J. Phycol. 46, 743–750 (2010) Ó 2010 Phycological Society of America DOI: 10.1111/j.1529-8817.00851.x DNA BARCODING OF CHLORARACHNIOPHYTES USING NUCLEOMORPH ITS SEQUENCES1 Gillian H. Gile,2 Rowena F. Stern,2 Erick R. James, and Patrick J. Keeling3 Department of Botany, University of British Columbia, 3529-6270 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z4 Chlorarachniophytes are a small group of marine Chlorarachniophytes are a small group of marine photosynthetic protists. They are best known as photosynthetic protists belonging to the phylum examples of an intermediate stage of secondary Cercozoa in the supergroup Rhizaria (Bhattacharya endosymbiosis: their plastids are derived from green et al. 1995, Cavalier-Smith and Chao 1996, Keeling algae and retain a highly reduced nucleus, called a 2001, Nikolaev et al. 2004). They can take the form nucleomorph, between the inner and outer pairs of of amebae, coccoid cells, or flagellates, and many of membranes. Chlorarachniophytes can be challenging them alternate among all of these forms and varia- to identify to the species level, due to their small tions thereof during their complex life cycles size, complex life cycles, and the fact that even (Fig. 1). A major distinguishing feature of the chlor- genus-level diagnostic morphological characters are arachniophytes is their retention of a nucleomorph. observable only by EM. Few species have been for- Chlorarachniophytes acquired their plastid by the mally described, and many available culture collec- secondary endosymbiotic uptake of a green alga, tion strains remain unnamed. To alleviate this and the nucleomorph is a relict nucleus of that difficulty, we have developed a barcoding system endosymbiont that persists between the inner and for rapid and accurate identification of chlorarach- outer pairs of their four membrane-bound plastids niophyte species in culture, based on the internal (Ludwig and Gibbs 1989, Ishida et al. 1997, Rogers transcribed spacer (ITS) region of the nucleomorph et al. 2007). Chlorarachniophyte nucleomorph rRNA cistron. Although this is a multicopy locus, genomes are invariably composed of three chromo- encoded in both subtelomeric regions of each chro- somes ranging in size from roughly 100 to 200 kbp mosome, interlocus variability is low due to gene and bearing rRNA cistrons at each of the chromo- conversion by homologous recombination in this some ends (Silver et al. 2007). The nucleomorph region. Here, we present barcode sequences for has attracted the attention of molecular evolutionary 39 cultured strains of chlorarachniophytes (>80% biologists as a window into processes of endosymbi- of currently available strains). Based on barcode otic organelle integration, such as endosymbiotic data, other published molecular data, and informa- gene transfer, genome compaction, and development tion from culture records, we were able to recom- of protein-targeting systems (Gilson and McFadden mend names for 21 out of the 24 unidentified, 1996, 2002, Archibald et al. 2003, Rogers et al. 2004, partially identified, or misidentified chlorarachnio- Gilson et al. 2006, Gile and Keeling 2008). As a result, phyte strains in culture. Most strains could be knowledge about chlorarachniophytes is heavily assigned to previously described species, but at least biased toward their molecular evolution: the entire two to as many as five new species may be present chloroplast and nucleomorph genomes of Bigelowiella among cultured strains. natans, the model chlorarachniophyte for molecular studies, have been sequenced (Gilson et al. 2006, Key index words: Bigelowiella; Chlorarachnion;cul- Rogers et al. 2007). ture collections; Gymnochlora; internal transcribed The type species, Chlorarachnion reptans Geitler, spacer; Lotharella; Norrisiella; Partenskyella was first described in 1930 by Lothar Geitler, but Abbreviations: bp, base pairs; ITS, internal there were no further studies of chlorarachnio- transcribed spacer phytes until it was rediscovered, described more fully with EM, and brought into culture by Hibberd and Norris in 1981 (Geitler 1930, Hibberd and Norris 1984). Today, seven genera and 12 species have been described, of which nine species from five genera are represented among the 48 strains in 1 public culture collections worldwide. Chlorarachnio- Received 15 May 2009. Accepted 28 April 2010. phyte genera are defined mainly by the morphology 2These authors contributed equally to this work. 3Author for correspondence: e-mail [email protected]. of the bulbous pyrenoid that protrudes from the 743 744 GILLIAN H. GILE ET AL. Fig. 1. Differential interference contrast light micrographs illustrating a diversity of chlorarachniophyte cell types. (A) Chlorarachnion reptans CCCM 449 ameboid cells, (B) Bigelowiella longifila characteristic ameboid cell with single extended filopodium, (C) Gymnochlora stellata CCMP 2057 ameboid cells, (D and E) Lotharella oceanica CCMP 622 flagellate cells, (F) C. reptans CCCM 449 coccoid cell, (G) Lotharella amoeboformis CCMP 2058 ameboid cell, (H) C. reptans CCMP 238 ameboid cell, (I) Bigelowiella natans CCMP 621 flagellate cell, (J) Lotharella vacuolata CCMP 240 thick-walled resting cysts (a sealed pore is visible in the wall of one cell). All scale bars represent 10 lm. plastid toward the center of the cell and is capped morphology in Cryptochlora E. Caldero´n et Schnetter on the cytoplasmic side by a carbohydrate storage (Calderon-Saenz and Schnetter 1987, 1989), and vesicle. The pyrenoid can have a shallow cleft the genus Bigelowiella Moestrup (Moestrup and caused by an invagination of the inner pair of plas- Sengco 2001) was defined by its dominant life-cycle tid membranes, as in Norrisiella S. Ota et Ishida (Ota stage being flagellate cells (pyrenoids of Bigelowiella et al. 2007a); a deep cleft, as in Lotharella Ishida et are invaded by a shallow cleft, as in Norrisiella). Y. Hara (Ishida et al. 1996, 2000, Ota et al. 2005, The principle behind DNA barcoding is to enable 2009a); or a deep cleft that encloses the nucleo- accurate species identification through a short sig- morph, as in Chlorarachnion (Hibberd and Norris nature sequence of DNA (Hebert et al. 2003a). 1984). Otherwise, the pyrenoid can be invaded with Where feasible, barcoding has the advantage of small tubular invaginations of the innermost plastid allowing nonexperts to accurately and rapidly iden- membrane, as in Gymnochlora Ishida et Y. Hara tify species even when diagnostic morphological fea- (Ishida et al. 1996), or it may be lacking entirely, tures are missing (as for immature or damaged as in Partenskyella S. Ota, Vaulot et Ishida (Ota et al. specimens), misleading (as for common convergent 2009b). Two genera are exceptions to this morphologies, such as coccoid green algae), or scheme: there are no EM data concerning pyrenoid not easily observable (as for simple microscopic BARCODING CHLORARACHNIOPHYTES 745 organisms). Chlorarachniophytes exemplify the lat- MATERIALS AND METHODS ter two points in that in their coccoid or flagellate forms they can easily be mistaken for other green- DNA extraction, PCR, and sequencing. Chlorarachniophyte pigmented algae [CCMP 621 B. natans was initially cultures (Table S1 in the supplementary material) were either purchased or donated from the Provasoli-Guillard National misidentified as Pedinomonas minutissima, a chloro- Center for Culture of Marine Phytoplankton (CCMP, West phyte (Moestrup and Sengco 2001)], and their diag- Boothbay Harbor, ME, USA); the Roscoff Culture Collection nostic features can be observed only by EM. (RCC, Roscoff, France); the Plymouth Culture Collection of Moreover, a great many chlorarachniophyte cultures Marine Algae (PCC, Plymouth, UK); the Culture Collection of in available collections have not been identified to Algae at the University of Texas (UTEX, Austin, TX, USA); the the species level and for many years were automati- Culture Collection of Algae, Philipps-University Marburg (Marburg, Germany); the Canadian Centre for the Culture of cally assigned to the type genus Chlorarachnion. Microorganisms (CCCM, Vancouver, Canada); and the Micro- A barcoding system for chlorarachniophytes would bial Culture Collection at the National Institute for Environ- therefore be a useful tool for clarifying the existing mental Studies (NIES, Tsukuba, Japan). Chlorarachniophyte diversity of cultures as well as accurately distinguish- cells from 10 mL of culture were harvested by centrifugation ing chlorarachniophyte species from each other and at 1,150g in an Eppendorf 5415 D benchtop centrifuge from other groups of algae. (Eppendorf, Hamburg, Germany) and subjected to two rounds of snap-freeze treatment with liquid nitrogen. DNA was The most commonly used DNA sequence marker extracted using the DNeasy Plant mini kit (Qiagen, Mississauga, for barcoding is the 5¢ end of the cytochrome c oxi- ON, Canada) according to the manufacturer’s instructions. dase I (cox1) gene. This locus can effectively dis- Previously published primers (Inagaki et al. 1998) failed to criminate species in many animal groups (Hebert amplify the most commonly employed locus for barcoding, the et al. 2003b), as well as red algae (Saunders 2005), 5¢ region of cytochrome c oxidase subunit 1 (cox1) under brown algae (Lane et al. 2007, Kucera and Saun- various reaction conditions. Because cox1 sequences are pub- ders 2008), diatoms (Evans et al. 2007), and the cil- licly available for only two chlorarachniophytes and only two other Cercozoa, primer optimization for this locus in the iate