Environmental PCR surveys of small-subunit (SSU) rDNA sequences are powerful approximations for the overall diversity of microbial eukaryotes (protists) living in specific marine habitats. However, many environmental DNA sequences generated from these approaches have unknown cellular origins because they are not closely related to other sequences that were generated directly from fully characterized, identified organisms. The unidentified sequences from marine environments tend to belong to poorly understood groups of apicomplexan parasites, especially gregarines. Single-cell PCR (SC-PCR) approaches on newly discovered gregarines provide the evidence necessary for determining the cellular identities of SSU rDNA sequence clades. In this study, the trophozoites of four novel gregarine morphotypes were isolated from the intestines of two different species of capitellid polychaetes collected from the eastern Pacific Ocean (British Columbia, Canada). The trophozoites of each morphotype were characterized using light microscopy, scanning electron microscopy, and SSU rDNA sequences amplified from four, single-cell isolates from each of the four novel morphotypes described in this study (16 new SSU rDNA sequences in total). Molecular phylogenetic analyses demonstrated five robust subclades within a more inclusive clade that contained all 16 new sequences and 5 environmental SSU rDNA sequences. The combination of SC-PCR approaches, molecular phylogenetic analyses, and comparative morphology (1) illustrate the utility of SC-PCR approaches for distinguishing between different gregarine species, (2) demonstrate the cellular identity of a previously unidentified environmental SSU rDNA sequence clade, and (3) enable us to establish four new species within one novel genus: Polyplicarium lacrimae n. gen. et sp. (type species), P. curvarae n. gen. et sp., P. translucidae n. gen. et sp. |
