John Bunge

Estimating the Number of Species: A Review

How many kinds are there? Suppose that a population is partitioned into C classes. In many situations interest focuses not on estimation of the relative sizes of the classes, but on estimation of C itself. For example, biologists and ecologists may be interested in estimating the number of species in a population of plants or animals, numismatists may be concemed with estimating the number of dies used to produce an ancient coin issue, and linguists may be interested in estimating the size of an authors vocabulary. In this article we review the problem of statistical estimation of C. Many approaches have been proposed, some purely data-analytic and others based in sampling theory. In the latter case numerous variations have been considered. The population may be finite or infinite. If finite, samples may be taken with replacement (multinomial sampling) or without replacement (hypergeometric sampling), or by Bernoulli sampling; if infinite, sampling may be multinomial or Bernoulli, or the sample may be th...

Polymerase chain reaction primers miss half of rRNA microbial diversity.

The rRNA approach is the principal tool to study microbial diversity, but it has important biases. These include polymerase chain reaction (PCR) primers bias, and relative inefficiency of DNA extraction techniques. Such sources of potential undersampling of microbial diversity are well known, but the scale of the undersampling has not been quantified. Using a marine tidal flat bacterial community as a model, we show that even with unlimited sampling and sequencing effort, a single combination of PCR primers/DNA extraction technique enables theoretical recovery of only half of the richness recoverable with three such combinations. This shows that different combinations of PCR primers/DNA extraction techniques recover in principle different species, as well as higher taxa. The majority of earlier estimates of microbial richness seem to be underestimates. The combined use of multiple PCR primer sets, multiple DNA extraction techniques, and deep community sequencing will minimize the biases and recover substantially more species than prior studies, but we caution that even this—yet to be used—approach may still leave an unknown number of species and higher taxa undetected.

Protistan microbial observatory in the Cariaco Basin, Caribbean. I. Pyrosequencing vs Sanger insights into species richness

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80 000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36 000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100 000–500 000 species), suggesting a degree of protistan endemism.

Microeukaryote Community Patterns along an O2/H2S Gradient in a Supersulfidic Anoxic Fjord (Framvaren, Norway)

ABSTRACT To resolve the fine-scale architecture of anoxic protistan communities, we conducted a cultivation-independent 18S rRNA survey in the superanoxic Framvaren Fjord in Norway. We generated three clone libraries along the steep O2/H2S gradient, using the multiple-primer approach. Of 1,100 clones analyzed, 753 proved to be high-quality protistan target sequences. These sequences were grouped into 92 phylotypes, which displayed high protistan diversity in the fjord (17 major eukaryotic phyla). Only a few were closely related to known taxa. Several sequences were dissimilar to all previously described sequences and occupied a basal position in the inferred phylogenies, suggesting that the sequences recovered were derived from novel, deeply divergent eukaryotes. We detected sequence clades with evolutionary importance (for example, clades in the euglenozoa) and clades that seem to be specifically adapted to anoxic environments, challenging the hypothesis that the global dispersal of protists is uniform. Moreover, with the detection of clones affiliated with jakobid flagellates, we present evidence that primitive descendants of early eukaryotes are present in this anoxic environment. To estimate sample coverage and phylotype richness, we used parametric and nonparametric statistical methods. The results show that although our data set is one of the largest published inventories, our sample missed a substantial proportion of the protistan diversity. Nevertheless, statistical and phylogenetic analyses of the three libraries revealed the fine-scale architecture of anoxic protistan communities, which may exhibit adaptation to different environmental conditions along the O2/H2S gradient.

Bacterial diversity in a glacier foreland of the high Arctic

Over the past 100 years, Arctic temperatures have increased at almost twice the global average rate. One consequence is the acceleration of glacier retreat, exposing new habitats that are colonized by microorganisms whose diversity and function are unknown. Here, we characterized bacterial diversity along two approximately parallel chronosequences in an Arctic glacier forefield that span six time points following glacier retreat. We assessed changes in phylotype richness, evenness and turnover rate through the analysis of 16S rRNA gene sequences recovered from 52 samples taken from surface layers along the chronosequences. An average of 4500 sequences was obtained from each sample by 454 pyrosequencing. Using parametric methods, it was estimated that bacterial phylotype richness was high, and that it increased significantly from an average of 4000 (at a threshold of 97% sequence similarity) at locations exposed for 5 years to an average of 7050 phylotypes per 0.5 g of soil at sites that had been exposed for 150 years. Phylotype evenness also increased over time, with an evenness of 0.74 for 150 years since glacier retreat reflecting large proportions of rare phylotypes. The bacterial species turnover rate was especially high between sites exposed for 5 and 19 years. The level of bacterial diversity present in this High Arctic glacier foreland was comparable with that found in temperate and tropical soils, raising the question whether global patterns of bacterial species diversity parallel that of plants and animals, which have been found to form a latitudinal gradient and be lower in polar regions compared with the tropics.

Protistan microbial observatory in the Cariaco Basin, Caribbean. II. Habitat specialization

This is the second paper in a series of three that investigates eukaryotic microbial diversity and taxon distribution in the Cariaco Basin, Venezuela, the oceans largest anoxic marine basin. Here, we use phylogenetic information, multivariate community analyses and statistical richness predictions to test whether protists exhibit habitat specialization within defined geochemical layers of the water column. We also analyze spatio-temporal distributions of protists across two seasons and two geographic sites within the basin. Non-metric multidimensional scaling indicates that these two basin sites are inhabited by distinct protistan assemblages, an observation that is supported by the minimal overlap in observed and predicted richness of sampled sites. A comparison of parametric richness estimations indicates that protistan communities in closely spaced—but geochemically different—habitats are very dissimilar, and may share as few as 5% of total operational taxonomic units (OTUs). This is supported by a canonical correspondence analysis, indicating that the empirically observed OTUs are organized along opposing gradients in oxidants and reductants. Our phylogenetic analyses identify many new clades at species to class levels, some of which appear restricted to specific layers of the water column and have a significantly nonrandom distribution. These findings suggest many pelagic protists are restricted to specific habitats, and likely diversify, at least in part due to separation by geochemical barriers.

Comparing the Hyper‐Variable V4 and V9 Regions of the Small Subunit rDNA for Assessment of Ciliate Environmental Diversity

The hyper‐variable V4 and V9 regions of the small subunit (SSU) rDNA have been targeted for assessing environmental diversity of microbial eukaryotes using next generation sequencing technologies. Here, we explore how the genetic distances among these short fragments compare with the distances obtained from near full‐length SSU‐rDNA sequences by comparing all pairwise estimates, as well as within and among species of ciliates. Results show that pairwise distances from V4 more closely match the near full‐length SSU‐rDNA and are more comparable with previous studies based on much longer SSU‐rDNA fragments, then pairwise distances from V9. Thus, studies that use the V4 will estimate similar values of phylotype richness and community structure as would have been estimated using the full‐length SSU‐rDNA.

Spatio-temporal variations in protistan communities along an O2/H2S gradient in the anoxic Framvaren Fjord (Norway)

Despite its relevance for ecology and biodiversity, the stability of spatial microeukaryote diversity patterns in time has received only little attention using gene-based strategies, and there is little knowledge about the relation of spatial vs. temporal variation. We addressed this subject by investigating seasonal fluctuations in protistan communities in three ecologically distinct marine habitats. We analyzed 3360 eukaryote small subunit rRNA gene sequences collected along an O(2)/H(2)S gradient in a Norwegian fjord in order to reveal shifts in protistan community composition and structure in three different seasons. In all nine clone libraries, ciliates and stramenopiles accounted for the largest proportion. Yet, as expected, at the phylotype level, the protistan communities from distinct habitats differed significantly, with the number of shared phylotypes between two habitats being as low as 18%. This confirmed previous notions that environmental factors along the stratification gradient shape biodiversity patterns. Surprisingly, the intrahabitat community composition and structure varied at a comparable order of magnitude over time, with only 18-28% phylotypes shared within the same habitat. Our study demonstrates that the consideration of local fluctuations in microeukaryote diversity over time offers additional information for diversity surveys and can significantly contribute to the revelation of spatial protistan community patterns.

Environmental rRNA inventories miss over half of protistan diversity

BackgroundThe main tool to discover novel microbial eukaryotes is the rRNA approach. This approach has important biases, including PCR discrimination against certain rRNA gene species, which makes molecular inventories skewed relative to the source communities. The degree of this bias has not been quantified, and it remains unclear whether species missed from clone libraries could be recovered by increasing sequencing efforts, or whether they cannot be detected in principle. Here we attempt to discriminate between these possibilities by statistically analysing four protistan inventories obtained using different general eukaryotic PCR primers.ResultsWe show that each PCR primer set-specific clone library is not a sample from the community diversity but rather from a fraction of this diversity. Therefore, even sequencing such clone libraries to saturation would only recover that fraction, which, according to the parametric models, varies between 17 ± 4% to 49 ± 10%, depending on the set of primers. The pooled data is thus qualitatively richer than individual libraries, even if normalized to the same sequencing effort.ConclusionThe use of a single pair of primers leads to significant underestimation of the true community richness at all levels of taxonomic hierarchy. The majority of available protistan rRNA gene surveys likely sampled less than half of the target diversity, and might have completely missed the rest. The use of multiple PCR primers reduces this bias but does not necessarily eliminate it.

Employee participation in pollution reduction: Preliminary analysis of the Toxics Release Inventory

Abstract Can the amount of toxic waste released into the environment by manufacturing facilities be reduced by formally involving employees in pollution prevention? Pursuant to the Pollution Prevention Act of 1990, the U.S. Environmental Protection Agency began requiring that manufacturers report human resource management strategies related to “source reduction” (reduction of waste at the source), as part of the Toxics Release Inventory (TRI). These strategies often involve employee participation in some form. Here we report the preliminary findings of an ongoing study of the effect of employee participation on source reduction, based on the 1991–1992 TRI database. We find that manufacturers using a certain combination of three formal employee participation practices had triple the reduction in emissions of manufacturers using none of these practices. Manufacturers that combined these practices with external pollution prevention assistance showed comparable or better results. Finally we discuss competing predictors of source reduction, and future research directions.