Analysis of Photosynthetic Picoeukaryote Diversity at Open Ocean Sites in the Arabian Sea Using a PCR Biased Towards Marine Algal Plastids
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AQUATIC MICROBIAL ECOLOGY Vol. 43: 79–93, 2006 Published May 3 Aquat Microb Ecol Analysis of photosynthetic picoeukaryote diversity at open ocean sites in the Arabian Sea using a PCR biased towards marine algal plastids Nicholas J. Fuller1, Colin Campbell1, David J. Allen1, Frances D. Pitt1, Katrin Zwirglmaier1, Florence Le Gall2, Daniel Vaulot2, David J. Scanlan1,* 1Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK 2Station Biologique de Roscoff, UMR 7144 CNRS et Université Pierre et Marie Curie, BP74, 29682 Roscoff, Cedex, France ABSTRACT: Marine photosynthetic picoeukaryotes (PPEs), representing organisms <3 µm in size, are major contributors to global carbon cycling. However, the key members of the PPE community and hence the major routes of carbon fixation, particularly in the open ocean environment, are poorly described. Here, we have accessed PPE community structure using the plastid encoded 16S rRNA gene. Plastid 16S rRNA genes were sequenced from 65 algal cultures, about half being PPEs, representing 14 algal classes. These included sequences from 5 classes where previously no such sequences from cultured representatives had been available (Bolidophyceae, Dictyochophyceae, Eustigmatophyceae, Pelagophyceae and Pinguiophyceae). Sequences were also obtained for 6 of the 7 (according to 18S rRNA gene sequence) prasinophyte clades. Phylogenetic analysis revealed plastids from the same class as clustering together. Using all the obtained sequences, as well as plastid sequences currently in public databases, a non-degenerate marine algal plastid-biased PCR primer, PLA491F, was developed to minimize amplification of picocyanobacteria, which often dominate numerically environmental samples. Clone libraries subsequently constructed from the pico-sized fraction from 2 open ocean sites in the Arabian Sea, revealed an abundance of 16S rRNA gene clones phylogenetically related to chrysophytes, whilst prymnesiophyte, clade II prasinophyte (Ostreococcus-like) and pelagophyte clones were also well represented. The finding of a wealth of novel clones related to the Chrysophyceae highlights the utility of a PCR biased towards marine algal plastids as a valuable complement to 18S rDNA based studies of PPE diversity. KEY WORDS: Photosynthetic picoeukaryotes · Plastid 16S rRNA · Arabian Sea · PCR Resale or republication not permitted without written consent of the publisher INTRODUCTION production by the picophytoplankton in Californian coastal waters. That this contribution to C fixation is Marine phytoplankton are responsible for about half much greater than that of the far more numerous of the global carbon fixation (Field et al. 1998). A sig- cyanobacteria Prochlorococcus and Synechococcus, nificant proportion of this fixed carbon derives from appears largely due to their greater cell size and rates the eukaryotic component <3 µm in size, the photosyn- of cell-specific C uptake (Li 1994). thetic picoeukaryotes (PPEs). For example, a study in Compared with the prokaryotic component of the the central North Atlantic Ocean showed that photo- picophytoplankton the PPE community is highly synthetic eukaryotes, despite accounting for only 10% diverse, comprising members from virtually every of the ultraphytoplankton by cell number, were algal class. Indeed, several new algal classes with responsible for 68% of the primary production (Li picoplanktonic representatives have been described in 1995). More recently, Worden et al. (2004) showed that recent years, such as Pelagophyceae (Andersen et al. PPEs were responsible for, on average, 76% of net C 1993), Bolidophyceae (Guillou et al. 1999a) and Pin- *Corresponding author. Email: [email protected] © Inter-Research 2006 · www.int-res.com 80 Aquat Microb Ecol 43: 79–93, 2006 guiophyceae (Kawachi et al. 2002). Yet despite the targeted 18S rRNA gene oligonucleotides do not evident ecological significance of PPEs, relatively little necessarily allow analysis purely of ‘autotrophs’ since is known of their diversity in the marine environment, several algal classes, e.g. the Dinophyceae and Cryp- particularly in the open ocean. This has been attrib- tophyceae, contain representatives whose sole mode uted mainly to difficulties in identification by light of nutrition is heterotrophy (though these do lack microscopy. Only recently, with the advent of mole- plastids). By focusing on the plastid 16S rRNA gene, cular techniques, has picoeukaryote diversity begun to however, one targets specifically the phototrophic be revealed (for a recent review see Moreira & Lopez- community. Garcia 2002). Thus, phylogenetic studies based on 18S 16S rRNA gene sequences have been used exten- rRNA gene sequence analysis are now beginning to sively to analyse prokaryotic diversity in both cultures show the extent of taxonomic diversity within this and natural environments and hence a large number of group of organisms (Diez et al. 2001, Lopez-Garcia these sequences now exist (Cole et al. 2003). However, et al. 2001, Moon-van der Staay et al. 2001, Stoeck & the generally low numerical abundance of PPEs, and Epstein 2003, Romari & Vaulot 2004). Such studies, the fact that oligotrophic open ocean environments are using ‘universal’ 18S rRNA gene primers (which target dominated numerically by heterotrophic bacteria and both [photo]autotrophs and heterotrophs) and clone the picocyanobacteria Prochlorococcus and Synecho- library construction from environmental samples have coccus, has meant that PPEs are generally poorly demonstrated the presence of PPEs affiliated with reflected in clone libraries constructed using universal many different algal classes including the Bacillario- 16S rRNA gene primers (Giovannoni et al. 1990, phyceae, Bolidophyceae, Chrysophyceae, Cryptophy- Britschgi & Giovannoni 1991, Fuhrman et al. 1993). ceae, Dictyochophyceae, Dinophyceae, Eustigmato- Those libraries that have yielded substantial numbers phyceae, Glaucocystophyceae, Pelagophyceae, Prasi- of plastid clones have tended to be derived from nophyceae and Prymnesiophyceae (Díez et al. 2001, marine coastal waters or from open ocean regions con- Romari & Vaulot 2004, Savin et al. 2004). Amongst taining elevated nutrients where PPEs are highly these classes prasinophyte, prymnesiophyte and cryp- abundant (Rappé et al. 1995, Brown & Bowman 2001, tophyte sequences were found to be particularly abun- Wilmotte et al. 2002). Although more specific PCR dant at a French coastal site in the English channel primers are currently available, capable of recognising (Romari & Vaulot 2004). Indeed, the development of generally ‘oxygenic phototrophs’, i.e. both plastids and 18S rRNA gene oligonucleotides targeting specific cyanobacteria (Nübel et al. 1997, West et al. 2001), algal classes, and used in combination with fluorescent the fact that these primers target picocyanobacteria, in situ hybridisation technology has confirmed that organisms dominating most marine water columns, prasinophytes, particularly of the order Mamiellales, means very few plastid clones appear in libraries using dominate picoeukaryote communities in both coastal this primer pair (Fuller & Scanlan unpubl. data). Simi- and nutrient rich marine environments e.g. coastal lar problems have arisen using the functional gene waters of northern France (Not et al. 2002, 2004) and psbA (encoding the D1 protein of photosystem II) nutrient rich waters of the Norwegian and Barents which has also been used for analysing marine pico- Seas (Not et al. 2005). phytoplankton diversity (Zeidner et al. 2003). Thus, Although these 18S rRNA gene based studies show although these authors were able to detect prasino- excellent promise for describing PPE diversity, the fact phyte, prymnesiophyte, cryptophyte and bacillario- that these ‘universal’ primers target both (photo)- phyte sequences from the Red Sea, Mediterranean autotrophic and heterotrophic components of the Sea, and central North Pacific Ocean, this was amongst <3 µm fraction means that clone libraries constructed a plethora of picocyanobacterial sequences. Further- using these primers require extensive screening to more, the relative dearth of 16S rRNA gene sequences assess the diversity solely of the autotrophic compo- from cultured isolates (and more so for psbA sequen- nent. In addition, significant differences in 18S rRNA ces) has made it often impossible to identify the envi- gene copy number occur in microalgae, ranging from ronmental clones which arise, even to the class level. 1 copy in Nannochloropsis to over 1000 copies in To overcome these problems we report here the se- some nanoplanktonic dinoflagellates (Zhu et al. 2005) quencing of over 60 algal 16S rRNA genes to consider- whilst PCR bias may be significant with primers used ably ‘enrich’ the 16S rRNA gene database with cul- for this gene, as evidenced by little difference in tured isolates, so that virtually every known algal class marine eukaryote community structure in light and can now be identified. Using these derived sequences dark enrichment cultures, possibly due to selective we have designed a new PCR primer, PLA491F, biased amplification of heterotrophic over photosynthetic towards recognising marine algal plastids over pico- organisms irrespective of relative cell abundance cyanobacteria. We demonstrate that this primer, in (Guillou et al. unpubl.). Furthermore, even specifically combination with a previously described oxygenic- Fuller et al.: Photosynthetic picoeukaryote diversity 81 phototroph biased primer (OXY1313R) (West et