A notable feature of this and other clone library analyses from G.I. environments is the number of sequences that do not cluster with any known species. Sequences from nonruminant feces fell within the genera Bacteroides and Prevotella, while most sequences from ruminant hosts did not cluster with any known species. Both the range of sequence identity with the closest known species (87 to 91%) and the interclade identity range (81 to 94%) suggest that taxonomic diversity exists among ruminant sequences. Representatives of these groups must be isolated before phenotypic characterization or classification can be determined.
Within the genus Bacteroides, cloned sequences were closely related to known species, reflecting the greater number of cultivated representatives in this genus. This in turn reflects a greater emphasis placed on the study of the human fecal flora relative to other animal hosts. Many cultivated Bacteroides species, particularly B. vulgatus, B. uniformis, B. thetaiotaomicron, and B. stercoris, may not provide useful targets for fecal source discrimination assays because of the many similar or identical sequences from nonhuman hosts.
Phylogenetic resolution may be limited by the use of partial 16S rRNA gene sequences in a comparative analysis (23
). However, since the primers used here were designed for aquatic fecal source identification, they were constrained by the need to exclude amplification of closely related aquatic bacteria such as Cytophaga
). Bootstrap values compared favorably with those in the earlier study by Paster and colleagues (31
), in which full-length sequences were used.
Twenty-eight percent of the cloned sequences were identified as potential chimeras, fewer than the 32% identified by Wang and Wang (43
) in a study analyzing chimera frequency in 16S rRNA gene sequences from a mixed genomic population. They used PCR cycling parameters similar to those used in this study (30 cycles). Robison-Cox and colleagues (35
) proposed comparing trees inferred from half sequences to identify chimeric sequences. This method and the CHECK_CHIMERA program reliably identify chimeras formed between distantly related sequences (23
); we eliminated such chimeras from our analyses. Any chimeras remaining in our analyses were formed between sequences too closely related to perturb placement of the chimera halves in trees; these would not affect our conclusions on host distributions. In addition, the probability of formation of identical chimeras in different gene libraries is low (10
). Thus, recovery of clusters containing closely related or identical sequences from different libraries and hosts provides evidence that these clusters are not of chimeric origin.
Several evolutionary hypotheses could explain the existence of both cosmopolitan and endemic host distributions of fecal bacteria. Endemic distributions could occur among host species with limited physical contact and, thus, no horizontal transmission of fecal bacteria. Several studies have provided evidence for endemism when geographic barriers inhibit dispersal of bacterial populations (18
). If fecal bacteria within a host species had diversified in the time since host species diverged, each host species might contain unique types. In this case, we would expect to see types endemic to distantly related hosts such as humans and gulls. Instead, we see that humans and gulls share closely related Bacteroidales
A striking example of endemic distribution of fecal Bacteroidales
is provided by ruminants, which share unusual clades and do not have the common groups shared by other host species. The unique ruminant digestive system may provide a different way for these organisms to make a living than those inhabiting nonruminant hosts. Populations of fecal bacteria endemic to host species may be the result of evolution in different types of digestive systems (29
). Alternatively, the evolution of different G.I. systems may have been influenced by the microbial populations themselves (34
Cosmopolitan distributions could occur with frequent horizontal transmission of fecal bacteria among hosts with similar digestive systems. Sequences from multiple hosts were nearly identical to B. vulgatus
, B. thetaiotaomicron
, B. uniformis
, B. fragilis
, and B. stercoris
, suggesting that these Bacteroides
species are cosmopolitan with respect to host species. The “bushy tips” at branch termini within the genus Bacteroides
also suggest a cosmopolitan population structure, in which adaptations to new host environments represent species “ecotypes” (1
). Humans share proximity with domestic pets, making frequent occurrence of horizontal transmission of fecal bacteria likely. Gulls inhabit beaches, picnic areas, and landfills, where contact with human and domestic pet excrement may occur.
-related clones exhibited more varied host distribution patterns than Bacteroides
-related clones. A cluster of human and domestic pet Prevotella
sequences was not closely related to any known species but again showed a cosmopolitan host distribution. Prevotella
-related pig sequences clustered together, separate from other host species, as did the horse sequences, suggesting a more endemic distribution pattern. Pig-only and horse-only clades were used to identify unique sequences for these hosts, and PCR primers designed from these clades did not amplify DNA from nontarget hosts. However, this approach was not always successful, most likely because coverage of clone libraries was low. For example, a primer designed from a clade that appeared to be elk-specific amplified cattle fecal sequences (data not shown), probably because cattle clone library representation was lacking in that group. The overlap in domestic pet and human sequences also precluded the development of a unique dog or cat marker. The representation issue when using clone library sequences has led us to look for supplemental methods, including subtractive hybridization, to identify host-specific fecal markers in new hosts (8a
The 16S rRNA gene sequence analysis of fecal Bacteroidales revealed both endemic and cosmopolitan distributions among the eight hosts used in the study. The evolution of host-enteric bacterial interactions is complex and will certainly not be understood in the context of a single gene. Multiple interactions and sources of gene flow result in evolutionary trajectories that may not be predictable. However, the evolutionary consequences of these host distribution patterns have potential for practical application in the field of fecal source identification.