Diversity of Oligotrichia and Choreotrichia Ciliates in Coastal Marine Sediments and in Overlying Plankton

Elucidating the relationship between ciliate communities in the benthos and the plankton is critical to understanding ciliate diversity in marine systems. Although data for many lineages are sparse, at least some members of the dominant marine ciliate clades Oligotrichia and Choreotrichia can be found in both plankton and benthos, in the latter either as cysts or active forms. In this study, we developed a molecular approach to address the relationship between the diversity of ciliates in the plankton and those of the underlying benthos in the same locations. Samples from plankton and sediments were compared across three sites along the New England coast, and additional subsamples were analyzed to assess reproducibility of methods. We found that sediment and plankton subsamples differed in their robustness to repeated subsampling. Sediment subsamples (i.e., 1-g aliquots from a single ∼20-g sample) gave variable estimates of diversity, while plankton subsamples produced consistent results. These results indicate the need for additional study to determine the spatial scale over which diversity varies in marine sediments. Clustering of phylogenetic types indicates that benthic assemblages of oligotrichs and choreotrichs appear to be more like those from spatially remote benthic communities than the ciliate communities sampled in the water above them.Planktonic ciliates provide a critical trophic link between the microbial and macroscopic components of the pelagic food web, and the subclasses Choreotrichia and Oligotrichia are the most abundant ciliate groups in this environment (46). One key to understanding the diversity and ecology of Choreotrichia and Oligotrichia is the relationship between benthic and planktonic forms. While the ciliates in these two groups are predominantly swimmers (54), there is crossover between benthic and pelagic environments for many species. Some taxa are described as epibenthic, living in the layer of water just above the sediment (16, 54), some have the capacity to live attached to sediment particles for a period and then become free-swimming (21), and a large number of taxa within these two groups spend a portion of their life cycles in dormancy, persisting in the sediments in cyst form (22, 23, 25, 35, 36, 39, 40, 41, 43, 49, 51). An accurate assessment of ciliate dynamics in the plankton requires careful study of both benthic and pelagic environments and the extent of coupling between the two environments.The role of the cyst in the life cycle of marine planktonic ciliates is particularly critical for understanding their distribution, evolutionary history, and ecology (6) as cysts provide a mechanism for dormancy during periods of poor environmental conditions. Relatively few marine ciliate species have been directly studied to determine conditions for encystment and excystment, period of dormancy (22, 23, 25, 26, 43), and role of the encystment cycle in the ecology of the organism (36). Moreover, studies on the conditions related to encystment and excystment in ciliates reveal different patterns and potential causes depending on the species (22, 23, 25, 26, 36, 43). While some data link the cycle of encystment with environmental factors such as light (23), temperature (23, 25, 26), and presence of food (22), other data suggest a temporal/seasonal cycling independent of external environmental conditions (26, 36, 43).A further factor limiting our understanding of the role of cysts in the life cycle of ciliates is identification based on the limited morphological features of the cysts, which are highly convergent (4, 17). In the case of ciliates that encyst within a lorica, as in the tintinnids, this is less of a problem (45), but for aloricate species, identification is not certain without direct observation of excystment (41, 48). Hence, morphological surveys of ciliates in benthic environments frequently capture members of the Oligotrichia and Choreotrichia (19, 31, 52, 53, 54) but are often limited to identification at the genus level using morphological approaches.More is known about planktonic ciliates, where morphology provides a wealth of data (11) and where molecular studies have revealed tremendous diversity, with many rare haplotypes (10). We define distinct sequences at the small-subunit (SSU) ribosomal DNA (rDNA) locus as haplotypes to remain conservative in our approach to identifying operational taxonomic units (OTUs) because ciliates have an unusual genome structure with high chromosome copy number, which potentially could generate multiple sequence types for the same locus within an organism or within a species. Planktonic ciliates show high molecular diversity at the SSU rDNA locus (10, 24), and primer sequences have been developed to detect ciliates from environmental samples within the subclasses Choreotrichia and Oligotrichia (10). Ciliates from these subclasses sampled across three coastal locations comprised distinct assemblages, with a few ubiquitous and abundant haplotypes (10) and many singletons (haplotypes unique to a particular sample).This study lays the groundwork for an alternative to morphological methods for analyzing benthic assemblages of oligotrichs and choreotrichs and comparing them to assemblages in the overlying water. Our goal was to compare levels of genetic diversity between sediment and plankton samples as a means of assessing the potential of methods for monitoring exchange between these two communities. There are two main questions addressed in this study: (i) are the two environments, plankton and sediment, comparable in robustness to repeated sampling using PCR, cloning, and sequencing and (ii) what is the relationship between genetic diversity of oligotrich and choreotrich ciliate communities sampled in marine sediments and in the plankton?To investigate the first question, we designed resampling experiments in plankton and sediment collections to test spatial heterogeneity as well as the robustness of repeated PCR cloning and sequencing for capturing diversity. Using two plankton samples collected by different means from the same time and place, we compared the similarity of subsamples in this environment to the similarity between separate subsamples of sediment collected at the same time and place. Additionally, we resampled DNA extracted from each of the two environments and investigated the reproducibility of repeated PCR cloning and sequencing between environmental types.To investigate the second question, we compared the diversity in sediment samples collected in the Gulf of Maine and Long Island Sound in May 2005 to previously published data from plankton samples collected at the same times and locations (10). Cluster analyses of the communities in sediment and plankton were used to determine the degree of coupling between the benthic and pelagic forms of Oligotrichia and Choreotrichia. The predicted result would be that the ciliate community observed in the plankton represents a subset of the diversity found in the benthic community, including cysts, beneath it. While the community in the plankton for many oligotrichs and choreotrichs would change depending on prevailing environmental conditions, predation, and chance, the benthic community, which includes encysted planktonic forms, should represent the longer-term diversity in a given region.