Intertidal benthic macrofauna of rare rocky fragments in the Amazon region

Rock fragment fields are important habitat for biodiversity maintenance in coastal regions, particularly when located in protected areas dominated by soft sediments. Researches in this habitat have received surprisingly little attention on the Amazon Coast, despite rock fragments provide refuges, nursery grounds and food sources for a variety of benthic species. The present survey describes the mobile macroinvertebrate species composition and richness of the intertidal rocky fragments in Areuá Island within the “Mãe Grande de Curuçá” Marine Extractive Reserve (RESEX) on the Brazilian Amazon Coast. Samples were collected during the dry (August and November 2009) and rainy seasons (March and May 2010) on the upper and lower intertidal zone, using a 625cm2 quadrat. At each season and intertidal zone, macroinvertebrate samples were collected along four transects (20m each) parallel to the waterline, and within each transect two quadrats were randomly sampled. Macroinvertebrates were identified, density determined, and biomass values obtained to characterize benthic diversity from the rocky fragments. The Jackknife procedure was used to estimate species richness from different intertidal zones during the dry and rainy seasons. Macrofaunal community comprised 85 taxa, with 17 “unique” taxa, 40 taxa were common to both intertidal zones and seasons, and 23 taxa have been recorded for the first time on the Brazilian Amazon Coast. Species richness was estimated at 106±9.7 taxa and results suggest that sampling effort was representative. Polychaeta was the most dominant in species number, followed by Malacostraca and Gastropoda. Regarding frequency of occurrence, Crustacean species Dynamenella tropica, Parhyale sp. and Petrolisthes armatus were the most frequent representing >75% of frequency of occurrence and 39 taxa were least frequent representing <5% of frequency of occurrence. Occurrence of crustaceans and polychaetes were particularly noteworthy in all intertidal zones and seasons, represented by 15 and 13 taxa, respectively. The most representative class in abundance and biomass was Malacostraca that represented more than half of all individuals sampled, and was dominated by Petrolisthes armatus. The latter was one of the most frequent, numerous and higher biomass species in the samples. In general, results indicated greater richness and biomass in the lower zone. Additionally, richness and density increase during the rainy season. Rock fragment fields in Areuá Island are rich in microhabitats and include a diverse array of species in a limited area. Our results underline the importance of rock fragment fields in Areuá Island for the maintenance of biodiversity in the Amazon Coast. Rev. Biol. Trop. 62 (1): 69-86. Epub 2014 March 01.

Rocky shorelines are commonly found worldwide, forming extensive coastlines or patches along the coast (Cruz- Motta et al., 2010).Organisms of several phyla responsible for the maintenance of ecosystem functioning dwell in this important environment (Little & Kitching, 2000;Menge & Branch, 2001), and many species (e.g.algae, mussels, oysters) can be of considerable social and economic value.Rocky shores are susceptible to many impacts, and have increasingly been the focus of research and monitoring programs for biodiversity conservation (Thompson, Crowe, & Hawkins, 2002).
Rock fragment fields (comprised of varying rock sizes) are considered as an intermediate environment between soft-sediment (e.g.sand or mud) and rocky substrate (e.g.rocky shores) (Lewis, 1964;Little & Kitching, 2000).These intermediate substrates generally provide three microhabitats: the upper and lower rock surfaces and the substrate below the rocks (Motta, Underwood, Chapman, & Rossi, 2003), creating a diverse microhabitat for the establishment of a large number of species that are rarely found in other habitats (Chapman, 2002a).Thus, such three-dimensionally complex system may function as island patches for many benthic organisms (Londono-Cruz & Tokeshi, 2007), and may promote coexistence of species from rocky and soft substrates, which lead to increasing richness and diversity in these heterogeneous environments (Cusson & Bourget, 1997).
The rocky fragments are uncommon fields compared with other rocky shorelines, but highly important to local diversity (Cruz-Motta, 2005;Le Hir & Hily, 2005;Motta et al., 2003).In tropical regions, there are more studies on the structure of macroinvertebrates from coastal soft shores than rocky ones (Alongi, 1989(Alongi, , 1990;;Neves & Valentin, 2011), largelly because tropic shores are dominated by softsediment habitats.The Brazilian Amazon Coast (BAC) consists mainly of mangrove forests (Souza Filho, 2005), which are habitats dominated by soft sediments with muddy and sandy areas, and few patches of rocky formations (Franzinelli, 1982(Franzinelli, , 1990)).In a review conducted by Rosa Filho & Lopes (2005), no study had described the benthic macroinvertebrate diversity on rocky fields in the BAC, and to the best of our knowledge, hitherto this lack of information continues.
Measuring species richness and diversity in various habitats is a useful tool for conservation action planning of the marine and coastal biodiversity (Amaral & Jablonski, 2005;Gray, 1997).Therefore, the present study describes the species richness and composition of macroinvertebrates from rare intertidal rocky fragment habitats in Areuá Island, Northeast of Pará State, located at the Marine Extractive Reserve (RESEX) "Mãe Grande de Curuçá" on the BAC.In addition, we provide a brief account of the taxa dominance and distributional data between intertidal zones and climate seasons.

Study site:
The study was carried out in Areuá Island (00º35'08.65"S -47º50'51.97"W) located in the lower Curuçá River Estuary, within the "Mãe Grande de Curuçá" Marine Extractive Reserve in Curuçá City, Northeastern Pará State, on the Brazilian Amazon Coast (BAC).The region is under super-humid equatorial climate, with average temperature around 27ºC (SUDAM, 1984) and annual rainfall between 2 400mm and 3 330mm (Moraes, M.N. Costa, A.C.L. Costa, & M.H. Costa, 2005).There are two defined seasons: a rainy season typically from January to June, and a dry season from July to December, with annual averages of 1 657mm and 487mm, respectively (Moraes et al., 2005).Areuá Island extends over an area of 1.34km² with a variety of coastal habitats that are dominated by mangrove forests (mainly Rhizophora mangle and Avicennia germinans, and some patches of Laguncularia racemosa), sandy and muddy beaches, tidal flats, and a few patches of saltmarshes and rocky fragments.This region has a strong marine and freshwater influence with semi-diurnal tides showing largest spring amplitudes of more than 4m.The rock fragment fields are parallel to the shoreline and cover an area of ca.0.25km².It is located approximately 170m from the minimum low water mark, and during high water spring tides, the fragments are completely immersed to a depth of ca.1m.This habitat comprises mixed substrates of pebbles, cobbles and boulders, fragments of laterites, with fixed, semi-fixed or detached rocks in sandy-muddy substrate.Rocky fragments have bare surfaces often with algal or biofilm/detritus cover.Furthermore, surfaces are colonized by sessile (e.g.barnacles, anemones), sedentary (e.g.polychaetes, bivalves) and mobile fauna (e.g.crabs, amphipods, isopods, gastropods).
Data collection: Samples were collected on rocky fragments of the upper and lower intertidal zones during the dry (August and November 2009) and the rainy seasons (March and May 2010).On each month, four transects (20m each) parallel to the water line were randomly distributed on each intertidal zone examined.Along these transects, two quadrats of 25x25cm (625cm²) were randomly sampled with a distance of at least 1m between replicates.A total of 64 samples were collected.All mobile benthic organisms were collected manually at three microhabitats: the upper and lower surfaces of the rocks and in the sediment below down to 10cm deep whenever possible.The borders of the quadrats were enclosed by "tulle" cloth to avoid escaping organisms.Samples were sieved using a 0.5mm mesh-size sieve, stored in buffered 4% formalin solution in seawater and stained with Bengal Rose.Benthic samples were maintained in plastic bags and transported within plastic drums to the laboratory.Samples were transfer to 70% ethanol in the laboratory after sorting and benthic macroinvertebrates were identified to the lowest taxonomic level possible using specific identification keys for each taxa.Benthic organisms were counted and wet weights were obtained to estimate biomass on an analytical balance accurate to 0.0001g.Wet weight of gastropods and bivalves were obtained without shell, except for extreme small shell species.To estimate biomass: (1) all species from samples with <20% frequency of occurrence were weighted; and (2) at least 15% of replicates were weighted in species with >20% frequency of occurrence.The weight of the remaining samples was estimated from number of individuals.Classification and nomenclature were checked on identification keys (Blake, Hilbig, & Scott, 1995, 1996, 1997;Mugnai, Nessimian, & Baptista, 2010;Rios, 1994;Ruppert, Fox & Barnes, 2005) and taxonomic information available on-line, from the World Register of Marine Species (WoRMS-http://www.marinespecies.org),and the Index to Organism Names (ION-http://www.organismnames.com).All reported species were deposited in the Crustacean and Malacofauna Collection at the Museu de Zoologia da Universidade de São Paulo (MZUSP) and the Invertebrate Collection at the Museu Paraense Emílio Goeldi (MPEG).The Brazilian Environmental Protection Institute (IBAMA) supplied the collecting permits (n° 16346-3, 16346-4 and 16346-5).
Due to limited description available for the species in the region, benthic macroinvertebrates were classified as morphospecies when species-level identification was not possible.Nevertheless, morphological distinctions were well-defined and hereafter will be treated as taxa.Characterizations of the benthic macroinvertebrate assemblages in relation to intertidal level and sampling period were carried out using taxa richness, density, biomass and taxa dominance.The Jackknife richness estimate was obtained by the Ecological Methodology Program (Kenney & Krebs, 2000): Ŝ=S obs +k(n-1/n), where S obs is the observed total number of species present in n quadrats; k is the number of unique species (species found only once) and n is total number of quadrats sampled (Krebs, 1999).Jackknife is one of the best richness estimator, with good prediction of less biased estimates for small number of samples (Colwell & Coddington, 1994).The results of these estimates were expressed with 95% Confidence Interval: Ŝ±CI.Taxa richness, abundance and biomass of benthic macroinvertebrates were analysed using a three-factor mixed model ANOVA (Analysis of Variance) to test for differences among intertidal zones (2 levels, fixed and orthogonal), seasons (2 levels, fixed and orthogonal) and months (2 levels, random and nested within seasons).Homogeneity of variances was checked by means of Cochran's test and data were transformed when appropriate (Underwood, 1997).Additionally, Student-Newman-Keuls (SNK) tests were used for a posteriori comparison of the means, following the recommendations of Underwood (1997).

RESULTS
The present study recorded 4 Phyla, 7 Classes and 85 macroinvertebrates taxa on the rocky fragments in Areuá Island (Table 1).Richness among samples varied from 2 (Upper zone; August) to 35 (Lower zone; March) taxa.
According to Jackknife estimates, 81% of the total richness (106±9.7 taxa) in the intertidal rocky fragments were sampled.The observed richness in the upper and lower tidal zones were lower than confidence limits for all months sampled (Fig. 1).Nonetheless, in the upper and lower zones at least 72% and 67% of the total richness estimated in each collecting period was sampled, respectively, indicating that sampling effort was representative.Polychaeta was the most diverse class with 32 taxa, followed by Malacostraca and Gastropoda with 24 and 14 taxa, respectively.A total of 17 taxa were found only once among samples (Table 1).The taxa with highest occurrence frequency are shown in figure 3, and particularly high occurrences were observed in Crustacean taxa such as Dynamenella tropica, Parhyale sp. and Petrolisthes armatus that represented more than 75% in frequency of occurrence.The less frequent taxa, representing less than 5% frequency of occurrence, were 14 polychaetes, 11 crustaceans, 5 gastropods, 4 bivalves, 3 hexapoda and 2 oligochaetes (Table 1).In addition, 15 crustacean and 13 polychaete taxa occurred in both intertidal zones and seasons (Table 1).
Some species were comprised of many individuals or few individuals with large biomass (Table 1), while others showed only a few individuals ("rarity of species", Table 1).P. armatus was one of the most representative in frequency (77% of all quadrats) (Fig. 3) and dominant in abundance (ranging from 16 to 1 600ind./m²)and biomass (ranging from 0.15 to 117.36g/m²).This species was found in all intertidal zones and sampling periods.
Besides P. armatus, only Capitella capitata, Isolda pulchella, Syllis sp., D. tropica, Parhyale sp., Sphenia fragilis, Thaisella trinitatensis and Nemertea were found in all intertidal zones and sampling periods (months).In addition, some taxa were unique for the specific rocky intertidal zone and/or season (Table 1) mainly in the lower zone during the rainy season (e.g.Hydroides sp., Pettiboneia sp., Petricolaria serrata, Hexapanopeus cf.paulensis, Melita sp. and other taxa).Furthermore, the unique taxa in the lower zone during the dry season were Portunidae, Pugilina morio, Arabella sp. and Phyllodocidae sp.A. Besides,  Taxa richness and biomass of macroinvertebrates differed between intertidal zones (Table 2) and in general, richness and biomass on the lower zone was higher (Fig. 4A, C; SNK: p<0.05).In addition, there was a significantly higher taxa richness and density on March than on May during the rainy season (Fig. 4A-B, SNK: p<0.01).

DISCUSSION
Our results show that areas of rock fragments in Areuá Island can be considered of high conservation priority for the BAC as they represent habitats for several macroinvertebrate species.It is likely that the area has endemic species, but expertise of the  species-level identification and more studies on description for benthic species in the region are necessary.The number of species in the present study is likely underestimated as taxonomic identification to species level is a common problem to researches on marine invertebrates (more details in Amaral & Jablonski, 2005).Many taxonomic groups were classified as morphospecies, showing a large representation of different taxa for rocky fragments in Areuá Island.
The complex three-dimensional structure of rocky fragments supplies shelter, refuge, and enhances feeding and reproduction (Cruz-Motta, 2005;Le Hir & Hily, 2005).In addition, it favours high taxon richness and the occurrence of different taxonomic groups that directly influence the biodiversity patterns.As a result, the functioning and regulation of ecological process in the ecosystem may be affected (Lanari & Coutinho, 2010;Srivastava & Vellend, 2005).Thus, the taxonomic dominance of Polychaeta and Crustacea in the present study is similar to rocky fragments from other coastal regions worldwide, although in some temperate regions (Canada, Ireland and Chile) and Jervis Bay-Australia, Mollusca was the most diverse and dominant phylum.Differences may be related to different environmental conditions on macrofaunal assemblage's composition and taxonomic richness or substrate type sampled.In rocky habitats, dominance in abundance and/or biomass by Malacostraca, Polychaeta and Gastropoda (Chapman, 2002b;Motta et al., 2003) is a common pattern.In the present study, Malacostraca dominated both abundance and biomass, and was comprised of small species that normally have a high number of individuals (e.g.amphipods and isopods) and large species that stood out in terms of biomass (e.g.crabs).
The dominance of Polychaeta and Crustacea is also very common to other habitats in the BAC (Table 3).This pattern may be partially attributed to the diverse feeding habits found in these taxonomic groups and the stress tolerance to natural environmental fluctuation in salinity, temperature and dissolved oxygen (Elliott & Quintino, 2007;Gray & Elliott, 2009) in the region.The tropical organisms have ecophysiological adaptability to salinity changes providing ontogenetic adaptation and distribution to the effects of salinity fluctuations in the environment (Chung, 2001).The diversity of food resources by constant input of detritus from mangrove ecosystems in the BAC is also important to the diversity and organisms coexistence (Lacerda et al., 2002).
The highest representativeness of the porcelain crab P. armatus in rock fragments is also common to others regions.In Marapanim River Estuary, located 24km East from the present study, P. armatus has been found in all intertidal zones and sampling periods, where density in excess of 3800ind./m² has been recorded (D.C.Silva, 2011).This species, as well as other Porcellanidae are found usually in high abundance on rocky fragments, living in dense aggregations under rocks and coexisting with other organisms in this habitat (Emparanza, 2007;Hollebone & Hay, 2007;Masunari & Dubiaski-Silva, 1998).
In addition, our results suggest a positive effect of rainy season on benthic invertebrate richness and density, when highest taxa numbers and density were observed, particularly on March.There were no differences in mean biomass within sampling seasons, but some taxa showed highest total biomass on the rainy season than on the dry season (e.g.Alitta succinea, Nereis oligohalina, Clibanarius vittatus, Sphenia fragilis).However, our data differ from those found on other habitats in the BAC.Benthic invertebrate richness was higher in the dry season on Ajuruteua sand beach and Caeté River Estuary (Rosa Filho, Almeida, & Aviz, 2009;Silva, Rosa Filho, S.R. Souza, & Souza Filho, 2011); and higher during the  Taylor, 2006 transition between rainy and dry season on saltmarshes of Canela Island, off the Caeté River Estuary (Braga, Beasley, & Isaac, 2009).These differences may be associated to larval supply, recruitment and settlement processes (López & Coutinho, 2008;Schiel, 2004) which are largely influenced by different features of each habitat (e.g.oceanic circulation and coastal currents).Rock fragments are considered habitats of high richness, abundance and endemism of benthic organisms and represent important habitats for coastal biodiversity conservation (Cusson & Bourget, 1997;Londono-Cruz & Tokeshi, 2007), comparable to oceanic islands that are susceptible to the extinction risks.The rock fragment areas in Brazil are still poorly known, in spite of their occurrence between Areuá Island-Curuçá-Northern Brazil (00º35'08.65"S -47º50'51.97"W, present study) and Farol Island-Matinhos-Southeastern Brazil (25º50'55.13"S -48º32'09.13"W).The scarce studies available focused generally on few specific groups (mainly epibenthic organisms) and complete macrobenthic diversity of rock fragments remains largely unknown.
The present study is the first record of benthic assemblages on rocky substrate in the BAC.This research also represents an important contribution for understanding global patterns of macroinvertebrates diversity of rock fragments.Although the rocky fragments in Areuá Island are an uncommon habitat on the BAC, this survey recorded high taxonomic diversity, which highlights the importance of this habitat for maintenance and conservation of coastal biodiversity.The Areuá Island is located in the Mãe Grande Extractive Reserve created in 2002 which continues to have no management plan, an indispensable tool for the sustainable use and conservation of natural resources.This study will, therefore, provide relevant baseline information for the design and creation of a future monitoring program in the region.

Fig. 1 .
Fig. 1.Estimate taxon richness (Jackknife)±Confidence interval (95%) and Observed richness on the rocky fragments in Areuá Island during the sampling periods.The terms lower and upper refer to lower and upper intertidal zone, respectively.

Fig. 2 .
Fig. 2. Abundance and biomass (%) of macrozoobenthos taxonomic groups in the Areuá Island on rocky fragments sampled between August 2009 and May 2010.Total abundance was 10 762 individuals and total biomass was 223.32g.
zone during rainy season, Nassarius polygonatus and Munnidae were common; and in the upper zone during dry season larvae Lepidoptera, larvae Hymenoptera, Anthuridae, Enchytraeidae and Tubificidae sp.2 were found.

Fig. 4 .
Fig. 4. Means (±SE) of density (A), biomass (B) and taxa richness (C) on the lower and upper zones from intertidal rocky fragments on Areuá Island during sampling periods.

TABLE 1
List of benthic macroinvertebrates species from rocky fragments in the Areuá Island, considering density and biomass in the intertidal zones (Low-L and Upper-U) and seasons (Dry-D and Rainy-R)

TABLE 2
Analysis of variance on the effects of Zone (Zo, fixed and orthogonal), Season (Se, fixed and orthogonal) and Month (Mo, random and nested within Season) to taxa richness, total density and total biomass of benthic macroinvertebrates from rocky fragments in the Areuá Island *p<0.05; **p<0.01;ns=Not significant.

TABLE 3
Total taxonomic richness observed in this study of the rocky fragments on Areuá Island and other habitats on the Brazilian Amazon Coast (BAC) and rocky shores of various coastal regions worldwide