Comparative Analysis of the Methanogen Diversity in Horse and Pony by UsingmcrAGene and Archaeal 16S rRNA Gene Clone Libraries

Comparative analysis of methanogen compositions in the feces of horse and pony was carried out by constructing theα-subunit of methyl coenzyme-M reductase (mcrA) gene and 16S ribosomal RNA gene (16S rRNA) clone libraries. ThemcrAclone library analysis indicated that Methanomicrobiales was predominant in both horse and pony. Furthermore, most of the clones of the 16S rRNA gene library showed that Methanomicrobiales was also predominant in horse and pony, but the LIBSHUFF analysis showed that the horse and pony libraries were significantly different (P<0.05). Most of operational taxonomic units (OTUs) showed low similarity to the identified methanogens in both themcrAand the 16S rRNA clone libraries. The results suggest that horse and pony harbor unidentified and novel methanogens in their hindgut. The methanogen population was higher in horse than in pony; however, the anaerobic fungal population was similar in horse and pony. The methanogen diversity was different between two breeds ofEquus caballus.

Distinct Patterns of Picocyanobacterial Communities in Winter and Summer in the Chesapeake Bay

ABSTRACT In the Chesapeake Bay, picocyanobacteria were usually 100-fold less abundant in winter than in summer. However, little is known about how picocyanobacterial populations shift between winter and summer in the bay. This is due mainly to undetectable winter picocyanobacterial populations in bacterial 16S rRNA clone libraries. In this study, the winter and summer picocyanobacterial populations in the bay were detected using picocyanobacterium-specific primers and were compared based on the analysis of rRNA internal transcribed spacer sequences. Temperature was found to be the dominant environmental factor controlling picocyanobacterial populations in the Chesapeake Bay. In the summer, marine cluster B Synechococcus dominated the upper bay, while a unique cluster, CB1 (marine cluster A [MC-A] Synechococcus), made up the vast majority in the middle and lower bay. In the winter, the picocyanobacteria shifted to completely different populations. Subclades CB6 and CB7, which belong to MC-A Synechococcus and Cyanobium, respectively, made up the entire winter picocyanobacterial populations in the bay. Interestingly, the winter members in subclade CB6 clustered closely with Synechococcus CC9311, a coastal strain known to have a greater capacity to sense and respond to changing environments than oceanic strains.

Phylogenetic Comparisons of Bacterial Communities from Serpentine and Nonserpentine Soils

ABSTRACT I present the results of a culture-independent survey of soil bacterial communities from serpentine soils and adjacent nonserpentine comparator soils using a variety of newly developed phylogenetically based statistical tools. The study design included site-based replication of the serpentine-to-nonserpentine community comparison over a regional scale (∼100 km) in Northern California and Southern Oregon by producing 16S rRNA clone libraries from pairs of samples taken on either side of the serepentine-nonserpentine edaphic boundary at three geographical sites. At the division level, the serpentine and nonserpentine communities were similar to each other and to previous data from forest soils. Comparisons of both richness and Shannon diversity produced no significant differences between any of the libraries, but the vast majority of phylogenetically based tests were significant, even with only 50 sequences per library. These results suggest that most samples were distinct, consisting of a collection of lineages generally not found in other samples. The pattern of results showed that serpentine communities tended to be more similar to each other than they were to nonserpentine communities, and these differences were at a lower taxonomic scale. Comparisons of two nonserpentine communities generally showed differences, and some results suggest that the geographical site may control community composition as well. These results show the power of phylogenetic tests to discern differences between 16S rRNA libraries compared to tests that discard DNA data to bin sequences into operational taxonomic units, and they stress the importance of replication at larger scales for inferences regarding microbial biogeography.

PCR-Induced Sequence Artifacts and Bias: Insights from Comparison of Two 16S rRNA Clone Libraries Constructed from the Same Sample

ABSTRACT The contribution of PCR artifacts to 16S rRNA gene sequence diversity from a complex bacterioplankton sample was estimated. Taq DNA polymerase errors were found to be the dominant sequence artifact but could be constrained by clustering the sequences into 99% sequence similarity groups. Other artifacts (chimeras and heteroduplex molecules) were significantly reduced by employing modified amplification protocols. Surprisingly, no skew in sequence types was detected in the two libraries constructed from PCR products amplified for different numbers of cycles. Recommendations for modification of amplification protocols and for reporting diversity estimates at 99% sequence similarity as a standard are given.