Copepods and ambient water samples were collected at Sta. SY-C (18°13.199′N, 109°28.799′E) near Luhuitou coral waters in the Sanya Bay on May 21, 2011 at three time points (6:00, 12:00, 18:00) (Fig. 1). Copepods were collected using a plankton net (505 μm) and were fixed immediately in neutral Lugol’s solution at 2% final concentration. Ambient water samples were also collected using a Niskin bottle and 500 mL was fixed immediately. The samples were covered with black bags and taken to the laboratory for next analysis.

Water samples were gently mixed by inversion and 50 mL subsample was taken into a 50 mL centrifuge tube. Then the tube was kept for >24 h and concentrated to 1 mL according to the Utermöhl Settling method. From the concentrated samples plankton were identified and enumerated in a Sedgwick-Rafter counting chamber under an Olympus BX51 microscope.

Dominant copepod species from different time were identified under stereomicroscope and sorted. The sorted copepod samples were serially rinsed thoroughly with autoclaved 0.45 µm filtered seawater for >3 times and then with sterilized water for several times, examined under stereomicroscope to ensure no attachment of other visible organisms on the surface body, and homogenized in a microfuge tube using a disposable micro-pestle. The homogenates were re-suspended in 500 µL DNA buffer (1% SDS, 100 mmol/L EDTA, and 200 µg/mL proteinase K) and incubated for 3 d at 55°C for thorough cell lysis and DNA from all samples were extracted. 18S rDNA of copepod samples were PCR amplified using the copepod-excluding eukaryote-inclusive primer set (CEEC) and the PCR products were purified, cloned and 30–50 clones for each sample were picked randomly and sequenced (Hu et al., 2014).

Sequences obtained were searched against the GenBank database using the Basic Local Alignment Search Tool. The best hits were aligned with new sequences obtained in this study using CLUSTAL W (1.8) after the primer sequences were trimmed. Then the alignment results were exported to MEGA 4.0 and a Neighbor Joining (NJ) tree was inferred from the aligned dataset.

A percentage of each sequence from the whole clone library of each copepod sample was used to estimate the relative diet proportion. The percentage was then recorded to calculate diet diversity, which was described by food niche breadth (B), a reciprocal of Simpson's diversity index, following Levins (1968):

where P_i is the relative abundance of prey i and n is the total taxa of all the preys in the diet of copepod.

Diet niche overlap (O) was also calculated by the percentage of overlapped diet species between different copepod species (Pianka, 1973):

where x and y denote different species of copepod.

Bipartite network was also used based on percentage of each diet on species-level, to intuitively reflect main dietary specialization and overlaps of different copepods, and visualization was performed with the R package “bipartite”.

Microscopic analysis of ambient water identified 35 phytoplankton species totally, including diatom, dinoflagellates, chrysophyta and cyanobacteria (Table 1). Diatom dominated the community both in species numbers and cell density with 68.4% and 74.2%, 85% and 95.9%, 85% and 52.6% in morning, midday and night, respectively. And the cell density presented an increase trend in time series morning (9.7×10³ cells/L), midday (25.4×10³ cells/L) and night (73.3×10³ cells/L). Skeletonema sp. was dominant group both in morning and midday, while Trichodesmium erythraeum occupied almost half of the total abundance (46.5%) in night. Rhizosolenia sp. was also abundant group in midday and night.

Different composition patterns were also observed for copepod community in different time, 13, 10 and 19 species were identified in morning, midday and night, respectively. The highest density of copepods (38.1 ind./m³) presented in night, being much higher than that in morning (8.26 ind./m³) and midday (7.93 ind./m³), respectively. As for the species community, small copepods (<2 mm), such as Paracalanus, Acrocalanus, Temora, Centropages and Acartia, showed similar but lower densities in morning, midday and night, but occupied almost 95% of total community in midday. Large copepods in genus of Labidocera, Pontella, Calanopia and Undinula appeared only in night and dominated the copepod community. Subeucalanus subcrassus was the dominant species in all three times, and other species with different sizes, such as Neocalanus tenuicornis, Acrocalanus gibber, Temora turbinate, Acrocalanus gibber, Acartia negligens and Calanopia elliptica, which were sorted out for diets analysis, were also abundant in different community.

The 18S rDNA clone libraries were constructed for diet analyses of all these six species of copepod from different time points. Different taxa (1–10) of preys were revealed by randomly sequenced clones for each library. Chao1 estimates indicated that the actual numbers of taxa were similar with sequenced taxa, showing adequate coverage of diversity (Table 2). Diverse diets were detected for all the copepods as shown by the wide phylogenetic range and taxonomic distribution of these prey species (Fig. 2), including phytoplankton (e.g., diatom, dinoflagellate, green algae), plant, protozoa and different groups of metazoan (e.g., cnidarian, tunicate, crustacean). Metazoan was among the most abundant and diverse groups of prey items, with a variety of crustacean preys, such as Erythrops (Mysis), Processidae, and Galathea (coral shrimp). Other metazoan included ctenophora, cnidaria (jellyfish), tunicate (appendicularia), and Echinodermata (brittle star). Diatoms were also among the most diverse groups of prey items, including Coscinodiscus, Chaetoceros, Skeletonema, Proboscia and Nitzschia, which were all microscopic identified in the ambient water. Relatively, dinoflagellates were less abundant prey items only for some copepod species (e.g., S. subcrassus), with Karenia, Syndinium and two unclassified species being detected. Land plant was also abundant prey items for some copepod species (e.g., C. elliptica, T. turbinate, S. subcrassus), with Hordeum, Ficus and Cucurbita being detected. A species of green algae (Nannochloris sp.) was also detected in C. elliptica. Some other organisms, such as Fungus and unclassified Alveolata, were also detected in some copepod species.

The diets composition of spatially co-occurring six species were different from each other as shown by the wide taxonomic distribution of these prey species and diverse phylogenetic affiliations that received strong bootstrap supports (Fig. 2). Large copepod S. subcrassus fed mostly on protozoa and dinoflagellates, and N. tenuicornis diet consisted of a large fraction of metazoan and fungus. While another large copepod C. elliptica, which was the most abundant species in night, exhibited a much wider prey spectrum, including diatom, dinoflagellates, green algae and plant, ctenophore and a large amount of crustacean. Relatively, small copepods showed much lower diet diversity. Acartia negligens consumed a large fraction of metazoan (e.g. cnidarian, appendicularia) and a little diatom (Coscinodiscus sp.), while large amount of fungus and plant were detected in A. gibber and T. turbinate. Generally, metazoan, plant and phytoplankton were common prey items for these copepods, but with different preference in different populations. For instance, metazoan dominated the diet of A. negligen with two species of cnidarian, and C. elliptica’s with a large fraction of crustacean and a species of ctenophore, respectively. Proboscia indica and Skeletonema tropicum, which were abundant in ambient water, were detected in the diet of S. subcrassus and C. elliptica, respectively.

Food niche breadth (B) of six copepod species differed greatly from each other. Calanopia elliptica showed the highest B index of 10.48, followed by A. negligens with a value of 4. The B index for other species varied from 1.0 to 2.2.

On the other hand, although temporally co-exiting copepod populations sharing the same restricted food resources, diet differentiation was also observed. Thirty-eight feeding events were observed totally by the bipartite network analysis (Fig. 3). All these copepod species showed very limited food overlap as demonstrated by low (<0.2) diet niche overlap (O) index (Table 3). The three species from morning showed no overlap with each other in diet composition, with an O index of 0. Copepods from midday and night showed very limited diet overlap as the O index ranging from 0.04 to 0.07.

Diverse diet items were detected from the clone libraries of six copepods with 35 taxa totally, including not only common food reported by previous studies, such as diatoms, dinoflagellates, chlorophyta and protozoan, but also diet species uncovered recently by new methods, such as metazoan and land plants. Metazoan and land plants were the most common prey items among six copepod species analyzed here, followed by diatom, and this was consistent to some extent with previous study conducted in the same area in the Sanya Bay (Hu et al., 2015). Diatom was abundance group in prey items but with relatively low percentage of clone libraries. Skeletonema tropicum, Chaetoceros sp. and Proboscia indica were also identified in ambient water. However, among all the prey items detected, metazoan exhibited highest species diversity and relative abundance, with different phyla (e.g., Ctenophhora, Ophionereididae, Crustacea). It had been confirmed that copepod could feed on metazoan by both gut content analysis (Schnetzer and Steinberg, 2002) and molecular detection (Hu et al., 2014, 2015). Most of the crustacea detected here were from Malacostraca, such as Erythrops, Nikoides, Coralliocaris, Galathea and Platyxanthus, which might be the eggs or larva. In the Sanya Bay of spring (April to June), almost all the aquatic organisms were in spawning or breeding stage, and planktonic larvae could reach a percentage of 25% in zooplankton community with diverse species (e.g., Mysidacea larva, Luciferinae larva and Zoea larva) (Yin et al., 2004). Ophiuroids were also common benthic echinoderms in coral reef ecosystem (Lewis and Bray, 1983) and had only a spawning period during spring to summer (Yokoyama et al., 2008). Another group of metazoan worth noting was Hydrozoa (jellyfish), which was considered to be predators of copepod (Ishii and Tanaka, 2001), indicating complex trophic relationships among zooplankton in coastal waters. Land-plant detritus were another abundant food resource for copepods, and this was consisted with our previous results which found that copepods consumed large amounts of land plant detritus as a supplementary food source when faced with food limited situation (Hu et al., 2015). Widened food spectrum of these copepods could help to reduce food completion to some extent, and this might be important strategy for copepod populations to acquire food for their community development in complex natural environment (Leising et al., 2005; Kiørboe et al., 2010).

Copepods differed in both diet diversity and food preference in our results. The highest diversity was observed in larger copepod populations in night, with 18 and 4 taxa in C. elliptica and S. subcrassus respectively. This was consistent with previous studies that copepods would increase feeding activity from dusk, which could decrease the danger of predation (Saito and Taguchi, 1996; Calliari and Antezana, 2001). While there were also some species which showed enhanced feeding in daytime, such as S. subcrassus in this study with 6 taxa detected in midday (Wong et al., 1991). Subeucalanus subcrassus fed mostly on protozoa and dinoflagellates, while C. elliptica and A. negligens consumed a large fraction of metazoan. Plant dominated the diet of T. turbinata and protozoa (Rhizaria) dominated the diet of N. tenuicornis.

The spatially co-exiting species in our study could be divided into different ecological groups according to their ecological habits and distribution characters. Calanopia elliptica and A. negligens were subtropical coastal epipelagic species, and S. subcrassus and T. turbinata were warm water nearshore groups, while N. tenuicornis was widely-distributed warm water pelagic group (Yin et al., 2004). It is obvious that coastal population groups, such as C. elliptica, T. turbinata and A. negligens consumed large amount of metazoan and plant, which might originate from organic detritus, indicating a detritivorous feeding of these omnivorous copepods. These copepods were considered to be omnivores and they might apply a filter feeding during the day time, as the time-averaged fluid signal and the consequent predation risk is much less for copepods >1 mm using this feeding pattern (Kiørboe et al., 2010). We also found that the population structure varied by copepod size-fractions. The abundance of small copepods (<2 mm), such as Paracalanus, Acrocalanus, Temora and Acartia, showed little variations from morning, midday and night, but they occupied a large fraction (>50%) of the total biomass in midday, while large copepods (>2 mm) were more abundant and diverse in night. Dagg (1977) measured starvation time of different copepods, and showed that small coastal species, such as Acartia spp., were more vulnerable to starvation than larger ones (such as Pseudocalanus minutus and Calanus finmarchicus), so they might reside within specific environment to reduce energy consumption.

As the sampling site was reported to have high suspended matter and terrigenous input accounted for 44.5% of total sediments, primary production was much lower and might be insufficient to support zooplankton biomass (Zhao et al., 2013). Lombard et al. (2013) found that copepod can detect chemical trails originating from sinking marine snow particles, indicating that organic detritus in the water column might be potential sources of food supplements for coastal copepods like the coastal groups detected in this study. While as the most important dominant copepod in the Sanya Bay, S. subcrassus exhibited a more flexible feeding pattern due to its high capacity of migration and ambush feeding, indicating that organic detritus might be important supplementary food sources for omnivorous coastal populations (Lee et al., 2012). As the only pelagic group, N. tenuicornis consumed mostly unknown rhizaria, which was not detected in other copepods, indicating they might occupy different ecological niche through food partitioning even if spatially collocated in the same environment.

Moreover, copepods sampled from the same time also showed different food preference. Sequences of metazoan and protozoa were abundant in diets of S. subcrassus and N. tenuicornis in the morning, while T. turbinate consumed large amount of plant. Dominant small copepod, A. negligens, in midday community also consumed a large amount of organic detritus from metazoan (e.g., tunicate, cnidaria), while the larger population of S. subcrassus mainly fed on dinoflagellate. Even though the night community was dominated by two large species with similar size (S. subcrassus and C. elliptica), they showed apparent differences in diets diversity. The food partitioning here might be important strategy for the stabilization and survival of copepod assemblage (Guisande et al., 2003). In the morning, all the three copepods showed lowest diet diversity and none of the dominant phytoplankton in ambient water were detected for prey. This was consistent with previous conclusions that copepod would decrease feeding activity since dawn, especially pigment prey which would make them more visible to predators (Ishii, 1990). While in night, the diversity of all dominant populations was high, as the copepod biomass was highest in night and the strengthened feeding competition make the copepod more selective to acquire nutritional needs.

Variations of the phytoplankton community in our results suggested a changing food environment in the study area during the sampling time in the Sanya coastal waters. Copepod was reported to be capable of switching feeding pattern to better utilize the food resources in such conditions. Subeucalanus subcrassus copepods here could switch their feeding behavior to corporate with co-occurring copepods in food allocation, with a more herbivorous feeding at midday and relatively carnivorous feeding in morning and night. Subeucalanus subcrassus was dominant species during the sampling time and they had strong ability to migrate throughout the whole water column in the Sanya Bay (Yin et al., 2004). When faced with harsh competition and relatively lower predation risk in night, they consumed mainly protozoa by ambush feeding, while in midday with high predation risk they fed on more diverse preys by filter feeding. Different feeding mode will generate different fluid signal and hence exposes the grazers differently to predators. For such large copepods as S. subcrassus, fluid signal and the consequent predation risk is much less by filter feeding than ambush feeding (Kiørboe et al., 2010). It was clear from the results that S. subcrassus consumed a large amount of dinoflagellates in midday, even though diatom dominated the phytoplankton. The discrepancy between preys detected and ambient phytoplankton community might indicate selective feeding of S. subcrassus, consisting with that Carrasco and Perissinotto (2011) demonstrated copepod was more selective in complex coastal ecosystem and hence support food partitioning between species (Pagano et al., 2003). These feeding switching and selectivity might be vital mechanism to ensure S. subcrassus being the most important ecological groups throughout the year in the Sanya Bay (Mackas et al., 1993, Yin et al., 2004).

The variations of dominant species like S. subcrassus in community will bring different extent of feeding competition for co-occurring populations, as copepods exhibit a wide array of foraging behaviors across many spatial scales, depending on copepod species and ambient food environment (Leising et al., 2005). The selective differences amongst copepods could well avoid inter-specific competition within assemblage temporally appeared in the same water column (Arroyo et al., 2007; Laakmann et al., 2009). Such varied food niche differentiation pattern in field might be an important adaptive mechanism for copepod to survival in complex environment like the Sanya coastal water here.