Aibin Zhan

Invasion genetics of the Ciona intestinalis species complex: from regional endemism to global homogeneity.

Determining the degree of population connectivity and investigating factors driving genetic exchange at various geographical scales are essential to understanding population dynamics and spread potential of invasive species. Here, we explore these issues in the highly invasive vase tunicate, Ciona intestinalis, a species whose invasion history has been obscured by its poorly understood taxonomy and population genetics. Recent phylogenetic and comparative genomic studies suggest that C. intestinalis is a cryptic species complex consisting of at least three species. We reconstructed phylogenies based on both mitochondrial (cytochrome c oxidase subunit 3—NADH dehydrogenase subunit 1 region and NADH dehydrogenase subunit 4 gene) and nuclear (internal transcribed spacer 1) sequences, results of which support four major phylogroups corresponding to the previously reported spA, spB and Ciona spp. (spC) as well as an undescribed cryptic species (spD). While spC and spD remain restricted to their native ranges in the Mediterranean Sea and Black Sea, respectively, the highly invasive species (spA and spB) have disjunct global distributions. Despite extensive interspecific divergences, we identified low phylogeographical structure within these two invasive species. Haplotype network analyses revealed comparatively limited mutation steps among haplotypes within each species. Population genetic analyses based on two mtDNA fragments and eight unlinked microsatellites illustrated relatively low population differentiation and high population connectivity at both regional and continental scales in the two invasive species. Human‐mediated dispersal coupled with a high potential for natural dispersal is probably responsible for the observed genetic homogeneity.

Fine-Scale Population Genetic Structure of Zhikong Scallop ( Chlamys farreri ): Do Local Marine Currents Drive Geographical Differentiation?

Marine scallops, with extended planktonic larval stages which can potentially disperse over large distances when advected by marine currents, are expected to possess low geographical differentiation. However, the sessile lifestyle as adult tends to form discrete “sea beds” with unique population dynamics and structure. The narrow distribution of Zhikong scallop (Chlamys farreri), its long planktonic larval stage, and the extremely hydrographic complexity in its distribution range provide an interesting case to elucidate the impact of marine currents on geographical differentiation for marine bivalves at a fine geographical scale. In this study, we analyzed genetic variation at nine microsatellite DNA loci in six locations throughout the distribution of Zhikong scallop in the Northern China. Very high genetic diversity was present in all six populations. Two populations sampled from the same marine gyre had no detectable genetic differentiation (FST = 0.0013); however, the remaining four populations collected from different marine gyres or separated by strong marine currents showed low but significant genetic differentiation (FST range 0.0184–0.0602). Genetic differentiation was further analyzed using the Monmonier algorithm to identify genetic barriers and using the assignment test conducted by software GeneClass2 to ascertain population membership of individuals. The genetic barriers fitting the orientation of marine gyres/currents were clearly identified, and the individual assignment analysis indicated that 95.6% of specimens were correctly allocated to one of the six populations sampled. The results support the hypothesis that significant population structure is present in Zhikong scallop at a fine geographical scale, and marine currents can be responsible for the genetic differentiation.

Construction of microsatellite-based linkage maps and identification of size-related quantitative trait loci for Zhikong scallop (Chlamys farreri).

We constructed the microsatellite-based linkage maps using 318 markers typed in two F(1) outbred families of Zhikong scallop (Chlamys farreri). The results showed an extremely high proportion (56.2%) of non-amplifying null alleles and a high ratio (30%) of segregation distortion. By aligning different individual-based linkage maps, 19 linkage groups were identified, which are consistent with the haploid chromosome number of Zhikong scallop. The integrated linkage map contains 154 markers covering 1561.8 cM with an average intermarker spacing of 12.3 cM and 77.0% of genome coverage. We found that the heterogeneity in recombination rate was not determined by sexes but by different individuals on 18 linkage regions. The phenotypic marker of general shell colour was placed on LG4, which was flanked by microsatellite markers CFLD064 and CFBD055. Four size-related traits including shell length (SL), shell width (SW), shell height (SH) and gross weight (GW) were analysed to identify the putative quantitative trait loci (QTL). Under the half-sib model, using dam as common parent, three, two, two and one QTL affecting SL, SW, SH and GW exceeded the genome-wide thresholds respectively. While using sir as common parent, a larger number of QTL were detected for these four traits: four, five, three and two for SL, SW, SH and GW respectively. The single QTL explained 3.7-19.2% of the phenotypic variation. The linkage map and the QTL associated with economic traits will provide useful information for marker-assisted selection of Zhikong scallop.

Performance comparison of genetic markers for high-throughput sequencing-based biodiversity assessment in complex communities

Metabarcode surveys of DNA extracted from environmental samples are increasingly popular for biodiversity assessment in natural communities. Such surveys rely heavily on robust genetic markers. Therefore, analysis of PCR efficiency and subsequent biodiversity estimation for different types of genetic markers and their corresponding primers is important. Here, we test the PCR efficiency and biodiversity recovery potential of three commonly used genetic markers – nuclear small subunit ribosomal DNA (18S), mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (mt16S) – using 454 pyrosequencing of a zooplankton community collected from Hamilton Harbour, Ontario. We found that biodiversity detection power and PCR efficiency varied widely among these markers. All tested primers for COI failed to provide high‐quality PCR products for pyrosequencing, but newly designed primers for 18S and 16S passed all tests. Furthermore, multiple analyses based on large‐scale pyrosequencing (i.e. 1/2 PicoTiter plate for each marker) showed that primers for 18S recover more (38 orders) groups than 16S (10 orders) across all taxa, and four vs. two orders and nine vs. six families for Crustacea. Our results showed that 18S, using newly designed primers, is an efficient and powerful tool for profiling biodiversity in largely unexplored communities, especially when amplification difficulties exist for mitochondrial markers such as COI. Universal primers for higher resolution markers such as COI are still needed to address the possible low resolution of 18S for species‐level identification.

Microsatellite markers derived from bay scallop Argopecten irradians expressed sequence tags

When data mining was performed on the National Center for Biotechnology Information database, a total of 2038 sequences from five different expressed sequence tag libraries were registered. Eighty sequences (3.9%) were found to contain 91 microsatellites. Clustering analysis indicated that 23 sequences of these expressed sequence tags fell into five clusters and that the remaining 57 sequences were independent. The di- and tri-nucleotide repeat motifs accounted for approximately 62.1% of the total microsatellites. The most abundant dinucleotide microsatellite was TA, followed by GA and CA, and the trinucleotide microsatellites GAT and GGT showed a high abundance. Nineteen sequences representing di-, tri-, tetra- and penta-nucleotides motifs were chosen for the design of polymerase chain reaction (PCR) primers. Of primer pairs, 16 successfully amplified scorable PCR products and 11 revealed polymorphism, with the average polymorphic information content value of 0.5082 and 3.1 alleles per locus. A transferability analysis on three other related scallop species, Chlamys farreri, Chlamys nobilis and Patinopecten yessoenssis, showed that only 1 of 16 primer pairs could amplify PCR products with the expected size in Chlamys nobilis.

Looking at both sides of the invasion: patterns of colonization in the violet tunicate Botrylloides violaceus

Understanding the ecological and evolutionary forces that shape the genetic structure of invasive populations and facilitate their expansion across a large spectrum of environments is critical for the prediction of spread and management of ongoing invasions. Here, we study the dynamics of postestablishment colonization in the colonial ascidian Botrylloides violaceus, a notorious marine invader. After its initial introduction from the Northwest Pacific, B. violaceus spread rapidly along the Pacific and Atlantic coasts of North America, impacting both aquaculture facilities and natural ecosystems. We compare genetic diversity and patterns of gene flow among 25 populations (N = 679) from the West and East coasts, and evaluate the contribution of sexual vs. asexual reproduction to this species’ invasion success using data from the mitochondrial cytochrome c oxidase subunit I (COI) gene and 13 nuclear polymorphic microsatellite loci. Our results reveal contrasting patterns of spread in the coastal waters of North America. While the West coast was colonized by noncontiguous (long‐distance) dispersal, the East coast invasion appears to have occurred through contiguous (stepping‐stone) spread. Molecular data further indicate that although dispersal in colonial ascidians is predominantly achieved through sexually produced propagules, aquaculture practices such as high‐pressure washing can facilitate fragmentation and potentially exacerbate infestations and spread via asexual propagules. The results presented here suggest that caution should be used against the general assumption that all invasions, even within a single species, exhibit similar patterns of colonization, as highly contrasting dynamics may transpire in different invaded ranges.

Complex genetic patterns in closely related colonizing invasive species

Anthropogenic activities frequently result in both rapidly changing environments and translocation of species from their native ranges (i.e., biological invasions). Empirical studies suggest that many factors associated with these changes can lead to complex genetic patterns, particularly among invasive populations. However, genetic complexities and factors responsible for them remain uncharacterized in many cases. Here, we explore these issues in the vase tunicate Ciona intestinalis (Ascidiacea: Enterogona: Cionidae), a model species complex, of which spA and spB are rapidly spreading worldwide. We intensively sampled 26 sites (N = 873) from both coasts of North America, and performed phylogenetic and population genetics analyses based on one mitochondrial fragment (cytochrome c oxidase subunit 3–NADH dehydrogenase subunit I, COX3-ND1) and eight nuclear microsatellites. Our analyses revealed extremely complex genetic patterns in both species on both coasts. We detected a contrasting pattern based on the mitochondrial marker: two major genetic groups in C. intestinalis spA on the west coast versus no significant geographic structure in C. intestinalis spB on the east coast. For both species, geo-graphically distant populations often showed high microsatellite-based genetic affinities whereas neighboring ones often did not. In addition, mitochondrial and nuclear markers provided largely inconsistent genetic patterns. Multiple factors, including random genetic drift associated with demographic changes, rapid selection due to strong local adaptation, and varying propensity for human-mediated propagule dispersal could be responsible for the observed genetic complexities.

Rare biosphere exploration using high-throughput sequencing: research progress and perspectives

Identification of rare species and mapping their distributions is crucial for understanding natural species distributions and causes and consequences of accelerating species declines. However, detection of rare species in both terrestrial and especially aquatic communities typically dominated by numerous microscopic species (i.e. rare biosphere) represents a formidable technical challenge. Rapid advances in high-throughput sequencing (HTS) technologies have revolutionized biodiversity studies in the rare biosphere, and also stimulated associated debates. Here we summarize research progress, discuss debates and problems, and propose possible solutions and future studies to address these issues. In addition, we provide take-home messages for experimental design and data interpretation when utilizing HTS techniques for rare biosphere exploration in ecology and conservation biology.

Past and present: Phylogeography of the Bufo gargarizans species complex inferred from multi-loci allele sequence and frequency data

Using multi-locus DNA sequence and frequency data, we examined the phylogeographic patterns of the Asian toad Bufo gargarizans species complex. A total of 166 individuals from 56 sites were genotyped for one mitochondrial locus (516 base pairs, ND2 gene) and five nuclear intron loci (Sox9-2, Rho-3, CCNB2-3, UCH-2, DBI-2; 250-350 base pairs each). We conducted a phylogenetic analysis of the allele sequence data to construct gene trees for each individual locus using Bayesian inference, a multi-locus phylogeny based on all five nuclear loci using POFAD, and a phylogenetic network using the NeighborNet algorithm. Furthermore, we used population-genetic analysis of the allele frequency data to reveal ongoing processes, including an analysis of molecular variance (AMOVA), a Bayesian assignment analysis, and a non-equilibrium Bayesian method for estimating recent migration rate. Our phylogenetic analyses showed that the observed divergence in the B. gargarizans species complex likely dated back to 7-8 million years ago. Repeated range expansions during the inter-glacial periods of Pleistocene likely established the current distribution of genetic diversity, although historical vicariant events were still evidenced. Both the west and the southeast regions may have served as refugia during the glaciation, and the range expansion was in general from west to northeast. Additionally, we detected strong ongoing migration both from west to the other regions and from south to north, which likely represents a meta-population dynamic that has emerged over the past ∼10,000 years. The morphologically identified species Bufo tibetanus is not genetically diagnosable, and therefore should be considered a junior synonym of B. gargarizans.

Reproducibility of pyrosequencing data for biodiversity assessment in complex communities

High-throughput sequencing is rapidly becoming a popular method to profile complex communities and has generated deep insights into community biodiversity. However, the reproducibility of this method for biodiversity assessment remains largely unexplored. Here we evaluated reproducibility by analysing 454 pyrosequenced biological replicates of two complex plankton communities collected from one freshwater port and one marine port. We also tested whether reproducibility potentially influences biodiversity estimates, notably - and -diversity. Our evaluation of reproducibility revealed a complex scenario, having both technical and biological significance. At the Operational Taxonomic Unit (OTU) level, reproducibility was 100% for high-abundance OTUs (>100 sequences), although it was lower for low-abundance OTUs, and sometimes 88% of irreproducible OTUs had high sequence similarity to existing records, suggesting that some singletons may reflect rare lineages/genotypes in communities. However, spurious amplification of distantly related taxonomic groups generated mainly low-abundance OTUs that were characterized by low reproducibility. At a broad taxonomic level (i.e. order level), reproducibility decreased as the abundance of OTUs decreased and was particularly low for distantly related taxonomic groups such as algae and protists that were not the targets of our zooplankton biodiversity survey. At a lower taxonomical level (i.e. family-level), overall reproducibility was high (>80%) for crustaceans, the dominant group in zooplankton samples. Therefore, we suggest that random variation during both sample collection and sequencing processes can be responsible for low reproducibility. Our analyses also suggest that random sampling processes may influence both - and -diversity estimates. Our results add to growing evidence that caution needs to be applied when designing and interpreting experiments utilizing high-throughput sequencing data for biodiversity assessments. Technical replicates are needed to statistically correct intra-sample variation, while field-based replicate samples are desirable to substantiate results. An overestimation of species diversity can occur when OTUs are uniquely characterized by spuriously amplified sequences and errors/artifacts. Therefore, careful management of low-abundance OTUs is required to reveal unique/rare lineages. Our results suggest that further studies are needed to determine the ecological significance of low-abundance OTUs in complex communities.