Effects of experimental choices and analysis noise on surveys of the "rare biosphere".

When planning a survey of 16S rRNA genes from a complex environment, investigators face many choices including which primers to use and how to taxonomically classify sequences. In this study, we explored how these choices affected a survey of microbial diversity in a sample taken from the aerobic basin of the activated sludge of a North Carolina wastewater treatment plant. We performed pyrosequencing reactions on PCR products generated from primers targeting the V1-V2, V6, and V6-V7 variable regions of the 16S rRNA gene. We compared these sequences to 16S rRNA gene sequences found in a whole-genome shotgun pyrosequencing run performed on the same sample. We found that sequences generated from primers targeting the V1-V2 variable region had the best match to the whole-genome shotgun reaction across a range of taxonomic classifications from phylum to family. Pronounced differences between primer sets, however, occurred in the "rare biosphere" involving taxa that we observed in fewer than 11 sequences. We also examined the results of analysis strategies comparing a classification scheme using a nearest-neighbor approach to directly classifying sequences with a naïve Bayesian algorithm. Again, we observed pronounced differences between these analysis schemes in infrequently observed taxa. We conclude that if a study is meant to probe the rare biosphere, both the experimental conditions and analysis choices will have a profound impact on the observed results.

Nuclear and mitochondrial diversification in two native California minnows: insights into taxonomic identity and regional phylogeography.

Diversification of the California icthyofauna has been greatly influenced by a complex geomorphological history and past fluctuations in climate regimes. This complex history has resulted in areas of high endemism for a number of taxa. Here we present data on the two species in the genus Lavinia, the California roach (Lavinia symmetricus) and hitch (Lavinia exilicauda), that are widespread throughout the region. Individuals were sequenced at two mitochondrial DNA fragments and genotyped at eight microsatellite loci. Mitochondrial DNA indicated the presence to two highly divergent clades representing roach from the Gualala and Pit Rivers, which diverged from all other Lavinia approximately 3-6 MYA. Support was also foundfor roach from the Navarro River, Tomales Bay region, the Red Hills region, and the Russian River-Clear Lake basin. We found no evidence for any geographical groupings of mtDNA haplotypes for roach and hitch from the Monterey Bay region and the Sacramento-San Joaquin River drainages. Additionally roach and hitch from these two areas could not be readily distinguished by mtDNA data. Analysis of microsatellite DNA recovered all groupings found in the mtDNA analysis and was able to separate out roach, hitch, and all currently recognized subspecies of each species. These results indicate that hybridization may obscure the phylogenetic/phylogeographic informativeness of mtDNA in this group. Additionally, these results suggest that differentiation in this group occurs at the river basin level and that the described genetic entities constitute distinct units and should be conserved as such.

Eptatretus strickrotti n. sp. (Myxinidae): first hagfish captured from a hydrothermal vent.

A single hagfish (Myxinidae, Eptatretus) specimen was recently captured at a hydrothermal vent site on the East Pacific Rise (38 degrees S). This is the first capture of a member of the jawless fishes (agnathans) from a hydrothermal vent site. The specimen differs from all congeners by the very slender body (depth 2.9% of total length), the paired and median ventral nasal sinus papillae, and the presence of 10 afferent branchial arteries on the medial ventral aorta. It is further unique because of a combination of the following features: slime pore counts; paired dorsal nasal sinus papillae; 12 gill pouches and gill apertures; posterior left side of body widely separated from pharyngocutaneous duct; 3/2 multicusp configuration; ventral aorta bifurcated anteriorly between 2nd and 3rd gill pouches (counted from the snout toward the heart); and pink coloration. The specimen is here described as a new species named Eptatretus strickrotti. Molecular 16S rRNA data places this new species as the basal-most species of Eptatretus, providing important new insight to the evolution of hagfishes as a whole.