Protist, Vol. 164, 643–659, xx 2013 http://www.elsevier.de/protis Published online date xxx ORIGINAL PAPER Morphology, Genome Plasticity, and Phylogeny in the Genus Ostreococcus Reveal a Cryptic Species, O. mediterraneus sp. nov. (Mamiellales, Mamiellophyceae) a,b c d a,b Lucie Subirana , Bérangère Péquin , Stéphanie Michely , Marie-Line Escande , a,b,2 a,b e a,b Julie Meilland , Evelyne Derelle , Birger Marin , Gwenaël Piganeau , a,b a,b a,b,1 Yves Desdevises , Hervé Moreau , and Nigel H. Grimsley a CNRS, UMR7232 Laboratoire de Biologie Intégrative des Organisms Marins (BIOM), Observatoire Océanologique, Banyuls-sur-Mer, France b UPMC Univ Paris 06, Observatoire Océanologique, Banyuls-sur-Mer, France c Québec-Océan, Département de Biologie and Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Québec (QC) G1 V 0A6, Canada d INRA UMR1319, AgroParisTech, Micalis, Bimlip, Bât. CBAI, BP 01, 78850 Thiverval-Grignon, France e Biozentrum Köln, Botanisches Institut, Universität zu Köln, Zülpicher Str. 47b, 50674 Köln, Germany Submitted December 4, 2011; Accepted June 18, 2013 Monitoring Editor: David Moreira Coastal marine waters in many regions worldwide support abundant populations of extremely small (1-3 ␮m diameter) unicellular eukaryotic green algae, dominant taxa including several species in the class Mamiellophyceae. Their diminutive size conceals surprising levels of genetic diversity and defies classical species’ descriptions. We present a detailed analysis within the genus Ostreococcus and show that morphological characteristics cannot be used to describe diversity within this group. Karyotypic analyses of the best-characterized species O. tauri show it to carry two chromosomes that vary in size between individual clonal lines, probably an evolutionarily ancient feature that emerged before species’ divergences within the Mamiellales. By using a culturing technique specifically adapted to members of the genus Ostreococcus, we purified >30 clonal lines of a new species, Ostreococcus mediterraneus sp. nov., previously known as Ostreococcus clade D, that has been overlooked in several studies based on PCR-amplification of genetic markers from environment-extracted DNA. Phylogenetic analyses of the S-adenosylmethionine synthetase gene, and of the complete small subunit ribosomal RNA gene, including detailed comparisons of predicted ITS2 (internal transcribed spacer 2) secondary structures, clearly support that this is a separate species. In addition, karyotypic analyses reveal that the chromosomal location of its ribosomal RNA gene cluster differs from other Ostreococcus clades. © 2013 Elsevier GmbH. All rights reserved. Key words: Chromosome; karyotype; culture; ribosomal gene; barcode; picoeukaryote; ITS2. 1 Corresponding author; 2 Current address: CNRS, UMR6112, LPG-BIAF, Bio-Indicateurs Actuels et Fossiles, Université Angers 2 Boulevard Lavoisier, 49045 Angers CEDEX 01, France e-mail [email protected] (N.H. Grimsley). © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.protis.2013.06.002 644 L. Subirana et al. Introduction Rodriguez et al. 2005). This analysis strongly sug- gested a complex of cryptic species, the initial Although bacteria represent the largest biomass species O. tauri belonging to the clade C, although of marine life, photosynthetic eukaryotic protists this hypothesis could not be tested experimentally nevertheless account for a large proportion of the since the sexual cycle is unknown. The complete oceans’ primary production, largely due to their genome of O. tauri was then analysed (Derelle rapid turnover time (Li et al. 1992; Stockner 1988). et al. 2006). When a second strain isolated from Within the extremely diverse protistan marine com- Pacific was described (now known as O. lucimari- munity, picoeukaryotes of the Chlorophyta are nus, type member of clade A), its karyotype and its present worldwide, and can represent a high genome sequence were clearly too divergent for it proportion of eukaryotic plankton, particularly in to interbreed with O. tauri, leading to its designa- coastal regions (Gross 1937; Knight-Jones and tion as O. lucimarinus (Palenik et al. 2007; without Walne 1951; Lovejoy 2007; Manton and Parke valid taxonomic description, this name is currently 1960; Worden et al. 2004; see Massana 2011 a ‘nomen nudum’). Many other strains were then for a recent review). The abundance of partic- isolated from various oceanic areas, and their ular species or ecotypes may be governed by ribosomal gene sequences confirmed their classi- their adaptations to the local environment, and fication in four clades. These clades may represent cryptic species may exist (Cardol et al. 2008; groups with differing environmental adaptations, Foulon et al. 2008; Jancek et al. 2008; Lovejoy clade B strains being “low light” adapted strains, and Potvin 2010; Rodriguez et al. 2005; Worden whereas the others may be “high light” or “poly- et al. 2009), but little information exists about their valent light” strains (Rodriguez et al. 2005). This population structures and why such sympatric com- association between clades and adaptations was munities of cryptic species of phytoplankton exist. confirmed recently by Demir-Hilton et al. (2011) Grimsley et al. (2010) showed that genetic recom- who also showed that co-occurrence of both eco- bination between strains, a hallmark of sexual types (which we now know are probably different reproduction in the large sense, must be inferred species) at the same geographical location is rare, in the genealogy of wild-type O. tauri cultures to and that factors explaining clade distribution were explain the distribution and sequences of neutral more complex than irradiance alone. They pro- genetic markers. We are particularly interested in posed that these two “low light” and “high light” the Mamiellophyceae (Marin and Melkonian 2010) “ecotypes” might better be described as oceanic because (i) they are distributed worldwide, as and coastal clades/species, respectively. Until now, documented by sequence data from analyses of the two clades C and D have only been found in environmental DNA extractions, especially for the the Mediterranean whereas strains of the two other genera Bathycoccus, Micromonas and Ostreococ- clades A and B have been isolated from various cus (Massana 2011; Slapeta et al. 2006; Vaulot oceanic origins. et al. 2008; Viprey et al. 2008) in the order Mamiel- In order to describe the population structure lales, (ii) several complete genome sequences of of a species, numerous wild-type individuals or species from this group are available (reviewed in clones should be isolated to assess the level Piganeau et al. 2011a) permitting detailed phyloge- of intraspecific polymorphism. Different, indepen- netic and evolutionary comparisons to be made, (iii) dently isolated, wild-type clones of Ostreococcus they can be grown easily as clonal cultures, facilitat- spp. may belong to any one of four phylogenetically ing genetic and physiological analyses. Numerous distinct clades that certainly represent different examples of these three genera are maintained in species, because the extent of the differences culture collections (Vaulot et al. 2004). seen in their genome characteristics such as the The genus Ostreococcus was initially described level of sequence homology (Jancek et al. 2008; using one Mediterranean strain, Ostreococcus tauri Palenik et al. 2007) or the number and size of (Chrétiennot-Dinet et al. 1995; Courties et al. chromosomes, preclude the possibility of conven- 1994). Subsequently, several more strains pre- tional meiosis occurring. Since they may show no senting a similar morphology were isolated, but clear phenotypical differences, even when exam- DNA sequence analysis of their small subunit ined by electron microscopy, molecular markers ribosomal RNA gene including the more variable must be used to describe individuals and to deter- ITS sequences (two internal transcribed spacer mine whether intraspecific genetic exchanges, the regions, separating the three rRNA genes) revealed hallmark of a biologically defined species, occur. that the genus Ostreococcus should be divided in We have already isolated several clonal lines of O. four clades A, B, C and D (Guillou et al. 2004; tauri and inferred that sexual recombination occurs Species Diversity in Unicellular Marine Green Algae 645 by genetic analyses of the segregation of DNA sequence polymorphisms between strains in the genealogy of 19 strains from a natural population (Grimsley et al. 2010). Sexual cycles have not yet been described in the Mamiellophyceae, but the frequency of meiosis is probably as low as it is in wild yeasts (Tsai et al. 2008). This would not be surprising because the genetic systems of these species are rather similar, involving haploid uni- cellular organisms, most likely with defined mating types, dispersed over geographically large regions, often at relatively low cell densities, that grow mainly by clonal division. Here, we present the isolation and culture of more than 30 new clade D strains of Ostre- ococcus, previously thought to be rare, using techniques that specifically favour the growth of very small autotrophic picoeukaryotes. We make detailed electron microscopical examina- tions of individual strains from different clades of Ostreococcus, characterize their karyotypes by pulsed field electrophoresis (PFGE), and present a detailed comparison of ITS2 secondary structures. These morphological and molecular analyses led us to propose