Distribution of living coccolithophores in eastern Indian Ocean during spring intermonsoon

We studied the biodiversity of autotrophic calcareous coccolithophore assemblages at 30 locations in the Eastern Equatorial Indian Ocean (EEIO) (80°–94°E, 6°N–5°S) and evaluated the importance of regional hydrology. We documented 26 species based on the identification of coccospheres and coccoliths, respectively. The coccolithophore community was dominated by Gephyrocapsa oceanica, Emiliania huxleyi, Florisphaera profunda, Umbilicosphaera sibogae, and Helicosphaera carteri. The abundance of coccoliths and coccospheres ranged from 0.2 × 103 to 160 × 103 coccoliths l−1 and 0.2 × 103 to 68 × 103 cells l−1, averaged 23 × 103 coccoliths l−1 and 9.4 × 103 cells l−1, respectively. Biogenic PIC, POC, and rain ratio mean values were 0.50 μgC l−1, 1.047 μgC l−1, and 0.10 respectively. High abundances of both coccoliths and coccospheres in the surface ocean layer occurred on the north of the equator. Vertically, the great majority of coccoliths and coccospheres were concentrated in water taken from depths of <75 m. The ratios between the number of coccospheres and free coccoliths indicated that coccoliths experience different levels of dissolution when transported to deep water. Abundant coccolithophores mainly occurred at the west of 90°E, which is in accordance with the presence of Wyrtki jets. Patterns of coccolithosphores and of coccoliths have been reflected in hydrological processes.

Coccolithophores are thrived in the photic water column. They are the unicellular microalgal flagellates with diverse life cycles that (alternating diploid -haploid stage) belongs to marine nanoplankton 1,2 . Life phase transitions can easily occur in natural assemblages when nutrient level changes 3 . The coccolithophore cell is surrounded by one to several layers of coccoliths. Coccolithophores are globally distributed and contribute up to 10% of the global phytoplankton biomass [4][5][6][7][8][9] . In its dual functions of biomineralization and photoautotrophy, the coccolithophore community influences the global carbon cycle, sulphur cycle and other oceanographic parameters 3,10 . Inorganic calcareous coccoliths can serve as a ballast for organic carbon sequestration in the deep ocean [11][12][13] . As a consequence, the PIC/POC (particulate inorganic carbon to particulate organic carbon = "rain ratio"), is a factor modulating the biomineralization on the export of organic production. Coccolithophore assemblages are sensitive to climate variability 14,15 . The increased concentration of CO 2 used to combined with other factors (e.g., nutrient elements, pH, irradiance, temperature) and stimulate the fixation of cell organic carbon by photosynthesis, thus the effect diminishing the rain ratio of coccolithophores [16][17][18][19] . These calcifying nanoplankton are negatively affected by ocean acidification with decreased availability of carbonate, especially in colder water realms 20,21 . The response of coccolithophore ecophysiology to environmental change has aroused much concern 22 . When detached coccoliths are exported to the deep sediment, they provide an ideal tool to record paleoenvironmental change, e.g. sea-surface temperatures, mixed layers and nutriclines 6,[23][24][25] . Coccolithophore geographical distributions interact with physicochemical characteristics, thus making them useful in paleoenvironmental sediment SCientifiC RepORTS | (2018) 8:12488 | DOI: 10.1038/s41598-018-29688-w records − 26 . Coccolithophore community structure and ecological distributions in the Atlantic Ocean have been documented by Brown and Yoder 5 , Baumann et al. 27 , Kinkel et al. 28 , and Shutler et al. 29 . Pacific Ocean studies have included by 9McIntyre et al. 30 , Okada and Honjo 31,32 , Okada and McIntyre 33 , Houghton and Guptha 34 , Saavedra-Pellitero et al. 35,36 , and López-Fuerte et al. 37 .
The Indian Ocean is the world's third largest ocean basin, and it is strongly influenced by the South Asian monsoon system. The warm seawater area in the eastern equatorial Indian Ocean (EEIO) is a large region that influences worldwide climatology and El Niño/Southern Oscillation (ENSO) events 38,39 . The Indian Ocean dipole is another oceanic phenomenon influencing global oceanographic circulation 40 . Surface currents in the EEIO are seasonally dynamic due to the monsoon forces. Unlike most other ocean basins, the Indian Ocean experiences semiannual reversal of prevailing currents 41,42 . Many prevailing currents, however, persist in the EEIO during the summer and winter monsoon periods. These include the Equatorial undercurrent and the South Java Current 39,43 . Ocean currents also can exist throughout the year. One example is the Indonesian Through Flow (ITF), which is the passageway connecting the Pacific Ocean and Indian Ocean 44 . In the spring and fall inter monsoon periods, many surface circulations disappear, and Wyrtki jets (WJs) are the only semi-annual currents present at the equator. The equatorial Indian Ocean is controlled by the eastward WJs (also known as Equatorial Jets) 45 .
Recently the studies on coccolithophores in the Indian Ocean have been relatively compared in Atlantic and Pacific Ocean studies. In the Indian Ocean the studies of coccolihophore have been made by Young 46 52 , about the nanofossil or living species biogeography in the monsoon season. Relatively few studies have evaluated the occurrence of living coccolithophores in the water column during the intermonsoon period in the eastern Indian Ocean. Our three main objectives were to (1) document the abundance, diversity and geographical patterns of living coccolithophores; (2) explain the variations occurring in nano flora assemblages; (3) correlate these variations to regional hydrographic parameters.

Results
Hydrographic features. The present investigation area is crossed by diverse hydrographic gradients as seen from the vertical profiles of temperature and salinity (data not shown). The temperature increased southwards along longitudinal section (Fig. 1a). Notably, there was an interesting phenomenon at St. I306 with the lowest temperature and highest salinity. High temperature and highly saline waters from the west equatorial zone were advected into the east equatorial zone (Fig. 1a,b). The temperature-salinity (T-S) curve can be divided into three regions: high temperature & low salinity surface water, intermediate temperature & salinity water, and low temperature & high salinity deep water (Fig. 1c). During the spring monsoon transition period, the water column was well stratified and quite stable, which is mainly attributed to weak wind-driven surface circulation compared to the monsoon period. Due to the well-stratified water column, the spring intermonsoon was considered to be the most oligotrophic period 53 .   Tables S2 and S3).
Distribution and diversity pattern. The H′ and J values for coccospheres were slightly higher than the corresponding values of coccoliths ( Supplementary Fig. S10). The horizontal distributions of dominant coccoliths and coccospheres were shown in Supplementary Figs S2 and 3. The greatest abundance of coccolith was noticed in three regions: south of Sri Lanka, easternmost Sri Lanka, and southernmost area. There was a peculiar oceanographic phenomenon at St. I316 characterized by surface lowest temperature and highest salinity, where the coccoliths of U. sibogae and H. carteri were predominant. Abundance was relatively low in the equatorial region. In contrast to the coccoliths, coccospheres were more homogeneous in their horizontal distributions (Fig. 2).  Fig. S6). Most of them reached peak values at the 50 m water layer, except for E. huxleyi and H. carteri, which peak values were located in the 200 m and 100 m water layers. Coccosphere were increased from the surface towards the middle water and then decreased towards the bottom water (Sup. Fig. S7). The ratios between coccospheres and free coccoliths were charted vertically through the depth profiles (Fig. 4). The ratio values basically coincided with coccosphere abundance. The ratio reached a maximum at 40 m layer along sections A and C. The ratio along section B exhibited a differed trend and its maximum was present at the surface layer. The ratio along section D was similar to that along section C. We presumed that coccospheres disintegrated into coccoliths after sinking at a short distance, then the coccoliths dissolved as the depths increased to about 100 m and the pH decreased. The ratio decreased to its minimum, 0.03 at a depth of 200 m, where attenuation of photosynthetically active radiation is estimated to have been 1%, which is unfavourable for the coccosphere proliferation. PIC, POC, and rain ratios. The mean PIC, POC, and rain ratios were 0.002~10.008, 0.498 μgC l − , 0.001~6.100, 1.047 μgC l −1 , and 0.093~9.439, 0.990, respectively. The surface distributions and depth-integrated patterns of PIC, POC, and rain ratio were shown in Supplementary Fig. S8. We found a dominance of Oolithotus fragilis and G. oceanica in the biogenic PIC. Unlike PIC, POC was mainly contributed by cells of U. sibogae and U. irregularis. The pattern of PIC and POC appeared to be similar. The surface water around Sri Lanka section displayed two peaks. In the case of the integral value, PIC and POC were preferentially distributed to the west of 90°E. The depth averaged-rain ratio peak occurred at 80°E-85°E (Sup. Fig. S8 55,56 . ANOSIM analysis revealed remarkable difference (Global R = 0.85, p = 0.001) among group classification with the exception of Group b-d and Group c-d whose R value < p value 57 . It is accepted that Global R-value larger than 0.5 accounts for significant difference among groups 58 . Apparently, localities were basically classified along transects (e.g. Group c included the equatorial localities), whereas some exceptions existed (Fig. 5). Besides, MDS bubble plots for first six dominant coccospheres were presented in Fig. 5. It is apparently stated that the Groups a and b were mainly composed by dominant coccosphere G. oceanica, F. profunda, E. huxleyi and A. robusta. While Group c was primarily contributed by species U. sibogae and U. irregularis. Considering Group d only contained two localities, G. oceanica dominated the whole group. The SIMPER results were shown in Supplementary  Table S4. It showed that the contribution rate of the dominant was coccospheres in each group.

Discussion
The surface water of eastern Sri Lanka (around St. I 104 A) had the greatest coccolith and coccosphere richness and abundance. The biodiversity indices were much lower around the waters of Sri Lanka (Sup. Fig. S9), suggesting that the local water in that system has lacked ecosystem stability. Therefore, coccosphere aggregations exhibited more diversity than coccoliths. This finding was consistent with that of Guptha et al. 6 . The physical distributions of coccolithophore assemblages in relation to the temperature-salinity were also shown (Figs 6 and 7). The coccoliths represented by G. oceanica, U. sibogae, H. carteri and H. hyalina were concentrated in the surface layer characterized by high temperature and low salinity. Furthermore, E. huxleyi was predominantly distributed in the intermediate layer with moderate temperature and salinity. The coccospheres, F. profunda and E. huxleyi were mainly found in the deeper euphotic layer where the DCM was located. U. irregularis and U. sibogae has greater abundances in the surface layer, confirming their preference for oligotrophic conditions. The peculiar oceanographic phenomenon at St. I316, characterized by the lowest surface temperature and the highest surface salinity, was occupied predominately by coccoliths of U. sibogae and H. carteri (Sup. Fig. S2). F. profunda was distributed only below 50 m at St. I316, indicating a stratified and stable water locally. This peculiar hydrology was therefore not caused by upwelling but may have been produced by lateral advection. It is very hard to identify what kinds of currents created this peculiar biophysical distribution after all, water currents are not prosperous during the intermonsoon. The POC pattern can be represented by coccosphere abundance. Varied allocation to calcification produced dissimilarities in the PIC/POC ratios. Large rain ratio values around Sri Lanka waters predicted a mineral ballast with a strong drawdown of biological carbon towards the deep seafloor 59,60 . We suggest that the rain ratio is of great importance in predicting biomineralization and photosynthetic production 12,61 .
Many coccolithophore indicator species were collected in this study although several were uncommon. G. oceanica is a representative dominant species that shows a preference for eutrophic water 62 . In the surface distribution of G. oceanica, both coccoliths and coccospheres were predominantly distributed in the easternmost waters of Sri Lanka. This may be due to the eutrophic water derived from the highly productive Andaman Sea which was linked to the Bay of Bengal through narrow channels 63,64 . The coccosphere of U. irregularis was only common in the equatorial zone, indicating oligotrophic water conditions overthere 65 . In the Indian Ocean, eight species of Florisphaera were discovered in deep water 66 . We found only one species of Florisphaera (F. profunda) and were typically occurred in the disphotic layer below at 100 m. As an inhabitant of deep water, F. profunda hardly occurred on the surface water layer unless associated with upwelling. Maxima of among the coccoliths of U. sibogae and H. carteri were found at St. I316 suggesting that these species showed affinities to low temperature and high salinity in water. The cosmopolitan taxa, Calcidiscus leptoporus, was detected and its coccoliths has peaked at a depth of 200 m at St. I705. C. leptoporus is sparsely distributed in the water column, whereas it predominates in the coccolithophore flora of the sediment owing to its resistance to disintegration 67 . Biogenic coccoliths are considered as an important carbon sink and experience different levels of dissolution in the context of varied hydrological condition 68 .
Coccolithophore abundance was relatively low during the low wind transition period compared to previous studies conducted during the monsoon period in the EEIO. The low abundance is due to the gentle associated with light winds and low nutrient availability during the spring intermonsoon season leading to low primary productivity and biomass in the EEIO 69 . The coccolithophores in surface water were most abundant in the northeast area where pockets of low-salinity water plume occur (Fig. 1). This resulted from the inflow of less saline water into the equatorial Indian Ocean from the Bay of the Bengal and Andaman Seas 70,71 . The outflows derived from the surface water of the Andaman Sea become concentrated between the south Nicobar Islands and Sumatra 72 . In contrast, a highly saline water tongue was observed along the equatorial Indian Ocean (west of 90°E), indicating that Wyrtki jets (WJs) prevailed during the spring intermonsoon period. There was consistency in the coccolithophore distribution pattern at the equator. The maximum abundance along the section west of 90°E was probably caused by inflow from WJs considering their ability to alter the oceanic layer structure. PCA was carried out to examine the relationships among the environmental variables, with the most abundant coccolithophores superposed on the PC1-PC2 hyperspace (Fig. 7). Coccolithophore abundance was driven primarily by temperature, salinity, density. The abundance of coccolithophorid phytoplankton will usually correspond to high Chla levels. The clustering of environmental data from sample locations reflected the grouping of species data (except for a few isolated points). The most abundant species were shown above each locality symbol (Fig. 7). The first three principal components (PC1, PC2, PC3) were extracted based on eigenvalues larger than 1 and explained 42%, 24%, and 20.2% of the variation, respectively. The cumulative variances of the three components were reached up to 86.2% (PC3 not shown). The eigenvectors of all five principal components were shown in Supplementary Table S5. The results of PCA indicated that salinity, density, and pico-Chla has a positive relationship with PC1, whereas a close correlation occurred in Group B that was dominated by E. huxleyi and G. oceanica. Similarly, temperature, Chla, micro-Chla and nano-Chla were positively correlated to PC2. Groups C and D, characterized by U. irregularis, were associated with high temperature. The majority of localities in Group

Conclusions
The coccolithophore assemblage in the EEIO during the spring intermonsoon season was primarily comprised of the coccoliths (in order of mean abundance) such as G. oceanica, E. huxleyi, U. sibogae, H. carteri, and H. hyalina and the coccospheres F. profunda, G. oceanica, E. huxleyi, U. irregularis, and U. sibogae based on dominance index. The abundance of coccoliths and coccospheres ranged from 0.19 × 10 3~1 61 × 10 3 coccoliths l −1 and 0.19 × 10 3~6 8 × 10 3 cells l −1 , with an average value of 23. × 10 3 coccoliths l −1 and 9.4 × 10 3 cells l −1 , respectively. The mean values of the biogenic PIC, POC, and the rain ratio were 0.50 μg C l −1 , 1.0 μg C l −1 , and 0.10, respectively. From the ratio of coccosphere and free coccolith, we can see that coccolith experienced different levels of dissolution when transported to the deep water. The rain ratio was considered to be of great importance in predicting biomineralization and photosynthetic production so relative biovolume and carbon biomass were calculated and used to derive the values of PIC, POC and rain ratio.
The horizontal distributions of coccolithophores exhibited three patches: south of Sri Lanka, easternmost Sri Lanka, and southernmost area. An unusual phenomenon was observed at the surface water of St. I316. Vertically, coccoliths abundance was restricted to the water column west of 90°E, exactly consistent with WJs appearance region. The localities and coccosphere were ordered by MDS and all samples were clustered into four groups in the EEIO. The coccolithophore abundance in this study was relatively low and resulting from the weak winds and minimal nutrient upwelling compared to previous studies that were conducted during the summer or winter monsoon seasons. During the spring intermonsoon period, no significant oceanic circulation occurred in the EEIO except for WJs. We inferred that, in the study area, different coccolithophore species had specific environmental preferences. Thus, coccolithophore species are good indicators of oceanographic changes in the EEIO. PCA was used to study the correlation between environmental variables, indicating positive or negative relationships with nanofloral species. Coccosphere distribution was highly correlated to specific environmental variables. This was shown by the MDS ordination of response variables and PCA ordination of explanatory variables. Coccolithophores can be used as dynamic indicators of the upper ocean for their sensitivity to environmental changes. Obtaining knowledge of specific cellular physiological behaviour related to global change variables will be a future challenge Future studies are required involving laboratory experiments using axenic cultures of coccolithophores, and cell POC and other chemical parameters need to be measured to refine existing algorithms of POC:cell volume ratios, allowing better evaluation of in situ POC, PIC and other chemical parameters in the future.

Materials and Methods
Survey area and sampling strategy. An initial investigation cruise was conducted in the eastern equatorial Indian Ocean (EEIO) (80°~94°E, 6°N~5°S) (Fig. 8) onboard R/V "Shiyan 1" from March 10 th through April 9 th , 2012. Seawater was collected at eight depths from the surface to 200 m using Niskin bottles on a rosette sampler (Sea-Bird SBE-911 Plus V2). At all the stations, temperature and salinity profile data were determined in situ with the attached sensors system (conductivity-temperature-depth, CTD) (Supplementary Table S1).
Coccolithophore analysis. Coccolithophore samples 400-500 ml were filtered with a mixed cellulose membrane (25 mm, 0.22 μm) using a Millipore filter system connected to a vacuum pump under <100 mm Hg filtration pressure as soon as the seawater was collected onboard. After drying at room temperature in plastic Petri dishes, the filters were cut and subsequently mounted on glass slides with neutral balsam for a polarized microscope (Motic, BA300POL.) examination 74 . Totally at least 400 fields were counted by the standard of 30 coccospheres and 50 coccoliths were enumerated under a light microscope. The coccolithophore biomass (POC) was then calculated following the formula in Sun et al. 74 . One litter of seawater samples were gently filtered through 47 mm 0.45 μm polycarbonate filter for qualitative diagnosis under scanning electron microscope (SEM).
Size-fractionated Chla analysis. Chlorophyll a (Chla) samples 800 ml were serially filtered using the same filtration system (vacuum <200 mm Hg) through 20 μm × 20 mm silk net (micro-class), 2 μm × 20 mm nylon membrane (nano-class) and 0.7 μm × 20 mm Whatman GF/F filters (pico-class). After filtration, Chla membranes were immediately wrapped with aluminium foil and stored in a freezer −20 ed in alu and 0.7 μm × 20 mm and the measurements were made using the fluorescence method of Parsons et al. 75 . The primary data were displayed at (Supp. Table S6).
Estimation of coccolith calcite, coccosphere carbon biomass. The cell size biovolume was evaluated from geometric models 76 and then converted into carbon biomass (i.e. coccolithophore organic carbon, particulate organic carbon, POC, hereafter) using the formula of Eppley et al. and Guo et al. 77,78 . Cellular dimension was measured under SEM by scanning 20 individuals. Measured dimensions of most common species were found to be similar to those recorded in previous studies. Therefore, the determinations of common species calcite-CaCO 3 (i.e. coccolithophore inorganic carbon, particulate inorganic carbon, PIC, hereafter) masses were based on k s values (shape factor) and maximum length (diameter, μm) were recorded in previous studies 79,80 . The PIC/POC value is a potential rain ratio, which expresses the carbonate flux export to the outside of the euphotic zone. As for the irregularly shaped coccolithophores which biovolume has rare records, nearly 33% of the species (e.g. Michaelsarsia elegans, Reticulofenestra sessilis) were estimated with geometric models using SEM pictures from the literature, websites, and this study 47,[81][82][83] . The website can be access from: http://ina.tmsoc.org/Nannotax3/ index.html. It is noted that organic carbon was calculated with the exception of Gladiolithus flabellatus and Reticulofenestra sessilis by the reason of insufficient records from SEM data.

Multivariate analysis.
Box-whisker plots were prepared by the Golden Software Grapher 10.3.825 (LLC, Colorado, USA) (https://support.goldensoftware.com/hc/en-us/categories/115000653847-Grapher). Cluster analysis and non-metric multidimensional scaling 84 on coccosphere data (after square root transformation) were simultaneously implemented using the program package PRIMER 6.0 (Plymouth Routines In Multivariate Ecological Research, developed at the Plymouth Marine Laboratory, United Kingdom, http:// www.primer-e.com/). Prior to the above operations, the raw data were square root transformed. Then, principal component analysis (PCA) considering Euclidean distance was employed after data transformation and normalization. Significance testing was performed using the Analysis of Similarities (ANOSIM). In the Similarity Percentages-Species Contributions that the Percentages Routine (SIMPER) program was used for evaluating the contribution of each species to their sample group. All analyses were conducted to visualize the relations between the data abundance of phytoplankton and specific environmental factors. The spatial distribution of coccolithophores and hydrologic data were analyzed using freeware package Ocean Data View (ODV) 4.7.6 (https:// odv.awi.de/) 85 . Coccolithophore identification guiding lines. The coccolithophore identification is principally guided by the rules and features of light microscopic pictures and scanning electronic microscopic pictures of published references 82,83,86 , and the specialized website http://www.mikrotax.org/Nannotax3/index.php?dir=Coccolithophores. Also, the species are classified based on the four general niches of coccolithophore: upwelling water species, oligotrophic water species, deep water dwellers, and miscellaneous species 87,88 .