, except for cox1 which starts with CGA. The Bayesian phylogenetic analysis was performed using a dataset matrix containing 13 PCGs concatenated from the mitogenomes of 14 Saturniidae species. The monophyly of the five Actias species was highly supported and Antheraea was inferred as the sister group of Actias.

Complete mitochondrial genome of Casmara patrona (Lepidoptera: Oecophoridae).

The complete mitochondrial genome of Casmara patrona (Lepidoptera: Oecophoridae) was sequenced for a future phylogenetic study of Lepidoptera. The circle genome of the moth is 15,393 bp in length with a pronounced base bias of A + T (79.3%), containing 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and a putative control region. The coxI gene had a CGA start codon as most lepidopteran species, other PCGs use the typical ATN codons. All PCGs end with the complete stop codon TAA. Phylogenetic analyses showed that the monophyly of Oecophoridae was highly supported based on the concatenated sequence of the 13 PCGs. In addition, Oecophoridae and Xyloryctidae had the closest relationship.

Gut Microbiota Plasticity Influences the Adaptability of Wild and Domestic Animals in Co-inhabited Areas.

Due to the increased economic demand for livestock, the number of livestock is increasing. Because of human interference, the survival of wild animals is threatened in the face of competition, particularly in co-inhabited grazing pastures. This may lead to differences in the adaptability between wild and domestic animals, as well as nutritional deficiencies in wild animals. The gut microbiota is closely associated with host health, nutrition, and adaptability. However, the gut microbiota diversity and functions in domestic and wild animals in co-inhabited areas are unclear. To reveal the adaptability of wild and domestic animals in co-inhabited areas based on gut microbiota, we assessed the gut microbiota diversity. This study was based on the V3-V4 region of 16S rRNA and gut microbiota functions according to the metagenome analysis of fresh fecal samples in wild goitered gazelles (Gazella subgutturosa) and domestic sheep (Ovis aries) in the Qaidam Basin. The wild and domestic species showed significant differences in alpha- and beta-diversities. Specifically, the alpha-diversity was lower in goitered gazelles. We speculated that the nutritional and habitat status of the goitered gazelles were worse. The gut microbiota functions in the gazelles were enriched in metabolism and cellular processes based on the KEGG database. In summary, we reasoned that gut microbiota can improve the adaptability of goitered gazelles through energy maintenance by the functions of gut microbiota in the face of nutritional deficiencies. These findings highlight the importance of gut microbiota diversity to improve the adaptability of goitered gazelles, laying a foundation for the conservation of wild goitered gazelles. In addition, we further provide management suggestions for domestic sheep in co-inhabited grazing pastures.

Complete mitochondrial genome of Linoclostis gonatias (Lepidoptera: Xyloryctidae).

The complete mitochondrial genome of Linoclostis gonatias (Lepidoptera: Xyloryctidae) is 15,528 bp in length, containing 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and a putative control region. Except for cox1 starts with CGA, all other PCGs use the typical ATN codons. Most of the PCGs end with the complete stop codon TAA, whereas cox2 terminates with the incomplete stop codon T. The BI analysis was performed using a dataset matrix containing 13 PCGs concatenated from the mitogenomes of Gelechioidea species. The monophyly of Xyloryctidae was highly supported. In addition, Oecophoridae was inferred as the sister group of Xyloryctidae.

Lithobius (Ezembius) hualongensis sp. nov. and Lithobius (Ezembius) sui sp. nov. (Lithobiomorpha, Lithobiidae), two new species of centipede from northwest China.

Lithobius (Ezembius) hualongensissp. nov. and Lithobius (Ezembius) suisp. nov. (Lithobiomorpha, Lithobiidae) recently discovered from the Qinghai-Tibet Plateau, China are described. Morphologically, the two new species are very similar but can be distinguished by the number of coxosternal teeth: L. (E.) hualongensissp. nov. has 2 + 2 while L. (E.) suisp. nov. has 3 + 3. The two new species resemble L. (E.) multispinipesPei et al., 2016, from the Xinjiang Autonomous Region, but can be readily distinguished by having the Tömösváry's organ slightly larger than the adjoining ocelli rather than smaller, 3 + 3 spurs on female gonopods versus 2 + 2, and the simple terminal claw of female gonopods with a small triangular protuberance on the basal ventral side versus simple, without a small triangular protuberance on the basal ventral side. We also compare the main morphological characters of the two new species with the other Lithobius (Ezembius) species known in Qinghai Province. A key to the Chinese species of Ezembius is presented.

Maxent modeling for predicting the spatial distribution of three raptors in the Sanjiangyuan National Park, China.

Upland buzzard (Buteo hemilasius), Saker falcon (Falco cherrug), and Himalayan vulture (Gyps himalayensis) are three common large raptors in the Sanjiangyuan National Park (SNP), China's first national park. Among them, Upland buzzard and Saker falcon play a significant role in controlling plateau rodent populations and reducing the transmission of pathogens carried by rodents. The Himalayan vulture can provide services for the redistribution and recycling of nutrients in the ecosystem, and play an irreplaceable role in the celestial burial culture of Tibetans in China. Exploring their habitat suitability is important for the protection of the three raptors. Our research was based on the current distribution of Upland buzzard, Saker falcon, and Himalayan vulture that we had extensively surveyed in the Sanjiangyuan National Park from 2016 to 2017. Combined with the correlation analysis of environmental variables, we utilized maximum entropy model (MaxEnt) to evaluate and compare the habitat suitability of the three species in the Sanjiangyuan National Park. Elevation, climate, and human disturbance factors, which had direct or indirect effects on species survival and reproduction, were all included in the model. Among them, elevation was the most important environmental variables affecting the suitability of habitats of three species. Temperature-related factor was another important predictor. The high (>60%) suitable habitat areas for Upland buzzard, Saker falcon, and Himalayan vulture were 73,017.63, 40,732.78, and 61,654.33 km2, respectively, accounted for 59.32%, 33.09%, and 50.08% of the Sanjiangyuan National Park and their total suitable area (i.e., the sum area of high and moderate habitats) reached 96.07%, 60.59%, and 93.70%, respectively. Besides, the three species have overlapping areas for the suitable habitats, which means that overlapping areas should be highly valued and protected. Therefore, understanding the distribution of suitable habitats of the three raptors can provide useful information and reasonable reference for us to put forward suggestions for their protection and regional management.

Evolutionary Rates of Bumblebee Genomes Are Faster at Lower Elevations.

The importance of climate in determining biodiversity patterns has been well documented. However, the relationship between climate and rates of genetic evolution remains controversial. Latitude and elevation have been associated with rates of change in genetic markers such as cytochrome b. What is not known, however, is the strength of such associations and whether patterns found among these genes apply across entire genomes. Here, using bumblebee genetic data from seven subgenera of Bombus, we demonstrate that all species occupying warmer elevations have undergone faster genome-wide evolution than those in the same subgenera occupying cooler elevations. Our findings point to a critical biogeographic role in the relative rates of whole species evolution, potentially influencing global biodiversity patterns.

The roles of calving migration and climate change in the formation of the weak genetic structure in the Tibetan antelope (Pantholops hodgsonii).

Geographical barriers and distance can reduce gene exchange among animals, resulting in genetic divergence of geographically isolated populations. The Tibetan antelope (Pantholops hodgsonii) has a geographical range of approximately 1600 km across the Qinghai-Tibet Plateau, which comprises a series of tall mountains and big rivers. However, previous studies indicate that there is little genetic differentiation among their geographically delineated populations. To better understand the genetic structure of P. hodgsonii populations, we collected 145 samples from the 3 major calving regions, taking into consideration their various calving grounds and migration routes. We used a combination of mitochondrial sequences (Cyt b, ATPase, D-loop and COX I) to investigate the genetic structure and the evolutionary divergence of the populations. Significant, albeit weak, genetic differentiation was detected among the 3 geographical populations. Analysis of the genetic divergence process revealed that the animals gradually entered a period of rapid genetic differentiation approximately 60 000 years ago. The calving migration of P. hodgsonii cannot be the main cause of their weak genetic structure because this cannot fully homogenize the genetic pool. Instead, the geological and climatic events as well as the coupling vegetation succession process during this period have been suggested to greatly contribute to the genetic structure and the expansion of genetic diversity.

Two new species of Lithobius on Qinghai-Tibetan plateau identified from morphology and COI sequences (Lithobiomorpha: Lithobiidae).

Lithobius (Ezembius) longibasitarsussp. n. and Lithobius (Ezembius) datongensissp. n. (Lithobiomorpha: Lithobiidae), recently discovered from Qinghai-Tibet Plateau, China, are described. A key to the species of the subgenus Ezembius in China is presented. The partial mitochondrial cytochrome c oxidase subunit I barcoding gene was amplified and sequenced for eight individuals of the two new species and the dataset was used for molecular phylogenetic analysis and genetic distance determination. Both morphology and molecular data show that the specimens examined should be referred to Lithobius (Ezembius).

Genetic differentiation of the oriental rat flea, Xenopsylla cheopis, from two sympatric host species.

BACKGROUND: The oriental rat flea (Xenopsylla cheopis), which infests several mammals, primarily rats (Rattus spp.), is the most notorious vector of human plague. In this study, we measured the genetic differentiation among populations of fleas from the Asian house rat (Rattus tanezumi) and the brown rat (R. norvegicus) using microsatellite markers in order to investigate the extent of host-switching in this parasite. RESULTS: We developed 11 polymorphic microsatellite loci for our study, nine of which showed high potential for inbreeding. AMOVA showed that the majority (84.07%, P < 0.001) of the variation was derived from within populations, followed by variation among groups (14.96%, P < 0.001); in contrast, variation within groups of populations was nearly absent (0.97%, P > 0.05). Analyses of the pairwise fixation index revealed that most of the ten allopatric population pairs but none of the five sympatric population pairs were significantly differentiated. Moreover, based on genetic structure clustering analysis, there was obvious differentiation between allopatric populations but not between sympatric population pairs. CONCLUSIONS: These results indicate the presence of frequent migrations of the oriental rat flea between the sympatric Asian house rat and brown rat, causing a high rate of gene flow and limited genetic differentiation. We suggest that there is no clear boundary limiting the migration of oriental rat fleas between the two hosts, and thus both rat species should be monitored equally for the purposes of plague prevention and control.

Variation of the Bovidae (Mammalia: Artiodactyla) mitochondrial DNA control region and their phylogenetic relationship.

The control region is the major noncoding segment of animal mitochondrial DNA. The mammal family Bovidae comprises all artiodactyl ungulates. To infer the organization and variation of Bovidae mitochondrial DNA control region, the complete control region sequences of 91 species were analysed. The control region ranged from 677 bp (Saiga tatarica) to 1329 bp (Oryx dammah) in length and can be separated into three domains among these species. The control region has the same flanking gene order from tRNApro to tRNAPhe. Genetic distances between species ranged from 0.44% (between Bos javanicus and Bos taurus) to 24.05% (between Syncerus caffer and Gazella subgutturosa). The average genetic distances among the species within the genera varied from 2.78% (Procapra) to 22.07% (Hemitragus). The average genetic distances showed significantly negative correlation with ts/tv. The maximum-likelihood method was used to construct a phylogenetic tree. Members of Bovinae appear in basal position among the Bovidae lineage. Analysis of control region genes supported the hypothesis of polyphyly for Antilopinae.

The complete mitochondrial DNA sequence of the Chinese red pika (Ochotona erythrotis).

In this study, we undertook the first complete Ochotona erythrotis mitochondrial genome. The genome sequence was 16,663 bp in length, including the typical structure of 22 transfer RNA genes, 13 protein-coding genes, 2 ribosomal RNA genes, and the non-coding control region. The overall base composition of O. erythrotis mitogenome is 31.8% A, 26.0% T, 28.6% G, and 13.6% C, with a high A + T content of 57.8%.