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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Protist, Vol. 163, 91–104, January 2012 http://www.elsevier.de/protis Published online date 15 April 2011 ORIGINAL PAPER Lotharella reticulosa sp. nov.: A Highly Reticulated Network Forming Chlorarachniophyte from the Mediterranean Sea a,b,1 a Shuhei Ota , and Daniel Vaulot a UPMC (Paris 06) and CNRS, UMR 7144, Station Biologique de Roscoff, Place Georges Tessier, 29682 Roscoff, France b Marine Biology, Department of Biology, University of Oslo, P. O . Box 1066 Blindern, NO-0316 Oslo, Norway Submitted December 1, 2010; Accepted February 16, 2011 Monitoring Editor: Michael Melkonian A new chlorarachniophyte Lotharella reticulosa sp. nov. is described from a culture isolated from the Mediterranean Sea. This strain is maintained as strain RCC375 at the Roscoff Culture Collection, France. This species presents a multiphasic life cycle: vegetative cells of this species were observed to be coccoid, but amoeboid cells with filopodia and globular suspended cells were also present in the life cycle, both of which were not dominant phases. Flagellate cells were also observed but remained very rare in culture. The vegetative cells were 9-16 ␮m in diameter and highly vacuolated, containing several green chloroplasts with a projecting pyrenoid, mitochondria, and a nucleus. The chloroplast was surrounded by four membranes possessing a nucleomorph in the periplastidial compartment near the pyrenoid base. According to ultrastructural observations of the pyrenoid and nucleomorph, the present species belongs to the genus Lotharella in the phylum Chlorarachniophyta. This taxonomic placement is consistent with the molecular phylogenetic trees of the 18S rRNA gene and ITS sequences. This species showed a unique colonization pattern. Clusters of cells extended cytoplasmic strands radially. Then, amoeboid cells being born proximately moved distally along the cytoplasmic strand like on a “railway track”. Subsequently the amoeboid cell became coccoid near the strand. In this way, daughter cells were dispersed evenly on the substratum. We also observed that the present species regularly formed a structure of filopodial nodes in mid-stage and later-stage cultures, which is a novel phenotype in chlorarachniophytes. The unique colonization pattern and other unique features demonstrate that RCC375 is a new chlorarachniophyte belonging to genus Lotharella, which we describe as Lotharella reticulosa sp. nov. © 2011 Elsevier GmbH. All rights reserved. Key words: chlorarachniophytes; Lotharella; Mediterranean Sea; RCC375; RCC376; taxonomy. Introduction Chlorarachniophytes are a small algal group of 1 autotrophic or mixotrophic marine coccoid, flagel- Corresponding author; fax +47 22 85 44 38 e-mail [email protected] (S. Ota). late, and amoeboid cells with green plastids (e.g., © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.protis.2011.02.004 Author's personal copy 92 S. Ota, D. Vaulot Ishida et al. 2007). Under the current classifi- as DNA barcoding in accordance with Gile et al. cation system, twelve species and eight genera (2010). have been described, all classified in the Chlo- rarachniophyceae (Calderon-Saenz and Schnetter 1987; Dietz et al. 2003; Geitler 1930; Hibberd Results and Norris 1984; Ishida et al. 1994, 1996, 2000, 2011; Moestrup and Sengco 2001; Ota et al. Morphology 2005, 2007a, 2007b, 2009a, 2009b), and a thir- teenth species will be added soon (Ota et al. Coccoid cells were the predominant life stage in 2011). Molecular phylogenetic studies have shown the culture (Fig. 1A-C). The coccoid cells were ␮ that chlorarachniophytes belong to Rhizaria, which 9-16 m in diameter (mean = 13.2 ␮m, n = 30), set- include foraminiferans, radiolarians, and cerco- tle onto the bottom of a culture vessel, and were zoans, most of which are heterotrophic (e.g., Bass not easily re-suspended after settlement. The coc- et al. 2005; Burki et al. 2010; Cavalier-Smith and coid cells usually possessed a single large vacuole, Chao 2003; Keeling 2001). Chlorarachniophytes, but non-vacuolated cells were also observed in however, acquired photosynthesis secondarily and culture (Fig. 1A). Each cell had two to eight, or possess four-membrane chloroplasts with a nucle- sometimes more, green bilobed chloroplasts and omorph, i.e. a reduced algal endosymbiont nucleus each chloroplast possessed a projecting pyrenoid that resides in the periplastidial compartment that was located between the chloroplast lobes (Hibberd and Norris 1984; Ludwig and Gibbs (Fig. 1B). In early or old stage cultures, chloro- 1989; McFadden et al. 1994). Recent phylogenetic plasts lacking pyrenoids were sometimes observed studies suggest that the host components of chlo- (Fig. 1A). The chloroplasts were located in the rarachniophytes are closely related to cercozoans, parietal region of the cell (Fig. 1B). Occasionally, notably Metromonadea (Filosa) as suggested by an aperture in the lorica (cell wall) was observed Bass et al. (2009) and the secondary plastids orig- through which some cells extended their filopodium inated from a green alga (e.g., Ishida et al. 1999; (Fig. 1C). In middle or older aged cultures, the Rogers et al. 2007; Takahashi et al. 2007). vegetative cell sometimes possessed a vacuole The generic delimitation of the chlorarachnio- containing reddish particles (Supplementary Figure phytes, except for Cryptochlora (Calderon-Saenz S1 for color micrograph). Some large cells (25- and Schnetter 1987) for which ultrastructural data 33 ␮m in diameter) occurred in old cultures; such are unavailable, is defined by a set of ultrastruc- cells always possessed red particles in a vac- tural and life cycle characteristics as follows: (1) uole (Fig. 1E; Supplementary Figure S1 for color ultrastructure of pyrenoid, (2) nucleomorph posi- micrograph). Sometimes the large cells possessed tion, and (3) dominant cell-type stage (Ishida et al. one or more filopodia (Fig. 1F). Fluorescence 1996, 2011; Ota et al. 2007b, 2009b). The life cycle microscopic observation showed that large cells patterns are mainly used as specific diagnostic fea- usually possessed one nucleus and many chloro- tures (Dietz et al. 2003; Ota et al. 2005, 2007a, plasts (Fig. 1G, H). The nature of the cytoplasm in 2009b). the large cells was similar to the normal vegeta- The Roscoff Culture Collection (RCC) contains tive cells or sometimes more granular. These large several chlorarachniophyte cultures (currently ca. coccoid cells also settled onto the substratum like 10 strains), most of which were collected from the normal vegetative cells. Mediterranean Sea during the PROSOPE (PRO- Amoeboid cells were observed in early and ductivité des Systèmes Océaniques PElagiques) mid-stage of cultures (Fig. 1I-N). The amoe- cruise in 1999. These strains are mostly unchar- boid cells were solitary, naked, and variable in acterized and their taxonomic positions are not form, possessing several fine filopodia of vary- ␮ known. Strain RCC365 was recently described as ing length. Amoeboid body (8-11 m in diameter Partenskyella glossopodia, which is the first pico- when rounded; mean = 9.4, n = 10) was lanceolate planktonic chloarachniophyte (Ota et al. 2009b). to narrowly elliptic (Fig. 1I, J) when cells were Here we provide morphological and ultrastruc- migrating along the filopodia, or slightly polygo- tural characterization of another strain, RCC375, nal, circular to roughly elliptical (Fig. 1K–N). We using light and transmission electron microscopy, occasionally observed suspended globular cells and formally describe it as a new chlorarachnio- (11-25 ␮m in diameter; mean = 19.2, n = 9) in mid phyte species, Lotharella reticulosa sp. nov. We or later stage cultures, which were never dominant also determined nucleomorph internal transcribed (Fig. 1O-R). This cytoplasmic structure was differ- spacer (ITS) sequences of RCC375 and RCC376 ent from the normal vegetative cell; cell cytoplasm Author's personal copy A New Chlorarachniophyte, Lotharella reticulosa 93 Figure 1. Light micrographs of Lotharella reticulosa sp. nov. A. Coccoid cells. Projecting pyrenoids (arrows) are visible. Arrowheads indicate chloroplasts lacking pyrenoids. B. Surface view of coccoid cells, showing bilobed chloroplasts possessing a projecting pyrenoid (arrows). C. Filopodium extending through an aperture of a lorica (arrow). D. Large cells (arrows). E. A large cell possessing a red particle (arrowhead). F. A large cell possessing a filopidium (arrowheads). G, H. Differential interference contrast and fluorescence microscopy images of the DAPI stained large cell. I-N. Amoeboid cells, showing varying shapes: narrowly elliptic/linear form (I, J) or slightly polygonal, circular to roughly elliptical (K–N). O. A globular cell. Arrowhead indicates reddish particles P. Surface view of the globular cell, showing cytoplasm is extremely granular and chloroplasts