mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the alpha and beta diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.

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Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities

The Ribosomal Database Project (RDP) Classifier, a naive Bayesian classifier, can rapidly and accurately classify bacterial 16S rRNA sequences into the new higher-order taxonomy proposed in Bergey's Taxonomic Outline of the Prokaryotes (2nd ed., release 5.0, Springer-Verlag, New York, NY, 2004). It provides taxonomic assignments from domain to genus, with confidence estimates for each assignment. The majority of classifications (98%) were of high estimated confidence (≥95%) and high accuracy (98%). In addition to being tested with the corpus of 5,014 type strain sequences from Bergey's outline, the RDP Classifier was tested with a corpus of 23,095 rRNA sequences as assigned by the NCBI into their alternative higher-order taxonomy. The results from leave-one-out testing on both corpora show that the overall accuracies at all levels of confidence for near-full-length and 400-base segments were 89% or above down to the genus level, and the majority of the classification errors appear to be due to anomalies in the current taxonomies. For shorter rRNA segments, such as those that might be generated by pyrosequencing, the error rate varied greatly over the length of the 16S rRNA gene, with segments around the V2 and V4 variable regions giving the lowest error rates. The RDP Classifier is suitable both for the analysis of single rRNA sequences and for the analysis of libraries of thousands of sequences. Another related tool, RDP Library Compare, was developed to facilitate microbial-community comparison based on 16S rRNA gene sequence libraries. It combines the RDP Classifier with a statistical test to flag taxa differentially represented between samples. The RDP Classifier and RDP Library Compare are available online at http://rdp.cme.msu.edu/.

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Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy

We introduce here a new method for computing differences between microbial communities based on phylogenetic information. This method, UniFrac, measures the phylogenetic distance between sets of taxa in a phylogenetic tree as the fraction of the branch length of the tree that leads to descendants from either one environment or the other, but not both. UniFrac can be used to determine whether communities are significantly different, to compare many communities simultaneously using clustering and ordination techniques, and to measure the relative contributions of different factors, such as chemistry and geography, to similarities between samples. We demonstrate the utility of UniFrac by applying it to published 16S rRNA gene libraries from cultured isolates and environmental clones of bacteria in marine sediment, water, and ice. Our results reveal that (i) cultured isolates from ice, water, and sediment resemble each other and environmental clone sequences from sea ice, but not environmental clone sequences from sediment and water; (ii) the geographical location does not correlate strongly with bacterial community differences in ice and sediment from the Arctic and Antarctic; and (iii) bacterial communities differ between terrestrially impacted seawater (whether polar or temperate) and warm oligotrophic seawater, whereas those in individual seawater samples are not more similar to each other than to those in sediment or ice samples. These results illustrate that UniFrac provides a new way of characterizing microbial communities, using the wealth of environmental rRNA sequences, and allows quantitative insight into the factors that underlie the distribution of lineages among environments.

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UniFrac: a New Phylogenetic Method for Comparing Microbial Communities

Statistics for Spatial Data.

Both the magnitude and the urgency of the task of assessing global biodiversity require that we make the most of what we know through the use of estimation and extrapolation. Likewise, future biodiversity inventories need to be designed around the use of effective sampling and estimation procedures, especially for 'hyperdiverse' groups of terrestrial organisms, such as arthropods, nematodes, fungi, and microorganisms. The challenge of estimating patterns of species richness from samples can be separated into (i) the problem of estimating local species richness, and (ii) the problem of estimating the distinctness, or complementarity, of species assemblages. These concepts apply on a wide range of spatial, temporal, and functional scales. Local richness can be estimated by extrapolating species accumulation curves, fitting parametric distributions of relative abundance, or using non-parametric techniques based on the distribution of individuals among species or of species among samples. We present several of these methods and examine their effectiveness for an example data set. We present a simple measure of complementarity, with some biogeographic examples, and outline the difficult problem of estimating complementarity from samples. Finally, we discuss the importance of using 'reference' sites (or sub-sites) to assess the true richness and composition of species assemblages, to measure ecologically significant ratios between unrelated taxa, to measure taxon/sub-taxon (hierarchical) ratios, and to 'calibrate' standardized sampling methods. This information can then be applied to the rapid, approximate assessment of species richness and faunal or floral composition at 'comparative' sites.

Estimating Terrestrial Biodiversity through Extrapolation

To determine the significance of differences between clonal libraries of environmental rRNA gene sequences, differences between homologous coverage curves, CX(D), and heterologous coverage curves, CXY(D), were calculated by a Cramer-von Mises-type statistic and compared by a Monte Carlo test procedure. This method successfully distinguished rRNA gene sequence libraries from soil and bioreactors and correctly failed to find differences between libraries of the same composition.

Quantitative Comparisons of 16S rRNA Gene Sequence Libraries from Environmental Samples

The population dynamics of a long-lived conifer

We investigated the demography and spatial pattern of an old-growth longleaf pine population using a large plot in which all trees of at least 2 cm in dbh were mapped and tagged for individual recognition. The population was of uneven age and size; tree size correlated positively with tree age. Large or old trees were only loosely aggregated, forming a background matrix that filled the forest. In contrast, juvenile trees were highly aggregated and were located in areas of low adult densities. Recruitment within this population thus appears to occur primarily within open spaces created by the deaths of large trees. Variable time lags may occur before the colonization of open spaces, however, because of temporal variation in seed production and the occurrence of summer ground fires. The absence of large gaps within the age-class distribution indicated that recruitment within the mapped plot has occurred frequently for at least the past 250 years. Neither age- nor size-class distributions, however, were stab...

The Population Dynamics of a Long-Lived Conifer (Pinus palustris)

--The common practice of expressing community structure in terms of indices of diversity and evenness involves a serious loss of information. Differences attributable to the accumulation of species with increasing area are ignored, differences in the density of individuals are often masked by other factors, and many combinations of species richness and relative abundance can produce the same value of the index. As an alternative we suggest (1) comparing species richness by standardizing samples either to equal numbers of individuals or to the number of individuals expected on equal-sized plots, and (2) expressing the relative abundance of species as a graph of their relative abundances arranged in a decreasing array. We present an analysis of bird census data based on the proposed methods, and we include comparisons with applications of four indices commonly used in ecology, the Shannon-Weaver index of diversity, the J' evenness index, the inverse of Simpson's measure of concentration, and Hill's evenness index. For 37 Breeding Bird Censuses taken in various terrestrial habitats across the United States and Canada, the proposed methods reveal some very general relationships about the organization of bird communities in different habitats. Equal-sized areas of mature deciduous forest and secondgrowth habitats may be equally species rich (14-24 species with •> 1 breeding territory per 6 ha); the density of individuals (territorial pairs) is generally higher in deciduous forest habitats, and the relative abundance of bird species shows more dominance (less evenness) in the deciduous forest. Mixed coniferous-deciduous forests and dense young deciduous forests have fewer species than mature eastern deciduous forests or second-growth abitats (9-16 and 7-10 species per 6 ha, respectively), although the density of individuals is approximately equal to that in second-growth habitats. Coniferous forests are species-poor (5-8 per 6 ha), and the density of territorial pairs is low (8-12 per 6 ha compared with 40-70 in deciduous forests). Although the proposed methods require assumptions that need to be evaluated carefully, we are optimistic that they will have other useful applications in the analysis of arian communities. Received 8 October 1980, accepted 15 April 1981. A large literature has developed in ecology presenting descriptive analyses of biotic assemblages (Dennis et al. 1979, Patil and Taille 1979). Although one should not infer mechanisms of community regulation from such studies (Pielou 1975), certain patterns recur in vertebrate communities in different habitats (Palmgren 1930, Udvardy 1957, MacArthur and MacArthur 1961, Williams 1964, MacArthur 1964, Karr and Roth 1971, Wiens 1973, Willson 1974), climates (Bock and Lepthien 1974, Rotenberry 1978), seasons (Rotenberry et al. 1979), and geographic areas (Pianka 1966, Recher 1969, Karr 1971, Tramer 1974, Cody 1975, Short 1979). One methodological problem with much of this literature and with community ecology generally since the early 1960's is the expression of community structure in terms of indices of diversity and evenness. Indices such as H' [-5; p/log Pi] (Shannon and Weaver 1949, Margalef 1958) and J' [H'/log s] (Pielou 1966a) confound important parameters that should be defined as precisely as possible and examined separately before communities are compared. These are (1) the number of species (species richness), (2) their relative abundance (evenness), (3) the number of individuals or territorial pairs, and (4) the area sampled. To combine any of these variables into a single statistic assures that the relative effects of the contributing parameters cannot be determined. The same value of the index can result from various com785 The Auk 98: 785-800. October 1981 786 JAMES AND RATHBUN [Auk, Vol. 98 binations of values of the parameters (Pielou 1975). Here, we recommend that data be standardized to either equal numbers of individuals (in this case, territorial pairs) or equal-sized areas before comparisons are attempted. We propose that rarefaction and relative abundance curves be used as an alternative to diversity indices. In addition to clarifying components of biological interest, these methods avoid many of the mathematical deficiencies of the application of indices. We will examine the community structure of a large set of breeding bird censuses on the basis of the traditional methods and then present four graphic displays of the results of rarefactions (Figs. 1-3) and relative abundance curves (Fig. 4).

Rarefaction, Relative Abundance, and Diversity of Avian Communities

We evaluated a commercial point-of-care circulating cathodic antigen (POC-CCA) test for assessing Schistosoma mansoni infection prevalence in areas at risk. Overall, 4,405 school-age children in Cameroon, Cote d'Ivoire, Ethiopia, Kenya, and Uganda provided urine for POC-CCA testing and stool for Kato-Katz assays. By latent class analysis, one POC-CCA test was more sensitive (86% versus 62%) but less specific (72% versus ~100%) than multiple Kato-Katz smears from one stool. However, only 1% of POC-CCA tests in a non-endemic area were false positives, suggesting the latent class analysis underestimated the POC-CCA specificity. Multivariable modeling estimated POC-CCA as signifi- cantly more sensitive than Kato-Katz at low infection intensities (< 100 eggs/gram stool). By linear regression, 72% prevalence among 9-12 year olds by POC-CCA corresponded to 50% prevalence by Kato-Katz, whereas 46% POC-CCA prevalence corresponded to 10% Kato-Katz prevalence. We conclude that one urine POC-CCA test can replace Kato-Katz testing for community-level S. mansoni prevalence mapping.

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Stephen L. Rathbun

Papers

Quantitative Comparisons of 16S rRNA Gene Sequence Libraries from Environmental Samples

The population dynamics of a long-lived conifer

The Population Dynamics of a Long-Lived Conifer (Pinus palustris)

Rarefaction, Relative Abundance, and Diversity of Avian Communities

A five-country evaluation of a point-of-care circulating cathodic antigen urine assay for the prevalence of Schistosoma mansoni.