ORIGINAL RESEARCH Discovery of a Katablepharis sp. in the Columbia River estuary that is abundant during the spring and bears a unique large ribosomal subunit sequence element Peter Kahn, Lydie Herfort, Tawnya D. Peterson & Peter Zuber Center for Coastal Margin Observation & Prediction and Institute of Environmental Health, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Mail code HRC3, Portland, Oregon 97239 Keywords Abstract Columbia River coastal margin, heterotrophic protist diversity, Katablepharis CRE, unique Heterotrophic protists play significant roles in pelagic food webs as bacterivor- sequence element. ous and herbivorous consumers. However, heterotrophic protists—unlike auto- trophic ones—are often difficult to track since they tend to lack features such Correspondence as photosynthetic pigments that allow for remote sensing or for bulk character- Peter Kahn, Institute of Environmental Health ization. Difficulty in the identification of heterotrophic protists has often and Center for Coastal Margin Observation & resulted in lumping them into broad groups, but there is a strong need to Prediction and, Oregon Health & Science develop methods that increase the spatial and temporal resolution of observa- University, Portland, OR 97239. Tel: (001) 503 748 1643; Fax: (001) 503 tions applied to particular organisms in order to discover the drivers of popula- 748 1464; tion structure and ecological function. In surveys of small subunit rRNA, gene E-mail: [email protected] (SSU) sequences of microbial eukaryotes from the Columbia River to the Paci- fic Ocean, the heterotrophic flagellate Katablepharis sp. were found to dominate Funding Information protist assemblages (including autotrophic and heterotrophic fractions) in the This study was carried out within the context spring, prior to the freshet. We discovered a 332 base pair unique sequence ele- of the Science and Technology Center for ment (USE) insertion in the large subunit rRNA gene (28S) that is not present Coastal Margin Observation & Prediction (CMOP) supported by the National Science in other katablepharids or in any other eukaryote. Using this USE, we were able Foundation (grant number OCE-0424602). to detect Katablepharis within mixed assemblages in river, estuarine, and oce- anic samples and determine spatial and temporal patterns in absolute abun- Received: 7 April 2014; Revised: 16 June dance through quantitative PCR and fluorescence in situ hybridization. Given 2014; Accepted: 11 July 2014 their high abundance and repeatable temporal patterns of occurrence, we hypothesize that the Columbia River Estuary Katablepharis (Katablepharis CRE) MicrobiologyOpen 2014; 3(5): 764–776 plays an important role in estuarine biogeochemical and ecosystem function. doi: 10.1002/mbo3.206 Introduction cal features or characteristic pigments. As a result, they have often been placed into broad groups, such as “het- Heterotrophic protists play significant roles in pelagic erotrophic nanoflagellates” (2–20 lm), which could food webs as bacterivorous and herbivorous consumers include a wide range of organisms of different taxonomic (Pomeroy 1974; Azam 1983), as food sources for organ- groups bearing different metabolic potentials and that isms at higher trophic levels such as metazoans (Gifford play varied roles in aquatic food webs. The collection of 1991), and as remineralizers of essential nutrients such as established cultures of heterotrophic protists is also not nitrogen and phosphorus (Caron et al. 1990). Heterotro- likely to be representative of the dominant cells in the phic protists, particularly small cells (<20 lm), are often environment, as organisms that are easily cultured are difficult to assign taxonomically using light or electron often found at low abundance in natural assemblages microscopy, as many of them lack distinctive morphologi- (Lim et al. 1999). Since the advent of culture-independent 764 ª 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. P. Kahn et al. Katablepharis CRE, Genetic Marker and Ecology molecular-based techniques, numerous studies (Diez et al. in the fate of particulate organic matter in the Columbia 2001; Lopez-Garcia et al. 2001; Moon-van der Staay River estuary. This critical gap in knowledge prevents the et al. 2001; Stoeck et al. 2003; Bass and Cavalier-Smith identification of trophic linkages within the aquatic food 2004) have begun to report unexpectedly high levels of web, which could be used to inform management decisions. diversity within the autotrophic, mixotrophic, and hetero- Higher spatial and temporal resolution of monitoring for trophic protist populations in diverse aquatic environ- taxa of interest, as well as improved estimates of protist ments. For example, these studies have uncovered several diversity, are necessary in order to determine the drivers of novel lineages within the bacterivorous marine strameno- population structure as well as biogeochemical and ecosys- piles that can comprise a large proportion of protist pop- tem function. ulations and can be responsible for up to 10% of A molecular approach can offer valuable new insights bacterivory and nutrient remineralization in the upper into protist assemblage structure and diversity in the ocean (Massana et al. 2006). Columbia River coastal margin, particularly for the hetero- The Columbia River coastal margin, which includes trophs. To evaluate heterotrophic protist assemblages, freshwater, brackish, and saline environments along a small subunit (SSU) rRNA gene clone libraries were ana- river-to-ocean continuum, is an ideal place to investigate lyzed for water samples collected at salinities of ~0 and ~15 the diversity of heterotrophic protists because of the in the Columbia River estuary, and salinities of ~28–31 in broad range of environmental conditions that exist over a the plume in April and August 2007 and in April, July, and relatively narrow geographical region. The Columbia September 2008. Further analysis of the large subunit River coastal margin provides habitats for a wide variety (LSU) rRNA gene sequences was conducted for the of ecologically and economically important species, such highly dominant heterotrophic flagellate in the mid-salinity as salmonids and various types of shellfish (Roegner et al. water SSU rRNA gene clone libraries in both April 2007 2011). The Columbia River estuary is characterized by and 2008, a unique katablepharid henceforth referred to large amounts of allochthonous detritus from the adjacent as Katablepharis CRE (Columbia River Estuary). This river and ocean that is the primary source of organic analysis uncovered a 332 base pair unique sequence matter driving ecosystem processes, with allochthonous element (USE) within the D2 region of the LSU that shows organic matter supporting up to 84% of secondary pro- no significant similarity to any LSU sequences in the duction by the estuarine microbial populations (Simens- National Center for Biotechnology Information (NCBI) tad et al. 1990). Yet, the overwhelming majority of database and displays an elevated GC content compared to research conducted on protist assemblages in the Colum- its associated SSU and LSU rRNA sequences (data retrieved bia River (including the saline and freshwater reaches of on 10 January 2014). The presence and diversity of this ele- the estuary) thus far has been focused on the autotrophic ment were further examined to answer the following fraction, which is dominated by freshwater diatoms research questions: (Haertel et al. 1969; Frey et al. 1984; Lara-Lara et al. 1 What is the spatial and temporal distribution of organ- 1990; Small et al. 1990; Sullivan et al. 2001). isms bearing this unique element amongst Katableph- Previous studies on heterotrophs in the Columbia River aris CRE and other katablepharids in the Columbia estuary have shown that the mesozooplankton (0.2–2 mm) River coastal margin? are composed primarily of freshwater, oligohaline, and 2 Is this unique element found in any other organisms in polyhaline forms (Haertel et al. 1969; Simenstad et al. the Columbia River coastal margin and/or elsewhere? 1990), while particle-attached bacteria accounted for up to 3 Can the unique element be used as a taxonomic mar- 90% of heterotrophic bacterial activity and are an impor- ker to facilitate ecological studies of Katablepharis CRE? tant part of the estuarine food web (Crump et al. 1998). One study (Crump and Baross 1996) does suggest that Methods nanoflagellates and oligotrich ciliates are the most common form of heterotrophic protist in the estuarine turbidity Sample acquisition maximum, but that study was limited in spatial and tempo- ral extent. Relatively little is known about the composition Samples for SSU sequence analysis were collected in the and ecological role of heterotrophic protists in the river, Columbia River coastal margin along the river-to-ocean estuary, and plume environments, despite their likely gradient from sites with three distinct salinities in April importance in organic matter transformations within aqua- 2007 and 2008. Figure 1 and Table 1 provide the details of tic food webs that link microbial activity and higher trophic location, salinity, temperature, and depth for all samples levels (Sherr and Sherr 1994, Arndt et al. 2000). Given the used for SSU sequence analysis. Water was collected