Status of the phylogenetic diversity census of ruminal microbiomes

RESEARCH ARTICLE Status of the phylogenetic diversity census of ruminal microbiomes Minseok Kim1, Mark Morrison1,2 & Zhongtang Yu1 1 Department of Animal Sciences, The Ohio State University, Columbus, OH, USA; and 2CSIRO Livestock Industries, St Lucia, Qld, Australia Received 16 September 2010; revised 25 November 2010; accepted 2 December 2010. Final version published online 12 January 2011. DOI:10.1111/j.1574-6941.2010.01029.x Editor: Julian Marchesi MICROBIOLOGY ECOLOGY Keywords 16S rRNA gene; rrn; OTUs; rarefaction analysis; ruminal microbiomes. Abstract In this study, the collective microbial diversity in the rumen was examined by performing a meta-analysis of all the curated 16S rRNA gene (rrn) sequences deposited in the RDP database. As of November 2010, 13 478 bacterial and 3516 archaeal rrn sequences were found. The bacterial sequences were assigned to 5271 operation taxonomic units (OTUs) at species level (0.03 phylogenetic distance) representing 19 existing phyla, of which the Firmicutes (2958 OTUs), Bacteroidetes (1610 OTUs) and Proteobacteria (226 OTUs) were the most predominant. These bacterial sequences were grouped into more than 3500 OTUs at genus level (0.05 distance), but only 180 existing genera were represented. Nearly all the archaeal sequences were assigned to 943 species-level OTUs in phylum Euryarchaeota. Although clustered into 670 genus-level OTUs, only 12 existing archaeal genera were represented. Based on rarefaction analysis, the current percent coverage at species level reached 71% for bacteria and 65% for archaea. At least 78 218 bacterial and 24 480 archaeal sequences would be needed to reach 99.9% coverage. The results of this study may serve as a framework to assess the significance of individual populations to rumen functions and to guide future studies to identify the alpha and global diversity of ruminal microbiomes. Introduction The rumen has evolved to digest various plant materials by a complex microbiome consisting of bacteria, archaea, protozoa and fungi. Within this microbiome, bacteria are the dominant domain and make the greatest contribution to digestion and conversion of feeds to short-chain fatty acids and microbial proteins (Hobson & Stewart, 1997). Ruminal archaea are mostly methanogens that belong to phylum Euryarchaeota. Utilizing the carbon dioxide (CO2) and hydrogen (H2) produced from bacterial fermentation, these methanogens produce methane, a potent greenhouse gas that is implicated in global warming (Janssen & Kirs, 2008). Both bacterial and archaeal populations can be affected by many factors, such as species and age of hosts, diets, feeds, feed additives, seasons and geographic regions (Tajima et al., 2001a; Zhou et al., 2009). Numerous efforts have been made to optimize rumen functions by enhancing feed digestion, improving conversion of dietary nitrogen to microbial proteins, and reducing methane emission and nitrogen excretion by manipulating the ruminal microbiome through dietary means. Although limited success has been achieved, FEMS Microbiol Ecol 76 (2011) 49–63 few of these dietary manipulations achieved persistent effects without negatively affecting overall rumen functions (van Nevel & Demeyer, 2004; Calsamiglia et al., 2007; Patra & Saxena, 2009). The lack of sufficient understanding of the ruminal microbiome is one of the major knowledge gaps that hinder effective enhancement of rumen functions (Firkins & Yu, 2006). The ruminal microbiome, as other microbiomes, was investigated primarily using cultivation-based methods for many decades until the 1980s, when 16S rRNA gene-targeted analysis was applied (Stahl et al., 1988). Cultured bacteria and archaea both helped in defining some of the important metabolisms underpinning rumen functions (Hobson & Stewart, 1997); however, it soon became evident that most of the rumen microorganisms escaped laboratory cultivation (Whitford et al., 1998). Thereafter, most studies attempted to characterize the ruminal microbiome by phylogenetic analysis of 16S rRNA gene (rrn) sequences recovered in clone libraries by direct PCR amplification (reviewed by Edwards et al., 2004; Deng et al., 2008). Some DNA-based studies focused on the microorganisms present in rumen fluid (e.g. Tajima et al., 2000, 2007; Ozutsumi 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c Correspondence: Zhongtang Yu, Department of Animal Sciences, The Ohio State University, 2029 Fyffe Road, Columbus, OH 43210, USA. Tel.: 11 614 292 3057; fax: 11 614 292 2929; e-mail: 50 c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Materials and methods Sequence data collection and phylogenetic analysis As of November 2010, the RDP database (Release 10, Update 22) was searched for rrn sequences of rumen origin using the search terms ‘rumen’ and ‘ruminal’ to collect sequences of both ruminal bacteria and archaea. Sequences of ‘suspect quality’ were excluded using the option of ‘Quality’ in the RDP database. All the found sequences were examined to confirm their rumen origin, and those of nonrumen origin were removed manually. To ensure the rrn sequences of cultured ruminal bacteria and archaea were included in our analysis, databases of the American Type Culture Collection (ATCC), Culture Collection, University of Göteborg (CCUG), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and Japan Collection of Microorganisms (JCM) were searched using the same search terms ‘rumen’ and ‘ruminal’. The rrn sequences of those isolates were added to the above sequence selections if they were not found in the initial search in RDP. The sequences for total bacteria, bacterial isolates, total archaea and archaeal isolates were downloaded separately from the RDP database in aligned format with common gaps removed. The navigation tree for each of these sequence datasets was also downloaded. Each of the sequence datasets and the associated navigation tree were imported into ARB, a software environment that can store, manage and analyze rrn sequences (Ludwig et al., 2004). Taxonomic trees with the Bergey’s taxonomy applied were generated from the imported navigation trees using the ARB program. All the rrn sequences of both ruminal bacteria (Z274 bp) and archaea (Z200 bp) were aligned against the rrn Greengenes database (DeSantis et al., 2006). The resulting aligned sequences were inserted into the Greengenes database ARB tree to generate a detailed phylogenetic tree using the positional variance by parsimony method (Ludwig et al., 2004). The detailed phylogenetic tree generated in this study is available from the corresponding author. Diversity estimate Numbers of operation taxonomic units (OTUs) were calculated for total bacteria, total archaea and major groups of bacteria using the MOTHUR program (Schloss et al., 2009). Briefly, the aligned sequences for each gro (...truncated)