Characterization of highly polymorphic microsatellite markers for the chinese monal (Lophophorus lhuysii, Galliformes) using Illumina MiSeq sequencing.

BACKGROUND: The Chinese monal (Lophophorus lhuysii, Galliformes) is a vulnerable and endemic bird from southwestern China. To better protect this species and increase its population size, genetic markers are urgently needed for investigation and conservation of both wild and captive populations. METHODS AND RESULTS: By using next-generation sequencing, we developed and characterized markers for seven microsatellite loci of the Chinese monal. PCR examination and statistical analysis indicated that these microsatellites exhibited moderate to high levels of polymorphism, with the expected heterozygosity and polymorphic information content ranging from 0.578 to 0.858 and from 0.540 to 0.841, respectively. Cross-species genome comparison further suggests that these microsatellites are a feature of certain galliform species rather than being specific to the Chinese monal. CONCLUSION: A combination of the seven highly polymorphic loci may provide a fundamental genetic toolkit to assess genetic backgrounds and will contribute to design conservation plan, breeding management and other possible studies of the Chinese monal and other evolutionarily related species in the future.

Field investigation- and dietary metabarcoding-based screening of arthropods that prey on primary tea pests.

Predatory natural enemies play key functional roles in biological control. Abundant predatory arthropod species have been recorded in tea plantation ecosystems. However, few studies have comprehensively evaluated the control effect of predatory arthropods on tea pests in the field. We performed a 1-year field investigation and collected predatory arthropods and pests in the tea canopy. A total of 7931 predatory arthropod individuals were collected, and Coleosoma blandum (Araneae, Theridiidae) was the most abundant species in the studied tea plantation. The population dynamics between C. blandum and four main tea pest species (Aleurocanthus spiniferus, Empoasca onukii, Ectropis grisescens, and Scopula subpunctaria) were established using the individual number of predators and pests in each month. The results showed that C. blandum appeared to co-occur in the tea canopy with A. spiniferus, Em. onukii, and Ec. grisescens in a longer period. The prey spectrum of C. blandum was further analyzed using DNA metabarcoding. Among prey species, A. spiniferus, Em. onukii, and Ec. grisescens were included, and the relative abundance and positive rates of target DNA fragments of A. spiniferus were greater than that of other two pests. Combined with the high dominance index of C. blandum, co-occurrence between C. blandum and A. spiniferus in time and space and high positive rate and relative abundance of target DNA fragments of A. spiniferus, C. blandum was identified to prey on A. spiniferus, and C. blandum may be an important predator of A. spiniferus. Thus, C. blandum has potential as a biological control agent of A. spiniferus in an integrated pest management strategy.

Transcriptome analyses of potential regulators of pre- and post-ovulatory follicles in the pigeon (Columba livia).

To identify the dominant genes controlling follicular maturation, ovulation and regression for pigeon, we used RNA-seq to explore the gene expression profiles of pre- and post-ovulatory follicles of pigeon. We obtained total of 4.73million (96% of the raw data) high-quality clean reads, which could be aligned with 20282 genes. Gene expression profile analysis identified 1461 differentially expressed genes (DEGs) between the pre- (P4) and post-ovulatory follicles (P5). Of these, 843 genes were upregulated, and 618 genes were down-regulated. Furthermore, many DEGs were significantly enriched in some pathways closely related to follicle maturation, ovulation and regression, such as ECM-receptor interaction, vascular smooth muscle contraction, progesterone-mediated oocyte maturation, phagosome. Importantly, the DGEs in ECM-receptor interaction pathway included COL1A1 , COL1A2 , COL4A1 , COL4A2 , ITGA11 , ITGB3 and SDC3 , in the progesterone-mediated oocyte maturation pathway involved CDK1 , CDC25A , CCNB3 , CDC20 and Plk1 , and in the vascular smooth muscle contraction covered CALD1 , KCNMA1 , KCNMB1 , CACNA1 , ACTA2 , MYH10 , MYL3 , MYL6 , MYL9 , closely related to promoting follicular maturation and ovulation in pre-ovulatory follicles. Moreover, it seems that the lysosomal cathepsin family has a decisive role in the regression of early stage of post-ovulatory follicle. Taken together, these data enrich the research of molecular mechanisms of pigeon follicular activities at the transcriptional level and provide novel insight of breeding-related physiology for birds.

Differences in microbiome composition and transcriptome profiles between male and female Paederus fuscipes harbouring pederin-producing bacteria.

Pederin, a group of antitumor compounds, is produced by an endosymbiotic bacterium of Paederus fuscipes. Pederin content differed between male and female P. fuscipes, but the reason why these differences are maintained remains unexplored. Here, the pederin-producing bacteria (PPB) infection rate in P. fuscipes was investigated. Furthermore, we assessed the microbiota structure differences in male and female P. fuscipes harbouring PPB and sequenced the transcriptome of both sexes to shed light on genes of interest. Of the 625 analysed beetles (275 females, 350 males), 96.36% of females and 31.14% of males were positive for PPB infection. PPB accounted for 54.36%-82.70% of the bacterial population in females but showed a much lower abundance in males (0.92%-3.87%). Reproductive organs possessed the highest PPB abundance compared with other parts of females, but no such relationships existed in males. Moreover, we provide the first transcriptome analysis of male and female P. fuscipes harbouring PPB and identified 8893 differentially expressed unigenes. Our results indicated that the pederin content difference between males and females might be caused by the PPB density difference in hosts. The biosequence data would be helpful for illustrating the mechanism that regulates PPB density in P. fuscipes.

A comparative analysis of spider prey spectra analyzed through the next-generation sequencing of individual and mixed DNA samples.

As one of the most abundant predators of insects in terrestrial ecosystems, spiders have long received much attention from agricultural scientists and ecologists. Do spiders have a certain controlling effect on the main insect pests of concern in farmland ecosystems? Answering this question requires us to fully understand the prey spectrum of spiders. Next-generation sequencing (NGS) has been successfully employed to analyze spider prey spectra. However, the high sequencing costs make it difficult to analyze the prey spectrum of various spider species with large samples in a given farmland ecosystem. We performed a comparative analysis of the prey spectra of Ovia alboannulata (Araneae, Lycosidae) using NGS with individual and mixed DNA samples to demonstrate which treatment was better for determining the spider prey spectra in the field. We collected spider individuals from tea plantations, and two treatments were then carried out: (1) The DNA was extracted from the spiders individually and then sequenced separately (DESISS) and (2) the DNA was extracted from the spiders individually and then mixed and sequenced (DESIMS). The results showed that the number of prey families obtained by the DESISS treatment was approximately twice that obtained by the DESIMS treatment. Therefore, the DESIMS treatment greatly underestimated the prey composition of the spiders, although its sequencing costs were obviously lower. However, the relative abundance of prey sequences detected in the two treatments was slightly different only at the family level. Therefore, we concluded that if our purpose were to obtain the most accurate prey spectrum of the spiders, the DESISS treatment would be the best choice. However, if our purpose were to obtain only the relative abundance of prey sequences of the spiders, the DESIMS treatment would also be an option. The present study provides an important reference for choosing applicable methods to analyze the prey spectra and food web compositions of animal in ecosystems.

Characterization of the complete mitochondrial genome of Ectropis grisescens (Lepidoptera, Geometridae).

Ectropis grisescens (Lepidoptera, Geometridae) is one of the main leaf-eating pests in tea plantations in China. In this study, the complete mitochondrial genome of this species was sequenced and assembled. The total length of the mitochondrial genome of E. grisescens was 15,794 bp (GenBank accession No. MW337302). The base composition was 41.26% for A, 39.49% for T, 7.92% for G, and 11.33% for C. The circular mitogenome contained 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. Phylogenetic analysis performed using 13 protein-coding genes of 15 species of Geometridae and an out-group Pieris melete (Lepidoptera, Pieridae) showed that E. grisescens is closely related to species of E. obliqua, and this is consistent with the morphological identification.

Comparison of the Microsatellite Distribution Patterns in the Genomes of Euarchontoglires at the Taxonomic Level.

Microsatellite or simple sequence repeat (SSR) instability within genes can induce genetic variation. The SSR signatures remain largely unknown in different clades within Euarchontoglires, one of the most successful mammalian radiations. Here, we conducted a genome-wide characterization of microsatellite distribution patterns at different taxonomic levels in 153 Euarchontoglires genomes. Our results showed that the abundance and density of the SSRs were significantly positively correlated with primate genome size, but no significant relationship with the genome size of rodents was found. Furthermore, a higher level of complexity for perfect SSR (P-SSR) attributes was observed in rodents than in primates. The most frequent type of P-SSR was the mononucleotide P-SSR in the genomes of primates, tree shrews, and colugos, while mononucleotide or dinucleotide motif types were dominant in the genomes of rodents and lagomorphs. Furthermore, (A)n was the most abundant motif in primate genomes, but (A)n, (AC)n, or (AG)n was the most abundant motif in rodent genomes which even varied within the same genus. The GC content and the repeat copy numbers of P-SSRs varied in different species when compared at different taxonomic levels, reflecting underlying differences in SSR mutation processes. Notably, the CDSs containing P-SSRs were categorized by functions and pathways using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes annotations, highlighting their roles in transcription regulation. Generally, this work will aid future studies of the functional roles of the taxonomic features of microsatellites during the evolution of mammals in Euarchontoglires.

Transcriptome analyses to reveal the dynamic change mechanism of pigeon magnum during one egg-laying cycle.

We analyzed the transcriptome of pigeon magnum in three stages (C1: pre-ovulation, C2: post-ovulation, C3: 5-6 days after ovulation) to elucidate the molecular and cellular events associated with morphological changes during the laying cycle. We observed that C1 was highly developed, apoptosis rate was highest in C2, and C3 attained the smallest size. Through RNA-sequencing, we obtained 54,764,938 (97.2%) high-quality clean reads that aligned to 20,767 genes. Gene expression profile analysis showed the greatest difference between C1 and C3; 3966 differentially expressed genes (DEGs) were identified, of which 2250 genes were upregulated and 1716 genes were downregulated in C1. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that protein processing and transport activities were prominent in C1, and upregulated genes included those related to signal recognition particle (SRP), signal recognition particle receptor (SRPR), translocon, GRP78, RRBP1, TRAP, TRAM1, and OST. Egg white protein-related gene expression was highest, with OVALY being the most highly expressed. In C2, apoptosis-related gene expression was higher than in C1, and fatty acid metabolism was active, which may be correlated with magnum tissue regression. Collagen- and laminin-related gene expression was prominent in C1 and C3, indicating roles in egg white protein generation and magnum reconstruction. PR gene expression was highest and exhibited drastic change in the three groups, indicating that PR and its regulation may be involved in changes in magnum morphology and function. Through the identification and functional analysis of DEGs and other crucial genes, this may contribute to understand the egg white protein production, magnum tissue regression, and magnum regeneration mechanisms.

Comparison of microsatellite distribution patterns in twenty-nine beetle genomes.

Simple sequence repeats (SSRs) represent an important source of genetic variation that provides a basis for adaptation to different environments in organisms. In this study, we examined the distribution patterns of SSRs in twenty-nine beetle genomes and carried out Gene Ontology (GO) analysis of CDSs embedded with perfect SSRs (P-SSRs). The results demonstrated that imperfect SSRs (I-SSRs) represented the most abundant SSR category in beetle genomes and in different genomic regions (CDS, exon, and intron regions). The numbers of P-SSRs, I-SSRs, compound SSRs, and variable number tandem repeats were positively correlated with beetle genome size, whereas neither the frequency nor the density of the SSRs was correlated with genome size. Moreover, our results demonstrated that common genomic features of P-SSRs within the same suborder or family of Coleoptera were rare. Mono-, di-, tri-, or tetranucleotide SSRs were the most abundant P-SSR categories in beetle genomes. The preferred predominant repeat motif among the mononucleotide P-SSRs was (A)n, but the most frequent repeat motifs for other length classes varied differentially among these genomes. Furthermore, the P-SSR type with the highest GC content differed in the beetle genomes and in different genomic regions. CV (coefficient of variability) analysis demonstrated that the repeat copy numbers of P-SSRs presented relatively higher variation in introns than in CDSs and exons. The GO terms of CDSs containing P-SSRs for molecular functions were mainly enriched in "binding" and "transcription". Our findings will be useful for studying the functional roles of microsatellite heterogeneity in beetle adaptation.

Molecular characterization, mRNA gene expression, and antimicrobial activity of 2 new cathelicidin genes in goose.

Cathelicidins represent a major group of host defense peptides (HDPs) that share a highly conserved cathelin-like domain. In birds, this gene family has been identified in many species. However, no information was available in the goose until now. In this study, we present the molecular characterization of 2 goose cathelicidin genes, namely goose CATH2 and goose CATH3, for the first time. The complete cDNA of goose CATH2 and goose CATH3 were 571 bp and 573 bp in length, respectively, and the deduced amino acid sequences exhibited high similarity with other avian cathelicidins. Furthermore, evolutionary analyses indicated that all known cathelicidins form 3 distinct clusters from reptiles, while the oldest cathelicidin member, which is known as CATHB1, is very likely absent in the goose genome. Meanwhile, highly expressed goose CATH2 and goose CATH3 were also observed in primary and secondary lymphoid tissues, same as the observations in other avian species. In addition, chemically synthesized mature peptides of the 2 cathelicidins exerted optimal antimicrobial abilities to a range of gram-negative and gram-positive bacteria. The discovery and characterization of goose cathelicidins complete the knowledge for goose HDPs and might contribute to understanding the evolution of avian cathelicidins as well as for the development of antibacterial agents.

High-yield grass Pennisetum sinese Roxb plantation and organic manure alter bacterial and fungal communities structure in an ecological agriculture farm.

Pennisetum sinese Roxb (P. sinese) is an efficient and economic energy crop for its high productivity, and has been well studied in its application in phytoremediation and fodder production. However, little is known about how P. sinese plantation and fermented manures of P. sinese-feed livestock affect the composition of soil bacterial and fungal communities. In this study, 16S rRNA/ITS1 gene-based Illumina Miseq sequencing was employed to compare the bacterial and fungal community structure among soils that had been subjected to uncultivated control (CK), 2-year P. sinese plantation (P), and P. sinese plantation combined with the use of organic manures (P-OM) in a "P. sinese-breeding industry" ecological agriculture farm. The results found microbial communities were altered by P. sinese plantation and fertilization. The P. sinese plantation resulted in increased Actinobacteria and Planctomycetes abundance. Comparatively, significant increased abundance of Chloroflexi, Firmicutes, Nitrospirae, and Euryarchaeota, and genes related with nitrogen and carbon metabolic pathways based on PICRUSt prediction was observed in P-OM soil. Fungal compositions suggested a markedly increased abundance of Ascomycota in P soil. Potential organic matter decomposers Candida, Thermoascus, and Aspergillus were enriched in P soil, indicating the enhanced role of fungi in litter decomposition. Redundancy analysis suggested that soil properties (NH4+-N, total nitrogen, organic matter content, and soil water content) significantly correlated with the changes of microbial compositions (P < 0.05). These results highlight the divergence of microbial communities occurs during P. sinese-based plantation, implying functional diversification of soil ecosystem in P. sinese fields.

Interactive spatial scale effects on species distribution modeling: The case of the giant panda.

Research has shown that varying spatial scale through the selection of the total extent of investigation and the grain size of environmental predictor variables has effects on species distribution model (SDM) results and accuracy, but there has been minimal investigation into the interactive effects of extent and grain. To do this, we used a consistently sampled range-wide dataset of giant panda occurrence across southwest China and modeled their habitat and distribution at 4 extents and 7 grain sizes. We found that increasing grain size reduced model accuracy at the smallest extent, but that increasing extent negated this effect. Increasing extent also generally increased model accuracy, but the models built at the second-largest (mountain range) extent were more accurate than those built at the largest, geographic range-wide extent. When predicting habitat suitability in the smallest nested extents (50 km2), we found that the models built at the next-largest extent (500 km2) were more accurate than the smallest-extent models but that further increases in extent resulted in large decreases in accuracy. Overall, this study highlights the impacts of the selection of spatial scale when evaluating species' habitat and distributions, and we suggest more explicit investigations of scale effects in future modeling efforts.

Genomic Identification and Expression Analysis of the Cathelicidin Gene Family of the Forest Musk Deer.

The forest musk deer (Moschus berezovskii) is a small-sized artiodactyl species famous for the musk secreted by adult males. In the captive population, this species is under the threat of infection diseases, which greatly limits the increase of individual numbers. In the present study, we computationally analyzed the repertoire of the cathelicidin (CATHL) family from the genome of forest musk deer and investigated their expression pattern by real-time PCR. Our results showed that the entire genome of forest musk deer encodes eight cathelicidins, including six functional genes and two pseudogenes. Phylogenetic analyses further revealed that all forest musk deer cathelicidin members have emerged before the split of the forest musk deer and cattle and that forest musk deer CATHL3L2 and CATHL9 are orthologous with two cattle pseudogenes. In addition, the gene expression results showed that the six functional genes are not only abundantly expressed in the spleen and lung, but are also differently expressed in response to abscesses, which suggests that forest musk deer cathelicidins may be involved in infections. Taken together, identification and characterization of the forest musk deer cathelicidins provide fundamental data for further investigating their evolutionary process and biological functions.

Genome-wide analysis of the ovodefensin gene family: Monophyletic origin, independent gene duplication and presence of different selection patterns.

Ovodefensins (OvoDs) represent a group of cysteine-rich host defense peptides that are abundant in the egg white. Recent studies have found that ovodefensins are specific to birds and reptiles. However, the entire repertoire and evolutionary relationships of this gene family have not been thoroughly elucidated to date. Following our cross-species and genome-wide computational study, a total of 94 ovodefensin genes with multiple novel cysteine sequence motifs were identified from 22 phylogenetically divergent species. Phylogenetic analysis suggests that a large number of OvoDs evolved by gene duplication after species divergence. Furthermore, the OvoD genes in each species trend to be clustered densely in a syntenic region flanked by the XKR6 and MTMR9 genes, indicating that they are of monophyletic origin and appear to have emerged via independent gene duplication events in snakes, turtles, crocodiles, birds and the green lizard. Furthermore, positive selection sites are located primarily in the mature peptide region of the turtle, lizard and snake OvoD genes. Moreover, the duplicate OvoDAs in birds seem to be maintained in almost identical sequences and functions by strong purifying selection. Genome-wide identification and analyses of the OvoD gene family may greatly improve our understanding of the potential evolutionary relationship scenario of the OvoD gene family. Continued sequence mining and functional studies of OvoDs will be helpful in shedding light on the relationships between OvoDs and other defensin-related gene families.

Distribution patterns and variation analysis of simple sequence repeats in different genomic regions of bovid genomes.

As the first examination of distribution, guanine-cytosine (GC) pattern, and variation analysis of microsatellites (SSRs) in different genomic regions of six bovid species, SSRs displayed nonrandomly distribution in different regions. SSR abundances are much higher in the introns, transposable elements (TEs), and intergenic regions compared to the 3'-untranslated regions (3'UTRs), 5'UTRs and coding regions. Trinucleotide perfect SSRs (P-SSRs) were the most frequent in the coding regions, whereas, mononucleotide P-SSRs were the most in the introns, 3'UTRs, TEs, and intergenic regions. Trifold P-SSRs had more GC-contents in the 5'UTRs and coding regions than that in the introns, 3'UTRs, TEs, and intergenic regions, whereas mononucleotide P-SSRs had the least GC-contents in all genomic regions. The repeat copy numbers (RCN) of the same mono- to hexanucleotide P-SSRs showed significantly different distributions in different regions (P < 0.01). Except for the coding regions, mononucleotide P-SSRs had the most RCNs, followed by the pattern: di- > tri- > tetra- > penta- > hexanucleotide P-SSRs in the same regions. The analysis of coefficient of variability (CV) of SSRs showed that the CV variations of RCN of the same mono- to hexanucleotide SSRs were relative higher in the intronic and intergenic regions, followed by the CV variation of RCN in the TEs, and the relative lower was in the 5'UTRs, 3'UTRs, and coding regions. Wide SSR analysis of different genomic regions has helped to reveal biological significances of their distributions.

Role of AMPK in the expression of tight junction proteins in heat-treated porcine Sertoli cells.

Hyperthermia can cause dysfunction of the tight junctions (TJs) in testes. Adenosine 5'-monophosphate-activated protein kinase (AMPK) participates in the regulation of TJs in testis. However, whether AMPK regulates the expression of TJ proteins in the response of Sertoli cells to heat treatment remains unknown. We subjected Sertoli cells from 3-week-old piglets to heat treatment (43 °C, 30 min), which decreased cell viability, and increased the early apoptosis rate. These effects were reversible and the cells gradually recovered to normal viability at 48 h post-heat treatment. Expression of TJ proteins (claudin 11, JAMA, occludin, and ZO1) was detected in immature porcine Sertoli cells. The mRNA and protein levels of TJ proteins significantly decreased at 1 h after heat exposure, but recovered with increasing recovery time. Additionally, the expression of claudin 11 in the cytoplasm was also markedly decreased by heat treatment. AMPK phosphorylation, the cellular ATP level, and Ca2+/calmodulin-dependent protein kinase kinase B (CaMKKB) level, but not the liver kinase B1 (LKB1) level, were downregulated by heat treatment. More importantly, activation or overexpression of AMPK, which is a regulator of the assembly of TJs, partially rescued the heat treatment-induced downregulation of TJ proteins. By contrast, AMPK knockdown using small interfering RNA (siRNA) further decreased the expression levels of TJ proteins. In addition, claudin 11 was almost undetectable post heat treatment. Collectively, this study demonstrated that heat treatment could reversibly perturb the expression of TJ proteins in immature porcine Sertoli cells by inhibiting the AMPK signaling pathway.

Long-term distribution and habitat changes of protected wildlife: giant pandas in Wolong Nature Reserve, China.

Changes in wildlife habitat across space and time, and corresponding changes in wildlife space use, are increasingly common phenomenon. It is critical to study and understand these spatio-temporal changes to accurately inform conservation strategy and manage wildlife populations. These changes can be particularly large and complex in areas that face pressure from human development and disturbance but are also under protection and/or restoration regimes. We analyzed changes in space use and habitat suitability of giant pandas in Wolong Nature Reserve, China, over three decades using kernel density, spatio-temporal analysis of moving polygons (STAMP), and MaxEnt methods, and data from three national censuses. Between 2001 and 2012, there was a slight retraction in total range, and more area of significant space use decreases than increases. Habitat suitability varied spatially and temporally, with a 4.1% decrease in average suitability between 1987 and 2001 and a 3.5% increase in average suitability in between 2001 and 2012. Elevation and bamboo were the most important habitat predictors across the three censuses. Human and natural disturbance variables such as distance to household and the distance to landslide variable in the 4th census were also important predictors, and likely also negatively influenced important habitat variables such as bamboo and forest cover. We were able to measure changes in space utilization and habitat suitability over a large time scale, highlighting the achievements and challenges of giant panda conservation. Long-term monitoring of the changes in distribution and habitat of threatened species, and an analysis of the drivers behind these changes such as undergone here, are important to inform the management and conservation of the world's remaining wildlife populations.

Modeling activity patterns of wildlife using time-series analysis.

The study of wildlife activity patterns is an effective approach to understanding fundamental ecological and evolutionary processes. However, traditional statistical approaches used to conduct quantitative analysis have thus far had limited success in revealing underlying mechanisms driving activity patterns. Here, we combine wavelet analysis, a type of frequency-based time-series analysis, with high-resolution activity data from accelerometers embedded in GPS collars to explore the effects of internal states (e.g., pregnancy) and external factors (e.g., seasonal dynamics of resources and weather) on activity patterns of the endangered giant panda (Ailuropoda melanoleuca). Giant pandas exhibited higher frequency cycles during the winter when resources (e.g., water and forage) were relatively poor, as well as during spring, which includes the giant panda's mating season. During the summer and autumn when resources were abundant, pandas exhibited a regular activity pattern with activity peaks every 24 hr. A pregnant individual showed distinct differences in her activity pattern from other giant pandas for several months following parturition. These results indicate that animals adjust activity cycles to adapt to seasonal variation of the resources and unique physiological periods. Wavelet coherency analysis also verified the synchronization of giant panda activity level with air temperature and solar radiation at the 24-hr band. Our study also shows that wavelet analysis is an effective tool for analyzing high-resolution activity pattern data and its relationship to internal and external states, an approach that has the potential to inform wildlife conservation and management across species.

Integrative taxonomy helps to reveal the mask of the genus Gynandropaa (Amphibia: Anura: Dicroglossidae).

Species of the genus Gynandropaa within the family Dicroglossidae are typical spiny frogs whose taxonomic status has long been in doubt. We used integrative methods, involving morphological and molecular analyses, to elucidate the phylogenetic relationships, and to determine identities and the geographic distribution of each valid species. We obtained DNA sequence data of 5 species of Gynandropaa (complete sequences of the mitochondrial NADH dehydrogenase subunit 2 [ND2] gene, and 890 bp of 12S rRNA and 16S rRNA partial sequences) from 37 localities (including the topotypes of 5 described species) and constructed Bayesian and maximum-likelihood trees to examine the patterns of phylogeography. A total of 28 morphological variables were taken on 624 specimens. Three clades with clear geographic patterns were recognized: clade C (from south-western Sichuan Province and central Yunnan Province), clade E (western Guizhou Province and eastern to central Yunnan Province) and clade W (western to southern Yunnan Province). Integrating morphological characteristics and distribution information, the clades W, E and C represent Gynandropaa yunnanensis, G. phrynoides and G. sichuanensis, respectively. We draw the following conclusions: (i) the taxon G. phrynoides, formerly evaluated as a junior synonym of G. yunnanensis, is revalidated herein at the rank of species; (ii) G. liui is a junior synonym of G. sichuanensis; and (iii) G. yunnanensis is a valid species while G. bourreti is probably a subspecies of G. yunnanensis, with the distribution range from Vietnam to southern Yunnan Province. This study clears up the taxonomic status of Gynandropaa and provides important information for understanding the evolution and conservation of these spiny frogs.

[Community structure of phycophyta and evaluation of water quality in Sichuan section of Jialing River].

In order to understand the characteristics of the phycophyta community structure and water quality in Sichuan section of Jialing River, water samples were collected from 12 sites along the section in dry season (January) and rainy season (September), with the phycophyta species composition, Shannon diversity index (H'), Pielou evenness index (E), and Margalef richness index (d) analyzed. A total of 171 phycophyta species (including variety) were collected, belonging to 8 phyla, 42 families, and 95 genera, among which, Bacillariophyta, Chlorophyta, and Cyanophyta were the dominant groups. In dry season, the mean cell density of the phycophyta was 14.71 x 10(4) ind x L(-1), being the highest at sites JX (28.33.4 x 10(4) ind x L(-1)) and HYZ (25.40 x 10(4) ind x L(-1)), and diatom species had a higher richness than the others. In rainy season, the mean cell density was only 10.78 x 10(4) ind x L(-1), being the lowest (3.31 x 10(4) ind x L(-1)) at site QJ, and the species richness of chlorophyta and cyanobateria had somewhat increase. In the whole section, the mean d, H', and E of the phycophyta were 2.35, 1.60, and 0.31 in dry season, and 2.57, 2.09, and 0.39 in rainy season, respectively. Our results indicated that there were significant differences in the spatiotemporal distribution patterns of the community structure, cell density, diversity index, and evenness index of phycophyta in Sichuan section of Jialing River. The water quality of this section was overall belonged to mesosaprobic, being better at sites JX and SX (belonged to oligosaprobic or beta-mesosaprobic), but worse at sites CX, HYZ, XZ, and QJ (belonged to alpha-mesosaprobic).