Boreal Tintinnid Assemblage in the Northwest Pacific and Its Connection with the Japan Sea in Summer 2014

Tintinnids are planktonic ciliates that play an important role in marine ecosystem. According to their distribution in the world oceans, tintinnid genera were divided into several biogeographical types such as boreal, warm water, austral and neritic. Therefore, the oceanic tintinnid assemblage could be correspondingly divided into boreal assemblage, warm water assemblage and austral assemblage. The purpose of this study was to investigate the characteristics of boreal tintinnid assemblage in the Northwest Pacific and the Arctic, and to identify the connection between boreal tintinnid assemblage and neighboring assemblages. Surface water samples were collected along a transect from the East China Sea to the Chukchi Sea in summer 2014. According to the presence of boreal genera and warm water genera, three tintinnid assemblages (the East China Sea neritic assemblage, the Japan Sea warm water assemblage, and the boreal assemblage) were identified along the transect. The boreal assemblage extended from the Chukchi Sea to the waters north of the Sōya Strait. Densities peaks occurred at stations in the two branches of the Alaska Current and decreased both northward and southward. The densities were <10 ind./dm3 at most stations in Arctic region. The dominant genera (Acanthostomella, Codonellopsis, Parafavella, and Ptychocylis) accounted for 79.07±29.67% (n = 49) of the abundance in the boreal assemblage. The densities of the dominant genera covaried with strongly significant positive correlations. Tintinnids with lorica oral diameter of 22–26 μm and 38–42 μm were dominant and contributed 67.35% and 15.13%, respectively, to the total abundance in the boreal assemblage. The distribution and densities of tintinnids in the study area suggest that the Sōya Strait might be a geographical barrier for tintinnids expansion.

Introduction along the transect, the distances between the adjoining stations were in the range of 60-480 km (on average 180±90 km, n = 67, Fig 1). No specific permissions were required to collect water samples because the stations were located in international waters. The field studies did not involve or impact on any endangered or protected species. The stations in the Japan Sea were in the path of the Tsushima Current, a branch of the Kuroshio Current, which influences the East China Sea and the Japan Sea, while the stations north of the Sōya Strait were influenced by two major North Pacific currents (the Oyashio Current and the Alaska Current) and currents flowing from the Bering Sea to the Chukchi Sea [19][20][21][22].
At each station, 80 dm 3 of surface (5 m depth) seawater was collected using an underway sampling system. The water was then gently filtered through a small net (mesh pore size 10 μm). The samples in the cod end of the net (about150 cm 3 ) were transferred into sample bottles and immediately fixed with Lugol's solution (1% final concentration). The samples were placed at cool, dark environment for preservation. At the same time, the temperature and salinity of the surface seawater and chlorophyll a (Chl a) concentration were measured using a CTD sampling system (Seabird 21).
In the laboratory, one subsample (25 cm 3 or a larger volume if tintinnids were scarce) from each concentrated sample was settled in an Utermöhl counting chamber for at least 24 h and examined using an Olympus IX 71 inverted microscope (100× or 400×). At least 20 individuals (if possible) of each species were photographed and measured. Tintinnid species were identified based on lorica morphology and size according to the literatures [23][24][25]. SPSS version 16 statistical software was used to perform a correlation analysis.  [19], Longhurst [20], Senjyu [21] and Steele et al. [22].  (Fig 2).The surface water salinity increased gradually from the East China Sea to the Japan Sea, and then remained at a high level (30.88-34.66) between the Japan Sea and the Bering Strait. The salinity dropped at St. 53 and remained at a low level (<27.38) in the Chukchi Sea (Fig 2). According to the T-S diagram, the water mass along the transect could be divided into East China Sea Water, Japan Sea Water, Subarctic Pacific Water and Coastal Arctic Water. St. 1-St. 3 located in East China Sea Water with high temperature but the salinity was relatively low. The stations between St. 4 and St. 15 located in the Japan Water with high temperature and high salinity. St. 53-St. 68 located in the Coastal Arctic Water with low temperature and low salinity (Fig 3).
The surface Chl a concentration ranged from 0.06 μg/dm 3

Species richness, densities of tintinnids and their relationship with environmental factors
In total, 54 tintinnid species from 21 genera were identified (Figs 4 and 5; Table 1). There was an undetermined species ( Fig 5) with hyaline, cylindro-conical in shape, and open at both ends. The lorica length was about 44.88±3.97 μm, the LOD was 10.70±1.19 μm, and the aboral diameter was 3.62±0.77 μm (n = 20). We discussed it as a separate undetermined species and genus in this study. Thirty three species occurred at less than five stations and most (22) of them had low densities (<1 ind./dm 3 ; Table 1).
The tintinnid species richness at individual stations ranged from 0 to 12, and was relatively high (3-12 species per station) in the northeastern East China Sea, the southwestern Japan Sea, and the northern Bering Sea. Tintinnid species richness declined after St. 55 (<4 species per station; Fig 2). Correlation analysis identified significant positive correlations between tintinnid species richness and densities and temperature, salinity, and Chl a concentration (all P<0.01; Table 2). Tintinnid species richness and densities were very low when the temperature was <5°C, but were high when the temperature was between 8°C and 14°C. Tintinnid species richness and densities increased as salinity increased. Tintinnid species richness initially increased with an increase in Chl a concentration and then stabilized, while densities increased steadily as Chl a concentration increased (Fig 6).

Distribution patterns of tintinnid genera
Among the 21 tintinnid genera, there were 2 boreal genera, 8 cosmopolitan genera, 4 neritic, and 3 warm water genera. Two genera and the undetermined species were not grouped into any biogeographical type ( Table 1). The occurrence of each tintinnid genus by station was shown in Fig 7. Five genera (Ascampbelliella, Climacocylis, Coxliella, Protorhabdonella, and Stenosemella) were found at only one or two stations and their densities were <1 ind./dm 3 (Table 1). Density distribution of other genera were shown in Fig 8. Proplectella and Rhabdonella were warm water genera, Proplectella contained one species (Proplectella expolita) and Rhabdonella contained two species (Rhabdonella cornucopia and Rhabdonella sp.). They occurred at stations in the Japan Sea (Fig 8).   Favela, Leprotintinnus, Helicostomella and Tintinnopsis were neritic genera. Favella occurred at 4 stations in the Japan Sea and the East China Sea. Leprotintinnus and Helicostomella occurred at coastal stations east of the Kamchatka Peninsula and the Chukchi Sea. Tintinnopsis was found at coastal stations along the whole transect (Fig 8).
Parafavella and Ptychocylis belonged to boreal genera. Ptychocylis obtusa was the only species in genus Ptychocylis. It occurred north of the Sōya Strait (St. ). Nine tintinnid species of the genus Parafavella were identified. The distribution of Parafavella faceta and P. jorgenseni was similar. Both species were first detected at St. 17. Their densities initially increased along the transect, then decreased in the Arctic. P. elegans occurred in the Bering Strait with low densities of <0.66 ind./dm 3 (Table 1). P. denticulata occurred between St. 28 and St. 40 where most tintinnid species of the genus Parafavella were highly abundant. Most species in Parafavella only occurred at stations north of the Sōya Strait with the exception that P. pacifica was only found in the northern Japan Sea (Fig 9).

Tintinnid assemblages division
We used the occurrence of boreal genera and warm water genera as objective criteria of tintinnid assemblage division.   Neither warm water genera nor boreal genera tintinnid was identified at the two stations (St. 1 and St. 2) in the East China Sea. We considered the tintinnid assemblage at these two stations as East China Sea neritic assemblage. Warm water genera occurred in the Japan Sea (St. 4 to St. 15). At the stations between St. 11 and St. 15, warm water genera and P. pacifica belonging to boreal genera mixed. In the T-S diagram, these stations belong to the Japan Sea Water with high temperature (Fig 3). Therefore the tintinnid assemblage at St. 4 to St. 15 was

Characteristics of boreal assemblage
The boreal assemblage covered the Subarctic Pacific Water and Coastal Arctic Water and has a difference in densities in the two waters. Total densities decreased sharply at stations in the Coastal Arctic Water and was <10 ind./dm 3 at most stations north of the St. 49 (Figs 2 and 10).   (Fig 11).
With maximum densities of 478.09, 445.16, 229.87, and 59.88 ind./dm 3 , respectively, the genera Acanthostomella, Codonellopsis, Parafavella, Ptychocylis, had higher maximum densities than other genera. On average, they contributed 79.07±29.67% (n = 49) to the abundance in the boreal assemblage (Fig 12). They also had consecutive distributions. The distribution patterns of Acanthostomella, Codonellopsis, Parafavella, Ptychocylis, and the undetermined species were similar. They centered in the Bering Sea with extensions both southward and northward. Their high densities centered in the Bering Sea and north of the Okhotsk Sea (St .18-St. 41) and decreased both southward and northward. The undetermined species occurred between St. 16 and St. 47 with densities of 0.27-23.43 ind./dm 3 (Fig 8). The correlations among densities of them were strongly significant positive (all P<0.01; Table 3; Fig 13).
According to whether neritic genera tintinnid occurred, the boreal assemblage could be divided into two parts: boreal assemblage without neritic genera (B1 assemblage) and boreal assemblage with neritic genera (B2 assemblage). There were 12 species with 7 LOD size-classes in B1 assemblage, and these species were considered as core boreal species. In the B1 assemblage, two species (A. norvegica and C. frigida) with a LOD of 22-26 μm accounted for 69.65% (49.16% and 20.49%, respectively) of the total abundance. The second highest contributor to abundance (13.16%) was P. jorgenseni with a LOD of 38-42 μm (Fig 11). Core boreal species had high species richness at stations between St. 29 and St. 42, and decreased northward and southward, and extremely low in Arctic region (Fig 10).
Twenty nine species with 14 LOD size-classes were found in B2 assemblage. Among them, 11 species were core boreal species. Eighteen other species (added species) occurred in B2 assemblage, including 12, 4, and 1 species belong to neritic, cosmopolitan, and boreal genera, respectively. Though the number of species and LOD size-classes increased in the B2 assemblage, the most abundant species in B2 assemblage were similar with those in B1 assemblage: species in LOD of 22-26 μm and 38-42 μm contributed to 63.74% and 18.22% to total abundance, respectively (Fig 11). The added species increased species richness in B2 assemblage, especially at stations in the Coastal Arctic Water. However, compared with core boreal species, densities of the added species were low and made up a small portion of total abundance at most stations in B2 assemblage. High portion mainly occurred at stations in the Arctic water where the total densities were low (Fig 10). If the core species were excluded, the added species with LOD size-classes of 34-38 μm, 18-22 μm, and 30-34 μm were dominant and accounted for 43.88%, 25.51%, and 16.51% of abundance in B2 assemblage, respectively (Fig 11).

Hydrography and assemblages division
The division of tintinnid assemblage corresponded well with water mass division by T-S diagram. The East China Sea neritic assemblage and the Japan Sea warm water assemblage located in the East China Sea and the Japan Sea, respectively. The boreal assemblage was in the waters north of the Sōya Strait where the T-S diagram identified two water masses (Subarctic Pacific Water and Coastal Arctic Water). The transect in this study passed through the following biological provinces: the North Pacific Epicontinental Sea Province (the Bering Sea and the Okhotsk Sea), the Pacific Subarctic Gyre Province, the Kuroshio Current Province (the Japan Sea), and the China Sea Coastal Province [20,26]. Our division of the East China Sea neritic assemblage and the Japan Sea warm water assemblage corresponded with the China Sea Coastal Province and the Kuroshio Current Province, respectively. The boreal assemblage in this research covered the North Pacific Epicontinental Sea Province and the Pacific Subarctic Gyre Province [20,26]. Our result was in consistent with Priede [26] who described the region north of the westerly current in the Northern Hemisphereas the polar (or boreal) biome. In the present study we used the term boreal to describe the assemblages in this biome.
The Japan Sea warm water assemblage was at the brim of the warm water assemblage. The East China Sea neritic assemblage only had 2 stations. Therefore these two assemblages in our study were not typical. The transect covered the boreal assemblage from its north end to south end. Our research is the first study of the boreal assemblage in a transect across the assemblage.

Characteristics of the boreal assemblage
There were very few previous studies of tintinnids in the Arctic [17,18] and subarctic Pacific [17] regions. The study in subarctic Pacific [17] region only gave total densities and species names of dominant species in 15 stations.
The densities of tintinnids in the boreal assemblage had a sharp decrease from the Subarctic Pacific Water to Coastal Arctic Water (Figs 2 and 10). This decrease in densities were consistent with findings of previous studies [17,18]. Meanwhile, all tintinnid species occurred in the Chukchi Sea had been reported at other areas other than Arctic [17,18]. Therefore tintinnids might intrude into the Chukchi Sea through the Bering Strait by the help of current from the Bering Sea [19]. But most tintinnids cannot adapt to the extreme environment in the Chukchi Sea, this caused the low densities and tintinnid species richness in the Arctic region [17]. This also proved that there was no indigenous tintinnid species in the Arctic Ocean [17]. This is different from the case in Antarctic. Some species in genera Cymatocylis, laackmanniella, and codonellopsis only distributed in Antarctic region south of 58°S, and were considered as Antarctic indigenous tintinnids [10].
The positions of the high densities (St. ) were corresponding with the two branches of the Alaska Current located in the Bering Sea. But we did not have current data, we were not sure whether the high densities were related with the current branches at the time of sampling. Previous study showed that tintinnid destiny was high in the area where water masses mixed [12]. Therefore we thought the high densities were caused by the mixing of branches of the Alaska Current and the Oyashio Current.
It was reported that Ptychocylis urnula or Salpingella faurei was the most abundant species in Arctic region [18], while the species in genera Acanthostomella, Codonellopsis, Ptychocylis, Parafavella, and Tintinnopsis were the most abundant in subarctic Pacific [17]. The similar abundant genera were found in our study with Acanthostomella, Codonellopsis, Ptychocylis, and Parafavella accounted for 79.07±29.67% (n = 49) of abundance at stations in the boreal assemblage (Fig 12).
One undetermined species that has not been reported in previous study was found in this study. According to its lorica characteristics, it may belong to the genus Salpingella. But it was different with S. acuminata or S. faurei that had been reported in the Arctic [18], and subarctic regions [17].
The distribution patterns of Acanthostomella, Codonellopsis, Parafavella, Ptychocylis, and the undetermined species were similar (Fig 13). Those genera was similar to "species group" proposed by Fager and Mcgowan [27]. Their positive correlation might be because that they had similar reactions to properties of the environment.
LOD is a valuable characteristic of tintinnid feeding activity. The maximal prey size ingested is about 45% of the LOD, while the preferred prey size is about 25% of the LOD [4]. Species in two LOD size-classes (22-26 and 38-42 μm) contributed 82.81% to the total abundance in B1 assemblage (Fig 11). This means that the prey size was limited to a small number of size-classes. Species in those two LOD size-classes were still the main contributors to total abundance in B2 assemblage, but the number of LOD size-classes increased. This indicated that the added species in B2 assemblage had different prey size-classes with the core species in boreal assemblage.
Boreal assemblage in our study was at the downstream of the Alaska Current, the characteristics of the assemblage in this transect should also apply to the area in the east part of the Alaska Current and to the entire Bering Sea.

Obstruction of the Sōya Strait to tintinnid expansion
The Sōya Strait is about 40 km long and 20-40 m deep. The boreal assemblage and the Japan Sea warm water assemblage were separated by the Sōya Strait, which might be a natural geographic barrier for tintinnid species. Species including Dadayiella ganymedes, Eutintinnus stramentus, and Proplectella expolita were identified in the Japan Sea but were not found north of the Sōya Strait. Acanthostomella norvegica and Codonellopsis frigida mainly distributed north of the Sōya Strait. S. faurei was reported to be the most abundant species in the Arctic region [18]. In this study, it was identified in areas south of the Japan Sea and in the Northwest Pacific, but were not found in the northern Japan Sea or the Okhotsk Sea. We did not know the connection of the two distribution areas.
Lorica morphology of some tintinnid species might vary following the environmental change, especially species in genus Parafavella [28]. The variation of tintinnid lorica morphology made species classification inaccurate just based on lorica morphology. In our study, Parafavella pacifica was the only species in genus Parafavella occurred in the Japan Sea warm water assemblage. It may be an intraspecific variation of the species north of the Sōya Strait. The lorica shape changed to adapt to the Japan Sea environment.
As the Sōya Strait is a natural geographic barrier, it was not possible to observe mixing between warm water and boreal tintinnid assemblages where the Oyashio and Kuroshio Currents meet. There are limited biological data available for this transitional zone created by the mixing of the Oyashio and Kuroshio Currents [29]. D. ganymedes was distributed the farthest north from the East China Sea into the Japan Sea, whereas Steenstrupiella spp. were confined to the south. This pattern was consistent with the intrusion of the Kuroshio Current into the East China Sea [30]. Therefore, we suggest that D. ganymedes is the most likely species to exist in the region east of Japan where the Kuroshio and Oyashio Currents meet, whereas Steenstrupiella spp. are likely to be the first to disappear.

Conclusions
With large space coverage and high resolution in station distribution, we identified three tintinnid assemblages (East China Sea neritic, Japan Sea warm water, and boreal assemblages) along the study transect. The tintinnid assemblage north of the Sōya Strait was boreal assemblage. There were peaks in densities at stations approximately between the two branches of the Alaska Current. The densities decreased both northward and southward. In Arctic region, the densities were extremely low (<10 ind./dm 3 at most stations). Acanthostomella, Codonellopsis, Parafavella, and Ptychocylis were the dominant genera in this boreal assemblage. There were strong positive correlations between the densities of these four genera. In coastal waters, more species occurred and made the boreal assemblage much more complex both in tintinnid composition and LOD size-classes. The Sōya Strait forms a physical barrier between the boreal assemblage and the Japan Sea warm water assemblage.
Supporting Information S1 File. All original data. (XLS)