Macrofouler Community Succession in South Harbor, Manila Bay, Luzon Island, Philippines During the Northeast Monsoon Season of 2017–2018

Home , Hydroides elegans, Port of Manila

Philippine Journal of Science 148 (3): 441-456, September 2019 ISSN 0031 - 7683 Date Received: 26 Mar 2019

Macrofouler Community Succession in South Harbor, Manila Bay, Luzon Island, Philippines during the Northeast Monsoon Season of 2017–2018

Claire B. Trinidad1, Rafael Lorenzo G. Valenzuela1, Melody Anne B. Ocampo1, and Benjamin M. Vallejo, Jr.2,3*

1Department of Biology, College of Arts and Sciences, University of the Philippines Manila, Padre Faura Street, Ermita, Manila 1000 Philippines 2Institute of Environmental Science and Meteorology, College of Science, University of the Philippines Diliman, Diliman, Quezon City 1101 Philippines 3Science and Society Program, College of Science, University of the Philippines Diliman, Diliman, Quezon City 1101 Philippines

Manila Bay is one of the most important bodies of water in the Philippines. Within it is the Port of Manila South Harbor, which receives international vessels that could carry non-indigenous macrofouling species. This study describes the species composition of the macrofouling community in South Harbor, Manila Bay during the northeast monsoon season. Nine fouler collectors designed by the North Pacific Marine Sciences Organization (PICES) were submerged in each of five sampling points in Manila Bay on 06 Oct 2017. Three collection plates from each of the five sites were retrieved every four weeks until 06 Feb 2018. Identification was done via morphological and CO1 gene analysis. A total of 18,830 organisms were classified into 17 families. For the first two months, Amphibalanus amphitrite was the most abundant taxon; in succeeding months, polychaetes became the most abundant. This shift in abundance was attributed to intraspecific competition within barnacles and the recruitment of polychaetes. Diversity and richness values increased across all sites, which are commonly observed in primary succession events, while evenness values were low due to the dominance of Amphibalanus amphitrite and polychaetes. New macrofouling species in Manila Bay were reported: Barbatia foliata, Membranipora sp., a stylochid flatworm, a venerid clam, and hesionid, phyllodocid, and cirratulid polychaetes. More importantly, non-indigenous species were observed: Mytilopsis sp., Mytella charruana, Brachidontes pharaonis, Hydroides elegans, and the North Pacific giant flatworm Kaburakia excelsa. These species are potentially invasive and may alter the ecosystem of Manila Bay. Thus, it is recommended to further monitor the seasonally variable macrofouling community of South Harbor to observe annual succession patterns and to use DNA barcoding techniques more extensively for identification of macrofoulers – especially the polychaete taxa – to the species level and rapid early detection of potentially invasive species.

Keywords: biological invasion, DNA barcoding, fouling, species abundance, species diversity, species evenness, species composition

*Corresponding Author: [email protected]

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INTRODUCTION and human health; therefore, continuous monitoring of a locality’s fouling community is necessary. Manila Bay supports the food, livelihood, employment, and recreation needs of 23 million Filipinos residing on Artificial structures in ports and harbors are sites of its coasts – making it one of the most important bodies of biological invasion; hence, these are appropriate for water in the Philippines (Prudente et al. 1994, 1997; Kim monitoring new introductions of non-indigenous and et al. 2011; Jacinto et al. 2006; PEMSEA and MBEMP- potentially invasive fouling species (Ruiz et al. 2009). MBIN 2007; Greenpeace 2013). Fisheries, aquaculture, and Globally, monitoring of fouling communities in ports, shipping are major economic activities along the coasts of coasts, aquaculture sites, and marinas has been conducted Bataan, Cavite, and other provinces of Luzon surrounding over the past decades (Ramadan et al. 2006, Kocak et the bay (PEMSEA and MBEMP-MBIN 2007, Kim et al. 1999, Brankevich et al. 1988, George and Thomas al. 2011). The port of Manila is the busiest port in the 1979, Menon et al. 1977). In addition, the effects of Philippines (PPA 2010). The port is composed of the North seasonal variation on fouling assemblages are also well and South Harbors; the North Harbor is for local inter- established in international waters (Sahu et al. 2015, island shipping, while the South Harbor is for international Lin and Shao 2002, Emara and Belal 2004, Swami and shipping (Jacinto et al. 2006). International vessels transport Udhayakumar 2010). Locally, monitoring of the fouling macrofouling organisms between localities (Farrapeira et al. community in Manila Bay has only begun within the past 2007, Darbyson et al. 2009, Davidson et al. 2010). five years (Ocampo et al. 2014, Vallejo et al. 2017) and studies on seasonal variation of the fouling community Marine biofouling is a process in the marine environment, have never been conducted. Clearly, there is a demand which refers to colonization by micro- and macro- for further studies to define the fouling community of organisms on surfaces immersed in water (Bressy and Manila Bay. Hence, this study satisfied the following Lejars 2014, Ramadan et al. 2006, George and Thomas objectives: (1) identified indigenous and non-indigenous 1989, Salama et al. 2017). The colonization process macrofoulers via morphological and CO1 gene analysis; is composed of biochemical, bacterial, and eukaryotic (2) determined abundance and relative abundance of all phases (Wahl 1989, Chambers et al. 2006, Dobretsov et al. observed macrofoulers; and (3) observed changes in the 2013). Globally, billions of dollars are spent every year to species composition, relative abundance, community combat the negative effects of biofouling (Venugopalam diversity, and evenness of the fouling community during 2015). Settlement of fouling organisms on ships can lead the northeast monsoon period. Therefore, this study to increased fuel consumption, deterioration of coatings, served as a monitoring action for the early detection and corrosion, while settlement on water purifying stations of potentially invasive macrofoulers and, at the same and other ocean-based infrastructure damages underwater time, provided novel observations on the changes in sensors, heat exchangers, and membranes (Bressy and South Harbor’s fouling community during the northeast Lejars 2014, Okamura et al. 2010, Venugopalam 2015, monsoon period. Early detection of potentially invasive Rosenhahn 2010). Biofouling on ship hulls, aquaculture, macrofoulers – coupled with knowledge of the seasonal and ballast waters are the major players in the spread of variation in the community – will help create profiles, fouling species across the globe (Wasson et al. 2005, Galil models, and successional trends in characterizing the 2000, Maclsaac et al. 2001, Naylor et al. 2001). Other fouling community of Manila Bay. A well-characterized secondary vectors that transport fouling organisms are fouling community will aid future studies on Manila recreational boat uses and floating artificial objects in Bay’s macrofouler ecology, reproduction, succession, and the oceans (Murray et al. 2011, Zabin et al. 2014, Thiel development in order to create appropriate solutions for and Gutow 2005). managing the undesirable consequences of biofouling. When an introduced fouling organism is transported This is important for the Philippines and the Port of Manila and established into an indigenous fouling community, to meet its obligations on antifouling and ballast water it may pose harm to the fouling species in the area if the management for the International Maritime Organization introduced organism becomes an invasive species (Carlton conventions, which the government acceded to in 2018. 1996, Ruiz et al. 1999). Invasive fouling species may out-compete native organisms due to their reproductive capacity and tolerance for a wide range of environmental conditions (Spotorno-Oliveira et al. 2016, Yuan et al. MATERIALS AND METHODS 2016, Cai et al. 2014). Introduction of new foreign fouling species may also serve as a pathway for the spread of Study Site infectious diseases affecting marine produce and human The study site was a man-made marina of the Manila health (Mooney et al. 2005). Thus, fouling can have Ocean Park (MOP), situated within the South Harbor of detrimental impacts on aquaculture, the maritime industry, Manila Bay (Figure 1).

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Figure 1. Map showing the five sampling sites in MOP, South Harbor, Manila Bay, Philippines.

Five sampling sites were randomly selected from the pool Fouler Collector Assembly and Setup of possible sites in MOP. A site was considered a possible The collectors designed by PICES were used as fouling sampling site if it was accessible to the researchers and if it collectors and monitoring instruments. Four circular had railings (≥10 m in length) to which fouler collectors could collection plates (plastic Petri plates), 9 cm in diameter be tied. Nine fouler collectors were deployed at each sampling and 15 mm in height, were fastened to a 30 cm round point and GPS coordinates of each point were obtained (Table plastic bucket lid using cable ties. The collection plates 1). A total of 45 collectors were deployed in the marina. were fastened equidistantly from each other. A hole was

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Table 1. GPS coordinates of the present study’s sampling sites. Site GPS Coordinates 1 14°34'49.3"N 120°58'19.8"E 2 14°34'47.4"N 120°58'19.2"E 3 14°34'45.1"N 120°58'19.7"E 4 14°34'43.9"N 120°58'2 1.1"E 5 14°34'42.1"N 120°58'23.5"E drilled in the middle of the plastic bucket lid wherein a ≈15 m nylon rope was passed through (Figure 2). Figure 3. Diagram of deployed fouler collectors in a sampling site. One week before deployment, all assembled fouler collectors were submerged in sterilized Manila Bay seawater from MOP for seven days of soak time. On the Specimen Collection, Sampling Design, and day of deployment, one end of the rope was tied to the Monitoring of Water Quality Parameters railings while the other end was tied to a cement weight Specimen collection lasted 116 days during the Oct 2017 (≥2 kg) that kept the fouler collectors submerged 1 m – Feb 2018 northeast monsoon season. The sampling unit below the lowest tide level (Figure 3). The lowest tide is the PICES collector. Fouler collectors were deployed level was determined via the National Mapping and in Oct 2017 and were retrieved every four weeks until Resource Information Authority tides and currents table Feb 2018. During retrieval, three fouler collectors were for 2017–2018. Each fouler collector was placed 1 m apart randomly selected from each sampling site, after which a from each other within each sampling site. randomly selected collection plate from each of the three fouler collectors was obtained. This plate was replaced by a clean plate (Figure 4). Therefore, a total of 15 collection plates were taken every retrieval session. Each collected plate was sealed in a plastic bag then brought to the laboratory to identify and quantify individual fouling species. In addition, pH, temperature (ºC), dissolved oxygen (DO; mg L–1), and salinity (psu) were measured on a weekly basis during the study period between 3:00 and 4:00 PM by submerging a multi-parameter probe at the level of the fouler collectors.

Species Identification Each collection plate was examined by transferring its contents into a ceramic bowl (≈10 cm in diameter) containing treated salt water. The plate contents were suspended, which allowed the researchers to isolate small soft-bodied fouling organisms such as polychaetes. Using forceps, small suspended organisms were transferred into saltwater-containing Petri plates and observed under a dissecting microscope. Macrofauna such as crustaceans and mollusks were also transferred from the bowl into Petri plates. Organisms were identified and distinguished based on morphology. Published literature on marine biological invasions (e.g., Vallejo et al. 2017); online databases (e.g., World Register of Marine Species, Sea Life Base and Marine Species Identification Portal); and expert opinions were employed to confirm the identity of observed organisms. Samples for genetic analysis were packed in Figure 2. The PICES collector utilized in this ice and immediately brought to the Institute of Biology at study (from Vallejo et al. 2017). the University of the Philippines in Diliman, Quezon City.

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Figure 4. Schematic of sampling design.

Those examined for morphological diagnostics were fixed totaling to a final reaction volume of 25 µL. The optimized with 10% buffered seawater formalin. CO1 gene analysis PCR profile for the venerid clam and the mytilid Mytella was employed for mytilid and venerid bivalve samples to charruana is as follows: one cycle of initial denaturation determine species identity. Collections were identified at at 94.0 °C for 2 min, 40 cycles of final denaturation at least to the family level and at most to the species level. 94.0 °C for 30 s, annealing at 45.0 °C for 30 s, extension All identified taxa were photographed alongside a 1 mm at 65.0 °C for 1 min, one cycle of final extension at 72 scale for documentation. Furthermore, species abundance °C for 5 min, and storage at 5.0 °C for ∞. PCR products was determined by counting all individuals belonging to were loaded with Invitrogen BlueJuice™ 10x gel loading the same taxon, and relative abundance was calculated buffer and run alongside KAPA™ universal 100bp by expressing the abundance of a taxon as a fraction of DNA ladder on 1% agarose gel with ethidium bromide the total number of macrofoulers quantified during the and were visualized via a UV trans-illuminator and gel retrieval session. As invasive mollusks were a research documentation system. Luminescent bands corresponding focus in Manila Bay biofouling research, collected mytilid with the known size of the CO1 gene (≈600–700bp) were and venerid mollusks were DNA barcoded. excised from the gel. The CO1 gene was extracted from the excised gel via the Thermo Scientific™ GeneJet gel extraction kit. The extracted CO1 gene was sequenced at DNA Extraction, Gene Amplification, Gene Macrogen International Laboratories and was assembled Extraction, Gene Sequencing, and BLASTn using the Staden™ package for gene assembly. Assembled DNA was extracted from all bivalve samples via the CO1 gene sequences were then aligned via the nucleotide Purelink™ genomic DNA mini kit for DNA extraction. Basic Local Alignment Tool (BLASTn) with nucleotide The DNA concentration was determined via a Nanodrop™ sequences from the GenBank database. spectrophotometer for DNA and RNA quantitation. Cytochrome oxidase 1 (CO1) was the amplified gene from the extracted genomic DNA templates. CO1 gene Statistical Analyses of Species Abundance Trends amplification was done using the primer pair designed Microsoft Excel was used to tabulate all physicochemical for Lepidoptera by Hebert et al. (2004): LEP-F1, and species abundance data. The same program was 5’-ATTCAACCAATCATAAAGATAT-3’ and LEP-R1, used to compute for the following community indices: 5’-TAAACTTCTGGATGTCCAAAAA-3’. Each PCR Shannon Wiener’s Diversity index, Simpson’s Diversity reaction mix contained 15.125 µL of double distilled H2O, index, and Pielou’s evenness index. For statistical 5 µL of Bioline MyTaq™ 5x reaction buffer with dNTPs, analysis, the program used was IBM Statistical Package 1 µL each of the forward and reverse primers (LEP-R1 for Social Science (SPSS) Version 16.0 and JASP- and LEP-F1), 1.25 µL of 5% DMSO, 0.5 µL of Bioline™ Stats and the statistical level was set at P < 0.05. The 50mM MgCl2, 0.125 µL of Bioline MyTaq™, 5 µ/µL physicochemical parameters among the five study sites DNA polymerase, and 4 µL of 10 ng/µL DNA template – were tested for normality and homogeneity of variance via

445 Philippine Journal of Science Trinidad et al.: Macrofouler Community Vol. 148 No. 3, September 2019 Succession in Manila Bay the Kolmogorov-Smirnov test and the Levene’s test. Sites ± SD of 8.07 ± 0.2. DO was observed to fluctuate over that were found to be normal and homogenous were tested the four-month period. DO measurements had a mean ± for significant differences in physicochemical parameters SD value of 5.83 mg/L ± 0.9. Salinity was observed to be via analysis of variance (ANOVA) and subsequently relatively constant in all sites. Salinity measurements had a with Tukey’s HSD post hoc test when a significant mean value of 30.93 psu ± 2.09. All physicochemical data difference was present. Some sites presented non-normal except for salinity in sites 2 and 3 (p = 0.047 and p = 0.036 distribution; therefore, significant differences among these respectively) showed normal and homogenous measures. site’s physicochemical parameters were detected via the Analysis of variance showed that temperature and DO did Kruskal-Wallis test. Two-factor ANOVA with replication not display a significant difference between sites [F (4, was used to determine between month differences 70) = 0.120, p = 0.975; and F (4, 70) = 0.657, p = 0.624, in community species abundances of the dominant respectively] and the Kruskal-Wallis non-parametric test species forming the biogenic matrix abundances. To showed no significant difference in salinity across the sites meet the assumptions of normality, the raw counts were [X2(4) = 0.315, p = 0.989]. However, the pH measurements transformed according to the equation: in site 1 were found to be significantly different from the other sites via ANOVA and subsequently, Tukey’s post ln(A)= ln(c + 1) (1) hoc comparisons [F (4, 70) = 4.036, p = 0.005]. where A is the abundance observed and c is the count of each species observed. The transformation was enough Fouling Species Composition and Species to normalize the distribution. ANOVA calculations were Abundance Trends of the Biogenic Matrix done in JASP-Stats. A total of 18,830 organisms were identified and quantified during the four-month study period. Observed organisms The following most abundant taxa were included in were classified into 17 families – two from Class the model: Amphibalanus amphitrite, Mytilopsis sallei, Bivalvia, three from Class Malacostraca, eight from Class Mytella charruana, Modiolus metcalfei, Musculista sp., Polychaeta, and one from each from Classes Maxillopoda, Hydroides elegans, Nereiidae, Spionidae, Hesionidae, and Anthozoa, Gymolaemata, and Rhabditophora. Table 2 Anemonia majano. Taxa with extremely low frequencies presents the species composition with corresponding < 5 individuals per month were not included in analysis. abundances observed over the four-month study period (Table 2). The model is: For the first two months, the cirriped Amphibalanus Y =+µ τ ++ ς() τς + ε (2) iml m l m l iml amphitrite was most abundant with a relative abundance of 87% and 72%, respectively. However, in the succeeding where I = count per species in each sampling unit, m = two months, polychaetes became most abundant with month, and l = location in the marina. relative abundances of 70% and 71%, respectively. It was also notable that bivalve settlement was first observed on To investigate the role of the possible effect on the the second-month plates and then drastically decreased abundance of previously reported MNIS, Mytella in the succeeding months. Decapods, cnidarians, charruana and Mytilopsis sallei mussels with changes platyhelminths, and bryozoans were also observed in low in the species abundances and species composition of abundances during collections (Figure 5). the biogenic substrate species and indigenous mussel species, ANCOVA was used with sampling month as Table 3 shows the results of the ANOVA. the fixed factor and the biogenic and indigenous species abundances as covariates. The results suggest that community abundances change with time. As there is sufficient replication in each site and month of sampling, the results suggest that variability in species abundances is not significant in collectors in each RESULTS location in each month. However, the interaction between month and species abundances is significant. Physicochemical Parameters For the ANCOVA on abundances, the results are presented The observed physicochemical parameters showed in Tables 4a and b. Significant factors are in bold font. pronounced variations over the four-month study period from Oct 2017 to Feb 2018. The temperature gradually Tables 4a and b show ANCOVA results on Mytilopsis decreased over the four-month period with a mean ± SD and Mytella abundances with biogenic substrate species value of 28.35 °C ± 1.19. pH was observed to be slightly as covariates. basic in all sites during all collection dates with a mean

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Table 2. Number of individuals from all observed taxa during the four-month study. Taxa Observed number of individuals 31d 58d 88d 116d Cirripedia Amphibalanus 2229 2564 1577 1611 amphitrite Decapoda Portunidae 0 5 1 1 Penaeidae 0 0 1 7 Xanthidae 1 3 1 1 Figure 5. Fouling species composition and relative abundance after 31, 58, 88, and 116 days of immersion in Manila Bay Bivalvia from 06 Oct 2017 to 06 Feb 2018. Mytilopsis sp. 0 344 178 170 Modiolus 0 0 13 32 metcalfei The ANCOVA results suggest that in the observation period, Perna viridis 0 0 1 4 the month is significant as a fixed factor for Mytilopsis Mytella 0 0 6 2 sallei, which suggests its abundances increases over time. charruana Its significant covariate is Modiolus metcalfei. This would Brachidontes 0 0 0 1 suggest that M. metcalfei is a possible competitor or enabler exustus in Mytilopsis recruitment. For Mytella, the month is not a Veneridae 0 0 0 106 significant fixed factor since the species does not recruit Barbatia foliata 0 0 0 5 in large numbers during the northeast monsoon season. Musculista 0 2 1 0 However, M. metcalfei and Perna viridis are possible senhousia competitors and so are significant covariates. This is Polychaeta suggested by previous studies on the biological invasion Hydroides elegans 94 104 7 18 of Mytella in Manila Bay. Hesionidae 1 3 16 15 Several trends were observed in the relative abundance Nereididae 3 24 200 402 of each taxon across the four-month study period. All Polynoidae 0 3 1 28 polychaete taxa except for Hydroides elegans increased Phyllodocidae 0 0 0 2 in abundance over the four-month period. Notably, those Dorvilleidae 42 178 2645 2296 from the families Nereididae, Dorvilleidae, and Spionidae Spionidae 184 341 1216 2044 substantially increased in abundance during the transition Cirratulidae 0 0 0 31 from the 58d to the 88d immersion period. However, the Cnidaria opposite occurred for H. elegans wherein its abundance decreased during the transition. Within the bivalve Anemonia 0 11 15 40 manjano group, Mytilopsis sp. was the most abundant throughout Bryozoa the sampling period; however, its abundance abruptly decreased during the transition from the 58d to the 88d Membranipora sp. 1 0 0 0 panels. It is also notable that on the 116d panel, a member Platyhelminthes of the family Veneridae was first detected and occurred Stylochidae 0 0 0 1 in substantial abundance. Anemonia manjano was first detected on the 58d panel and was observed to increase

Table 3. ANOVA results testing for the effect of species abundances over time.

Source of Variation SS df MS F P-value Fcrit Month 200.0277 3 66.67591 131.1683 6.24E–68 2.617136 Species abundances 1581.665 12 131.8054 259.2945 2.7E–253 1.765469 Interaction 328.7551 36 9.132085 17.96511 1.34E–77 1.433116 Within month 370.0593 728 0.508323 Total 2480.507 779

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Tables 4a and b. ANCOVA results on Mytilopsis and Mytella in abundance through the 116d panel. Brachidontes abundances with biogenic substrate species as covariates. exustus, Barbatia foliata, Phyllodocidae, Cirratulidae, a. ANCOVA – dependent variable: Mytilopsis Membranipora sp., and Stylochidae were observed only once during plate collection and occurred in relatively Sum of Mean Cases df F p squares square minute numbers. Mytella charruana was identified by means of DNA barcoding technique via its CO1 gene Month 42.470 3 14.157 17.611 <0.001 sequence. BLASTn search results were conclusive in Amphibalanus 0.187 1 0.187 0.233 0.632 confirming its identity as Mytella charruana. However, amphitrite BLASTn search results showed insufficient percentage Hydroides 2.815 1 2.815 3.502 0.068 identity match between the unknown venerid CO1 elegans sequence and reported GenBank sequences, thus limiting Hesionidae 3.171 1 3.171 3.945 0.053 the identification to the family level. Nereididae 0.199 1 0.199 0.247 0.621 Dorvilleidae 0.003 1 0.003 0.003 0.955 Species Diversity and Evenness Spionidae 0.063 1 0.063 0.079 0.781 The estimates of diversity indices (Shannon-Weiner index, Modiolus Simpson’s Index of diversity showed an increasing trend 16.527 1 16.527 20.560 <0.001 metcalfei over time (Figure 6). However, Pielou’s evenness value Perna viridis 1.217 1 1.217 1.514 0.225 was observed to be relatively low. It can also be noted Mytella that species richness and total abundance significantly 0.814 1 0.814 1.012 0.320 charruana increased during the period of study. Musculista 0.246 1 0.246 0.305 0.583 senhousia Anemonia 0.865 1 0.865 1.076 0.305 manjano Residual 36.172 45 0.804

b. ANCOVA – dependent variable: Mytella Sum of Mean Cases df F p squares square Month 0.312 3 0.104 2.105 0.113 Amphibalanus 0.023 1 0.023 0.458 0.502 Hydroides sp. 0.003 1 0.003 0.066 0.798 Hesionidae 0.005 1 0.005 0.097 0.757 Nereididae 0.007 1 0.007 0.137 0.713 Figure 6. The biofouling community diversity indices showed an Dorvilleidae 0.002 1 0.002 0.031 0.861 increasing diversity trend over time, while evenness values were relatively low. 7.748e– Spionidae 3.833e–6 1 3.833e–6 0.993 5 Modiolus 0.213 1 0.213 4.315 0.044 metcalfei Rank Abundance Curves From the rank abundance curves (Figure 7) depicting the Perna viridis 0.562 1 0.562 11.361 0.002 relative abundance vs. abundance ranks of fouling species, Musculista 0.002 1 0.002 0.039 0.844 it can be noted that there was a substantial increase in the senhousia number of fouling organisms observed from the 31d to the Anemonia 3.256e–4 1 3.256e–4 0.007 0.936 116d immersion period, thus increasing species richness manjano over time. It can also be noted that species evenness Mytilopsis 0.006 1 0.006 0.125 0.726 increased as shown by the decrease in the steepness of PICES_ 0.045 1 0.045 0.904 0.347 the gradients. Collector Residual 2.177 44 0.049 Canonical Correspondence Analysis Note: Type III Sum of Squares The CCA biplot reveals the shift in the community from the Oct–Nov 2017 to the Jan–Feb 2018 periods, thereby confirming ecological succession. The most significant physical environmental factors that correlate with the

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Figure 7. Rank abundance curves showing relative abundance vs. abundance ranks of fouling organisms after 31, 58, 116, and 88 days of immersion in Manila Bay, Philippines from Oct 2017 to Feb 2018.

CCA axis 1 is significantly correlated (p < 0.000001) to the environmental factors under a Monte Carlo simulation (n = 10,000). Among the taxa observed, the following species have the strongest correlation (loading > 0.7) with CCA axis 1: Hydroides elegans, Membranipora, and Phyllocidae. While these taxa have low abundances, their appearances at certain months have contributed to a large variance in the correlations. The taxa that are abundant and form part of the biogenic matrix like Amphibalanus amphitrite have lower loadings (0.4) on CCA axis 1 since they are observed on all months, although their abundances may decrease.

Figure 8. CCA biplot showing a shift in community composition and structure during the 2017–2018 northeast monsoon season. DISCUSSION The sampling unit is the PICES collector. Legend: blue dot (Oct 2017), triangle (Nov 2017), square (Dec 2017), diamond (Jan 2018), and green dot (Feb 2018). Physicochemical Parameters All values of physicochemical parameters from the waters of South Harbor were within the standard values set by shift in community composition and species abundance the Philippine Department of Environment and Natural are temperature and DO. Resources and the normal salinity values reported by Vallejo (2012). However, it was observed that the pH of The CCA axis 1 in the context of ecological succession site 1 is significantly different from that of all the other represents the temporal changes in species composition sites. This disparity in pH level may be attributed to over time. Th first CCA axis accounts for 92.31% of the more organic wastes observed in Site 1, which may have variance (Table 5). On this axis, the environmental factors released acidic carbon dioxide into the water during their that are strongly correlated are temperature (0.709) and decomposition process (Chang et al. 2009). The water DO (–0.453). The second CCA axis accounts for 5.482% temperature was also observed to decrease and this may of the variance. The environmental factors correlated with be attributed to the declining atmospheric temperature this axis are salinity (0.258) and pH (–0.19).

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Fouling Community Composition and Ecological Table 5. Loadings and variation on the first two canonical correlation axes. Succession The present study reports barnacles, polychaetes, Axis 1 Axis 2 and bivalves as the major constituents of the fouling Month –0.0742868 0.044062 community, while bryozoans, decapods, cnidarians, and Amphibalanus 0.46538 0.026718 platyhelminths were scarcely observed. The same was Portunidae 0.231618 –0.12549 reported in Suez Canal, Egypt; northern Taiwan; and Penaeidae –0.388419 –1.31231 Mangalore and Kalpakkam, India (Emara and Belal 2004, Lin and Shao 2002, Menon et al. 1977, Sahu et al. 2011). Xanthidae 0.742334 0.147195 Alpheaus –0.358476 0.972022 Changes in the relative abundance of individual foulers Mytilopsis sallei 0.0489051 0.124737 and in the community composition of the fouling assemblage in South Harbor, Manila Bay during the Modiolus metcalfei –0.464827 0.043207 northeast monsoon period were observed. Such variations Perna viridis –0.349993 –0.41324 in a fouling community’s composition may be attributed Mytella charruana –0.415771 –0.21993 to seasonal influences and/or environmental pressures Geukensia –0.395945 0.523248 (Emara and Belal 2004, Lin and Shao 2002, Sahu et al. 2015, Greene and Grizzle 2006, Swami and Udhayakumar Brachidontes pharoanis –0.335915 –0.79739 2010, Ramadan et al. 2006, Kocak et al.1999); this study Musculista senhousia –0.261764 0.398209 focuses on the former during the northeast monsoon Barbatia foliata –0.410689 –1.4483 period. During the same period in northern Taiwan, Hydroides elegans 0.947931 –0.43275 it was reported that immersed plates were relatively Hesionidae –0.166437 –0.04167 lower in species richness when compared to those plates immersed during the southeast monsoon period (Lin and Nereididae –0.50645 –0.10618 Shao 2002). Low fouling densities were also observed Polynoidae –0.414722 –0.36684 during the northeast monsoon period in the Mumbai Phyllodocidae –1.16628 –0.22544 harbor and the Kalpakkam waters of India (Swami and Dorvilleidae –0.510141 0.062274 Udhayakumar 2010; Sahu et al. 2011, 2015). Hence, the Spionidae –0.231314 –0.11884 fouling assemblage observed in the present study and the previous study by Ocampo et al. (2014) may not Cirratulidae –0.541217 –0.58089 represent the climax community of fouling assemblages Anemonia manjano –0.344469 –0.03831 in Manila Bay. Nevertheless, the previous and present Membranipora sp. 0.865055 –0.6831 studies have reported several new fouling organisms and Kaburakia excelsa –0.335915 –0.79739 the latter provides novel information on the variation of fouling community during the northeast monsoon period Temp 0.709235 0.015476 in Manila. pH 0.14964 –0.19605 Salinity 0.200017 0.258436 Barnacles, solely represented by Amphibalanus amphitrite, were the most abundant macrofouler on the 31d (Oct– DO –0.453606 –0.09151 Nov 2017) and 58d (Oct–Dec 2017) plates. Factors that Axis Eigenvalue % variance may have contributed to barnacle abundance are (1) its 1 0.19449 92.31 perennial breeding pattern of year-round recruitment in 2 0.01155 5.482 tropical marine environments and (2) the observed positive CCA axis 1 is significantly correlated (p < 0.000001) to the environmental factors correlation between light intensity in the water column and under a Monte Carlo simulation (n = 10,000). barnacle abundance (George and Thomas 1979, Sahu et al. 2011, Nair et al. 1988, Brankevich et al. 1988, Menon et al. 1977, Sasikumar et al. 1989). The present study’s sites were well-illuminated throughout the study period, annually observed in Manila from Oct 2017 to Feb 2018. which may have provided optimal conditions for barnacle In addition, the relatively constant salinity levels during settlement. They are the first to recruit on the settling plate. the study may be attributed to the lack of rainfall observed during sampling. During the transition from the 58d to the 88d panels, a shift was observed in the most abundant organism (see Figure 4). During the first two months (31d and 58d), Amphibalanus amphitrite was the most abundant macrofouler but in the succeeding two months (88d

450 Philippine Journal of Science Trinidad et al.: Macrofouler Community Vol. 148 No. 3, September 2019 Succession in Manila Bay and 116d), the polychaetes exceeded Amphibalanus the detected clam is a new venerid species. The present amphitrite in abundance. The same trend was observed in work also reports the presence of a cnidarian, a bryozoan, northern Taiwan after three months of immersion during and a flatworm in South Harbor. the fall-winter season (corresponding with this study’s 58d to 88d period), where more than half of the fouling The changes in species composition and abundances assemblages were dominated by polychaetes (Lin and are reflected in the CCA, with the first canonical Shao 2002). Similarly, Sahu et al. (2011) reported that the axis representing variation over time (Ter Braak and peak settlement of polychaetes was observed in January Verdonschot 1995, Prach et al. 2007). This is consistent in India’s Kalpakkam waters – corresponding with the with the hypothesis of ecological succession. The main present study’s 88d panels. Additionally, Sudhendradev environmental factors that correlate with these are and Muthuraman (1988) reported that polychaete temperature and salinity. As the northeast monsoon abundance was higher in fouling assemblages during weakens in February, water temperatures rise. Lower DO the northeast monsoon period in the Gulf of Mannar; levels at the start of the northeast monsoon are consistent moreover, Satheesh and Wesley (2008) and Rajagopal with the observed hypoxia in the bay due to the upwelling et al. (1990) reported high polychaete abundance even of anoxic bottom water (Jacinto et al. 2011). As the during pre-monsoon and post-monsoon seasons in India’s monsoon weakens, primary productivity increases and Kudankulam and Edaiyur towns, respectively. With all with it DO levels. these considered, polychaetes – in general – have been found to have high relative abundance during the northeast Non-indigenous and Invasive Species monsoon period in both foreign tropical waters – in A number of recruited species in Manila Bay are agreement with our findings in South Harbor. considered to be potentially invasive and non-indigenous to the Philippines. These non-indigenous species may Bivalves first appeared on the 58d (Oct–Dec 2017) panels pose serious ecological damage to the community due to with a relative abundance of 10%, which then decreased (1) their lack of predators in the community, (2) ability to 3% and 5% in the 88d (Oct 2017 – Jan 2018) and the to survive in a wide range of environmental parameters, 116d (Oct 2017 – Feb 2018) collections. In terms of and (3) ability to reproduce a large number of offspring overall abundance, the bivalves only ranked third next to (Zenetos et al. 2016). the cirripeds and polychaetes. Some identified invasive bivalve species were Mytilopsis On the 116d collection, a venerid clam was detected sallei, Mytella charruana, and Brachidontes pharoanis. and occurred as the second most abundant bivalve with Mytilopsis sallei is native to the Caribbean islands and 106 counts. This is the first report of a venerid clam the Gulf of Mexico (Tan 2012). It is distributed in West occurring in Manila Bay waters. Interestingly, after the Africa, eastern Pacific, Fiji, Japan, Taiwan, Hong Kong, DNA barcoding protocol, the detected venerid clam’s CO1 China, the Philippines, Thailand, Singapore, Malaysia, gene sequence showed no adequate match in the Genbank and India where it is considered non-native and oftentimes database. The hit with the highest % identity match was invasive. Risk of further introduction remains high due to Clausinella isabellina with 80%, which is insufficient and the presence of M. sallei in Asian and Indian ports. In Asia, non-conclusive to ascertain the venerid’s identity. This this species is dominant on intertidal and subtidal hard indicates that the detected organism’s CO1 gene sequence surfaces in sheltered, estuarine harbors and aquaculture has not yet been reported and uploaded to GenBank or that

Figure 9. The present study reports new macrofoulers in Manila Bay (A–G): Barbatia sp., Venerid clam, Membranipora sp., stylochid flatworm and cirratulid, phyllodocid, and hesionid polychaetes. Potentially invasive and invasive species were also observed (H–L): Mytilopsis sallei, Mytella charruana (H–J), Spionidae, and Hydroides elegans.

451 Philippine Journal of Science Trinidad et al.: Macrofouler Community Vol. 148 No. 3, September 2019 Succession in Manila Bay farms (Tan and Morton 2006). Mytella charruana was this environmental issue relevant and urgent (Peh 2009). reported first by Vallejo et al. (2017). The species can There have been many proposed ways on how to prevent tolerate salinities ranging from 2 to 40 psu (Yuan et al. the introduction of alien species in the region, but the most 2016) and temperatures ranging from 6 to 36 °C (Brodsky efficient way to combat the problem is early detection and et al. 2011). M. charruana was also reported as invasive prevention. Marine invasions continue to increase globally in Florida, USA (Boudreaux et al. 2006). In Yundang (Cohen and Carlton 1998); thus, concerns regarding the lagoon, Xiamen, China, the invasive Mytilopsis sallei impacts of non-indigenous species are continuously was observed to reduce the species diversity index of growing (Pimentel et al. 2000, 2005; Grosholz 2002). the fouling macrofauna; thus, M. sallei was considered a pest in the area (Cai et al. 2014). Similarly, Brachidontes Species Diversity pharaonis is considered invasive in Egypt, Syria, northern Total abundance values were observed to increase Cyprus, Greece (Egeu Sea), Croatia (Northern Adriatic), over time – from 2,555 observed organisms on the 31d and the Sicilian coast due to its formation of extensive collection to 6,817 on the 116d collection. Also, taxonomic mats in midlittoral sites and patches in the subtidal parts richness substantially increased during the study (Table 3) – consequently displacing native species (Fernandez from eight taxa on the 31d collection to 22 taxa on the 116d 2017). Coral reefs near Suez were reported to have been collection. These observed patterns correspond with most replaced by mats of B. pharaonis. along with coralline primary successional sequences where total abundance red algae and Enteromorpha sp. It originates from the and species richness increase over time as new species Indian Ocean and is widely spread throughout the Red colonize a new substrate (Brown et al. 2007, Sahu et al. Sea (Fernandez 2017). 2015, Greene and Grizzle 2006). A number of polychaetes surveyed in Manila Bay are Observed Shannon-Weiner diversity (H’) values ranged also potentially invasive and non-indigenous to the from 0.51 (on 31d) to 1.57 (on 116d). This notable increase Philippines. Some of which are from the Spionidae along in diversity is in agreement with the observed increase with Hydroides elegans. Some members of the Spionidae, in total abundance and richness during the study (Table which occurred in relatively large numbers, may be 2). On the other hand, the values of Simpson’s index considered non-indigenous to the Philippines and many of ranged from 0.23 to 0.74, with only a slight increase its possible genera are considered invasive in many parts during the last two collection periods. Simpson’s index of the world. A member of this family – Marenzelleria values range from 0 to 1, with 1 representing perfect neglecta, commonly known as the red gilled mud worm – evenness (all species present in equal number). All is native only in the Atlantic Coast in North America. It is observed Simpson’s diversity values were less than 1, now widely distributed throughout Europe – specifically hence indicating imperfect evenness (Olopade and Rufai in Denmark, Estonia, Finland, Germany, Netherlands, 2014). This is justified by the large discrepancy in terms Norway, Poland, Sweden, and Central Russia – where of abundance between taxa (Table 2). To further support it is considered to be invasive (Parnoja and Kotta 2009). this, Pielou’s evenness index (J) was computed. Pielou’s Another fouling organism, H. elegans, is considered an index is simply the Shannon-Weiner diversity index (H’) invasive species in many harbor areas around the world. divided by the natural logarithm of species richness, taking This species grows mainly on man-made structures where into consideration the relative abundances of the species native organisms do not thrive. It could also dominate recruited. Observed Pielou’s index values substantially hard substrata on the intertidal zone (Schwan et al. 2015), increased from 0.234 (on 31d) to 0.399 (on 58d) and then consequently displacing other organisms. Polychaetes to 0.470 (on 88d). However, it increased only slightly have been transported by artificial substrates and to 0.486 during the 116d collection as was observed in commercial shipping around the world’s oceans. Around the Simpson’s index. Low evenness values indicate that 15% of all known polychaete genera are included in this some species are dominant (Alatalo 1981, Routledge phenomenon. The Spionidae and Serpulidae account for 1980). This can be observed in Figure 4 where during the highest number of alien species with 53 species and every collection, cirripeds and/or polychaetes dominated 46 species, respectively (Çinar 2012). The giant flatworm in terms of relative abundance. This is common in most Kaburakia excelsa (Platyhelminthes: Callioplanidae) is fouling communities where barnacles, polychaetes, and non-indigenous and is originally recorded from the North bivalves are among the most abundant taxonomic groups Pacific coastlines of Alaska to Northern California. This is due to their life history patterns (Menon et al. 1977, Sahu the first time in the Philippines that it has been recorded. et al. 2015, Ocampo et al. 2014, Bernardo and Torres It is predatory on mussel recruits. 2015, Pati et al. 2011, Ramadan et al. 2006). In addition, Introduction of invasive species in Asia is likely cirripeds and polychaetes may have also dominated due to detrimental to the rich biodiversity of the region, making the conditions of Manila Bay that favored their settlement, growth, and reproduction over other foulers. The presence

452 Philippine Journal of Science Trinidad et al.: Macrofouler Community Vol. 148 No. 3, September 2019 Succession in Manila Bay of chemical contaminants and garbage pollution in Manila and Dorvilleidae. CCAs confirmed this community Bay may have also played a role in providing a selective composition shift, with temperature and DO as the environment for the members of the fouling community, major environmental correlates. Their dominance is as the bay serves as a catchment of Metro Manila’s sewage also a common occurrence in fouling assemblages due wastes (Uttah et al. 2013, Chang et al. 2009). to their reproductive traits as reported in the literature. Furthermore, the Manila Bay port environment is likely In order to better visualize the changes in fouling to have favored their settlement, reproduction, and growth community richness, evenness, and abundance, Whittaker’s more than others. Significantly, the study reported new rank abundance curve (Figure 5) was applied. This graph macrofoulers in South Harbor, Manila Bay: Hesionidae, ranks the fouling species from least abundant to most Phyllodicidae, Cirratulidae, Barbatia foliata, Veneridae, abundant and represents both species richness and evenness Membranipora sp., Kaburakia excelsa, and Stylochidae. in a single figure. The width of the curve represents species More importantly, potentially invasive macrofoulers richness, while the slope represents evenness providing were detected: Mytilopsis sallei, Mytella charruana, a more complete picture of the community structure. A Brachidontes sp., Hydroides elegans, and Spionidae. thicker width is observed with higher species richness and For the non-indigenous mussels Mytilopsis and Mytella, narrower width for lower species richness. A horizontal line ANCOVA suggests that indigenous mussels like Perna represents a completely evenly distributed species, while virdis and Barbatia foliata as possibly competitors to the a steeper curve indicates less evenly distributed species recruitment of non-indigenous bivalves. This supports (Sreedevi et al. 2014). The relatively steep slopes observed previous studies on their ecological interactions. These in the Whittaker’s rank abundance curves during the study organisms can potentially be detrimental to the Manila (Figure 6) indicated low fouling species evenness in Manila Bay ecosystem and its economic assets. It is, therefore, Bay, as indicated by diversity and evenness indices (Figure recommended to continuously monitor the seasonally 5). More importantly, it can be noticed that the steepness variable macrofouling community of South Harbor of the curve became gradual over time – in agreement with during other seasons to observe annual succession the observed increasing J values. The curves were observed patterns in species composition and allow early detection to be gradual due to the increasing taxa and abundance of potentially invasive organisms. It is also highly of organisms observed on the fouler plates (see Table recommended that a DNA barcoding technique be 2). Finally, the rank abundance curve revealed that there employed in more observed taxa to rapidly confirm the were common species, namely Amphibalanus amphitrite, identities of morphologically complex macrofouling Dorvillidae, and Pionidae – which were dominating the species. recruitment plates. Some taxa were found to be rare or have low abundances. These are Membranipora sp., Polynoidae, Portunidae, Penaeidae, Perna viridis, Mytella charruana, Kaburakia excelsa, Brachidontes pharaonis, Barbatia sp., ACKNOWLEDGMENTS Phyllodocidae, and Xanthidae. The authors of this work acknowledge the facilities, equipment, reagents, and manpower provided by MOP through Ms Desiree Gestiada; the Biogeography, CONCLUSION AND Environment, Evolution, and Climate Laboratory of the RECOMMENDATIONS Institute of Environmental Science and Meteorology, University of the Philippines (UP) Diliman; and the DNA The present study observed changes in species composition Barcoding Laboratory from the Institute of Biology, UP of the macrofouling community in South Harbor, Manila Diliman through the kindness of Prof. Ian Fontanilla, Bay during the 2017–2018 northeast monsoon season PhD. This paper is based on the Bachelor of Science and detected potentially invasive species within the in Biology thesis of Claire Bellen Trinidad and Rafael community. 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