Tetraselmis Indica (Chlorodendrophyceae, Chlorophyta), a New Species Isolated from Salt Pans in Goa, India

Eur. J. Phycol. (2013), 48(1):61–78

Tetraselmis indica (Chlorodendrophyceae, Chlorophyta), a new species isolated from salt pans in Goa, India

MANI ARORA1,2, ARGA CHANDRASHEKAR ANIL1, FREDERIK LELIAERT3, JANE DELANY2 AND EHSAN MESBAHI2

1CSIR-National Institute of Oceanography, Dona Paula, Goa, 403004, India 2School of Marine Science and Technology, Newcastle University, Newcastle-Upon-Tyne NE1 7RU, UK 3Phycology Research Group, Biology Department, Ghent University, Krijgslaan 281 S8, 9000 Ghent, Belgium

(Received 14 June 2011; revised 28 July 2012; accepted 23 August 2012)

A new species of Tetraselmis, T. indica Arora & Anil, was isolated from nanoplankton collected from salt pans in Goa (India) and is described based on morphological, ultrastructural, 18S rRNA gene sequence and genome size data. The species is characterized by a distinct eyespot, rectangular nucleus, a large number of Golgi bodies, two types of ﬂagellar pit hairs and a characteristic type of cell division. In nature, the species was found in a wide range of temperatures (48°C down to 28°C) and salinities, from hypersaline (up to 350 psu) down to marine (c. 35 psu) conditions. Phylogenetic analysis based on 18S rDNA sequence data showed that T. indica is most closely related to unidentiﬁed Tetraselmis strains from a salt lake in North America.

Key words: Chlorodendrophyceae; green algae; molecular phylogeny; morphology; pit hairs; Prasinophyceae; taxonomy; Tetraselmis indica; ultrastructure

Introduction (Melkonian, 1990). Cells generally have a single chloroplast, which includes a single conspicuous eye- The Chlorodendrophyceae is a small class of green spot and a pyrenoid (only in Tetraselmis). Sexual algae, comprising the genera Tetraselmis and reproduction is unknown in the class. Some species Scherffelia (Massjuk & Lilitska, 2006; Leliaert et al., form vegetative thick-walled cysts, which may be 2012). Although traditionally considered as members extensively sculptured (McLachlan & Parke, 1967; of the prasinophytes, these unicellular ﬂagellates Norris et al., 1980; Sym & Pienaar, 1993). share several ultrastructural features with the core Most Chlorodendrophyceae are found as plank- Chlorophyta (Trebouxiophyceae, Ulvophyceae and tonic or benthic organisms in marine environments, Chlorophyceae), including closed mitosis and a phyco- where they sometimes occur in dense populations plast (Mattox & Stewart, 1984; Melkonian, 1990; Sym causing blooms in tidal pools or bays. A number of & Pienaar, 1993). A phylogenetic relationship with core

Downloaded by [National Inst of Oceanography ] at 03:04 14 March 2013 species occur in freshwater habitats (John et al., Chlorophyta has been confirmed by molecular data 2002). Some species have been described as endo- (Fawley et al., 2000; Guillou et al., 2004; Marin, 2012). symbionts of marine animals, including Tetraselmis The best-known members of the class are quadri- convolutae which is a facultative symbiont of the flagellate unicells, but some species of Tetraselmis acoel flatworm Symsagittifera (Convoluta) roscoffen- (originally considered to belong to the genus sis (Parke & Manton, 1965; Provasoli et al., 1968; Prasinocladus) may form stalked colonies during Serodio et al., 2011), and an undescribed Tetraselmis some stage of the life cycle (Proskauer, 1950; Norris species that has been isolated from the radiolarian et al., 1980; Sym & Pienaar, 1993). The motile cells of Spongodrymus. Chlorodendrophyceae are generally laterally com- Tetraselmis and Scherffelia are two relatively small pressed, and bear four equal and homodynamic fla- genera. Scherffelia, a genus of about 10 described gella, which emerge from an anterior pit of the cell. species, differs from Tetraselmis in lacking pyrenoids The cells are typically covered by a theca, which is a (Melkonian & Preisig, 1986). Molecular data from thin cell wall formed by extracellular fusion of scales several species will be needed to test if the two genera (Manton & Parke, 1965; Sym & Pienaar, 1993). The form separate clades (Marin, 2012). Scherffelia dubia flagella are covered by hairs and pentagonal scales has been extensively used as a model organism for Correspondence to: Arga Chandrashekar Anil. E-mail: acanil@ examining the structure and formation of the cytoske- nio.org leton, endomembrane system, cell wall and flagella

(Becker et al., 1996, 2001; Wustman et al., 2004). very high in these shallow salt pan pools and the species Tetraselmis includes about 26 currently accepted spe- thrives well even in these conditions. For example, salinity in cies, including taxa previously assigned to the genera the salt pan from which T. indica was isolated reached as high Platymonas, Prasinocladus and Aulacochlamys as 350 psu and as low as 35 psu, while the temperature ranged (Norris et al., 1980; Sym & Pienaar, 1993). between 48.2°C and 28.5°C. Traditional species circumscriptions were based on A sample of the dark green water between the salt lumps in the pan was collected and diluted five-fold with autoclaved light microscopical (LM) characteristics, such as cell seawater. Unialgal clonal cultures were established by dilut- size and shape, structure of anterior cell lobes, chlor- ing the enriched crude culture and micropipetting single cells. oplast morphology, position of the stigma, and shape These cultures were grown and maintained at the National and position of the pyrenoid (West, 1916; Kylin, 1935; Institute of Oceanography, India, in f/2 medium without Carter, 1938; Margalef, 1946; Proskauer, 1950; silicate (Guillard & Ryther, 1962) at 25°C, with a photon Butcher, 1952, 1959). Many of these features were flux density of 80 µmol photons m−2 s−1, and a 16 : 8 h light : found to be variable and hence not useful for species dark cycle. identification. More recently, electron microscopical characters, including the ultrastructure of the pyrenoid Light and confocal microscopy and flagellar hair scales, have been proposed to distin- guish between species (Parke & Manton, 1965; For light microscopy (LM), living cells were observed using McLachlan & Parke, 1967; Melkonian, 1979; an Olympus BX 51 microscope equipped with an Olympus Melkonian & Robenek, 1979; Norris et al., 1980; DP70 digital camera system and Image-Pro software. Cells Hori et al., 1982, 1983, 1986; Throndsen & Zingone, were also observed under an Olympus FluoView 1000- 1988; Becker et al., 1990, 1994; Marin et al., 1993, Confocal laser scanning microscope equipped with a multi- line Argon laser. 1996; Marin & Melkonian, 1994). Although many Tetraselmis species are relatively well characterized morphologically and ultrastructu- Transmission electron microscopy rally, correct species assignment is arduous because of Cells were primarily fixed by rinsing several times in 2% complex methodologies for cellular characterization glutaraldehyde (TAAB, Aldermaston, Berks) in seawater (e.g. the need for electron microscopic observations). containing 0.1 M cacodylate buffer (pH 7.0). The cells were DNA sequence analysis provides a reliable and more post-fixed overnight in 1% cold osmium tetroxide (Agar convenient tool for species delimitation in the genus. Scientific, Stansted, Essex) in 0.1 M cacodylate buffer, rinsed Genetic diversity has been studied based on 18S in buffer for 10 min and then dehydrated in an acetone series rDNA sequence data, especially from temperate (30 min each in 25, 50 and 75% acetone, followed by 100% regions (Lee & Hur, 2009). Diversity of Tetraselmis acetone for 1 h at room temperature). Following dehydration, in the tropics has been much less explored. Several cells were impregnated using an epoxy resin kit (TAAB) for Tetraselmis species are economically important as 1 h each with 25, 50 and 75% resin (in acetone), followed by they are ideal for mass cultivation because of their 100% resin for 1 h, with rotation overnight. The embedding medium was then replaced with fresh 100% resin at room euryhaline and eurythermal nature (Butcher, 1959; temperature and the cells transferred 5 h later to an embed- Fabregas et al., 1984). The genus is widely used in ding dish for polymerization at 60°C overnight. aquaculture facilities as feed for juvenile molluscs, Sections were cut with a diamond knife mounted on a shrimp larvae and rotifers (Kim & Hur, 1998; Park RMC MT-XL ultramicrotome. The sections were stretched & Hur, 2000; Cabrera et al., 2005; Azma et al., 2011). with chloroform to eliminate compression and mounted on

Downloaded by [National Inst of Oceanography ] at 03:04 14 March 2013 In addition, high lipid-containing strains have poten- pioloform (Agar Scientific) filmed copper grids. Sections tial to be used in biofuel production (Laws & Berning, were stained for 20 min in 2% aqueous uranyl acetate 1991; Montero et al., 2011; Grierson et al., 2012 ). (Leica UK, Milton Keynes) and lead citrate (Leica). The A survey of the nanoplankton from salt pans in Goa, grids were examined using a Philips CM 100 Compustage India, revealed the presence of a hitherto undescribed (FEI) transmission electron microscope (TEM) and digital species of Tetraselmis. The aim of this paper was to images were collected using an AMT CCD camera (Deben) characterize this new species through light, electron at the Electron Microscopy Research Services facility, and confocal microscopy, and assess its phylogenetic Newcastle University. relationship by DNA sequence analysis. In addition genome size was estimated using flow cytometry. Scanning electron microscopy For scanning electron microscopy (SEM), cultured cells were Materials and methods sampled during the late exponential growth phase and fixed Collection and culturing in 2% glutaraldehyde (TAAB) in cacodylate and seawater. Cells were allowed to settle on poly-l-lysine coated cover- Material was collected from a pool in salt pans at Panaji, Goa, slips. The coverslips bearing the cell sample were placed in a India. The marine salt pans in this region are systems of coverslip holder and dehydrated in an ethanol series (10 min interconnected ponds, in which there is a discontinuous sali- each in 25, 50 and 75% ethanol, followed by two 15 min steps nity gradient. Salinity and maximum temperature become in 100% ethanol at room temperature). The coverslips were Tetraselmis indica sp. nov. 63

dried in a critical point dryer (BalTec, Reading, UK) and identified strains of official culture collections), in addition to subsequently mounted on stubs with a silver DAG and car- the single available sequence of Scherffelia, and six treboux- bon disc (Agar Scientific). Finally, the cells were sputtered iophycean sequences as outgroups. The alignments used for with gold using a Polaron SEM coating unit and observed this paper are available in the Supplementary information. using a Stereoscan S40 Scanning Electron Microscope Sequences of the two datasets were aligned using (Cambridge Instruments, UK) at the Electron Microscopy MUSCLE (Edgar, 2004), and inspected visually in BioEdit Unit of the Department of Biomedical Science, Newcastle 7.0.5.3 (Hall, 1999). Evolutionary models for the two align- University. ments were determined by the Akaike Information Criterion in JModeltest (Posada, 2008). Both datasets were analysed under a GTR + I + G model with maximum likelihood (ML) Flow cytometry using RAxML (Stamatakis et al., 2008) and Bayesian infer- The DNA content of the species was estimated by flow ence (BI) using MrBayes v3.1.2 (Ronquist & Huelsenbeck, cytometry. Nuclei were released by the injection of 50 µl of 2003). Bayesian inference analyses consisted of two parallel Tetraselmis culture into 450 µl of nuclei isolation buffer runs of four incrementally heated chains each, and 5 million (NIB, described by Marie et al., 2000) twice diluted with generations with sampling every 1000 generations. distilled water. Five microlitres of Micromonas pusilla Convergence of log-likelihood and model parameters, (Mamiellophyceae) culture CCAP 1965/4 (CCMP 1545) checked in Tracer v. 1.4 (Rambaut & Drummond 2007), were added as an internal standard of known genome size was achieved after c. 50 000 generations for both datasets. (15 Mb) (Moran & Armbrust, 2007). The nucleic acid spe- A burn-in sample of 500 trees, well beyond the point of cific stain SYBR Green I (Molecular Probes) was added at a convergence, was removed before constructing the majority final dilution of 1 : 10 000 of the commercial solution. rule consensus tree. Samples were incubated for 15 min before analysis by flow −1 cytometry. Samples were run at a rate of 10 µl min on a Results FACS Aria II flow cytometer (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA) equipped Light microscopical examination of the water with a 488 nm excitation laser and a standard filter setup. revealed a dominance of motile Tetraselmis cells The data were acquired on a logarithmic scale due to the large along with diatoms, including Amphora and Pseudo- difference in genome size between the sample and the refer- nitzschia. Based on the sampling location, this species ence, and the DNA concentration was estimated according to likely prefers hypersaline environments, although it the method proposed by Marie et al. (2000). cannot be excluded that it also occurs in other marine habitats. A clonal strain was examined by LM. A schematic presentation of cells is presented in differ- Molecular phylogenetic analysis ent orientations (Fig. 1), as well as micrographs taken Cultures were grown in 50-ml flasks for 1–2 weeks and cells using LM (Figs 2–11) and SEM (Figs 12–17). recovered by centrifugation at 7000 × g for 10 min. Genomic DNAwas extracted from cell pellets using an Invisorb® Spin Food Kit II, according to the manufacturer’s instructions. The Cell body morphology and ultrastructure fi 18S rRNA gene was ampli ed using universal eukaryotic Cells are slightly compressed, 10–25 µm long, primers (Medlin et al., 1988) and a QIAGEN Fast Cycling 7–20 µm broad and 6.5–18 µm thick. A broad lateral PCR Kit (Qiagen, USA) and Eppendorf Mastercycler PCR fi view shows the cell shape to be oval with the posterior machine (Eppendorf Scienti c, USA). Dye terminator – sequencing using the same primers as in the amplification part wider than the anterior (Figs 2 4), and when step and an Applied Biosystems 3730xl DNA Analyzer viewed from the narrow side, cells are elliptical

Downloaded by [National Inst of Oceanography ] at 03:04 14 March 2013 (Applied Biosystems, USA) were used to obtain nucleotide (straight in the middle with a markedly curved base sequences. The 18S rDNA sequence of 1706 bp has been and apex) (Figs 5, 6). Cells have distinct creases (Figs deposited in GenBank as accession HQ651184. 11, 14 and video S1 of the supplementary material) Two datasets were created for phylogenetic analyses. The that extend along much of the length of the cell wall. ﬁrst one was assembled to assess the phylogenetic position of Cells contain a single lobed chloroplast and a nucleus the new species within the Chlorophyta. This alignment located near the ﬂagellar base (Fig. 18). The chloro- consisted of 44 18S rDNA sequences representing a broad plast is yellow-green in colour, cup-shaped. There is a range of Chlorophyta (Leliaert et al., 2012; Marin, 2012) and conspicuous orange-red eyespot or stigma (Fig. 3). two Streptophyta (Chlorokybus and Mesostigma), which Two large rhizoplasts are present per cell, one asso- were selected as outgroups. A second 18S dataset was used ciated with each pair of basal bodies. Each rhizoplast to examine the phylogenetic position of the new species within the Chlorodendrophyceae with more precision. This branches immediately adjacent to the basal body pairs alignment was created as follows. First, all 18S sequences of (Fig. 18). The nucleus is positioned in the anterior half – Chlorodendrophyceae available in GenBank were down- of the cell and is shield shaped (Fig. 18), 6.5 8.5 µm loaded, aligned using MUSCLE (Edgar, 2004) and a neigh- long and 3.5–4 µm broad, with a characteristic apical bour-joining tree was created using MEGA v5 (Tamura et al., groove and a basal arch as seen in longitudinal 2011). Based on this tree, a reduced alignment was created by sections. The nucleolus is very prominent (Fig. 18). selecting 29 representative sequences from the main clades of A large pyrenoid is located in the chloroplast (Fig. Tetraselmis (with a preference for sequences obtained from M. Arora et al. 64

Fig. 1. Schematic drawings of cells of Tetraselmis indica in broad lateral view, narrow lateral view and apical view, indicating the position and arrangement of major cell components.

19), posterior to the nucleus and is traversed from Numerous mitochondria are present (Fig. 23). several directions by cytoplasmic invaginations. The Electron dense structures are present in the periphery pyrenoid matrix measures 2.6–2.8 × 3.4–3.6 µm in of resting cells (Fig. 24) which appear as orange red longitudinal section and is surrounded by many globules under LM, possibly representing haemato- biconvex or concave starch grains (Fig. 19). In addi- chrome bodies. tion to the starch grains appressed to the pyrenoid, the chloroplast contains numerous starch grains in Flagella and flagellar aperture the stroma (Figs 20, 21); the stigma is located at the level of the pyrenoid. Dictyosomes (Fig. 23), usually The four anterior flagella emerging from the thecal slit 2–8 in number are positioned in a circle near the in the bottom of the apical depression are slightly anterior end of the nucleus and sometimes on the shorter than the cell. The flagellar pit is deep (Figs sides. The endoplasmic reticulum is widely distribu- 27–30), up to about 0.5–1.8 µm, and grooved. It is ted allowing the dictyosome forming face to be turned covered with two types of well-developed pit hairs at in a different direction in relation to the nucleus. distinct positions (Figs 29–31); the first type is present Downloaded by [National Inst of Oceanography ] at 03:04 14 March 2013

Figs 2–11. Light micrographs of T. indica in broad lateral view. 2. Cell with emerging flagella. 3. The positions of the nucleus (upper arrow), pyrenoid (lower arrow) and eyespot (red-brown granules) are visible. 4. Resting cell. 5, 6. Narrow lateral view. 7, 8. Narrow lateral view of a cell attaching to the glass slide via the flagella. 9, 10. Posterior view. 11. Confocal micrograph of a cell showing flagellar aperture (left arrow) and distinct creases (e.g. at right arrow). Scale bars = 10 µm. Tetraselmis indica sp. nov. 65

Figs 12–17. SEM of T. indica. 12. Scatter of cells at low magniﬁcation. 13. Narrow lateral view. 14. Broad lateral view. 15. Posterior view with scales (arrow). 16. Dividing cells that are still enclosed by the parent cell wall. 17. Individual cells. Scale bars = 200 µm (Fig. 12) or 10 µm (Figs 13–17).

on the floor of the cavity and is striated, thick and rod end. They may resume movement in a new direction shaped (Figs 30, 31), while the second type is present without pause. The cells show marked phototaxis. at the extension of the wall bordering the flagellar slit and is curly (Figs 29–31). The flagella emerge from Reproduction the cell in two pairs, each pair ± parallel to the longitudinal flatter sides of the cell in the root position. The Vegetative reproduction is by transverse division of partners of the flagellar pair remain close to one the protoplast into two (Figs 25, 26), three or four another as they emerge from the pit, thereby lying on daughter cells within the parental theca. Cells some- the middle part of the flat side of the cell (Fig. 18). The times divide asymmetrically. Nucleolar and nuclear flagella are hairy and blunt ended and covered by a division are followed by cytoplasmic division. layer of pentagonal scales that are aligned in a com- Daughter cells often develop flagella while still sur- pact layer next to the plasmalemma (Fig. 34). The rounded by the parental theca. flagellar scales in this layer have a low rim, a raised The alga survives during unfavourable periods as central point, and a tetragonal electron translucent cysts, which are capable of rejuvenation and normal zone on the floor of the scale surrounding the protu- development with the return of favourable conditions berance (Fig. 31). The scales are arranged in long- (Figs 35–40). Apical papilla can be seen when the itudinal rows. Each flagellum also bears rod-shaped walls are cast off. Sexual reproduction has not been scales or ‘man scales’ and fine hairs (Figs 31–34). observed. Downloaded by [National Inst of Oceanography ] at 03:04 14 March 2013

Cell covering Genome size A theca of two layers covers the cell body, except for Genome size was estimated by flow cytometry and the flagellar grooves. Each of the two layers of the compared to an internal standard (Micromonas theca has a complex architecture (Fig. 20). A slit at the pusilla CCAP 1965/4, genome size 15 Mbp) base of the flagellar pit becomes everted and appears (Fig. 41). One peak was observed for T. indica of c. as a very short papilla when the walls are cast off (Figs 90 Mbp. 35–40).

Phylogenetic analysis Swimming behaviour Analysis of the Chlorophyta 18S rDNA sequence Under LM, cells may swim for a few minutes before alignment firmly placed T. indica in the settling and attaching to the slide via the flagella. Cells Chlorodendrophyceae clade (Figs 42, 43). Tetraselmis usually swim rapidly in an almost straight line or in a was recovered as paraphyletic with respect to slightly curved path, with the flagella at the forward Scherffelia. Analysis of the Chlorodendrophyceae alignment resulted in a monophyletic Tetraselmis M. Arora et al. 66

Figs 18–26. Thin sections of T. indica, TEM. 18. Detail of a cell sectioned vertically showing the characteristic shield-shaped nucleus Downloaded by [National Inst of Oceanography ] at 03:04 14 March 2013 (N) with its apical groove a basal arch and nucleolus (No),and the pyrenoid (P), chloroplast, vacuoles (V) and rhizoplast (R). 19. The pyrenoid (P) showing cytoplasmic channels. 20. Periphery of cell showing the two layers of the theca (two right arrows) surrounding the cell membrane (left arrow). 21. Starch grain (S) in a chloroplast surrounded by mitochondria (e.g. M) and endoplasmic reticulum (ER, arrows). 22. Eyespot (E). 23. Golgi bodies (G, arrows) and endoplasmic reticulum (ER) distributed on both sides of lobes, with nucleus (N) and a mitochondrion (M). 24–26. Dividing cells. Scale bars = 2 µm (Figs 18, 24–26), 500 nm (Figs 19, 21–23), or 100 nm (Fig. 20).

clade (although with low support) in which T. indica divergent from other Tetraselmis sequences with was placed on a long branch and close to two unidenti- p distances up to 0.080, which exceeds sequence diver- ﬁed strains from the Great Salt Lake, Utah (USA). The gence among the other Tetraselmis strains (excluding 18S sequences from Utah, which were only 1358 bp our new species and the strains from Utah) (maximum (GenBank GQ243429) and 1255 bp (GenBank p distance 0.040). The exact phylogenetic position of FJ546704) long, differed from the sequence from Goa the Indian–American clade was uncertain, with only by 10–12 bp, corresponding to an uncorrected p low support (ML bootstrap value 56%, Bayesian pos- distance of 0.006–0.007. Sequences of T. indica and terior probability 0.91) for a sister relationship with T. the North American strains were found to be very cordiformis. Tetraselmis indica sp. nov. 67

Discussion Marin & Melkonian, 1994). This taxonomic frame- work allows a detailed comparison of morphological The taxonomy and morphology of the genus and ultrastructural features between the isolate from Tetraselmis have been relatively well studied (Parke Goa and described Tetraselmis species (Table 1). The & Manton, 1965; McLachlan & Parke, 1967; presence of several distinct morphological features, in Melkonian, 1979; Melkonian & Robenek, 1979; combination with the results of the molecular phylo- Norris, 1980; Norris et al., 1980; Hori et al., 1982, genetic analyses, supports the recognition of a new 1983, 1986; Throndsen & Zingone, 1988; Becker species of Tetraselmis. et al., 1990, 1991, 1994; Marin et al., 1993, 1996; Downloaded by [National Inst of Oceanography ] at 03:04 14 March 2013

Figs 27–34. Thin sections of T. indica, TEM. 27. Apical view of a cell showing the aperture through which flagella emerge. 28. Broad lateral view of the cell (anterior pointing downwards). 29. Transverse section close to the very top of the cell through the apical aperture, showing the four flagella with their 9 + 2 microtubular pattern, and hairs present on the walls bordering the flagellar pit (e.g. at arrow). 30, 31. Flagellar aperture and detail in longitudinal section, showing the characteristic three types of hairs (at arrows). 32– 34. Oblique, longitudinal and cross-sections of flagella, showing the three types of scales (arrows); the scales of the outer layer overlap the gap between the scales of the inner layer and the two types of hair scales are present outside these two layers. Scale bars = 2 µm (Figs 27, 28), 500 nm (Fig. 30), or 100 nm (Figs 29, 31–34). M. Arora et al. 68

Figs 35–37. Tetraselmis indica: stages of transformation of a motile cell into a resting phase, LM. The only pore or opening in the wall is the slit at the base of ﬂagellar pit; this part becomes everted and appears as a very short papilla when the walls are cast off. Scale bars = 5 µm.

Figs 38–40. Tetraselmis indica, resting cells. 38, 39. LM showing formation of an apical papilla in resting cells. 40. TEM showing concentric rings of discarded walls. Scale bars = 10 µm (Figs 38, 39) or 2 µm (Fig. 40).

Tetraselmis indica Arora & Anil, sp. nov. formam loborum cellulae subsequitur. Nucleus in cellulae parte anteriore, peltatus, 6.5–8.5 µm longus, 3.5– Figs 1–40 4 µm latus, cum apicali canaliculo proprio suo et basi DESCRIPTIO: Algae planktonicae marinae, praeferens formae fornicatae apparentibus in sectionibus longitu- habitat hypersalinae. Cellulae in statu monadoide pler- dinalibus multis; nucleolus maxime prominet. umque paulum compressae, 10–25 µm longae, 7–20 Pyrenoides in cellulae posteriore situm, matrix pyre- µm latae, 6–18 µm altae, bilateraliter symmetricae, a noidis per canaliculos invasa e cytoplasmate oriundos, latere latiore ovales, faciebus latioribus sulco antico matrix pyrenoidalis magna, 2.6–2.8 × 3.4–3.6 µm sec- transverse conjunctis; a latere angustiore ellipticae. tione longitudinali, circumdata granulis amylaceis Species habet rugas distinctas. Chloroplasti ﬂavovir- multis plerumque biconvexis, aliquando latere uno entes, cyathiformes, lobis a dorso; forma chloroplasti concavis. Stigma unicum (interdum stigmata pluria) Downloaded by [National Inst of Oceanography ] at 03:04 14 March 2013

Fig. 41. Flow cytometry analysis of the DNA content of a cell nuclei of T. indica, compared to that of Micromonas pusilla CCAP 1965/4, which has a genome size of 15 Mbp. Relative DNA content (x-axis), no. of events (y-axis). Tetraselmis indica sp. nov. 69

Scenedesmus obliquus X56103 90/1 Ankistrodesmus stipitatus X56100

-/.84 Pseudoschroederia antillarum AF277649 Chlamydomonas reinhardtii AB511834 80/1 AF395438 98/1 Pteromonas angulosa Chlorophyceae Dunaliella parva M62998

55/.90 55/- Stigeoclonium helveticum FN824371 Chaetopeltis orbicularis U83125 -/.80 Oedogonium cardiacum U83133 Marsupiomonas pelliculata HE610136 70/.96 -/.99 100/1 Pedinomonas minor HE610132 Pedinophyceae Chlorochytridion tuberculatum HE610134 Ignatius tetrasporus AB110439 53/.99 Oltmannsiellopsis viridis D86495 Ulvophyceae -/59 100/1 Ulothrix zonata AY 2 7 8 2 17 Halochlorococcum moorei AY19 812 2 -/.82 -/.98 Xylochloris irregularis EU105209 Parietochloris alveolaris EU878373 Microthamnion kuetzingianum Z28974 -/.80 52/.98 Choricystis minor AY 7 6 2 6 0 5 Watanabea reniformis X73991 Trebouxia impressa Z21551 Trebouxiophyceae -/.87 100/1 Prasiola crispa AJ416106 Lobosphaera tirolensis AB006051 Chlorella vulgaris X13688

61/1 80/1 Planctonema sp AF387148 Oocystis solitaria AF228686 61/- 66/1 Tetraselmis indica sp. nov. 100/1 Tetraselmis cordiformis HE610130 Tetraselmis marina HE610131 Chlorodendrophyceae

53/- Tetraselmis striata X70802 Scherffelia dubia X68484

Picocystis salinarum DQ267705 CCMP1205 U40921 67/.98 coccoid prasinophyte Nephroselmis pyriformis X75565 79/1 Pycnococcus provasolii AF122889 Monomastix sp FN562447 80/1 prasinophytes Dolichomastix tenuilepis FN562449 52/.94 Ostreococcus tauri Y15814 52/.92 Crustomastix stigmatica AJ629844 Pyramimonas parkeae FN562443 Prasinoderma coloniale AB058379 Mesostigma viride AJ250109 93/1 Streptophyta (outgroup) Chlorokybus atmophyticus M95612 0.05 subst./site

Fig. 42. Maximum likelihood (ML) tree of the Chlorophyta inferred from 18S rDNA sequences, showing the phylogenetic position of Tetraselmis indica within the Chlorodendrophyceae. ML bootstrap values (> 50) and Bayesian inference (BI) posterior probabilities (> 0.80) are indicated at the branches, respectively. Downloaded by [National Inst of Oceanography ] at 03:04 14 March 2013 aurantiacum conspicuum infra pyrenoidem positum. green, cup-shaped, dorsoventrally lobed, the shape of Corpuscula Golgiana plerumque 2–8, in circulo the chloroplast closely following the shape of the cell locata prope partem nuclei anteriorem et aliquando lobing. Nucleus in the anterior half of the cell, shield- ab lateribus. Reticulum endoplasmaticum late diffu- shaped, 6.5–8.5 µm long and 3.5–4 µm broad with a sum. Mitochondria multa et admodum aucta. characteristic apical groove and a basal arch, both Propagatio non-sexualis ﬁssione est effecta; cellulae visible in many longitudinal sections; nucleolus very ﬁliales in theca parentis sunt bina vel terna vel prominent. Cells containing a pyrenoid located in the quaterna. posterior third of the cell; pyrenoid matrix traversed from several directions by cytoplasmic canaliculi, pyr- DESCRIPTION: Marine planktonic green alga, preferring enoid matrix large, 2.6–2.8 × 3.4–3.6 µm in longitudi- high salinities. Motile cells usually slightly nal section, surrounded by many starch grains which compressed, 10–25 µm long, 7–20 µm wide, 6–18 are mostly biconvex and sometimes concave on one µm thick, bilaterally symmetrical, oval in shape side; one or sometimes several conspicuous orange-red when viewed from broad side, with a single apical eyespots are located below the level of the pyrenoid. furrow passing from one broad face to the other; cells Dictyosomes usually 2–8, positioned in a circle near elliptical when viewed from the narrow side. The the anterior end of the nucleus and sometimes on the species has distinct creases. Chloroplasts yellow- sides. Endoplasmic reticulum widely distributed. M. Arora et al. 70

T. chuii strain SAG1.96, JN903999 freshwater T. chuii strain Ifremer.Argenton, DQ207405 brackish marine T. suecica strain KMMCC P4 / CCAP 66/22 AFJ559377 hypersaline Tetraselmis chuii / suecica clade T. suecica strain KMMCC P9 FJ559381 99/1 T. chuii strain KMMCC P39 / CCAP 8/6, FJ559401

T. tetrathele strain KMMCC P2, FJ517749

59/.89 T. hazennii strain KMMCC P1 / UTEX 171, FJ517748

Tetraselmis sp strain KMMCC P31, FJ559394

Tetraselmis sp strain KMMCC P57, GQ917221

91/1 T. striata strain KMMCC P58, GQ917220

96/1 T. striata strain KMMCC P62, FJ559398 Tetraselmis striata / convolutae clade

T. convolutae strain NEPCC 208, U05039

T. striata strain PCC 443, X70802

T. carteriiformis strain KMMCC P13, FJ559385 50/1 T. carteriiformis strain KMMCC P12, FJ559384 100/1 Tetraselmis carteriiformis / subcordiformis clade T. subcordiformis strain KMMCC P6, FJ559380 Chlorodendro- T. marina strain CCMP 898, HE610131 Tetraselmis marina phyceae

99/1 T. astigmatica strain CCMP 880, JN376804 Tetraselmis astigmatica -/.86 T. “kochiensis” isolate KSN2002, AJ431370

radiolarian symbiont host: Spongodrymus.257, AF166379 100/1 symbiont of the radiolarian Spongodrymus radiolarian symbiont host: Spongodrymus.331, AF166380

100/1 Tetraselmis sp strain MBIC 11125 AB058392

Uncultured clone BL010625.1, AY425300

100/.98 Tetraselmis sp strain RCC 500, AY425299

Uncultured clone KRL01E42, JN090902 100/1 Tetraselmis cordiformis T. cordiformis strain SAG 26.82, HE610130

56/.91 Tetraselmis indica sp. nov. strain MA2011, HQ651184 100/1 Tetraselmis sp isolate GSL026, GQ243429

100/1 Tetraselmis sp isolate GSL018, FJ546704 Scherffelia dubia strain SAG 17.86, X68484

Trebouxia impressa Z21551

Chlorella vulgaris X13688

Lobosphaera tirolensis AB006051 Trebouxiophyceae (outgroup) Watanabea reniformis X73991

-/.91 Xylochloris irregularis EU105209 59/.96 0.05 subst./site Parietochloris alveolaris EU878373

Fig. 43. Maximum likelihood (ML) tree of the Chlorodendrophyceae inferred from 18S rDNA sequences, showing the phylogenetic position of Tetraselmis indica. ML bootstrap values (> 50) and Bayesian inference (BI) posterior probabilities (> 0.80) are indicated at the branches, respectively. Species names are adopted from GenBank or the culture collections. Strain or sample information, and Downloaded by [National Inst of Oceanography ] at 03:04 14 March 2013 habitat type (freshwater/brackish/marine/hypersaline) is provided for each sequence.

Mitochondria well developed and many. Asexual Tetraselmis indica can be distinguished from other reproduction by fission resulting in two, three or four Tetraselmis species on the basis of several features daughter cells within the parental theca. including its hypersaline habitat, the structure and Estimated genome size: 90 Mbp. position of the eyespot, nuclear shape, the kinds and positions of flagellar cavity hairs, numerous dictyo- HOLOTYPE: Permanently resin-embedded strain depos- somes, sequence divergence and overall cell appear- ited in the National Facility for Marine Cyanobacteria, ance (Table 1). Daughter cells often develop flagella Bharathidasan University, Tiruchirappalli, Tamil Nadu, while still surrounded by the parental theca. This 620024, India. (Accession Number BDU GD001). feature is almost unique within the genus Tetraselmis TYPE LOCALITY: Salt pan near Mandovi Bridge, Panaji, and is a major aspect of the diagnosis. Notably, such a Goa (15.500623°N, 73.849046°E). type of cell division is only known from two other species of Tetraselmis, T. subcordiformis and T. impel- HABITAT: Hypersaline to marine (preferring hypersa- lucida (Stewart et al., 1974; Trick, 1979). Typically, line conditions); up to now known only from the type eight Golgi bodies are observed in apical cell sections, locality. with a few more on the sides of the nucleus, which is Downloaded by [National Inst of Oceanography ] at 03:04 14 March 2013 ersli nias.nov. sp. indica Tetraselmis

Table 1. Comparison of various species of the genus Tetraselmis. ND: information not available (certain species were described before the emergence of electron microscopy as a tool to describe ultrastructure, hence information is not available for those characteristics). Some species of Tetraselmis, such as T. kochiensis, T. micropapillata and T. tetrabrachia have not been included because detailed morphological characteristics are lacking.

Known geographical Cell Starch grains Nuclear shape, distribution and shape Apical aperture surrounding the position and Species the type of habitat and size hairs Pyrenoid matrix pyrenoid Golgi bodies Eyespot Chloroplast cell division References

T. indica India (Goa); Slightly compressed, 2 types of hairs, Large, irregular in Convex, irregu- 2–8, located Situated below the Cup shaped with 4 Present in the anterior This study Arora & Anil hypersaline to elliptical to oval in both are abundant. shape, penetrated larly scattered around the level of pyrenoid, anterior lobes, half of the cell, rectangu- marine outline, 10–25 × 7–20 by cytoplasmic flagellar very conspicuous, more than 8 lobed lar, shield shaped with an × 6.5–18 µm. Distinct strands base, a few red-orange posteriorly apical groove and a basal creases divide the cell may be pre- arch. Cells develop fla- into longitudinal sent next to gella while surrounded by segments the nucleus the parental theca. Cells occasionally divide asymmetrically T. alacris Butcher Europe and North Compressed, cuneate Abundant;single Spherical Concave convex 2, located Not conspicuous, Finely lobed Spherical Butcher America; marine in outline, 9–14 µm × type around the up to 2 µm in dia- (1959), Hori 7–10.5 µm flagellar meter, located at et al. (1986) base the level of pyrenoid T. apiculata Europe (France); Slightly compressed, ND Large, spherical ND ND Situated towards Deeply bilobed at ND Butcher (Butcher) Butcher marine broadly elliptical to the anterior por- the anterior end (1959) narrowly oval, 7.5– tion of cell 10.5 × 6.5 × 4.5–5µm T. arnoldii Russia, western Broad side elliptical ND Basal, Spherical, ND ND Conspicuous, Cup shaped, Centrally placed. Proshkina- (Proshkina- Ukraine and to oval, narrow side located in the small, anterior, bilobed at the Lavrenko Lavrenko) Norris, Spain); marine stretched oval, pos- posterior part of subcircular anterior end (1945), Ettl Hori & Chihara terior side wider, cell (1983), Norris 12–15 × 9.6–12.5 µm et al. (1980) T. ascus (Proskauer) Pacific coast of Plants colonial and Hairs absent Large, circular Lens-shaped 5, surround- Conspicuous, Large, forming 4 Spherical and large Proskauer Norris, Hori & North America colony forming an starch grains ing the basal located in the lobes (1950), Hori Chihara and Japan; mar- aseptate stalk, cells body. anterior third of & Chihara ine, growing den- elliptical, 19–30 µm × cell (1974), sely on rocks or 8–16 µm Tanimoto & on shells Hori (1975), Hori et al. (1983) T. astigmatica Norris Pacific coast of Spherical, 11–19 × Sparse, single Large, located in Lens shaped 2–4, sur- Not present Large, invagi- Lobed anteriorly Hori et al. & Hori North America; 7–16 µm type the posterior part rounding the nated with cyto- (1982) brackish or mar- of the cell basal body plasmic canaliculi ine (salt marsh) in the posterior part

(continued) 71 Downloaded by [National Inst of Oceanography ] at 03:04 14 March 2013 .Arora M.

Table 1. Continued tal et Known

geographical Cell Starch grains Nuclear shape, . distribution and shape Apical aperture surrounding the position and Species the type of habitat and size hairs Pyrenoid matrix pyrenoid Golgi bodies Eyespot Chloroplast cell division References

T. bolosiana Norris, Spain; marine Compressed, ND Small, spherical, ND ND Conspicuous, red, Anteriorly ND Margalef Hori & Chihara obovate, 15–22 × located in the elongated bilobed, and then (1946), Norris 10–16 × 6–10 µm posterior part of irregular and et al. (1980) cell perforated T. chuii (chui) Europe and N Compressed, ellipti- Abundant,single Small, irregular in Concave convex 2 Large, conspicu- Finely lobed Lobed anteriorly Butcher Butcher America; marine cal to obovate, 12–16 type shape with angu- ous, located in the (1959), Hori et ×7–10 µm lar outline upper region of al. (1986) pyrenoid T. contracta (N. UK; marine Compressed, broadly ND Basal, medium, ND ND Central to ante- Two large and two ND Carter (1937), Carter) Butcher elliptical, 25 × 17 × 11 oval or irregular. rior, small, small apical lobes Butcher µm. conspicuous (1959) T. convolutae (Parke Europe and Japan; Compressed, shape Hairs absent Conspicuous, 2–4 Concave 2–4 Exceptionally Yellow green, Central, immediately Butcher & Manton) Butcher marine variable, occasionally µm, in posterior towards large, 1–2.3 µm, companulate, with anterior to the pyrenoid (1959), Parke curved, 8–13 × 6–10 third of body, pyrenoid pale orange-red, four lobes extend- & Manton ×4–6 µm. Theca not appearing oval to oblong, ing forward from (1967) stratiﬁed eccentric located in anterior just behind the third of body in middle of body one of the plastid lobes T. cordiformis (N. Cosmopolitan; Compressed, Hairs absent Large, penetrated Biconvex 2–4, around Stigma located in Large, highly reti- Spherical, lobed Stein (1878), Carter) Stein brackish and fresh obovate, 17–19 × from all directions the basal the middle of one culate in the pos- Hori et al. waters 13–16 × 8–11 µm with canaliculi or body of the cell’s broad terior region (1982) cytoplasmic complex sides strands T. desikacharyi France; marine Not compressed, Hairs absent Located poster- Concave convex 2(–4), Very large, 2–3 Cup shaped and Irregular in shape, non- Marin et al. Marin, Hoef-Emden elliptical in broad lat- iorly, surrounded parabasal µm in diameter, divided into > 6 spherical (1996) & Melkonian eral view, 11–13 × by a closed starch located in the lobes anteriorly 9–12 × 7–10 µm sheath and pene- anterior part of trated by cyto- cell plasmic channels T. fontiana Europe: Balearic Cells compressed, ND Located in the ND ND Conspicuous, 4 anterior lobes ND Margalef (Margalef) Norris, Islands and Spain oval, 14–20 × 8–12 × posterior third of small, subcircular, and 4 short pos- (1946); Norris Hori & Chihara 5–6µm cell, rounded, sur- red, located adja- terior lobes et al. (1980), rounded by amy- cent to pyrenoid Ettl (1983) loid (starch) cells (continued) 72 Downloaded by [National Inst of Oceanography ] at 03:04 14 March 2013 ersli nias.nov. sp. indica Tetraselmis

Table 1. Continued

T. gracilis (Kylin) Europe; marine Compressed, broadly ND Conspicuous, Concave con- ND Large, conspicu- Uniformly and ND Butcher Butcher to narrowly elliptical, sub-basal, large, vex, large starch ous, red orange, markedly rugose, (1959) 8–9 × 5.5–7.5 × 5–6.5 spherical with a grains situated in the yellow green, µm U-shaped starch anterior half of the axile sheath cell and well above pyrenoid T. hazeni Butcher Europe: Spain, Compressed, ellipti- ND Basal, cup shaped, ND ND Small, sub-cen- Bright green, cup ND Butcher and USA; marine cal to oval, 13–17 × rather large tral, usually situ- shaped, with 4 (1959) 7–8×4–5µm ated in the upper anterior lobes but part of the non posterior pyrenoid T. helgolandica Helgoland; Compressed, oval, ND Sub-central to Large ND Stigma 3–6, Bright green, ND Butcher (Kylin) Butcher marine 21–24 × 14–15 × 7–9 sub-basal, con- scattered axile, with a sinus (1959) µm spicuous, reaching down to spherical pyrenoid, a shorter posterior lobe, and two longitudinal lateral lobes T. impellucida Puerto Rico; Slightly compressed, Sparse, single Inconspicuous by Pyrenoid lack- ND Stigma pale, Yellow green, cup Large, rounded McLachlan & (McLachlan & marine shape variable, 14–23 type light microscopy, ing a starch shell orange-red, shaped, covering Parke (1967) Parke) Norris, Hori & ×8–17 µm lying posterior to usually single but the peripheral Chihara nucleus, pene- may be multiple, region with a slit trated by cyto- irregular in out- from cell apex to plasmic canaliculi line, position vari- middle of the able but never in body anterior part of the body T. incisa (Nygaard) Russia, Ukraine; Slightly compressed, ND Lacks a pyrenoid ND ND Conspicuous, ND Central Nygaard Norris, Hori & marine 13.5–17.5 × 10.5– round, small (1949), Norris Chihara1 13.5 × 7–9µm et al. (1980), Ettl (1983), Massjuk & Lilitskaya (1999), Massjuk & Lilitska (2006)

(continued) 73 Downloaded by [National Inst of Oceanography ] at 03:04 14 March 2013 .Arora M.

Table 1. Continued

Known

geographical Cell Starch grains Nuclear shape, al et distribution and shape Apical aperture surrounding the position and Species the type of habitat and size hairs Pyrenoid matrix pyrenoid Golgi bodies Eyespot Chloroplast cell division References .

T. inconspicua Europe; marine Slightly compressed, ND Basal, very small, ND ND Conspicuous, in Anterior two ND Butcher Butcher oval in front, elliptical globular, with a the region of pyr- lobed to the centre (1959) in lateral view, 4.5–7 continuous starch enoid, reddish of the cell × 4.5–6 × 3.5–4µm sheath orange T. levis Butcher England; marine Compressed, ovate, Poorly developed Small, irregular in Biconvex 2 Same size as of Finely lobed Non-spherical Hori et al. 9–12 × 6–7.5 µm and scanty shape with angu- pyrenoid, located (1986) lar outline at about the same level T. maculata Butcher Europe, collected Slightly compressed, ND Basal, medium or Small ND Stigma large, con- Yellow green, ND Butcher from salt marsh ovate in front, ellipti- small, usually spicuous, at least rugose or ﬁnely (1959) pools and appar- cal in lateral view, 8–9 with a discontinu- half the size of, granular, anterior ently not com- × 5.5–7.5 × 5–6.5 µm ous starch sheath and close to pyre- two lobed, sinus mon; marine noid, irregularly wide, reaching up rounded, diffuse, to pyrenoid orange T. marina Europe, North Plants unicellular or Hairs absent Large, almost Concave on the Usually 5, in Stigma conspicu- Massive cup Irregular, with a lobe Norris et al. (Cienkowski) Norris, America and colonial with a septate spherical side adjacent to a circle near ous, located per- shaped, located penetrating the pyrenoid (1980), Hori et Hori & Chihara Japan; marine stalk, cells elliptical, the pyrenoid the anterior ipherally at a level peripherally with matrix. Longitudinal al. (1983) 16–20 × 7–8µm matrix end of the between the 4 anterior lobes, division nucleus nucleus and the irregularly lobed pyrenoid posteriorly T. mediterranea France; marine Cells ﬂattened dorsi- ND Spherical to ellip- ND ND Stigma small, Coat-shaped, on Positioned in the anterior Lucksch (Lucksch) Norris, ventrally, rounded tical, in the rear conspicuous, at the one side a nar- end before the middle part (1932), Ettl & Hori & Chihara base, 15–25 × 10–20 third of cell the same position row column of the cell Ettl (1959), µm as pyrenoid releases that Norris et al. passes until to the (1980), Ettl rear end of the cell (1983) T. rubens Butcher Europe; marine Compressed, 8–11 × ND Basal, medium, Concave convex ND Conspicuous, in Green, anterior ND Butcher 5–8 × 4.5–5 µm. globular with a U- the anterior to deeply 2 lobed, (1959) (Similar to T. verru- like starch sheath middle region, sinus long and cosa except reddish orange, dense, narrow, presence due to large of haematochrome) haematochrome T. striata Butcher Europe and Japan; Compressed Poorly developed Small, circular ND 2 Conspicuous, lar- ND Irregular Hori et al. marine elliptical, 7–11 × and scanty ger than pyrenoid (1986) 5.5–7.2 µm matrix, located lateral to the pyrenoid

(continued) 74 Downloaded by [National Inst of Oceanography ] at 03:04 14 March 2013 ersli nias.nov. sp. indica Tetraselmis

Table 1. Continued

T. subcordiformis Norway; marine Compressed, ND Sub-central to Large ND In lower part of Bright green, ND Butcher (Wille) Butcher elliptical, 11–17 × sub-basal, large, the cell near the axile, a shorter (1959) 8–10 µm spherical, pyrenoid posterior lobe and conspicuous two lateral lobes T. suecica (Kylin) Widely distribu- Compressed, ellipti- Single type Spherical, large Concave convex 2 Not conspicuous Cup shaped, Spherical Hori et al. Butcher ted; brackish, cal to obovate, 6–11 × usually simple, (1986) marine 4–8.5 µm rarely bilobed at the posterior part T. tetrathele (West) Europe, widely Compressed, ND Pyrenoid conspic- Large ND Sub-median, Bright green, axile ND West (1916), Butcher distributed and elliptical, 10–16 × uous, large, sub- usually situated in with a narrow Carter (1937), common; marine 8–11 × 4.2–5µm central to sub- the region of sinus reaching to Butcher basal, spherical upper part of pyr- pyrenoid, a (1959) enoid, large, red shorter posterior orange lobe and two lateral lobes T. verrucosa Butcher Europe and Japan; Compressed, ellipti- Poorly covered Spherical, sub- Concave on the Usually 2, Conspicuous and Bright, yellow- Irregularly lobed at the Butcher marine cal in front view with with hairs or bare, basal, or at times side adjacent to rarely three variable in posi- green, massive, posterior part, the lobe (1959), Hori et a deep apical furrow single type central, small, pyrenoid matrix tion but usually with 2 anterior invading the pyrenoid al. (1983) in lateral view, 8.5–10 with a starch located at or lobes near the ×6–6.5 × 4.5–6 µm. sheath of uniform above the middle pyrenoid and 4 or Warty appearance due outline of the cell, orange more sublobes in to irregularly scat- anterior region, tered plastids, starch not lobed grains and other irre- posteriorly gular refractive bodies T. wettsteinii Gulf of Naples; Cells strongly com- Hairs absent 2 or more asym- ND Usually 2 A faint eyespot Single green Present at the anterior part Schiller (Schiller) Throndsen marine pressed, heart shaped, metrically posi- located eccentri- chloroplast of the cell (1913), Ettl et broader than long, tioned pyrenoids cally in the middle al. (1959), 7–9×11–12 µm. surrounded by of the cell Throndsen et Cells have a charac- starch sheath al. (1988) teristic median yel- lowish accumulation body

1 This species has been transferred to the genus Scherffelia,asScherffelia incisa (Nygaard) Massjuk & Lilitska. 75 M. Arora et al. 76

much more than the number reported in other Acknowledgements Tetraselmis taxa (which generally have 2–4 Golgi This document is an output from the UKIERI (UK– bodies). A widely distributed endoplasmic reticulum INDIA Education and Research Initiative) project allows the Golgi bodies to be more widely placed entitled ‘Development of Methodology for Biological fl and allows exibility in the direction of their forming Assessment of Ballast Water Management Systems’ faces. funded by the British Council, the UK Department for Although the taxonomy of the genus has been well Education and Skills (DfES), Office of Science and studied, a comprehensive systematic revision of the Innovation, the FCO, Scotland, Northern Ireland, genus, combining morphological and molecular data Wales, GSK, BP, Shell and BAE for the benefit of the of a large number of species and isolates, is lacking. India Higher Education Sector and the UK Higher Such a study will be indispensable to assess the valid- Education Sector. The views expressed are not necessa- ity of morphology-based species circumscriptions and rily those of the funding bodies. We wish to convey our to examine possible morphological variation across gratitude to Christine Campbell from the CCAP algal taxa. Phylogenetic studies based on 18S sequence culture collection for comparing this new organism with data have shown that different morphospecies (e.g. the existing strains and to Dr Gary Caldwell for the algal T. chuii, T. hazenii, T. suecica and T. tetrathele) cluster culturing facility. We express our sincere thanks to in a single clade of nearly identical sequences, indica- Professor Jakob Wisse, for his kind help in preparing tive of intraspecific morphological variation (Lee & the Latin version of the diagnosis and to Marina Hur, 2009; present study). However, it is likely that Tumanina for English translation of the Ukrainian spe- – these 18S clades may in fact comprise multiple species descriptions. We thank our colleagues at CSIR cies as 18S sequences have been shown to be too National Institute of Oceanography (NIO) and conservative to assess planktonic eukaryotic diversity Newcastle University, especially V. D. Khedekar, Ian Harvey, Nithyalakshmy Rajarajan, Sneha Naik, Carol (Piganeau et al., 2011). More variable molecular mar- ’ kers, such as the rDNA internal transcribed spacer Barnett, Ravidas Naik, Priya D Costa and Jonathan Rand for their help and support. This work was sup- regions or protein coding genes (Verbruggen et al., ported by the Council of Scientific and Industrial 2007; Leliaert et al., 2009; McManus & Lewis, 2011; Research (CSIR), India and British Council, UK. This Friedl & Rybalka, 2012; Krienitz & Bock, 2012) will is a NIO contribution no. 5321. be needed to assess species boundaries within Tetraselmis. Our phylogenetic analyses showed a very close rela- Supplementary information fi tionship between T. indica and unidenti ed Tetraselmis The following supplementary material is available for strains from the Great Salt Lake, Utah (USA) (Posewitz this article, accessible via the Supplementary Content et al., unpublished GenBank data). The position of this tab on the article’s online at http://dx.doi.org/10.1080/ clade could not be determined with high certainty, 09670262.2013.768357 although low support was provided for a sister relation- Supplementary Video, showing the position of the ship with T. cordiformis (the type species of flagellar groove with respect to distinct creases and the Tetraselmis). As has been revealed in previous phylo- overall appearance of the cell of T. indica. genetic studies (e.g. Guillou et al., 2004), the position Nexus files of the alignments used in the phyloge- of Scherffelia dubia is unstable based on 18S rDNA netic analysis. sequence data. Our analysis of the Chlorophyta align-

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