Characterization of phytoplankton biodiversity in tropical shipwrecks off the coast of Pernambuco, Brazil

(Characterization of phytoplankton biodiversity in tropical shipwrecks off the coast of Pernambuco – Brazil). The phytoplankton community was studied around two artifi cial reefs (shipwrecks Servemar-X and Servemar-I), located off the coast of Pernambuco, northeastern Brazil, aiming to identify and thus, better describe these environments. Water samples were collected with Nansen bottle at three depths (surface, mid-column and bottom) for chlorophyll a analysis and salinity; temperature and water transparency were also measured. To collect phytoplankton samples, a diver using SCUBA equipment carried out phytoplankton hauls with a 20 μm net around the shipwrecks, about 1.5 m from the bottom for approximatly 3 minutes. Chlorophyll a concentrations at the bottom varied between 0.61 and 5.97 mg.m-3, with an average that indicates a mesotrophic environment. Temperature and salinity registered small seasonal variation, while water transparency showed a seasonal spatial pattern positively related to Chl-a rates. As regards the phytoplankton community, diatoms were the most representative group in the samples, followed by dinofl agellates, and among the species, the cyanobacteria Trichodesmium thiebautii Gomont ex Gomont prevailed quantitatively. The ecological importance of these ecosystems was confi rmed by the high diversity and evenness indexes, with the infl uence of coastal waters playing an essential role in phytoplankton structure and diversity.


Introduction
On the northeastern coast of Brazil, where oligotrophic waters prevail, there is growing interest in creating artifi cial reefs.Studies have revealed that such structures are indicated for the improvement of the ocean environment, even in areas of low productivity (White et al. 1990).Further aims of such actions are to create protected areas, increase fi sh stocks and offer leisure activities, such as diving and sport fi shing (Seaman & Seaman 2000).
As the Brazilian state with the largest number of shipwrecks (Santos & Passavante 2007) and calm, warm, transparent waters throughout most of the year, the sinking of ships as artifi cial reefs has helped to strengthen the diving and ecotourism industries in Pernambuco.Within a few months on the ocean fl oor, these sunken vessels become coated with a variety of marine organisms (from barnacles to algae) that attract small fi sh, which, in turn, lure larger predators (Grossman et al. 1997).
Despite indications of environmental degradation in some shipwrecks, there have been no studies of these artifi cial reefs detailing the impact such structures have on the marine environment.Moreover, surveys regarding the biomass, ecology and taxonomy of phytoplankton in artifi cial reefs are scarce in the world as a whole.In Brazil, studies have focused on investigating fi sh assemblages and the development of sessile biota associated with these structures (Jardeweski & Almeida 2005;Krohling et al. 2006;Brotto & Zalmon 2007).
The phytoplankton community is extremely important for the open sea environment, as it contributes most of the organic carbon available to pelagic food chains (Reynolds 2006).The continual documentation of phytoplankton biomass and species composition can provide an invaluable record of water quality and useful information for better understanding of other types of communities and ecosystems in general (Harris 1986).In this context, chlorophyll a (Chl-a) has proven to be an excellent indicator of trophic states and a useful tool in the environmental management of coastal areas (Häkanson 1994;Passavante & Feitosa 2004).
As investigations into the phytoplankton community of artifi cial reefs are rare, the aim of the present study was to contribute to understanding the role of these structures in the marine environment and obtain a better characterization of phytoplankton biodiversity in tropical shipwrecks.

Materials and methods
The two shipwrecks studied (both tugboat vessels) have the following structural characteristics: Servemar-X -19 m in length, sunk in January 2002, 12.5 km from the coast (08º07'19"S/034º45'46"W) at a depth of 25 m; Servemar-I -approximately 20 m in length, sunk in June 2004, 9.7 km from the coast (08º06'28"S/034º46'79"W) at a depth of 23 m.The distance between the two wrecks is 2.9 km (Fig. 1).Before sinking, both vessels underwent thorough preparation, which strictly followed the guidelines of Brazilian Navy norm NORMAM-07 (Diretoria de Portos e Costas 1998).
Sampling was carried out through dives with SCUBA equipment in the morning shift in both the rainy season (May, June, July 2005) and dry season (November 2005, December 2005 and January 2006), with a total of 12 dives (6 for each shipwreck).Water samples were collected with Nansen bottles at three depths in the water column at each site (surface, mid-column and bottom -alongside the shipwreck).Up to one L of water from the samples was fi ltered through glass fi ber fi lters (Whatman, 47 mm diameter and 0.45 μm pore size) and analyzed by spectrophotometry for determining Chl-a concentration (UNESCO 1966).
For the evaluation of hydrological parameters, water samples were also collected from each of the aforementioned depths for for determining temperature and salinity.The Aladdin-Pro dive computer was used to determine temperature (ºC), with readings at each sampling depth.Salinity was determined using a refractometer (Hanna Instruments).A Secchi disk was used to determine water transparency.Data regarding precipitation, wind direction and wind intensity were obtained from the Center for Weather Forecasting and Climatic Studies through the Data Collection Platform of Recife, located about 15 km from the area of the shipwrecks.Tide height was obtained from the Directorate of Hydrography and Navigation of the Brazilian Navy.
Phytoplankton was collected from around the shipwrecks using a net with a 20-μm mesh (Fig. 2).Due to the physical exertion of the sampling method, hauls lasted less than three minutes, starting at the prow at about 1.5 m from the bottom and 0.5 m from the shipwreck.The collected material was placed in a recipient containing 4% buffered formaldehyde.
Species identifi cation was based on morphological characters, using specifi c literature on phytoplankton systematics, biology and ecology.Frequency of occurrence was calculated based on the method described by Mateucci & Colma (1982).Relative abundance was determined using the method described by Lobo & Leighton (1986).Diversity (H') and evenness (J') were calculated using the Shannon-Weaver (1963) and Pielou (1975) indexes.Species richness was determined as the number of taxa per sample.
For the principal component analysis, hydrological and precipitation data were added to the Chl-a matrix.Species with over 50% frequency were submitted to unweighted pair group method (UPGMA) cluster analysis using the Bray-Curtis index with the NTSYS 2.2 program.Analysis of variance (ANOVA) was applied using the BioEstat 3.0 program to determine signifi cant differences (p < 0.05) between shipwrecks and sampling depths.

Results
In the rainy season, precipitation 48 h prior to all sampling days was recorded [maximal volume of 90 mm in June (326 mm for the entire month), 5.25 mm in May and 3 mm in July].In the dry season, rainfall 48 h prior to sampling only occurred in December [maximal volume of 90 mm (100 mm for the entire month)] (Fig. 3).Wind direction exhibited a slight tendency toward southeasterly winds in the rainy season (average velocity = 5.4 m/s) and a predominance of northeasterly winds in the dry season (average velocity = 3.9 m/s).
Secchi vanishing-point depths ranged from six m in May to 17 m in January around Servemar-I and from 10 m in June to 19 m in January around Servemar-X.The difference between the two shipwrecks was non-signifi cant (p = 0.5625).However, there was a signifi cant difference between seasons (p = 0.0102), with an increase of water transparency in the dry season (Fig. 4).
There was no difference in thermal stability of the water column between seasons (p = 0.1660).Seasonal variation in temperature was 2.2 ºC at Servemar-I and 1.7 ºC at Servemar-X.Mean salinity was approximately 35‰, ranging from 33 to 38‰ at Servemar-I, with no statistically signifi cant differences between seasons (p = 0.5144) or shipwrecks (p = 0.3210) (Fig. 5).
Chl-a concentration at the bottom (alongside the shipwrecks) ranged from 0.61 to 5.97 mg.m -3 at Servemar-X, with the minimum recorded in November, maximum recorded in July and January and a mean value of 3.67 mg.m -3 .At Servemar-I, Chl-a values were lower than those found   at Servemar-X (minimum of 1.30 mg.m -3 in November, maximum of 5.09 mg.m -3 in January and mean value of 2.74 mg.m -3 ).At the surface and mid-column, mean values were respectively 1.98 and 1.33 mg.m -3 at Servemar-X and 2.23 and 1.51 mg.m -3 at Servemar-I (Fig. 6).ANOVA revealed no signifi cant differences in phytoplankton biomass between the two shipwrecks (bottom) (p = 0.5869), between surface and bottom (Servemar-X, p = 0.1089; Servemar-I, p = 0.5002) or between seasons (p = 0.6924).
Principal component analysis revealed a positive relationship between phytoplankton biomass and water transparency.On the other hand, these variables had a negative relationship to precipitation and salinity (Tab.1).
The microphytoplankton was composed of 56 infrageneric taxa, 21 generic taxa and three classes.
Regarding species richness, Servemar-X had a higher average number of species than Servemar-I (21 species/ month versus 14 species/month).Of the 80 taxa identifi ed, 38 were found exclusively in the Servemar-X samples, 13 were found exclusively in the Servemar-I samples and 29 were found at both shipwrecks (Fig. 7).
Phytoplankton diversity and evenness were generally high, which may be related to the variability and high number of species at the shipwrecks.The lowest diversity values occurred in July at Servemar-X, which was related to the high number of Trichodesmium thiebautii found in the samples, accounting for 79.95% of the overall sample.At Servemar-I, Trichodesmium thiebautii accounted for 90.38% of the overall sample.Evenness was also low in June at Servemar-X; of the 22 species identifi ed, three accounted for 87.73% of the overall sample: Heliotheca thamensis (66.52%),Pseudonitzshia delicatissima (13.28%) and Trichodesmium thiebautii (7.94%).In July, Trichodesmium thiebautii accounted for 79.95% and 90.38% of relative abundance at Servemar-X and Servemar-I, respectively (Fig. 8).

Discussion
Mean Chl-a concentrations in the present study were high -more than half of those reported for shelf waters off northeastern Brazil (Passavante et al. 1987/89;Passavante & Feitosa 1995;Ekau & Knoppers 1996;Medeiros et al. 1999).Based on the Häkanson (1994) classifi cation, these values indicate mesotrophic environments near the shipwrecks.Despite the lack of a statistically signifi cant difference between sampling depths, the greater concentration of phytoplankton biomass at the bottom may be associated with the existence of the shipwrecks, with a greater concentration of Chl-a at this depth.
While Chl-a concentration in waters of the continental shelf may be higher at the bottom (Medeiros et al. 1999), the comments of Seaman & Seaman (2000) regarding turbid currents are pertinent to explaining this particular situation alongside the shipwrecks.Once settled on the ocean fl oor,  artifi cial reefs become obstacles to currents, which undergo detours, even on a small scale, with a consequent stirring up of sediment.This mechanism allows nutrient recycling and regeneration in the water column, which is necessary for phytoplankton growth (Rezende & Brandini 1997).Thus, the bottom-up effect seems to be the main factor for the high rates of phytoplankton biomass alongside the shipwrecks.As the ocean fl oor is moved by currents and the water is clear, allowing light to reach the bottom, these structures offer ideal conditions for phytoplankton growth.This may explain why phytoplankton diversity, biomass and production are higher in areas of natural or artifi cial reefs, such as sunken ships, although on a much smaller scale than that of large structures (Wilding & Sayer 2002).
Water transparency in the present study exhibited seasonal variation, with higher values in the summer.On the continental shelf off the coast of Pernambuco, previous studies have reported such seasonality, with Secchi values greater than 25 m (Resurreição et al. 1996).Regarding the Servemar-I, which is located closer to the coast, the variation in water transparency was 13 m, whereas the variation was seven m at Servemar-X.This clearly reveals the infl uence of continental runoff.Although the shipwrecks are rather distant from the coast, in situ observations (especially during the rainy season) indicate that discharge from the Capibaribe River can reach distances of over 9 km from the coast, which is confi rmed by decrease in the photic layer around Servemar-I as well as amount of waste (plastic bags, bottles and vegetation of continental origin -e.g., Eichornia crassipes).Eskinazi-Leça et al. (1997) report the infl uence of this discharge on phytoplankton growth, with the waters of the Capibaribe River reaching four miles from the Port of Recife.
The slight variation in temperature and salinity values in the areas of the shipwrecks was expected and had no infl uence on phytoplankton growth.A salinity of 33‰ at Servemar-I in May may be associated with rain and freshwater inputs.
The phytoplankton community around the shipwrecks was characterized by a mixture of oceanic and coastal species, with considerable infl uence from the mainland, where water from Pina Basin meets oligotrophic oceanic waters.An indication of this are the freshwater species Oscillatoria perornata Skuja, Stichosiphon sansibaricus Geitler and Fragilaria capucina Desmazières, which accounted for 3.6% of the overall sample, as well as the occurrence of Euglenophyceae.Morphological analyses of these algae revealed the absence of pigment, indicating that they may have been brought by the currents and tended not to survive in the waters of the continental shelf.Estuarine species (which live in brackish water), such as Gyrosigma balticum and Phormidium retizzi Gomont, were found, possibly originating from inshore locations across the Pina Basin, and accounted for 7.2% of the taxa.Oceanic and neritic species corresponded to 69.6% of the sample and tychoplankton accounted for 19.6%.
The predominance of diatoms at both shipwrecks may be explained by the characteristics of the group, which is considered the largest of the marine phytoplankton, with wide geographic distribution, high growth rates and the ability to tolerate variations in environmental conditions (Bold & Wynne 1985).Most of the species correspond to what has been recorded for coastal waters off Brazil (Fenandes & Brandini 2004;Souza et al. 2008).Asterionellopsis glacialis is a common, widespread Atlantic near-shore and estuarine species (Johnson & Allen, 2005) that is frequent and abundant in other coastal regions as well (Videau et al. 1998;Varela & Prego 2003).
The metallic structures of the shipwrecks may also have a positive effect on the development of microfl ora.Studies have demonstrated the particular importance of iron in the development of marine phytoplankton as a potential factor in the regulation of phytoplankton growth in coastal and oceanic regions (Martin & Gordon 1988;Martin 1994).Laboratory experiments have proven that addition of iron increases the formation of diatomic chains up to sixfold for the genera Chaetoceros and Peseudonitzschia (Graham & Wilcox 2000).
Some tychoplankton, such as Bacillaria paxillifera and Nitzschia longissima Brèbisson, may also be associated with the shipwrecks, attached to the structure as a periphyton and eventually detaching and integrating the phytoplankton community.Silva (1982) reports the importance of these organisms on the continental shelf off the coast of Pernambuco.With time, the development of a shipwreck as a reef ecosystem contributes to the natural enrichment of the water, creating ideal conditions for the growth of this kind of species.
The abundance of dinoflagellates was greater at Servemar-X than at Servemar-I, especially the genus Ceratium Schrank.The species of this genus have cosmopolitan distribution from eurythermal and euryhaline areas of oceanic and neritic waters (Smalley & Coats 2002).Off Pernambuco, the coastal water is less rich in terms of Ceratium species (Koening & Lira 2005).With increasing distance from the coast, the decrease in relative abundance of diatoms is followed by an increase in dinofl agellates (Zhou et al. 2008).
Found in 91.6% of the samples, Trichodesmium thiebautii is a colonial, nitrogen-fi xing, fi lamentous cyanobacterium, responsible for a significant portion of total primary production of oceanic surface waters (Marañón et al. 2000).On the Brazilian coast, a number of studies have recorded Trichodesmium erythraeum Ehrenberg causing occasional blooms (Satô et al. 1963/64;Medeiros et al. 1999;Rosevell-Silva 2005;Rörig 1998).Clayton (2000) found an abundant population of T. thiebautii in samples collected from the Gulf of Mexico in an area with several artifi cial reefs (oil rigs).This researcher reports that colonies were commonly found in parallel bunches, which was also observed in the present study.In the Atlantic Ocean, high Trichodesmium abundance has been found to be correlated with shallow, mixed-layer depths and high estimated iron deposition on the ocean fl oor (Tyrrell et al. 2003).
Servemar-X had greater species richness in the rainy period, whereas Servemar-I had greater species richness in the dry season.Spatial fl uctuations in species richness have been described for the coast of Pernambuco (Resurreição et al. 1996;Eskinazi-Leça et al. 1997), with phytoplankton blooms occurring in autumn and winter more than fi ve miles offshore.Closer to the mainland, freshwater inputs decrease light penetration in the rainy season; thus, phytoplankton rates increase only in spring and summer, when the water is clear.
Diversity and evenness values confi rm the ecological importance of these ecosystems.Equilibrium among taxa indicates stable conditions in the area, which is characteristic of open sea environments.Moreover, the occurrence of coastal species reveals the connection between land and ocean environments, with strong interaction between these communities.

Conclusion
There is evidence that shipwrecks are associated with high concentrations of chlorophyll a on the ocean fl oor, with turbid currents and a bottom-up effect, acting as potential factors for phytoplankton growth alongside sunken vessels.However, the infl uence of enriched coastal waters is a key factor in phytoplankton structure and the local food chain, contributing to the high diversity of species found at the shipwrecks.
The shipwreck situated farther off the coast had a higher concentration of chlorophyll a, greater number of taxa and greater species richness and diversity than the wreck located closer to shore, which was more affected by continental runoff, as evidenced by the occurrence of freshwater species, such as Oscillatoria perornata, Stichosiphon sansibaricus and Fragilaria capucina.Moreover, distance from the coast led to two different seasonal patterns, with an increase in phytoplankton biomass and richness in the rainy season at Servemar-X and in the dry season at Servemar-I.
The sampling method around the shipwrecks using SCUBA equipment and plankton nets proved successful and can be extended to other, even deeper areas, including natural reefs.The use of a small mesh size (< 65 μm) will increase diver effort during hauls, thereby slowing down the collection procedure.Strong currents can also hinder sampling.Sampling with several divers at the same site is not recommended.Moreover, divers should avoid swimming close to the bottom and stirring up the sediment.

Figure 1 .
Figure 1.Map of the study area, continental shelf of Pernambuco (Northeast Coast of Brazil) and location of the shipwrecks Servemar-X and Servemar-I (Source: GoogleEarth).

Figure 2 .
Figure 2. Diver using SCUBA equipment and plankton net carried out the hauls around the shipwrecks Servemar-X and Servemar-I.

Figure 3 .
Figure 3. Pluviometric data registered 48h before sampling days (black bars) and total per month (grey bars) on the coast of Pernambuco.

Figure 4 .
Figure 4. Water transparency and tide height recorded at the moment of sampling in the shipwrecks Servemar-X and Servemar-I.

Figure 6 .
Figure 6.Chlorophyll a record in the shipwrecks Servemar-X and Servemar-I.

Figure 7 .
Figure 7.Total number of phytoplankton species found in the samples of shipwrecks Servemar-X and Servemar-I.

Table 1 .
PCA coeffi cient and correlation of environmental, hydrological and biological variables in the shipwrecks Servemar-X and Servemar-I.