Morphology, Ultrastructure and Molecular Phylogeny of Cyst-Producing Caladoa Arcachonensis Gen

Home , Durinskia, Kryptoperidiniaceae

European Journal Of Phycology Archimer April 2019, Volume 54 Issue 2 Pages 235-248 https://doi.org/10.1080/09670262.2018.1558287 https://archimer.ifremer.fr https://archimer.ifremer.fr/doc/00489/60112/

Morphology, ultrastructure and molecular phylogeny of cyst-producing Caladoa arcachonensis gen. et sp. nov. (Peridiniales, Dinophyceae) from France and Indonesia

Luo Zhaohe 1, Mertens Kenneth 2, *, Nezan Elisabeth 2, Gu Li 1, Pospelova Vera 3, Thoha Hikmah 4, *, Gu Haifeng 1

1 Minist Nat Resources, Inst Oceanog 3, Xiamen 361005, Peoples R China. 2 IFREMER, LER BO, Stn Biol Marine, Pl Croix,BP40537, F-29185 Concarneau, France. 3 Univ Victoria, Sch Earth & Ocean Sci, OEASB A405,POB 1700 16 STN CSC, Victoria, BC V8W 2Y2, Canada. 4 Indonesian Inst Sci, Res Ctr Oceanog, Jakarta 14430, Indonesia.

* Corresponding authors: Kenneth Mertens, email address : [email protected] ; Hikmah Thoha, email address : [email protected]

Abstract :

The dinoflagellate order Peridiniales encompasses several well circumscribed families. However, the family level of some genera, such as Bysmatrum and Vulcanodinium, has remained elusive for many years. Four Peridinium-like strains were established from the Atlantic coast of France and North Sulawesi, Indonesia through cyst germination or isolation of single cells. The cyst-theca relationship was established on specimens from the French Atlantic. Their morphologies were examined using light, scanning and transmission electron microscopy. The cells were characterized by a much larger epitheca relative to the hypotheca, a large anterior sulcal (Sa) plate deeply intruding the epitheca and a small first anterior intercalary plate. The plate formula was identified as Po, cp, X, 4′, 3a, 7′′, 6C, 5S, 5′′′, 2′′′′, shared by Apocalathium, Chimonodinium, Fusiperidinium and Scrippsiella of the family Thoracosphaeraceae but the configuration of Sa plate and anterior intercalary plates is different. Transmission electron microscopy showed that the eyespot was located within a chloroplast comprising two rows of lipid globules and thus belongs to type A. All four strains were classified within a new genus Caladoa as C. arcachonensis gen. et sp. nov. Small subunit ribosomal DNA (SSU rDNA), partial large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer ribosomal DNA (ITS rDNA) sequences were obtained from all strains. Genetic distance based on ITS rDNA sequences between French and Indonesian strains reached 0.17, suggesting cryptic speciation in C. arcachonensis. The maximum likelihood and Bayesian inference analysis based on concatenated data from SSU and LSU rDNA sequences revealed that Caladoa is monophyletic and closest to Bysmatrum. Our results supported that Caladoa and Bysmatrum are members of the order Peridiniales but their family level remains to be determined. Our results also support that Vulcanodinium is closest to the family Peridiniaceae.

Keywords : Bysmatrum, cyst, dinoflagellate, eyespot, Peridiniales, plate overlap

Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site. European Journal of Phycology Page 4 of 47

1 4 2 3 4 45 Introduction 5 6 7 46 The dinoflagellate order Peridiniales is characterized by a symmetrical first apical plate 8 9 47 and two more or less symmetrical antapical plates (Fensome et al., 1993). Peridiniales 10 11 12 48 currently encompasses several families including Blastodiniaceae, Heterocapsaceae, 13 14 49 Kryptoperidiniaceae, Peridiniaceae, Peridiniopsidaceae, Protoperidiniaceae, 15 16 For Peer Review Only 17 50 Thoracosphaeraceae and Zooxanthellaceae (Gottschling et al., 2017). The morphological 18 19 20 51 features in some of these families are well circumscribed, e.g. Peridiniopsidaceae has six 21 22 52 cingular plates and less than two anterior intercalary plates (Gottschling et al., 2017); 23 24 25 53 Protoperidiniaceae has three cingular plates and one transitional plate (Fensome et al., 26 27 54 1993). In other families, however, morphological features are not well defined. For 28 29 30 55 instance, Kryptoperidiniaceae includes species with one or two anterior intercalary plates 31 32 33 56 (Pienaar et al., 2007; Saburova et al., 2012; You et al., 2015), but some genera (e.g. 34 35 57 Galeidinium M.Tamura & T. Horiguchi) do not show any thecal plate pattern (Tamura et 36 37 38 58 al., 2005). The family Thoracosphaeraceae has unified the subfamily Calciodinelloideae 39 40 59 and the order Thoracosphaerales, but its emended description was not provided by 41 42 43 60 Elbrächter et al., (2008). 44 45 46 61 Information on the evolutionary history and cell ultrastructure are important for 47 48 62 systematics, even at the family level. For instance, Kryptoperidiniaceae includes a small 49 50 51 63 group of dinoflagellates hosting a tertiary endosymbiont derived from a diatom 52 53 64 (Horiguchi & Takano, 2006; Kretschmann et al., 2018). Kryptoperidiniaceae currently 54 55 56 65 includes Durinskia S. Carty & E.R. Cox, Blixaea M. Gottschling, Galeidinium, 57 58 4 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 5 of 47 European Journal of Phycology

1 5 2 3 4 66 Kryptoperidinium Lindemann, and Unruhdinium M. Gottschling. All these genera share a 5 6 7 67 type D eyespot (Tamura et al., 2005; You et al., 2015; Yamada et al., 2017) that was 8 9 68 likely derived from the original dinoflagellate chloroplast (Moestrup & Daugbjerg, 2007). 10 11 12 69 The woloszynskioid dinoflagellates families Tovelliaceae and Borghiellaceae are 13 14 70 characterized by a type C eyespot (composed of pigment globules not bound by 15 16 For Peer Review Only 17 71 membranes outside of the chloroplast) (Lindberg et al., 2005) and by a type B eyespot 18 19 20 72 (intraplastidic with bricklike crystals overlying the chloroplast), respectively (Moestrup et 21 22 73 al., 2009). In addition to the eyespot, peduncle microtubules were also suggested to be 23 24 25 74 one of the diagnostic features to separate Peridiniopsidaceae from Peridiniaceae 26 27 75 (Gottschling et al., 2017). 28 29 76 30 31 77 Among 2000 extant dinoflagellate species, only 13% to 16% of the motile stages 32 33 34 78 have been related to resting cysts (Head, 1996). Cyst morphologies are often considered 35 36 37 79 to be useful at interspecific level, e.g. Protoperidinium Bergh produces a number of cysts 38 39 80 classified within different cyst genera (Gu et al., 2015), but also at the generic level, e.g. 40 41 42 81 cysts of Biecheleria Ø. Moestrup, K. Lindberg & N. Daugbjerg are spherical and 43 44 82 bearnumerous short processes (Moestrup et al., 2009), or even at the subfamily level, e.g. 45 46 47 83 Calciodinelloideae is characterized by possessing calcareous cysts (Elbrächter et al., 48 49 50 84 2008). 51 52 85 Molecularly, more than 90% of peridinialean species can be reliably placed into one 53 54 55 86 of the families of Peridiniales (Gottschling et al., 2017), but some genera (e.g. 56 57 58 5 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 6 of 47

1 6 2 3 4 87 Bysmatrum M.A. Faust & K.A. Steidinger, Vulcanodinium E. Nézan & N. Chomérat) 5 6 7 88 have not been identified to the family level yet, although they display a peridinialean 8 9 89 tabulation. Vulcanodinium was reported to be closely related with Peridinium cinctum 10 11 12 90 (O.F. Müller) C.G. Ehrenberg based on LSU sequences (Nézan & Chomérat, 2011) and 13 14 91 concatenated data from SSU, ITS and LSU sequences (Luo et al., 2018), but whether 15 16 For Peer Review Only 17 92 Vulcanodinium is also a member of Peridiniaceae is not clear. The phylogenetic position 18 19 20 93 of Bysmatrum is elusive too: it is close to Peridinium C.G. Ehrenberg with low support in 21 22 94 the phylogeny based on concatenated data from SSU, ITS and LSU sequences (Anglès et 23 24 25 95 al., 2017; Luo et al., 2018), but sometimes it is closer to Prorocentrum C.G. Ehrenberg 26 27 96 (Gottschling et al., 2012; Dawut et al., 2018). 28 29 30 97 To clarify the phylogenetic positions of Vulcanodinium and Bysmatrum, sequences 31 32 33 98 from closely related species of these two genera are essential. In the present study, four 34 35 99 strains of Peridinium-like species were established from the Atlantic coast of France and 36 37 38 100 North Sulawesi, Indonesia by incubating cysts or isolating single cells. The four cultured 39 40 101 strains were examined morphologically and ultrastructurally, the latter with an emphasis 41 42 43 102 on the eyespot. In addition, small subunit ribosomal DNA (SSU rDNA), partial large 44 45 46 103 subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer ribosomal DNA 47 48 104 (ITS rDNA) sequences were determined for the cultured strains and molecular phylogeny 49 50 51 105 was inferred using concatenated SSU and LSU rDNA sequences. 52 53 106 54 55 56 57 58 6 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 7 of 47 European Journal of Phycology

1 7 2 3 4 107 Material and methods 5 6 7 8 108 Sample collection and treatment 9 10 11 109 Seagrass and sand samples were collected from the seabed by scuba divers near Manado 12 13 14 110 and Bitung, North Sulawesi of Indonesia on July 31 and August 1, 2016 and placed into 15 16 For Peer Review Only 17 111 bottles containing seawater from the same location. The samples were stirred vigorously 18 19 112 to detach the epibenthic cells and the suspension settled in a composite settling chamber. 20 21 22 113 The settled material was rinsed with filtered seawater and transferred into a 23 24 114 polycarbonate bottle. Single Peridinium-like cells were isolated from this material and 25 26 27 115 the strains TIO339 and TIO340 were established (Table 1). 28 29 30 116 Sediment sampling was done using an Ekman grab on April 15 and 19, 2016 in a 31 32 117 shallow natural reservoir used for oyster storage before commercialization (water depth 33 34 35 118 1.2–1.5 m). The small basin is located in Arcachon Bay in the Gironde region of 36 37 119 southwestern France. The top 2 cm of sediment were sliced off and stored in the dark at 4 38 39 40 120 °C until further treatment. Approximately 5 g of wet sediment was mixed with 20 mL of 41 42 43 121 filtered seawater and stirred vigorously to dislodge detrital particles. The settled material 44 45 122 was subsequently sieved through 120 μm and 10 μm filters. Single cysts were isolated 46 47 48 123 with a micropipette using an inverted Eclipse TS100 (Nikon, Tokyo, Japan) microscope, 49 50 124 alternatively small quantity of sediment was directly incubated in small containers with 51 52 53 125 f/2-Si medium (Guillard & Ryther, 1962) at 20ºC, 90 μE·m–2·s–1 under a 12:12 h light: 54 55 56 57 58 7 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 8 of 47

1 8 2 3 4 126 dark cycle. The strains TIO278 and TIO282 were established using direct incubation 5 6 7 127 (Table 1). 8 9 10 128 Morphological study of motile cells with microscopy 11 12 13 14 129 15 16 For Peer Review Only 17 130 Live cells were examined and photographed using a Zeiss Axio Imager light microscope 18 19 131 (Carl Zeiss, Göttingen, Germany) equipped with a Zeiss Axiocam HRc digital camera. 20 21 22 132 The size of 30 cells was measured using Axiovision (4.8.2 version) software at ×1000 23 24 133 magnification. To observe the shape and location of the nucleus, cells were stained with 25 26 27 134 1:100,000 SYBR Green (Sigma Aldrich, St. Louis, USA) for 1 min, and photographed 28 29 30 135 using the Zeiss fluorescence microscope with a Zeiss-38 filter set (excitation BP 470/40, 31 32 136 beam splitter FT 495, emission BP 525/50). Chloroplast autofluorescence microscopy 33 34 35 137 was carried out on live cells using the above mentioned microscope equipped with a 36 37 138 Chroma filter cube (emission filter ET480/20x, dichromatic mirror AT505dc, suppression 38 39 40 139 filter AT515lp), and digitally photographed using a Zeiss Axiocam HRc digital camera. 41 42 43 140 For scanning electron microscopy (SEM), mid-exponential batch cultures of four 44 45 141 strains were concentrated by a Universal 320 R centrifuge (Hettich-Zentrifugen, 46 47 48 142 Tuttlingen, Germany) at 850 g for 10 min at room temperature. Cells were fixed with 49 50 143 2.5% glutaraldehyde for 3 h at 8ºC, rinsed with Milli-Q water twice and post-fixed with 51 52 53 144 1% OsO4 overnight at 8ºC in a tube. The supernatant was removed and the settled cells 54 55 56 145 were transferred to a coverslip coated with poly-L-lysine (molecular weight 57 58 8 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 9 of 47 European Journal of Phycology

1 9 2 3 4 146 70,000–150,000). The cells attached to the cover slip were rinsed in Milli-Q water twice. 5 6 7 147 The samples were then dehydrated in a graded ethanol series (10, 30, 50, 70, 90 and 3× in 8 9 148 100%, 10 min at each step), critical point dried (K850 Critical Point Dryer, 10 11 12 149 Quorum/Emitech, West Sussex, UK), sputter-coated with gold, and examined with a 13 14 150 Zeiss Sigma FE (Carl Zeiss Inc., Oberkochen, Germany) scanning electron microscope. 15 16 For Peer Review Only 17 151 Labelling of tabulation follows a modified Kofoid system (Fensome et al., 1993), and the 18 19 20 152 sulcal plate labelling is according to Balech (1980). 21 22 23 153 Transmission electron microscopy (TEM) 24 25 26 27 154 Mid-exponential batch cultures of strain TIO278 (France) were fixed in 2.5% 28 29 30 155 glutaraldehyde in phosphate buffer saline (PBS, 0.1 M at pH 7.4) for 1 h, concentrated by 31 32 156 centrifugation and then washed three times with the same PBS for 10 min each. They 33 34 35 157 were post-fixed in 1% OsO4 overnight at 4°C and washed three times with the same PBS 36 37 158 for 10 min each. The cells were then dehydrated through a graded ethanol series (10, 30, 38 39 40 159 50, 70, 95, 3× in 100%, 10 min at each step). The pellet was embedded in Spurr’s resin 41 42 43 160 (Spurr, 1969) and sectioned with a Reichert Ultracut E microtome (Leica, Vienna, 44 45 161 Austria), mounted on Formvar-coated grids, stained with uranyl acetate and lead citrate, 46 47 48 162 and observed in a JEOL JEM-100 transmission electron microscope (JEOL, Tokyo, 49 50 163 Japan). 51 52 53 54 55 56 57 58 9 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 10 of 47

1 10 2 3 4 164 PCR amplifications and sequencing 5 6 7 8 165 The total algal DNA was extracted from 10 mL of exponentially growing cultures using a 9 10 166 MiniBEST Universal DNA Extraction Kit (Takara, Tokyo, Japan) according to the 11 12 13 167 manufacturer’s protocol. PCR amplifications were carried out using 1×PCR buffer, 50 14 15 µM dNTP mixture, 0.2 μM of each primer, 10 ng of template genomic DNA, and 1 U of 16 168 For Peer Review Only 17 18 169 ExTaq DNA Polymerase (Takara, Tokyo, Japan) in 50 μL reactions. The SSU rDNA was 19 20 21 170 amplified using the primers of PRIMER A/PRIMER B (Medlin et al. 1988). The LSU 22 23 171 rDNA was amplified using the primers of D1R/28-1483R (Scholin et al., 1994; 24 25 26 172 Daugbjerg et al., 2000). The total ITS1–5.8S–ITS2 was amplified using ITSA/ITSB 27 28 29 173 primers (Adachi et al., 1996). The thermal cycle procedure was 4 min at 94ºC, followed 30 31 174 by 30 cycles of 1 min at 94ºC, 1 min at 45ºC, 1 min at 72ºC, and final extension of 7 min 32 33 34 175 at 72ºC with a Mastercycler (Eppendorf, Hamburg, Germany). The PCR product was 35 36 176 purified using a SanPrep Column DNA Gel Extraction Kit (Sangon Biotech, Shanghai, China) 37 38 39 177 and sequenced directly in both directions on an ABI PRISM 3730XL (Applied 40 41 42 178 Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. Newly 43 44 179 obtained sequences were deposited in GenBank with accession numbers MK012070 to 45 46 47 180 MK012084. 48 49 50 181 Sequence alignment and phylogenetic analysis 51 52 53 54 182 Newly obtained sequences (SSU and partial LSU rDNA) were incorporated into a 55 56 57 58 10 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 11 of 47 European Journal of Phycology

1 11 2 3 4 183 systematically representative set of dinoflagellates available in GenBank. Sequences were 5 6 7 184 aligned using MAFFT v7.110 (Katoh & Standley, 2013) online program 8 9 185 (http://mafft.cbrc.jp/alignment/server/) with default settings. Alignments were manually 10 11 12 186 checked with BioEdit v. 7.0.5 (Hall, 1999). For Bayesian inference (BI), the program 13 14 187 jModelTest (Posada, 2008) was used to select the most appropriate model of molecular 15 16 For Peer Review Only 17 188 evolution with Akaike Information Criterion (AIC). Bayesian reconstruction of the data 18 19 20 189 matrix was performed using MrBayes 3.2 (Ronquist & Huelsenbeck, 2003) with the 21 22 190 best-fitting substitution model (GTR+G). Four Markov chain Monte Carlo (MCMC) 23 24 25 191 chains ran for 2,000,000 generations, sampling every 100 generations. The first 10% of 26 27 192 burn-in trees were discarded. A majority rule consensus tree was created in order to 28 29 30 193 examine the posterior probabilities of each clade. Maximum likelihood (ML) analyses 31 32 33 194 were conducted with RaxML v7.2.6 (Stamatakis, 2006) on the T-REX web server (Boc et 34 35 195 al., 2012) using the model GTR+G. Node support was assessed with 1000 bootstrap 36 37 38 196 replicates. 39 40 197 Multiple ITS1-5.8S-ITS2 sequences of new strains were aligned using MAFFT v7.110 41 42 43 198 (Katoh & Standley, 2013) online program with default settings. Completed alignments 44 45 46 199 were saved as NEXUS files and imported into PAUP*4b10 software (Swofford, 2002) to 47 48 200 estimate divergence rates using simple uncorrected pairwise (p) distance matrices. 49 50 51 201 52 53 202 Results 54 55 56 203 Caladoa Z. Luo, K.N. Mertens & H.F. Gu gen. nov. 57 58 11 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 12 of 47

1 12 2 3 4 204 DIAGNOSIS: Cells with a plate formula of Po, cp, X, 4′, 3a, 7′′, 6C, 5S, 5′′′, 2′′′′. A 5 6 7 205 large anterior sulcal plate deeply intrudes the epitheca. Chloroplasts with a stalked 8 9 206 pyrenoid and a type A eyespot is present. Cells differ from Chimonodinium, 10 11 12 207 Fusiperidinium, Apocalathium, Scrippsiella, Vulcanodinium and Bysmatrum in the size 13 14 208 and location of Sa plate and from Peridinium by the presence of an additional cingular 15 16 For Peer Review Only 17 209 plate. 18 19 20 210 ETYMOLOGY: The epithet Caladoa is named after António José Calado, who 21 22 211 carried out outstanding work on freshwater dinoflagellate taxonomy. 23 24 25 212 TYPE SPECIES: Caladoa arcachonensis Z. Luo, K.N. Mertens & H.F. Gu 26 27 213 28 29 30 214 Caladoa arcachonensis Z. Luo, K.N. Mertens & H.F. Gu sp. nov. (Figs. 1–26) 31 32 33 215 DIAGNOSIS: Cells are 16.7–23.5 µm long and 13.1–19.4 µm wide. The cells have 34 35 216 a rounded epitheca and hypotheca with an epitheca: hypotheca length ratio of 1.5–1.8. 36 37 38 217 The thecae display a plate formula of Po, cp, X, 4′, 3a, 7′′, 6C, 5S, 5′′′, 2′′′′. A large 39 40 218 anterior sulcal plate deeply intrudes in the epitheca. Plate 1a is about half the size of 3a 41 42 43 219 and one quarter the size of 2a. Each cell has a single reticulated chloroplast with one 44 45 46 220 stalked pyrenoid. An eyespot comprising two rows of lipid globules is situated within a 47 48 221 chloroplast and belongs to type A. The nucleus is spherical and located posteriorly. Cysts 49 50 51 222 are proximate with a chasmic archeopyle. 52 53 223 HOLOTYPE: SEM stub of strain TIO278 designated as TIO201803 (illustrated in 54 55 56 224 Fig. 9) and deposited at Third Institute of Oceanography, State Oceanic Administration, 57 58 12 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 13 of 47 European Journal of Phycology

1 13 2 3 4 225 Xiamen 361005, China. 5 6 7 226 TYPE LOCALITY: Arcachon Bay, France (1°4'0.0'' W, 44°38'11.0''N); Collection 8 9 227 date: 15 and 19 April 2016. 10 11 12 228 ETYMOLOGY: The epithet arcachonensis is named after the type locality, 13 14 229 Arcachon Bay. 15 16 For Peer Review Only 17 230 HABITAT: Benthic sand-dwelling or epiphytic on seagrass. 18 19 20 231 DISTRIBUTION: Brackish and marine coastal water of French Atlantic (21.3–23 21 22 232 psu), North Sulawesi, Indonesia. 23 24 25 233 GENBANK ACCESSION NUMBER SEQUENCES: MK012071 (SSU rDNA), 26 27 234 MK012076 (LSU rDNA) and MK012081 (ITS rDNA) of strain TIO278. 28 29 30 235 31 32 33 34 236 Morphology 35 36 37 237 Cysts of Caladoa arcachonensis, isolated from Arcachon Bay, France surface sediment 38 39 40 238 samples are spherical or ovoid in shape, proximate and with a well-defined posteriorly 41 42 43 239 located cingulum (Fig. 1). They are 23.9–28.1 µm long (mean = 26.6 ± 2.3 μm, n = 3) 44 45 240 and 19.9–24.4 µm wide (mean = 22.0 ± 2.3μm, n = 3). Each cyst contains an endospore, 46 47 48 241 green to brown granules, a pyrenoid and the red eyespot. Two cysts were isolated, but 49 50 242 only one germinated, displaying a chasmic archeopyle on the epitheca (Fig. 2). The cyst 51 52 53 243 wall is transparent and it appears to be smooth (Fig. 3). Sometimes, small detrital 54 55 56 244 particles can be attached to the cyst wall surface. The germinated cell had a pronounced 57 58 13 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 14 of 47

1 14 2 3 4 245 eyespot in the sulcal area (Fig. 4). The cysts withstand palynological treatment. 5 6 7 246 Cells of the French strain TIO278 are 16.7–23.5 µm long (mean = 19.8 ± 2.2 μm, n 8 9 247 = 30) and 13.1–19.4 µm wide (mean = 16.4 ± 1.8 μm, n = 30). The cells have a rounded 10 11 12 248 epitheca and hypotheca with a much larger epitheca (Fig. 5). There is a ring-like pyrenoid 13 14 249 located in the epicone and one pronounced orange eyespot in the sulcal area (Fig. 6). 15 16 For Peer Review Only 17 250 There is a single chloroplast forming a network in the periphery of the cell ('c' in Fig. 7). 18 19 20 251 The nucleus is rounded and located posteriorly ('N' in Fig. 8). 21 22 252 The thecae have a plate formula of Po, cp, X, 4′, 3a, 7′′, 6C, 5S, 5′′′, 2′′′′ (Figs. 9–16). 23 24 25 253 The epitheca: hypotheca length ratio in the dorsal side is 1.5–1.8 (mean = 1.6 ± 0.1, n = 26 27 254 7). Thecal pores with a diameter of 0.1–0.2 μm are randomly scattered throughout the 28 29 30 255 thecal plates (Figs. 10, 11, 15). The epithecal plates are symmetrical in arrangement 31 32 33 256 (bipesoid). There are four apical plates. Plate 1′ and 3′ are six-sided and are nearly 34 35 257 symmetrical (Figs. 13, 14). Plate 2′ and 4′ are seven-sided and similar in size, much 36 37 38 258 larger than the other apical plates (Figs. 13, 14). There are three anterior intercalary plates 39 40 259 (1a, 2a and 3a), of which 1a and 3a are pentagonal and 2a is hexagonal. Plate 1a is about 41 42 43 260 half the size of 3a and one quarter the size of 2a (Fig. 14). There are seven precingular 44 45 46 261 plates among which the fourth is four-sided and smallest, whereas the others are 47 48 262 five-sided (Figs. 9, 10, 13, 14). The cingulum is 1.6–2.8 µm wide (mean = 2.3 ± 0.4 μm, 49 50 51 263 n = 12), situated in the low part of the cell and descended ca. half its width (Fig. 9). The 52 53 264 cingulum comprises six plates. The cingular plates are similar in size except C1 is 54 55 56 265 relatively smaller (Figs. 9, 10, 15). The apical pore complex is tear-shaped comprising a 57 58 14 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 15 of 47 European Journal of Phycology

1 15 2 3 4 266 round pore plate (Po), a round apical pore covered by a cover plate (cp) and an elongated 5 6 7 267 canal plate (X) (Figs. 13, 14). There are five postcingular plates, among them the third 8 9 268 and fifth postcingular plates (3′′′, 5′′′) are five-sided whereas the other plates are 10 11 12 269 four-sided (Fig. 12). The antapical plates (1′′′′, 2′′′′) are five-sided and similar in size (Fig. 13 14 270 12). 15 16 For Peer Review Only 17 271 There are five sulcal plates. The large anterior sulcal plate (Sa) intrudes the epitheca 18 19 20 272 (Fig. 16). The left sulcal (Ss) is elongated and narrow. The median sulcal (Sm) is small 21 22 273 and always masked by the sulcal list emerging from the right sulcal plate (Sd). The 23 24 25 274 posterior sulcal plate (Sp) is seven-sided and has no contact with the cingulum (Figs. 15, 26 27 275 16). A peduncle was not observed. Schematic drawings showing the plate patterns of 28 29 30 276 Caladoa arcachonensis are provided in Figs. 17–20. Plate overlap (Figs. 19, 20) was 31 32 33 277 identified individually for each suture by inspecting cells with visible growth bands or by 34 35 278 internal theca views (Figs. 9–16). The third precingular and postcingular plates and the 36 37 38 279 fourth cingular plate were identified as the keystone plates (i.e., plates overlapping all 39 40 280 their neighbors). 41 42 43 281 Longitudinal sections through the cell of TIO278 show a large spherical nucleus in 44 45 46 282 the hypotheca, a stalked pyrenoid in the epitheca, a single chloroplast in the periphery 47 48 283 and an eyespot in the sulcal region (Figs. 21, 22). The nucleus consists of condensed 49 50 51 284 chromosomes with a smooth nuclear envelope. The chloroplast is enveloped by three 52 53 285 membranes and the thylakoids are grouped in threes to form lamellae (Fig. 23). The 54 55 56 286 stalked pyrenoid is surrounded by a starch sheath without intrusion of lamellae (Fig. 24). 57 58 15 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 16 of 47

1 16 2 3 4 287 Many diamond-shaped trichocysts were observed beneath the thecate wall (Fig. 23). The 5 6 7 288 eyespot is located within a chloroplast comprising two rows of ~ 20 lipid globules in each 8 9 289 row (Figs. 25, 26). The lipid globules are ~ 100–150 nm in diameter. A microtubular 10 11 12 290 strand of the peduncle and a pusular system were not observed but an intensive search 13 14 291 was not carried out. 15 16 For Peer Review Only 17 292 Cells of the Indonesian strain TIO339 are 13.2–18.9 µm long (mean = 16.5 ± 1.5 μm, n 18 19 20 293 = 24) and 10.7–15.2 µm wide (mean = 12.9 ± 1.1 μm, n = 29). The cells show a spherical 21 22 294 nucleus in the hypotheca, an orange eyespot in the sulcal region and a single reticulated 23 24 25 295 chloroplast with a doughnut-shaped pyrenoid in the epitheca (Figs. 27, 28). Cell 26 27 296 morphology of strain TIO339 is indistinguishable from strain TIO278 (Figs. 29–34). 28 29 30 31 297 Molecular analysis and phylogeny 32 33 34 35 298 The French strains TIO278 and TIO282 shared identical SSU, LSU and ITS rDNA 36 37 299 sequences, and the Indonesian strains TIO339 and TIO340 shared identical SSU, LSU 38 39 40 300 and ITS rDNA sequences as well. Strain TIO278 differed from TIO339 at 13 positions 41 42 43 301 (SSU rDNA, 99.2% similarity out of 1724 bp), 61 positions (LSU rDNA, 95.5% 44 45 302 similarity out of 1331 bp), and 131 positions (ITS rDNA, 79.8% similarity out of 648 bp). 46 47 48 303 The genetic distance based on ITS rDNA sequences was 0.17 between French and 49 50 304 Indonesian strains. 51 52 53 305 The maximum likelihood (ML) and Bayesian inference (BI) analysis based on 54 55 56 306 combined SSU rDNA and partial LSU rDNA sequences yielded similar phylogenetic 57 58 16 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 17 of 47 European Journal of Phycology

1 17 2 3 4 307 trees. The BI tree was illustrated in Fig. 35. The order Peridiniales is well resolved (0.9 5 6 7 308 BPP/55 BS) encompassing the families Peridiniaceae, Peridiniopsidaceae, 8 9 309 Thoracosphaeraceae, Kryptoperidiniaceae, and the genera Caladoa, Bysmatrum and 10 11 12 310 Vulcanodinium as well. The genus Caladoa was monophyletic with maximal support (1.0 13 14 311 BPP/100 BS), which formed a sister clade of Bysmatrum with maximal support. They 15 16 For Peer Review Only 17 312 were closest to a well resolved clade (0.90 BPP/93 BS) formed by Peridiniaceae and 18 19 20 313 Vulcanodinium rugosum E. Nézan & N. Chomérat with a low Bayesian posterior 21 22 314 probability (<0.7 BPP) and moderate ML bootstrap support (58 BS). 23 24 25 315 26 27 316 Discussion 28 29 30 31 317 Morphology 32 33 34 35 318 To determinate plate homologies, the plate overlap pattern was investigated. Plate 36 37 319 overlap pattern of strain TIO278 (Figs. 19, 20) is generally consistent with other 38 39 40 320 dinoflagellates, following a trend from dorsal to ventral and from equatorial to poles 41 42 43 321 (Netzel & Dürr, 1984). The third precingular plate is identified as the keystone plate in 44 45 322 our strain, also previously reported for Parvodinium marciniakii J. Kretschmann, P. 46 47 48 323 Owsianny, A.Z. Zerdoner & M. Gottschling by Kretschmann et al. (2018) and 49 50 324 Parvodinium umbonatum (F. Stein) S. Carty by Luo et al. (2018). In contrast, the fourth 51 52 53 325 precingular plate was the keystone plate in many other species of Peridiniales including 54 55 56 326 Bysmatrum subsalsum (C.H. Ostenfeld) M.A. Faust & K.A. Steidinger and 57 58 17 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 18 of 47

1 18 2 3 4 327 Vulcanodinium rugosum (Luo et al., 2018), Scrippsiella acuminata (C.G. Ehrenberg) J. 5 6 7 328 Kretschmann, M. Elbrächter, C. Zinssmeister, S. Soehner, M. Kirsch, W.-H. Kusber & M. 8 9 329 Gottschling (Kretschmann et al., 2015), Peridinium cinctum (Dürr, 1979), 10 11 12 330 Fusiperidinium wisconsinense (S. Eddy) F.M.G. McCarthy, H. Gu, K.N. Mertens & C. 13 14 331 Carbonell-Moore (McCarthy et al., 2018), Heterocapsa triquetra (C.G. Ehrenberg) F. 15 16 For Peer Review Only 17 332 Stein (Tillmann et al., 2017), Parvodinium travinskii J. Kretschmann, P. Owsianny, A.Z. 18 19 20 333 Zerdoner & M. Gottschling, Parvodinium mixtum Wołoszyńska ex J. Kretschmann, P. 21 22 334 Owsianny, A.Z. Zerdoner & M. Gottschling (Kretschmann et al., 2018) and Parvodinium 23 24 25 335 umbonatum (Elbrächter & Meyer, 2001). In addition, Caladoa has a primtegulate series 26 27 336 that touches the terttegulate series, which can also be found in Scrippsiella Balech ex 28 29 30 337 A.R.Loeblich III, Fusiperidinium F.M.G. McCarthy, H. Gu, K.N. Mertens & C. 31 32 33 338 Carbonell-Moore, Parvodinium, and Heterocapsa F. Stein, but is different from 34 35 339 Bysmatrum, Vulcanodinium, Peridinium and Protoperidinium, where both series are 36 37 38 340 separated (partly) by the sectegulate series. 39 40 341 The central ventral plate of strain TIO278 (denoted here by Sa) is overlapped by all 41 42 43 342 adjacent epithecal plates including the first apical plate 1′. This ventral plate is identified 44 45 46 343 as homologous with the anterior sulcal plate (Sa) instead of a precingular plate because 47 48 344 plate 1′ is generally overlapped by precingular plates (Dürr & Netzel, 1974; Fensome et 49 50 51 345 al., 1993). The plate pattern of strain TIO278 was thus determined as Po, cp, X, 4′, 3a, 7′′, 52 53 346 6C, 5S, 5′′′, 2′′′′. This plate formula is reminiscent of several previously described genera, 54 55 56 347 including the freshwater genera Chimonodinium S.C. Craveiro, A.J. Calado, N. 57 58 18 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 19 of 47 European Journal of Phycology

1 19 2 3 4 348 Daugbjerg, Gert Hansen & Ø. Moestrup and Fusiperidinium (Craveiro et al., 2011; 5 6 7 349 McCarthy et al., 2018), freshwater-brackish genus Apocalathium S.C. Craveiro, N. 8 9 350 Daugbjerg, Ø. Moestrup & A.J. Calado (Craveiro et al., 2016), and brackish-marine 10 11 12 351 genera Bysmatrum, Scrippsiella and Vulcanodinium (Nézan & Chomérat, 2011; Luo et 13 14 352 al., 2016; Luo et al., 2018). The strain TIO278 can be separated from these genera 15 16 For Peer Review Only 17 353 primarily based on the size and location of Sa plate, which is large and intrudes deeply in 18 19 20 354 the epitheca. Such kind of Sa plate was only reported in marine Heterocapsa (Tillmann et 21 22 355 al., 2017) and Laciniporus Saburova & N. Chomérat (Saburova & Chomérat 2018), 23 24 25 356 freshwater Parvodinium (Carty, 2008) and Palatinus S. Craveiro, A. Calado, N. 26 27 357 Daugbjerg & Ø. Moestrup (Craveiro et al., 2009). A similar plate was observed in 28 29 30 358 Sphaerodinium cracoviense J. Wołoszyńska (Craveiro et al., 2010), but the authors 31 32 33 359 expressed some doubts whether it should be considered as a Sa plate. Interestingly, such a 34 35 360 type of deeply intruding Sa with similar overlap with the 1′ has also been recorded for the 36 37 38 361 fossil cyst Susadinium? pinna (Below) Lentin & Williams by Below (1987, as Dodekovia 39 40 362 pinna Below) described from the Lower Jurassic (Toarcian), a species assigned to the 41 42 43 363 Heterocapsaceae (Fensome et al., 1993); this suggests that this type of Sa is a primitive 44 45 46 364 evolutionary trait, not present in genera that have presumably evolved later, such as 47 48 365 Protoperidinium. 49 50 366 51 52 367 53 54 55 368 Strain TIO278 differs from Chimonodinium in the presence of a pyrenoid and lack of a 56 57 58 19 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 20 of 47

1 20 2 3 4 369 peduncle (Craveiro et al., 2011), from Scrippsiella in the size of the first cingular plate 5 6 7 370 (Luo et al., 2016) and it differs from Fusiperidinium, Apocalathium and Vulcanodinium 8 9 371 in the presence of an eyespot (Nézan & Chomérat, 2011; Craveiro et al., 2016; McCarthy 10 11 12 372 et al., 2018), as well as it differs from Bysmatrum in the configuration of anterior 13 14 373 intercalary plates. Plates 2a and 3a are separated in Bysmatrum, but they touch in strain 15 16 For Peer Review Only 17 374 TIO278. Strain TIO278 shares a similar Sa plate with Heterocapsa and Laciniporus, but 18 19 20 375 it differs in the plate pattern. Only two anterior intercalary plates are present in 21 22 376 Heterocapsa and Laciniporus and an eyespot is not observed (Tillmann et al., 2017, 23 24 25 377 Saburova & Chomérat 2018). Strain TIO278 shares a type A eyespot with Peridinium but 26 27 378 differs in the number of cingular plates (six versus five), therefore, a new genus Caladoa 28 29 30 379 is established to incorporate TIO278 and related strains. 31 32 33 380 Caladoa arcachonensis is morphologically close to the freshwater “Peridinium 34 35 381 subtatranum” (invalidly described), Apocalathium aciculiferum var. inerme (J. 36 37 38 382 Wołoszyńska) Ø. Moestrup & A.J. Calado and Chimonodinium lomnickii var. minus (J. 39 40 383 Wołoszyńska) Ø. Moestrup based on the plate pattern and a large Sa plate, but it differs 41 42 43 384 in the relative size of anterior intercalary plates and the size of the apical pore 44 45 46 385 (Wołoszyńska, 1916, Wołoszyńska, 1937, Wołoszyńska, 1952, Moestrup & Calado, 47 48 386 2018). Caladoa arcachonensis is also morphologically close to Chimonodinium lomnickii 49 50 51 387 (J. Wołoszyńska) S.C. Craveiro, A.J. Calado, N. Daugbjerg, Gert Hansen & Ø. Moestrup 52 53 388 and Chimonodinium lomnickii var. wierzejskii (J. Wołoszyńska) S.C. Craveiro, A.J. 54 55 56 389 Calado, N. Daugbjerg, Gert Hansen & Ø. Moestrup but differs in the size and location of 57 58 20 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 21 of 47 European Journal of Phycology

1 21 2 3 4 390 the Sa and relative size of anterior intercalary plates (Craveiro et al., 2011, Moestrup & 5 6 7 391 Calado, 2018). Caladoa arcachonensis differs from Peridinium gutwinskiiJ. 8 9 392 Wołoszyńska and Peridinium bipes F. Stein in the number of cingular plates and 10 11 12 393 relative size of anterior intercalary plates (Boltovskoy, 1989; Lee et al., 2006), and differs 13 14 394 from Peridinium allorgei M. Lefèvre in the cell shape and relative size of anterior 15 16 For Peer Review Only 17 395 intercalary plates (Moestrup & Calado 2018). Caladoa arcachonensis differs from 18 19 20 396 Apocalathium malmogiense (G. Sjöstedt) S.C. Craveiro, N. Daugbjerg, Ø. Moestrup & 21 22 397 A.J. Calado in a much larger epitheca and relative size of anterior intercalary plates 23 24 25 398 (Craveiro et al., 2016). 26 27 399 Phylogeny and genetic differentiation 28 29 30 400 The only morphological difference between French strain TIO278 and Indonesian strain 31 32 33 401 TIO339 is the cell size, therefore, they are considered to be the same morphospecies. 34 35 402 These strains however inhabit temperate and tropical waters, respectively, and are likely 36 37 38 403 to differ in their eco-physiological traits. These two strains also have a genetic distance, 39 40 404 based on ITS sequences, as much as 0.17, and that is much larger than the threshold value 41 42 43 405 (0.04) for differentiating dinoflagellate species at interspecific level (Litaker et al., 2007), 44 45 46 406 therefore suggesting the existence of cryptic species. The French strain has a cyst stage, 47 48 407 but whether the Indonesian strain produces cysts is yet to be determined. 49 50 51 408 Our molecular phylogeny supports the classification of Caladoa within the order 52 53 409 Peridiniales, consistent with the morphological features characteristic of Peridiniales, 54 55 56 410 such as a symmetrical 1′ plate and two symmetrical antapical plates. The close 57 58 21 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 22 of 47

1 22 2 3 4 411 relationship between Caladoa and Bysmatrum, however, is surprising. Bysmatrum shows 5 6 7 412 an asymmetrical 1′ plate and two somewhat asymmetrical antapical plates that even do 8 9 413 not fit to the definition of Peridiniales (Fensome et al., 1993). However, Bysmatrum and 10 11 12 414 Caladoa share identical plate pattern and most importantly, they share a type A eyespot 13 14 415 (located within the chloroplast and consisted of several two rows of opaque globules) 15 16 For Peer Review Only 17 416 (Moestrup & Daugbjerg, 2007; Dawut et al., 2018). A type A eyespot appears to be a 18 19 20 417 common feature of Peridiniales, reported also in Scrippsiella acuminata and 21 22 418 Chimonodinium lomnickii (Craveiro et al., 2011), Palatinus apiculatus (C.G. Ehrenberg) 23 24 25 419 S. Craveiro, A. Calado, N. Daugbjerg & Ø. Moestrup (Craveiro et al., 2009), Naiadinium 26 27 420 polonicum (J. Wołoszyńska) S. Carty (Craveiro et al., 2015), and Peridinium willei H. 28 29 30 421 Huitfeldt-Kaas (Moestrup & Daugbjerg, 2007). Bysmatrum can also have a type B 31 32 33 422 eyespot but this is a modified type A eyespot, with the difference only in the presence of 34 35 423 an overlying brick like crystals (Luo et al., 2018). Caladoa and Bysmatrum both have 36 37 38 424 proximate cysts that are similar to the motile cells (Anglès et al., 2017; Luo et al., 2018). 39 40 425 A new family is possible for Caladoa and Bysmatrum in view of their morphological 41 42 43 426 similarity, but evidence from more closely related species is needed. 44 45 46 427 The close relationship between Vulcanodinium and Peridinium sensu stricto has been 47 48 428 reported before based on the LSU rDNA sequences (Nézan & Chomérat, 2011), 49 50 51 429 concatenated data from SSU, ITS and LSU rDNA sequences (Luo et al., 2018), and 52 53 430 supported here again based on concatenated data from SSU and LSU rDNA sequences. It 54 55 56 431 is worth noting that the closest relative of Peridinium is a brackish species, in contrast 57 58 22 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 23 of 47 European Journal of Phycology

1 23 2 3 4 432 with the suggestion that marine and freshwater species are usually not closely related 5 6 7 433 (Logares et al., 2007). However, Vulcanodinium has six cingular plates compared to five 8 9 434 in Peridinium sensu stricto (Boltovskoy, 1975; Nézan & Chomérat, 2011), and 10 11 12 435 Vulcanodinium does not have a type A eyespot whereas Peridinium cinctum and P. willei 13 14 436 have one (Spector & Triemer, 1979; Moestrup & Daugbjerg, 2007). The assignment of 15 16 For Peer Review Only 17 437 Vulcanodinium to Peridiniaceae is possible depending on the emendation of Peridiniaceae. 18 19 20 438 Multi-gene phylogenies or phylotranscriptomics should provide more evidence for a 21 22 439 definite assignment. 23 24 25 440 26 27 441 Acknowledgements 28 29 30 442 We thank two anonymous reviewers for constructive suggestions that improved the 31 32 33 443 manuscript greatly. Claire Meteigner and Myriam Rumèbe are acknowledged for 34 35 444 collecting samples from Arcachon Bay. 36 37 38 445 Funding 39 40 446 This work was supported by the National Key Research and Development Program of 41 42 43 447 China (2016YFE0202100), National Natural Science Foundation of China (41676117, 44 45 46 448 41806154), and China-ASEAN Maritime Cooperation Fund. 47 48 449 49 50 51 450 Author contributions 52 53 451 Z. Luo: light microscopy, electron microscopy and drafting manuscript; K.N. Mertens: 54 55 56 452 original concept, drafting and editing manuscript, incubation experiment, light 57 58 23 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 24 of 47

1 24 2 3 4 453 microscopy; E. Nézan: drafting and editing manuscript; Li Gu: electron microscopy; Vera 5 6 7 454 Pospelova: palynological treatment, drafting and editing manuscript; H. Thoha: sampling 8 9 455 and editing manuscript; H. Gu: original concept and editing of manuscript. 10 11 456 12 13 457 References 14 15 16 458 ADACHI, M., SAKOFor, Y. & Peer ISHIDA, Y. (1996).Review Analysis of AlexandriumOnly (Dinophyceae) 17 18 459 species using sequences of the 5.8S ribosomal DNA and internal transcribed 19 20 21 460 spacer regions. Journal of Phycology, 32: 424–432. 22 23 NGLÈS EÑÉ ARCÉS UGLIÈ ECHI AMP ATTA 24 461 A , S., R , A., G , E., L , A., S , N., C , J. & S , C.T. 25 26 462 (2017). Morphological and molecular characterization of Bysmatrum subsalsum 27 28 29 463 (Dinophyceae) from the western Mediterranean Sea reveals the existence of 30 31 464 cryptic species. Journal of Phycology, 53: 833–847. 32 33 34 465 BALECH, E. (1980). On the thecal morphology of dinoflagellates with special emphasis on 35 36 37 466 circular and sulcal plates. Anales del Centro de Ciencias del Mar y Limnologia, 38 39 467 Universidad Nacional Autonomia de Mexico, 7: 57–68. 40 41 42 468 BELOW, R. (1987). Evolution und Systematik von Dinoflagellaten-Zysten aus der 43 44 469 Ordnung Peridiniales. I. Allgemeine Grundlagen und Subfamilie 45 46 47 470 Rhaetogonyaulacoideae (Familie Peridiniaceae). Palaeontographica Abteilung B, 48 49 50 471 205: 1–164. 51 52 472 BOC, A., DIALLO, A.B. & MAKARENKOV, V. (2012). T-REX: a web server for inferring, 53 54 55 473 validating and visualizing phylogenetic trees and networks. Nucleic Acids 56 57 58 24 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 25 of 47 European Journal of Phycology

1 25 2 3 4 474 Research, 40: W573–W579. 5 6 7 475 BOLTOVSKOY, A. (1975). Peridinium cinctum (Müller) Ehrenberg. Physis secc. B, 34: 8 9 476 73–84. 10 11 12 477 BOLTOVSKOY, A. (1989). Thecal morphology of the dinoflagellate Peridinium gutwinskii. 13 14 478 Nova Hedwigia, 49: 369–380. 15 16 For Peer Review Only 17 479 CARTY, S. (2008). Parvodinium gen. nov. for the Umbonatum group of Peridinium 18 19 20 480 (Dinophyceae). Ohio Journal of Science, 108: 103–107. 21 22 481 CRAVEIRO, S., CALADO, A.J., DAUGBJERG, N. & MOESTRUP, Ø. (2009). Ultrastructure and 23 24 25 482 LSU rDNA-based revision of Peridinium group palatinum (Dinophyceae) with 26 27 483 the description of Palatinus gen. nov. Journal of Phycology, 45: 1175–1194. 28 29 30 484 CRAVEIRO, S.C., MOESTRUP, Ø., DAUGBJERG, N. & CALADO, A.J. (2010). Ultrastructure 31 32 33 485 and large subunit rDNA-based phylogeny of Sphaerodinium cracoviense, an 34 35 486 unusual freshwater dinoflagellate with a novel type of eyespot. Journal of 36 37 38 487 Eukaryotic Microbiology, 57: 568–585. 39 40 488 CRAVEIRO, S.C., CALADO, A.J., DAUGBJERG, N., HANSEN, G. & MOESTRUP, Ø. (2011). 41 42 43 489 Ultrastructure and LSU rDNA-based phylogeny of Peridinium lomnickii and 44 45 46 490 description of Chimonodinium gen. nov. (Dinophyceae). Protist, 162: 590–615. 47 48 491 CRAVEIRO, S.C., DAUGBJERG, N., MOESTRUP, Ø. & CALADO, A.J. (2015). Fine-structural 49 50 51 492 characterization and phylogeny of Peridinium polonicum, type species of the 52 53 493 recently described genus Naiadinium (Dinophyceae). European Journal of 54 55 56 494 Protistology, 51: 259–279. 57 58 25 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 26 of 47

1 26 2 3 4 495 CRAVEIRO, S.C., DAUGBJERG, N., MOESTRUP, Ø. & CALADO, A.J. (2016). Studies on 5 6 7 496 Peridinium aciculiferum and Peridinium malmogiense (= Scrippsiella hangoei): 8 9 497 comparison with Chimonodinium lomnickii and description of Apocalathium gen. 10 11 12 498 nov. (Dinophyceae). Phycologia, 56: 21–35. 13 14 499 DAUGBJERG, N., HANSEN, G., LARSEN, J. & MOESTRUP, Ø. (2000). Phylogeny of some of 15 16 For Peer Review Only 17 500 the major genera of dinoflagellates based on ultrastructure and partial LSU rDNA 18 19 20 501 sequence data, including the erection of three new genera of unarmoured 21 22 502 dinoflagellates. Phycologia, 39: 302–317. 23 24 25 503 DAWUT, M., SYM, S.D., SUDA, S. & HORIGUCHI, T. (2018). Bysmatrum austrafrum sp. 26 27 504 nov. (Dinophyceae), a novel tidal pool dinoflagellate from South Africa. 28 29 30 505 Phycologia, 57: 169–178. 31 32 33 506 DÜRR, G. & NETZEL, H. (1974). The fine structure of the cell surface in Gonyaulax 34 35 507 polyedra (Dinoflagellata). Cell and Tissue Research, 150: 21–41. 36 37 38 508 DÜRR, G. (1979). Elektronenmikroskopische Untersuchungen am Panzer von 39 40 509 Dinoflagellaten: II. Peridinium cinctum. Archiv für Protistenkunde, 122: 88–120. 41 42 43 510 44 45 46 511 ELBRÄCHTER, M. & MEYER, B. (2001). Plate pattern variability and plate overlap in a 47 48 512 clonal culture of the freshwater dinoflagellate Peridinium umbonatum Stein 49 50 51 513 species complex (Dinophyceae). Neues Jahrbuch für Geologie und 52 53 514 Paläontologie/Abhandlungen, 219: 221–227. 54 55 56 515 ELBRÄCHTER, M., GOTTSCHLING, M., HILDEBRAND-HABEL, T., KEUPP, H., KOHRING, R., 57 58 26 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 27 of 47 European Journal of Phycology

1 27 2 3 4 516 LEWIS, J., MEIER, S.K.J., MONTRESOR, M., STRENG, M. & VERSTEEGH, G.J.M. 5 6 7 517 (2008). Establishing an agenda for calcareous dinoflagellate research 8 9 518 (Thoracosphaeraceae, Dinophyceae) including a nomenclatural synopsis of 10 11 12 519 generic names. Taxon, 57: 1289–1303. 13 14 520 FENSOME, R.A., TAYLOR, F.J.R., NORRIS, G., SARJEANT, W.A.S., WHARTON, D.I. & 15 16 For Peer Review Only 17 521 WILLIAMS, G.L. (1993). A classification of fossil and living dinoflagellates. 18 19 20 522 Micropaleontology Special Publication, 7: 1–245. 21 22 523 GOTTSCHLING, M., SOEHNER, S., ZINSSMEISTER, C., JOHN, U., PLÖTNER, J., SCHWEIKERT, 23 24 25 524 M., ALIGIZAKI, K. & ELBRÄCHTER, M. (2012). Delimitation of the 26 27 525 Thoracosphaeraceae (Dinophyceae), including the calcareous dinoflagellates, 28 29 30 526 based on large amounts of Ribosomal RNA sequence data. Protist, 163: 15–24. 31 32 33 527 GOTTSCHLING, M., KRETSCHMANN, J. & ČALASAN, A.Ž. (2017). Description of 34 35 528 Peridiniopsidaceae, fam. nov. (Peridiniales, Dinophyceae). Phytotaxa, 299: 36 37 38 529 293–296. 39 40 530 GU, H., LIU, T. & MERTENS, K. (2015). Cyst-theca relationship and phylogenetic 41 42 43 531 positions of Protoperidinium (Peridiniales, Dinophyceae) species of the sections 44 45 46 532 Conica and Tabulata, with description of Protoperidinium shanghaiense sp. nov. 47 48 533 Phycologia, 54: 49–66. 49 50 51 534 GUILLARD, R.R.L. & RYTHER, J.H. (1962). Studies of marine planktonic diatoms. I. 52 53 535 Cyclotella nana Hustedt and Detonula confervacea Cleve. Canadian Journal of 54 55 56 536 Microbiology, 8: 229–239. 57 58 27 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 28 of 47

1 28 2 3 4 537 HALL, T.A. (1999). BioEdit: a user-friendly biological sequence alignment editor and 5 6 7 538 analysis program for Windows 95/98/NT. pp. 95–98. 8 9 539 HEAD, M.J. (1996). Modern dinoflagellate cysts and their biological affinities. In 10 11 12 540 Palynology: principles and applications (Jansonius, J. &McGregor, D.C., editors), 13 14 541 1197–1248. American Association of Stratigraphic Palynologists Foundation, 15 16 For Peer Review Only 17 542 Dallas. 18 19 20 543 HORIGUCHI, T. & TAKANO, Y. (2006). Serial replacement of a diatom endosymbiont in 21 22 544 the marine dinoflagellate Peridinium quinquecorne (Peridiniales, Dinophyceae). 23 24 25 545 Phycological Research, 54: 193–200. 26 27 546 KATOH, K. & STANDLEY, D.M. (2013). MAFFT multiple sequence alignment software 28 29 30 547 version 7: improvements in performance and usability. Molecular Biology and 31 32 33 548 Evolution, 30: 772–780. 34 35 549 KRETSCHMANN, J., ELBRÄCHTER, M., ZINSSMEISTER, C., SOEHNER, S., KIRSCH, M., 36 37 38 550 KUSBER, W.-H. & GOTTSCHLING, M. (2015). Taxonomic clarification of the 39 40 551 dinophyte Peridinium acuminatum Ehrenb.,≡ Scrippsiella acuminata, comb. 41 42 43 552 nov. (Thoracosphaeraceae, Peridiniales). Phytotaxa, 220: 239–256. 44 45 46 553 KRETSCHMANN, J., ŽERDONER, ČALASAN, A. & GOTTSCHLING, M. (2018). Molecular 47 48 554 phylogenetics of dinophytes harboring diatoms as endosymbionts 49 50 51 555 (Kryptoperidiniaceae, Peridiniales), with evolutionary interpretations and a focus 52 53 556 on the identity of Durinskia oculata from Prague. Molecular Phylogenetics and 54 55 56 557 Evolution, 118: 392–402. 57 58 28 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 29 of 47 European Journal of Phycology

1 29 2 3 4 558 LEE, J.-J., JANG, S.-H., LEE, J.-H. & LEE, J.-H. (2006). Morphology and ecology of 5 6 7 559 Peridinium bipes var. occultatum Lindem.(Dinophyceae) forming freshwater red 8 9 560 tides in Korean dam reservoirs. Algae, 21: 433–443. 10 11 12 561 LINDBERG, K., MOESTRUP, Ø. & DAUGBJERG, N. (2005). Studies on woloszynskioid 13 14 562 dinoflagellates I: Woloszynskia coronata re-examined using light and electron 15 16 For Peer Review Only 17 563 microscopy and partial LSU rDNA sequences, with description of Tovellia gen. 18 19 20 564 nov. and Jadwigia gen. nov. (Tovelliaceae fam. nov.). Phycologia, 44: 416–440. 21 22 565 LITAKER, W.R., VANDERSEA, M.W., KIBLER, S.R., REECE, K.S., STOKES, N.A., LUTZONI, 23 24 25 566 F.M., YONISH, B.A., WEST, M.A., BLACK, M.N.D. & TESTER, P.A. (2007). 26 27 567 Recognizing dinoflagellate species using ITS rDNA sequences. Journal of 28 29 30 568 Phycology, 43: 344–355. 31 32 33 569 LOGARES, R., SHALCHIAN-TABRIZI, K., BOLTOVSKOY, A. & RENGEFORS, K. (2007). 34 35 570 Extensive dinoflagellate phylogenies indicate infrequent marine-freshwater 36 37 38 571 transitions. Molecular Phylogenetics and Evolution, 45: 887–903. 39 40 572 LUO, Z., MERTENS, K.N., BAGHERI, S., AYDIN, H., TAKANO, Y., MATSUOKA, K., 41 42 43 573 MCCARTHY, F. MG & GU, H. (2016). Cyst-theca relationship and phylogenetic 44 45 46 574 positions of Scrippsiella plana sp. nov. and S. spinifera (Peridiniales, 47 48 575 Dinophyceae). European Journal of Phycology, 51: 188–202. 49 50 51 576 LUO, Z., LIM, Z.F., MERTENS, K.N., GURDEBEKE, P., BOGUS, K., CARBONELL-MOORE, C., 52 53 577 VRIELINCK, H., LEAW, C.P., LIM, P.T., CHOMÉRAT, N., LI, X. & GU, H. (2018). 54 55 56 578 Morpho-molecular diversity and phylogenetic relationship of Bysmatrum species 57 58 29 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 30 of 47

1 30 2 3 4 579 (Dinophyceae) from the South China Sea and France. European Journal of 5 6 7 580 Phycology, 53: 318–335. 8 9 581 MCCARTHY, F.M., GU, H., MERTENS, K.N., CARBONELL-MOORE, C., KRUEGER, A.M., 10 11 12 582 TAKANO, Y. & MATSUOKA, K. (2018). Transferring the freshwater dinoflagellate 13 14 583 Peridinium wisconsinense (Dinophyceae) to the family Thoracosphaeraceae, with 15 16 For Peer Review Only 17 584 the description of Fusiperidinium gen. nov.. Phycological Research, 66: 137–168. 18 19 20 585 MEDLIN, L., ELWOOD, H.J., STICKEL, S. & SOGIN, M.L. (1988). The characterization of 21 22 586 enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene, 71: 23 24 25 587 491–499. 26 27 588 MOESTRUP, Ø. & DAUGBJERG, N. (2007). On dinoflagellate phylogeny and classification, 28 29 30 589 in: Brodie, J. et al. (Ed.) (2007). Unravelling the algae: the past, present, and 31 32 33 590 future of algal systematics. The Systematics Association Special Volume Series, 34 35 591 75: 215–230. 36 37 38 592 MOESTRUP, Ø., LINDBERG, K. & DAUGBJERG, N. (2009). Studies on woloszynskioid 39 40 593 dinoflagellates IV: The genus Biecheleria gen. nov.. Phycological Research, 57: 41 42 43 594 203–220. 44 45 46 595 MOESTRUP, Ø. & CALADO, A.J. (2018). Dinophyceae. In Freshwater Flora of Central 47 48 596 Europe (Büdel, B., Gärtner, G., Krienitz, L. & Schagerl, M., editors), 6: 1–560, 49 50 51 597 Springer, Berlin. 52 53 598 NÉZAN, E. & CHOMÉRAT, N. (2011). Vulcanodinium rugosum gen. et sp. nov. 54 55 56 599 (Dinophyceae), un nouveau dinoflagellé marin de la côte méditerranéenne 57 58 30 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 31 of 47 European Journal of Phycology

1 31 2 3 4 600 française. Cryptogamie-Algologie, 32: 3–18. 5 6 7 601 NETZEL, H. & DÜRR, G. (1984). Dinoflagellate cell cortex. In Dinoflagellates (Spector, 8 9 602 D.L., editor), Academic Press Inc., Orlando, Florida, 43–105. 10 11 12 603 POSADA, D. 2008. jModelTest: phylogenetic model averaging. Molecular Biology and 13 14 604 Evolution, 25: 1253–1256. 15 16 For Peer Review Only 17 605 PIENAAR, R.N., SAKAI, H. & HORIGUCHI, T. (2007). Description of a new dinoflagellate 18 19 20 606 with a diatom endosymbiont, Durinskia capensis sp. nov. (Peridiniales, 21 22 607 Dinophyceae) from South Africa. Journal of Plant Research, 120: 247–258. 23 24 25 608 RONQUIST, F. & HUELSENBECK, J.P. (2003). MrBayes 3: Bayesian phylogenetic inference 26 27 609 under mixed models. Bioinformatics, 19: 1572–1574. 28 29 30 610 SABUROVA, M., CHOMÉRAT, N. & HOPPENRATH, M. (2012). Morphology and SSU rDNA 31 32 33 611 phylogeny of Durinskia agilis (Kofoid & Swezy) comb. nov. (Peridiniales, 34 35 612 Dinophyceae), a thecate, marine, sand-dwelling dinoflagellate formerly classified 36 37 38 613 within Gymnodinium. Phycologia, 51: 287–302. 39 40 614 SABUROVA M. & CHOMÉRAT, N. (2018). Laciniporus arabicus gen. et sp. 41 42 43 615 nov.(Dinophyceae, Peridiniales), a new thecate, marine, sand‐dwelling 44 45 46 616 dinoflagellate from the northern Indian Ocean (Arabian Sea). Journal of 47 48 617 Phycology, doi: 10.1111/jpy.12783. 49 50 51 618 SCHOLIN, C.A., HERZOG, M., SOGIN, M. & ANDERSON, D.M. (1994). Identification of 52 53 619 group- and strain-specific genetic markers for globally distributed Alexandrium 54 55 56 620 (Dinophyceae). II. Sequence analysis of a fragment of the LSU rRNA gene. 57 58 31 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 32 of 47

1 32 2 3 4 621 Journal of Phycology, 30: 999–1011. 5 6 7 622 SPECTOR, D. & TRIEMER, R. (1979). Ultrastructure of the dinoflagellate Peridinium 8 9 623 cinctum f. ovoplanum. I. Vegetative cell ultrastructure. American Journal of 10 11 12 624 Botany, 66: 845–850. 13 14 625 SPURR, A.R. (1969). A low-viscosity epoxy resin embedding medium for electron 15 16 For Peer Review Only 17 626 microscopy. Journal of Ultrastructure Research, 26: 31–43. 18 19 20 627 STAMATAKIS, A. (2006). RAxML-VI-HPC: maximum likelihood-based phylogenetic 21 22 628 analyses with thousands of taxa and mixed models. Bioinformatics, 22: 23 24 25 629 2688–2690. 26 27 630 SWOFFORD, D. (2002). Sinauer Associates; Sunderland, MA: 2002PAUP*. Phylogenetic 28 29 30 631 analysis using parsimony (* and other methods), version 4 b10. 31 32 33 632 TAMURA, M., SHIMWIA, S. & HORIGUCKI, T. (2005). Galeidinium rugatum gen. et sp. nov. 34 35 633 (Dinophyceae), a new coccoid dinoflagellate with a diatom endosymbiont. 36 37 38 634 Journal of Phycology, 41: 658–671. 39 40 635 TILLMANN, U., HOPPENRATH, M., GOTTSCHLING, M., KUSBER, W.-H. & ELBRÄCHTER, M. 41 42 43 636 (2017). Plate pattern clarification of the marine dinophyte Heterocapsa triquetra 44 45 46 637 sensu Stein (Dinophyceae) collected at the Kiel Fjord (Germany). Journal of 47 48 638 Phycology, 53: 1305–1324. 49 50 51 639 Wołoszyńska, J. (1916). Polskie Peridineae słodkowodne. – Polnische Süsswasser 52 53 640 -Peridineen. Bulletin International de l’Academie des Sciences de Cracovie, 54 55 56 641 Classe des Sciences Mathématiques et Naturelles, série B: Sciences Naturelles, 57 58 32 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 33 of 47 European Journal of Phycology

1 33 2 3 4 642 1915: 260–285. 5 6 7 643 WOŁOSZYŃSKA, J. (1937). Die Algen der Tatraseen und Tümpel. III. Peridineen im 8 9 644 Winterplankton einiger Tatraseen. Archiwum Hydrobiologji i Rybactwa, 10: 10 11 12 645 188–196 13 14 646 WOŁOSZYŃSKA, J. (1952). Bruzdnice Tatr i Karpat Wschodnich [Peridineae montium 15 16 For Peer Review Only 17 647 Tatrensium et Carpathorum Orientalium]. Acta Societatis Botanicorum Poloniae, 18 19 20 648 21: 311–316. 21 22 649 YAMADA, N., SYM, S.D. & HORIGUCHI, T. (2017). Identification of highly divergent 23 24 25 650 diatom-derived chloroplasts in dinoflagellates, including a description of 26 27 651 Durinskia kwazulunatalensis sp. nov. (Peridiniales, Dinophyceae). Molecular 28 29 30 652 Biology and Evolution, 34: 1335–1351. 31 32 33 653 YOU, X., LUO, Z., SU, Y., GU, L. & GU, H. (2015). Peridiniopsis jiulongensis, a new 34 35 654 freshwater dinoflagellate with a diatom endosymbiont from China. Nova 36 37 38 655 Hedwigia, 100: 313–326. 39 40 656 41 42 43 657 Figure captions 44 45 46 658 Figs. 1–4. Light micrographs of cysts and cells of Caladoa arcachonensis from France. 47 48 659 Fig. 1. Ventral view showing the outline and a post-equatorial cingulum of a living 49 50 51 660 cyst with a pyrenoid (p) and eyespot (arrow). Fig. 2. The cyst in Fig. 1 upon 52 53 661 germination showing the chasmic archeopyle (arrow). Fig. 3. The cyst in Fig. 1 54 55 56 662 upon germination showing granular surface. Fig. 4. Ventral view of the germinated 57 58 33 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 34 of 47

1 34 2 3 4 663 cell from the cyst in Fig. 1, showing a red eyespot in the sulcal area (e). Scale bars 5 6 7 664 = 5 μm. 8 9 665 10 11 12 666 Figs. 5–8. Light micrographs of cells of Caladoa arcachonensis strain TIO278 from 13 14 667 France. Fig. 5. Ventral view showing a rounded epitheca and hypotheca with a 15 16 For Peer Review Only 17 668 post-equatorial cingulum. Fig. 6. Lateral view showing a ring like pyrenoid (p) and 18 19 20 669 a red eyespot (arrow). Fig.7. Epifluorescence image of a cell in ventral view 21 22 670 showing the single reticulate chloroplast (c) in the periphery of the cell. Fig. 8. 23 24 25 671 Epifluorescence image of a SYBR Green-stained cell showing a spherical nucleus 26 27 672 (N). Scale bars = 5 μm. 28 29 30 673 31 32 33 674 Figs. 9–12. Scanning electron micrographs of vegetative cells of Caladoa arcachonensis 34 35 675 strain TIO278 from France. Fig. 9. Ventral view showing the first apical plate (1′), 36 37 38 676 anterior sulcal plate (Sa), right sulcal plate (Sd), posterior sulcal plate (Sp), and the 39 40 677 first three cingular plates (C1 – C3). Fig. 10. Dorsal view showing three anterior 41 42 43 678 intercalary plates (1a – 3a), three precingular plates (3′′ – 5′′), three cingular plates 44 45 46 679 (C3 – C5) and two postcingular plates (3′′′, 4′′′). Fig. 11. Ventral-lateral right view 47 48 680 showing two precingular plates (6′′, 7′′), sixth cingular plate (C6) and fifth 49 50 51 681 postcingular plate (5′′′). Fig. 12. Dorso-antapical view showing five postcingular 52 53 682 plates (1′′′ – 5′′′) and two antapical plates (1′′′′, 2′′′′) of similar size. Scale bars = 5 54 55 56 683 μm. 57 58 34 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 35 of 47 European Journal of Phycology

1 35 2 3 4 684 5 6 7 685 Figs. 13–16. Scanning electron micrographs of vegetative cells of Caladoa 8 9 686 arcachonensis strain TIO278 from France. Fig. 13. Ventro-apical view showing 10 11 12 687 four apical plates (1′ – 4′), the anterior sulcal plate (Sa), the first anterior intercalary 13 14 688 plate (1a) and three precingular plates (1′′, 2′′, 7′′). Fig. 14. Dorso-apical view 15 16 For Peer Review Only 17 689 showing three apical plates (2′ – 4′), three anterior intercalary plates (1a – 3a) and 18 19 20 690 five precingular plates (2′′ – 6′′). Fig. 15. Internal view of the hypotheca showing 21 22 691 six cingular plates (C1 – C6) and right sulcal plate (Sd), left sulcal plate (Ss), 23 24 25 692 median sulcal plate (Sm) and posterior sulcal plate (Sp). Fig. 16. Sulcal area 26 27 693 showing the first cingular plate (C1) and plates Sa, Sd, Sp and Ss (arrow). Scale 28 29 30 694 bars = 5 μm. 31 32 33 695 34 35 696 Figs. 17–20. Schematic drawings of thecal plate patterns of Caladoa arcachonensis. Fig. 36 37 38 697 17. Ventral view. Fig. 18. Dorsal view. Fig. 19. Apical view showing plate overlap 39 40 698 patterns. Fig. 20. Antapical view showing plate overlap patterns. 41 42 43 699 44 45 46 700 Figs. 21–26. Transmission electron micrographs of vegetative cells of Caladoa 47 48 701 arcachonensis strain TIO278 from France. Figs. 21, 22. Longitudinal sections 49 50 51 702 through the cell showing a large nucleus (N), a stalked pyrenoid (p), a single 52 53 703 chloroplast (c) in the periphery of the cell and an eyespot (arrow). Fig. 23. The 54 55 56 704 chloroplast showing the thylakoids grouped in threes to form lamellae and several 57 58 35 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 36 of 47

1 36 2 3 4 705 trichocysts (arrow). Fig. 24. Detail of the stalked pyrenoid (p) with dense matrices 5 6 7 706 and a surrounding starch (s). Figs. 25, 26. The eyespot (e) located within a 8 9 707 chloroplast (c) comprising two rows of globular lipids. Scale: Figs. 21, 22 = 5 μm, 10 11 12 708 Figs. 23–26 = 1 μm. 13 14 709 15 16 For Peer Review Only 17 710 Figs. 27–28. Light micrographs of vegetative cells of Caladoa arcachonensis strain 18 19 20 711 TIO339 from Indonesia. Fig. 27. Lateral view of a living cell showing a pyrenoid 21 22 712 (p), a nucleus (N) and an eyespot (arrow). Fig. 28. Dorwal view of a living cell 23 24 25 713 showing a single chloroplast forming a network. Scale bars = 5 μm. 26 27 714 28 29 30 715 Figs. 29–34. Scanning electron micrographs of vegetative cells of Caladoa 31 32 33 716 arcachonensis strain TIO339 from Indonesia. Fig. 29. Ventro-antapical view 34 35 717 showing the anterior sulcal plate (Sa), the right sulcal plate (Sd), posterior sulcal 36 37 38 718 plate (Sp), two postcingular plates (1′′′, 5′′′) and two antapical plates (1′′′′, 2′′′′). Fig. 39 40 719 30. Dorsal view showing three precingular plates (4′′ – 6′′), three cingular plates 41 42 43 720 (C4 – C6) and three postcingular plates (3′′′– 5′′′). Fig. 31. Dorso-apical view 44 45 46 721 showing four precingular plates (2′′ – 5′′), three anterior intercalary plates (1a – 3a). 47 48 722 Fig. 32. Antapical view showing four postcingular plates (2′′′ – 5′′′), plate Sp and 49 50 51 723 two antapical plates (1′′′′, 2′′′′) of similar size. Fig. 33. Internal view of the 52 53 724 hypotheca showing six cingular plates (C1 – C6). Fig. 34. The sulcus showing Sa, 54 55 56 725 Sd, Sp and left sulcal plate (arrow). Scale bars = 5 μm. 57 58 36 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 37 of 47 European Journal of Phycology

1 37 2 3 4 726 5 6 7 727 Fig. 35. Phylogeny of Caladoa inferred from concatenated SSU and partial LSU rDNA 8 9 728 sequences using maximum likelihood (ML). New sequences are indicated in bold. 10 11 12 729 Branch lengths are drawn to scale, with the scale bar indicating the number of 13 14 730 nucleotide substitutions per site. Numbers on branches are statistical support values 15 16 For Peer Review Only 17 731 to clusters on the right of them (left: ML bootstrap support (BS) values; right: 18 19 20 732 Bayesian posterior probabilities (BPP). Bootstrap support values >50% and 21 22 733 Bayesian posterior probabilities above 0.7 are shown. Asterisk indicates maximal 23 24 25 734 support (ML BS = 100% and BPP = 1.00). 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 37 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 38 of 47

1 38 2 3 4 5 6 Table 1 Strains of Caladoa arcachonensis examined in the present study, including collection data, locations and sources 7 8 Species Strains Longitude Latitude Collectiton date Sources Collection locality 9 TIO278, 15 and 19 April Caladoa arcachonensis 1°4'0.0'' W 44°38'11.0''N Sediment Arcachon Bay, France 10 TIO282 2016 11 Caladoa arcachonensis TIO339 124°51'6.4''E 1°35'44.8''N 2016.07.31 Seagrass Manado, Indonesia 12 For Peer Review Only 13 Caladoa arcachonensis TIO340 125°13'38.6''E 1°32'3.7''N 2016.08.01 Coral sands Bitung, Indonesia 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 38 44 45 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 39 of 47 European Journal of Phycology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Figs. 1–4. Light micrographs of cysts and cells of Caladoa arcachonensis from France. Fig. 1. Ventral view 44 showing the outline and a post-equatorial cingulum of a living cyst with a pyrenoid (p) and eyespot (arrow). 45 Fig. 2. The cyst in Fig. 1 upon germination showing the chasmic archeopyle (arrow). Fig. 3. The cyst in Fig. 46 1 upon germination showing granular surface. Fig. 4. Ventral view of the germinated cell from the cyst in Fig. 1, showing a red eyespot in the sulcal area (e). Scale bars = 5 μm. 47 48 170x193mm (300 x 300 DPI) 49 50 51 52 53 54 55 56 57 58 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 40 of 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Figs. 5–8. Light micrographs of cells of Caladoa arcachonensis strain TIO278 from France. Fig. 5. Ventral 45 view showing a rounded epitheca and hypotheca with a post-equatorial cingulum. Fig. 6. Lateral view 46 showing a ring like pyrenoid (p) and a red eyespot (arrow). Fig.7. Epifluorescence image of a cell in ventral 47 view showing the single reticulate chloroplast (c) in the periphery of the cell. Fig. 8. Epifluorescence image 48 of a SYBR Green-stained cell showing a spherical nucleus (N). Scale bars = 5 μm. 49 80x94mm (300 x 300 DPI) 50 51 52 53 54 55 56 57 58 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 41 of 47 European Journal of Phycology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figs. 9–12. Scanning electron micrographs of vegetative cells of Caladoa arcachonensis strain TIO278 from 46 France. Fig. 9. Ventral view showing the first apical plate (1′), anterior sulcal plate (Sa), right sulcal plate 47 (Sd), posterior sulcal plate (Sp), and the first three cingular plates (C1 – C3). Fig. 10. Dorsal view showing three anterior intercalary plates (1a – 3a), three precingular plates (3′′ – 5′′), three cingular plates (C3 – 48 C5) and two postcingular plates (3′′′, 4′′′). Fig. 11. Ventral-lateral right view showing two precingular plates 49 (6′′, 7′′), sixth cingular plate (C6) and fifth postcingular plate (5′′′). Fig. 12. Dorso-antapical view showing 50 five postcingular plates (1′′′ – 5′′′) and two antapical plates (1′′′′, 2′′′′) of similar size. Scale bars = 5 μm. 51 52 170x205mm (300 x 300 DPI) 53 54 55 56 57 58 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 42 of 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Figs. 13–16. Scanning electron micrographs of vegetative cells of Caladoa arcachonensis strain TIO278 from 40 France. Fig. 13. Ventro-apical view showing four apical plates (1′ – 4′), the anterior sulcal plate (Sa), the 41 first anterior intercalary plate (1a) and three precingular plates (1′′, 2′′, 7′′). Fig. 14. Dorso-apical view 42 showing three apical plates (2′ – 4′), three anterior intercalary plates (1a – 3a) and five precingular plates 43 (2′′ – 6′′). Fig. 15. Internal view of the hypotheca showing six cingular plates (C1 – C6) and right sulcal 44 plate (Sd), left sulcal plate (Ss), median sulcal plate (Sm) and posterior sulcal plate (Sp). Fig. 16. Sulcal area showing the first cingular plate (C1) and plates Sa, Sd, Sp and Ss (arrow). Scale bars = 5 μm. 45 46 170x173mm (300 x 300 DPI) 47 48 49 50 51 52 53 54 55 56 57 58 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 43 of 47 European Journal of Phycology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Figs. 17–20. Schematic drawings of thecal plate patterns of Caladoa arcachonensis. Fig. 17. Ventral view. 42 Fig. 18. Dorsal view. Fig. 19. Apical view showing plate overlap patterns. Fig. 20. Antapical view showing plate overlap patterns. 43 44 179x191mm (300 x 300 DPI) 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 44 of 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Figs. 21–26. Transmission electron micrographs of vegetative cells of Caladoa arcachonensis strain TIO278 34 from France. Figs. 21, 22. Transverse section through the cell showing a large nucleus (N), a stalked 35 pyrenoid (p), a single chloroplast (c) in the periphery of the cell and an eyespot (arrow). Fig. 23. The chloroplast showing the thylakoids grouped in threes to form lamellae and several trichocysts (arrow). Fig. 36 24. Detail of the stalked pyrenoid (p) with dense matrices and a surrounding starch (s). Figs. 25, 26. The 37 eyespot (e) located within a chloroplast (c) comprising several rows of globular lipids. Scale: Figs. 21, 22 = 38 5 μm, Figs. 23–26 = 1 μm. 39 40 170x138mm (300 x 300 DPI) 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 45 of 47 European Journal of Phycology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 Figs. 27–28. Light micrographs of vegetative cells of Caladoa arcachonensis strain TIO339 from Indonesia. 27 Fig. 27. Lateral view of a living cell showing a pyrenoid (p), a nucleus (N) and an eyespot (arrow). Fig. 28. 28 Dorwal view of a living cell showing a single chloroplast forming a network. Scale bars = 5 μm. 29 80x47mm (300 x 300 DPI) 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] European Journal of Phycology Page 46 of 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 Figs. 29–34. Scanning electron micrographs of vegetative cells of Caladoa arcachonensis strain TIO339 from 30 Indonesia. Fig. 29. Ventro-antapical view showing the anterior sulcal plate (Sa), the right sulcal plate (Sd), 31 posterior sulcal plate (Sp), two postcingular plates (1′′′, 5′′′) and two antapical plates (1′′′′, 2′′′′). Fig. 30. 32 Dorsal view showing three precingular plates (4′′ – 6′′), three cingular plates (C4 – C6) and three 33 postcingular plates (3′′′– 5′′′). Fig. 31. Dorso-apical view showing four precingular plates (2′′ – 5′′), three anterior intercalary plates (1a – 3a). Fig. 32. Antapical view showing four postcingular plates (2′′′ – 5′′′), 34 plate Sp and two antapical plates (1′′′′, 2′′′′) of similar size. Fig. 33. Internal view of the hypotheca showing 35 six cingular plates (C1 – C6). Fig. 34. The sulcus showing Sa, Sd, Sp and left sulcal plate (arrow). Scale bars 36 = 5 μm. 37 38 170x118mm (300 x 300 DPI) 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected] Page 47 of 47 European Journal of Phycology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Fig. 35. Phylogeny of Caladoa inferred from concatenated SSU and partial LSU rDNA sequences using 46 maximum likelihood (ML). New sequences are indicated in bold. Branch lengths are drawn to scale, with the 47 scale bar indicating the number of nucleotide substitutions per site. Numbers on branches are statistical support values to clusters on the right of them (left: ML bootstrap support (BS) values; right: Bayesian 48 posterior probabilities (BPP). Bootstrap support values >50% and Bayesian posterior probabilities above 0.7 49 are shown. Asterisk indicates maximal support (ML BS = 100% and BPP = 1.00). 50 51 196x254mm (300 x 300 DPI) 52 53 54 55 56 57 58 59 60 URL: http:/mc.manuscriptcentral.com/tejp Email: [email protected]