First Report of the Marine Benthic Dinoﬂagellate Bysmatrum subsalsum from Korean Tidal Pools

: Dense patches were observed in the tidal pools of the southern area of Korea. To clarify the causative organisms, the cells were collected and their morphological features were examined using light and scanning electron microscopy (SEM). In addition, after establishing strains for the cells the molecular phylogeny was inferred with concatenated small subunit (SSU) and large subunit (LSU) rRNA sequences. The cells were characterized by a nucleus in the hypotheca, strong reticulations in thecal plates, the separation of plates 2a and 3a, the tear-shaped apical pore complex, an elongated rectangular 1a plate and the absence of the right sulcal list. The thecal plate formula was Po, X, 4 (cid:48) , 3a, 7”, 6c, 4S, 5 (cid:48)(cid:48)(cid:48) , 2 (cid:48)(cid:48)(cid:48)(cid:48) . Based on these morphological features, the cells were identiﬁed as Bysmatrum subsalsum. In the culture, the spherical cysts of B. subsalsum without thecal plates were observed. Molecular phylogeny revealed two ribotypes of B. subsalsum are identiﬁed; The Korean isolates were nested within the ribotype B consisting of the isolates from China, Malaysia and the French Atlantic, whereas the ribotype A includes only the isolates from the Mediterranean Sea. In the phylogeny, B. subsalsum and B. austrafrum were grouped. This can be supported by the morphological similarity between the two species, indicating that the two species may be conspeciﬁc, however B. subsalsum may distinguish from B. austrafrum, because of differences in the types of eyespots reported in previous studies. These ﬁndings support the idea that there is cryptic diversity within B. subsalsum.

Since the morphological descriptions of Bysmatrum species by Faust and Steidinger [1], Anglès et al. [7] documented the morphological details and molecular phylogeny of B. subsalsum collected from different locations of the Mediterranean Basin, and concluded that the strains of B. subsalsum are morphologically indistinguishable but genetically distinct, probably due to the differences in habitat, physiology or life-history traits. Since then, Luo et al. [8] documented that B. subsalsum strains from Malaysia and the French Atlantic formed a subclade (ribotype B) that can be distinguished from a clade (ribotype A) consisting of stains from the Mediterranean Sea and that there is cryptic diversity within B. subsalsum, based on the genetic distance shown in ITS sequences among Bysmatrum species [7]. This indicates that additional molecular data (including morphological descriptions) are needed to clarify the cryptic diversity of B. subsalsum, with strains established from water bodies of various geographical regions.
Bysmatrum subsalsum is a cosmopolitan species [10], and its occurrences have been recorded in the Aral Sea, the Mediterranean Sea, the Caribbean Sea, the Gulf of Mexico, the French and Portuguese Atlantic coast and the East and South China Sea [7,8]. However, in the Korean coastal area, this species has not been found, and only B. gregarium (as B. caponii) isolated from plankton samples has been recorded, with its morphology and molecular information [9]. In the tidal pools of the southern area of Korea, we observed dense patches caused by unidentified organisms. These were isolated, and strains were established and the morphological features were examined using light and scanning electron microscopy (SEM). The observations revealed that the species was identical to B. subsalsum. In this study, we describe the morphological details of Korean strains of B. subsalsum and report on their molecular characterization, based on concatenated small subunit (SSU) and large subunit (LSU) rRNA gene sequences.

Sampling and Culture
In June 2017, dense patches of phytoplankton were observed in the tidal pools of Geoje Island (34 • 59 34.3" N, 128 • 41 44.1" E) and Jeju Island (33 • 29 26.5" N, 126 • 25 12.8" E), Korea. Water temperature and salinity were 23.0 • C and 34 psu in the tidal pool of Geoje Island, and 10.5 • C and 24.5 psu in the tidal pool of Jeju Island, respectively. The water samples from the pools were collected using the 50 mL conical tubes and were then transported to the laboratory for observation of the causative organism. In the laboratory, the single mass colonies from the samples were isolated using a glass micropipette on an inverted microscope (Eclipse Ts2R, Nikon, Tokyo, Japan), and transferred into a 24-well tissue plate containing f/2 medium adjusted to a salinity of 32. The isolated colonies were maintained at 24 • C in a 12:12 LD cycle under a photon irradiance of 100 µmol photons·m −2 ·s −1 . After sufficient growth, the cells were transferred to a culture flask. A monoclonal culture was successfully established from the cells collected in the tidal pool of Geoje Island and deposited in the Library of Marine Samples, Korea Institute of Ocean Science and Technology, as strain number LIMS-PS-2685 (=MABIK PD00002006). The isolate from the tidal pool of Jeju Island was also established as a strain; however, it is currently unavailable because of unexpected cell death.
For scanning electron microscopy, a 20 mL aliquot of a dense culture was fixed in glutaraldehyde and paraformaldehyde with a final concentration of 2% (w/v). The aliquot containing fixed cells was filtered through a polycarbonate membrane filter (5 µm pore size), without applying additional pressure and rinsed three times with distilled water to remove the salt. After rinsing, the sample was dehydrated in an ethanol series (10,30,50,70, 90 and 100% ethanol, followed by two 100% ethanol steps) and dried using a critical point dryer (BAL-TEC, CPD 300, Balzers, Germany). Finally, the sample was coated with gold-palladium and examined using a field emission-scanning electron microscope (JSM-7610F, Jeol, Japan).

Phylogenetic Analysis
The sequences were aligned using MAFFT in Geneious plugin with default settings, and some regions manually adjusted using the Geneious Prime. The phylogenetic analysis includes all the published sequences of Bysmatrum species. A total of 50 sequences of the order Peridinales were selected, while the family Peridiniaceae and Protoperidiniaceae were used as outgroups. The sequence alignments of SSU and LSU rRNA were concatenated with introduced gaps. The phylogenetic tree for the concatenated sequence alignment was inferred using maximum likelihood (ML) analyses via RAxML version 8 [13], and using Bayesian inference (BI) through MrBayes version 3.2 [14]. The general time reversible (GTR) model with parameters accounting for γ-distributed rate variation across sites (G) was used in all analyses, taking into account 6-class gamma. The GTR+G substitution model was selected using the Akaike information criterion (AIC) as implemented in jModelTest version 2.1.4 [15]. Bootstrap analyses for ML were carried out with 1000 replicates to evaluate statistical reliability. The Markov chain Monte Carlo (MCMC) method for BI was used with four runs for 10 million generations, sampling every 100 generations. The first 10% of trees were deleted as burn-in, and a majority rule consensus tree was constructed to examine the posterior probabilities of each clade. The final trees were visualized with FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 5 January 2020)).  SEM observation revealed that the cells have a plate formula of Po, Cp, X, 4 , 3a, 7", 6C, 4S, 5 , 2 ( Figure 2). The thecal plates were covered with strong reticulations (Figure 2a-e). Apical pore complex (APC) was tear-shaped and included a Po plate, a round cover plate (Cp) and a canal plate (X) with thick margins formed by the raised borders of the apical plates (Figure 2f). The first apical plate (1 ) was pentagonal and asymmetrical with shorter anterior sutures than the posterior ones, and surrounded five plates: 2 , 4 , 1 , 7 and Sa (Figure 2a,d,e). Three intercalary plates (1a, 2a and 3a) were similar in size, and 1a and 2a contacted each other; however, 3a was separated from 1a and 2a. (Figure 2b,c,e). Plate 1a was elongate and rectangular, whereas plates 2a and 3a were hexagonal and pentagonal, respectively (Figure 2b,c,e). The precingular plates were symmetrically distributed (Figure 2e). The first precingular plate (1 ) was pentagonal and smaller than the others (Figure 2a,e). The cingulum was deeply excavated and descended by about one cingulum width (Figure 2a-d). Six cingulum plates were observed; plates C1, C2 and C3 were much smaller than the C4, C5 and C6 plates (Figure 2a  Spherical cysts were observed under culture conditions (Figure 3). The cysts were 29.5-34.6 µm in diameter (n = 25). The cyst was grayish in color, and an orange accumulation body was visible (Figure 3a-c). The cyst wall was smooth, without any distinguishing features on the surface (Figure 3d). Spherical cysts were observed under culture conditions (Figure 3). The cysts were 29.5-34.6 µm in diameter (n = 25). The cyst was grayish in color, and an orange accumulation body was visible (Figure 3a-c). The cyst wall was smooth, without any distinguishing features on the surface (Figure 3d).

Molecular Phylogeny
Bayesian inference (BI) and maximum likelihood (ML) based on the concatenated SSU and LSU rRNA gene sequences yielded similar phylogenetic trees. The genus Bysmatrum was a monophyletic group, with maximum support (Figure 4). Bysmatrum arenicola was the base of Bysmatrum species, and B. subsalsum was branched from B. gregarium, with moderate supports (BT/PP = 68/0.92). Two ribotypes (ribotype A and B) of B. subsalsum were identified; ribotype A was comprised only of strains from the Mediterranean Sea with maximum support (BT/PP = 100/1.00), whereas ribotype B included the strains from China, Malaysia, the French Atlantic and Korea. Bysmatrum austrafrum (HG321) was nested between the two ribotypes. In ribotype B, the Korean strains of B. subsalsum were closely related to the Malaysian strain (TBBYS03) (BT/PP = 80/1.00).

Molecular Phylogeny
Bayesian inference (BI) and maximum likelihood (ML) based on the concatenated SSU and LSU rRNA gene sequences yielded similar phylogenetic trees. The genus Bysmatrum was a monophyletic group, with maximum support (Figure 4). Bysmatrum arenicola was the base of Bysmatrum species, and B. subsalsum was branched from B. gregarium, with moderate supports (BT/PP = 68/0.92). Two ribotypes (ribotype A and B) of B. subsalsum were identified; ribotype A was comprised only of strains from the Mediterranean Sea with maximum support (BT/PP = 100/1.00), whereas ribotype B included the strains from China, Malaysia, the French Atlantic and Korea. Bysmatrum austrafrum (HG321) was nested between the two ribotypes. In ribotype B, the Korean strains of B. subsalsum were closely related to the Malaysian strain (TBBYS03) (BT/PP = 80/1.00).

Morphological Comparisons of Korean Isolates of Bysmatrum subsalsum with other Isolates of B. Subsalsum, and B. Austrafrum
According to Anglès et al. [7] and Luo et al. [8], the genetic sequences of B. subsalsum show large intraspecific differences, clustering two well-differentiated clades (ribotype A and B). The two clades of B. subsalsum based on SSU and LSU sequences were also shown in this study, and the Korean isolates of B. subsalsum were nested in the ribotype B and clustered with the isolates from China, Malaysia and the French Atlantic (Figure 4). The Korean isolates of B. subsalsum were morphologically characterized by the separation of plates 2a and 3a, the tear-shaped APC, an elongated rectangular 1a plate and the general morphology, such as cell size, plate ornamentation and nucleus position, which coincides with that of B. subsalsum in previous studies (Luo et al. [8] and reference therein). Luo et al. [8] reported the differences in the number of sulcal lists among strains of B. subsalsum in two clades; the French strain has the right sulcal list (Three sucal lists), whereas the Malaysian strains are characterized by the absence of the right sulcal list (Two sulcal lists). In Korean strains, the right sulcal list was not present. In addition, the number of sulcal lists varied among specimens collected from other geographical regions [16][17][18][19]. Anglès et al. [7] concluded that despite a certain degree of morphological variation (such as cell

Morphological Comparisons of Korean Isolates of Bysmatrum subsalsum with Other Isolates of B. subsalsum, and B. austrafrum
According to Anglès et al. [7] and Luo et al. [8], the genetic sequences of B. subsalsum show large intraspecific differences, clustering two well-differentiated clades (ribotype A and B). The two clades of B. subsalsum based on SSU and LSU sequences were also shown in this study, and the Korean isolates of B. subsalsum were nested in the ribotype B and clustered with the isolates from China, Malaysia and the French Atlantic (Figure 4). The Korean isolates of B. subsalsum were morphologically characterized by the separation of plates 2a and 3a, the tear-shaped APC, an elongated rectangular 1a plate and the general morphology, such as cell size, plate ornamentation and nucleus position, which coincides with that of B. subsalsum in previous studies (Luo et al. [8] and reference therein). Luo et al. [8] reported the differences in the number of sulcal lists among strains of B. subsalsum in two clades; the French strain has the right sulcal list (Three sucal lists), whereas the Malaysian strains are characterized by the absence of the right sulcal list (Two sulcal lists). In Korean strains, the right sulcal list was not present. In addition, the number of sulcal lists varied among specimens collected from other geographical regions [16][17][18][19]. Anglès et al. [7] concluded that despite a certain degree of morphological variation (such as cell size, APC morphology and size, and cingulum displacement), cells from the two clades of B. subsalsum exhibit similar morphological characteristics. Luo et al. [8] also confirmed the morphological similarities in the Malaysian and French strains of B. subsalsum. Consequently, the prominent morphological characteristics for clarifying the two clades of B. subsalsum still remain unclear.
Recently, Luo et al. [20] recorded B. austrafrum between two clades of B. subsalsum, with strong support in the phylogeny, which is in agreement with our result. Bysmatrum austrafrum was first described by Dawut et al. [6]. This species has a typical plate of Po, X, 4 , 3a, 7", 6C, 4S, 5 , 2 for the genus Bysmatrum and is morphologically characterized by dimensions of 25-45 µm long and 20-42.5 µm wide, arranged reticulation in the thecal plates, the possession of an equatorially positioned cingulum and a cingulum displaced by a distance exceeding its own width. Based on these morphological features, Dawut et al. [6] reported that B. austrafrum is similar to B. subsalsum, and concluded that B. austrafrum can be distinguished from B. subsalsum by differences in cell size, the shape of the APC and apical plate 1 . However, cell sizes recorded in B. austrafrum were recorded in the other specimens of B. subsalsum (see Table 2 in Luo et al. [8]), and the shapes of the APC and apical plate 1 of B. austrafrum quite resembled those of Korean isolates and the specimens of B. subsalsum recorded by Luo et al. [8]. In addition, although Dawut et al. [6] did not consider the absence or presence of the right sulcal list, B. austrafrum does not have the right sulcal list in their description. Consequently, it is quite difficult to distinguish B. subsalsum from B. austrafrum, based on their morphological features. Nevertheless, different types of eyespots have been reported in B. subsalsum and B. austrafrum [6,8]. Based on the types of eyespots suggested by Moestrup and Daugbjerg [21], B. subsalsum has the Type B eyespot, whereas B. austrafrum presents the Type A eyespot. This may be a useful characteristic for distinguishing B. subsalsum from B. austrafrum. However, as the Type A eyespot has not been reported in other Bysmatrum species, such as B. granulosum and B. gregarium, more isolates of B. austrafrum need to be examined for clarifying the type of eyespot within Bysmatrum species.

Morphology of Bysmatrum subsalsum Cyst
Cyst morphology can be helpful to understand the diversity within the genus (e.g., Li et al. [22]). In Bysmatrum species, a cyst-theca stage relationship has only been established through germination experiments for Mediterranean B. subsalsum [7]. Two types of cysts of B. subsalsum have been described in culture and sediments; in the culture spherical to ovoidal cysts without any ornamentations were observed [7,19], whereas cysts with the typical plate pattern, which are morphologically similar to vegetative cells of B. subsalsum, were identified in sediments [8,23]. In our study, the spherical cysts were also observed from the culture. Bysmatrum subsalsum from the Mediterranean Sea (ribotype A) could produce both cyst types in culture [7,19], and from the French strain of B. subsalsum (ribotype B), only cysts with the thecal plate were described [8]. In previous studies, differences in cyst types have been recorded between cultures and natural sediments. For example, unarmored dinoflagellate Margalefidinium polykrikoides (formerly Cochlodinium polykrikoides) produced two different types of cyst (a spherical cyst without ornamentation in culture and an ornamented cyst in sediments) [24,25]. Tang and Gobler [24] suggested that the ornaments and spines of M. polykrikoides might be caused by biotic or chemical processes in sediments. However, this does not seem to be in accordance with B. subsalsum, because two different types of cysts were reported in sediments.
Similar morphological features between cyst and vegetative cell of unarmored dinoflagellate M. polykrikoides have been reported [26,27], and the cyst was identified as a temporary cyst that can be the short-term stage. The temporary cyst of M. polykrikoides is surrounded by a transparent and thin hyaline membrane, indicating in sediments the unarmored temporary cysts may be destroyed because of geochemical processes related to organic matter degradation or the attack of viruses or bacteria into the cell. However, temporary cysts with thecal plates (armored cyst) (such as cyst of B. subsalsum) may be protected from the environmental conditions in sediments. If so, the cyst with typical thecal plates of B. subsalsum from sediments or culture may be temporary, because the resting cysts of dinoflagellate usually have distinct morphological features that can be distinguished from their vegetative cell (e.g., Matsuoka and Fukuyo [28]).

Phylogenetic Position of Bysmatrum subsalsum
In the phylogenetic tree, the Korean isolates within B. subsalsum were nested in the ribotype B consisting of the isolates from China, Malaysia and the French Atlantic, whereas the ribotype A includes only the isolates from the Mediterranean Sea. Iwataki et al. [29] documented that in the phylogenetic tree for M. polykrikoides isolates, the ribotypes can be useful for characterizing the geographical distribution pattern of M. polykrikoides. In our study, the ribotype A also represents the isolates of B. subsalsum from the Mediterranean Sea, which is in agreement with the conclusion by Iwataki et al. [29]. However, the ribotype B is a mixture of isolates originated from different geographic regions, although it mostly includes the Asian isolates. This indicates that the cells originating from France might be transferred from the Asian areas. Benthic dinoflagellates such as Bysmatrum species usually have a restricted distribution, possibly because of benthic, epiphytic behavior. Nevertheless, B. susalsum has been reported from various samples, including plankton samples, and their resting cysts are also present in the sediments (Luo et al. [8] and reference therein). This suggests that the vegetative cells and resting cysts of B. subsalsum can artificially be introduced into other coastal areas, possibly caused by ballast ship waters (e.g., Hallegraeff [30]).
Since the discovery of two ribotypes of B. subsalsum, Luo et al. [20] identified B. austrafrum between two clades of B. subsalsum in a phylogenetic tree based on concatenated SSU and LSU rDNA sequences, which is in agreement with our result. In the phylogenetic tree, B. subsalsum and B. austrafrum were grouped, with strong support (BT/PP = 95/1.00) (Figure 4), indicating that the two species may be conspecific. This result can be supported by morphological similarity between the two species. However, as there are differences in the types of eyespots between the two species, B. subsalsum can also be a species that distinguished from B. austrafrum. If so, it is possible that B. subsalsum is not monophyletic. This finding supports the idea that there is cryptic diversity within B. subsalsum [7].

Environmental Conditions in Relation to the Growth of Bysmtrum subsalsum
Although Bysmatrum species are considered benthic dinoflagellates, the occurrence of B. subsalsum has been reported in the plankton, with floating detritus and on sand and macroalgae [18]. Luo et al. [8] recorded B. subsalsum in plankton and sediment samples, and Anglès et al. [7] found B. subsalsum in mangrove detritus and salt marshes. In Korean isolates of B. subsalsum, the species is considered to inhabit tidal pools, and B. austrafrum was also discovered in tidal pools in South Africa [6]. In comparison with B. subsalsum, other Bysmatrum species, such as B. teres, B. granulosum, B. arenicola and B. gregarium have been reported in the restricted habitats (Luo et al. [8] and reference therein). This indicates that B. subsalsum in contrast to other Bysmatrum, may have broad environmental tolerance and a worldwide distribution.
Environmental conditions in relation to the growth of B. subsalsum have been rarely reported. Anglès et al. [7] documented that, based on the occurrence of B. subsalsum reported in previous studies, the species has a wide salinity tolerance and is able to grow under a wide range of sea water temperatures, with a preference for salinities > 30 and temperatures > 20 • C. López-Flores et al. [31] recorded that the cell abundances of B. subsalsum (as Scrippsiella subsalsa) decreased sharply in October, when salinity reached values > 30 and water temperature was low (15.6 • C). In this study, however, the dense patch of B. subsalsum was observed at 10.5 • C, indicating that it has a wider temperature tolerance than previously known. Further studies are also needed to clarifying the morphology (vegetative cell and cyst) of the genus Bysmatrum and, in particular, of B. subsalsum.