Whole-Genome Sequencing of Individuals from a Founder Population Identifies Candidate Genes for Asthma

Asthma is a complex genetic disease caused by a combination of genetic and environmental risk factors. We sought to test classes of genetic variants largely missed by genome-wide association studies (GWAS), including copy number variants (CNVs) and low-frequency variants, by performing whole-genome sequencing (WGS) on 16 individuals from asthma-enriched and asthma-depleted families. The samples were obtained from an extended 13-generation Hutterite pedigree with reduced genetic heterogeneity due to a small founding gene pool and reduced environmental heterogeneity as a result of a communal lifestyle. We sequenced each individual to an average depth of 13-fold, generated a comprehensive catalog of genetic variants, and tested the most severe mutations for association with asthma. We identified and validated 1960 CNVs, 19 nonsense or splice-site single nucleotide variants (SNVs), and 18 insertions or deletions that were out of frame. As follow-up, we performed targeted sequencing of 16 genes in 837 cases and 540 controls of Puerto Rican ancestry and found that controls carry a significantly higher burden of mutations in IL27RA (2.0% of controls; 0.23% of cases; nominal p = 0.004; Bonferroni p = 0.21). We also genotyped 593 CNVs in 1199 Hutterite individuals. We identified a nominally significant association (p = 0.03; Odds ratio (OR) = 3.13) between a 6 kbp deletion in an intron of NEDD4L and increased risk of asthma. We genotyped this deletion in an additional 4787 non-Hutterite individuals (nominal p = 0.056; OR = 1.69). NEDD4L is expressed in bronchial epithelial cells, and conditional knockout of this gene in the lung in mice leads to severe inflammation and mucus accumulation. Our study represents one of the early instances of applying WGS to complex disease with a large environmental component and demonstrates how WGS can identify risk variants, including CNVs and low-frequency variants, largely untested in GWAS.     

One Bacterial Cell,One Complete Genome

While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200–900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.     

Estimates of Genetic Differentiation Measured by F(ST) Do Not Necessarily Require Large Sample Sizes When Using Many SNP Markers

Population genetic studies provide insights into the evolutionary processes that influence the distribution of sequence variants within and among wild populations. F_ST is among the most widely used measures for genetic differentiation and plays a central role in ecological and evolutionary genetic studies. It is commonly thought that large sample sizes are required in order to precisely infer F_ST and that small sample sizes lead to overestimation of genetic differentiation. Until recently, studies in ecological model organisms incorporated a limited number of genetic markers, but since the emergence of next generation sequencing, the panel size of genetic markers available even in non-reference organisms has rapidly increased. In this study we examine whether a large number of genetic markers can substitute for small sample sizes when estimating F_ST. We tested the behavior of three different estimators that infer F_ST and that are commonly used in population genetic studies. By simulating populations, we assessed the effects of sample size and the number of markers on the various estimates of genetic differentiation. Furthermore, we tested the effect of ascertainment bias on these estimates. We show that the population sample size can be significantly reduced (as small as n = 4–6) when using an appropriate estimator and a large number of bi-allelic genetic markers (k>1,000). Therefore, conservation genetic studies can now obtain almost the same statistical power as studies performed on model organisms using markers developed with next-generation sequencing.     

Contextualising the Last Survivors: Population Structure of Marine Turtles in the Dominican Republic

Nesting by three species of marine turtles persists in the Dominican Republic, despite historic threats and long-term population decline. We conducted a genetic survey of marine turtles in the Dominican Republic in order to link them with other rookeries around the Caribbean. We sequenced a 740_bp fragment of the control region of the mitochondrial DNA of 92 samples from three marine turtle species [hawksbill (n = 48), green (n = 2) and leatherback (n = 42)], and incorporated published data from other nesting populations and foraging grounds. The leatherback turtle (Dermochelys coriacea) in the Dominican Republic appeared to be isolated from Awala-Yalimapo, Cayenne, Trinidad and St. Croix but connected with other Caribbean populations. Two distinct nesting populations of hawksbill turtles (Eremochelys imbricata) were detected in the Dominican Republic and exhibited interesting patterns of connectivity with other nesting sites and juvenile and adult male foraging aggregations. The green sea turtle (Chelonia mydas) has almost been extirpated from the Dominican Republic and limited inference could be made from our samples. Finally, results were compared with Lagrangian drifting buoys and published Lagrangian virtual particles that travelled through the Dominican Republic and Caribbean waters. Conservation implications of sink-source effects or genetic isolation derived from these complex inter-connections are discussed for each species and population.     

Vertical and Horizontal Genetic Connectivity in Chromis verater,an Endemic Damselfish Found on Shallow and Mesophotic Reefs in the Hawaiian Archipelago and Adjacent Johnston Atoll

Understanding vertical and horizontal connectivity is a major priority in research on mesophotic coral ecosystems (30–150 m). However, horizontal connectivity has been the focus of few studies, and data on vertical connectivity are limited to sessile benthic mesophotic organisms. Here we present patterns of vertical and horizontal connectivity in the Hawaiian Islands-Johnston Atoll endemic threespot damselfish, Chromis verater, based on 319 shallow specimens and 153 deep specimens. The mtDNA markers cytochrome b and control region were sequenced to analyze genetic structure: 1) between shallow (<30 m) and mesophotic (30–150 m) populations and 2) across the species'' geographic range. Additionally, the nuclear markers rhodopsin and internal transcribed spacer 2 of ribosomal DNA were sequenced to assess connectivity between shallow and mesophotic populations. There was no significant genetic differentiation by depth, indicating high levels of vertical connectivity between shallow and deep aggregates of C. verater. Consequently, shallow and deep samples were combined by location for analyses of horizontal connectivity. We detected low but significant population structure across the Hawaiian Archipelago (overall cytochrome b: Φ_ST = 0.009, P = 0.020; control region: Φ_ST = 0.012, P = 0.009) and a larger break between the archipelago and Johnston Atoll (cytochrome b: Φ_ST = 0.068, P<0.001; control region: Φ_ST = 0.116, P<0.001). The population structure within the archipelago was driven by samples from the island of Hawaii at the southeast end of the chain and Lisianski in the middle of the archipelago. The lack of vertical genetic structure supports the refugia hypothesis that deep reefs may constitute a population reservoir for species depleted in shallow reef habitats. These findings represent the first connectivity study on a mobile organism that spans shallow and mesophotic depths and provide a reference point for future connectivity studies on mesophotic fishes.     

Meta-Analysis Indicates That the European GWAS-Identified Risk SNP rs1344706 within ZNF804A Is Not Associated with Schizophrenia in Han Chinese Population

Recent genetic association studies have implicated several candidate susceptibility variants for schizophrenia among general populations. Rs1344706, an intronic SNP within ZNF804A, was identified as one of the most compelling candidate risk SNPs for schizophrenia in Europeans through genome-wide association studies (GWASs) and replications as well as large-scale meta-analyses. However, in Han Chinese, the results for rs1344706 are inconsistent, and whether rs1344706 is an authentic risk SNP for schizophrenia in Han Chinese is inconclusive. Here, we conducted a systematic meta-analysis of rs1344706 with schizophrenia in Chinese population by combining all available case-control samples (N = 12), including a total of 8,982 cases and 12,342 controls. The results of our meta-analysis were not able to confirm an association of rs1344706 A-allele with schizophrenia (p = 0.10, odds ratio = 1.06, 95% confidence interval = 0.99–1.13). Such absence of association was further confirmed by the non-superiority test (p = 0.0003), suggesting that rs1344706 is not a risk SNP for schizophrenia in Han Chinese. Detailed examinations of individual samples revealed potential sampling bias in previous replication studies in Han Chinese. The absence of rs1344706 association in Han Chinese suggest a potential genetic heterogeneity in the susceptibility of schizophrenia on this locus and also demonstrate the difficulties in replicating genome-wide association findings of schizophrenia across different ethnic populations.     

Individual Identification and Genetic Variation of Lions (Panthera leo) from Two Protected Areas in Nigeria

This survey was conducted in two protected areas in Nigeria to genetically identify individual lions and to determine the genetic variation within and between the populations. We used faecal sample DNA, a non-invasive alternative to the risky and laborious task of taking samples directly from the animals, often preceded by catching and immobilization. Data collection in Yankari Game Reserve (YGR) spanned through a period of five years (2008 –2012), whereas data in Kainji Lake National Park (KLNP) was gathered for a period of three years (2009, 2010 and 2012). We identified a minimum of eight individuals (2 males, 3 females, 3 unknown) from YGR and a minimum of ten individuals (7 males, 3 females) from KLNP. The two populations were found to be genetically distinct as shown by the relatively high fixation index (F_ST  = 0.17) with each population exhibiting signs of inbreeding (YGR F_IS  = 0.49, KLNP F_IS  = 0.38). The genetic differentiation between the Yankari and Kainji lions is assumed to result from large spatial geographic distance and physical barriers reducing gene flow between these two remaining wild lion populations in Nigeria. To mitigate the probable inbreeding depression in the lion populations within Nigeria it might be important to transfer lions between parks or reserves or to reintroduce lions from the zoos back to the wild.     

Metagenomic Sequencing of an In Vitro-Simulated Microbial Community

Background

Microbial life dominates the earth, but many species are difficult or even impossible to study under laboratory conditions. Sequencing DNA directly from the environment, a technique commonly referred to as metagenomics, is an important tool for cataloging microbial life. This culture-independent approach involves collecting samples that include microbes in them, extracting DNA from the samples, and sequencing the DNA. A sample may contain many different microorganisms, macroorganisms, and even free-floating environmental DNA. A fundamental challenge in metagenomics has been estimating the abundance of organisms in a sample based on the frequency with which the organism''s DNA was observed in reads generated via DNA sequencing.

Methodology/Principal Findings

We created mixtures of ten microbial species for which genome sequences are known. Each mixture contained an equal number of cells of each species. We then extracted DNA from the mixtures, sequenced the DNA, and measured the frequency with which genomic regions from each organism was observed in the sequenced DNA. We found that the observed frequency of reads mapping to each organism did not reflect the equal numbers of cells that were known to be included in each mixture. The relative organism abundances varied significantly depending on the DNA extraction and sequencing protocol utilized.

Conclusions/Significance

We describe a new data resource for measuring the accuracy of metagenomic binning methods, created by in vitro-simulation of a metagenomic community. Our in vitro simulation can be used to complement previous in silico benchmark studies. In constructing a synthetic community and sequencing its metagenome, we encountered several sources of observation bias that likely affect most metagenomic experiments to date and present challenges for comparative metagenomic studies. DNA preparation methods have a particularly profound effect in our study, implying that samples prepared with different protocols are not suitable for comparative metagenomics.     

Population Genetics and the Effects of a Severe Bottleneck in an Ex Situ Population of Critically Endangered Hawaiian Tree Snails

As wild populations decline, ex situ propagation provides a potential bank of genetic diversity and a hedge against extinction. These programs are unlikely to succeed if captive populations do not recover from the severe bottleneck imposed when they are founded with a limited number of individuals from remnant populations. In small captive populations allelic richness may be lost due to genetic drift, leading to a decline in fitness. Wild populations of the Hawaiian tree snail Achatinella lila, a hermaphroditic snail with a long life history, have declined precipitously due to introduced predators and other human impacts. A captive population initially thrived after its founding with seven snails, exceeding 600 captive individuals in 2009, but drastically declined in the last five years. Measures of fitness were examined from 2,018 captive snails that died between 1998 and 2012, and compared with genotypic data for six microsatellite loci from a subset of these deceased snails (N = 335), as well as live captive snails (N = 198) and wild snails (N = 92). Surprisingly, the inbreeding coefficient (F_is) declined over time in the captive population, and is now approaching values observed in the 2013 wild population, despite a significant decrease in allelic richness. However, adult annual survival and fecundity significantly declined in the second generation. These measures of fitness were positively correlated with heterozygosity. Snails with higher measures of heterozygosity had more offspring, and third generation offspring with higher measures of heterozygosity were more likely to reach maturity. These results highlight the importance of maintaining genetic diversity in captive populations, particularly those initiated with a small number of individuals from wild remnant populations. Genetic rescue may allow for an increase in genetic diversity in the captive population, as measures of heterozygosity and rarified allelic richness were higher in wild tree snails.     

Endometriosis Is Associated with Rare Copy Number Variants

Endometriosis is a complex gynecological condition that affects 6–10% of women in their reproductive years and is defined by the presence of endometrial glands and stroma outside the uterus. Twin, family, and genome-wide association (GWA) studies have confirmed a genetic role, yet only a small part of the genetic risk can be explained by SNP variation. Copy number variants (CNVs) account for a greater portion of human genetic variation than SNPs and include more recent mutations of large effect. CNVs, likely to be prominent in conditions with decreased reproductive fitness, have not previously been examined as a genetic contributor to endometriosis. Here we employ a high-density genotyping microarray in a genome-wide survey of CNVs in a case-control population that includes 2,126 surgically confirmed endometriosis cases and 17,974 population controls of European ancestry. We apply stringent quality filters to reduce the false positive rate common to many CNV-detection algorithms from 77.7% to 7.3% without noticeable reduction in the true positive rate. We detected no differences in the CNV landscape between cases and controls on the global level which showed an average of 1.92 CNVs per individual with an average size of 142.3 kb. On the local level we identify 22 CNV-regions at the nominal significance threshold (P<0.05), which is greater than the 8.15 CNV-regions expected based on permutation analysis (P<0.001). Three CNV''s passed a genome-wide P-value threshold of 9.3×10⁻⁴; a deletion at SGCZ on 8p22 (P = 7.3×10⁻⁴, OR = 8.5, Cl = 2.3–31.7), a deletion in MALRD1 on 10p12.31 (P = 5.6×10⁻⁴, OR = 14.1, Cl = 2.7–90.9), and a deletion at 11q14.1 (P = 5.7×10⁻⁴, OR = 33.8, Cl = 3.3–1651). Two SNPs within the 22 CNVRs show significant genotypic association with endometriosis after adjusting for multiple testing; rs758316 in DPP6 on 7q36.2 (P = 0.0045) and rs4837864 in ASTN2 on 9q33.1 (P = 0.0002). Together, the CNV-loci are detected in 6.9% of affected women compared to 2.1% in the general population.     

Associations of Two Common Genetic Variants with Breast Cancer Risk in a Chinese Population: A Stratified Interaction Analysis

Recent genome-wide association studies (GWAS) have identified a series of new genetic susceptibility loci for breast cancer (BC). However, the correlations between these variants and breast cancer are still not clear. In order to explore the role of breast cancer susceptibility variants in a Southeast Chinese population, we genotyped two common SNPs at chromosome 6q25 (rs2046210) and in TOX3 (rs4784227) in a case-control study with a total of 702 breast cancer cases and 794 healthy-controls. In addition, we also evaluated the multiple interactions among genetic variants, risk factors, and tumor subtypes. Associations of genotypes with breast cancer risk was evaluated using multivariate logistic regression to estimate odds ratios (OR) and their 95% confidence intervals (95% CI). The results indicated that both polymorphisms were significantly associated with the risk of breast cancer, with per allele OR = 1.35, (95%CI = 1.17–1.57) for rs2046210 and per allele OR = 1.24 (95%CI = 1.06–1.45) for rs4784227. Furthermore, in subgroup stratified analyses, we observed that the T allele of rs4784227 was significantly associated with elevated OR among postmenopausal populations (OR = 1.44, 95%CI 1.11–1.87) but not in premenopausal populations, with the heterogeneity P value of P = 0.064. These findings suggest that the genetic variants at chromosome 6q25 and in the TOX3 gene may play important roles in breast cancer development in a Chinese population and the underlying biological mechanisms need to be further elucidated.     

Joint analysis of multiple metagenomic samples

The availability of metagenomic sequencing data, generated by sequencing DNA pooled from multiple microbes living jointly, has increased sharply in the last few years with developments in sequencing technology. Characterizing the contents of metagenomic samples is a challenging task, which has been extensively attempted by both supervised and unsupervised techniques, each with its own limitations. Common to practically all the methods is the processing of single samples only; when multiple samples are sequenced, each is analyzed separately and the results are combined. In this paper we propose to perform a combined analysis of a set of samples in order to obtain a better characterization of each of the samples, and provide two applications of this principle. First, we use an unsupervised probabilistic mixture model to infer hidden components shared across metagenomic samples. We incorporate the model in a novel framework for studying association of microbial sequence elements with phenotypes, analogous to the genome-wide association studies performed on human genomes: We demonstrate that stratification may result in false discoveries of such associations, and that the components inferred by the model can be used to correct for this stratification. Second, we propose a novel read clustering (also termed "binning") algorithm which operates on multiple samples simultaneously, leveraging on the assumption that the different samples contain the same microbial species, possibly in different proportions. We show that integrating information across multiple samples yields more precise binning on each of the samples. Moreover, for both applications we demonstrate that given a fixed depth of coverage, the average per-sample performance generally increases with the number of sequenced samples as long as the per-sample coverage is high enough.     

Using High-Throughput Sequencing to Leverage Surveillance of Genetic Diversity and Oseltamivir Resistance: A Pilot Study during the 2009 Influenza A(H1N1) Pandemic

Background

Influenza viruses display a high mutation rate and complex evolutionary patterns. Next-generation sequencing (NGS) has been widely used for qualitative and semi-quantitative assessment of genetic diversity in complex biological samples. The “deep sequencing” approach, enabled by the enormous throughput of current NGS platforms, allows the identification of rare genetic viral variants in targeted genetic regions, but is usually limited to a small number of samples.

Methodology and Principal Findings

We designed a proof-of-principle study to test whether redistributing sequencing throughput from a high depth-small sample number towards a low depth-large sample number approach is feasible and contributes to influenza epidemiological surveillance. Using 454-Roche sequencing, we sequenced at a rather low depth, a 307 bp amplicon of the neuraminidase gene of the Influenza A(H1N1) pandemic (A(H1N1)pdm) virus from cDNA amplicons pooled in 48 barcoded libraries obtained from nasal swab samples of infected patients (n  =  299) taken from May to November, 2009 pandemic period in Mexico. This approach revealed that during the transition from the first (May-July) to second wave (September-November) of the pandemic, the initial genetic variants were replaced by the N248D mutation in the NA gene, and enabled the establishment of temporal and geographic associations with genetic diversity and the identification of mutations associated with oseltamivir resistance.

Conclusions

NGS sequencing of a short amplicon from the NA gene at low sequencing depth allowed genetic screening of a large number of samples, providing insights to viral genetic diversity dynamics and the identification of genetic variants associated with oseltamivir resistance. Further research is needed to explain the observed replacement of the genetic variants seen during the second wave. As sequencing throughput rises and library multiplexing and automation improves, we foresee that the approach presented here can be scaled up for global genetic surveillance of influenza and other infectious diseases.     

Genetic Diversity and Population Structure of Sitodiplosis mosellana in Northern China

The wheat midge, Sitodiplosis mosellana, is an important pest in Northern China. We tested the hypothesis that the population structure of this species arises during a range expansion over the past 30 years. This study used microsatellite and mitochondrial loci to conduct population genetic analysis of S. mosellana across its distribution range in China. We found strong genetic structure among the 16 studied populations, including two genetically distinct groups (the eastern and western groups), broadly consistent with the geography and habitat fragmentation. These results underline the importance of natural barriers in impeding dispersal and gene flow of S. mosellana populations. Low to moderate genetic diversity among the populations and moderate genetic differentiation (F _ST = 0.117) between the two groups were also found. The populations in the western group had lower genetic diversity, higher genetic differentiation and lower gene flow (F _ST = 0.116, Nm = 1.89) than those in the eastern group (F _ST = 0.049, Nm = 4.91). Genetic distance between populations was positively and significantly correlated with geographic distance (r = 0.56, P<0.001). The population history of this species provided no evidence for population expansion or bottlenecks in any of these populations. Our data suggest that the distribution of genetic diversity, genetic differentiation and population structure of S. mosellana have resulted from a historical event, reflecting its adaptation to diverse habitats and forming two different gene pools. These results may be the outcome of a combination of restricted gene flow due to geographical and environmental factors, population history, random processes of genetic drift and individual dispersal patterns. Given the current risk status of this species in China, this study can offer useful information for forecasting outbreaks and designing effective pest management programs.     

Parallel Tagged Next-Generation Sequencing on Pooled Samples – A New Approach for Population Genetics in Ecology and Conservation

Next-generation sequencing (NGS) on pooled samples has already been broadly applied in human medical diagnostics and plant and animal breeding. However, thus far it has been only sparingly employed in ecology and conservation, where it may serve as a useful diagnostic tool for rapid assessment of species genetic diversity and structure at the population level. Here we undertake a comprehensive evaluation of the accuracy, practicality and limitations of parallel tagged amplicon NGS on pooled population samples for estimating species population diversity and structure. We obtained 16S and Cyt b data from 20 populations of Leiopelma hochstetteri, a frog species of conservation concern in New Zealand, using two approaches – parallel tagged NGS on pooled population samples and individual Sanger sequenced samples. Data from each approach were then used to estimate two standard population genetic parameters, nucleotide diversity (π) and population differentiation (F_ST), that enable population genetic inference in a species conservation context. We found a positive correlation between our two approaches for population genetic estimates, showing that the pooled population NGS approach is a reliable, rapid and appropriate method for population genetic inference in an ecological and conservation context. Our experimental design also allowed us to identify both the strengths and weaknesses of the pooled population NGS approach and outline some guidelines and suggestions that might be considered when planning future projects.     

Distribution and Medical Impact of Loss-of-Function Variants in the Finnish Founder Population

Exome sequencing studies in complex diseases are challenged by the allelic heterogeneity, large number and modest effect sizes of associated variants on disease risk and the presence of large numbers of neutral variants, even in phenotypically relevant genes. Isolated populations with recent bottlenecks offer advantages for studying rare variants in complex diseases as they have deleterious variants that are present at higher frequencies as well as a substantial reduction in rare neutral variation. To explore the potential of the Finnish founder population for studying low-frequency (0.5–5%) variants in complex diseases, we compared exome sequence data on 3,000 Finns to the same number of non-Finnish Europeans and discovered that, despite having fewer variable sites overall, the average Finn has more low-frequency loss-of-function variants and complete gene knockouts. We then used several well-characterized Finnish population cohorts to study the phenotypic effects of 83 enriched loss-of-function variants across 60 phenotypes in 36,262 Finns. Using a deep set of quantitative traits collected on these cohorts, we show 5 associations (p<5×10⁻⁸) including splice variants in LPA that lowered plasma lipoprotein(a) levels (P = 1.5×10⁻¹¹⁷). Through accessing the national medical records of these participants, we evaluate the LPA finding via Mendelian randomization and confirm that these splice variants confer protection from cardiovascular disease (OR = 0.84, P = 3×10⁻⁴), demonstrating for the first time the correlation between very low levels of LPA in humans with potential therapeutic implications for cardiovascular diseases. More generally, this study articulates substantial advantages for studying the role of rare variation in complex phenotypes in founder populations like the Finns and by combining a unique population genetic history with data from large population cohorts and centralized research access to National Health Registers.     

Robust estimation of microbial diversity in theory and in practice

Quantifying diversity is of central importance for the study of structure, function and evolution of microbial communities. The estimation of microbial diversity has received renewed attention with the advent of large-scale metagenomic studies. Here, we consider what the diversity observed in a sample tells us about the diversity of the community being sampled. First, we argue that one cannot reliably estimate the absolute and relative number of microbial species present in a community without making unsupported assumptions about species abundance distributions. The reason for this is that sample data do not contain information about the number of rare species in the tail of species abundance distributions. We illustrate the difficulty in comparing species richness estimates by applying Chao''s estimator of species richness to a set of in silico communities: they are ranked incorrectly in the presence of large numbers of rare species. Next, we extend our analysis to a general family of diversity metrics (‘Hill diversities''), and construct lower and upper estimates of diversity values consistent with the sample data. The theory generalizes Chao''s estimator, which we retrieve as the lower estimate of species richness. We show that Shannon and Simpson diversity can be robustly estimated for the in silico communities. We analyze nine metagenomic data sets from a wide range of environments, and show that our findings are relevant for empirically-sampled communities. Hence, we recommend the use of Shannon and Simpson diversity rather than species richness in efforts to quantify and compare microbial diversity.     

Genetic Structure,Diversity and Long Term Viability of a Medicinal Plant,Nothapodytes nimmoniana Graham. (Icacinaceae), in Protected and Non-Protected Areas in the Western Ghats Biodiversity Hotspot

Background and Question

The harvesting of medicinal plants from wild sources is escalating in many parts of the world, compromising the long-term survival of natural populations of medicinally important plants and sustainability of sources of raw material to meet pharmaceutical industry needs. Although protected areas are considered to play a central role in conservation of plant genetic resources, the effectiveness of protected areas for maintaining medicinal plant populations subject to intense harvesting pressure remain largely unknown. We conducted genetic and demographic studies of Nothapodytes nimmoniana Graham, one of the extensively harvested medicinal plant species in the Western Ghats biodiversity hotspot, India to assess the effectiveness of protected areas in long-term maintenance of economically important plant species.

Methodology/Principal Findings

The analysis of adults and seedlings of N. nimmoniana in four protected and four non-protected areas using 7 nuclear microsatellite loci revealed that populations that are distributed within protected areas are subject to lower levels of harvesting and maintain higher genetic diversity (H_e = 0.816, H_o = 0.607, A = 18.857) than populations in adjoining non-protected areas (H_e = 0.781, H_o = 0.511, A = 15.571). Furthermore, seedlings in protected areas had significantly higher observed heterozygosity (H_o = 0.630) and private alleles as compared to seedlings in adjoining non-protected areas (H_o = 0.426). Most populations revealed signatures of recent genetic bottleneck. The prediction of long-term maintenance of genetic diversity using BOTTLESIM indicated that current population sizes of the species are not sufficient to maintain 90% of present genetic diversity for next 100 years.

Conclusions/Significance

Overall, these results highlight the need for establishing more protected areas encompassing a large number of adult plants in the Western Ghats to conserve genetic diversity of economically and medicinally important plant species.     

Invasion Pathway of the Ctenophore Mnemiopsis leidyi in the Mediterranean Sea

Gelatinous zooplankton outbreaks have increased globally owing to a number of human-mediated factors, including food web alterations and species introductions. The invasive ctenophore Mnemiopsis leidyi entered the Black Sea in the early 1980s. The invasion was followed by the Azov, Caspian, Baltic and North Seas, and, most recently, the Mediterranean Sea. Previous studies identified two distinct invasion pathways of M. leidyi from its native range in the western Atlantic Ocean to Eurasia. However, the source of newly established populations in the Mediterranean Sea remains unclear. Here we build upon our previous study and investigate sequence variation in both mitochondrial (Cytochrome c Oxidase subunit I) and nuclear (Internal Transcribed Spacer) markers in M. leidyi, encompassing five native and 11 introduced populations, including four from the Mediterranean Sea. Extant genetic diversity in Mediterranean populations (n = 8, N _a = 10) preclude the occurrence of a severe genetic bottleneck or founder effects in the initial colonizing population. Our mitochondrial and nuclear marker surveys revealed two possible pathways of introduction into Mediterranean Sea. In total, 17 haplotypes and 18 alleles were recovered from all surveyed populations. Haplotype and allelic diversity of Mediterranean populations were comparable to populations from which they were likely drawn. The distribution of genetic diversity and pattern of genetic differentiation suggest initial colonization of the Mediterranean from the Black-Azov Seas (pairwise F _ST = 0.001–0.028). However, some haplotypes and alleles from the Mediterranean Sea were not detected from the well-sampled Black Sea, although they were found in Gulf of Mexico populations that were also genetically similar to those in the Mediterranean Sea (pairwise F _ST = 0.010–0.032), raising the possibility of multiple invasion sources. Multiple introductions from a combination of Black Sea and native region sources could be facilitated by intense local and transcontinental shipping activity, respectively.