eDNA-based detection reveals invasion risks of a biofouling bivalve in the world's largest water diversion project.

Environmental DNA (eDNA) has increasingly been used to detect rare species (e.g., newly introduced nonindigenous species) in both terrestrial and aquatic ecosystems, often with distinct advantages over traditional methods. However, whether water eDNA signals can be used to inform invasion risks remains debatable owing to inherent uncertainties associated with the methods used and the varying conditions among study systems. Here, we sampled eDNA from canals of the central route of the South-to-North Water Diversion Project (hereafter SNWDP) in China to investigate eDNA distribution and efficacy to inform invasion risks in a unique lotic system. We first conducted a total of 16 monthly surveys in this system (two sites in the source reservoir and four sites in the main canal) to test if eDNA could be applied to detect an invasive, biofouling bivalve, the golden mussel Limnoperna fortunei. Second, we initiated a one-time survey in a sub-canal of the SNWDP using refined sampling (12 sites in ~22 km canal) and considered a few environmental predictors. We found that detection of target eDNA in the main canal was achieved up to 1100 km from the putative source population but was restricted to the warmer months (May-November). Detection probability exhibited a significant positive relationship with average daily minimum air temperature and with water temperature, consistent with the expected spawning season. eDNA concentration in the main canal generally fluctuated across months and sites and was generally higher in warmer months. Golden mussel eDNA concentration in the sub-canal decreased significantly with distance from the source and with increasing water temperature and became almost undetectable at ~22 km distance. Given the enormity of the SNWDP, golden mussels may eventually expand their distribution in the main canal, with established "bridgehead" populations facilitating further spread. Our findings suggest an elevated invasion risk of golden mussels in the SNWDP in warm months, highlighting the critical period for spread and, possibly, management.

Genomic analyses reveal three phylogenetic species and their evolutionary histories in the big-headed turtle.

The critically endangered big-headed turtle (Platysternon megacephalum) is currently classified into three subspecies. However, the classification is still controversial and their evolutionary histories are still unclear. Here, multiple genetic analyses consistently revealed three phylogenetic groups with substantial genetic divergences and distinct demographic histories, suggesting three phylogenetic species (P. megacephalum, P. peguense, and Baise clade). Phylogeographical analyses revealed that the Red River plains and Guangxi basins are largely coincident with the boundaries between the three phylogenetic species, highlighting the key role of lowland areas in driving speciation in the big-headed turtle. The Baise clade is characterized by high-linkage disequilibrium but the lowest effective population size, indicating that the cryptic phylogenetic species is more vulnerable to human activities and environmental disturbance, and urgently needs more protection. Our findings provide fundamental insights into the taxonomy and scientific conservation of the family Platysternidae.

Genomic insights into the genetic basis of eagle-beak jaw, large head, and long tail in the big-headed turtle.

The big-headed turtle (Platysternon megacephalum) is an endemic chelonian species in Asia. Unlike most other turtles in the world, P. megacephalum is characterized with eagle-beak jaw, large head, and long tail. Although these unique characteristics are well recognized, the underlying genetic basis remains largely elusive. Here, we performed comparative genomic analysis between P. megacephalum and other representative species, aiming to reveal the genetic basis of the unique morphological features. Our results revealed that the eagle-beak jaw is most likely enabled by combined effects of expansion of SFRP5, extraction of FGF11, and mutation of both ZFYVE16 and PAX6. Large head is supported by mutations of SETD2 and FGRF2 and copy number variations of six head circumference modulation-related genes (TGFBR2, Twist2, Rdh10, Gas1, Chst11, and SNAP25). The long tail is probably involved in a genetic network comprising Gdf11, Lin 28, and HoxC12, two of which showed a consistent expression pattern with a model organism (mice). These findings suggest that expansion, extraction, and mutation of those genes may have profound effects on unique phenotypes of P. megacephalum.

Semi-automatic Framework for Voxel Human Deformation Modeling.

BACKGROUND: With the advancement of computer and medical imaging technologies, a number of high-resolution, voxel-based, full-body human anatomical models have been developed for medical education, industrial design, and physics simulation studies. However, these models are limited in many applications because they are often only in an upstanding posture. OBJECTIVE: To quickly develop multi-pose human models for different applications. A semi-automatic framework for voxel deformation is proposed in the study. METHODS: This paper describes a framework for human pose deformation based on three-dimensional (3D) medical images. The voxel model is first converted into a surface model using a surface reconstruction algorithm. Second, a deformation skeleton based on human bones is defined, and the surface model is bound to the skeleton. The bone Glow algorithm is used to assign weights to the surface vertices. Then, the model is deformed to the target posture by using the Smoothed Rotation Enhanced As-Rigid-As-Possible (SR-ARAP) algorithm. Finally, the volume-filling algorithm is applied to refill the tissues into the deformed surface model. RESULTS: The proposed framework is used to deform two standing human models, and the sitting and running models are developed. The results show that the framework can successfully develop the target pose. When compared to the results of the As-Rigid-As-Possible algorithm, SR-ARAP preserves local tissues better. CONCLUSION: The study proposes a frame for voxel human model deformation and improves the local tissue integrity during deformation.

Disentangling the Pelodiscus axenaria complex, with the description of a new Chinese species and neotype designation for P. axenaria (Zhou, Zhang amp; Fang, 1991).

We describe a new species from the Pelodiscus axenaria complex from Hunan and Jiangxi Provinces, China. Also, the application of the name P. axenaria (Zhou, Zhang Fang, 1991) is clarified by designating a neotype for this species. Besides its genetic divergence, the new species differs from all other Pelodiscus species, including the two other taxa constituting the complex (P. axenaria, P. huangshanensis), in the following combination of morphological traits: (1) small adult size, <15 cm carapace length; (2) carapace distinctly keeled, more or less strongly tuberculated, usually olive clay-coloured and adorned with greenish black marbling; (3) plastron yellowish white, typically immaculate except for a blurred-edged blotch behind each axilla that does not extend to the entoplastron and some slight black suffusion along its anterior border; (4) underside of the leathery margin of the carapace with varying amounts of dark pigmentation; (5) head olive clay-coloured with numerous black splotches; (6) chin grey brown with pale stipples, throat dark grey, finely spotted with black; (7) neck with a wide yellow lateral band stretching from the tympanum posteriorly, which tends to fade with age; (8) entoplastron boomerang-shaped, the amount of bending of the transverse bar between the two posteriolaterally directed rami >90.

Complementary genomic and epigenomic adaptation to environmental heterogeneity.

While adaptation is commonly thought to result from selection on DNA sequence-based variation, recent studies have highlighted an analogous epigenetic component as well. However, the relative roles of these mechanisms in facilitating population persistence under environmental heterogeneity remain unclear. To address the underlying genetic and epigenetic mechanisms and their relationship during environmental adaptation, we screened the genomes and epigenomes of nine global populations of a predominately sessile marine invasive tunicate, Botryllus schlosseri. We detected clear population differentiation at the genetic and epigenetic levels. Patterns of genetic and epigenetic structure were significantly influenced by local environmental variables. Among these variables, minimum annual sea surface temperature was identified as the top explanatory variable for both genetic and epigenetic variation. However, patterns of population structure driven by genetic and epigenetic variation were somewhat distinct, suggesting possible autonomy of epigenetic variation. We found both shared and specific genes and biological pathways among genetic and epigenetic loci associated with environmental factors, consistent with complementary and independent contributions of genetic and epigenetic variation to environmental adaptation in this system. Collectively, these mechanisms may facilitate population persistence under environmental change and sustain successful invasions across novel environments.

Local environment-driven adaptive evolution in a marine invasive ascidian (Molgula manhattensis).

Elucidating molecular mechanisms of environment-driven adaptive evolution in marine invaders is crucial for understanding invasion success and further predicting their future invasions. Although increasing evidence suggests that adaptive evolution could contribute to organisms' adaptation to varied environments, there remain knowledge gaps regarding how environments influence genomic variation in invaded habitats and genetic bases underlying local adaptation for most marine invaders. Here, we performed restriction-site-associated DNA sequencing (RADseq) to assess population genetic diversity and further investigate genomic signatures of local adaptation in the marine invasive ascidian, Molgula manhattensis. We revealed that most invasive populations exhibited significant genetic differentiation, low recent gene flow, and no signal of significant population bottleneck. Based on three genome scan approaches, we identified 109 candidate loci potentially under environmental selection. Redundancy analysis and variance partitioning analysis suggest that local environmental factors, particularly the salinity-related variables, represent crucial evolutionary forces in driving adaptive divergence. Using the newly developed transcriptome as a reference, 14 functional genes were finally obtained with potential roles in salinity adaptation, including SLC5A1 and SLC9C1 genes from the solute carrier gene (SLC) superfamily. Our findings confirm that differed local environments could rapidly drive adaptive divergence among invasive populations and leave detectable genomic signatures in marine invaders.

Effect of incubation temperature and substrate moisture on embryonic development, hatchling phenotypes and post-hatching growth in the Reeves' Turtle, Mauremys reevesii.

BACKGROUND: Reeves' Turtles (Mauremys reevesii) are economically important in aquaculture in China. Understanding the effects of incubation temperature and substrate moisture on embryos and hatchlings is of great significance for improving the artificial culture of M. reevesii. However, available studies have not yet determined the thermal and hydric optima for M. reevesii eggs, and the potential interaction between the two factors. METHODS: In this study, eggs of M. reevesii were incubated at five temperature levels (23, 26, 29, 32 and 35 °C, fluctuation range ± 0.5 °C). In each temperature level, there were three substrate moisture levels (1:0.5, 1:0.9 and 1:1.2, weight ratio of vermiculite to water). Thus, a total of 15 combinations of temperature and moisture were used to examine the effects of incubation temperature and substrate moisture on incubation duration, hatching success, hatchling phenotypes, post-hatching growth and hatchling survival. RESULTS: Substrate moisture did not significantly affect most development parameters (except incubation duration and carapace width of hatchlings). Eggs incubated at low moisture level (1:0.5) had a longer incubation duration and produced hatchlings with smaller carapace widths than those incubated at medium (1:0.9) or high (1:1.2) moisture levels. Incubation temperature had a significant effect on incubation duration, hatching success, hatchling phenotypes and hatchling survival. Incubation duration decreased as incubation temperature increased. Eggs incubated at 23, 26 and 29 °C showed higher hatching success than those incubated at 32 and 35 °C. Hatchlings incubated at 32 °C were smaller in body size and mass than those incubated at 23, 26 and 29 °C. At 12 months of age, incubation temperature had no long-lasting effect on body mass, but hatchlings incubated at 23 and 35 °C had lower survival rates than those incubated at 26, 29 and 32 °C. For the development of embryos and hatchlings, the interaction between incubation temperature and substrate moisture was not significant. CONCLUSIONS: Our results indicate that incubation temperature has a significant influence on the development of embryos and hatchlings of M. reevesii, while substrate moisture only significantly affects the incubation duration and carapace width of hatchlings. The combination of an incubation temperature of 29 ± 0.5 °C and a substrate moisture level of 1:1.2 represented optimal incubation conditions in this experiment. Such incubation conditions are helpful in obtaining higher hatching success, shorter incubation duration and higher survival rates for this aquaculture species.

Phylogenetic analyses of distantly related clades of bent-toed geckos (genus Cyrtodactylus) reveal an unprecedented amount of cryptic diversity in northern and western Thailand.

Cyrtodactylus species are the most diverse of the geckos and are widely distributed in Southeast Asia, including Thailand. However, their patterns of distribution, especially in northern and western parts of Thailand, remain unknown because few Cyrtodactylus species in these regions have been described. Thus, a data set of mitochondrial NADH dehydrogenase 2 (ND2) gene and flanking tRNAs from Cyrtodactylus found in northern and western Thailand, including contiguous areas, was assembled to elucidate phylogenetic relationships and identify the distribution patterns of these geckos. The results showed four well-supported clades, a northwestern clade (A), a northern clade (B), a western clade (C), and a special clade characterized by specific morphological features (D). Clades A-C were grouped with strong support by the geography of their localities from northern Thailand (Mae Hong Son and Chiang Mai Provinces) along the Tenasserim mountain ranges to Phang-Nga Province, Thailand. Clade D is a distinct clade of Cyrtodactylus species characterized by a tuberculate and prehensile tail and distributed widely in mainland Southeast Asia. Overall, the results suggest a pattern of geographic separation and distribution of Cyrtodactylus in northern and western Thailand. Additionally, this study provides evidence of a hidden biodiversity of Cyrtodactylus in these regions.

Integrating and updating wildlife conservation in China.

China has about 11% of the world's total wildlife species, so strengthening China's wildlife conservation is of great significance to global biodiversity. Despite some successful cases and conservation efforts, 21.4% of China's vertebrate species are threatened by human activities. The booming wildlife trade in China has posed serious threat to wildlife in China and throughout the world, while leading to a high risk of transmission of infectious zoonotic diseases. China's wildlife conservation has faced a series of challenges, two of which are an impractical, separated management of wildlife and outdated protected species lists. Although the Wildlife Protection Law of China was revised in 2016, the issues of separated management remain, and the protected species lists are still not adequately revised. These issues have led to inefficient and overlapping management, waste of administrative resources, and serious obstacles to wildlife protection. In this article, we analyze the negative effects of current separated management of wildlife species and outdated protected species lists, and provide some suggestions for amendment of the laws and reform of wildlife management system.

Anthropogenic pollutant-driven geographical distribution of mesozooplankton communities in estuarine areas of the Bohai Sea, China.

Mesozooplankton communities in marine ecosystems are mainly influenced by both anthropogenic pollutants (e.g. nutrients and heavy metals) and natural variables (e.g. temperature, salinity and geographic distance). To achieve a deeper understanding of the effects of anthropogenic pollutants on mesozooplankton communities, we analyzed the community structure of mesozooplankton from 91 stations representing five typical estuarine regions in the Bohai Sea and assessed the relative importance of anthropogenic pollutants and natural variables by using multiple statistical approaches. Cd was identified as the leading pollutant for observed community variation among the five regions, followed by NH4-N and COD. Redundancy analysis (RDA) model demonstrated that mesozooplankton communities were largely determined by both anthropogenic pollutants and natural variables, and the indicator species of mesozooplankton also varied when responding to different factors. Variance partitioning analysis showed both anthropogenic pollutants and natural variables posed significant influences (ANOVA, P < 0.05) on the mesozooplankton community structure, but the explanatory power of anthropogenic pollutants overrode the natural variables. These observations highlighted the importance of anthropogenic pollutants in the shifts of zooplankton structures among different regions. Our results obtained in this study provided new insights into the mechanism of the influence of anthropogenic pollutants on mesozooplankton communities in estuarine areas.

Biological consequences of environmental pollution in running water ecosystems: A case study in zooplankton.

Biodiversity in running water ecosystems such as streams and rivers is threatened by chemical pollution derived from anthropogenic activities. Zooplankton are ecologically indicative in aquatic ecosystems, owing to their position of linking the top-down and bottom-up regulators in aquatic food webs, and thus of great potential to assess ecological effects of human-induced pollution. Here we investigated the influence of water pollution on zooplankton communities characterized by metabarcoding in Songhua River Basin in northeast China. Our results clearly showed that varied levels of anthropogenic disturbance significantly influenced water quality, leading to distinct environmental pollution gradients (p < 0.001), particularly derived from total nitrogen, nitrate nitrogen and pH. Redundancy analysis showed that such environmental gradients significantly influenced the geographical distribution of zooplankton biodiversity (R = 0.283, p = 0.001). In addition, along with the trend of increasing environmental pollution, habitat-related indicator taxa were shifted in constituents, altering from large-sized species (e.g. arthropods) in lightly disturbed areas to small-sized organisms (e.g. rotifers and ciliates) in highly disturbed areas. All these findings clearly showed that anthropogenic activity-derived water pollution significantly influenced biological communities. Thus, biotic consequences of human-induced environmental pollution in running water ecosystems should be deeply investigated. More importantly, the findings of biotic consequences should be well integrated into existing monitoring programs to further assess impacts of anthropogenic disturbance, as well as to advance the management of running water ecosystems for conservation and ecological restoration.

Distribution patterns of dinoflagellate communities along the Songhua River.

BACKGROUND: Dinoflagellates have the potential to pose severe ecological and economic damages to aquatic ecosystems. It is therefore largely needed to understand the causes and consequences of distribution patterns of dinoflagellate communities in order to manage potential environmental problems. However, a majority of studies have focused on marine ecosystems, while the geographical distribution patterns of dinoflagellate communities and associated determinants in freshwater ecosystems remain unexplored, particularly in running water ecosystems such as rivers and streams. METHODS: Here we utilized multiple linear regression analysis and combined information on species composition recovered by high-throughput sequencing and spatial and environmental variables to analyze the distribution patterns of dinoflagellate communities along the Songhua River. RESULTS: After high-throughput sequencing, a total of 490 operational taxonomic units (OTUs) were assigned to dinoflagellates, covering seven orders, 13 families and 22 genera. Although the sample sites were grouped into three distinctive clusters with significant difference (p < 0.05) in environmental variables, OTUs-based dinoflagellate communities among the three clusters showed no significant difference (p > 0.05). Among all 24 environmental factors, two environmental variables, including NO3-N and total dissolved solids (TDS), were selected as the significantly influential factors (p < 0.05) on the distribution patterns of dinoflagellate communities based on forward selection. The redundancy analysis (RDA) model showed that only a small proportion of community variation (6.1%) could be explained by both environmental (NO3-N and TDS) and dispersal predictors (watercourse distance) along the River. Variance partitioning revealed a larger contribution of local environmental factors (5.85%) than dispersal (0.50%) to the total variation of dinoflagellate communities. DISCUSSION: Our findings indicated that in addition to the two quantifiable processes in this study (species sorting and dispersal), more unquantifiable stochastic processes such as temporal extinction and colonization events due to rainfall may be responsible for the observed geographical distribution of the dinoflagellate community along the Songhua River. Results obtained in this study suggested that deeper investigations covering different seasons are needed to understand the causes and consequences of geographical distribution patterns of dinoflagellate biodiversity in river ecosystems.

Chemical oxidants affect byssus adhesion in the highly invasive fouling mussel Limnoperna fortunei.

Biofouling by the invasive mussel species Limnoperna fortunei has caused severe negative consequences in various freshwater ecosystems, but there is still a lack of effective antifouling strategies for this species, particularly in drinking water treatment systems where many existing strategies cannot be used. Chemical oxidants have been proposed to potentially control fouling mussels by influencing 3,4­dihydroxyphenylalanine oxidation involved in byssus adhesion. To test this potential control strategy, L. fortunei adults were exposed to four oxidants: sodium hypochlorite (NaClO), potassium permanganate (KMnO4), hydrogen peroxide (H2O2) and chloramine T (CAT) at concentrations of 0.5, 1.0, 3.0 and 5.0 mg L-1 for one week. The results showed that low concentrations of KMnO4 stimulated foot protein secretion, which was beneficial to byssus production (number and length). NaClO and H2O2, but not KMnO4 and CAT, significantly inhibited byssus production and reduced breaking force, causing an increased byssus shedding rate. However, only NaClO dissolved byssus and altered the failure location from the byssal thread to adhesive plaque, even when exposed at low concentrations, showing its great impacts on byssus adhesion. Further analysis of polyphenoloxidase activities and gene expression profiles of LfBP-1, LfFP-2 and LfBP-3 revealed that chemical oxidants affected byssus adhesion by altering Dopa oxidation and foot protein gene expressions. This study, therefore, suggests that a low concentration of NaClO can be used as an alternative and environmentally friendly chemical for controlling L. fortunei biofouling by weakening byssus adhesion.

Adaptive shifts of bacterioplankton communities in response to nitrogen enrichment in a highly polluted river.

Anthropogenic activity-mediated nutrient pollution, especially nitrogen enrichment, poses one of the major threats to river ecosystems. However, it remains unclear how and to which extent it affects aquatic microbial communities, especially in heavily polluted rivers. In this study, a significant environmental gradient, particularly nitrogen gradient, was observed along a wastewater receiving river, the North Canal River (NCR). The pollution level was highest, moderate, and lowest in the up-, middle, and down-streams, respectively. The community composition of bacterioplankton transitioned from being Betaproteobacteria-dominated upstream to Gammaproteobacteria-dominated downstream. Copiotrophic groups, such as Polynucleobacter (Betaproteobacteria) and Hydrogenophaga (Betaproteobacteria), were dominant in the upstream. Multiple statistical analyses indicated that total nitrogen (TN) was the most important factor driving the adaptive shifts of community structure. Analyses of co-occurrence networks showed that the complexity of networks was disrupted in the up- and middle streams, while enhanced in the downstream. Our findings here suggested that microbial interactions were reduced in response to the aggravation of nutrient pollution. Similar to these changes, we observed significant dissimilarity of composition of functional groups, with highest abundance of nitrogen metabolism members under the highest level of nitrogen enrichment. Further analyses indicated that most of these functional groups belonged to Betaproteobacteria, suggesting the potential coupling of community composition and function diversity. In summary, adaptive shifts of bacterioplankton community composition, as well as species interactions, occurred in response to nutrient pollution in highly polluted water bodies.

Genome-Wide Identification, Characterization and Expression Analyses of Heat Shock Protein-Related Genes in a Highly Invasive Ascidian Ciona savignyi.

Biological response to rapid changing environments is an outstanding research question in ecology and evolution. Biological invasions provide excellent "natural" experiments to study such a complex response process, as invaders often encounter rapidly changing environments during biological invasions. The regulation of heat shock proteins (Hsp) is a common pathway responsible for various environmental stresses; however, the comprehensive study on Hsp system across the whole genome and potential roles in determining invasion success are still largely unexplored. Here, we used a marine invasive model ascidian, Ciona savignyi, to investigate transcriptional response of Hsp-related genes to harsh environments. We identified 32 genes, including three Hsp20, six Hsp40, ten Hsp60, eight Hsp70, three Hsp90, one Hsp100, and one heat shock transcription factor (Hsf), across the whole genome of C. savignyi. We further characterized gene structure and protein motifs, and identified potential heat shock elements (HSEs) in promoters of Hsp genes. The expression analysis showed that most Hsp genes, but not all, were involved in transcriptional response to temperature and salinity challenges in a duration- and stress-specific pattern, and the maximum amplitude of induction occurred in Hsp70-4 after 1-h of high temperature treatment. However, the Hsf gene was scarcely induced and limited interactions were predicted between Hsp and Hsf genes. Our study provide the first systematic genome-wide analysis of Hsp and Hsf family in the marine invasive model ascidian, and our results are expected to dissect Hsp-based molecular mechanisms responsible for extreme environmental adaptation using Ciona as a model system.

Geographical distribution of zooplankton biodiversity in highly polluted running water ecosystems: Validation of fine-scale species sorting hypothesis.

Dispersal, rather than species sorting, is widely recognized as the dominant driver for determining meta-community structure at fine geographical scales in running water ecosystems. However, this view has been challenged by a recently proposed "fine-scale species sorting hypothesis," where community structure can be largely determined by an environmental gradient formed by local pollution at fine scales. Here, we tested this hypothesis by studying community composition and geographical distribution of metazoan zooplankton in a heavily polluted river-the North Canal River in the Haihe River Basin, China. Analysis of similarity (ANOSIM) showed that the community composition of metazoan zooplankton differed significantly (p = .001) along the environmental gradient. Ammonium (NH4-N) was the leading factor responsible for changes in zooplankton community structure and geographical distribution, followed by total dissolved solid (TDS), Na, dissolved oxygen (DO) and temperature (T). Variation partitioning revealed a larger contribution of environmental variables (21.6%) than spatial variables (1.1%) to the total explained variation of zooplankton communities. Our results support that species sorting, rather than dispersal, played a key role in structuring communities. Threshold Indicator Taxa ANalysis (TITAN) also revealed significant change points at both taxon and community levels along the gradient of NH4-N, providing further support for the influence of environmental variables on zooplankton communities. Collectively, we validate the fine-scale species sorting hypothesis when an environmental gradient exists in running water ecosystems at fine geographical scales. However, future studies on interactions between pollutants and zooplankton communities are still needed to better understand mechanisms responsible for the meta-community dynamics.

Byssus Structure and Protein Composition in the Highly Invasive Fouling Mussel Limnoperna fortunei.

Biofouling mediated by byssus adhesion in invasive bivalves has become a global environmental problem in aquatic ecosystems, resulting in negative ecological and economic consequences. Previous studies suggested that mechanisms responsible for byssus adhesion largely vary among bivalves, but it is poorly understood in freshwater species. Understanding of byssus structure and protein composition is the prerequisite for revealing these mechanisms. Here, we used multiple methods, including scanning electron microscope, liquid chromatography-tandem mass spectrometry, transcriptome sequencing, real-time quantitative PCR, inductively coupled plasma mass spectrometry, to investigate structure, and protein composition of byssus in the highly invasive freshwater mussel Limnoperna fortunei. The results indicated that the structure characteristics of adhesive plaque, proximal and distal threads were conducive to byssus adhesion, contributing to the high biofouling capacity of this species. The 3,4-dihydroxyphenyl-α-alanine (Dopa) is a major post-transnationally modification in L. fortunei byssus. We identified 16 representative foot proteins with typical repetitive motifs and conserved domains by integrating transcriptomic and proteomic approaches. In these proteins, Lfbp-1, Lffp-2, and Lfbp-3 were specially located in foot tissue and highly expressed in the rapid byssus formation period, suggesting the involvement of these foot proteins in byssus production and adhesion. Multiple metal irons, including Ca2+, Mg2+, Zn2+, Al3+, and Fe3+, were abundant in both foot tissue and byssal thread. The heavy metals in these irons may be directly accumulated by L. fortunei from surrounding environments. Nevertheless, some metal ions (e.g., Ca2+) corresponded well with amino acid preferences of L. fortunei foot proteins, suggesting functional roles of these metal ions by interacting with foot proteins in byssus adhesion. Overall, this study provides structural and molecular bases of adhesive mechanisms of byssus in L. fortunei, and findings here are expected to develop strategies against biofouling by freshwater organisms.

Genome-wide single nucleotide polymorphisms (SNPs) for a model invasive ascidian Botryllus schlosseri.

Invasive species cause huge damages to ecology, environment and economy globally. The comprehensive understanding of invasion mechanisms, particularly genetic bases of micro-evolutionary processes responsible for invasion success, is essential for reducing potential damages caused by invasive species. The golden star tunicate, Botryllus schlosseri, has become a model species in invasion biology, mainly owing to its high invasiveness nature and small well-sequenced genome. However, the genome-wide genetic markers have not been well developed in this highly invasive species, thus limiting the comprehensive understanding of genetic mechanisms of invasion success. Using restriction site-associated DNA (RAD) tag sequencing, here we developed a high-quality resource of 14,119 out of 158,821 SNPs for B. schlosseri. These SNPs were relatively evenly distributed at each chromosome. SNP annotations showed that the majority of SNPs (63.20%) were located at intergenic regions, and 21.51% and 14.58% were located at introns and exons, respectively. In addition, the potential use of the developed SNPs for population genomics studies was primarily assessed, such as the estimate of observed heterozygosity (H O ), expected heterozygosity (H E ), nucleotide diversity (π), Wright's inbreeding coefficient (F IS ) and effective population size (Ne). Our developed SNP resource would provide future studies the genome-wide genetic markers for genetic and genomic investigations, such as genetic bases of micro-evolutionary processes responsible for invasion success.

Conventional versus real-time quantitative PCR for rare species detection.

Detection of species in nature at very low abundance requires innovative methods. Conventional PCR (cPCR) and real-time quantitative PCR (qPCR) are two widely used approaches employed in environmental DNA (eDNA) detection, though lack of a comprehensive comparison of them impedes method selection. Here we test detection capacity and false negative rate of both approaches using samples with different expected complexities. We compared cPCR and qPCR to detect invasive, biofouling golden mussels (Limnoperna fortunei), in samples from laboratory aquaria and irrigation channels where this mussel was known to occur in central China. Where applicable, the limit of detection (LoD), limit of quantification (LoQ), detection rate, and false negative rate of each PCR method were tested. Quantitative PCR achieved a lower LoD than cPCR (1 × 10-7 vs. 10-6 ng/µl) and had a higher detection rate for both laboratory (100% vs. 87.9%) and field (68.6% vs. 47.1%) samples. Field water samples could only be quantified at a higher concentration than laboratory aquaria and total genomic DNA, indicating inhibition with environmental samples. The false negative rate was inversely related to the number of sample replicates. Target eDNA concentration was negatively related to distance from sampling sites to the water (and animal) source. Detection capacity difference between cPCR and qPCR for genomic DNA and laboratory aquaria can be translated to field water samples, and the latter should be prioritized in rare species detection. Field environmental samples may involve more complexities-such as inhibitors-than laboratory aquaria samples, requiring more target DNA. Extensive sampling is critical in field applications using either approach to reduce false negatives.