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.

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.

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.

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.

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.

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.

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.

DNA barcodes and insights into the phylogenetic relationships of Corvidae (Aves: Passeriformes).

DNA barcoding has become a promising tool for species identification and phylogeny in a wide range of animal taxa using mitochondrial cytochrome c oxidase subunit I (COI). The Corvidae (Aves: Passeriformes) is a species rich and morphologically diverse family. In the present study, we analyzed the COI barcodes of 39 species from 12 genera of Corvidae. COI gene was also used to examine phylogenetic relationships of Corvidae. Every species possessed a barcode distinct from that of other species. Kimura two-parameter distances were calculated between species barcodes. The average genetic distance between the species was 22 times higher compared to the average genetic distance within species. Maximum likelihood method was used to construct a phylogenetic tree. All the species could be discriminated by their distinct clades in the phylogenetic tree. COI gene data provided good evidence for the monophyly of the Corvidae. Members of Cyanopica and Pyrrhocorax were the first to split from the Corvidae lineage. Analysis of COI genes supported the others genera fell into two clades. DNA barcoding is an effective molecular tool for Corvidae species identification and phylogenetic inference.

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.

A quadratic correlation between long-term mean group size and group density in a cooperatively breeding passerine.

Both mean group size (MGS) and mean group density (MGD) are critical indices to characterize a population of cooperatively breeding birds. When a population reaches its carrying capacity, both long-term MGS and long-term MGD will remain relatively stable. However, there has been little study of how these two variables relate. The Masked laughingthrush Garrulax perspicillatus is a cooperatively breeding bird living in fragmented habitats. During 2010 and 2012-2016, we used song playback to observe and confirm the group sizes and territory ranges of the birds and the data of bird presence to determine habitat suitability. By grouping the nearest territories according to their geographical coordinates, we divided the whole study area into 12 subareas and the whole population into 12 subpopulations. Then, we calculated both MGS and MGD for different time durations for each subpopulation. Finally, using MGD as independent variable and MGS as the dependent variable, we explored the correlations between MGS and MGD by fitting quadratic functions and modeling quadratic regression. Both MGS and MGD were averaged for different time durations and were cross-related. Our results show that the MGS for more than 2 years significantly correlated with MGD for more than 3 years in a reverse parabolic shape, differing from that of short-term effects. Our findings suggest that long-term MGD is a better predictor of long-term habitat quality and that long-term MGS is determined by long-term habitat quality in Masked Laughingthrushes. Based on above findings, we can infer that: (1) Long-term habitat quality determines the long-term MGS, but it sets no prerequisite for the status and source of group members; (2) Long-term MGS in certain populations is adapted to the corresponding level of long-term habitat quality, it facilitates us to predict the helper effects on current or future survival or reproduction in different situations. These findings and inferences are both helpful for us to understand the evolution of cooperative breeding.

Structure and variation of the Fringillidae (Aves: Passeriformes) mitochondrial DNA control region and their phylogenetic relationship.

The control region is the major noncoding segment of animal mitochondrial DNA. To infer the structure and variation of Fringillidae mitochondrial DNA control region, the entire control region sequences of 25 species were analyzed. The length of the control region sequences was very conserved (1230 ± 18) and can be separated into three domains. The frequency of both substitutions and gaps was highest in the third domain and lowest in the central region. The control region has the same flanking gene order from tRNAGlu to tRNAPhe. Genetic distances between species ranged from 1.80% (between Carduelis pinus and Carduelis spinus) to 25.34% (between Carduelis sinica and Coccothraustes vespertinus). The average genetic distances among the species within the genera varied from 5.11% (Leucosticte) to 14.31% (Carpodacus). The average genetic distances showed insignificantly negative correlation with ts/tv. Domain III is the most variable of the three domains among all the genera. The control region of Fringillidae contains a putative TAS element and the highly conserved CSB-1, and F, E, D, C boxes. However, neither CSB-2 nor CSB-3 could be unambiguously identified in the Fringillidae. The maximum likelihood method was used to construct a phylogenetic tree. Control region analysis demonstrated that some currently recognized genera may be polyphyletic, including Carpodacus, Carduelis and Serinus.

DNA barcoding and phylogeny of Calidris and Tringa (Aves: Scolopacidae).

The avian genera Calidris and Tringa are the largest of the widespread family of Scolopacidae. The phylogeny of members of the two genera is still a matter of controversial. Mitochondrial cytochrome c oxidase subunit I (COI) can serve as a fast and accurate marker for the identification and phylogeny of animal species. In this study, we analyzed the COI barcodes of thirty-one species of the two genera. All the species had distinct COI sequences. Two hundred and twenty-one variable sites were identified. Kimura two-parameter distances were calculated between barcodes. Neighbor-joining and maximum likelihood methods were used to construct phylogenetic trees. All the species could be discriminated by their distinct clades in the phylogenetic trees. The phylogenetic trees grouped all the species of Calidris and Tringa into different monophyletic clade, respectively. COI data showed a well-supported phylogeny for Calidris and Tringa species.

Organization and variation of the Tetraonidae (Aves: Galliformes) mitochondrial DNA control region.

The mitochondrial DNA control region is the most polymorphic region of the mitochondrial genome. To infer the organization and variation of Tetraonidae mitochondrial DNA control region, the entire control region sequences of 18 species were analyzed. The length of the control region sequences ranged from 1127 bp (Bonasa sewerzowi) to 1156 bp (Centrocercus minimus). The average genetic distances among the species within the genera varied from 0.53% (Tympanuchus) to 9.42% (Bonasa). The average genetic distances showed insignificantly negative correlation with ts/tv. Five conserved sequence boxes in the domain II of Tetraonidae sequences were identified. The alignment of the Tetraonidae five boxes and CSB-1 sequences showed a few sequence variations. The results indicated that the genus Dendragapus might not be monophyletic.

Determination of the complete mitogenome of Spotted Dove, Spilopelia chinensis (Columbiformes: Columbidae).

The Spotted Dove Spilopelia chinensis (Columbiformes: Columbidae) is widely distributed in Southeast Asia. In the present study, we investigated the complete mitochondrial genome of S. chinensis and the mitogenome is 16,964 bp in length, consists of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a non-coding control region, with the base composition of A 31.1%, G 13.9%, T 23.9%, and C 32.1%. A single extra base "C" at position 174 is inserted in gene Nd3, similar to some other birds and turtles. The phylogenetic relationships using partitioned Bayesian inference based on the 12 concatenated PCGs indicated that ((((Spilopelia, Columba) Ectopistes) (Geotrygon (Leptotila, Zenaida))) Hemiphaga). While, more complete mitogenome sequences should be determined to confirm the phylogenetic status of Spilopelia within family Columbidae.

DNA barcoding and molecular phylogeny in Ranidae.

The family Ranidae has the widest distribution compared with other frog family. The phylogeny of the Ranidae is still a matter of dispute. In the present study, we analyzed the COI barcodes of 29 species from six genera belonging to the family Ranidae. Twenty-seven species (93.10% of all the species) were correctly identified by their DNA barcodes. Pelophylax lessonae and Pelophylax ridibundus shared the same one barcode sequence. Kimura two-parameter distances were calculated between barcodes. Pair-wise comparisons among-species were distributed from 0.16% (between Pelophylax lessonae and Pelophylax esculenta) to 29.13% (between Rana warszewitschii and Rana dybowskii). The average genetic distance between species was 28 times higher than the average genetic distance within species. The neighbor-joining method was used to construct a phylogenetic tree, which grouped all the genera into two divergent clades. The results indicated that some currently recognized genera of Ranidae may not be monophyletic. COI gene data supported the hypothesis of polyphyly for Rana, Amolops, Babina, and Hylarana. DNA barcoding is an effective molecular tool for Ranidae species identification and phylogenetic inference.

Structure and variation of the Anseriformes mitochondrial DNA control region.

The control region is the major non-coding segment of animal mitochondrial DNA. To infer the structure and variation of Anseriformes mitochondrial DNA control region, the control region sequences of 52 species were analyzed. The length of the control region sequences ranged from 968 bp (Chenonetta jubata) to 1335 bp (Anseranas semipalmata) and can be separated into three domains. There is a deletion of 100-130 bp in Anatinae, compared to other groups of Anserinae. The average genetic distances among the species within the genera varied from 4.14% (Anser) to 10.58% (Cygnus). The average genetic distances showed insignificantly negative correlation with ts/tv. Domain I is the most variable among the three domains among all the genera. Five conserved sequence boxes in the domain II of Anseriformes sequences were identified. The alignment of the Anseriformes five boxes sequences showed considerable sequence variation. CSB-1, -2 and 3 were not found in the Anseriformes. Maximum-likelihood method was used to construct a phylogenetic tree, which grouped all of the genera into four divergent clades. Anseranas + Chauna and Dendrocygna were identified as early offshoots of the Anatidae. All the remaining taxa fell into two clades that correspond to the two subfamilies Anserinae and Anatiane.

DNA barcoding and phylogenetic relationships in Anatidae.

Mitochondrial cytochrome c oxidase subunit I (COI) has been used as a powerful marker in a variety of phylogenetic studies. According to studies of bird species, the 694-bp sequence of the mitochondrial gene encoding COI is extremely useful for species identification and phylogeny. In the present study, we analyzed the COI barcodes of 79 species from 26 genera belonging to the Anatidae family. Sixty-six species (83.54%) of the species were identified correctly from their DNA barcodes. The remaining 13 species shared barcodes sequences with closely related species. Kimura two-parameter (K2P) distances were calculated between barcodes. The average genetic distance between species was 41 times higher compared to the average genetic distance within species. Neighbor-joining method was used to construct a phylogenetic tree, which grouped all of the genera into three divergent clades. Dendrocygna and Nomonyx + Oxyura were identified as early offshoots of the Anatidae. All the remaining taxa fell into two clades that correspond to the two subfamilies Anserinae and Anatiane. Based on our results, DNA barcoding is an effective molecular tool for Anatidae species identification and phylogenetic inference.