New Dinoflagellate Species Protoperidinium Haizhouense Sp. Nov

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Phycological Research 2014; 62: 109–124

New dinoﬂagellate species Protoperidinium haizhouense sp. nov. (Peridiniales, Dinophyceae), its cyst-theca relationship and phylogenetic position within the Monovela group

Tingting Liu,1 Haifeng Gu,1* Kenneth Neil Mertens2 and Dongzhao Lan1 1Third Institute of Oceanography, SOA, Xiamen, China, and 2Research Unit for Palaeontology, Ghent University, Ghent, Belgium

which are thecate and reportedly heterotrophic (Gómez SUMMARY et al. 2012). Protoperidinium species play an important role in the marine ecosystem as they feed on a The number of cingular plates has been used to differ- variety of dinoflagellates and diatoms (e.g. Jacobson & entiate Protoperidinium from Peridinium and related Anderson 1986; Naustvoll 2000; Menden-Deuer et al. genera. Protoperidinium is characterized by the pres- 2005). The taxonomic history of Protoperidinium is ence of three cingular plates plus a transitional plate complicated and several authors have reviewed it in (3C+t). However, many Protoperidinium species have detail (e.g. Taylor 1976; Abé 1981; Harland 1982; been described that exhibit different cingular plate Fensome et al. 1993). tabulations. How these species should be classified There are three complementary ways to study the within the genus remains unclear. To address this classification of Protoperidinium species: through mor- question, the phylogenetic relationship of four phological study of the thecate stage, morphological Protoperidinium species, with three or four cingular study of the resting cysts produced during the sexual plates and lacking a transitional plate, were examined phase of the life cycle (Gribble et al. 2009) and more in relationship to other Protoperidinium species. These recently through molecular phylogenetics. The identifi- four species were germinated from cysts deposited cation of the thecate stage of Protoperidinium species in surface sediments collected from the East China is based on the size, shape, presence of apical and/or Sea, the Bohai Sea and the Yellow Sea. Three of the antapical horns/spines, displacement of the cingulum isolated species, P. tricingulatum, P. americanum and and particularly the tabulation pattern (e.g. Hoppenrath P. parthenopes, were described previously. The fourth et al. 2009). The latter has been the prime focus in is here described as P. haizhouense sp. nov. with the classification of Protoperidinium species, and two plate formula Po, X, 4′, 3a, 7′′, 3C, 6S, 5′′′,2′′′′. major classifications of the genus Protoperidinium were Differences in the cyst stages of these four species, proposed (Balech 1974; Abé 1981). which can be taxonomically informative, were com- For the first major classification system by Balech pared. Partial large subunit ribosomal DNA sequences (1974), the focus has been mainly on the number of were obtained by single-cell polymerase chain reaction. cingular plates, the number of anterior intercalary Maximum-likelihood and Bayesian inference showed plates, the shape of the second anterior intercalary that these four species, P. fukuyoi and Islandinium (2a) plate (quadra, penta or hexa), the number of minutum form a monophyletic clade with maximal precingular plates and the shape of the first apical plate support. The genus as a whole, however, appeared (1′ ortho, meta or para). On an intergeneric level, polyphyletic. Our results suggest that the presence/ Balech (1974) used the consistency in the number of absence of a transitional plate is significant in the cingular plates to classify 231 marine species with phylogeny of Protoperidinium. three cingular plates and a transitional plate (3C+t) in the genus Protoperidinium, leaving the freshwater Key words: archeopyle, cysts, large subunit ribosomal species (with five to six cingular plates and without a DNA, Protoperidinium americanum, Protoperidinium transitional plate, 5–6C) in the genus Peridinium. Next, parthenopes, Protoperidinium tricingulatum, Protoperi- in accordance with the classic work by Jörgensen dinium, transitional plate.

*To whom correspondence should be addressed. INTRODUCTION Email: [email protected] Communicating Editor: M. Hoppenrath. Protoperidinium Bergh is one of the largest dinoﬂagel- Received 8 April 2013; accepted 23 September 2013. late genera with 280 species (Gómez 2012), all of doi: 10.1111/pre.12041

(1912), Balech (1974) used the number of anterior P. bolmonense Chomérat et Couté, P. tricingulatum intercalary plates and precingular plates to classify the Kawami, van Wezel, Koeman et Matsuoka, P. americanum genus Protoperidinium into three subgenera: the sub- (Gran et Braarud) Balech, P. parthenopes Zingone genus Protoperidinium, which has seven precingular et Montresor, Archaeperidinium saanichi Mertens, plates and three anterior intercalary plates, the subge- Yamaguchi, Kawami et Matsuoka and P. fukuyoi Mertens, nus Minuscula, which has six precingular plates and Head, Pospelova et Matsuoka were later also included in three anterior intercalary plates and the subgenus this group (Chomérat & Couté 2008; Kawami & Matsuoka Archaeperidinium, which has seven precingular plates 2009; Kawami et al. 2009; Mertens et al. 2013). and two anterior intercalary plates. Additionally the Within the Monovela group, the cysts also have subgenus Testeria was erected by Faust (2006) to distinct shape. Cysts of Archaeperidinium minutum accommodate species with seven precingular plates (Ribeiro et al. 2010; Mertens et al. 2012b) and and one anterior intercalary plate and no apical pore Archaeperidinium saanichi (Mertens et al. 2012b) complex. have 2a archeopyles. On the other hand, cysts of The second classification system by Abé (1981) P. americanum, P. parthenopes, P. tricingulatum, and on the other hand used a different subgeneric P. fukuyoi have compound archeopyles involving three classification and subdivided Peridinium into three apical plates (Lewis & Dodge 1987; Kawami & subgenera (i.e. Protoperidinium, Mesoperidinium and Matsuoka 2009; Kawami et al. 2009; Mertens et al. Veroperidinium) based on the configuration and shape 2013). Similar archeopyle types can be observed in of the posterior sulcal (Sp) plate. These subgenera were cysts belonging to the cyst-defined genus Islandinium further informally divided into groups and further (Head et al. 2001; Pospelova & Head 2002; Potvin divided in sections based on different characteristics of et al. 2013), and Islandinium minutum (Harland et the theca. Reid) Head, Harland et Matthiessen is phylogenetically The cysts produced by Protoperidinium species are related to the Monovela group (Mertens et al. 2013; diverse in morphology and have been considered taxo- Potvin et al. 2013). nomically informative. The use of cysts in classification Three subgroups within the Monovela group were within the genus Protoperidinium was first proposed by recognized on the basis of thecal morphology, cyst Harland (1982) who stressed the importance of the morphology and molecular phylogenetic data: the anterior intercalary plates, which essentially corre- Americanum, Minutum and Monovelum subgroups sponds to the archeopyle of many Protoperidinium (Mertens et al. 2013; Matsuoka & Kawami 2013). Here species (Harland 1982). we describe a new Protoperidinium species that The rise of molecular techniques has provided an belongs to the Monovela group, lacks a transitional important accessory tool in establishing phylogenies. plate and has a cyst with a compound archeopyle. We The constructed molecular phylogenies have largely explore the systematic importance of the transitional supported the classification of species into discrete plate, and the archeopyle type and compare with taxonomic sections as proposed by Jörgensen (1912) a phylogeny based on large subunit (LSU) rDNA (based on the shape of the 1′ and 2a plate) (Yamaguchi sequences. et al. 2006), but did not support the justification of subgenera within Protoperidinium as proposed by Jörgensen (1912) (based on the number of anterior MATERIALS AND METHODS intercalary plates) (Gribble & Anderson 2006; Yamaguchi et al. 2006; Ribeiro et al. 2010) and Abé Sampling, treatment and germination (1981) (based on the shape of Sp plate). experiment Over the last few years, a particular group of Protoperidinium species, the so-called Monovela group, Surface sediment samples were collected with small has received increased interest. Thecate stages in this boats from six sites (Fig. 1, geographical coordinates group were characterized by a flagellar fin covering the are provided in Table 1) using a grab sampler sulcal area, a large anterior sulcal plate (Sa) and a V-shaped deployed in the Yellow Sea, the Bohai Sea and East Sp, and this group was informally classified within Abé’s China Sea from April 2011 to May 2011. Samples subgenus Veroperidinium (Abé 1981). P. monovelum were stored in the dark at 4°C until further treatment. (Abé) Balech was designated as the type species, and About 5 g wet sediments were mixed with filtered sea- P. minutum (Kofoid) Loeblich III (now reinstated as water with a salinity of 30 psu in a 20 mL beaker and Archaeperidinium minutum Jörgensen; see Yamaguchi sonicated for 1 min (JY96 Xinzhi, Ningbo, China). The et al. 2011), P. monospinum (Paulsen) Zonneveld et Dale, water slurry was sieved with filtered seawater through P. fusiforme (Abé) Kawami et Matsuoka, P. mutsuensis a20μm nylon mesh. The fraction retained by the (Abé) Balech and P. asymmetricum (Abé) Balech were also sieve was resuspended with 1 mL filtered seawater, included in Monovela group. P. vorax Siano et Montresor, and used for cyst isolation.

Fig. 1. Map of sampling stations, dark circles (1–6) refer to the stations where sediment samples were taken.

Cysts were isolated with a micropipette under a Table 1. Location of sampling stations, their collection area, microscope BX-51 (Olympus, Tokyo, Japan) into 96 μ geographic coordinates and water depth well plates with each well containing 200 L f/2-Si medium (Guillard & Ryther 1962). The plates were − − Station Location Latitude Longitude Water incubated at 20°C, 90 μmol photons · m 2 ·s1 under a depth (m) light:dark cycle of 12:12 h and examined daily with an 1 Yellow Sea 36°00′50.58″ N 120°15′39.84″ E 9.4 inverted microscope AE31 (Motic, Xiamen, China). 2 Yellow Sea 36°04′58.68″ N 120°18′07.02″ E 9.8 3 Yellow Sea 34°48′45.77″ N 119°31′37.64″ E 15.0 Morphology and microscopy 4 East China Sea 29°31′0.12″ N 122°36′47.88″ E 44.0 5 East China Sea 26°34′21.21″N 119°56′50.91″ E 12.0 Both cells and cysts were transferred onto glass slides ′ ″ ′ ″ 6 Bohai Sea 38°56 45.93 N 117°57 16.45 E 16.0 individually upon germination and examined under an

Olympus BX51 light microscope with a digital camera P. haizhouense, P. tricingulatum, P. americanum and (Qimaging, Burnaby, Canada) at × 400 magnification. P. parthenopes as has been commonly observed for Calcofluor white was used to discern plates in vegeta- many other dinoflagellates (Gribble & Anderson 2007; tive cells following the method of Fritz and Triemer Miranda et al. 2012). A single sequence of each species (1985). was aligned with those of related species available For observation by scanning electron microscopy in GenBank with ‘BioEdit’ v7.0.0 first (Hall 1999), (SEM), cells and cysts were fixed with buffered and then with Mafft (Katoh et al. 2005) (http:// glutaraldehyde at a final concentration of 2.5% v/v for mafft.cbrc.jp/alignment/server/). The ciliate of Neospora 1 h. They were transferred to a coverslip coated with caninum was selected as the outgroup. Sequences avail- poly-L-lysine (molecular weight 70 000–150 000) for able in GenBank were not cut and the length of the final 30 min and then washed for 10 min in a 1:1 solution of alignment is 1892 bp. Additionally, all clonal sequences distilled water and filtered seawater, followed by a of P. haizhouense, P. tricingulatum, P. americanum and second wash in distilled water for 10 min. The samples P. parthenopes were aligned with those of related were then dehydrated in a series of ethanol (10, 30, species with ‘BioEdit’ v7.0.0 first (Hall 1999), and then 50, 70, 90% and 3× 100%, 10 min at each step), with Mafft (Katoh et al. 2005) (http://mafft.cbrc.jp/ critical point dried (K850 Critical Point Dryer, Quorum/ alignment/server/). Peridinium willei was selected as the Emitech, West Sussex, UK), sputter-coated with gold, outgroup. and examined using LEO 1530 Gemini SEM (Zeiss/ The optimal model was chosen using JmodelTest LEO, Oberkochen, Germany). The Kofoidian system (Posada 2008). A general time reversible model (GTR (Taylor 1980; Fensome et al. 1993) was used for the +I + G) was selected by AIC. Maximum likelihood-based designation of the thecal plate formula. analyses were conducted using ‘RAxML’ v7.2.6 (Stamatakis 2006) with the best-fitting substitution Single-cell PCR amplification model on the T-REX web server (Boc et al. 2012). One thousand bootstraps were carried out. A Bayesian Identified cells were rinsed several times in sterilized reconstruction of the data matrix was performed with distilled water, and broken by squeezing the coverslip MrBayes 3.0b4 (Ronquist & Huelsenbeck 2003) using above, and then transferred into a PCR tube. The single the best-fitting substitution model. Four Markov chain cell was used as the template to amplify about 1430 bp Monte Carlo chains ran for 5 million generations, sam- of the LSU rDNA (D1-D6 domains), using the primers pling every 1000 generations with burn in of 10%. A D1R (Scholin et al. 1994) and 28-1483R (Daugbjerg majority rule consensus tree was created in order to et al. 2000). A 50 μL PCR cocktail containing 0.2 μM examine the posterior probabilities of each clade. forward and reverse primer, PCR buffer, 50 μMdATP, dTTP, dCTP, dGTP, 1U of Taq DNA polymerase (Takara, RESULTS Dalian, China) was subjected to 35 cycles using a Mastercycler PCR (Eppendorf, Hamburg, Germany). Morphology The PCR protocol was as follows: initial denaturation for 3.5 min at 94°C, followed by 35 cycles of 50 s dena- Four species were identified after germination experi- turation at 94°C, 50 s annealing at 45°C, and 80 s ments and are described below. Three of these species extension at 72°C, plus a final extension of 10 min were described previously and the fourth was described at 72°C. as a new species. The amplified products were run on a 1% agarose gel. Positive bands were excised and purified using a Species descriptions DNA extraction kit (Sangon, Shanghai, China). The purified PCR products were then cloned into PUC-m Division DINOFLAGELLATA (Bütschli 1885) Fensome vectors (Sangon, Shanghai, China). One to eight clones et al. 1993. were picked up from each cell and sequenced in both Class DINOPHYCEAE Pascher 1914. directions using the ABI Big-Dye dye-terminator tech- Order PERIDINIALES Haeckel 1894. nique (Applied Biosystems, Foster City, CA, USA), Genus Protoperidinium Bergh 1881. according to the manufacturer’s recommendations. Species: Protoperidinium haizhouense T. Liu, H. Gu Sequences were deposited in GenBank with accession et Mertens sp. nov. (Figs 14–19). numbers from KF651012 to KF651051. HOLOTYPE: SEM stub TIO2012PER03 deposited at the Third Institute of Oceanography, SOA, Xiamen Phylogenetic analysis 361005, China. ICONOTYPE: Figures 14–19. There was allelic sequence variation in the partial LSU TYPE LOCALITY: Haizhou Bay, Lianyungang, China rDNA sequences obtained from individual cells of (34°48′45.77″ N, 119°31′37.64″ E).

ETYMOLOGY: ‘haizhouense’ is derived from Haizhou (Fig. 4). The Sd plate is almost rectangular and has a Bay and refers to the geographic area in which the type small wing on the left side, and does not contact the material was collected. cingulum. The Ss plate is rectangular and larger than the Sd plate. The Sm plate is long and narrow, located between Sd and Ss. The Spa plate is small, triangular, and Diagnosis often hidden by the wing of plate Sd (Fig. 10). The Sp Motile cells are subspherical, 20–30 μm long and plate is V-shaped and its posterior end forms a small 20–25 μm wide. The plate tabulation is Po, X, 4′, 3a, indentation between the two antapical plates (1′′′′ and 7′′, 3C, 6S, 5′′′,2′′′′. Plates 1a and 3a are pentagonal, 2′′′′) (Fig. 12). The two antapical plates are wide and whereas plate 2a is heptagonal, much larger and is pentagonal; 1′′′′ and 2′′′′ are symmetrical and of nearly situated mostly in the left part of the theca. The cin- equal size (Fig. 13). The typical plate pattern of gulum consists of three plates without a transitional P. haizhouense is illustrated in Figures 14–19. plate. The posterior sulcal plate (Sp) is V-shaped and its posterior end forms a small indentation between the two antapical plates. Cysts of P. haizhouense are Motile stage dimensions μ ± μ = spherical and brown with a diameter of 25–30 μm, 20–30 m long (mean: 27.4 3.0 m, n 14) and μ ± μ = densely packed with numerous processes 1–3 μm long. 20–25 m wide (mean: 24.9 2.9 m, n 14). The archeopyle is theropylic, formed by a slit corresponding to the parasutures of 2′–4′. Cyst stage description Cysts of P. haizhouense are spherical and dark brown, and densely packed with numerous short, unor- Motile stage description namented processes (Figs 20–22). The processes are Motile cells of P. haizhouense are subspherical without solid, and generally acuminate (Fig. 23), but variable apical and antapical horns (Fig. 2). The cells contain lipid distal ends can be observed at times (Fig. 24). The bodies and are greenish in color. The plate tabulation is process base is circular. The archeopyle is theropylic, Po, X, 4′, 3a, 7′′, 3C, 6S, 5′′′,2′′′′ (Figs 3–9). The cingu- formed by a slit corresponding to the parasutures of lum is located in the equatorial part of the cell and does three apical plates (2′–4′) (Figs 25–28). Live cysts not show displacement (Figs 2,4). The thecal plates are have greenish cell contents. smooth except for numerous pores surrounded by circular rims, that are scattered on the thecal surface including Cyst stage dimensions the apical pore complex (Fig. 5). The apical pore complex The central body diameter is 25–30 μm (mean: 27.9 ± comprises an elliptical pore plate (Po) and a small, rec- 2.3 μm, n = 14), the processes are 1–3 μm long. tangular X platelet (Fig. 5). The ﬁrst apical plate (1′)is asymmetrical, four-sided (ortho-plate), in contact with the Cyst stage distribution X platelet and the anterior sulcal plate (Sa) (Fig. 4). 2′ Cysts of P. haizhouense were found at stations 1–3 is subpentagonal, whereas 3′ and 4′ are subhexagonal. (Fig. 1) in the Yellow Sea. Fourteen cysts were incu- Three anterior intercalary plates are present in the dorsal bated and each gave rise to a single motile cell. side. Plates 1a and 3a are pentagonal and small, whereas plate 2a is heptagonal, much larger and markedly Comments extended to the left part of the theca, and touches 2′′–5′′, The geological preservability of these cysts was 1a, 3a and 3′ (Figs 6–8). 1′′,2′′,5′′ and 6′′ are pentago- demonstrated by their ability to withstand palynological nal, whereas 3′′,4′′ and 7′′ are quadrangular (Figs 4,6–9). treatment (use of cold HCl and HF, following recom- The cingulum consists of three plates and there is no mendations by Mertens et al. 2012a). transitional plate. The ﬁrst cingular plate (C1) is the small- est and its suture (labeled Y sensu Indelicato & Loeblich III 1986) touches the beginning of 2′′ and the end of 1′′′ Protoperidinium tricingulatum Kawami, (Figs 4,10). The C2 plate is the largest occupying the van Wezel, Koeman et Matsuoka whole dorsal part of the cingulum (Figs 6–9). The suture (labeled X sensu Indelicato & Loeblich III 1986) between Motile stage description C2 and C3 plates touches the end of 6′′ and the end of 4′′′ Motile cells of P. tricingulatum are subspherical (Fig. 9). 1′′′,3′′′ and 5′′′ are pentagonal, whereas 2′′′ and (Fig. 29) and slightly compressed in dorsoventral view 4′′′ are quadrangular (Figs 9–11,13). The sulcal plates (Fig. 30). The plate tabulation is Po, X, 4′, 2a, 7′′, consist of an Sa, a left sulcal plate (Ss), a right sulcal plate 3C,?S, 5′′′,2′′′′ (Figs 31–35). The cingulum is located in (Sd), a median sulcal plate (Sm), a posterior accessory the equatorial part of the cell and is slightly ascending. plate (Spa), and a posterior sulcal plate (Sp) (Figs 4, 1′ is of meta-type (Figs 31,32). 1a is pentagonal and 10–11). The Sa plate is quite big, intruding the left part located on the left lateral part of the theca (Fig. 32). 2a of the cingulum but not between the precingular plates is much larger and elongated, situated on the central

Figs 2–13. Motile cells of Protoperidinium haizhouense upon germination of cysts from Lianyungang. 2. A living cell showing the subspherical shape (light microscopy (LM)). 3. A calcofluor stained cell in apical view, showing the apical pore complex (arrow), four apical plates (1′–4′) and three intercalary plates (1a, 2a and 3a) (LM). 4. A calcofluor stained cell in ventral view, showing an ortho 1′ (the first apical plate), a large anterior sulcal plate (Sa) and the suture between C1 (the first cingular plate) and C2 plate (labeled as Y sensu Indelicato & Loeblich III 1986, arrow) (LM). 5. Detail of the apical pore complex, showing an elliptical pore plate (Po), a small, rectangular X platelet and a pore (arrow) (scanning electron microscopy (SEM)). 6–8. Calcofluor stained cells in dorsal view, showing pentagonal 1a and 3a plates, a large heptagonal 2a plate, and a large C2 plate occupying most part of the cingulum part (LM). 9. A calcofluor stained cell in left lateral view, showing the suture between C2 and C3 (labelled as X sensu Indelicato & Loeblich III 1986, arrow) located at the junction of the fourth and fifth postcingular plates (4′′′,5′′′) (LM). 10. Ventral view of a cell, showing a left sulcal plate (Ss), a right sulcal plate (Sd), a median sulcal plate (Sm), a posterior accessory plate (Spa), a posterior sulcal plate (Sp) and the suture between C1 and C2 (labelled as Y sensu Indelicato & Loeblich III 1986, arrow) located at the junction of 1′′′ and 2′′′ (SEM). 11. A calcofluor stained cell in antapical view, showing the Ss, Sd, Spa and V-shaped Sp plate (LM). 12. Detail of the Sp plate, showing a small indentation at its posterior end (arrow) and a pore inside (arrowhead) (SEM). 13. A calcofluor stained cell in antapical view, showing the two symmetrical antapical plates (1′′′′,2′′′′) with equal size (LM). part of the dorsal epitheca, and touches 2′–4′, 1a, 2′′–6′′ (Fig. 32). The C2 plate is the largest occupying the (Figs 33,34). The cingulum consists of three plates. whole dorsal part of the cingulum (Figs 33,34). The There is no transitional plate. C1 is wide and its suture suture between the plates C2 and C3 is collinear with with the C2 plate faces the right margin of 2′′ and 1′′′ the suture between the 4’” and 5’” plates (Fig. 31). We

Figs 14–19. Diagrams of thecal plates of Protoperidinium haizhouense. 14. Ventral view. 15. Dorsal view. 16. Apical view. 17. Antapical view. 18. Cingular plates. 19. Sulcal plates (Sa: anterior sulcal plate; Ss: a left sulcal plate; Sd: a right sulcal plate; Sm: a median sulcal plate; Spa: a posterior accessory plate; Sp: a posterior sulcal plate).

Figs 20–28. Living and empty cysts of Protoperidinium haizhouense from Lianyungang. 20. A living cyst full of granules (light microscopy (LM)). 21. A living cyst showing short and numerous processes (LM). 22. A living cyst showing short and numerous processes (scanning electron microscopy (SEM)). 23. Detail of the dominant acuminate processes on the cyst surface (SEM). 24. Terminals of processes with variable shape (SEM). 25– 27. Empty cysts, showing the theropylic archeopyle, formed by a slit corresponding to the parasutures of three apical plates (2′–4′) (LM). 28. Drawing of the archeopyle, showing the operculum formed by apical plates 2′–4′.

were unable to verify the exact number of sulcal plates. n = 11), and covered with numerous solid, smooth A large V-shaped Sp plate was observed, and 2′′′′ is processes of 5–7 μm long with capitate terminations remarkably larger than 1′′′′ (Fig. 35). (Fig. 36). The process base is hollow and the cross- section of the process base is circular. The archeopyle Motile stage dimensions is theropylic and traces the outer sutures of μ ± μ = 25–30 m long (mean: 26.8 1.8 m, n 11), plates 2′–4′ (Fig. 37). 25–30 μm wide (mean: 27.5 ± 1.6 μm, n = 11).

Cyst stage description Cyst stage distribution Cysts of P. tricingulatum are spherical and brown with Cysts of P. tricingulatum were only encountered a diameter of 25–30 μm (mean: 28.2 ± 1.7 μm, at station 6 (Fig. 1) in the Bohai Sea and are the

Figs 29–37. Light micrographs of motile cells and cysts of Protoperidinium tricingulatum. 29. A living cell in ventral view showing the subspherical shape. 30. A living cell in apical view showing compression in dorso-ventral view. 31. A calcofluor stained cell in right lateral view, showing a meta-type 1′, a large anterior sulcal plate (Sa) and the suture between C2 and C3 located at the junction of 4′′′ and 5′′′ (arrow). 32. A calcofluor stained cell in left lateral view, showing a meta-type 1′, a small pentagonal 1a and the suture between C1 and C2 located at the junction of 1′′′ and 2′′′ (arrow). 33. A calcofluor stained cell in dorsal view, showing a large, elongated 2a and a large C2 plate occupying most part of the cingulum. 34. A calcofluor stained cell in apical view, showing the large 2a and C2 plate, and the apical pore complex (arrow). 35. A calcofluor stained cell in antapical view, showing a left sulcal plate (Ss), a right sulcal plate (Sd), a V-shaped posterior sulcal plate (Sp), and two asymmetrical antapical plates (1′′′′,2′′′′) with unequal size. 36. A living cyst full of granules and covered with long spines. 37. An empty cyst showing the theropylic archeopyle, formed by a slit corresponding to the parasutures of three apical plates (2′–4′). dominant species there. Prior to our study, this plates are present on the dorsal side (Figs 40–42). 1a, species was not reported along the coast of China. 3a and 4a are pentagonal, whereas 2a is hexagonal. Eleven cysts were incubated and each yielded a The cingulum consists of four plates without a transi- motile cell. tional plate. We were unable to verify the exact number of sulcal plates. The Sa protrudes between 7′′,1′ and 1′′. A large and V-shaped Sp plate was observed Protoperidinium americanum (Fig. 43). (Gran et Braarud) Balech Motile stage description Motile stage dimensions Motile cells of P. americanum are subspherical and the 25–40 μm long (mean: 33.3 ± 6.2 μm, n = 3) and cell content is greenish in color (Fig. 38). The plate 25–35 μm wide (mean: 31.7 ± 5.3 μm, n = 3). tabulation is Po, X, 4′, 4a, 7′′, 4C,?S, 5′′′,2′′′′ (Figs 39–43). The cingulum is located in the equatorial Cyst stage description part of the cell and does not show displacement Cysts of P. americanum are spherical and pale-brown (Fig. 39). 1′ is of ortho-type (Fig. 39). Four intercalary with a diameter of 25–40 μm (mean: 33.4 ± 5.3 μm,

Figs 38–46. Light micrographs of motile cells and cysts of Protoperidinium americanum. 38. A living cell in ventral view showing the subspherical shape. 39. A calcofluor stained cell in ventral view, showing an ortho 1′. 40– 42. Calcofluor stained cells in dorsal view, showing pentagonal 1a and 3a plates, and hexagonal 2a and 4a plates. 43. A calcofluor stained cell in ventral view, showing a left sulcal plate (Ss), a right sulcal plate (Sd), a V-shaped posterior sulcal plate (Sp). 44. A living cyst with a round protoplasm, showing the periphragm (arrow). 45. An empty cyst showing the folding periphragm (arrow). 46. An empty cyst showing the archeopyle formed by a split along three apical plates (2′–4′).

n = 9) (Fig. 44). They are easily recognized by the dorsal part. We were unable to verify the exact number irregularly folded periphragm (Fig. 45). The archeopyle of sulcal plates. The Sp plate is large and V-shaped is composite corresponding to the apical pore complex (Fig. 53). (APC) and plates 2′–4′ (Fig. 46). The operculum always remained attached to the empty cyst. Motile stage dimensions 25–35 μm long (mean: 30.0 ± 3.6 μm, n = 8) and 25–32 μm wide (mean: 27.6 ± 2.7 μm, n = 8). Cyst distribution Cysts of P. americanum were found at stations 3–5 Cyst stage description (Fig. 1) in the Yellow Sea and East China Sea. Prior to Cysts of P. parthenopes are spherical and pale-brown in our study, this species was not reported in these two color with a diameter of 25–35 μm, including the areas. Only three of nine succeeded in producing motile periphragm (Fig. 54). The periphragm is thin, with an cells. average height of 1.0–4.5 μm (mean: 3.0 ± 1.0 μm, n = 12) and folding was not clearly observed. The archeopyle is saphopylic, and formed along the suture Protoperidinium parthenopes of plates 2′–4′ (Fig. 55). Zingone et Montresor Cyst distribution Motile stage description Cysts of P. parthenopes were found at stations 3 and 5 Motile cells of P. parthenopes are ovoid with a short (Fig. 1) in the Yellow Sea and East China Sea. Prior to apical horn (Fig. 47). The plate tabulation is Po, X, 4′, our study, this species was not reported along the coast 3a, 7′′, 4C,?S, 5′′′,2′′′′ (Figs 48–53). 1′ is rhomboid of China. Twelve cysts were incubated and eight yielded (ortho-type) (Fig. 48). Plate 1a is pentagonal and motile cells. located on the left side of the cell (Fig. 49). Plates 2a and 3a are hexagonal and situated in the mid-dorsal Molecular phylogenetics part of the epitheca (Figs 50–52). The cingulum consists of four plates without a transitional plate (Figs 49– Large subunit rDNA sequences were obtained from 51). Of these, C2 is the largest, occupying the right three cells of each of P. haizhouense, P. tricingulatum,

Figs 47–55. Light micrographs (LM) of motile cells and cysts of Protoperidinium parthenopes. 47. A living cell in ventral view showing the subspherical shape. 48. A calcofluor stained cell in ventral view, showing an ortho 1′, a large anterior sulcal plate (Sa), the suture between C1 and C2 plate (arrow). 49. A calcofluor stained cell in dorsal view, showing a pentagonal 1a plate and the suture between C1 and C2 plate (arrow). 50. A calcofluor stained cell in dorsal view, showing the two intercalary plates (2a, 3a) and the suture between C2 and C3 plate (arrow). 51. A calcofluor stained cell in dorsal view, showing the suture between C3 and C4 plate located at the junction of 4′′′ and 5′′′ (arrow). 52. A calcofluor stained cell in apical view, showing the apical pore complex (arrow), three apical plates (2′–4′) and three intercalary plates (1a–3a). 53. A calcofluor stained cell in ventral view, showing a left sulcal plate (Ss), a right sulcal plate (Sd), a V-shaped posterior sulcal plate (Sp). 54. A living cyst full of granules showing the periphragm (arrow). 55. An empty cyst showing the saphopylic archeopyle, formed by three apical plates (2′–4′).

P. americanum and P. parthenopes. All showed intra- groups of the Monovela group, that is, Archaeperidinium genomic polymorphism and the percentage of polymor- (=Minutum), Americanum and Monovelum,nexttothe phic sites was greatest in P. parthenopes (1.83%), benthic dinoﬂagellate Herdmania litoralis. The inclusion followed by P. haizhouense (1.47%), P. americanum of H. litoralis suggests that the Monovela group is (1.26%), and P. tricingulatum (0.9%). polyphyletic. The subgroup Americanum is further Maximum-likelihood (ML) and Bayesian inference composed of two clades: one consists of Islandinium generated two very similar trees that differed only in the minutum, and the other includes Protoperidinium positions of Diplopsalis lenticula and Preperidinium tricingulatum, P. fukuyoi, P. americanum, P. haizhouense meunieri. The family Protoperidiniaceae was well resolved and P. parthenopes (Fig. 56). consisting of four clades, that is, Protoperidinium sensu The phylogenetic tree constructed from multiple stricto clade, Oceanica clade, Diplopsalopsis clade and clonal sequences was very similar to trees constructed Monovela clade. All these clades were monophyletic and using a single sequence of each species, with the received maximal support except for the Diplopsalopsis exception of the position of the Diplopsalopsis clade. clade (Fig. 56). The Monovela clade appeared to be the P. tricingulatum and P. parthenopes appeared to be ancestor to the other three clades with moderate support monophyletic, but clonal sequences of P. americanum (100: ML bootstrap support, 0.7: Bayesian posterior prob- and P. haizhouense intermingled with each other (see abilities). The Monovela clade contained the three sub- Figure S1 in Supporting Information).

Fig. 56. A phylogenetic tree inferred from partial large subunit ribosomal DNA sequences using Bayesian inference. A single sequence of each species of Protoperidinium haizhouense, P. tricingulatum, P. americanum and P. parthenopes was selected. Branch lengths are drawn to scale, with the scale bar indicating the number of the substitutions per site. Numbers on branches are statistical support values to clusters on the right of them (Maximum-likelihood bootstrap support/Bayesian posterior probability). *indicates maximal support. Clades are labeled and marked with vertical lines on the right, with dashed lines indicating subgroups of the Monovela group. New sequences obtained in this study were indi- cated in bold font.

DISCUSSION The position of APC is in the middle of the anterior side of 3′ in P. haizhouense, in contrast to P. vorax where it is Morphology located in the right part of the anterior side of 3′ (Siano & There are only three other species with a similar cell shape Montresor 2005). and three anterior intercalary plates with an elongated 2a: Protoperidinium haizhouense is larger than P. P. vorax, P. bolmonense and P. asymmetricum (Abé) bolmonense, which is 18–22 μm long and 15– Balech (Table 2). P. haizhouense is slightly larger than 18 μm wide and more dorsoventrally ﬂattened P. vorax, which is 16–26 μm in length and 16–25 μmin (Chomérat & Couté 2008). P. haizhouense has pores on width (Siano & Montresor 2005), and has pores on the the APC, which are absent in P. bolmonense (Chomérat APC, cingular and sulcal plates, whereas P. vorax does not & Couté 2008). P. haizhouense also differs from show pores in these areas (Siano & Montresor 2005). The P. bolmonense in the shape and relative position of 2a, position of the suture between C2 and C3 touches the which is hexagonal and touches 2′′–4′′ (Chomérat & end of 6′′ and 4′′′ in P. haizhouense, while in P. vorax it Couté 2008). In addition, P. haizhouense has a small touches the end of 5′′ and 3′′′ (Siano & Montresor 2005). indentation in the posterior margin of Sp, which is not P. haizhouense has a seven-sided 2a, which touches 2′′– observed in P. bolmonense (Chomérat & Couté 2008). 5′′ and its margin of 2a with 1a is straight, whereas that of P. asymmetricum is larger (36 μm long and 34 μm P. vorax is hexagonal and touches 2′′–4′′ only and its wide) (Abé 1927). It has a 2a that is pentagonal and margin of 2a with 1a is bent (Siano & Montresor 2005). touches 2′′ and 3′′ only.

Table 2. Comparison of Protoperidinium haizhouense with related species

Species Protoperidinium Protoperidinium Protoperidinium Protoperidinium haizhouense vorax bolmonense asymmetricum

Shape of the cell Subspherical Spherical Rounded and Spherical dorsoventrally ﬂattened Length (μm) 20–30 16–26 18–22 36 Width (μm) 20–25 16–25 15–18 34 Apical pore complex (APC) Slightly asymmetric Slightly asymmetric Slightly asymmetric Asymmetric Cingular plates 3C 3C 3C 3C+t Sulcal plates 6S 5S 4(?)S 5S Ornamentation Sparse pores bordered by Absent Sparse pores bordered N.A. circular rims by circular rims Pores along margins Present Present Present Absent Pores on APC, cingular Present Absent Present (C, S) Absent (C) and sulcal (S) plates Type of the 1′ plate ortho ortho ortho ortho Position of the suture 6′′ and 4′′′ 5′′ and 3′′′ 6′′ and 4′′′ N.A. between C2 and C3 Anterior end of the Sd plate Rectangle-shaped Pointed Rectangle-shaped N.A. Shape of the Sp plate Triangular, with a protuberance Pointed Rectangle-shaped Rounded at its posterior end Anterior intercalary plates 3a 3a 3a 2a Shape of 2a plate Seven-sided Six-sided Six-sided N.A. Cyst shape (diameter) Spherical (25–30 μm) N.A. N.A. N.A. Ornamentation of cyst With spines 1–3 μm, solid, N.A. N.A. N.A. acuminate and capitate References Present study Siano and Chomérat and Abé (1927); Montresor (2005) Couté (2008) Balech (1974)

N.A., not acknowledged.

P. haizhouense differs from P. tricingulatum by Cells of Chinese P. americanum are identical to those possessing three anterior intercalary plates, whereas described by Lewis and Dodge (1987), and are very the latter species only has two (Kawami et al. similar in size (30–40 μm long, 28–38 μm wide). 2009). Moreover, antapical plates are symmetrical P. americanum cysts from the China Sea are small in P. haizhouense, but extremely asymmetrical in (25–40 μm), compared to those described by Lewis and P. tricingulatum (Kawami et al. 2009). Dodge (1987) (35–52 μm). The operculum remains Cysts produced by P. haizhouense are very similar to attached in the Chinese P. americanum cysts, whereas the cyst-defined species Islandinium brevispinosum the Scottish specimens quickly lost their operculum Pospelova et Head, which is smaller (central body (Lewis & Dodge 1987). However, empty cysts of diameter 18–25 μm) and some processes can be P. americanum from the field without operculum were shorter (0.3–3.0 μm) (Pospelova & Head 2002). The also observed. Cyst size was believed to be the most archeopyle of I. brevispinosum is clearly different: important characteristic to differentiate P. americanum saphopylic and formed by the loss of 2′–4′, while the (35–52 μm) and P. parthenopes (24–34 μm) (Kawami APC and canal plate remain attached to the cyst & Matsuoka 2009); however, our findings of P. (Pospelova & Head 2002). In view of the morphological americanum cysts with a smaller size (25–40 μm) differences between P. haizhouense and related suggest that germination is sometimes needed to differ- species, we describe it as a new species. entiate both species. Cells of Chinese P. tricingulatum are identical to The thecal morphology of Chinese P. parthenopes is those described by Kawami et al. (2009) in terms of identical to that described by Kawami and Matsuoka the shape of 1′, intercalary plates and antapical plates, (2009), and is similar in size (28–36 μm long, only the cells described by Kawami et al. (2009) are 25–35 μm wide) to that described by Zingone and slightly larger: 25–35 μm long and 30–36 μm wide. Montresor (1988), which is 30.0–38.8 μm in length The cysts of P. tricingulatum from China are identical to and 26.0–35.0 μm wide. Chinese P. parthenopes cysts those described by Kawami et al. (2009), although differ from those described by Kawami and Matsuoka their cysts are slightly larger in size (27–34 μm, mean (2009) in terms of the lower height of the periphragm 31 μm) in diameter with the length of the processes (5.4 μm for specimens described by Kawami & being 5–7 μm. Matsuoka 2009). Folding of periphragm is not evident

© 2013 Japanese Society of Phycology Protoperidinium haizhouense sp. nov. 121 in Chinese P. parthenopes cysts. It was difficult to Kawami & Matsuoka 2009; Kawami et al. 2009; differentiate cysts of P. parthenopes from those Mertens et al. 2013; Matsuoka & Kawami 2013), of P. americanum and germination was necessary to which can also be observed in the motile stage of unambiguously identify the species. I. minutum (Potvin et al. 2013). Our new species can be classified in the Americanum subgroup of the Molecular phylogeny Monovela group, and its close phylogenetic relationship with other species of the group support that the sulcal Our results support the classification of four well- area might be a useful taxonomic character (Abé resolved clades within Protoperidiniaceae based on 1981). LSU rDNA sequences (Mertens et al. 2013; Matsuoka Cyst morphology of the four species reported here & Kawami 2013), which is also consistent with all show a compound apical archeopyle, involving phylogenetic analyses based on small subunit (SSU) plates 2′–4′ (Lewis & Dodge 1987; Kawami & rDNA sequences (Mertens et al. 2013; Potvin et al. Matsuoka 2009; Kawami et al. 2009). In contrast, 2013; Matsuoka & Kawami 2013). All these clades archeopyle types of other Protoperidinium and exclusively encompass species with three cingular Archaeperidinium correspond to one or two of the ante- plates plus a transitional plate except the Monovela rior intercalary plates (Harland 1982; Mertens et al. Clade, which also includes species without a transi- 2012b; Potvin et al. 2013). A notable exception is tional plate. Those species that lack a transitional plate Protoperidinium thulesense (Balech) Balech, which has were included in the Americanum subgroup, which is a theropylic archeopyle (Matsuoka et al. 2006). Cysts consistent with previous reports based on SSU rDNA of the species Islandinium minutum and P. fukuyoi also sequences (Mertens et al. 2013; Matsuoka & Kawami have such a compound apical archeopyle (Head et al. 2013). 2001; Mertens et al. 2013; Potvin et al. 2013). The Several hundred copies of the ribosomal RNA locus inclusion of I. minutum and P. fukuyoi within the can be present in tandem in the nuclear genome Americanum subgroup based on SSU rDNA and LSU (Coleman 2007). LSU rDNA intragenomic polymor- rDNA sequences (present study; Mertens et al. 2013; phism has previously been reported in several Potvin et al. 2013) suggest that cysts are taxonomically Protoperidinium species (Gribble & Anderson 2007), informative. Cysts produced by the possibly related and also proved to be present in species shown in this species P. vorax and P. bolmonense, have not yet been study. The occurrence of intragenomic polymorphism reported. might be explained by a high rate of evolution in It is intriguing that a similar compound type of Protoperidinium (Gribble & Anderson 2007) or differ- archeopyle is recorded for the freshwater species ences between mutation rates and DNA repair rates Peridinium wisconsinense (McCarthy et al. 2011 and (Elder Jr & Turner 1995). Our phylogenetic analyses references therein). No information is currently avail- partly support the monophyly of polymorphic copies able on the phylogenetic position of this species. of each species (Gribble & Anderson 2007), but sequences from other faster evolving genes (e.g. inter- Theca characteristics of clade and nal transcribed spacer) might be adopted to differenti- evolutionary significance of the ate closely related species such as P. americanum and transitional plate P. haizhouense. The genus Protoperidinium is characterized by the Relation to the Monovela group presence of three cingular plates plus a transitional plate (3C+t) (Balech 1974). However, our new species, Species assigned to the Monovela group include P. haizhouense has three cingular plates and no Protoperidinium asymmetricum, P. mutsuensis, P. transitional plate, which was also observed for monospinum, Archaeperidinium minutum and Proto- P. tricingulatum, confirming previous observations by peridinium fusiforme based on a flagellar fin covering Kawami et al. (2009). Similar combinations of cingular the sulcal area, a large Sa and a V-shaped Sp (Abé plates were also observed in P. vorax and P. bolmonense 1981). The flagellar fin in the Monovela group is dif- (Siano & Montresor 2005; Chomérat & Couté 2008), ferent from the flagellar fin in the Diplopsalid group, and these species potentially belong to the same since it is rising from the left side of the right sulcal clade. The studied specimens of P. americanum and plate (Sd) (Kawami & Matsuoka 2009, Matsuoka P. parthenopes had four cingular plates and both lacked & Kawami 2013). Recently, P. fukuyoi, P. vorax, a transitional plate, confirming previous observations of P. tricingulatum, P. parthenopes, and P. americanum these species by Lewis and Dodge (1987) and Zingone were also included in this group because they share and Montresor (1988), respectively. similar sulcal plates, especially a large V-shaped Sp The transitional plate is suggested to have been (Siano & Montresor 2005; Chomérat & Couté 2008; formed by the cleavage of the anterior sulcal plate

Fig. 57. Hypothetical evolution of Peri- diniales, accompanied by reduction of cingular plates and cleavage of the anterior sulcal plate (Sa).

(Taylor 1980) and is often recorded in species of the P. bolmonense) is clarified, these Protoperidinium order Peridiniales, such as Scrippsiella Balech ex species in the Americanum subgroup might be trans- Loeblich III, Pentapharsodinium Indelicato et Loeblich ferred to another genus because the number of anterior III and Protoperidinium (Balech 1974; Indelicato & intercalary plates is not taxonomically significant above Loeblich III 1986; D’Onofrio et al. 1999), suggesting species level (Gribble & Anderson 2006; Yamaguchi that this plate might be an apomorphic character. et al. 2007; Ribeiro et al. 2010). Exclusively marine and heterotrophic species with three cingular plates and a transitional plate, formerly grouped in the family Congruentidiaceae (Fensome CONCLUSIONS et al. 1993) likely evolved from the freshwater Peridiniaceae, with five to six cingular plates, which The present study describes a new Protoperidinium thus encompassed a reduction in the number of species, P. haizhouense based on both thecal and cyst cingular plates (Taylor 1980) probably in the late Cre- morphology, which possesses three cingular plates, and taceous period (Fensome et al. 1993, p. 208). Such lacks a transitional plate. Phylogenetic analyses based early marine-freshwater transition is also suggested by on LSU rDNA sequences show that P. americanum, molecular data (Logares et al. 2007). During evolution P. tricingulatum, P. parthenopes, P. fukuyoi, I. minutum, the number of cingular plates might first have been as well as the new species, P. haizhouense,forma reduced to four and subsequently to three, while monophyletic clade (Americanum subgroup) within the cleavage of the Sa plate might have occurred separa- Monovela group, supporting the proposal that the tely (Fig. 57). This may explain the well resolved presence/absence of the transitional plate may be evolu- Americanum and Monovelum subgroup (all lack a tran- tionarily significant. Species of the Americanum subgroup sitional plate) based on LSU sequences (present study; are also characterized by cyst archeopyles which involve Mertens et al. 2013; Matsuoka & Kawami 2013) and three apical plates. Phylogenetic analysis of other SSU sequences (Mertens et al. 2013; Matsuoka & Protoperidinium species without a transitional plate Kawami 2013), which might represent the early ances- needs to be explored in future to fully understand their tors of other Protoperidinium species. Although the evolution. number of cingular plates was reduced, two sutures of ′′′ ′′′ the cingular series between the 4 and 5 plates ACKNOWLEDGMENTS (labeled X sensu Indelicato & Loeblich III 1986) and between the 1′′′ and 2′′′ plates (labeled Y sensu We thank the associate editor Dr Mona Hoppenrath for Indelicato & Loeblich III 1986) are present in all these constructive suggestions.This project was supported by species (present study; Lewis & Dodge 1987; Kawami the National Science Foundation of China (41376170). & Matsuoka 2009; Kawami et al. 2009). When the Kenneth Neil Mertens is a postdoctoral fellow of FWO molecular phylogeny of more species (e.g. P. vorax, Belgium.

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Phycol. 38: 593–601. online version of this article at the publisher’s web-site: Potvin, É., Rochon, A. and Lovejoy, C. 2013. Cyst-theca relationship of the arctic dinoflagellate cyst Islandinium Figure S1. A phylogenetic tree inferred from partial minutum (Dinophyceae) and phylogenetic position based LSU rDNA sequences using Bayesian inference (BI).