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European Journal Of Protistology Archimer October 2018, Volume 66 Pages 115-135 https://doi.org/10.1016/j.ejop.2018.09.002 https://archimer.ifremer.fr https://archimer.ifremer.fr/doc/00468/57970/

The affiliation of Hexasterias problematica and Halodinium verrucatum sp. nov. to ciliate cysts based on molecular phylogeny and cyst wall composition

Gurdebeke Pieter R. 1, *, Mertens Kenneth 2, Takano Yoshihito 3, Yamaguchi Aika 4, Bogus Kara 5, 6, Dunthorn Micah 7, Matsuoka Kazumi 3, Vrielinck Henk 8, Louwye Stephen 1

1 Univ Ghent, Dept Geol, Krijgslaan 281, B-9000 Ghent, Belgium. 2 Ifremer, Stn Biol Marine, LER BO, Pl Croix,BP40537, F-29185 Concarneau, France. 3 Inst East China Sea Res ECSER, 1-14 Bunkyo Machi, Nagasaki 8528521, Japan. 4 Kobe Univ, Res Ctr Inland Seas, Kobe, Hyogo 6578501, Japan. 5 Univ Nottingham, Sch Geog, Ctr Environm Geochem, Nottingham NG7 2RD, England. 6 Texas A&M Univ, Int Ocean Discovery Program, College Stn, TX 77845 USA. 7 Univ Kaiserslautern, Dept Ecol, Erwin Schrodinger St, D-67663 Kaiserslautern, Germany. 8 Univ Ghent, Dept Solid State Sci, Krijgslaan 281,S1, B-9000 Ghent, Belgium.

* Corresponding author : Pieter R. Gurdebeke, email address : [email protected]

Abstract :

Species in the genera Hexasterias and Halodinium have been recorded over the last decades as acritarchs in palynological and/or plankton studies. In paleoenvironmental studies, these resting stages are often interpreted as indicators of freshwater input. The biological affinity of these genera has never been definitely established. Here, a new species, Halodinium verrucatum sp. nov., is described and molecular evidence (single specimen SSU and LSU rDNA sequencing) reveals that both this new species and Hexasterias problematica, collected from sediment samples in the Skagerrak and Baltic Sea, are resting stages of prorodontid ciliates. Additionally, infrared spectroscopic analysis (micro-FTIR) of Hexasterias problematica and Halodinium spp. specimens indicates a carbohydrate-based composition of the cyst wall with evidence for nitrogen-containing functional groups. A similar composition is recorded for tintinnid loricae, which further supports the placement of Hexasterias and Halodinium as ciliate cysts, and the composition is consistent with the heterotrophic nature of ciliates. The morphologically similar species Radiosperma corbiferum has a comparable composition, suggesting a similar ciliate affinity and indicating the utility of micro-FTIR in understanding acritarch affinity. Hexasterias problematica typically occurs in coastal waters from temperate to arctic regions. Halodinium verrucatum sp. nov. is observed in temperate estuarine sediments in the northern hemisphere.

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. 2

Highlights

► Halodinium verrucatum sp. nov. is described, Hexasterias problematica is emended. ► LSU and SSU rDNA is obtained for Halodinium verrucatum and Hexasterias problematica. ► Molecular phylogeny and DAPI-staining demonstrate these are ciliate resting cysts. ► micro-FTIR of resting cysts and tintinid loricae supports this affiliation. ► The distribution and ecology of both palynomorphs is discussed.

Keywords : Acritarch, Ciliate cyst, FTIR, LSU-SSU rDNA, Prostomatida, Taxonomy

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. 119 120 42 Introduction 121 122 123 43 The informal group of Acritarcha was introduced by Evitt (1963, p. 300) to accommodate organic- 124 125 44 walled microfossil remains (i.e., palynomorphs) of apparently unicellular organisms whose 126 127 45 biological affinity is unknown and which are resistant to degradation and palynological treatment. 128 129 46 Acritarchs consist of a central cavity enclosed by a single- or multilayered wall and are extremely 130 131 47 variable in general shape, symmetry, structure, ornamentation and opening of the central cavity 132 133 48 (e.g., pore, slit, rupture, pylome). They occur in Proterozoic to modern sediments, and are 134 135 49 considered polyphyletic with diverse biological affinities (e.g., Servais 1996). Many palynomorphs 136 137 50 originally classified as acritarchs have since been reassigned to other groups, particularly 138 139 140 51 dinoflagellates (e.g., Dale 1977; Colbath and Grenfell 1995), chlorophytes (e.g., Aroui et al. 1999; 141 142 52 Kaźmierczak and Kremer 1999; Moczydłowska et al. 2011) and copepod eggs (e.g., McMinn et al. 143 144 53 1992; Van Waveren and Marcus 1993). For fossil species, arguments for these assignments are 145 146 54 based on morphological and ultrastructural observations through light microscopy (LM), scanning 147 148 55 electron microscopy (SEM) and transmission electron microscopy (TEM) (e.g., Colbath and 149 150 56 Grenfell 1995; Willman 2009) and chemical composition through spectroscopic methods (e.g., 151 152 57 Marshall et al. 2005; Javaux and Marshall 2006). For species in surface sediments, these are 153 154 58 complemented by cytological observations, life history information through incubation experiments 155 156 157 59 and molecular data obtained through methods such as single-cell polymerase chain reaction (PCR) 158 159 60 (e.g., Takano and Horiguchi 2005). Hexasterias problematica (syn. Polyasterias problematica ) and 160 161 61 species of the genus Halodinium are considered to be acritarchs and are often reported in surface 162 163 62 sediments. The former species is also known from plankton studies and Halodinium has an 164 165 63 extensive fossil record in the late Cenozoic. Hypotheses about their biological affinity have varied 166 167 64 widely but were never established conclusively. 168 169 65 Ciliates (phylum Ciliophora) are morphologically diverse heterotrophic unicellular 170 171 66 eukaryotes (protozoans) that occur in nearly all moist and aquatic environments from soils, 172 173 67 174 freshwater ponds and lakes to estuaries and the open ocean (e.g., Lynn 2008; Foissner et al. 2008). 175 176 177 3 178 179 68 An important synapomorphy of ciliates is nuclear dualism, in which a micro- and macronucleus can 180 181 182 69 be distinguished (e.g., Lynn 2008). Many of these mainly free-living forms are able to produce 183 184 70 cysts as part of their life cycle, often as a response to adverse conditions (Verni and Rosati 2011). 185 186 71 These cysts are often more resistant to destruction than any other stage of the life cycle and thus 187 188 72 offer higher potential for preservation and fossilization (Dunthorn et al. 2015). The class 189 190 73 Spirotrichea is particularly known to produce cysts (Reid and John 1978, 1983). In addition to 191 192 74 cysts, the tintinnid ciliates (Class Spirotrichea, Order Tintinnida) are known to have a long fossil 193 194 75 record due to their loricae, protective tubular structures that contain the ciliate body throughout the 195 196 76 life cycle (Dolan et al. 2013). The chemical composition of ciliate cyst walls is poorly known and 197 198 199 77 limited to a small number of species. Thus far, the major components are characterized as 200 201 78 (glyco)proteins and carbohydrates (Wang et al. 2017). Some ciliates are known to cause disease in 202 203 79 other organisms (e.g. Lynn 2008), and the management of, for example, sea itch, caused by the 204 205 80 parasitic ciliate Cryptocaryon irritans , is difficult because of the complexity of the life cycle 206 207 81 involving a resting cyst stage (Ma et al. 2017). Clearly, better understanding of ciliate life cycles 208 209 82 and resting cyst stages is of interest to workers in a broad range of fields. 210 211 83 In this study, living and empty Hexasterias problematica cysts were isolated from the 212 213 84 surface sediment of a low salinity site in SW Finland. Also, the new Halodinium species, H. 214 215 216 85 verrucatum sp. nov. is described from living and empty cysts isolated from surface sediments in a 217 218 86 brackish fjord within the Skagerrak, on the west coast of Sweden. Both species are shown, through 219 220 87 molecular phylogenetics based on single-cell polymerase chain reaction (PCR) of partial large 221 222 88 (LSU) and complete short (SSU) subunit ribosomal DNA (rDNA), to belong to the ciliate order 223 224 89 Prorodontida. Staining using the fluorescent 4´-6-diamidino-2-phenylindole (DAPI) was done to 225 226 90 confirm this placement. The geochemical composition of the cyst walls was analyzed through 227 228 91 micro-Fourier transform infrared (FTIR) spectroscopy and compared with loricae of tintinnid 229 230 92 ciliates and with morphologically similar palynomorphs. The species’ distributions are assessed 231 232 233 234 235 236 4 237 238 93 though a literature review and new observations, and the paleoenvironmental application is 239 240 241 94 evaluated. 242 243 95 244 245 96 Material and Methods 246 247 97 Sediment sampling, cyst extraction and microscopic observations 248 249 98 For the study of Hexasterias problematica , surface sediment was collected from Sällvik (28 250 251 99 October 2011), a freshwater-influenced low salinity estuary off southwestern Finland with a 252 253 100 halocline at 10 m depth and salinities up to 4.6 psu in deeper waters (e.g. Heiskanen and Tallberg 254 255 101 1999), using a Limnos gravity corer (Table 1, Fig. 1). For Halodinium verrucatum sp. nov., surface 256 257 258102 sediment was collected from station Strö 1, south of Havstensund (Skagerrak) using a boxcore 259 260103 (Table 1, Fig. 1). Additional samples from the Skagarrak and Brittany were collected for further 261 262104 observations (Table 1, Fig. 1). All samples were stored in sterile plastic bags at 4°C. 263 264105 About 0.5–1.0 cm 3 of wet sediment was immersed in filtered seawater and after 265 266106 ultrasonication (60 s, As One TM US2R ultrasonic bath), the sediment was rinsed using filtered 267 268107 seawater through a calibrated metallic-mesh Sanpo TM sieve with a 20 µm mesh. From this residue, 269 270108 the cyst fraction was separated using sodium polytungstate (SPT) at a density of 1.3 g cm -1 (Bolch 271 272 109 1997). For microscopic observations and geochemical measurements, residues were prepared using 273 274 275110 a palynological method similar to that described in Mertens et al. (2009a), involving hydrochloric 276 277111 and hydrofluoric acids and no oxidizing agents. 278 279112 The LM observations, measurements, and photography were made using a MRc5 camera 280 281113 mounted on a Zeiss Axio Imager A1 (400× and 1000× magnification) at Ghent University. For 282 283114 SEM observations, single cysts were picked from the residue and mounted on a glass slide or 284 285115 filtered on polycarbonate membrane filters (Millipore, Billerica, MA, USA, GTTP Isopore, 0.22 μm 286 287116 pore size), sputter coated with gold, and examined with a Zeiss SIGMA300 Gemini field emission 288 289 117 SEM at the Station de Biologie Marine (Ifremer, Concarneau, France). The terminology for 290 291 118 292 describing the morphology of Hexasterias and Halodinium is clarified in Fig. 2. 293 294 295 5 296 297 119 298 299 300120 Molecular analyses 301 302121 Four specimens of Hexasterias problematica were isolated for single-cell analysis from the Sällvik 303 304122 sediment sample. Two specimens of Halodinium verrucatum sp. nov. were isolated for single-cell 305 306123 PCR from the Strö 1 sediment sample of Skagerrak. Cysts were photographed and measured using 307 308124 an Olympus BX51 LM with a Nikon digital sight DS-1L 1 module with 100x oil immersion 309 310125 objectives. Subsequently, the cysts were transferred to an inverted LM, crushed with a fine glass 311 312 126 needle, and subsequently transferred into a 200 µL PCR tube containing 3 µL of Milli-Q water. 313 314 127 Sequences of partial LSU and SSU rDNA were determined from single cysts. In the first PCR 315 316 317128 round, the external primers (SR1 and LSU R2; Takano and Horiguchi 2005) were used with PCR 318 319129 mixtures of KOD-Plus-Ver. 2 Kit (Toyobo, Osaka, Japan) under PCR conditions: one initial cycle 320 321130 of denaturation at 94˚C for 2 min, followed by 35 cycles of denaturation at 94˚C for 30 s, annealing 322 323131 at 55˚C for 30 s, and extension at 72˚C for 2 min and final extension at 72˚C for 5 min. In the 324 325132 second PCR, five sets of primers (SR1-SR9, SR4-SR12, SR8-25F1R, SR12cF-25R1 and LSU D1R- 326 327133 LSU R2; Takano and Horiguchi 2005) were used with PCR mixtures of KOD-Plus-Ver. 2 Kit 328 329134 (Toyobo, Osaka, Japan), 0.5 µL of the first round PCR product as the DNA template, and the same 330 331 135 PCR conditions except that the extension at 72˚C was for 1 min. In the third PCR, five sets of 332 333 334136 primers (SR-1c-SR5, SR4-SR9, SR8-SR12, LSU D1R-25R1 and LSU D3A-LSU R2; Takano and 335 336137 Horiguchi 2005) were used with PCR mixtures of the TaKaRa EX taq system (Takara Bio Inc., 337 338138 Shiga, Japan), 0.5 µL of the second round PCR products as the DNA template, and the same PCR 339 340139 conditions except that the extension at 72˚C was for 30 s. PCR products were sequenced directly 341 342140 using the ABI PRISM BigDye Terminator Cycle Sequencing Kit (Perkin-Elmer, Foster City, CA, 343 344141 USA). 345 346142 347 348 143 Sequence alignments and phylogenetic analyses 349 350 351 352 353 354 6 355 356 144 After an initial analysis containing taxa from all major ciliate groups based on Dunthorn et al. 357 358 359145 (2014), a final sampling of 26 SSU-rDNA accessions from the Prostomatea, plus two outgroup 360 361146 species ( Furgasonia blochmanni and Platyophrya vorax ) was based on Yi et al. (2010). Nucleotides 362 363147 were aligned and ambiguous positions masked with GUIDANCE v1.1 (Penn et al. 2010a,b). The 364 365148 GTR-I-G evolutionary model was the best fit model selected by the Akaike information criterion 366 367149 (AIC) as implemented in jModeltest2 v0.1 (Darriba et al. 2012; Guindon and Gascuel 2003). 368 369150 Maximum Likehood (ML) analyses were carried out in RAxML-HPC v7.2.5 (Stamatakis et al. 370 371 151 2008), with bipartition support from 1000 multiparametic bootstrap replicates. 372 373 152 374 375 376153 DAPI Staining 377 378154 For DNA-specific fluorescent DAPI staining (Kapuscinski, 1995), a specimen of Hexasterias 379 380155 problematica was extracted from fresh sediments from the Vilaine Estuary (Brittany). The cyst was 381 382156 slightly crushed by pressing down the coverslip to allow the DAPI to intrude. Observation and 383 384157 imaging was done on a Olympus BX41 microscope with 100× oil immersion objectives (Ifremer, 385 386158 Concarneau). 387 388159 389 390 160 Geochemical analysis of cyst wall composition 391 392 393161 For micro-FTIR geochemical analysis of Halodinium spp., palynological residue was used from 394 395162 Skagerrak (Strö 1 station), Brittany (France) and the Irish Sea (see Mertens et al. 2009b and 396 397163 Gurdebeke et al., accepted, for sampling details), and from Brittany (France) and southern France 398 399164 for Hexasterias problematica (see Mertens et al. 2009b; Gurdebeke et al., accepted and Mertens et 400 401165 al. 2017, respectively for sampling details). For comparison, additional FTIR spectra were measured 402 403166 on specimens of the acritarch Radiosperma corbiferum from Brittany, the Irish Sea (Mertens et al. 404 405167 2009b; Gurdebeke et al., accepted for sampling details) and the Baltic Sea (see Mertens et al. 2012a 406 407 168 for details) and on loricae of the tintinnid ciliates Parafavella sp. from the Skagerrak (Strö 1 station, 408 409 169 410 mentioned above) and Stenosemella sp. from the German Wadden Sea (see Bogus et al. 2014 for 411 412 413 7 414 415 170 sampling details). Between palynological processing and the preparation for FTIR, these residues 416 417 418171 were stored in cool and dark conditions without adding conservation chemicals such as copper 419 420172 sulphate or fungicides. 421 422173 The palynological residues were treated as described in Gurdebeke et al. (accepted). They 423 424174 were ultrasonicated for 30 s and rinsed three times with organic solvents (methanol and 425 426175 dichloromethane, 1:1 in volume) and MilliQ water over a 20 µm mesh to remove polar and apolar 427 428176 compounds that might have adhered to the outside of the cyst walls. Visually clean individual cysts 429 430 177 were manually isolated, placed on an Au-coated mirror and dried. Specimens were analyzed with a 431 432 178 Bruker Hyperion 2000 microscope coupled to a Bruker Vertex 80v FTIR spectrometer at the 433 434 435179 Department of Solid State Sciences of Ghent University. Magnification of the microscope was set at 436 437180 15× and the aperture at 100×100 µm. The combination of detector (liquid N 2 cooled MCT detector), 438 439181 source (Globar) and beamsplitter (KBr) settings restrict the effective infrared spectral range 440 441182 examined to ~4000 –650 cm -1 . All presented spectra are recorded in reflection mode (deposits on the 442 443183 Au mirror), at a resolution of 2 cm -1 , and averaged over 100 scans. 444 445 184 Data were analyzed with OPUS© spectroscopy software (Bruker 2014). The presented 446 447 185 absorbance spectra were obtained after background (direct reflection on the Au mirror) subtraction, 448 449 450186 atmospheric correction and baseline correction using a rubberband correction method using 451 452187 polynomes. Functional groups were identified based on comparison with Colthup et al. (1990) and 453 454188 published data (e.g., Bogus et al. 2014; Cárdenas et al. 2004). 455 456189 457 458190 Results 459 460191 Species description 461 462192 Phylum Ciliophora Doflein, 1901 463 464 193 Class Prostomatea Schewiakoff, 1896 465 466 194 467 Order Prorodontida Corliss, 1974 468 469195 Genus Hexasterias Cleve, 1900 emend. 470 471 472 8 473 474 196 Synonymy . 475 476 477197 1900 Hexasterias problematica Cl. – Cleve, K. Sven. Vetenskapsakad. Handl. 32: 22 (original 478 479198 description of genus and species). 480 481199 1910 Polyasterias g.n. – Meunier, Microplancton, p. 87 (junior objective synonym; unjustified 482 483200 replacement name for Hexasterias ). 484 485201 Type species . Hexasterias problematica Cleve, 1910 486 487202 Improved diagnosis . Organic-walled cyst with bilayered wall and subspherical to flattened central 488 489 203 body from which coplanar radial processes arise from inner wall, are hollow and not connected 490 491 204 proximally or distally. Circular pylome in polar position, parallel to the process plane. 492 493 494205 Remarks . Cleve (1900) gave no formal description of the genus Hexasterias , which is why a 495 496206 diagnosis is provided here. Meunier (1910), who studied plankton from the Barents and Kara seas, 497 498207 proposed changing the genus name to Polyasterias , to emphasize that the number of projections is 499 500208 often not six (instead ranging from four to eight). This is an unjustified replacement and thus 501 502209 Polyasterias is an objective junior synonym of Hexasterias . 503 504210 Species classified in Hexasterias . Hexasterias problematica Cleve, 1900. A second species, 505 506211 Hexasterias spina-trifida, was described by Wright (1907, p. 9, Plate 3, Fig. 6), but it is not clear if 507 508 212 it has any affinity to H. problematica . Likewise for Polyasterias simplex of Kufferath (1950). 509 510 511213 512 513214 Hexasterias problematica Cleve ,1900 emend. 514 515215 Figs. 3a–c; Table 2. 516 517216 1887 ‘Röhrenstatoblast’ – Hensen, Ber. Komm. Wiss. Unters. Meere 5: 67, Plate IV, Fig. 27 (first 518 519217 illustration). 520 521218 1897 ‘Dornige cyste’ – Vanhöffen, Die Fauna und Flora Grönlands: 287, Plate VI, Fig. 3 (record 522 523219 from Egersund Fjord, Norway, with illustration). 524 525 220 1900 Hexasterias problematica Cl. – Cleve, K. Sven. Vetenskapsakad. Handl. 32: 22 (original 526 527 221 528 description, probably no type material available). 529 530 531 9 532 533 222 1904 ‘Ovum hispidum problematicum ’ (Clev.) Lohm. – Lohmann, Eier und sogenannte Cysten 534 535 536223 der Plankton-Expedition: 32, Pl. 5, Figs. 2, 4 (invalid by implication, as the genus was not 537 538224 validly published, see Williams et al. 2017, p. 920). 539 540225 1905 Xanthidium coronatum nov. sp. – Pavillard, Dissertation: 60, Pl. 3, Figs. 2–3 (invalid 541 542226 because the name was already occupied: Xanthidinium coronatum Ehrenberg, 1843, Verbr. 543 544227 und Einfluss des Mikrosk. Lebens in Süd- und Nord-Amerika, Pl. 4, Fig. 26). 545 546228 1907 Hexasterias problematica Cleve – Wright, Contrib. Can. Biol. Fish. 1: 8, Pl. 3, Fig. 5 547 548 229 (record from eastern Nova Scotia, with illustration). 549 550 230 1910 Polyasterias problematica (Cleve) pro parte – Meunier, Microplancton: 87, Pl. 5, Figs. 20– 551 552 553231 22 (redescription and combination with superfluous replacement name Polyasterias ). 554 555232 1919 Polyasterias problematica Meunier – Meunier, Mém. Mus. r. his. nat. Belg. 8: 40, Pl. 23, 556 557233 Figs. 19-20 (record from the Belgian coast, with illustration). 558 559234 1961 Polyasterias problematica (Cleve) Meunier – Bursa, J. Fish. Res. Bd. Canada 18: 76, Fig. 5 560 561235 (record from the Hudson Bay, with illustration). 562 563236 1962 Polyasterias problematica (Cleve) Meunier – Brunel, Phytoplancton de la Baie des 564 565237 Chaleurs: 211, Pl. 66 (record from the Baie des Chaleurs, with illustration). 566 567 238 1991 “Dinoquiste A-B” – Martínez-Hernández and Hernández-Campos, Palaeontología Mexicana 568 569 570239 57: 60, Pl. 4, Figs, 1-4 (record from the Gulf of California, with illustration). 571 572240 1997 Resting stage type C – Nehring, Bot. Mar. 40: 320, Fig. 36 (record from the German bight of 573 574241 the North Sea, with illustration). 575 576242 1998 Hexasterias problematica Cleve – Kunz-Pirrung, Ber. Polarforsch. 281: 98, Pl. 5, Fig. 3 577 578243 (record form the Laptev Sea, with illustration). 579 580244 1999 Polyasterias problematica (Cleve) Meunier – Bérard-Therriault et al., Guide de 581 582245 l’Identification: 323, Pl. 149, Fig. a (record from the Gulf of Saint-Lawrence, with 583 584 246 illustration). 585 586 587 588 589 590 10 591 592 247 2018 Polyasterias problematica – Kubiszyn and Svensen, Bot. Mar. 61: Fig. 2 (record from the 593 594 595248 Balsafjord, Norway, with illustration). 596 597249 Gene sequence. [to be inserted after acceptance] 598 599250 Original diagnosis (Cleve 1900, p. 22). “Flat disk, diameter 0.04 mm, with six, at the ends truncate 600 601251 and denticulate empty processes, twice as long as the radius of the disk.” 602 603252 Improved diagnosis . Cyst circular to subcircular outline in polar view (diameter ~100 µm) and 604 605253 flattened in lateral view. Central cyst wall portion (“central body”; average diameter 35 µm) 606 607 254 ornamented with four to nine radially arranged coplanar processes which are about as long as the 608 609 255 diameter of the central body (33 µm on average). Processes hollow and empty, attached to the inner 610 611 612256 wall, show longitudinal striations, are broad at the base, open up distally and terminate in numerous 613 614257 (~10 in intact specimens) denticulations which are sometimes recurved. A central circular pylome 615 616258 without operculum is present when there is no cell content, but inconspicuous elsewise. Cyst wall 617 618259 transparent and colorless. 619 620260 Studied material . Surface sediments from the Skagerrak, Baltic Sea and the Vilaine estuary 621 622261 (Brittany). 623 624262 Description . Table 2 gives detailed morphometrics of Hexasterias problematica . The number of 625 626 263 processes in most cases is six, but may vary from five to eight. The central body diameter and 627 628 629264 process length are correlated (R² = 0.85, Fig. 5). The cysts may be somewhat deformed from the 630 631265 coplanar configuration. In palynological preparations, the processes may be torn off, and the cyst is 632 633266 readily deformed. Cell contents include many small transparent to yellow lipid bodies (Fig. 3a–b). 634 635267 Micronucleus circular (3.7 µm in diameter) and macronucleus more irregular and elongated 636 637268 (10.3×12.7 µm) after DAPI staining (Fig. 6). 638 639269 Remarks . Meunier (1910) reports specimens with four processes, while Bursa (1961) reports nine. 640 641270 In some environments, e.g., Black Sea (Verleye et al. 2009; Mudie et al. 2010), the processes are 642 643 271 typically shorter (~13 µm in Verleye et al. 2009). Previous authors made various assumptions about 644 645 272 646 the affinity of Hexasterias problematica. Hensen (1887) proposed it is a bryozoan statoblast, and 647 648 649 11 650 651 273 Cleve (1900) considered it as an “unicellular alga”, without any further specification. Meunier 652 653 654274 (1910, 1919) rejected earlier propositions of algal affinity. Erdtman (1954) proposed a phytogenous 655 656275 origin based in cell content. Parke and Dixon (1964, p. 529; 1968, p. 815) assigned H. problematica 657 658276 to the Pterospermataceae (prasinophytes); however, in Parke and Dixon (1976), it was deleted from 659 660277 the Pterospermataceae, and they suggested it was possibly a desmid zygote. All of these revisions 661 662278 and changes have led to much confusion to this day, with some authors classifying H. problematica 663 664279 as a prasinophyte (e.g., Tappan 1980, p. 818), a tintinnid cyst (e.g., Mudie et al. 2010), and others as 665 666 280 incertae sedis (e.g., Bérard-Therriault et al. 1999). Hexasterias problematica sensu Pospelova et al. 667 668 281 (2010, Plate VIII, fig. 7) is a desmid. Cleve (1900) referred to chromatophores with an uncertain 669 670 671282 colour; here it cannot be assessed to what he was referring to. 672 673283 Occurrence and stratigraphic range . Hexasterias problematica Cleve, 1900 has been reported 674 675284 from both surface sediments and plankton samples. Kubiszyn and Svensen (2018) provided a 676 677285 distribution map for Hexasterias problematica , which is reproduced in Fig. 7 and extended with 678 679286 additional references from literature and personal observations. Besides the records presented by 680 681287 Kubiszyn and Svensen (2018), Hexasterias problematica is reliably recorded from Newfoundland 682 683288 (Lohmann 1904), Etang de Thau (southern France; Pavillard 1905), Nova Scotia (Wright 1907), 684 685 289 Gullmarn Fjord (Erdtman 1954), Gulf of California (Martínez-Hernández and Hernández-Campos 686 687 688290 1991), the German Bight of the North Sea (Nerhing 1997), the Beagle Chanel (Tierra del Fuego; 689 690291 Candel et al. 2012, 2013). Additional records that not substantiated by morphological data: the 691 692292 Faroe Islands (Ostenfeld 1903), Elbe River (Volk 1905), the west coast of Scotland (Herdman and 693 694293 Ridell 1911), central Atlantic Ocean (Gran 1912), Kola Gulf (Deriugin 1915), Thyborøn, 695 696294 (Denmark; Hansen-Ostenfeld 1916), British Coastal waters (Parke and Dixon 1964, 1968, 1976), 697 698295 Auray Estuary (Brittany; Paulmier 1972), southern Baltic Sea (Ringer 1973), Romanian coast of the 699 700296 Black Sea (Cărăuş 2002), offshore Walvis Bay (Mertens et al. 2009a), the Okhotsk Sea (Medvedeva 701 702 297 and Nikulina 2014), Vancouver Island fjords (Gurdebeke et al. 2018). Further observations were 703 704 705 706 707 708 12 709 710 298 made from the Vilaine Estuary (Brittany), Baltic Sea, German Wadden Sea, Harrington Sound 711 712 713299 (Bermuda Islands) and the East Chinese Sea (Mertens, Gurdebeke, unpublished data). 714 715300 The cysts are resistant to chemical degradation, shown by the fact that they withstand 716 717301 palynological treatment using strong (hydrochloric, hydrofluoric) acids (see Material and Methods). 718 719302 As a fossil, H. problematica is mostly known from late Holocene sediments, with the oldest 720 721303 occurrence from Kyuquot Sound (BC, Canada), around 6.2 ka BP (P. Gurdebeke, pers. obs.). They 722 723304 were also found in late Holocene sediments from the Laptev Sea (Kunz-Pirrung 1998) and 724 725 305 Saguenay Fjord (St-Onge et al. 1999), Santa Barbara Basin (Pospelova et al. 2006), the Aral Sea 726 727 306 (Sorrel et al. 2006), Black Sea (Verleye et al., 2009; Shumilovskikh et al. 2013) and Effingham 728 729 730307 Inlet (Bringué et al. 2016). 731 732308 733 734309 Genus Halodinium Bujak, 1984 735 736310 1984 Halodinium gen. nov.: Bujak, p. 196 (original description). 737 738311 Type species . Halodinium major Bujak, 1984 739 740312 Original diagnosis (Bujak, 1984). “Discoidal organic-walled microfossils with circular polar 741 742313 outline and irregular membranous flange. Central pylome present with lid often in place.” 743 744 314 Remarks . The genus Halodinium was erected by Bujak (1984), based on specimens isolated from 745 746 747315 Pleistocene North Pacific sediments. From that assemblage, he described H. major and H. minor . 748 749316 He considered these to be “Microphytoplankton incertae sedis”. Two other species have since been 750 751317 described: Halodinium scopaeum by Head (1993, p. 47) and Halodinium eiriksonii by Verhoeven et 752 753318 al. (2014, p. 45). Halodinium was suggested to be a rhizopod by de Vernal et al. (1989) and an 754 755319 incertae sedis alga by Head (1993). The four species of Halodinium have been identified globally in 756 757320 Neogene to modern sediments. The oldest published occurrence is H. eiriksonii from the Zanclean 758 759321 (early Pliocene) of Iceland (Verhoeven et al. 2014). In the absence of molecular data and incubation 760 761 322 experiments, the systematic positions of Halodinium and Hexasterias remained inconclusive and 762 763 323 764 they were classified as acritarchs by several authors (e.g., Kunz-Pirrung 1998, p. 98). 765 766 767 13 768 769 324 Species classified in Halodinium . Halodinium minor Bujak, Halodinium major Bujak, Halodinium 770 771 772325 scopaeum Head and Halodinium eiriksonii Verhoeven et al. 773 774326 775 776327 Halodinium verrucatum sp. nov. 777 778328 Figs. 3d–l; Fig 4, a–f; Table 3. 779 780329 Diagnosis . Bilayered, polar compressed cyst with circular to subcircular (average diameter ~70 µm) 781 782330 outline in polar view. Central body (average diameter ~57 µm) ornamented with low ridges forming 783 784 331 irregular reticulum; covered over the polar region with a thin, hyaline outer layer ornamented with 785 786 332 discrete, bifurcating sculptural elements appearing as verrucae in LM. Near margin of central body 787 788 789333 portion, outer layer forms a wrinkled, diaphanous collar-like flange with a varying width. Central 790 791334 pylome with thickened rim and without operculum. 792 793335 Studied material . Surface sediments from the Vilaine estuary (Brittany), the Skagerrak and the 794 795336 Baltic Sea. 796 797337 Stratigraphical horizon and type locality . Modern sediments from the Vilaine estuary (47.50°N, 798 799338 2.39°W, 6 m water depth), Brittany (sample BV5 of Mertens et al., 2009b). 800 801339 Type material . The holotype specimen, isolated from palynological residue BV5 and mounted on 802 803 340 SEM stub 16C05 (Fig. 4, a-e), has been deposited in the collection of the National Botanic Garden 804 805 806341 of Belgium (NBGB) (accession code BR-4516). 807 808342 Gene sequence . [to be inserted in proof] 809 810343 Zoobank registration number of Halodinium verrucatum . [to be inserted in proof] 811 812344 Etymology . The species-group name is derived from the Latin word verruca meaning wart, 813 814345 referring to the ornamentation as seen in light microscopy. 815 816346 Description . Detailed morphometrics are given in Table 3. The holotype has a maximum central 817 818347 body diameter 65.0 µm and a maximum diameter pylome of 12.1 µm; the width of its ambital 819 820 348 flange is 11.3 µm. Cyst with a strong polar compression and a circular to subcircular outline in 821 822 349 823 polar view (Fig. 4a–b). Cyst wall is bilayered, with the inner wall forming a central body (Fig. 2). 824 825 826 14 827 828 350 Inner wall of the discoidal cyst about 1.2 µm thick (N=3) and faintly ornamented with low ridges 829 830 831351 forming a reticulate pattern with varying mesh size (< 1 µm), only visible with high magnification 832 833352 (1000×) LM and SEM (Fig. 4d). Pylome central to the inner cyst wall, with slightly thickened rim 834 835353 and surrounded by a shallow circular depression of ~2 µm (Fig. 4d). The specimen with cell content 836 837354 illustrated in Fig. 3d–f, has a transparent and colorless operculum in place, but the fine structure is 838 839355 obscured by the cell content. Palynological specimens (e.g., Fig. 4) show no trace of an operculum. 840 841356 The hyaline outer layer is much thinner (~0.2 µm) than the inner layer and is ornamented with solid, 842 843 357 slender sculptural elements with a widened base. With medium magnification (400×, LM), they 844 845 358 often appear as verrucae (Fig. 3i-k) because they are recumbent (Fig. 4e), but higher LM 846 847 848359 magnifications and SEM reveal them as process-like sculptural elements. The discrete sculptural 849 850360 elements are of equal length (average of 1.7 µm, Table 3) and bifurcate at circa two third of their 851 852361 length (Fig. 4e). SEM reveals an occasional second bifurcation near the top of some sculptural 853 854362 elements (Fig. 4e). The sculptural elements are irregularly distributed and the distance between 855 856363 them averages 1.6 µm (Table 3). The cyst outer wall layer separates from the central body near the 857 858364 periphery and evolves into a folded, translucent outer flange producing a coarse reticulate 859 860365 appearance (Fig. 4b). The delicate outer flange is often observed as torn or bent with an irregular or 861 862 366 jagged appearance (Fig. 3h, Fig. 4a). The width of the outer flange observed on well-preserved 863 864 865367 specimens averages between 9.3 µm and 25.3 µm (Table 3). All observed specimens are pale and 866 867368 are difficult to observe under transmitted LM (Fig. 3). Sometimes, several specimens occur in 868 869369 clusters (Fig. 3g). Cell contents include many small transparent to yellow lipid bodies (Fig. 3d–f). 870 871370 Details on other organelles such as a central vacuole or the nuclear apparatus are lacking. 872 873371 Specimens from different studied populations are identical in all studies features. 874 875372 Comparison . Halodinium verrucatum sp. nov. is distinguished from all other Halodinium species 876 877373 by the ornamentation of the outer wall layer (Table 4). The diameter of the central body of 878 879 374 Halodinium major Bujak, 1984 and H. minor Bujak, 1984 are larger (104–116 µm and 46–63 µm, 880 881 375 882 respectively). Furthermore, their outer flange is unornamented. Halodinium scopaeum Head, 1993 883 884 885 15 886 887 376 is much smaller (average diameter central body: 21–24 µm), and possesses a smooth to scabrate 888 889 890377 ornamentation. Halodinium eirikssonii Verhoeven et al., 2014 has a smaller central body (23.9–27.4 891 892378 µm), a rugulate outer wall layer and a complex ambital flange. Species of the genus Cyclopsiella 893 894379 Drugg and Loeblich, 1967 possess a more reduced ambital flange and an eccentric pylome (e.g., 895 896380 Matsuoka and Head 1992). Cysts of Chattonella sp., as illustrated by Mastuoka and Iishi (2018), are 897 898381 hemispherical and lack a flange and ornamentation. 899 900382 Occurrence and stratigraphic range . Halodinium verrucatum n. sp. is described here from 901 902 383 modern sediments in the type locality (Brittany) and the Baltic Sea, and withstands palynological 903 904 384 treatment. This species has been recorded from modern sediments from the Skagerrak, Croatia, and 905 906 907385 Effingham Inlet (British Columbia) as well (unpublished data; see Fig. 7). 908 909386 910 911387 Phylogenetic position of Hexasterias problematica and Halodinium verrucatum sp. nov. based 912 913388 on LSU rDNA and SSU rDNA 914 915389 Identical SSU rDNA (1691 bp) and partial LSU rDNA (1230 bp) sequences were obtained from 916 917390 three out of four specimens of Hexasterias problematica (Accession numbers: [to be inserted in 918 919391 proof]). For Halodinium verrucatum sp. nov., SSU rDNA (1699 bp) was obtained from a single 920 921 392 specimen and identical LSU rDNA (1139 bp) from two specimens (Accession numbers: [to be 922 923 924393 inserted in proof]). The SSU sequences were used for phylogenetic analyses (Fig. 8), as there are 925 926394 more sequences available than for LSU. 927 928395 In the SSU phylogeny, Hexasterias problematica formed a well-supported clade with the 929 930396 planktonic, marine, prostome ciliate Balanion masanensis (Fig 7; Kim et al. 2007). This clade 931 932397 showed a sister relationship with another clade that includes Halodinium verrucatum sp. nov. and 933 934398 the prostome species Urotricha sp., Plagiocampa sp. and Cryptocaryon irritans Brown (Colorni 935 936399 and Burgess 1997; Foissner et al. 1994, 1999; Ma et al. 2017). Both clades formed a monophyletic 937 938 400 clade with 72% bootstrap support (Fig. 8). 939 940 401 941 942 943 944 16 945 946 402 Hexasterias and Halodinium micro-FTIR analyses 947 948 949403 Three specimens of Halodinium spp., three specimens of H. verrucatum sp. nov. and five specimens 950 951404 of Hexasterias problematica were measured (Fig. 9). See Table 5 for absorptions and assignments. 952 953405 The consistent absorption spectra for the same species from one location (e.g. the Halodinium 954 955406 verrucatum sp. nov. spectra from the Strö 6 station) indicates reproducibility of the method. The 956 957407 general consistency of spectra from one species from several locations indicates they truly reflect 958 959408 species composition. The remaining variation (e.g. specimen M4S13) is probably due to differing 960 961 409 environmental conditions (cf. Bogus et al. 2014; Mertens et al. 2015; Gurdebeke et al., accepted). 962 963 410 Consistently strong (and often sharp, e.g., M4S28) absorptions occur at ~3280 cm -1 (N-H 964 965 -1 -1 966411 stretch), with a shoulder at ~3400 cm (O-H stretch) and a minor peak around 3100 cm . 967 -1 968412 Absorptions indicative of aliphatic CH-stretching occur in the 3000–2800 cm region. 969 970413 The spectral region between 1800 and 1500 cm -1 dominates in all measured spectra, with 971 972414 specific maxima at 1620, 1570 and 1550 cm -1 and a distinct shoulder at 1640 cm -1 . Nitrogen- 973 974415 containing (amide) group absorptions are evident in the spectra, particularly the amide I and II 975 976416 bands around 1600 cm -1 , and minor amide III and V bands around 1312 and 690 cm -1 , respectively. 977 978417 The absorptions in the region between 1200 and 1000 cm -1 are indicative of C-O-C 979 980 418 asymmetric vibration (~1160 cm -1 ), glucose ring stretch (~1110 cm -1 ) and C-O stretching (~1040 981 982 -1 983419 cm ). They are indicative of polysaccharides but lacking diagnostic absorptions of β-linkage around 984 -1 985420 900 cm (Cárdenas et al. 2004; Kačuráková et al. 2002). 986 987421 There is variability in the composition of Halodinium spp. specimens (Fig. 9), in that several 988 989422 of the specimens showed relatively weak absorptions for C-O bonds, and the position of the 990 991423 strongest peak was either 1620 cm -1 ( Halodinium sp. from the Irish Sea and Brittany) or 1570 cm -1 992 993424 (H. verrucatum sp. nov. from the Skagerrak). These compositional differences may reflect 994 995425 interspecific or environmental differences. In earlier studies most Halodinium specimens from 996 997 426 Brittany and the Irish Sea were found to be H. minor . The Hexasterias problematica spectra also 998 999 1000 1001 1002 1003 17 1004 1005 427 show variability in the relative strength of the absorptions, specifically the specimen (M4S13) 1006 1007 -1 1008428 where the absorptions from 1160 to 1040 cm are stronger than in the others. 1009 1010429 The absorption spectra recorded for the tintinnid loricae ( Parafavella sp. and Stenosemella 1011 1012430 sp.) are very similar to Hexasterias and Halodinium . The two spectra of Parafavella sp. specimens 1013 1014431 from Skagerrak are more or less identical (Fig. 10); the spectrum of Stenosemella sp. differs in 1015 1016432 having more pronounced shoulders at 1640 cm -1 and 1550 cm -1 , and there are some differences in 1017 1018433 the spectral region around 1400 cm -1 . 1019 1020 434 Radiosperma corbiferum cysts (Fig. 10) again show similarities with Hexasterias and 1021 1022 435 Halodinium , with maximal absorptions at 1620 or 1570 cm -1 and a similar shape in the region 1023 1024 -1 1025436 around 1400 cm . There are some differences, however, for instance there is an additional small 1026 -1 -1 1027437 absorption around 3440 cm and relatively stronger absorptions in the region 1040–1160 cm , 1028 1029438 especially at 1040 cm -1 . 1030 1031439 1032 1033440 Discussion 1034 1035441 Phylogenetic implications and relation to acritarchs 1036 1037442 The rDNA sequences extracted from Hexasterias problematica and Halodinium verrucatum sp. 1038 1039 443 nov. reveal a phylogenetic placement within the ciliate order Prorodontida (Gao et al. 2016). The 1040 1041 1042444 genealogical structure and monophyly of the Colepidae, Prorodontidae, and Placidae (see Fig. 8) is 1043 1044445 in agreement with earlier studies (Gao et al. 2016; Lipscomb et al. 2012; Mordret et al. 2016; Zhang 1045 1046446 et al. 2014). The placement with the ciliates is further supported for Hexasterias problematica by 1047 1048447 the DAPI-staining, which shows a dual nuclear apparatus (Fig. 6), a derived character of the 1049 1050448 ciliates. This is in line with earlier observations of ciliate nuclei with DAPI staining (e.g., Bellec et 1051 1052449 al. 2014). Further observations of nuclear dualism should be made with the classic approach using 1053 1054450 TEM (e.g., Grimes 1973). 1055 1056 451 In this data set, Hexasterias problematica is most closely related to Balanion masanensis , 1057 1058 452 1059 described by Kim et al. (2007) from coastal waters off South Korea. Halodinium verrucatum sp. 1060 1061 1062 18 1063 1064 453 nov. is most closely related to the freshwater Urotricha sp. (EU024981) with which it forms a sister 1065 1066 1067454 clade to Plagiocampa sp. and the fish parasite Cryptocaryon irritans , both marine species (Wright 1068 1069455 and Colorni 2002; Zhang et al. 2014). The clade formed by these has been reported previously 1070 1071456 (Zhang et al. 2014), but the nature of the relationship with the Class Plagiopylea remains unclear 1072 1073457 (Gao et al. 2016). 1074 1075458 Within the free-living Ciliophora, the formation of resting cysts is common (see reviews in 1076 1077459 Guttiérez et al. 2001 and Verni and Rosati 2011), but detailed information on morphology and 1078 1079 460 physiology is only known for a very limited number of species (Verni and Rosati 2011). Most well- 1080 1081 461 described palynologically preservable cysts are assigned to the class Spirotrichea (Reid and John 1082 1083 1084462 1978, 1983). For the class Prostomatea, cysts have been described for Prorodon (Hiller and Bardele 1085 1086463 1988) and Cryptocaryon irritans (Ma et al. 2017). 1087 1088464 It is hypothesized that the palynomorphs Hexasterias problematica and Halodinium 1089 1090465 verrucatum sp. nov. represent cryptobiotic cyst stages of prorodontid ciliates. The classification of 1091 1092466 the Order Prorodontida is based on characteristics not present in cyst stages, such as the nature of 1093 1094467 the cytostome, kinetids and the brosse (Lynn 2008), and life cycle stages of a single species can be 1095 1096468 significantly heteromorphic (Hiller and Bardele 1988). Thus far, there are no morphological 1097 1098 469 observations of the ciliates that supposedly produce the cysts Hexasterias problematica and 1099 1100 1101470 Halodinium verrucatum sp. nov., and consequently the placement of these species within the order 1102 1103471 Prorodontida cannot be evaluated in light of diagnostic characteristics of their planktonic stages. 1104 1105472 Incubation experiments, which have been unsuccessful to date, or increased molecular taxon 1106 1107473 sampling could provide further insights. This situation stresses the importance of including cyst 1108 1109474 morphology in future ciliate species descriptions. 1110 1111475 The average size of Halodinium verrucatum and Hexasterias problematicum is within the 1112 1113476 range of other known ciliate cysts (see Table II in Verni and Rosati 2011). The disk-shaped 1114 1115 477 morphology, however, is unusual for ciliate cysts, which are typically globular or flask-shaped. This 1116 1117 478 1118 suggests that the morphological disparity within ciliate cysts might be larger than expected. The 1119 1120 1121 19 1122 1123 479 double cyst wall texture of Halodinium verrucatum sp. nov. agrees with the observation that ciliate 1124 1125 1126480 cyst walls in general differentiate between a thick inner and a thinner outer cyst wall (Matsusaka et 1127 1128481 al. 1989; Verni and Rosati 2011). TEM would be needed to evaluate the ultrastructure of the inner 1129 1130482 cyst wall, which in many cases is multi-layered in ciliate cysts (Verni and Rosati 2011). 1131 1132483 It is expected that morphologically similar, two-layered marine acritarchs with a pylome, 1133 1134484 such as the other Halodinium species, Radiosperma corbiferum , and Cyclopsiella (e.g. Matsuoka 1135 1136485 and Head 1992) may also be affiliated with the ciliates. An affinity with the ciliates has also been 1137 1138 486 tentatively proposed for other acritarchs with pylomes, such as species of Palaeostomocystis 1139 1140 487 (Roncaglia 2004; Warny et al. 2009). It should be noted, however, that other organic microfossils 1141 1142 1143488 with a circular opening exist (e.g., testate amoeba, desmids, cysts of the raphidophycean 1144 1145489 Chattonella ), but these differ in structure, composition and autecology. For many extinct acritarch 1146 1147490 species with a pylome structure, described from older sediments, a ciliate affinity may be 1148 1149491 considered, e.g. the Proterozoic Appendisphaera (Moczydłowska et al. 2011) or the Ordovician 1150 1151492 Velatasphaera (e.g., Miller and Williams 1988). However, because of the impossibility of obtaining 1152 1153493 molecular data for these species, combined observations of detailed morphology, wall structure, 1154 1155494 chemical composition and paleoecology would be needed to support this affiliation (Aroui et al. 1156 1157 495 1999; Marshall et al. 2005). 1158 1159 1160496 1161 1162497 Distribution and ecology of Hexasterias problematica and Halodinium verrucatum sp. nov. 1163 1164498 Specimens of Hexasterias problematica are typically recovered from plankton and surface 1165 1166499 sediments in estuarine and coastal areas in temperate zones of the northern and southern hemisphere 1167 1168500 (Fig. 7). Occurrences range from fresh to fully marine waters. Mudie (1992) linked the abundances 1169 1170501 in surface sediments with river/meltwater input. Indeed, sediment trap studies in British Columbia 1171 1172502 found a positive relationship between H. problematica abundance and freshwater input (Pospelova 1173 1174 503 et al. 2010; Price and Pospelova 2011). Consequently, H. problematica has been used as an 1175 1176 504 1177 indicator for freshwater input in paleoecological studies (e.g., Bringué et al. 2016). In all, it seems 1178 1179 1180 20 1181 1182 505 that H. problematica is a euryhaline species, occurring from fresh to marine environments, but with 1183 1184 1185506 maximal abundances in the case of lowered salinities (brackish water) due to freshwater discharge. 1186 1187507 The short (average of 16.2 µm, n = 4 in Verleye et al. 2009) processes noted for specimens from the 1188 1189508 Black Sea (e.g., Verleye et al. 2009; Mudie et al. 2010) suggest a possible covariance of process 1190 1191509 length with environmental variables such as salinity. Similar relationships were noted for 1192 1193510 dinoflagellate cyst species Lingulodinium machaerophorum (Mertens et al. 2009b; Verleye et al. 1194 1195511 2009) and Operculodinium centrocarpum (Mertens et al. 2011) and allow application of these 1196 1197 512 microfossils as a proxy for paleoenvironmental conditions (e.g., Mertens et al. 2012b; Bringué et al. 1198 1199 513 2016). The relation should be explored quantitatively for Hexasterias problematica . 1200 1201 1202514 For Halodinium , many authors do not distinguish species, preventing definite statements 1203 1204515 about the distributions of individual species. The sparse occurrence data of Halodinium verrucatum 1205 1206516 sp. nov. suggests it occurs in coastal estuarine environments with brackish marine salinities (Aure et 1207 1208517 al. 1993; Arapov et al. 2017). A similar ecology is often assigned to “ Halodinium sp.” in 1209 1210518 palynological studies (e.g. Matthiessen 1995) and to Halodinium minor in the sediment trap study 1211 1212519 of Price and Pospelova (2011). 1213 1214520 In the light of their phylogenetic position (Fig. 8), both Hexasterias and Halodinium are 1215 1216 521 grouped in a clade with mainly marine species, except for Urotricha sp., that is most closely related 1217 1218 1219522 to Halodinium verrucatum sp. nov. and was sequenced from lacustrine plankton (Auinger et al. 1220 1221523 2008). This large genus includes mostly freshwater but also marine species (e.g., Foissner et al. 1222 1223524 1999; Wilbert and Song 2008). 1224 1225525 The irregular flange rim and the monospecific clusters of specimens of Halodinium 1226 1227526 verrucatum sp. nov. (Fig. 3g) are not unlike the morphology described by Matsuoka and Head 1228 1229527 (1992) for Cyclopsiella , suggesting an attached, perhaps encrusting mode of life. 1230 1231528 1232 1233 529 Geochemical composition 1234 1235 1236 1237 1238 1239 21 1240 1241 530 The measured spectra bear some similarity to a carbohydrate-based macromolecule described from 1242 1243 1244531 heterotrophic dinoflagellates (Bogus et al. 2014; Mertens et al. 2015), e.g., the abundant sugar 1245 1246532 group component, specifically in Halodinium spp. from Brittany and the Irish Sea, and the amide 1247 1248533 functional groups. There are significant differences, however, suggesting that the composition here 1249 1250534 is not dinosporin. In particular, strong C-O bonds and evidence for a β-glycosidic linkage are 1251 1252535 lacking. The spectra also differ from chitin (Cárdenas et al. 2004), which was the cyst composition 1253 1254536 found by e.g. Foissner et al. (2005) and Verni and Rosati (2011) using a Van Wesselingh test. This 1255 1256 537 evidence supports the rejection by Agatha and Simon (2012) for a chitinous composition of ciliate 1257 1258 538 loricae and cysts. 1259 1260 1261539 The spectra of the loricae of the tintinnids Parafavella sp. and Stenosemella sp. indicate a 1262 1263540 comparable composition and corroborate the results of Agatha and Simon (2012), who evaluated 1264 1265541 tintinnid loricae using a multitude of methods, and suggested a nitrogen-rich proteinaceous 1266 1267542 composition. These authors also found, however, that polysaccharides are never the main 1268 1269543 component of tintinnid loricae. 1270 1271544 The compositional similarity of Halodinium verrucatum and Hexasterias problematica with 1272 1273545 the loricae supports their placement in the ciliates revealed by the genetic data. The scarce reports 1274 1275 546 on ciliate cyst wall chemistry (Izquierdo et al. 2000; Wang et al. 2017) report a composition of 1276 1277 1278547 mainly carbohydrates and (glyco)proteins. Wang et al. (2017) found, using electrophoresis 1279 1280548 combined with ionization mass spectrometry, that the protein composition within a single species is 1281 1282549 very diverse (42 proteins), a resolution that cannot be achieved by single specimen micro-FTIR. 1283 1284550 The chemical composition of both Hexasterias and Halodinium indicates that they are 1285 1286551 formed by heterotrophic organisms, evidenced by amide groups in the wall chemistry (see Table 5), 1287 1288552 a result also found for cysts of heterotrophic dinoflagellates (Bogus et al. 2014). This is also in 1289 1290553 accordance with their placement with the ciliates, which are nearly all heterotrophic (Lynn 2008). 1291 1292 554 This illustrates that cyst wall chemistry is a more reliable indicator for trophic preference of the 1293 1294 1295 1296 1297 1298 22 1299 1300 555 producing organism than are cyst color and autofluorescence, the approaches often applied with 1301 1302 1303556 dinoflagellate cysts (Brenner and Biebow 2001; Bogus et al. 2014). 1304 1305557 Finally, the acritarch Radiosperma corbiferum shows a similar composition, which would 1306 1307558 further support a ciliate affinity of acritarchs with a similar morphology, although genetic data is 1308 1309559 needed to confirm this. These results complement earlier studies applying spectroscopical 1310 1311560 techniques to fossil acritarchs to test hypotheses about their affinity (e.g., Arouri et al. 2000; 1312 1313561 Dhamelincourt et al. 2010). 1314 1315 562 1316 1317 563 5. Conclusions 1318 1319 1320564 Two palynomorphs, Hexasterias problematica , and a new species of the genus Halodinium , H. 1321 1322565 verrucatum sp. nov., were retrieved from surface sediments from Brittany, the Skagerrak and the 1323 1324566 Baltic Sea. Phylogenetic analysis using rDNA (LSU and SSU) revealed that these species belong to 1325 1326567 the ciliates (phylum Ciliophora), more specifically within the order Prorodontida. The ciliate nature 1327 1328568 is supported by the presence of a micro- and macronucleus. Both species represent cryptobiotic cyst 1329 1330569 stages of hitherto undescribed ciliate species. 1331 1332570 This placement is supported by the cyst wall chemistry, which, evaluated using micro-FTIR, 1333 1334 571 is found similar to that of loricae of the tintinnid ciliates Stenosemella sp. and Parafavella sp. The 1335 1336 1337572 cyst wall is carbohydrate-based and contains nitrogen-containing functional groups. The 1338 1339573 composition is in accordance with the heterotrophic nature of the ciliates. 1340 1341574 Hexasterias problematica and species of Halodinium have a global distribution, mainly in 1342 1343575 temperate coastal waters, and are often encountered in palynological studies. The affinity with the 1344 1345576 ciliates provides a better understanding of their ecological significance as an indicator for coastal 1346 1347577 conditions and freshwater influence. 1348 1349578 Finally, the results of this study highlight the potential utility of micro-FTIR in 1350 1351 579 understanding acritarch affinity, especially for fossil taxa for which molecular data are unavailable. 1352 1353 580 1354 As a test case, an acritarch with a similar morphology, here represented by Radiosperma 1355 1356 1357 23 1358 1359 581 corbiferum , was found to have a similar composition and possibly has a similar affinity, which 1360 1361 1362582 should be confirmed by molecular data. 1363 1364583 1365 1366584 Acknowledgements 1367 1368585 Veijo Kinnunen and Magnus Lindstrom are thanked for assistance with sampling Sällvik. Jaana 1369 1370586 Koistinen is thanked for help with shipping samples from Finland to Japan. Anna Godhe is thanked 1371 1372587 for providing surface sediment from the Skagerrak. Sabine Agatha is acknowledged for help with 1373 1374 588 identifications of tintinnid loricae. Zivana Nincevic is thanked for information about the sample 1375 1376 589 from Kastela Bay. 1377 1378 1379590 Funding: This work was supported by the Hercules Foundation (FWO, Flanders) [FT-IMAGER 1380 1381591 project - AUGE/13/16] and the National Science Foundation [OCE – 1326927]. 1382 1383592 1384 1385593 References 1386 1387594 Agatha, S., Simon, P., 2012. On the nature of the tintinnid loricae (Ciliophora: Spirotricha: 1388 1389595 Tintinnina): A histochemical, enzymatic, EDX, and high-resolution TEM study. Acta 1390 1391596 Protozool. 51, 1–19. 1392 1393 597 Arapov, J., Skejić, S., Bužančić, M. Bakrač, A., Vidjak, O., Bojanić, N., Ujević, I., Živana N. 1394 1395 1396598 Gladan, Ž.N., 2017. Taxonomical diversity of Pseudo-nitzschia from the Central Adriatic Sea. 1397 1398599 Phycol. Res. 65, 280–290. 1399 1400600 Aroui, K., Greenwood, P.F., Walter, M.R., 1999. A possible chlorophycean affinity of some 1401 1402601 Neoproterozoic acritarchs. Org. Geochem. 30, 1323–1337. 1403 1404602 Auinger, B.M., Pfandl, K., Boenigk, J., 2008. Improved methodology for identification of protists 1405 1406603 and microalgae from plankton samples preserved in Lugol’s iodine solution: Combining 1407 1408604 microscopic analysis with single-cell PCR. Appl. Environ. Microbiol. 74, 2505–2510. 1409 1410 605 Aure, J., Føyn, L., Pettersen, R., 1993. Environmental monitoring of Norwegian fjord 1975–1993. 1411 1412 606 1413 Fisken of Havet 12, 35 pp. 1414 1415 1416 24 1417 1418 607 Bellec, L., Maurer-Alcale, X.X., Kats, L.A., 2014. Characterization of the life cycle and 1419 1420 1421608 heteromeric nature of the macronucleus of the ciliate Chilodonella uncinata using 1422 1423609 fluorescence microscopy. J. Eukaryot. Microbiol. 61, 313-316. 1424 1425610 Bérard-Therriault, L., Poulin, M., Bossé, L., 1999. Guide d'identification du phytoplancton marin de 1426 1427611 l'estuaire et du golfe du Saint-Laurent incluant également certains protozoaires. Can. Spec. 1428 1429612 Publ. of Fish. Aquat. Sci. 128, 1–387. 1430 1431613 Bogus, K., Mertens, K.N., Lauwaert, J., Harding, I.C., Vrielinck, H., Zonneveld, K.A.F., Versteegh, 1432 1433 614 G.J.M., 2014. Differences in the chemical composition of organic-walled dinoflagellate 1434 1435 615 resting cysts from phototrophic and heterotrophic dinoflagellates. J. Phycol. 50, 254–266. 1436 1437 1438616 Bolch, C.J.S., 1997. The use of polytungstate for the separation and concentration of living 1439 1440617 dinoflagellate cysts from marine sediments. Phycologia 37, 472–478. 1441 1442618 Brenner, W.W., Biebow, N., 2001. Missing autofluorescence of recent and fossil dinoflagellate 1443 1444619 cysts – an indicator of heterotrophy? N. Jb. Geol. Paläont. Abh. 219, 229–240. 1445 1446620 Bringué, M., Pospelova, V., Calvert, S.E., Enkin, R.J., Lacourse, T., Ivanochko, T., 2016. High 1447 1448621 resolution dinoflagellate cyst record of environmental change in Effingham Inlet (British 1449 1450622 Columbia, Canada) over the last millennium. Palaeogeogr., Palaeoclimatol., Palaeoecol. 441, 1451 1452 623 787–810. 1453 1454 1455624 Bruker, 2014. OPUS Spectroscopy Software User Manual. Bruker Optik GmbH, Ettlingen. 1456 1457625 Brunel, J., 1962. Le phytoplancton de la Baie des Chaleurs. Contributions 91 du Ministere de la 1458 1459626 Chasse et des Pecheries, Montréal. pp. 365. 1460 1461627 Bujak, J.P., 1984. Cenozoic dinoflagellate cysts and acritarchs from the Bering Sea and northern 1462 1463628 North Pacific, DSDP Leg 19. Micropaleontology 30, 180–212. 1464 1465629 Bursa, A., 1961. Phytoplankton of the Calanus expeditions in Hudson Bay, 1953 and 1954. J. Fish. 1466 1467630 Res. Bd. Canada 18, 51–83. 1468 1469 1470 1471 1472 1473 1474 1475 25 1476 1477 631 Candel, M.S., Radi, T., de Vernal, A., Bujalesky, G., 2012. Distribution of dinoflagellate cysts and 1478 1479 1480632 other aquatic palynomorphs in surface sediments from the Beagle Channel, Southern 1481 1482633 Argentina. Mar. Micropaleontol. 96-97, 1-12. 1483 1484634 Candel, M.S., Borromei, A.M., Martinez, M.A., Bujalesky, G., 2013. Palynofacies analysis of 1485 1486635 surface sediments from the Beagle Channel and its application as modern analogues for 1487 1488636 Holocene records of Tierra del Fuego, Argentina. Palynology 37, 62-76. 1489 1490637 Cărăuş, I., 2002. Algae of Romania - A distributional checklist of actual algae. Studii şi Cercetări, 1491 1492 638 7, 694 pp. 1493 1494 639 Cárdenas, G., Cabrera, G., Taboada, E., Miranda, S.P., 2004. Chitin characterization by SEM, 1495 1496 13 1497640 FTIR, XRD, and C cross polarization/mass angle spinning NMR. J. Appl. Polym. Sci. 93, 1498 1499641 1876–1885. 1500 1501642 Cleve, P.T., 1900. The plankton of the North Sea, the English Channel, and the Skaggerrak in 1898. 1502 1503643 K. Sven. Vetenskapsakad. Handl. 32, 2–53. 1504 1505644 Colbath, G.K., Grenfell, H.R., 1995. Review of biological affinities of Paleozoic acid-resistant, 1506 1507645 organic walled eukaryotic algal microfossils (including “acritarchs”). Rev. Paleobot. Palynol. 1508 1509646 86, 287–314. 1510 1511 647 Colorni, A., Burgess, P., 1997. Cryptocaryon irritans Brown 1951, the cause of ‘white spot disease’ 1512 1513 1514648 in marine fish: An update. Aquar. Sci. Cons. 1, 217–238. 1515 1516649 Colthup, N.B., Daly, L.H., Wiberley, S.E., 1990. Introduction to Infrared and Raman Spectroscopy, 1517 1518650 3rd ed., Academic Press, San Diego, 547 pp. 1519 1520651 Dale, B., 1977. New observation on Peridinium faroense Paulsen (1905), and classification of small 1521 1522652 orthoperidinioid dinoflagellates. British Phycol. J. 12, 241–253. 1523 1524653 Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. jModelTest 2: more models, new 1525 1526654 heuristics and parallel computing. Nat. Methods 9, 772. 1527 1528 1529 1530 1531 1532 1533 1534 26 1535 1536 655 de Vernal., A., Goyette, C., Rodrigues, C.G., 1989. Contribution palynostratrigraphique 1537 1538 1539656 (dinokystes, pollen et spores) à la connaissance de la mer Champlain: Coupe de Saint-Césaire, 1540 1541657 Québec. Can. J. Earth Sci. 26, 2450–2464. 1542 1543658 Deriugin, K.M., 1915. La Faune du Golfe de Kola et les conditions de son existence. Mémoires de 1544 1545659 l’Académie Imperiale des Sciences de Petrograd 34. 1546 1547660 Dhamelincourt, M.-C., Vecoli, M., Mezzetti, A., Cesari, C. Versteegh, G., Riboulleau, A., 2010. 1548 1549661 Laser Raman micro-spectroscopy of Proterozoic and Palaeozoic organic-walled microfossils 1550 1551 662 (acritarchs and prasinophytes) from the Ghadamis Basin, Libya and Volta Basin, Ghana. 1552 1553 663 Spectroscopy 24, 207–212. 1554 1555 1556664 Dolan, J.R., Montagnes, D.J.S., Agatha, S., Coats, D.W., Stoecker, D.K., 2013. The Biology and 1557 1558665 Ecology of Tintinnid Ciliates. Wiley-Blackwell, 296 pp. 1559 1560666 Dunthorn, M., Otto, J., Berger, S.A., Stamatakis, A., Mahé, F., Romac, S., de Vargas, C., Audic, S. 1561 1562667 et al., 2014. Placing environmental next-generation sequencing amplicons from microbial 1563 1564668 eukaryotes into a phylogenetic context. Mol. Biol. Evol. 31, 993–1009. 1565 1566669 Dunthorn, M., Lipps, J.H., Dolan, J.R., Saab, M.A., Aescht, E., Bachy, C., Barría de Cao, M.S., 1567 1568670 Berger, H. et al., 2015. Ciliates — Protists with complex morphologies and ambiguous early 1569 1570 671 fossil record. Mar. Micropaleontol. 119, 1-6. 1571 1572 1573672 Erdtman, G., 1954. On pollen grains and dinoflagellate cysts in the Firth of Gullmarn, SW Sweden. 1574 1575673 Bot. Not. 2, 103-111. 1576 1577674 Evitt, W.R., 1963. A discussion and proposals concerning fossil dinoflagellates, hystrichospheres, 1578 1579675 and acritarchs, II. Proc. Natl. Acad. Sci. U. S. A. 49, 298–302. 1580 1581676 Foissner, W., Berger, H., Kohmann, F., 1994. Taxonomische und ökologische Revision der Ciliaten 1582 1583677 des Saprobiensystems, Band III: Hymenostomata, Prostomatida, Nassulida. 1584 1585678 Informationsberichte des Bayer. Landesamtes für Wasserwirtschaft, 1/94: 1-548. 1586 1587 679 Foissner, W., Herger, H., Schaumburg, J., 1999. Identification and Ecology of Limnetic Plankton 1588 1589 680 1590 Ciliates. Bavarian State Office for Water Management Reports, Issue 3/99, 793 pp. 1591 1592 1593 27 1594 1595 681 Foissner, W., Müller, H., Weisse, T., 2005. The unusual, lepidosome-coated resting cyst of Meres 1596 1597 1598682 corlissi (Ciliophora : Oligotrichea) : Light and scanning electron microscopy, cytochemistry. 1599 1600683 Acta Protozool. 44, 201-2015. 1601 1602684 Foissner, W., Chao, A., Katz, L.A., 2008. Diversity and geographic distribution of ciliates (Protista: 1603 1604685 Ciliophora). Biodivers. Conserv. 17, 345–363. 1605 1606686 Gao, F., Warren, A., Zhang, Q., Gong, J., Miao, M., Sun, P., Xu, D., Huang, J., Yi, Z., Song, W., 1607 1608687 2016. The all-data-based evolutionary hypothesis of ciliated protists with a revised 1609 1610 688 classification of the Phylum Ciliophora (Eukaryota, Alveolata). Sci. Rep. 6, 24874. 1611 1612 689 Gran, H.H., 1912. Pelagic plant life. In: Murray, J.J., Hjort, J., The Depth of the Ocean. McMillan, 1613 1614 1615690 London, 307–386. 1616 1617691 Grimes, G.W., 1973. Differentiation during encystment and excystment in Oxytricha fallax . J. 1618 1619692 Protozool. 20, 92-104. 1620 1621693 Guindon, S., Gascuel, O., 2003. A simple, fast, accurate algorithm to estimate large phylogenies by 1622 1623694 maximum likelihood. Syst. Biol. 52, 696–704. 1624 1625695 Gurdebeke, P.R., Mertens, K.N., Bogus, K., Marret, F., Chomérat, N., Vrielinck, H., Louwye, S. 1626 1627696 (accepted). Taxonomic re-investigation and geochemical characterization of Reid’s (1974) 1628 1629 697 species of Spiniferites from holotype and topotype material. Palynology. 1630 1631 1632698 DOI:10.1080/01916122.2018.1465735 1633 1634699 Gurdebeke, P.R., Pospelova, V., Mertens, K.N., Chana, J., Louwye, S., 2018. Diversity and 1635 1636700 distribution of dinoflagellate cysts in surface sediments from fjords of western Vancouver 1637 1638701 Island (British Columbia, Canada). Mar. Micropaleontol. 143, 12–29.. 1639 1640702 Guttiérez, J.C., Callejas, S., Borniquel, S., Benítez, L., Martín-Gonzalez, A., 2001. Ciliate 1641 1642703 cryptobiosis: a microbial strategy against environmental starvation. Int. Microbiol. 4, 151– 1643 1644704 157. 1645 1646 705 Hansen-Ostenfeld, C. 1916. De Danske Farvandes Plankton i aarene 1898-1901, phytoplankton og 1647 1648 706 1649 protozooer. Kgl. danske Vidensk. Selsk, 2, 298-378. 1650 1651 1652 28 1653 1654 707 Head, M. J., 1993. Dinoflagellates, sporomorphs, and other palynomorphs from the upper Pliocene 1655 1656 1657708 St. Earth beds of Cornwall, southwestern England. The Palaeontological Society Memoirs 31, 1658 1659709 62 pp. 1660 1661710 Heiskanen, A.-S., Tallberg, P., 1999. Sedimentation and particulate nutrient dynamics along a 1662 1663711 coastal gradient from a fjord-like bay to the open sea. Hydrobiologia 393, 127–140. 1664 1665712 Hensen, V., 1887. Ueber die Bestimmung des Plankton’s oder des im Meere treibende Materials an 1666 1667713 Pflanzen und Tieren. Fünfter Bericht der Kommission zur wissenschaftlichen Untersuchung 1668 1669 714 der deutschen Meere in Kiel für die Jahre 1882 bis 1886, 12–16, pp. 1–107. 1670 1671 715 Herdman, W.A., Ridell, W., 1911. Plankton of the West Coast of Scotland in relation to that of the 1672 1673 1674716 Irish Sea. Proc. Trans. Liverpool Biol. Soc. 25, 132-185. 1675 1676717 Hiller, S., Bardele, C.F., 1988. Prorodon aklitolophon n. spec. and the “Dorsal Brush” as a 1677 1678718 character to identify certain subgroups in the genus Prorodon . Arch. Protistenkd. 136, 213– 1679 1680719 236. 1681 1682720 Izquierdo, A., Martín-González, A., Guttiérez, J.C., 2000. Resting cyst wall glycoproteins isolated 1683 1684721 from two colpodid ciliates are glycine-rich proteins. Cell Biol. Int. 24, 115–119. 1685 1686722 Javaux, E.J., Marshall, C.P., 2006. A new approach in deciphering early protist paleobiology and 1687 1688 723 evolution: Combined microscopy and microchemistry of single Proterozoic acritarchs. Rev. 1689 1690 1691724 Palaeobot. Palynol. 139, 1–15. 1692 1693725 Kačuráková, M., Smith, A.C., Gidley, M.J., Wilson, R.H., 2002. Molecular interactions in bacterial 1694 1695726 cellulose composites studied by 1D FT-IR and dynamic 2D FT-IR spectroscopy. Carbohydr. 1696 1697727 Res. 337, 1145-1153. 1698 1699728 Kapuscinski, J., 1995. DAPI: A DNA-specific fluorescent probe. Biotech. Histochem. 70, 220-233. 1700 1701729 Kaźmierczak, J., Kremer, B., 1999. Spore-like bodies in some early Paleozoic acritarchs: Clues to 1702 1703730 chlorococcalean affinities. Acta Paleontol. Pol. 54, 541–551. 1704 1705 1706 1707 1708 1709 1710 1711 29 1712 1713 731 Kim, J.S., Jeong, H.J., Lynn, D.E., Park, J.Y., Lim, Y.W., Shin, W., 2007. Balanion masanensis n. 1714 1715 1716732 sp. (Ciliophora: Prostomatea) from the Coastal Waters of Korea: Morphology and Small 1717 1718733 Subunit Ribosomal RNA Gene Sequence. J. Eukaryot. Microbiol. 54, 482–494. 1719 1720734 Kubiszyn, A.M., Svensen, C., 2018. First record of a rare species, Polyasterias problematica 1721 1722735 (Prasinophyceae), in Balsafjord, northern Norway. Bot. Mar. 61, 421–428. 1723 1724736 Kunz-Pirrung, M., 1998. Aquatic palynomorphs: Reconstruction of Holocene sea-surface water 1725 1726737 masses in the eastern Laptev Sea. Ber. Polarfosch . 281, 1–117. 1727 1728 738 Lipscomb, D.L., Bowditch, B.M., Riordan, G.P., 2012. A molecular and ultrastructural description 1729 1730 739 of Spathidiopsis buddenbrocki and the phylogenetic position of the family Placidae 1731 1732 1733740 (Ciliophora). J. Eukaryot. Microbiol. 59, 67–79. 1734 1735741 Lohmann, H., 1904 Ergebnisse der Plankton-Expedition der Humboldt-Stiftung. Band IV, p. 32. 1736 1737742 Lynn, D.H., 2008. The Ciliated Protozoa. Characterization, Classification, and Guide to the 1738 1739743 Literature, 3 rd edition. Springer, 605 pp. 1740 1741744 Ma, R., Fan, X., Yin, F., Ni, B., Gu, F., 2017. Ultrastructural features of the tomont of 1742 1743745 Cryptocaryon irritans (Ciliophora: Prostomatea), a parasitic ciliate of marine fishes. 1744 1745746 Parasitology 144, 720–729. 1746 1747 747 Marshall, C.P., Javaux, E.J. Knoll, A.W., Walter, M.R., 2005. Combined micro-Fourier transform 1748 1749 1750748 infrared (FTIR) spectroscopy and micro-Raman spectroscopy of Proterozoic acritarchs: A 1751 1752749 new approach to Palaeobiology. Prec. Res. 138, 208–224. 1753 1754750 Martínez-Hernández, E., Hernández-Campos, H.E., 1991. Distribution des quistes de 1755 1756751 dinoflagelados y acritarchas en sedimentos Holocenicos del Golfo de California. 1757 1758752 Palaeontología Mexicana 57, 133 pp. 1759 1760753 Matsuoka, K., Head, M.J., 1992. Taxonomic revision of the Neogene marine palynomorphs 1761 1762754 Cyclopsiella granosa (Matsuoka) and Batiacasphaera minuta (Matsuoka), and a new species 1763 1764 755 of Pyxidinopsis Habib (Dinophyceae) from the Miocene of the Labrador Sea. In. Head, M. J. 1765 1766 1767 1768 1769 1770 30 1771 1772 756 and Wrenn, J. H. (Eds.) Neogene and Quaternary Dinoflagellate Cysts and Acritarchs. 1773 1774 1775757 American Association for Stratigraphic Palynologists Foundation, Dallas, 165–180. 1776 1777758 Matsuoka, K., Iishi, K., 2018. Marine and freshwater palynomorphs preserved in surface sediments 1778 1779759 of Osaka Bay, Japan. Bull. Osaka Mus. Nat. Hist . 72, 1-17. 1780 1781760 Matsusaka, T., Noguchi, O., Yonezawa, F., 1989. Cortical morphogenesis during encystment in a 1782 1783761 ciliate, Euplotes encysticus Yonezawa 1985. Europ. J. Protistol. 24, 133–137. 1784 1785762 Matthiessen, J., 1995. Distribution patterns of dinoflagellate cysts and other organic-walled 1786 1787 763 microfossils in recent Norwegian-Greenland Sea sediments. Mar. Micropaleontol. 24, 307– 1788 1789 764 334. 1790 1791 1792765 McMinn, A., Bolch, C., Hallegraeff, G., 1992. Cobricosphaeridium Harland and Sarjaent: 1793 1794766 Dinoflagellate cyst or copepod egg? Micropaleontology 38, 315–316. 1795 1796767 Medvedeva, L.A., Nikulina, T.V., 2014. Catalogue of freshwater algae of the southern part of the 1797 1798768 Russian Far East. Vladivostok: Dalnauka, 271 p. 1799 1800769 Mertens, K.N., Verhoeven, K., Verleye, T., Louwye, S., Amorim, A., Ribeiro, S., Deaf, A.S., 1801 1802770 Harding, I.C. et al., 2009a. Determining the absolute abundance of dinoflagellate cysts in 1803 1804771 recent marine sediments: The Lycopodium marker-grain method put to the test. Rev. 1805 1806 772 Palaeobot. Palynol., 157, 238-252. 1807 1808 1809773 Mertens, K.N., Ribeiro, S., Bouimetarhan, I., Caner, H., Nebout, N.C., Dale, B., de Vernal, A., 1810 1811774 Ellegaard, M. et al., 2009b. Process length variation in cysts of a dinoflagellate, 1812 1813775 Lingulodinium machaerophorum , in surface sediments: Investigating its potential as salinity 1814 1815776 proxy. Mar. Micropaleontol. 70, 54-69. 1816 1817777 Mertens, K.N., Dale, B., Ellegaard, M., Jansson, I.-M., Godhe, A., Kremp, A., Louwye, S., 2011. 1818 1819778 Process length variation in cysts of the dinoflagellate Protoceratium reticulatum , from surface 1820 1821779 sediments of the Baltic–Kattegat–Skagerrak estuarine system: a regional salinity proxy. 1822 1823 780 Boreas 40, 242-255. 1824 1825 1826 1827 1828 1829 31 1830 1831 781 Mertens, K.N., Bringué, M., Van Nieuwenhove, N., Takano, Y., Pospelova, V., Rochon, A., de 1832 1833 1834782 Vernal; A., Radi, T. et al., 2012a. Process length variation of the cyst of the dinoflagellate 1835 1836783 Protoceratium reticulatum in the North Pacific and Baltic-Skagerrak region: calibration as an 1837 1838784 annual density proxy and first evidence of pseudo-cryptic speciation. J. Quaternary Sci. 27, 1839 1840785 734-744. 1841 1842786 Mertens, K.N., Bradley, L.R., Takano, Y., Mudie, P.J., Marret, F., Aksu, A. E., Hiscott, R.N., 1843 1844787 Verleye, T.J. et al., 2012b. Quantitative estimation of Holocene surface salinity variation in 1845 1846 788 the Black Sea using dinoflagellate cyst process length. Quat. Sci. Rev. 39, 45-59. 1847 1848 789 Mertens, K.N., Wolny, J., Carbonell-Moore, C., Bogus, K., Ellegaard, M., Limoges, A., de Vernal, 1849 1850 1851790 A., Gurdebeke P.R. et al., 2015. Taxonomic re-examination of the toxic armored 1852 1853791 dinoflagellate Pyrodinium bahamense Plate 1906: Can morphology or LSU sequencing 1854 1855792 separate P. bahamense var. compressum from var. bahamense ? Harmful Algae 41, 1–24. 1856 1857793 Mertens, K.N., Gu, H., Pospelova, V., Chomérat, N., Nézan, E., Gurdebeke, P.R., Bogus, K., 1858 1859794 Vrielinck, H. et al., 2017. First record of resting cysts of the benthic dinoflagellate 1860 1861795 Prorocentrum leve in a natural reservoir in Gujan-Mestras, Gironde, France. J. Phycol. 53, 1862 1863796 1193–1205. 1864 1865 797 Meunier, A., 1910. Microplankton des Mers de Barents et de Kara. Duc d'Orléans. Campagne 1866 1867 1868798 arctique de 1907. Imprimerie scientifique Charles Bulens: Bruxelles. 355 pp. + atlas 1869 1870799 (XXXVII plates). 1871 1872800 Meunier, A., 1919. Microplankton de la mer Flamande. Mém. Mus. Hist. Nat. Belgique , 8, 59 pp.. 1873 1874801 Miller, M.A., Williams, G. L., 1988. Velatasphaera hudsonii gen. nov. et sp. nov., an Ordovician 1875 1876802 acritarch from Hudson Strain, North West Territories, Canada. Palynology 12, 121-127. 1877 1878803 Moczydłowska, M., Landing, E., Zang, W., Palacios, T., 2011. Proterozoic phytoplankton and 1879 1880804 timing of chlorophyte algae origins. Palaeontology 54, 721–733. 1881 1882 1883 1884 1885 1886 1887 1888 32 1889 1890 805 Mordret, S., Romac, S., Henry, N., Colin, S., Carmichael, M., Berney, C., Audic, S., Richter, D.J. et 1891 1892 1893806 al., 2016a. The symbiotic life of Symbiodinium in the open ocean within a new species of 1894 1895807 calcifying ciliata ( Tiarina sp.). ISME J. 10, 1424–1436. 1896 1897808 Mudie, P.J., 1992. Circum-arctic Quatematy and Neogene marine palynofloras: paleoecology and 1898 1899809 statistical analysis. M.J. Head, J.H. Wrenn (Editors), Neogene and Quatemary Dinoflagellate 1900 1901810 Cysts and Acritarchs. Am. Assoc. Stratigr. Palynol. Found., Dallas, pp. 347–390. 1902 1903811 Mudie, P.J., Marret, F., Rochon A., Aksu, A.E., 2010. Non-pollen palynomorphs in Black Sea 1904 1905 812 corridor. Veget. Hist. Archaeobot. 19, 531–544. 1906 1907 813 Nehring, S., 1997. Dinoflagellate resting cysts from Recent German coastal sediments. Bot. Mar., 1908 1909 1910814 40, 307-324. 1911 1912815 Ostenfeld, C.H., 1903. Phytoplankton from the sea around the Faröes. In Botany of the Faroës, 1913 1914816 based upon Danish investigations, part II, pp.558-612. 1915 1916817 Parke, M., Dixon, P.S., 1964. A revised check-list of British marine algae. J. mar. Biol. Ass. U.K. 1917 1918818 44, 499–542. 1919 1920819 Parke, M., Dixon, P.S., 1968. A revised check-list of British marine algae – Second revision. J. mar. 1921 1922820 Biol. Ass. U.K. 48, 783–832. 1923 1924 821 Parke, M., Dixon, P.S., 1976. Check-list of British marine algae – Third revision. J. mar. Biol. Ass. 1925 1926 1927822 U.K. 56, 527–594. 1928 1929823 Paulmier, G., 1972. Seston – phytoplankton et microphytobenthos en rivière d’Auray. Leur role 1930 1931824 dans le cycle biologique des huîtres ( Ostrea edulis L.). Thèse de Doctorat de l’Université de 1932 1933825 Provence, 142 pp. 1934 1935826 Pavillard, J., 1905. Recherches sur la flore pélagique (phytoplankton) de l'Étang de Thau, Impr. G. 1936 1937827 Firmin, Montane et Sicardi, 1905, 116 p. 1938 1939828 Penn, O., Privman, E., Ashkenazy, H., Landan, G., Graur, D., Pupko, T., 2010a. GUIDANCE: a 1940 1941 829 web server for asessing alignment confidence scores. Nucleic Acids Res. 38, W23–W28. 1942 1943 1944 1945 1946 1947 33 1948 1949 830 Penn, O., Privman, E., Landan, G., Graur, D. and Pupko, T., 2010b. An alignment confidence score 1950 1951 1952831 capturing robustness to guide tree uncertainty. Mol. Bio. Evol. 27, 1759–1767. 1953 1954832 Pospelova, V., Pedersen, T.F., de Vernal, A., 2006. Dinoflagellate cysts as indicators of climatic 1955 1956833 and oceanographic changes during the past 40 kyr in the Santa Barbara Basin, southern 1957 1958834 California. Paleoceanography 21, 16 pp. 1959 1960835 Popselova, V., Esenkulva, S., Johannessen, S.C., O’Brien, M.C., Macdonald, R.W., 2010. Organic- 1961 1962836 walled dinoflagellate cyst production, composition and flux from 1996 to 1998 in the central 1963 1964 837 Strait of Georgia (BC, Canada): A sediment trap study. Mar. Micropaleontol. 75, 17–37. 1965 1966 838 Price, A.M., Pospelova, V., 2011. High-resolution sediment trap study of organic-walled 1967 1968 1969839 dinoflagellate cyst production and biogenic silica flux in Saanich Inlet (BC, Canada). Mar. 1970 1971840 Micropaleontol. 80, 18–43. 1972 1973841 Reid, P.C., John, A.W. G., 1978. Tintinnid cysts. J. mar. Biol. Ass. U.K. 58, 551–557. 1974 1975842 Reid, P.C., John, A.W.G., 1983. Resting cysts in the Ciliate class Polyhymenophorea : Phylogenetic 1976 1977843 implications. J. Protozool. 30, 710–713. 1978 1979844 Ringer, Z., 1973. Phytoplankton of the southern Baltic Sea. Pol. Arch. Hydrobiol. 20, 371–378. 1980 1981845 Roncaglia, L., 2004. New acritarch species from Holocene sediments in central West Greenland. 1982 1983 846 Grana 43, 81–88. 1984 1985 1986847 Servais, T., 1996. Some considerations on acritarch classification. Rev. Palaeobot. Palynol. 93, 9– 1987 1988848 22. 1989 1990849 Shumilovskikh, L.S., Marret, F., Fleitmann, D., Arz, H.W., Nowaczyk, N. Behling, H., 2013. 1991 1992850 Eemian and Holocene sea-surface conditions in the southern Black Sea: Organic-walled 1993 1994851 dinoflagellate cyst record from core 22-GC3. Mar. Micropaleontol. 101, 146–160. 1995 1996852 Sorrel, P., Popescu, S.-M., Head, M.J., Suc, J.P. Klotz, S., Oberhänsli, H., 2006. Hydrographic 1997 1998853 development of the Aral Sea during the last 2000 years based on quantitative analysis of 1999 2000 854 dinoflagellate cysts. Palaeongeogr., Palaeoclimatol., Palaeoecol. 234, 304–327. 2001 2002 2003 2004 2005 2006 34 2007 2008 855 Stamatakis, A., Hoover, P., Rougemont, J., 2008. A fast boostrap algorithm for the RAxML web- 2009 2010 2011856 servers. Syst. Biol. 57, 758–771 2012 2013857 St-Onge, G., Leduc, J., Bilodeau, G., de Vernal., A., Devillers, R., Hillaire-Marcel, C., Loucheur, 2014 2015858 V., Mucci, A., Zhang, G., 1999. Caractérisation des sediments récents du Fjord de Saguenay 2016 2017859 (Québec) à partir de traceurs physiques, géochimiques, isotopiques et micropaléontologiques. 2018 2019860 Geog. Phys. Quat. 53, 339–350. 2020 2021861 Takano, Y., Horiguchi, T., 2005. Acquiring scanning electron microscopical, light microscopical 2022 2023 862 and multiple gene sequence data from a single dinoflagellate cell. J. Phycol., 42, 251–256. 2024 2025 863 Tappan, H., 1980. The Paleobiology of Plant Protists. Freeman, San Francisco, 977 pp. 2026 2027 2028864 Van Waveren, I.M., Marcus, N.H., 1993. Morphology of recent copepod egg envelopes from 2029 2030865 Turkey Point, Gulf of Mexico, and their implications for acritarch affinity. Spec. Pap.. 2031 2032866 Palaeont. 48, 111–124. 2033 2034867 Vanhöffen, E., 1897. Die Fauna und Flora Gronlands. Gronland-Expedition der Gesellschaft fur 2035 2036868 Erkunde zu Berlin 1891-1893 2, 1-383. 2037 2038869 Verhoeven, K., Louwye, S., Paez-Reyes, M., Mertens, K.N., Vercauteren, D., 2014. New acritarchs 2039 2040870 from the late Cenozoic of the southern North Sea Basin and the North Atlantic real. 2041 2042 871 Palynology 38, 38–50. 2043 2044 2045872 Verleye, T., Mertens, K.N., Louwye, S., Arz, H.W., 2009. Holocene salinity changes in the 2046 2047873 southwestern Black Sea: A reconstruction based on dinoflagellate cysts. Palynology 33, 77– 2048 2049874 100. 2050 2051875 Verni, F., Rosati, G., 2011. Resting cysts: A survival strategy in Protozoa Ciliophora. Ital. J. of 2052 2053876 Zool. 78, 134–145. 2054 2055877 Volk, R. 1905. Hamburgische Elb-Untersuchung VIII. Studien über die Einwirkung der 2056 2057878 Trockenperiode im Sommer 1904. Mitteilungen aus dem Naturhistorischen Museum in 2058 2059 879 Hamburg 23, 1-101. 2060 2061 2062 2063 2064 2065 35 2066 2067 880 Wang, B., Niu, T., Bhatti, M.Z., Chen, F., Wu, L., Chen, J., 2017. Identification of cyst wall 2068 2069 2070881 proteins of the hypotrich ciliate Euplotes encysticus using a proteomics approach. J. 2071 2072882 Microbiol. 55, 545-553. 2073 2074883 Warny, S., 2009. Species of the acritarch genus Palaeostomocystis Deflandre 1937: Potential 2075 2076884 indicators of neritic subpolar to polar environments in Antarctica during the Cenozoic. 2077 2078885 Palynology 33, 43-54. 2079 2080886 Wilbert, N., Song, W., 2008. A further study on littoral ciliates (Protozoa, Ciliophora) near King 2081 2082 887 George Island, Antarctica, with description of a new genus and seven new species. J. Nat. 2083 2084 888 Hist. 42, 979–1012. 2085 2086 2087889 Williams, G.L., Fensome, R.A., MacRae, R.A., 2017. The Lentin and Williams index of fossil 2088 2089890 dinoflagellate cysts, 2017 edition. AASP Conrib. Ser. 48, 1–1097. 2090 2091891 Willman, S., 2009. Morphology and wall structure of leiosphaeric and acanthomorphic acritarchs 2092 2093892 from the Ediacaran of Australia. Geobiology 7, 8–20. 2094 2095893 Wright, R.R., 1907. The plankton of eastern Nova Scotia waters. An account of floating organisms 2096 2097894 upon which young food-fishes mainly subsist. Contrib. Can. Biol. Fish. 1, 1–19. 2098 2099895 Wright, A.-D. G., Colorni, A., 2002. Taxonomic re-assignment of Cryptocaryon irritans , a marine 2100 2101 896 fish parasite. Europ. J. Protistol. 37, 375–378. 2102 2103 2104897 Yi, Z., Dunthorn, M., Song, W., Stoeck, T., 2010. Increased taxon sampling using both unidentified 2105 2106898 environmental sequences and identified cultures improves phylogenetic inference in the 2107 2108899 Prorodontida (Ciliophora, Prostomatea). Mol. Phylogenet. Evol. 57, 937–941. 2109 2110900 Zhang, Q., Yi, Z., Fan, X., Warren, A., Gong, J., Song, W., 2014. Further insights into the 2111 2112901 phylogeny of two ciliate classes Nassophorea and Prostomatea (Protista, Ciliophora). Mol. 2113 2114902 Phylogenet. Evol. 70, 162–170. 2115 2116 2117 2118 2119 2120 2121 2122 2123 2124 36 Fig. 1 . Map showing the five sampling sites (red dots) of surface sediments in this study.1.

Vilaine estuary, Brittany; 2. stations ‘Strö 1’ and Strö 6’, South of Havstenstund (Skagerrak,

Sweden); 3. Lysekil, station ‘Lyse 3’ (Skagerrak, Sweden); 4. Galterö (Skagerrak, Sweden);

5. Sällvik, Pojo Bay (Baltic Sea, Finland). Fig. 2a–e . Schematic drawing of Hexasterias problematica (a, b) and Halodinium verrucatum sp. nov. (c-e), depicting the morphological features discussed in text. Fig. 3a-l. Hexasterias problematica (a-c) and Halodinium verrucatum sp. nov. (d-l), in LM

(a-b, d-l) and SEM (c). a-b: cysts with cytoplasm from the Baltic Sea (Sällvik); c: empty cyst from the Skagerrak (Lyse 3); d-f : cysts with cytoplasm from the Skagerrak (Stro 1); g-l: empty cysts from the Skagerrak (Lyse 3), showing a cluster (g) and a specimen in polar view, obscured by amorphous organic matter. Fig. 4a-f. Halodinium verrucatum sp. nov., holotype from the Vilaine estuary (a-e) and specimen from Kaštela Bay, Croatia (f) , in SEM. a: oral view; b: oblique oral view; c: detail of wall rupture and sculptural elements; d: detail of pylome, with arrows indicating the texture of the inner wall (left) and the thickened rim of the pylome (right); e: detail of scultural elements. Scale bar is 20 µm unless otherwise indicated. Fig. 5. Correlation of central body diameter and process length of Hexasterias problematica . Fig. 6. DAPI-stained cell of Hexasterias problematica form the Vilaine Estuary (Brittany), showing micro (MI)- and macronucleus (MA). Fig. 7 . Map showing the distribution of Hexasterias problematica (red and small brown circles) and

Halodinium verrucatum sp. nov. (yellow squares). The occurrences of Hexasterias problematica are based on Kubiszyn & Svensen (2018), supplemented with additional published and personal observations (see section Occurrence and stratigraphic range in Species descriptions). The small brown circles represent occurrence data from OBIS, as in Kubiszyn & Svensen (2018). Fig. 8 . Phylogenetic placement of Hexasterias problematica and Halodinium verrucatum sp. nov. based on SSU rDNA sequences, with indication of GenBank accession numbers, existing classification (e.g., Gao et al. 2016) and ecological preferences (e.g., Lynn 2008). Fig. 9 . Normalized micro-FTIR absorbance spectra of Halodinium spp. (Irish Sea and

Brittany), Halodinium verrucatum sp. nov. (Strö 6) and Hexasterias problematica , with indication of the major absorptions (cf. Table 5). Specimen numbers (MxSy) refer to unique measurement codes. Fig. 10 . Normalized micro-FTIR absorbance spectra of ciliate loricae (German Wadden Sea and Skagerrak) and Radiosperma corbiferum (Brittany, Irish and Baltic Seas), with indication of the major absorptions (cf. Table 5). Specimen numbers (MxSy) refer to unique measurement codes. 1 2 3 Table 1. Region, site location, latitude, longitude and water depth of samples from Brittany, the 4 Skagerrak and the Baltic Sea used in this paper. 5 Latitude Longitude Water depth Region Sampling site 6 (°N) (°E) (m) 7 Brittany Vilaine Estuary 47.5 2.39 6 8 Baltic Sea Sällvik (Pojo bay, Finland) 60.02 23.48 34.5 9 10 Kattegatt Galterö 57.11 11.81 40.5 11 Skagerrak “Strö 1”, South of Havstensund (Sweden) 58.73 11.17 31 12 Skagerrak “Strö 6”, west of Strömstad (Sweden) 58.85 10.76 100 13 Skagerrak “Lyse 3” 58.34 11.36 29 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 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 1 2 3 Table 2. Morphometric data for Hexasterias problematica , based on material from Brittany 4 and the Skagerrak . 5 Arithmetic Median SD CV Min Max N 6 mean 7 Total diameter (µm) 102.2 99.1 14.9 14.58 82.8 130.2 12 8 Pylome diameter (µm) 22.4 22.1 1.8 8.04 18.9 25.5 9 9 Central body diameter (µm) 35.2 34.5 3.9 11.08 29.5 42.4 15 10 Process length (µm) 33.4 31.6 5.5 16.47 26.6 43.9 12 11 Process width (µm) 2.7 2.7 0.8 28.41 2 3.6 9 12 Number of processes 6.5 6 0.9 13.85 5 8 12 13 Number of denticulations 9.7 10 0.5 5.15 9 10 6 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 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 1 2 3 4 Table 3. Morphometric data for Halodinium verrucatum sp. nov. from Brittany and the Skagerrak 5 Arithmetic Median SD CV Min Max N 6 mean 7 Total diameter (µm) 70.2 67 8.5 12.1 60.4 87.5 21 8 Pylome diameter (µm) 14.2 13.7 2 14.1 10.5 18.5 24 9 Central body diameter (µm) 56.5 53.7 8.1 14.3 47 74 24 10 Flange width (µm) 13.4 12.4 3.9 29.1 9.3 25.3 15 11 Length of sculptural elements (µm) 1.7 1.7 0.2 9.4 1.6 2.1 10 12 Width of sculptural elements (µm) 0.13 0.12 0.02 15.4 0.1 0.18 15 13 Width of sculptural element basis (µm) 0.39 0.38 0.03 7.7 0.36 0.45 8 14 Distance between sculptural elements (µm) 1.6 1.6 0.3 16 1.3 2.3 21 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 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 1 2 3 Table 4. Comparison of diagnostic features of the species of Halodinium . 4 Species Halodinium major Halodinium minor Halodinium scopaeum Halodinium eiriksonii Halodinium verrucatum sp. nov. 5 6 Body diameter 104 - 116 µm 46 - 63 µm 21 - 24 µm 23.9 - 27.4 µm 47.0 (56.5) 74.0 µm 7 Pylome diameter 24 - 25 µm 11 - 22 µm 5.5 - 8.0 µm 4.6 - 8.8 µm 10.5 (14.2) 18.5 µm 8 Max. flange width 9 - 27 µm 4.5 - 9 µm 3 - 5.5 µm 1.6 - 6.8 µm 9.3 (13.4) 25.3 µm 9 Central body has a strong polar Discoidal with circular polar outline and Discoidal with circular polar outline and Small, polar compressed, with circular to polar compressed cyst with a circular to General shape compression and circular to subcircular 10 irregular membranous flange. irregular membranous flange. subcircular outline. subcircular outline in polar view. 11 outline in polar view. Bilayered, pale yellow-brown. 12 Bilayered, outer wall is appressed to the Periphragm much thinner than Wall - - inner wall over the greater part of the cyst Bilayered, 13 endophragm, to which it is appressed body. 14 except at ambitus. 15 Central body ornamented with low ridges Scabrate to smooth outer wall has a 16 Smooth or faintly scabrate. Periphragm forming an irregular reticulum, and is Nature of the wall The wall of the main body has a The wall of the main body has a smooth rugulate surface consisting of visible as small wrinkles over the surface covered over the polar region with a thin, 17 and ornamentation granulate or occasionally rugulate surface surface occasionally anastomosing wrinkle-like of the endocyst. hyaline outer layer ornamented with 18 ridges. discrete, bifurcating sculptural elements. 19 20 Central pylome with well-defined ridge on, or close to edge of pylome. 21 Central pylome present, with operculum Operculum often in place; circular with a Pylome located centrally and circular or often in place. The central pylome is A central pylome with a thickened rim; 22 Pylome smooth edge that is usually thickened, subcircular, with an entire and usually Circular without operculum. circular with a smooth edge that is operculum not observed. 23 although the thickening may occur a few unthickened margin. occasionally thickened. 24 micrometers away from the pylome on 25 the main body. Diophanous flange of approximately At the periphery of the cyst, the outer The outer layer forms near the margin of 26 The surrounding membrane extends The surrounding membrane extends constant width; its outer margin usually wall forms a collar-like ambital flange, the central body a wrinkled, diaphanous 27 Ambital flange around the main body and is thin, pale around the main body and is thin, pale ragged but may be partially entire on consisting of irregular septate or foldlike collar-like outer flange with a varying and often broken or torn and often broken or torn. 28 some specimens. structures. width. 29 Reference Bujak (1984) Bujak (1984) Head (1993) Verhoeven et al. (2013) This paper 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 1 2 3 Table 5. FTIR assignments of functional groups present in 4 Halodinium spp. and Hexasterias problematica (absorptions in 5 bold are shown on Figs. 9 and 10). 6 Wavenumber (cm -1 ) Assignment* Comments 7 ~3400 νOH shoulder 8 3280 νNH 9 3100 νNH 10 2970 νCH3 11 2950 νCH2 12 2860 νCH3 13 1640 νC=O 14 1620 νC=O Amide I 1580, 1550 νCN+ δNH Amide II 15 1420 δCH 16 2 1370 δCH + δC-CH 3 17 1312 νCN+ δNH Amide III 18 1255 δNH 19 1160 νC-O-C Ring 20 1110 νC-O 21 1040 νC-O 22 930 γCH3 23 860, 770 ρCH2 24 690 γNH Amide V * ν means bond stretching, δ means deformation, γ means 25 wagging and ρ means rocking of a bond within a molecule. 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 59