Reef foraminifera as bioindicators of coral reef health in southern South China Sea

Pulau Tioman is a famous tourist island off Peninsular Malaysia with beautiful coral reefs. This study aims to assess the health of the coral reefs surrounding Pulau Tioman based on the application of the Foraminifera in Reef Assessment and Monitoring Index (FI). Ten sampling sites around Pulau Tioman were studied with a total of 30 samples. Eight orders, 41 families, 80 genera, and 161 species of benthic foraminifera were identified. The agglutinated type of foraminifera constituted 2–8% of the total assemblages. Calcareous hyaline and porcelaneous groups represented 79% and 19% of the total assemblages, respectively. Symbiont-bearing taxa were the most common foraminifera. The results indicate that most of the sampling sites are conducive for coral reef growth with good recoverability from future stress to the ecosystem. However, several areas with higher coastal development and tourism have reduced water and sediment quality. Therefore, the limit on the number of visitors and tourists should be revised to enable coral growth and health. The FI values in this study showed a positive correlation with good water qualities and a negative correlation with organic matter enrichment. The FI is a good measure to assess the health of a coral reef and can be applied to other reef ecosystems around Malaysia.


Sediment characteristics and environmental condition of Pulau Tioman.
Most of the sediments in the study area can be classified as sandy with coarse-to medium-grained sand dominating the grain size percentages (Table 3). The study area near Kampung Mukut (B1-B3), where a fishing village is located, has coarser sediment. The study site in Batu Mambang (J1-J3) is dominated by finer sediment. The average percentage of organic matter was 3.35 ± 0.08% with a range of 1.31-5.89% (Table 3). The highest organic matter content was  www.nature.com/scientificreports/ recorded at E3 in the vicinity of Tekek Bay, a famous tourist site for snorkeling and diving. The lowest organic matter content was documented in Gelaber (C1), a sheltered headland that receives fewer tourists. The average bottom water temperature around Pulau Tioman was 29 °C with salinity of 33 PSU. The concentration of dissolved oxygen (DO) varied between 4.5 and 6.9 mg/L with the highest amount of DO recorded at B1 (Kampung Mukut). Meanwhile the water pH recorded was between 8.1 and 8.7.
Statistical analysis of benthic foraminiferal assemblages. The first and second axes of the Principal Component Analysis (PCA) explained 58.38% of the variations between the water quality and sediment quality variables (Fig. 2). In addition, the supplementary variables for the FI values indicated a positive correlation between these values and the dissolved oxygen, pH, salinity, and water temperature. Meanwhile, the FI values showed a negative correlation with organic matter, fine sand particles, and the mud composition.

Discussion
Foraminiferal assemblages in Pulau Tioman. The foraminifera assemblages in Pulau Tioman are dominated by rotaliid genera, such as Amphistegina, Calcarina, Operculina, and Peneroplis, with most species having a symbiotic relationship with diatom or algae (Appendix 1), which is similar to worldwide reef foraminifera distributions [16][17][18] . The foraminifera diversity around Pulau Tioman is slightly higher (H′ = 1.8-3.0) than those reported from the fringing reef environment in Brazil 19 . Amphistegina lessoni and Calcarina gaudichaudii, which are among the most widespread species found in Indo-Pacific waters 17    www.nature.com/scientificreports/ Amphistegina and Calcarina are common in reef environments worldwide 22,23 , especially in Southeast Asia 9,24,25 . Most living Amphistegina can be found attached to reef substrates, with less of a presence in sediment, and usually occur in high abundance in coral reef environments 9 . Owing to their ecological requirements and distribution, foraminifera assemblages are among the best candidates to monitor reef health. Opportunistic taxa, especially Ammonia, Elphidium, and Bolivina, are among those that are tolerant to environmental variations 11,13 . Hence, typically polluted marine conditions, with organic matter enrichment and reduced dissolved oxygen, allow these groups to opportunistically increase in abundance 15 .
In addition, the CCA indicated that environmental variables, such as the concentration of dissolved oxygen (mg/L), pH, salinity, and organic matter composition, had the most significant effect on the distribution of benthic foraminifera around Pulau Tioman (Fig. 3). Symbiont-bearing species, such as Calcarina spp., correlated well with the increase in the dissolved oxygen concentration, salinity, and pH. Previously, Prazeres et al. 26 suggested the need for calibration of the FI for the Indo-west Pacific where Calcarinidae occur. This is because Calcarina spp. usually thrive even in mesothrophic reefs 20 . Despite this concern, our study indicated that not only Calcarina spp. exhibits a good correlation with the water quality. Their relative abundance was significantly higher (> 15%) in Salang Bay, an area adjacent to where Shahbudin et al. 3 reported the highest percentages of live coral coverage. Therefore, based on the species-environment relationship assessment, we speculate that calibration of the FI was not necessary for our study area.
Ecological interpretation of FORAM index. The increase in construction and land reclamation activities around the east coast region has exposed many coral reef areas to high rates of sedimentation and a consequent reduction of the diversity of live corals including those in Pulau Tioman 3 . Additionally, active tourism related activities, such as trampling by divers or snorkelers and resuspension of sediment by boats has increased the mortality rate of corals 27,28 .
A majority of the FI values obtained in the waters around Pulau Tioman were greater than 4, which indicate that the water quality is favorable for reef growth and recovery 8,14 . Despite the excellent performance of the FI in numerous studies 8,16,29,30 , Prazeres et al. 26 have raised some concerns on the application of this index in new study areas. Therefore, to address this concern and reduce the bias associated with the application of the FI index, we performed PCA. Based on the PCA results (Fig. 2), the FI values in this study showed a positive correlation with good water qualities and a negative correlation with organic matter enrichment. Additionally, the FI values observed in east Pulau Tioman were > 5, which were higher than those observed in the west. This study, therefore, indicates that the reefs in east Pulau Tioman are more likely to survive and recover from future bleaching events www.nature.com/scientificreports/ compared to those on the west. Our finding are comparable to the study by Shahbudin et al. 3 , which reported that coral coverage in east Pulau Tioman was better than that in the west, where a higher percentage of dead corals was observed. The degraded reef conditions along the west coast of Pulau Tioman has been associated with rapid coastal development 31 , active tourism 32 , and boating activities 3 . Three stations (i.e., E3, F1, and G1) that recorded lower FI values (FI < 4) in this study are famous diving and snorkeling sites, and their reef conditions are similar to those established by Shahbudin et al. 3 and Akmal et al. 33 . These three stations are located near the jetty of the Pulau Tioman Marine Park, which also serves as the route for boat journeys to tourist spots for snorkeling and SCUBA diving activities. These relatively higher anthropogenic activities have led to an increase in the organic matter and nutrient concentrations in the water, which have created a less favorable environment for symbiont-bearing foraminifera and allowed stress-tolerant taxa to dominate 8,11 .
To understand the similarity of the foraminifera assemblages around Pulau Tioman, a Q-mode cluster analysis was conducted. The results indicated that the benthic foraminifera assemblages can be classified into four major groups (i.e., Group A, Group B, Group C, and Group D) (Fig. 4). Three of these groups (Group A, Group B, and Group C) represent foraminifera assemblages found on the western side of the island. Group A represents deep water conditions dominated by Numulites venosus and Operculina ammonoides. Both symbiont-bearing species that belong to the Nummulitidae family have been reported to have better growth rates in regions with low light 34 , thereby explaining their increase in abundance at the deeper reef slope area (> 18 m water depth) in west Pulau Tioman. The calculated FI for Group A (FI > 6) shows that the water quality conditions within these stations (F3 and G3) can be classified as oligotrophic, which is optimal for reef recovery 11,26 . Hence, despite the intensity of diving activity in Mesoh and Panuba Bay, the increased distance from the shoreline has reduced the anthropogenic impacts on coral reefs and supported healthy coral growth and recovery 35 . Meanwhile, Group B recorded a significant increase in the porcelaneous foraminifera group, with a higher abundance of stress-tolerant taxa, such as Ammonia tepida. In west Pulau Tioman, the high average composition of organic matter (3.81%) in the sediment serves as a food source for heterotrophic taxa and increases the number of stress tolerant species, such as Ammonia. The FI values for this group varied between 2.7 and 4.0, with the lowest values recorded in the vicinity of Mesoh, where high snorkeling and diving activities occur. The sheltered beach in Mesoh is among the most popular sites for locals and tourists to enjoy swimming and snorkeling. Based on the observations during field sampling, the water in F1 (Mesoh) is more turbid with numerous coral fragments present close to the shoreline. Group C represents foraminifera assemblages that are distributed in shallow waters (6-7 m depth) in west Pulau Tioman. This group has a relatively lower quantity of stress-tolerant taxa (< 5%), but a relatively higher quantity of heterotrophic species, such as Eponides, Discorbinella, and Textularia. The presence of abundant sources of food may have promoted the increase in the number of heterotrophic taxa but they are limited by the dominance of stress-tolerant taxa 11,13 . Finally, group D represents most of the stations located in east Pulau Tioman with an FI of 6-9, indicating good water conditions for reef growth and recovery. The reduced coastal development activities observed in east Pulau Tioman may have decreased the impact of sedimentation and allowed a greater diversity of live corals to thrive 3 .
This study indicates that the FI is an excellent low-cost monitoring tool that can aid in assessments of water quality surrounding coral reefs. This finding also implies that the FI can be used together with the coral reef health index to determine the conditions and status of coral reefs in the region. Similarly, these indices can be applied to other coral reef ecosystems around Malaysia to determine their health. The range of FI values reported here shows that most of the sampling sites around Pulau Tioman are conducive to coral reef growth and recovery after exposure to any future bleaching events or temporary damages to the ecosystem. Several sites (e.g., Mesoh and Tekek bays) with reduced FI values may not provide optimum conditions for reef recovery. Hence, marine park managers must closely monitor these touristic sites to prevent further coral reef deterioration. The carrying capacity of this ecosystem with respect to visitors and tourists at the Tioman Marine Park should be reviewed to determine optimum conditions for coral reef health, as indicated by the FI results.

Methods
Study site. The study was conducted at Pulau Tioman, Pahang, Malaysia (Fig. 5). Pulau Tioman is a tropical island situated in the southern South China Sea (SSCS) and is surrounded by extensive coral coverage, which hosts various marine species 36 . The island is influenced by two monsoonal systems: the northeast (November-March) and southwest monsoons (April-August) 37 . The maximum wave height during the northeast monsoon is ~ 4 m while during the southwest monsoon the height is < 1 m 38,39 . Pulau Tioman is surrounded by numerous coral reefs, with approximately 57-59 genera of hard corals distributed around the island 3 . Acropora, Montipora, and Porites are among the most common coral genera present around Pulau Tioman 3,27 . The extensive reef ecosystem surrounding Pulau Tioman is among the reasons why this island has been established as a National Marine Park by the Malaysian government 40 . The beauty of the island has attracted tourism-related activities since 1990 41 . The sheltered west coast of Pulau Tioman (Fig. 5) has become a suitable site for snorkeling and diving activities, as compared with the east side of the island. Therefore, the west coast of Pulau Tioman receives more tourists and is substantially more developed, with numerous resorts and housing areas distributed along the coast 3 . Tourist and recreational diving activities and coastal development may pose a threat to the surrounding reef ecosystem by reducing its resilience to climate change 6 . Therefore, the most viable management approach in the face of climate change is to reduce and monitor local stressors, such as coastal pollution 4 www.nature.com/scientificreports/ Sample collection. To assess the marine environmental health of Pulau Tioman, samples were collected from the coastal waters of the west and east sides of the island. A total of 10 sampling sites were selected based on the intensity of human activities and development along the coast of Pulau Tioman (Fig. 5). A transect of three sampling points was setup at each sampling site for a total of 30 sampling points around Pulau Tioman (Table 4). At each sampling site, three samples were collected along the 100-m transect laid perpendicular to the shore toward the reef slope. Along each transect, one sample was collected at 50-m intervals. Bulk sediment samples were collected by SCUBA divers using a scoop, which were stored in labelled plastic bags. Parameters, such as the water salinity (PSU), temperature (°C), pH, and water depth (m), were obtained in situ at each sampling station using a Hydrolab Quanta Multiparameter operated from a small vessel. All of the collected samples were transported to the Pulau Tioman marine park station in Mesoh for sorting. Sediment samples were divided into two components for foraminiferal and sedimentological analyses. Approximately 30 cm 3 of sediments were subsampled from the bulk samples for foraminiferal analysis and were fixed with 4% buffered formalin 43 . The remaining sediment samples were stored in zip-lock plastic bags, labelled, and brought back to the Central Laboratory of Universiti Malaysia Terengganu for sediment grain size analysis.
Laboratory analysis. The sediment samples for foraminiferal analysis were gently washed under running tap water over a 63-µm mesh sieve 11 . The residue on the 63-µm sieve was carefully transferred into a pre-labelled weighing boat before being dried in the oven at 40-50 °C overnight. The dried samples were later stored in plastic bags for analysis. Foraminiferal specimens were handpicked using a fine brush under a stereomicroscope. A total of 300 optimally preserved foraminifera tests were picked from every station 24 . If the samples contained less than 300 foraminifera, all of the specimens were picked 24 . Then, the specimens were sorted and mounted on micropaleontological cardboard slides. Foraminifera identification was performed based on the methods reported in Loeblich & Tappan 44 and other regional taxonomic manuscripts 24,45,46 .
The grain size analysis was conducted based on the dry-sieving method by Folk 47 . Approximately 100 g of dried sediment samples were sieved using a sieve set containing 4000-, 2000-, 1000-, 500-, 250-, 125-, and 63-µm sieves and the percentage of each sediment size fraction was determined. The organic matter content in the sediments was determined using the loss on ignition (LOI) method 48 . Prior to heating, 5 g of each sediment sample was weighed. The samples were gradually heated until they reached 500-550 °C, followed by ignition for 4 h, cooled to room temperature (30 °C), and the final weight was recorded. The percentages of organic matter were calculated based on the formula provided in Heiri et al. 49 . www.nature.com/scientificreports/ Data and statistical analyses. To avoid using reworked samples, only foraminiferal species with a relative abundance of more than 2% were maintained for further statistical analysis. Indices, such as Fisher's alpha diversity index (α), Shannon-Wiener species diversity (H′), and species evenness of Pielou (J′), were analyzed using the PAST (PAleontological STatistics) software version 3. Cluster analysis was performed to simplify the large datasets for easy recognition 50 . The Principal Component Analysis (PCA) was performed to investigate the relationships between the FI values and the sediment and water quality around Pulau Tioman. In addition, to investigate the relationships between the foraminifera species and the environment, canonical correspondence analysis (CCA) with a Monte Carlo permutation test was performed 51 . Foraminifera species with a relative abundance of > 10% in at least one sample were selected for the CCA and permutation test.
FORAM index. The FI was used to determine the health status of the reef at Pulau Tioman. The foraminifera species collected around Pulau Tioman were identified and segregated into functional groups (Table 5), as suggested by Hallock et al. 11 and Carnahan et al. 29 . The calculation of the FI was based on the equation proposed by Hallock et al. 11 . The values obtained from the calculation were interpreted based on Table 2. www.nature.com/scientificreports/

Data availability
All data generated or analyzed during this study are included in this article (and its Supplementary Information files).
Received: 13 November 2020; Accepted: 8 April 2021 Table 5. Functional Groups assigned to benthic foraminifera used in coral reef assessment 11,29 . *Full range of opportunistic genera under local conditions is not well known.