Morphology and Molecular Phylogeny of a New Marine, Sand‐dwelling Dinoflagellate Genus, Pachena (Dinophyceae), with Descriptions of Three New Species

Marine benthic dinoflagellates are interesting not only because some epiphytic genera can cause harmful algal blooms but also for understanding dinoflagellate evolution and diversification. Our understanding of their biodiversity is far from complete, and many thecate genera have unusual tabulation patterns that are difficult to relate to the diverse known phytoplankton taxa. A new sand‐dwelling genus, Pachena gen. nov., is described based on morphological and DNA sequence data. Three species were discovered in distant locations and are circumscribed, namely, P. leibnizii sp. nov. from Canada, P. abriliae sp. nov. from Spain, and P. meriddae sp. nov. from Italy. All species are tiny (about 9–23 μm long) and heterotrophic. Species are characterized by their tabulation (APC 4′ 3a 6′′ 5c 5s 5′′′ 2′′′′), an apical hook covering the apical pore, an ascending cingulum, and a sulcus with central list. The first anterior intercalary plate is uniquely “sandwiched” between two plates. The species share these features and differ in the relative sizes and arrangements of their plates, especially on the epitheca. The ornamentation of thecal plates is species‐specific. The new molecular phylogenies based on SSU and LSU rDNA sequences contribute to understanding the evolution of the planktonic relatives of Pachena, the Thoracosphaeraceae.

Marine benthic dinoflagellates are interesting not only because some epiphytic genera can cause harmful algal blooms but also for understanding dinoflagellate evolution and diversification. Our understanding of their biodiversity is far from complete, and many thecate genera have unusual tabulation patterns that are difficult to relate to the diverse known phytoplankton taxa. A new sanddwelling genus, Pachena gen. nov., is described based on morphological and DNA sequence data. Three species were discovered in distant locations and are circumscribed, namely, P. leibnizii sp. nov. from Canada, P. abriliae sp. nov. from Spain, and P. meriddae sp. nov. from Italy. All species are tiny (about 9-23 lm long) and heterotrophic. Species are characterized by their tabulation (APC 4 0 3a 6 00 5c 5s 5 000 2 0000 ), an apical hook covering the apical pore, an ascending cingulum, and a sulcus with central list. The first anterior intercalary plate is uniquely "sandwiched" between two plates. The species share these features and differ in the relative sizes and arrangements of their plates, especially on the epitheca. The ornamentation of thecal plates is species-specific. The new molecular phylogenies based on SSU and LSU rDNA sequences contribute to understanding the evolution of the planktonic relatives of Pachena, the Thoracosphaeraceae.
Benthic, sand-dwelling species seem to have morphological adaptations reflecting their life in the interstitial habitat, such as smooth (i.e., without striking extensions like wings, spines, or horns) and flattened cell shapes (Hoppenrath et al. 2014). Several taxa cover their apical pore with thecal extensions (e.g., Rhinodinium has a large apical hook; Murray et al. 2006); some Amphidiniopsis species and Herdmania have a small hook (Hoppenrath 2000b, Murray and Patterson 2002, Toriumi et al. 2002, Yamaguchi et al. 2011, Reñ e et al. 2020); Apicoporus has fingerlike projections (Sparmann et al. 2008); Laciniporus has a small flap-shaped projection (Saburova and Chom erat 2019); and Sinophysis has parallel upright projections (Hoppenrath 2000c, Chom erat 2016. Many thecate, benthic dinoflagellate taxa have unusual tabulation patterns that are difficult to relate to the known tabulations in planktonic taxa (Hoppenrath et al. 2014). For example, Madanidinium has no apical pore (Chom erat and Bilien 2014); Plagiodinium has no precingular or no apical plate series (Faust andBalech 1993, Wakeman et al. 2018), depending on interpretation; Thecadinium sensu stricto and Pseudothecadinium have only incomplete precingular plate series and other special plate arrangements ; Pseudadenoides is the only known genus with a complete posterior intercalary plate series (Hoppenrath et al. 2003. A species diversity survey of marine sandy sediments in British Columbia, Canada, revealed species richness including new taxa (Hoppenrath and Leander 2007, 2008, Sparmann et al. 2008, Hoppenrath et al. 2014, 2017M. Hoppenrath unpub. data).
Studies on benthic dinoflagellates from the Mediterranean Sea have mainly focused on epiphytic toxic species (Vila et al. 2001, Aligizaki and Nikolaidis 2006, Aligizaki et al. 2009, Penna et al. 2012, whereas sand-dwelling dinoflagellates have been poorly studied and information is scarce (Reñ e et al. 2020). Here, a new genus is described that was first discovered on the western shoreline of Vancouver Island, Canada and further species were recorded in Spanish and Italian Mediterranean Sea samples.

METHODS
Sampling, cell extractions, and microscopy. Sand samples from Canada were collected with a spoon during low tide at Pachena Beach (48°47 0 34.6″ N, 125°07 0 19.0″ W), Vancouver Island, British Columbia, in May and June 2005, April and June 2006, and May and June 2007. The sand samples were transported directly to the laboratory, and dinoflagellates were separated from the sand by extraction through a fine filter (mesh size 45 lm) using melting seawater ice (Uhlig 1964). Cells of the new taxon were observed directly with a Leica DMIL inverted microscope (Wetzlar, Germany) and isolated by micropipetting for the preparations described below. For differential interference contrast (DIC) light microscopy, pipetted cells were viewed with a Zeiss Axioplan 2 imaging microscope (Carl-Zeiss, Oberkochen, Germany) connected to a Leica DC500 color digital camera.
Mediterranean sediment samples from the Catalan Coast were obtained at Castelldefels Beach (41°15 0 37.0″ N; 1°55 0 48.8″ E) during spring and summer months from 2015 to 2017. Sediment samples from Sardinian beaches were obtained at Platamona Beach (40°49 0 27.1″ N; 8°31 0 36.4″ E) and La Speranza Beach (40°29 0 43.1″ N; 8°22 0 12.1″ E) during summer months in 2015 and 2018. Surface samples were taken by snorkeling at a depth of approximately 1.5-2 m with plastic bottles. The distance to the shore depended on the underwater slope of each beach. The sediments were kept at room temperature, in the dark, and immediately taken to the laboratory. Once there, cells were extracted from the sediment using the melting seawater-ice method (Uhlig 1964). Subsamples were fixed with Lugol's iodine or formaldehyde (2%) and preserved in the dark at 4°C. Alive and fixed samples from the Catalan coast were observed under a phase-contrast Leica DM-IRB inverted microscope (Leica Microsystems, Wetzlar, Germany) connected to a ProgRes C10 (Jenoptik Laser, Optik Systeme GmbH, Jena, Germany) digital camera. Cell measurements were conducted using the ProgRes Cap-turePro software (Jenoptik Laser; Optik Systeme GmbH). Live samples from the Sardinian coast were observed under a Zeiss 100 inverted microscope (Carl Zeiss, Oberkochen, Germany), equipped with DIC. Digital photos were taken using a Zeiss Axiocam (Carl Zeiss). Cell measurements were obtained from LM and SEM images using the ImageJ software (1.47v; W. Rasband, USA).
For scanning electron microscopy observations (1) the Canadian mixed-extraction samples were fixed overnight with acidic Lugol's solution. Cells were transferred onto a 5 lm polycarbonate membrane filter (Corning Separations Div., Acton, MA, USA), washed with distilled water, dehydrated with a graded series of ethanol, rinsed twice in hexamethyldisilazane (HMDS), and oven-dried at 65°C. Filters were mounted on stubs, sputter-coated with gold, and viewed under a Hitachi S4700 scanning electron microscope; (2) the fixed Mediterranean subsamples were filtered into a 3.0-DESCRIPTION OF PACHENA GEN. NOV. 5.0 lm polycarbonate filter, and washed in seawater and distilled water for 15 min. A subsequent dehydration was carried out in a 25, 50, 75, 90, 96, and 100% ethanol series for ca. 10 min. The final step of 100% ethanol was repeated twice. The filters were critical-point dried or rinsed twice in HMDS and dried for 5 min at 60°C. The dried filters were then mounted on stubs, sputter coated with gold-palladium. Catalan samples were examined with a HITACHI S-3500N scanning electron microscope (Hitachi High Technologies Corp., Tokyo, Japan) at the Servei de Microscopia Electr onica (ICM-CSIC) in Spain and a Tescan VEGA3 microscope (Elektronen-Optik-Service GmbH, Dortmund, Germany) in Germany. Sardinian samples were examined with a Tescan VEGA3 microscope (Elektronen-Optik-Service GmbH) in Germany.
Polymerase chain reactions and phylogenetic analyses. Canadian specimens isolated from a raw sample were washed with filtered (eukaryote-free) seawater and 100 cells deposited in 35 lL distilled water in a 1.5 mL Eppendorf tube (Dia-Med Lab Supplies Inc., Mississauga, ON, Canada) heated at 65°C and afterwards stored in a freezer. DNA amplification was carried out using 10 lL of this cell preparation and puRe-Taq Ready-To-Go PCR Beads (Amersham Biosciences, Piscataway, NJ, USA). The protocol using universal eukaryotic primers (PF1-R4) was described in Leander (2007, 2010). PCR products corresponding to the expected size were gel isolated and cloned into the pCR2.1 using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA). One clone from Pachena leibnizii sp. nov. was sequenced with ABI big-dye reaction mix (Applied Biosystems, Foster City, CA, USA) using the vector primers and internal primers oriented in both directions (See Table 1 for GenBank accession numbers).
Around 40 specimens from the Catalan Coast were isolated from a raw sample from May 2017, washed with filtered and autoclaved seawater, and transferred to a 1.5 mL Eppendorf tube containing 200 lL of seawater. Genomic DNA was extracted using a DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) following the manufacturer instructions. Two microliters of this extract was subjected to a first 25 lL PCR using EK-82F -28S-1611R primers with an amplification mixture containing 2 lL of 10X buffer (TaKaRa Bio), 1.5 mM MgCl 2 , 1 U of TaKaRa Taq DNA polymerase (TaKaRa Bio), 0.2 mM of each dNTP, and 0.4 mM of each primer. PCR amplification conditions were as follows: initial denaturation for 3 min at 95°C, followed by 6 cycles of 15 s at 95°C, 30 s at 58-53°C, decreasing 1°C each cycle, and 2 min at 72°C, and 34 additional cycles at annealing temperature of 52°C, followed by a final extension step for 5 min at 72°C. The resulting product was used as template for semi-nested PCRs to amplify the SSU and LSU rDNA regions independently, using primers EK-82F and EK-1520R, and 28S-1F -28S-1611R, respectively. Each amplification reaction contained 1 lL of template, 2.5 lL of 10X buffer (Invitrogen, Thermo Fisher Scientific Corp.) containing 15 mM MgCl 2 , 1.25 U of Platinum Taq DNA polymerase (Invitrogen, Thermo Fisher Scientific Corp.), 0.2 mM of each dNTP, and 0.4 mM of each primer. PCR conditions were as follows: initial denaturation for 2 min at 94°C, 35 cycles of 15 s at 94°C, 30 s at 55°C, and 1 min at 72°C, followed by a final extension step for 5 min at 72°C. A second semi-nested, using 1 lL of the previous PCR product as template was performed for each region using primers DIN-464F and EK-1520R, and 28S-1F and 28S-803R, respectively. Four lL of PCR products were electrophoresed in an agarose gel and visualized under UV illumination. Purification and Sanger sequencing was carried out by external services (Genewiz, Takeley, UK) using both forward and reverse primers. See Table 1 for GenBank accession numbers.
Several specimens from Platamona Beach, Italy were isolated from samples collected in June 2015 and 2018 and one specimen from La Speranza Beach, Italy, was isolated from a sample collected in June 2018. Isolated cells were washed in several drops of filtered seawater and transferred to a 0.2 mL PCR tubes containing 5 lL of lysis buffer (400 ng Á lL À1 Proteinase K and 0.005% SDS). Tubes were subjected to freezing at -20°C for 10 min, heating at 60°C for 30 min, and then at 95°C for 10 min to facilitate cell lysis. Resulting lysates were directly used as a template for amplification of SSU and LSU rDNA fragments.
The lysate from two specimens of Platamona (sample June 2015) was amplified for the LSU rDNA using D1R and D2C primers. The 50 lL PCR mixture contained 5 lL of 10X buffer, 1.5 lL of MgCl 2 , 0.25 lL of Hot start Taq DNA polymerase and 1 lL of dNTP 0.2 mM each (Qiagen), and 0.4 mM of each primer. PCR amplification conditions were as follows: initial denaturation for 5 min at 95°C, 40 cycles of 20 s at 95°C, 30 s at 55°C, and 1 min at 72°C, followed by a final extension step for 10 min at 72°C.
The lysate of one specimen from Platamona and one from La Speranza (samples June 2018) were used as template for amplification of SSU rDNA fragment using EukA and EukB primers. Each 50 lL PCR amplification reaction contained 5 lL of 10X buffer, 1.5 lL of MgCl 2 , and 0.25 lL of Hot start Taq DNA polymerase (Biotechrabbit), 1 lL of dNTP 0.2 mM each (Qiagen), and 0.4 mM of each primer. PCR conditions were as follows: initial denaturation for 5 min at 94°C, 34 cycles of 1 m at 94°C, 1:30 m at 55°C, and 2 min at 72°C, followed by a final extension step for 7 min at 72°C. The resulting product was used as template for nested PCRs using primers Dino18SF1 and 18S-comR1. Each PCR amplification reaction contained 5 lL of 10X buffer, 1.5 lL of MgCl 2 , and 0.25 lL of Hot start Taq DNA polymerase (Biotechrabbit), 1 lL of dNTP 0.2 mM each (Qiagen), 0.4 mM of each primer, and 1 lL of template. PCR conditions were as follows: initial denaturation for 5 min at 94°C, 29 cycles of 45 s at 94°C, 1 m at 55°C, and 3 min at 72°C, followed by a final extension step for 10 min at 72°C. All information regarding the primers used during the amplification protocols can be found in Table S1 in the Supporting Information.  Four lL of all PCR products were electrophoresed in an agarose gel and visualized under UV illumination. Purification and Sanger sequencing was carried out by external services (Macrogen Europe, Amsterdam, Netherlands in 2015 and Genoscreen, Lille, France in 2018) using both forward and reverse primers. See Table 1 for GenBank accession numbers.
The SSU and LSU rDNA sequences were aligned using MUSCLE (Edgar 2004) and viewed in Mesquite v3.11 (Maddison and Maddison 2015). Highly variable regions were eliminated using Gblocks (Castresana 2000, Talavera andCastresana 2007). The final alignments of the SSU and LSU rDNA dataset consisted of 48 with 1768 and 49 taxa with 1167 sites, respectively. The best-fit model for each dataset was chosen by ModelFinder in IQ-TREE under AICc (Trifinopoulos et al. 2016, Kalyaanamoorthy et al. 2017). Maximumlikelihood (ML) analyses were run with IQ-TREE using TIM2 + F + R4 and GTR+F+R5, as the model of evolution for the SSU and LSU rDNA, respectively. Bootstrap analyses were run for each dataset with 1000 replicates to evaluate statistical reliability. MrBayes v3.2.5 was used to perform Bayesian analyses (Ronquist and Huelsenbeck 2003) with the GTR + I + G and four Monte-Carlo-Markov chains starting from a random tree. For the SSU dataset a total of 2,500,000 and the first 6,250 trees were discarded; for the LSU rDNA dataset 1,000,000 generations were calculated and the first 2,500 trees in each run were discarded. Trees were sampled every 100 generations in both analyses. Posterior probabilities (PP) correspond to the frequency at which a given node was found in the post-burn-in trees.
Etymology: The genus is named after the sampling area of its first discovery, "Pachena Beach" near Bamfield, Vancouver Island, British Columbia, Canada.
Registration Description: Cells slightly dorsoventrally flattened, roughly oval to pentagonal in shape, 13-23 lm long, and 12-20 lm wide. Cells without chloroplasts. Epitheca and hypotheca are nearly of equal size, cingulum is ascending about one cingulum height, and sulcus is reaching the antapex. An apical hook covers the apical pore and points to the left dorsal cell side. It possesses a characteristic small, first anterior intercalary plate with only two plate borders that is sandwiched between the second anterior intercalary plate and the second precingular plate. Thecal plates are smooth or faintly ornamented except for the cingular and some sulcal plates that are always smooth.
Holotype: The SEM stub containing the type (specimen shown in Fig. 2A) is deposited at the dinoflagellate type collection in the Centre of Excellence for Dinophyte Taxonomy (CEDiT, Wilhelmshaven, Germany), which is part of the Herbarium Senckenbergianum Research: as funding body for institutes of the Leibniz Association the ministry enabled the renovation and relocation of the scientific collections and research infrastructure into the new Senckenberg buildings in Frankfurt/Main. This new research infrastructure allows safeguard Senckenberg's invaluable scientific collections, promotes Senckenberg's international visibility and scientific excellence, and opens new avenues for international collaboration and transfer of knowledge into the society.
Registration: http://phycobank.org/ 102114 Type locality: Pachena Beach, Vancouver Island, British Columbia, NE Pacific (48°47 0 34.6 0 ' N, Habitat: marine, benthic, sandy sediment Cells slightly dorsoventrally flattened, roughly oval to pentagonal in shape and small: 13.1-22.5 lm long and 11.5-20.0 lm wide (n = 20; Fig. 1, A-E). Cells are not pigmented but often contain a colored (green, orange to red) food body of varying size in the episome ( ). An apical hook covers the apical pore complex and points to the left dorsal cell side (Figs. 2, A, C and D and 3, G and H). The apical pore is not directly visible but seems to be surrounded by a rim (Fig. 3, G and H). It is unclear whether the apical pore is rounded (Fig. 3G) or slit like (Fig. 3H). The four apical plates are of very different size, with plates 2 0 and 3 0 being small and the hook being part of plate 4 0 (Figs. 2, C and D and 3, G and H). The three anterior intercalary plates are in contact to each other (Fig. 2, C and D). The characteristic small, first anterior intercalary plate (1a) has only two plate borders and is sandwiched between the second anterior intercalary plate (2a) and the second precingular plate (2 00 ; Figs. 2D and 3G). From the six precingular plates, plate 2 00 is large and 3 00 is noticeably narrow and relatively small located centrally on the dorsal side (Fig. 2, A-D). The hypothecal plates have an unusual asymmetric arrangement. The first (1 000 ) and fifth (5 000 ) postcingular plates are on the ventral side (Fig. 2, A and B) and the small, second (2 000 ) and third (3 000 ) postcingular plates are rectangular (Fig. 2, C-F). The fourth postcingular plate (4 000 ) is relatively large covering most of the right dorsal cell side (Fig. 2, C and F). The two antapical plates are of different size, with the first (1 0000 ) being very large (Fig. 2, C-F). The sulcus widens toward the posterior cell end and five sulcal plates were recognized. The anterior sulcal plate (Sa) is narrow, elongated, and extends into the epitheca, and has a short left posterior list (Figs. 2, A and B; 3, A-D). The narrow and elongated right sulcal plate (Sd) strikingly widens posteriorly through a wide, smooth, wing-like left list (Figs. 2, A and B; 3, B-F) that covers the sulcal center. The left (Ss) and middle (Sm) sulcal plates are mostly hidden and cannot be described in detail (Fig. 3, D-F Description: Cells slightly dorsoventrally flattened, roughly oval to pentagonal in shape, 16-21 lm long and 13-16 lm wide. Cells without chloroplasts. Epitheca and hypotheca are nearly of equal size, cingulum is ascending about one cingulum height, and sulcus is reaching the antapex. An apical hook covers the apical pore and points to the left dorsal cell side. It possesses a characteristic small, first anterior intercalary plate with only two plate borders that is sandwiched between the second anterior intercalary plate and the third precingular plate. Thecal plates are ornamented with small spines except for the cingular and some sulcal plates that are smooth. Holotype: The SEM stub containing the type (specimen shown on Fig. 4B) is deposited at the dinoflagellate type collection in the Centre of Excellence for Dinophyte Taxonomy (CEDiT, Wilhelmshaven, Germany), which is part of the Herbarium Senckenbergianum Frankfurt/M. (FR) with the designation CEDiT2019H106.
Molecular characterization: nuclear ribosomal SSU (MN707940) and LSU (MN703810) Etymology: The species is named after the daughter of the second author of this study, Abril Reñ e.
Registration: http://phycobank.org/102115 Type locality: Castelldefels beach, Catalonia, NW Mediterranean Sea (41°15 0 37″ N; 1°55 0 48.8″ E) Habitat: marine, benthic, sandy sediment Cells slightly dorsoventrally flattened, roughly oval to pentagonal in shape, and small: 16.4-21.3 lm long and 12.9-16.4 lm wide (n = 37; Fig. 1, F-I). Cells are not pigmented but often contain a colored (orange to red) food body of varying size in the episome (Fig. 1, F-I). The nucleus is located in the hyposome (Fig. 1, G and I). A pusule can be visible 802 in the hyposome (Fig. 1, F and H). Epitheca and hypotheca are nearly of equal size (Figs. 1, G-I and 4, A-F), the cingulum is ascending about one cingulum height, and the sulcus is reaching the antapex (Figs. 1F and 4, A and B). The thecal tabulation is APC 4 0 3a 6 00 5c 5s 5 000 2 0000 (Figs. 4 and 5). The thecal plates are ornamented with small spines except for the cingular and some sulcal plates that are smooth (Figs. 4, 5). In some cells cingular plates can have a faint bar-like ornamentation (Fig. 5, F and H). All plates have scattered thecal pores that are surrounded by a narrow rim (like a ring; Figs. 4, A-D and 5, A and H). An apical hook covers the apical pore complex and points to the left dorsal cell side (Fig. 4, A-F). The apical pore is not directly visible but seems to be surrounded by a raised rim (Fig. 5, B, D and E). The apical pore plate possesses a row of thecal pores at its margin   (Figs. 4, A, B, G and H and 5, G and H). The anterior sulcal plate (Sa) is narrow and elongated, with a spiny right margin and a short left posterior list (Figs. 4, A and B and 5, G and H). The narrow and elongated right sulcal plate (Sd) strikingly widens posteriorly through a wide, serrated, wing-like left list (Figs. 4, A and B, 5, G and H) that covers the sulcal center. The left (Ss) and middle (Sm) sulcal plates are mostly hidden and cannot be described in detail (Fig. 5, G and H). The posterior sulcal plate (Sp) is visible mainly between the two antapical plates and has no special structures (Figs. 4, A, B, G and H and 5, F-H).
Pachena meriddae sp. nov. Satta, Reñ e & Hoppenrath (Figs. 1, J and K, 6 and 7) Description: Cells slightly dorsoventrally flattened, pentagonal, with triangular epitheca and trapezoid hypotheca, 9-13 lm long and 6-10 lm wide. Cells without chloroplasts. Epitheca and hypotheca are nearly of equal size, cingulum is ascending about one cingulum height, and sulcus is reaching the antapex. An apical hook covers the apical pore and points to the left dorsal cell side. Epithecal plates are ornamented with ridges and some spines, hypothecal plates with spines, cingular and sulcal plates are smooth.
Holotype: The SEM stub containing the type (specimen shown on Fig. 6B)  Habitat: marine, benthic, sandy sediment Cells slightly dorsoventrally flattened, with triangular epitheca and trapezoid hypotheca, pentagonal in shape and small: 9.4-13.2 lm long and 6.3-10.2 lm wide (n = 20; Figs. 1, J and K and 6, A-F). Cells are not pigmented but often contain a colored (orange to red) food body of varying size in the episome (Fig. 1, J and K). The nucleus is located in the hyposome (Fig. 1J). A pusule can be visible centrally (Fig. 1K). Epitheca and hypotheca are nearly of equal size (Figs. 1, J and K and 6, A-F), the cingulum is ascending about one cingulum height (Fig. 6, A and B). The sulcus characteristically extends the end of the transverse furrow anteriorly and is reaching the antapex (Fig. 7, A-C). The thecal tabulation is APC 4 0 3a 6 00 5c 3 + s 5 000 2 0000 (Figs. 6 and 7). The epithecal plates are ornamented with ridges and some spines, the hypothecal plates with spines, and cingular and sulcal plates are smooth (Figs. 6 and 7). All plates have scattered thecal pores that are surrounded by a narrow rim (like a ring; Figs. 6, G and H and 7). An apical hook covers the apical pore complex and points to the left dorsal cell side (Fig. 6). The apical pore is not directly visible. The four apical plates are of very different size, with a narrow plate 1 0 , small plates 2 0 and 3 0 , and the hook being part of plate 4 0 (Fig. 7). The three anterior intercalary plates are in contact to each other (Fig. 7, B-D) but their shapes could not reliably be determined. Further observations are necessary to clarify whether plate 1a is triangular (Fig. 7C) or pentagonal (Fig. 7B) and not sandwiched between two plates. Plate 2a is in contact with plates 3 0 , 1a, 2 00 , 3 00 , 3 00 , and 3a (Fig. 7, B-D) but the outline needs verification. It is uncertain whether plate 3a is in contact with plate 3 0 (Fig. 7D) or not (Fig. 7B). To understand whether the anterior intercalary plates are very variable in this species much more cell observations are needed. From the six precingular plates, plate 2 00 is relatively large and 3 00 is noticeably smaller and narrower and located centrally on the dorsal side (Figs. 6,E,F and H and 7C). The hypothecal plates have an unusual asymmetric arrangement. The postcingular plates 1 000 and 5 000 are on the ventral side (Fig. 6, A-C) and the small plates 2 000 and 3 000 are rectangular (Fig. 6, D and E). The two antapical plates are of different size, with plate 1 0000 being very large and covering most of the dorsal hypotheca (Fig. 6, D-F). The sulcus widens toward the posterior cell end and only three sulcal plates were recognized. The anterior sulcal plate (Sa) is narrow and elongated, with a smooth ridge on the right margin and a short left posterior list (Fig. 6, A-C). The narrow and elongated right sulcal plate (Sd) optically extends the end of the transverse furrow anteriorly and it widens posteriorly through a wide, smooth left list that is posteriorly elongated and tapered (Fig. 6, B and C). A left (Ss) and middle (Sm) sulcal plate were not recognized. The posterior sulcal plate (Sp) is visible mainly between the two antapical plates (Fig. 6, A and B).
Phylogenetic relationships. SSU rDNA sequences were obtained for the three species described. The sequences corresponding to Pachena leibnizii show a 97.5% and a 98.1% pairwise similarity with those from P. abriliae (Castelldefels) and P. meriddae, respectively, and the sequences of these two latter species show a 96.2% pairwise similarity between them. Both sequences corresponding to P. abriliae were 99.8% similar. A phylogenetic tree inferred from SSU rDNA sequences (Fig. 8) showed that all of the Pachena sequences form a clade (94% BS / 1 BPP), where P. leibnizii and P. meriddae (100%/1) are more closely related to each other than to P. abriliae (100%/1). The Pachena clade clusters with Thoracosphaeraceae (Peridiniales) representatives, even though the clade shows low statistical support (63%/-). The Thoracosphaeraceae cluster includes

DISCUSSION
Morphology. Pachena is characterized by its tabulation (APC 4 0 3a 6 00 5c 5s 5 000 2 0000 ), the apical hook (part of the fourth apical plate) covering the apical pore and pointing to the left dorsal cell side, and the ascending cingulum (Fig. 10). The sulcus has an internal list in its center that is part of the anterior and right sulcal plate. The first anterior intercalary plate is uniquely sandwiched between two plates. The three species share these features and differ in the relative sizes and arrangements of their plates, especially on the epitheca (Fig. 10). The first apical plate is small and narrow in P. leibnizii and P. meriddae. Conversely, it is wide in P. abriliae. Consequently, the first precingular plate is wider in P. leibnizii and P. meriddae, than in P. abriliae. The second anterior intercalary plate is smaller in P. leibnizii than in P. abriliae and occupies a lateral position, rather than dorsal. Pachena leibnizii shows a large second precingular plate, like P. meriddae, while this plate is narrower in P. abriliae. The arrangement and shape of the remaining precingular plates is unique for each species. In particular, P. abriliae shows a distinctive elongated third precingular plate. The ornamentation of the thecal plates of the species is different. While P. leibnizii is faintly ornamented, P. abriliae shows small spines and P. meriddae ridges and some spines. In addition, there are slight variations among the species in the size ranges and cell shapes. Pachena leibnizii and P. abriliae are oval to pentagonal, with cell length between 13 and 22 lm, but P. meriddae is pentagonal, with triangular epitheca, and cells are smaller (<15 lm long).
Despite the relative asymmetric tabulation, especially of the hypotheca, and the absence of a canal plate in the apical pore complex, the genus is molecular phylogenetically related to peridinoid genera (Figs. 8 and 9; discussion below). The presence of anterior intercalary plates and five postcingular plates is typical for genera of the Peridiniales (Fensome et al. 1993). Most peridinoid taxa have seven precingular plates, but some have only six (e.g., Aduncodinium, Luciella, Pseudopfiesteria, Tyrannodinium; all pfiesteriaceans), which is usually characteristic for gonyaulacoids (Fensome et al. 1993, Hoppenrath 2017. Covering the apical pore with part of a thecal plate (hook-, finger-, and flap-like projection) is a special feature of some benthic sand-dwelling dinoflagellate taxa that evolved multiple times independently (Hoppenrath et al. 2014). In Rhinodinium, it is a prominent morphological character. The hook is part of the second apical plate and is pointing to the dorsal cell side (Murray et al. 2006). In contrast, Pachena has a hook that points to the left dorsal (to lateral) side and is part of the fourth apical plate, similar to the hook in Aduncodinium (Kang et al. 2015), Laciniporus (Saburova and Chom erat 2019), and Herdmania (e.g., Reñ e et al. 2020) that points to the left lateral side. The apical pore complex of Aduncodinium and Laciniporus comprises a canal plate that is typical for peridinoid taxa (Kang et al. 2015, Saburova and Chom erat 2019), a trait not detected for Pachena species. Aduncodinium further differs in having only two anterior intercalary plates and a relative symmetric tabulation (Kang et al. 2015). Laciniporus has also only two anterior intercalary plates, like Aduncodinium, seven precingular plates, and is photosynthetic (Saburova and Chom erat 2019). Herdmania differs from Pachena by its more symmetrical tabulation, especially of the hypotheca, seven precingular plates, and the sulcal construction (Hoppenrath 2000d, Yamaguchi et al. 2011, Reñ e et al. 2020. Eight Amphidiniopsis species have an apical hook, seven belonging to morphogroup 3 and one to morphogroup 1 (Reñ e et al. 2020). A hook originating from either the third or the fourth apical plate on the right lateral side and pointing to the left over the apical pore has been described for A. bulla, A. cristata, A. uroensis, A. elongata, A. hoppenrathae, A. korewalensis, and A. pectinaria of morphogroup 3 (Hoppenrath 2000b, Murray and Patterson 2002, Toriumi et al. 2002, Selina and Morozova 2017, Reñ e et al. 2020. Amphidiniopsis galericulata has a small hook as part of the first apical plate pointing dorsally (Hoppenrath 2000b). The apical hook morphology of Pachena species is most similar to that of Laciniporus arabicus (Saburova and Chom erat 2019).
The sulcal construction of Pachena resembles that of Amphidiniopsis. Striking is the central list that is mainly part of the right sulcal plate (Hoppenrath 2000b, Murray and Patterson 2002, Toriumi et al. 2002, Selina and Hoppenrath 2013, Selina and Morozova 2017, Reñ e et al. 2020. A second smaller part of this list is formed by the anterior sulcal plate. Species in morphogroup 3 of the Amphidiniopsis genus complex have a similar Sa-and Sd-plate arrangement to Pachena with the Sd-plate connecting to the end of the cingulum and the Sa-plate touching the last precingular, the first apical and the first precingular plate (Hoppenrath 2000b, Murray and Patterson 2002, Selina and Hoppenrath 2013, Selina and Morozova 2017, Reñ e et al. 2020. Also Herdmania has this internal sulcal list, but the anterior sulcal plate is in contact with the last cingular plate and the fifth postcingular plate different from Pachena (Yamaguchi et al. 2011, Reñ e et al. 2020. A complex sulcal list has been described as distinctive feature of Laciniporus (Saburova and Chom erat 2019). It is equivalent to these internal sulcal lists. The arrangement of the involved sulcal plates (Sa and Sd) is like for Pachena but the plate shapes are very different, wide, and short in Laciniporus and narrow and elongated in Pachena. A similar sulcal plate arrangement has been described for photosynthetic Scrippsiella species (e.g., Montresor and Zingone 1988, Janofske 2000, Zinssmeister et al. 2012, Kretschmann et al. 2015, Luo et al. 2016, Lee et al. 2019. In contrast to Pachena, the Sd-plate has a less pronounced list, the Sa-plate has no list and the Spplate is in contact with the first cingular plate in Scrippsiella species. The tide pool species Scrippsiella hexapraecingula possesses a peduncle and is likely mixotrophic Chihara 1983, Hoppenrath et al. 2014). Apocalathium is a putative photosynthetic sister clade to the pfiesteriaceans with sulcal plates like Scrippsiella and without microtubules associated with a peduncle (Larsen et al. 1995, Craveiro et al. 2017. Heterotrophic pfiesteriaceans (like Cryptoperidiniopsis, Luciella, Pfiesteria, Pseudopfiesteria, Tyrannodinium) also have a comparable sulcal construction, but with a plate specific for them, the peduncle cover plate (Litaker et al. 2005, Marshall et al. 2006, Steidinger et al. 2006, Mason et al. 2007, Calado and Craveiro 2009) that could be interpreted as Sd-plate. Its morphology is especially similar to the Sd-plate in Pachena. Nothing is known about the feeding behavior of Pachena yet. The species had colored food bodies in the episome like pfiesteriaceans. Comparing the Sd-plate 812 morphology to the peduncle cover plate, it can be hypothesized that Pachena is a benthic peduncle feeder, like the related Aduncodinium (Kang et al. 2015). A similar food body in the epi-or hyposome is characteristic for Cabra, Rhinodinium, and Roscoffia, benthic heterotrophic genera related to the family Podolampadaceae (Hoppenrath et al. 2014). Their feeding mode has not yet been documented. Planktonic Podolampadaceae are pallium feeders (Sch€ utt 1895, Carbonell-Moore 2004).
Three anterior intercalary plates can occur in the Peridiniales. The major Protoperidinium species (i.e., Protoperidinium sensu stricto, including the sections Protoperidinium, Avellana, Conica, Excentrica, and Divergentia) are known to have two or three anterior intercalary plates Horiguchi 2005, Yamaguchi et al. 2006). Among these sections Avellana and Excentrica have two anterior intercalary plates (Yamaguchi et al. 2006). These sections are not monophyletic in the Protoperidinium s.s. clade (Yamaguchi et al. 2006). It is assumed that the reduction of the number of anterior intercalary plates from three to two occurred in different lineages (Yamaguchi et al. 2006). Archaeperidinium has only two anterior intercalary plates, but the closely related Amphidiniopsis species (except morphogroup 2) and Herdmania have three anterior intercalary plates (Yamaguchi et al. 2011, Reñ e et al. 2020. Scrippsiella species have three anterior intercalary plates and similar sulcal plates, see above (e.g., Janofske 2000, Zinssmeister et al. 2012, Kretschmann et al. 2015. All Heterocapsa species, except for the type, have three anterior intercalary plates and are photosynthetic and characterized by specific body scales (Iwataki 2008, Tillmann et al. 2017. All these genera differ in the special arrangement of the three anterior intercalary plates and the shape of the 1a plate, plus possessing seven precingular plates. Nearly all species of Azadinium (family Amphidomataceae) possess three anterior intercalary plates (e.g., Tillmann et al. 2018, and references therein). This genus has four apical and six precingular plates like Pachena but Azadinium is distinct from it by the APC, the sulcal construction, a descending cingulum with six plates, and six postcingular plates (e.g., Tillmann et al. 2014Tillmann et al. , 2018.
Phylogenetic considerations. SSU rDNA sequences clearly separated the species in accordance with the morpho-species concept. Pachena leibnizii and P. meriddae formed a sister clade to P. abriliae, in agreement with their morphological similarities, mostly regarding the epithecal plate pattern.
Sequences from Pachena occupied a basal position within the Thoracosphaeraceae, even though this clade showed low statistical support. However, and as observed for the phylogenetically closely related sand-dwelling species Laciniporus arabicus (Saburova and Chom erat 2019), its phylogenetic position based on SSU rDNA sequences was unstable and varied depending on the taxon sampling used for phylogenetic inference. This situation is not unusual for benthic sand-dwelling species, which commonly represent diverging lineages clustering unrelated to other known (mostly planktonic) representatives, e.g., Madanidinium adanidinium loirii (Chom erat and Bilien 2014), Apicoporus spp. (Sparmann et al. 2008), or Plagiodinium belizeanum (Wakeman et al. 2018).
LSU rDNA sequences could only be obtained for Pachena abriliae and an unidentified Pachena specimen. However, their phylogenetic placement at the base of the Thoracosphaeraceae was more robust and in agreement with the position obtained in the phylogenetic analysis of SSU rDNA sequences. The molecular phylogenetic data suggest that planktonic Thoracosphaeraceae evolved from benthic taxa like Pachena and Laciniporus.
The Thoracosphaeraceae includes heterotrophic, autotrophic, and parasitic representatives, from marine and freshwater environments, and some showing noncalcareous or calcareous cysts (Elbr€ achter et al. 2008, Gottschling and Soehner 2013, Saburova and Chom erat 2019. Pachena could be the most basal taxon of the Thoracosphaeraceae with a heterotrophic nutrition and possible peduncle feeding, even though we could not verify the peduncle presence or prey source. Furthermore, the inability to establish cultures impeded the observation of cystproduction for Pachena representatives, unknown so far for heterotrophic sand-dwelling species. The sand-dwelling Laciniporus arabicus also occupies a basal position within the clade, and in this case, it is autotrophic and produces non-calcareous cysts. Diversity and biogeography. In this study, cells of a Pachena species were observed in a benthic sample from the German Bight, but they could not be identified to species (M. Hoppenrath, unpub. data). Also, in the Mediterranean Sea, cells were recorded that likely belong to one or two further undescribed species (M. Hoppenrath, A. Reñ e, C.T. Satta, unpub. data). Additional observations and data are needed for their delimitations. Selina (2016) documented a sand-dwelling species from Peter the Great Bay, Sea of Japan, and identified it as cf. Herdmania. Judged from the cell shape, the apical hook, cingulum path, and food body location (Selina 2016, p. 465, fig. 4, k and l) it could be a Pachena species. Thus, the genus shows a wide distribution, and several species probably coexist at the same location. However, the overall distribution of the different species remains to be determined.