Journal of Eukaryotic Microbiology ISSN 1066-5234 ORIGINAL ARTICLE Haptophyte Diversity and Vertical Distribution Explored by 18S and 28S Ribosomal RNA Gene Metabarcoding and Scanning Electron Microscopy Sandra Gran-Stadniczenko~ a,1, Luka Supraha b,1, Elianne D. Eggea & Bente Edvardsena a Department of Biosciences, University of Oslo, PO Box 1066 Blindern, Oslo 0316, Norway b Department of Earth Sciences, Uppsala University, Villavagen€ 16, Uppsala SE-75236, Sweden Keywords ABSTRACT Abundance; coccolithophores; high-throughput sequencing; Oslofjorden; Haptophyta encompasses more than 300 species of mostly marine pico- and phylogeny; richness. nanoplanktonic flagellates. Our aims were to investigate the Oslofjorden hapto- phyte diversity and vertical distribution by metabarcoding, and to improve the Correspondence approach to study haptophyte community composition, richness and propor- S. Gran-Stadniczenko,~ Department of tional abundance by comparing two rRNA markers and scanning electron Biosciences, University of Oslo, P. O. microscopy (SEM). Samples were collected in August 2013 at the Outer Box 1066 Blindern, Oslo 0316, Norway Oslofjorden, Norway. Total RNA/cDNA was amplified by haptophyte-specific Telephone number: +47-22-85-73-65; primers targeting the V4 region of the 18S, and the D1-D2 region of the 28S FAX number: +47-22-85-47-26; rRNA. Taxonomy was assigned using curated haptophyte reference databases e-mail: [email protected] and phylogenetic analyses. Both marker genes showed Chrysochromulinaceae and Prymnesiaceae to be the families with highest number of Operational Tax- 1Equal contribution. onomic Units (OTUs), as well as proportional abundance. The 18S rRNA data set also contained OTUs assigned to eight supported and defined clades con- Received: 15 September 2016; revised 6 sisting of environmental sequences only, possibly representing novel lineages December 2016; accepted December 7, from family to class. We also recorded new species for the area. Comparing 2016. coccolithophores by SEM with metabarcoding shows a good correspondence Early View publication January 29, 2017 with the 18S rRNA gene proportional abundances. Our results contribute to link morphological and molecular data and 28S to 18S rRNA gene sequences of hap- doi:10.1111/jeu.12388 tophytes without cultured representatives, and to improve metabarcoding methodology. THE protist division Haptophyta encompasses more than strong impact on coastal ecosystems through toxin pro- 300 morphospecies of mostly pico- and nanoplanktonic duction (Graneli et al. 2012; Moestrup 1994). flagellates (Edvardsen et al. 2016; Jordan et al. 2004; Being a large and diverse group, haptophytes commonly Thomsen et al. 1994). The group inhabits all seas and exhibit species- and even strain-specific physiological some also thrive in freshwater, exhibiting a high degree of traits, which ultimately define their ecological and biogeo- morphological, physiological and functional diversity (Jor- chemical performance (Edvardsen and Paasche 1998; Lan- dan and Chamberlain 1997). Haptophytes share common ger et al. 2006; Ridgwell et al. 2009). Therefore, structural features, notably the production of unmineral- identifying haptophytes to a low taxonomic level is of ized organic scales and possession of two flagella and a great importance in ecological surveys. Morphological haptonema, although the latter was lost in a few mem- identification to the species level is particularly difficult in bers (e.g. Isochrysidales). Haptophytes are major primary noncalcifying groups, which lack hard and mineralized producers in open oceans (Andersen et al. 1996; Liu et al. body parts, and can only be accurately identified by the 2009) and the calcifying coccolithophores play a key role time-consuming examination of organic scales using elec- in the biogeochemical carbon cycle (Holligan et al. 1993; tron microscopy (EM) (Edvardsen et al. 2011; Eikrem Iglesias-Rodrıguez et al. 2002; Robertson et al. 1994). In 1996; Eikrem and Edvardsen 1999). On the other hand, addition, blooms of noncalcifying haptophytes can have a scanning electron microscopy (SEM)-based methods allow © 2016 The Authors Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists 514 Journal of Eukaryotic Microbiology 2017, 64, 514–532 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Gran-Stadniczenko~ et al. Haptophyta Diversity and Distribution by Metabarcoding for precise analysis of taxonomic composition and species Previous metabarcoding studies of haptophytes (Bittner abundance of coccolithophore communities (Bollmann et al. 2013) and protists (Massana et al. 2015) comparing et al. 2002; Cros and Fortuno~ 2002; Young et al. 2003). DNA vs. RNA as templates did not find any significant dif- With the advance of molecular techniques, notably envi- ferences in community structure. However, Egge et al. ronmental sequencing of clone-libraries and metabarcoding (2013) found that RNA captured more of the diversity than using high-throughput sequencing (HTS), it has become with DNA where larger cells were favored. Using RNA possible to overcome some of the limitations of micro- may significantly reduce the bias due to variability in rDNA scopical analysis in investigating haptophyte communities copy numbers among taxonomic groups (Not et al. 2009). (Edvardsen et al. 2016). Most importantly, molecular Massana et al. (2015) found that Haptophyta were under- methods allow for detection of rare or fragile species that represented in DNA compared to RNA surveys (average commonly remain unnoticed in ecological surveys. A num- read ratio DNA/RNA was 7.4). In addition, compared to ber of studies specifically investigated haptophyte commu- DNA, RNA is thought to more accurately picture which nities using molecular methods (Bittner et al. 2013; Egge protists are metabolically active at the time of collection et al. 2015a,b; Liu et al. 2009), each of them further (Stoeck et al. 2007). improving the methodology and providing new insights Further, comparison of different studies is complicated about the diversity and distribution of the group. The due to use of different marker genes. Of both 18S and major shift in understanding the diversity of haptophytes 28S rRNA genes there are more than 600 different ref- was provided by the early clone-library studies (Moon-van erence sequences available in gene databases from cul- der Staay et al. 2000, 2001; Edvardsen et al. 2016 for tured material and environmental clone-libraries comprehensive list of studies), as well as HTS-based (Edvardsen et al. 2016). The number of described hapto- works of Bittner et al. (2013) and Egge et al. (2015a,b). phyte species for which there are reference sequences These studies identified an abundance of new haptophyte available are higher for the 18S than 28S rRNA gene sequences (OTUs) and new haptophyte lineages, which (96 vs. 76) (Edvardsen et al. 2016) which makes 18S a could not be assigned to a cultured and genetically charac- more useful marker for identifying species in an environ- terized taxon. This indicated that the haptophyte diversity mental sample. However, in haptophytes the 18S rRNA in the modern oceans was largely underestimated in previ- gene may differ in only a few base pairs between clo- ous microscopic investigations, and that there are many sely related species, and short variable regions used for morphospecies still to be described. HTS metabarcoding, such as the V4, may be identical However, despite the rigorous processing of data during (Bittner et al. 2013; Edvardsen et al. 2016). The 28S analysis of 454-reads, a significant portion of ribotypes rRNA gene has more variable regions than 18S, and may still represent chimeric sequences or amplification regions of the 28S such as the D1-D2 have therefore artefacts (Haas et al. 2011; Huse et al. 2010; Speksnijder been suggested to be more appropriate barcodes for et al. 2001). The lack of studies combining qualitative and distinguishing recently diverged species (Bittner et al. quantitative morphological analysis of haptophyte commu- 2013; Liu et al. 2009), although some intraspecies varia- nities with molecular approaches complicates the estima- tion may occur, which will overestimate species rich- tion of the actual species diversity and abundance. ness (Liu et al. 2009). As these two markers offer Conducting such investigations on haptophyte communi- complementary views of environmental haptophyte com- ties is difficult due to the mentioned methodological limita- munities, it is important to compare richness and taxo- tions in identifying noncalcifying species using a nomic composition of 18S and 28S metabarcoding data morphological approach. Therefore, coccolithophores are sets obtained from the same samples. To our knowl- arguably the most appropriate group of haptophytes for edge, no study has so far done such a rigorous compar- such comparative investigation. A study by Young et al. ison for haptophytes. (2014) aimed at comparing the molecular (environmental The aim of our study was to investigate the Oslofjorden sequencing of clone-libraries) approach based on the 28S haptophyte community diversity and vertical distribution rRNA gene with the morphological (LM and SEM) analysis by using HTS with