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Phycologia (2013) Volume 52 (2), 182–190

Published 5 March 2013

Morphology and phylogeny of Scrippsiella enormis sp. nov. and S. cf. spinifera (Peridiniales, Dinophyceae) from the China Sea HAIFENG GU*, ZHAOHE LUO, TINGTING LIU

AND

DONGZHAO LAN

Third Institute of Oceanography, SOA, Xiamen 361005, China GU H., LUO Z., LIU T. AND LAN D. 2013. Morphology and phylogeny of Scrippsiella enormis sp. nov. and S. cf. spinifera (Peridiniales, Dinophyceae) from the China Sea. Phycologia 52: 182–190. DOI: 10.2216/12-036.1 The genus Scrippsiella contains approximately 20 species that are widespread in coastal and oceanic areas. Classification of Scrippsiella traditionally relied on cyst morphology because the plate pattern was rather conserved. A new species, S. enormis sp. nov. was obtained by incubating a noncalcified cyst from sediments collected in the East China Sea. The vegetative cells consisted of a conical-convex epitheca and a round hypotheca with the plate formula of po, x, 4 0 , 3a, 7 00 , 6c (5cþt), 5s, 5000 , 2 0000 . It differed from other Scrippsiella species by possessing an asymmetrical 1 0 and generating noncalcareous, spherical cysts with paratabulation. Phylogenetic analyses based on internal transcribed spacer (ITS) and 5.8S rDNA sequences revealed that S. enormis was nested within the Calciodinellum clade. In addition, two strains of S. cf. spinifera (strains SSFC02, SSFC03) were obtained by incubating calcareous cysts from sediments collected in the South China Sea. They shared identical ITS sequence and formed a sister clade of the S. trochoidea species complex, suggesting little phylogenetic significance of antapical spines in Scrippsiella. KEY WORDS: Calcareous dinoflagellates, Cysts, ITS rRNA, Scrippsiella enormis, Scrippsiella spinifera

INTRODUCTION The Thoracosphaeraceae (Peridiniales, Dinophyceae) includes dinophytes that produce calcareous coccoid stages (cysts) during their life histories as well as their noncalcareous relatives (Elbrächter et al. 2008). Two systems are currently used to classify species of Thoracosphaeraceae: one is neontological, based on thecal plates, and the other is paleontological. The paleontologists use cyst characters, such as archeopyle/operculum morphology (Streng et al. 2004) and cyst wall ultrastructure (Keupp 1991), for identification. Unification of neontological and paleontological names has been proposed (Montresor et al. 2003; Elbrächter et al. 2008), but the cyst–theca relationship of many species remains to be clarified. Scrippsiella Balech ex Loeblich is one of the most important genera among the Thoracosphaeraceae. Scrippsiella species are abundant in plankton assemblages and some may form blooms (Honsell & Cabrini 1991; Hallegraeff 1992; Ishikawa & Taniguchi 1996). Moreover, Scrippsiella cysts are dominant in recent sediments of many coastal areas (Morquecho & Lechuga-Devéze 2005; Teodora Satta et al. 2010; Gu et al. 2011). Scrippsiella contains one fossil species and approximately 20 extant species (Balech 1963; Horiguchi & Chihara 1983; Indelicato & Loeblich 1985, 1986; D’Onofrio et al. 1999; Janofske 2000; Meier et al. 2002; Montresor et al. 2003; Attaran-Fariman & Bolch 2007; Gu et al. 2008). A coccoid stage is produced for as many as 18 Scrippsiella species. Two species, S. donghaienis H. Gu and S. hangoei (J. Schiller) J. Larsen, produce organic cysts (Kremp & Parrow 2006; Gu et al. 2008); whereas, the remaining species produce various calcareous cysts. Cysts * Corresponding author ([email protected]).

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are commonly ornamented with calcareous spines, e.g. S. trochoidea (F. Stein) A. R. Loeblich, S. trifida Lewis, S. regalis (Gaarder) Janofske, S. spinifera Honsell & Cabrini, S. precaria Montresor & Zingone, S. ramonii Montresor, and S. irregularis Attaran-Fariman & Bolch (Lewis 1991; Janofske 2000; Montresor et al. 2003). Conversely, the cysts of S. rotunda Lewis, S. crystallina Lewis, S. lachromosa Lewis, and S. infula (Deflandre) Montresor (¼ Calcigonellum infula Deflandre) are relatively smooth (Lewis 1991; Montresor et al. 2003; Head et al. 2006). In contrast to the variation in cyst forms, the thecal plate pattern of Scrippsiella is rather conserved and is even shared by species of Calciodinellum Deflandre and Pernambugia Janofske & Karwath (D’Onofrio et al. 1999; Meier et al. 2002; Gottschling et al. 2005b). The intercalary plates of most Scrippsiella species are symmetric (bipesioid); although, they are nonsymmetric (cinctioid) in several species (Montresor & Zingone 1988; Montresor 1995; Attaran-Fariman & Bolch 2007). The arrangement of the intercalary plates is an important feature for differentiation of the peridinioid species (Fensome et al. 1993). The phylogeny of Thoracosphaeraceae has been reported previously (D’Onofrio et al. 1999; Gottschling et al. 2005b, 2012). Use of the ultrastructure of the cyst wall as a character trait for the basic classification of calcareous dinoflagellates remains questionable. In contrast, species with combined archeopyles tend to group together (Gottschling et al. 2005b). Species with cinctioid tabulation are separated from bipesioid species (Attaran-Fariman & Bolch 2007; Gu et al. 2011; Zinssmeister et al. 2011). Although phylogenetic analyses of 11 Scrippsiella species have been performed based on molecular sequence data (Gottschling et al. 2005a; Gu et al. 2011), the systematic significance of cyst characters (e.g. calcareous vs noncalcareous, presence or absence of spines) remains unclear.

Gu et al.: S. enormis sp. nov. and S. cf. spinifera from the China Sea

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Figs 1–4. Scrippsiella enormis sp. nov., LM of vegetative cells (strain SSDH21). Scale bars ¼ 5 lm. Fig. 1. Ventral view of a cell with starch grains inside (arrows). N ¼ nucleus. Fig. 2. Ventral view of a cell showing a pyrenoid with starch sheath (arrow). Fig. 3. DAPI-stained cell in ventral view, showing a spherical nucleus (N) and many rod-like chloroplasts (arrows). Fig. 4. DAPI-stained cell in ventral view, showing a slightly elongated nucleus (N).

Herein we report a new Scrippsiella species from the East China Sea that produces noncalcareous cysts. Both theca and cyst morphology were examined in detail, and its internal transcribed spacer (ITS) region sequence was compared with those of other Scrippsiella species. We also report the phylogenetic position of S. cf. spinifera from the South China Sea.

MATERIAL AND METHODS Surface sediment samples were collected from the East China Sea (3080 0 0 00 N, 122844 0 48.1 00 E) on April 19, 2011, from which strain SSDH21 was established, and the South China Sea (21829 0 58.2 00 N, 108813 0 53.2 00 E) on May 4, 2011, from which strains SSFC02 and SSFC03 were established. The sediment samples were stored in the dark at 48C, after which approximately 5 g of wet sediment was mixed with 20 ml of filtered seawater and sonicated for 2 min (100 W) to dislodge detrital particles. The sample was then filtered through a 100-lm sieve and subsequently through a 20-lm sieve. The 20- to 100-lm fraction was resuspended in 1 ml of filtered seawater, and single cysts were isolated and washed through several drops of sterile seawater using drawn-out Pasteur pipettes. Cultures were established using single cysts, and cultures were maintained in f/2-Si medium (Guillard & Ryther 1962) at 208C and illuminated with 90 lmol photons m2 s1 under a 12:12 h L:D cycle (hereafter called ‘standard conditions’). Vegetative cells were examined using a Zeiss Axio Imager microscope (Carl Zeiss, Göttingen, Germany) that was equipped with differential interference illumination, epifluorescence and a Zeiss Axiocam HRc digital camera. For fluorescence microscopy, approximately 1 ml of live, healthy cell culture in midexponential growth phase was transferred to a 1.5-ml microcentrifuge tube, and 4 0 ,6-diamidino-2phenylindole dihydrochloride (DAPI) stain (Sigma-Aldrich, St. Louis, Missouri, USA) was added at a final concentration of 10 lg ml1. The cells were then incubated in the dark at

room temperature for 30 min. The cells were viewed and photographed through a Zeiss Filterset (emission: BP 365– 445; beamsplitter: FT 395). Thecal plates were discerned following the method of Fritz & Triemer (1985). For scanning electron microscopy, midexponential batch cultures were collected by centrifugation at 4500 3 g. The supernatant was removed and the cell pellet was resuspended in 60% ethanol for 1 h at 88C to strip off the outer cell membrane. The cells were centrifuged again and the pellet was resuspended in 40% seawater at 88C for 30 min. Next, the cell pellet was resuspended and fixed at 88C for 3 h with 2.5% glutaraldehyde prepared with f/2-Si medium. Cells were washed twice with f/2 medium and fixed at 88C overnight with 2% OsO4 prepared with filtered seawater. The supernatant was removed, and the cell pellet was applied to a coverslip coated with poly-L-lysine (molecular mass 70,000– 150,000 amu). The cells were allowed to adhere to the coverslip for 30 min, and then the cells were washed for 10 min in a 1:1 solution of distilled water and filtered seawater, followed by a second wash for 10 min using only distilled water. The samples were then dehydrated in an ethanol series (10%, 30%, 50%, 70%, 90%, and three times in 100%, 10 min at each step), critical point dried (K850 Critical Point Dryer, Quorum/Emitech, West Sussex, UK), sputter-coated with gold, and examined using LEO 1530 Gemini SEM (Zeiss/ LEO, Oberkochen, Germany). Plate terminology follows the modified Kofoidian system (Balech 1980). The scanning electron microscopy (SEM) stubs of strain SSDH21 have been deposited at Third Institute of Oceanography, SOA, with the designation of TIO2012PER01. Cysts generated in culture were fixed for 3 h at 88C with 2.5% glutaraldehyde prepared with f/2-Si medium. They were washed twice with f/2 medium and applied to a coverslip coated with poly-L-lysine (molecular mass 70,000– 150,000 amu). The cysts were allowed to adhere to the coverslip for 30 min and then washed for 10 min in a 1:1 solution of distilled water and filtered seawater, followed by a second wash for 10 min in only distilled water. The samples were dehydrated in an ethanol series (10%, 30%, 50%, 70%, 90%, and three times in 100%, 10 min at each step), critical

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Figs 13–16. Scrippsiella enormis sp. nov. Cysts produced in culture (strain SSDH21). Scale bars ¼ 5 lm. Fig. 13. LM of living cyst showing the red body (arrow). Fig. 14. SEM of cyst showing fine filament ornamentation. Fig. 15. SEM of cyst surface showing fine filaments and hidden paratabulation (arrow). Fig. 16. SEM of smooth cyst showing paratabulation.

point dried as above, sputter-coated with gold, and examined using LEO 1530 Gemini SEM. Total algal DNA was extracted from 10 ml of exponentially growing cultures using a plant DNA extraction kit (Sangon, Shanghai, China) according to the manufacturer’s protocol. The total ITS1–5.8S–ITS2 was amplified using ITSA and ITSB primers (Adachi et al. 1996). The PCR protocol was as follows: initial denaturation for 3.5 min at 948C, followed by 35 cycles of 50 s denaturation at 948C, 50 s annealing at 458C, and 80 s extension at 728C, plus a final extension of 10 min at 728C. PCR products were sequenced directly in both directions using the ABI Big-Dye dye-terminator technique (Applied Biosystems, Foster City, California, USA), according to the manufacturer’s recommendations. Multiple sequences were aligned using ‘MUSCLE’ (Edgar 2004) (http://www.ebi.ac.uk/Tools/msa/muscle/) with the default settings. Forty-one strains of Scrippsiella, Calciodinellum, and Pernambugia were included as ingroup and three species of Ensiculifera (Balech) emend. Matsuoka, Kobashi & Gains and Pentapharsodinium Indelicato & Loeblich as outgroups (Table S1). Maximum Likelihood (ML)-based analyses were conducted using ‘RAxML’ (Stamatakis 2006) (http://phylobench.vital-it.ch/raxml-bb/index.php). The Gamma model was selected and 100 bootstraps were carried out. The program JModeltest (Posada 2008) was used to select the most appropriate model of molecular evolution with Akaike information criterion (AIC). This test chose the general time-reversible (GTR) model of substitution (Ro-

driguez et al. 1990) following a gamma distribution shape parameter (0.4350) (GTR þ G). A Bayesian reconstruction of the data matrix was performed with MrBayes 3.0b4 (Ronquist & Huelsenbeck 2003) using the best-fitting substitution model (GTR þ G). Four Markov chain Monte Carlo (MCMC) chains ran for one million generations, sampling every 1000 generations. A majority rule consensus tree was created in order to examine the posterior probabilities of each clade.

RESULTS Scrippsiella enormis H. Gu sp. nov. Figs 1–16 DESCRIPTION : Epitheca conical, hypotheca rounded. Plate tabulation pattern po, x, 4 0 , 3a, 7 00 , 6c (5cþt), 5s, 5000 , 2 0000 . Vegetative cells 15.0–32.5 lm long and 10.0–30.5 lm wide. 1 0 asymmetrical. Cysts spherical (25.0–29.0 lm diameter) and noncalcareous. HOLOTYPE: SEM stub TIO2012PER01 deposited at Third Institute of Oceanography, SOA, Xiamen 361005, China. TYPE LOCALITY:

East China Sea (3080 0 0 00 N, 122844 0 48.1 00 E), 41-m

water depth. ETYMOLOGY : ‘Enormis’ means irregular, and refers to the asymmetry of the first apical plate.

The vegetative cells of Scrippsiella enormis (strain SSDH21) were 15.0–32.5 lm long (mean ¼ 21.8 6 5.1 lm,

Figs 5–12. Scrippsiella enormis sp. nov., SEM of vegetative cells (strain SSDH21). Fig. 5. Ventral view showing the asymmetrical first apical plate (1 0 ) and other plates. Scale bar ¼ 5 lm. Fig. 6. Apical view showing the canal plate (x) and an asymmetrical first apical plate (1 0 ). Scale bar ¼ 2 lm. Fig. 7. Lateral view showing the raised pore plate. Scale bar ¼ 5 lm. Fig. 8. Apical view showing the pentagonal 1a and 3a plates. Scale bar ¼ 5 lm. Fig. 9. Dorsal view showing the hexagonal 2a plate. Scale bar ¼ 5 lm. Fig. 10. Dorsal view showing the pentagonal 2a plate. Scale bar ¼ 5 lm. Fig. 11. Sulcal plates. Sa ¼ anterior sulcal plate, Sd ¼ right sulcal plate, Ss ¼ left sulcal plate, Sm ¼ median sulcal plate, Sp ¼ posterior sulcal plate. Scale bar ¼ 2 lm. Fig. 12. Antapical view of a cell, showing the two slightly unequal antapical plates. Scale bar ¼ 5 lm.

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Figs 17–24. Scrippsiella cf. spinifera. vegetative cells of LM and SEM (strain SSFC03). Fig. 17. LM, ventral view showing the large nucleus (N), three pyrenoids (P), and several antapical spines (arrow). Scale bar ¼ 5 lm. Fig. 18. SEM, ventral view showing the conical epitheca and the trapezoidal hypotheca with antapical spines (arrow). Scale bar ¼ 5 lm. Fig. 19. SEM, dorsal view showing the conical epitheca and the trapezoidal hypotheca. Scale bar ¼ 5 lm. Fig. 20. SEM, apical view showing the narrow and symmetrical first apical plate (1 0 ). Scale bar ¼ 5 lm. Fig. 21. SEM, dorsal view showing the hexagonal 2a plate. Scale bar ¼ 1 lm. Fig. 22. Fluorescence LM, calcofluor stained cell, in dorsal view, showing the pentagonal 2a plate. Scale bar ¼ 5 lm. Fig. 23. SEM of sulcal plates. Sa ¼ anterior sulcal plate, Sd ¼ right sulcal plate, Ss ¼ left sulcal plate, Sm ¼ median sulcal plate, Sp ¼ posterior sulcal plate. Scale bar ¼ 1 lm. Fig. 24. SEM, detail of thecal surface showing pores with concentric ridges (arrows). Scale bar ¼ 1 lm.

n ¼ 50) and 10.0–30.5 lm wide (mean ¼ 20.4 6 5.0 lm, n ¼ 50). The epitheca was conical and slightly convex medially; whereas, the hypotheca was rounded (Fig. 1). Rod-like chloroplasts and pyrenoids with starch sheath were visible under light microscopy (Figs 2, 3). The large nucleus was spherical to slightly elongated and occupied in the central part of the cell (Figs 3, 4). Large starch grains were observed in some cells (Fig. 1). The plate formula was po, x, 4 0 , 3a, 7 00 , 6c (5cþt), 5s, 5000 , 2 0000 (Figs 5–8). The apical pore complex (po) consisted of a round apical pore plate and a long canal plate (x) (Figs 5, 6). The pore plate was raised slightly. The first apical plate (1 0 ) was narrow and asymmetrical. The anterior part of the left side was about 50%–60% as long as that of the

right side (Fig. 5). Three intercalary plates (1a, 2a, and 3a) were present on the dorsal part of the epitheca. Plates 1a and 3a were pentagonal; whereas, plate 2a was hexagonal (Figs 8, 9). Plate 2a was occasionally pentagonal in shape (Fig. 10). Among 48 cells examined, 41 had a hexagonal shaped 2a plate, and seven had a pentagonal shaped one. The cingulum was displaced over about 2/3 of the girdle width. The first cingular plate was narrower than the others and was considered to be the transitional plate (t). The sulcus was deeply excavated and five sulcal plates were present. The anterior sulcal plate (Sa) was hook shaped; the posterior sulcal plate (Sp) was long and large, and it touched the


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plate 2a was pentagonal in shape (10 out of 40 cells) (Fig. 22). The first cingular plate was narrower than the others, and it was considered to be the transitional plate (t). The sulcus comprised five sulcal plates. The anterior sulcal plate (Sa) was five-sided; the posterior sulcal plate (Sp) was long and large, and it touched the cingulum (Fig. 23). There were numerous pores on the thecal surface, and each pore was surrounded by concentric ridges (Fig. 24). The cysts of Scrippsiella cf. spinifera were egg shaped and light brown and had a prominent red body. The cysts were around 30–40 lm long and 26–38 lm wide, and they were covered with slender calcareous spines, each of which was 3– 10 lm long (Fig. 25). Molecular phylogenetic analysis

Fig. 25. Scrippsiella cf. spinifera. LM of live cyst produced in culture showing the red body (arrow) and long spines. Strain SSFC03. Scale bar ¼ 5 lm.

cingulum (Fig. 11). The arrangement of the two antapical plates was slightly asymmetrical (Fig. 12). The cysts of Scrippsiella enormis generated in culture were noncalcareous and similar to the parent cyst. They were spherical with a diameter of 25.0–29.0 lm (mean ¼ 26.1 6 1.2 lm, n ¼ 20) and were filled with pale white and greenish granules. There was always a bright red body inside (Fig. 13). Most cysts were covered with fine filaments under SEM (Figs 14, 15). Paratabulation was observed on smooth cysts (Fig. 16). The archeopyle was not clear.

Scrippsiella cf. spinifera Figs 17–25 The vegetative cells of Scrippsiella cf. spinifera (strains SSFC02 and SSFC03) were 28–42 lm long and 18–30 lm wide. The epitheca was conical and larger than the trapezoidal hypotheca (Figs 17–19). A deep incised sulcus divided the antapical region of the hypotheca into two distinct lobes, and each lobe had one or two spines at its terminal part (Figs 18, 19). The plate formula was po, x, 4 0 , 3a, 7 00 , 6c (5cþt), 5s, 5000 , 2 0000 . The canal plate was about one half the length of plates 2 0 and 4 0 . Plate 1 0 was narrow and exhibited an ortho-arrangement (Fig. 20). Of the three intercalary plates, la and 3a were pentagonal in shape, and 2a was hexagonal in shape (Fig. 21); however, sometimes

Scrippsiella enormis strain SSDH21 shared 93.5% ITS sequence similarity with Scrippsiella sp. (strain SSND07), 90.1% with Scrippsiella sp. (strain SSND14), 83.3% with S. donghaienis (strain SSDH01) and 79.8% with Calciodinellum albatrosianum (Kamptner) Janofske & Karwath (strain M3417). Scrippsiella cf. spinifera strains SSFC02 and SSFC03 shared a nearly identical ITS sequence with strain NIES-684 from Japan (they differed at three positions). Scrippsiella cf. spinifera shared 89.6%, 79.2% similarity with S. trochoidea strains GeoB 214 and STXM01. ML and Bayesian analyses generated two similar trees that differed at only a few topologies. Scrippsiella enormis (strain SSDH21) and Scrippsiella spp. (strains SSND07 and SSND14) formed a group with strong support, and they were nested within the CAL clade (comprising species of Calciodinellum and some Scrippsiella spp.). The two strains of S. cf. spinifera (strains SSFC02, SSFC03) and strain NIES-684 also formed a group with strong support; it was a sister clade to the S. trochoidea species complex (Fig. 26).

DISCUSSION Scrippsiella enormis is morphologically similar to other Scrippsiella species, but it is unusual in possessing an asymmetrical plate 1 0 . The first apical plate (1 0 ) of this kind was previously reported for S. minima Gao & Dodge, S. lachrymosa, and S. tinctoria Indelicato & Loeblich, but there are obvious distinctions. For S. minima, the anterior part of the left side is approximately 85% as long as the right side, and it differs from S. enormis in possessing a large median sulcal plate (Sm) (Gao & Dodge 1991). In S. lachrymosa, the ratio of the anterior part of the left side to the right side is similar to S. enormis, but the anterior part of the right side is much shorter (Lewis 1991). Moreover, both S. minima and S. lachrymosa generate calcareous cysts (Gao & Dodge 1991; Lewis 1991), while the cysts in S. enormis are noncalcareous. Scrippsiella tinctoria has a much wider 1 0 and its pore plate is not raised (Indelicato & Loeblich 1985). Production of resting cysts by S. tinctoria is not known. Prior to this study, only two Scrippsiella species were reported to produce organic cysts: S. hangoei and S. donghaienis. Scrippsiella enormis differs from S. hangoei in the shape of plates 1 0 , 2a, and Sp. The latter species possesses

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Fig. 26. Phylogenetic tree inferred from ITS and 5.8S rDNA sequences based on maximum likelihood (ML) and Bayesian inference (BI). Ensiculifera and Pentapharsodinium were used as outgroups. Numbers on branches are statistical support values (MLbootstrap support/ Bayesian posterior probability). Bootstrap values .50% and posterior probabilities of 0.5 or above are shown. CAL ¼ clade of Calciodinellum and its relatives; PRE ¼ clade of Scrippsiella precaria and its relatives; LAC ¼ clade of Scrippsiella lachrymosa and its relatives. Scale bar ¼ 0.2 nucleotide substitutions per site.

a symmetrical 1 0 and a pentagonal 2a, and its Sp is relatively small and does not touch the cingulum (Larsen et al. 1995). In addition, cysts of S. hangoei are smooth and lack ornaments (Kremp & Parrow 2006). Scrippsiella enormis differs from S. donghaienis in the shape of plate 1 0 , and the latter species generates smooth cysts without a red accumulation body (Gu et al. 2008). Scrippsiella enormis also differs from strains SSND07 and SSND14 in the shape of 1 0 (Gu et al. 2008).

Both SSND07 and SSND14 have also been established from organic cysts, so they might represent undescribed species. Scrippsiella hangoei did not group with other Scrippsiella species in the phylogenetic trees (Gottschling et al. 2005a; present study). Scrippsiella hangoei possesses a pentagonal 2a and its Sp does not touch the cingulum (Larsen et al. 1995). Plate 2a can be variable in many Scrippsiella species, e.g. S. enormis (present study), S. spinifera (Honsell & Cabrini 1991;


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Table 1. Morphological comparison between Scrippsiella spinifera and S. cf. spinifera.

Species Scrippsiella spinifera

Cell width (lm)

Epitheca

Hypotheca

10

2a

30–52

21–36

conical

narrow, ortho-type

hexa (penta)

four-sided and narrow

0.10

28–42

18–30

conical

trapezoidal with 2–3 short spines trapezoidal with 1–2 short spines

narrow, ortho-type

hexa (penta)

five-sided and wide

0.13

1

Scrippsiella cf. spinifera2

1 2

Cingulum width/ cell length

Cell length (lm)

Sa

Honsell & Cabrini (1991). Present study.

present study), and S. sweeneyae (Balech 1959). These findings suggest that this plate is variable and that it may not be significant for systematic studies. On the other hand, the sulcal plates are rather conservative (Balech 1980). All Scrippsiella species except S. hangoei and S. saladense have a long Sp that touches the cingular plate. In contrast, species of Ensiculifera and Pentapharsodinium generally have a short Sp plate, with the only exception being Ensiculifera imariensis S. Kobayashi & Matsuoka. Ensiculifera imariensis appears to have evolved later than species with a short Sp (Gottschling et al. 2005b), which is consistent with the separation of S. hangoei from other Scrippsiella species (Fig. 26). Strains SSFC02 and SSFC03 are morphologically similar to S. spinifera, but they are slightly smaller in size and possess a five-sided Sa plate, a wider cingulum, and only one to two antapical spines (Table 1). Therefore, these strains are tentatively identified as S. cf. spinifera. Scrippsiella strain NIES-684 from Japan and strain CCCM280 (origin not available) have both been determined to be S. sweeneyae (Janofske 2000; Gottschling et al. 2005a). However, CCCM280 is clearly nested with the S. trochoidea species complex and is separated from strain NIES-684 (Gottschling et al. 2005a). Strain NIES-684 possesses a trapezoidal hypotheca and several short antapical spines (Masanobu Kawachi, personal communication) and is morphologically closer to S. spinifera (Honsell & Cabrini 1991) than S. sweeneyae (Balech 1959). Moreover, Strain NIES-684 shares a nearly identical ITS sequence to the Chinese S. cf. spinifera, indicating that it might have been misidentified. However, detailed morphology of strain NIES-684 is not available at the moment. Antapical spines are rare in Scrippsiella species but common in Protoperidinium (Bergh) Balech. Presence or absence of antapical spines was used to differentiate sections within Protoperidinium (Taylor 1976), which was supported by molecular phylogeny (Yamaguchi & Horiguchi 2005). Unlike S. hangoei, S. spinifera clustered with the S. trochoidea species complex, suggesting little phylogenetic significance of antapical spines in Scrippsiella.

ACKNOWLEDGEMENTS We are greatly indebted to Masanobu Kawachi (National Institute for Environmental Studies, Japan) for help with

identification of strain NIES-684. We thank Marc Gottschling, Ken-ichiro Ishida, and two anonymous reviewers for constructive suggestions. This project was supported by the National Scientific-Basic Special Fund (Grant No. 2009FY210400) and National Natural Science Foundation of China (Grant No. 0900081, 41101060).

SUPPLEMENTARY DATA Supplementary data associated with this article can be found online at http://dx.doi/10.2216/12-036.1.s1.

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Received 6 April 2012; accepted 15 November 2012 Associate Editor: Ken-ichiro Ishida