Ecological Indicators 11 (2011) 1123–1133 Contents lists available at ScienceDirect Ecological Indicators journal homepage: www.elsevier.com/locate/ecolind Connectivity between estuaries and marine environment: Integrating metrics to assess estuarine nursery function R.P. Vasconcelos a,∗ , P. Reis-Santos a , M.J. Costa a,b , H.N. Cabral a,b a b Centro de Oceanografia, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal Departamento de Biologia Animal, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal a r t i c l e i n f o Article history: Received 8 July 2010 Received in revised form 22 October 2010 Accepted 20 December 2010 Keywords: Nursery Effective juvenile habitat Juvenile fish Connectivity Estuary a b s t r a c t Natural or anthropogenic induced variations in estuaries and the dynamics of marine fish populations potentially promote differences in connectivity between estuaries and marine areas, i.e. in their importance as nursery grounds. Within this context, an integrated assessment of the differential nursery function of the main estuaries along the Portuguese coast for commercial fish species common sole Solea solea, Senegalese sole Solea senegalensis, flounder Platichthys flesus and sea bass Dicentrarchus labrax was performed through several indicators based on available data. Contribution of individual estuaries to marine subpopulations was measured with potential metrics (juvenile density, habitat quantity, juvenile number and habitat quality within estuaries) and effective metrics (estuarine source of young adults in marine environment measured via otolith elemental fingerprints). The relationship between the two types of metrics was also assessed. Estuaries identified as important nursery and/or effective juvenile habitat (EJH) differed with species and no single estuary was best for all, highlighting species-specific regulation of nursery function. Multiple species assessment of nursery and EJH function differed among estuaries. Management and conservation of estuaries should focus on sites with higher contributions to adult subpopulations of multiple species. The importance of defining precise scientific and management objectives was emphasized by the different rankings of estuaries obtained with nursery or EJH criteria. Potential and effective contribution of estuaries were not significantly correlated, but in a quantitative analysis juvenile densities and number of juveniles seem related with effective contribution in some species. An agreement between potential and effective contributions of estuaries is concurrent with the acknowledged minor role of juvenile stage processes in regulation of recruitment to adult subpopulations. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction The ecological and economical relevance of estuarine nursery function in supporting marine adult subpopulations of fishes has long raised interest (for reviews see Able, 2005; Beck et al., 2001). Use of segregated habitats during early life stages is a key ecological feature of these species: adults inhabit the marine environment and, after spawning, larvae/post-larvae/juveniles enter shallow coastal areas and estuaries, where they spend the first months or years of life, benefiting from high food availability, water temperature and refuge from predators, until returning to the marine environment (Gibson, 1994; Koutsikopoulos et al., 1989; Miller et al., 1985). ∗ Corresponding author. Tel.: +351 217500826; fax: +351 217500207. E-mail addresses: rpvasconcelos@fc.ul.pt (R.P. Vasconcelos), pnsantos@fc.ul.pt (P. Reis-Santos), mjcosta@fc.ul.pt (M.J. Costa), hcabral@fc.ul.pt (H.N. Cabral). 1470-160X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.ecolind.2010.12.012 Nevertheless, nursery function is likely to vary among estuaries and/or within them, partly as a result of estuarine colonization, habitat quality and quantity (Gibson, 1994; Rijnsdorp et al., 1992; van der Veer et al., 2000). Changes in these features are driven both by natural variability and anthropogenic actions (Courrat et al., 2009; Vasconcelos et al., 2007a). Estuaries are often acknowledged as nurseries for several species simultaneously (Elliott and Dewailly, 1995); even if a habitat or site has a weak nursery function for a particular species, it may play an important role for others. In addition, marine species which use estuaries as nursery areas have evolved meta-population structures that indicate hierarchical interactions between meta-estuarine systems (Ray, 2005). Recognized over a century ago, the nursery concept has generally been applied lato sensu to areas where fish density, growth or survival are enhanced (e.g. Désaunay et al., 1981; Gibson, 1994; Koutsikopoulos et al., 1989). However, these approaches do not contemplate whether habitats successfully transfer the higher juvenile biomass to adult populations (Beck et al., 2001). Recently, the ‘nursery’ concept was applied to sites/habitats which contribute 1124 R.P. Vasconcelos et al. / Ecological Indicators 11 (2011) 1123–1133 to habitats used by adults with relatively more recruits per unit area than others used by that species (Beck et al., 2001). In these sites/habitats, juvenile export and recruitment is maximized by successful combinations of: (1) juvenile density, (2) growth, (3) survival and (4) movement to the habitats used by adults. An innovative feature of this classification is the clear definition of nursery grounds and a testable hypothesis to pursue. More recently, the relevance of ‘effective juvenile habitats’ (EJH), i.e. sites/habitats with a higher overall contribution to adult populations, regardless of their unit area contribution, has also been proposed (Dahlgren et al., 2006). Both concepts recognize estuaries as a mosaic of habitats and the importance of their complexity and inter-connectivity. Dahlgren et al. (2006) aims to complement Beck et al. (2001) by departing from the management-oriented ‘per unit area contribution’ and focusing absolute dimensions of sites/habitats and their overall contributions. Hence, habitats disregarded as nurseries following Beck et al. (2001), that have a small per unit area contribution to adult populations, but are essential to sustain adult populations are valued. These definitions drove the paradigm shift from the classical approaches on nursery function, i.e. estimates of the potential contribution of juvenile sources of new recruits from putative nursery habitats to adult populations (e.g. Cabral et al., 2007; Désaunay et al., 1981; Le Pape et al., 2003a; Minello et al., 2003), and promoted the mandatory quantification of effective contribution of juvenile sources, stimulated by recent technological developments, rendering encouraging results on connectivity (e.g. Elsdon et al., 2008; Gillanders, 2002; Rooker et al., 2008). Understanding the processes occurring between juvenile and adult life stages is vital to improve our comprehension of connectivity and to ascertain to what extent changes in estuaries influence connectivity. The simultaneous assessment of multiple factors is required (Beck et al., 2001). Fishing surveys provide direct quantifications of juvenile density and distribution areas (e.g. Le Pape et al., 2003a; Rooper et al., 2004). Indirect quantifications of juvenile growth rates, such as marginal otolith increment width or nucleic acid based condition indices, produce reliable measures of individuals’ response to habitat quality (e.g. Amara et al., 2007; Kuropat et al., 2002). Quantifications of connectivity using otolith elemental composition as a natural tag of habitat use enable the retrospective identification of the juvenile source of adult fish (e.g. Brown, 2006; Gillanders and Kingsford, 1996; Thorrold et al., 2001). Some combined approaches have been applied, e.g. analysing juvenile density and condition (e.g. Rooker et al., 1997; Sogard, 1992), juvenile density and habitat area (e.g. Le Pape et al., 2003a; Riou et al., 2001; Rooper et al., 2004) or potential and effective contributions (Chittaro et al., 2009; Fodrie and Levin, 2008) and provided information on the interaction of the processes involved. However, knowledge on estuarine nursery function is mostly fragmented. Further comprehensive broad-scale multi-disciplinary approaches are required. There is worldwide interest in the evaluation of the ecological quality and integrity of coastal ecosystems. Whilst most biotabased approaches have focused on community structural features as indicators, such as benthic, crustacean and fish assemblages (Borja and Dauer, 2008; Jordan et al., 2010), the urgent need to move towards a functional and ecosystem approach has been greatly emphasized (de Jonge et al., 2006). Within this context, the present approach presents a novel and integrated assessment of multiple indicators to evaluate estuarine nursery function. Several metrics are combined to ascertain the potential and effective contribution of a set of estuaries to adult populations as well as analyse the relationship among indicators. An integrated review and assessment of available data for four commercial fish species (common sole Solea solea, Senegalese sole Solea senegalensis, flounder Platichthys flesus and sea bass Dicentrarchus labrax) which use estuaries along the Portuguese coast as nursery grounds is performed. Specifically, to determine: (1) if multi-species nursery and EJH function differed among a set of estuaries along this coast; (2) if estuaries identified as nursery or EJH were equivalent among species; and (3) if the nursery and EJH concepts had matching results. The relationship between potential and effective contributions of individual estuaries was evaluated by integrating information on juvenile density, habitat quantity, juvenile number, habitat quality for juveniles and on connectivity between estuaries and the marine environment. A broad-scale approach was defined and the analysis to each individual estuary comprised the main areas (and habitats they include) used by juveniles within the system, to account for ecosystem complexity. 2. Materials and methods 2.1. Study area Six main estuarine systems of the Portuguese coast (Douro, Ria de Aveiro, Mondego, Tejo, Sado and Mira) (Fig. 1) were selected for their importance as putative nursery areas for many fish species. They differ in hydrological and geomorphological features (Table 1) and as determined via multi-metric indices present different levels of: (1) natural vulnerability to disturbances (based on hydrological features); and (2) anthropogenic pressures (based on distinct pressure sources) (Vasconcelos et al., 2007a) (Table 1). 2.2. Multi-metric assessment of nursery function Contribution of each estuary to marine adult subpopulations of S. solea, S. senegalensis, P. flesus and D. labrax was quantified based on potential and effective metrics (Table 2). Potential metrics evaluate processes within each estuary, as estimates of the potential production of new recruits to adult populations. Specifically: juvenile density, habitat quantity, juvenile number and habitat quality for juveniles. Effective metrics quantify realized recruitment from an estuary to marine adult subpopulations. Specifically: number of adults which spent their juvenile life period in each estuary – expressed as overall contribution and as contribution per unit area (in both cases as % of all adults). Considering effective metrics, the estuary which overall contributes most to marine adult subpopulations is identified as EJH (following Dahlgren et al., 2006) whereas the estuary which contributes most per unit area is classified as nursery (following Beck et al., 2001). Multiple aspects of juvenile ecology of these four species in the selected estuaries as well as on their recruitment to marine environments were analysed. Previous studies were reassessed and integrated with new data, as follows. 2.2.1. Juvenile density and habitat quantity in estuaries 2.2.1.1. Background data. Density and distribution of juveniles of the four species within the selected estuaries was assessed through fishing surveys in July 2005 and 2006 (for additional details see Vasconcelos et al., 2010). In summary, several potential nursery sites were sampled within each estuary (two to six sites, depending on estuary size) using a 2-m wide beam trawl at night during ebb tide (ca. 10 replicate tows per site and season; tow duration ca. 10 min; mean area 862 m2 ). Sampling design comprised all main sites used by juveniles of these species within an estuary (and habitats they include) and was based on preliminary surveys covering the entire estuaries, as well as on previous knowledge on these systems. Sampling followed the beginning of estuarine colonization. Mean juvenile densities for each estuarine site were determined. In each estuary, sites where juveniles occurred in all sampling surveys and in most replicate tows were identified as important sites for juveniles of a species, i.e. pertinent to an evaluation of nursery function. As a measure of habitat quantity, total area of each site was R.P. Vasconcelos et al. / Ecological Indicators 11 (2011) 1123–1133 1125 Fig. 1. Study area. Location of sampled estuaries (open circles) and areas off the Portuguese coast (dotted circles) and fish species collected (SS – Solea solea, SN – Solea senegalensis, PF – Platichthys flesus and DL – Dicentrarchus labrax). Estuaries were sampled for juveniles and areas off the coast were sampled for young adults. Potential contribution of each estuary to the marine adult subpopulations was estimated for all present species in each system. Effective contribution was estimated through the identification of the estuarine juvenile source of young adults collected off the coast, for species marked with an asterisk (*). 1126 R.P. Vasconcelos et al. / Ecological Indicators 11 (2011) 1123–1133 Table 1 General characterization of main estuaries of the Portuguese coast. Variables presented are: total estuarine area (km2 ); percentage of intertidal area (%); natural vulnerability index (0 represents lowest and 1 highest) and anthropogenic pressures index (0 represents lowest and 1 highest), as in Vasconcelos et al. (2007a). Estuary Area (km2 ) Intertidal area (%) Natural vulnerability (index) Anthropogenic pressures (index) Douro Ria de Aveiro Mondego Tejo Sado Mira 10 74 10 320 180 5 11 87 64 40 44 42 0.54 0.62 0.67 0.33 0.58 0.78 0.47 0.42 0.28 0.76 0.49 0.14 determined with Geographic Information System software (ArcGIS 9) defined by the polygon covered by tows corrected to site natural features and areas with homogeneous environmental conditions between replicate tows. 2.2.1.2. New developments. Mean juvenile densities and habitat quantity per estuary were determined considering all important juvenile sites in the estuary, based on data for each site from Vasconcelos et al. (2010). Data from both sampling years were used, since sampled sites in both years showed similar densities and areas between years (see Vasconcelos et al., 2010). The total number of juveniles of a species in an estuary was determined based on mean juvenile densities and habitat quantity of the important sites for juveniles. 2.2.2. Habitat quality for juveniles in estuaries 2.2.2.1. Background data. Individual response of juveniles to habitat quality was assessed with RNA:DNA ratio (for additional details see Vasconcelos et al., 2009), a biochemical condition index commonly applied as a proxy of nutritional condition and growth rate: it reflects variations in protein synthesis rates, since RNA concentration increases with food availability and protein requirement whilst DNA somatic content remains relatively constant (Buckley et al., 1999). Briefly, age-0 juveniles collected in the sampled estuaries in July 2005 and 2006 were selected (ca. 20 individuals per species and site – one or two important sites for juveniles per estuary). RNA:DNA ratio was determined in fish muscle tissue through a fluorometric method (Caldarone et al., 2001 adapted by Fonseca et al., 2006). RNA:DNA results for each species were corrected to remove the effect of fish length and the mean for each estuarine site determined. 2.2.2.2. New developments. Based on Vasconcelos et al. (2009) RNA:DNA ratio for an estuary was determined as the mean of anal- Table 2 Summary of metrics used to assess nursery function of main estuaries of the Portuguese coast. Metric Description Potential contribution of estuaries (measured in the estuarine environment) Juvenile density Density of juveniles in important sites for juveniles in the estuary (individuals km−2 ) Habitat quantity Area of important sites for juveniles in the estuary (km2 ) Juvenile number Total number of juveniles in important sites for juveniles in the estuary (individuals) Habitat quality Condition of juveniles in important sites for juveniles in the estuary (RNA:DNA ratio) Effective contribution of estuaries (measured in the marine environment) Overall contribution of Number of adults identified to the estuary (% of the estuary to adult analysed adults) subpopulations Contribution per unit Number of adults identified to the estuary per unit area of the estuary to area of important sites for juveniles in the estuary adult subpopulations (% of analysed adults) ysed sites within the estuary and of both sampling years, since the pattern of RNA:DNA among estuaries was preserved between years. 2.2.3. Connectivity between juveniles in estuaries and adult subpopulations in marine areas 2.2.3.1. Background data. To measure effective contributions of the analysed estuaries to adult marine subpopulations, speciesspecific natural tags were established for the different estuaries based on otolith elemental fingerprints in juveniles (for additional details see Vasconcelos et al., 2007b) and subsequently applied to retrospectively identify the estuarine juvenile source of young adults from the marine environment (for additional details see Vasconcelos et al., 2008). The use of otolith chemical composition as a natural habitat tag relies on the following premises: (1) otoliths are deposited continually and metabolically inert; (2) chemical element deposition reflects the surrounding physical and chemical environment; (3) and tags differ at the scale of the study (Campana, 1999). Thus, the environmental history of a fish at a specific time period in the past can be tracked in the corresponding otolith portion (Brown, 2006). In the present context, marine adult fish that spent their juvenile period in a particular estuary are expected to have an otolith elemental composition which reflects that estuarine life period. Briefly and firstly, otolith elemental fingerprints in juveniles from the sampled estuaries were characterized, using age-0 juveniles collected in July 2005 (ca. 10 individuals per species and estuary – each species sampled in one important juvenile site per estuary) (Vasconcelos et al., 2007b). These fingerprints were considered representative of each estuary, since intra-estuarine differences have been shown to be reduced in comparison to inter-estuarine ones (Thorrold et al., 1998). The chemical composition (Li, Na, Mg, K, Mn, Cu, Zn, Sr, Ba and Pb) of whole sagittal otoliths was analysed using a solution based inductively coupled plasma mass spectrometer (ICP-MS). For each species, juveniles were classified to collection estuaries by Linear Discriminant Function Analysis (LDFA) of otolith composition data. High percentages of correct classifications in jacknife (leave-oneout) cross-validation were obtained (70.2% for S. solea, 92.0% for S. senegalensis, 89.3% for P. flesus and 77.1% for D. labrax). Subsequently, young adults were sampled off the Portuguese coast in July 2006 (ca. 10 individuals per species and coastal area, caught by local professional fishermen – each species sampled in four coastal areas) (Vasconcelos et al., 2008). Laser ablation ICP-MS was used to determine the chemical composition of the otolith section formed whilst fish were in a nursery area (i.e. corresponding to the otoliths sampled in age-0 juveniles). Each young adult was classified to an estuary as its juvenile source using the LDFA previously parameterized with the otolith composition of juveniles. Posterior group membership probabilities were above 0.8 for most individuals (71.1% for S. senegalensis, 77.8% for P. flesus and 65.5% for D. labrax) and lower for S. solea (only 6.1% of individuals above 0.7). The contribution of an estuary to marine adult subpopulations was determined as the percentage of sampled adults classified to it as the estuary where they spent their juvenile life period. R.P. Vasconcelos et al. / Ecological Indicators 11 (2011) 1123–1133 2.2.3.2. New developments. The overall contribution of an estuary to adult subpopulations was used as in Vasconcelos et al. (2008), i.e. percentage of sampled adults classified to it as the estuary where they spent their juvenile life period. In addition, this data was combined with habitat quantity of important juvenile sites in each estuary (from Vasconcelos et al., 2010): the number of adults assigned to an estuary was expressed per unit area and the percentage of sampled adults classified to each estuary recalculated. 2.2.4. Integrated data analysis and agreement among metrics Initially, fish species were analysed individually and the pattern of variability of each metric among the several estuaries explored. The relationship between potential and effective contributions of estuaries was also analysed. Potential metrics (e.g. juvenile density) were plotted vs effective metrics (e.g. overall effective contribution) using estuaries as data points. Relationships were tested using Spearman rank correlation coefficient for each pair of metrics and Kendall coefficient of concordance test among all metrics simultaneously. 2.2.5. Multimetric assessment of nursery function to multiple species Subsequently for each metric, a collective analysis of data obtained in the present paper on all fish species was conducted based on the sum of all species per estuary. Since RNA:DNA ratio is intrinsically related with species (Dahlhoff, 2004), these data were pre-treated for this purpose. Species’ condition data for the several estuaries were normalized in order to range from 0 to 1 (for each species the value for an estuary was subtracted of the species minimum and the result divided by the range). Multi-species condition in an estuary was then determined as the mean of all species. 3. Results 3.1. Multi-metric assessment of nursery function to individual species Estimated potential and effective contribution of estuaries varied with applied metrics (Fig. 2 ) and estuarine contributions were notably different among species. Higher densities of juvenile S. solea occurred in the Mondego and Mira estuaries (19.9 × 103 individuals km−2 and 7.3 × 103 individuals km−2 , respectively). Higher habitat quantity was found in Tejo and Ria de Aveiro (11.5 km2 and 9.2 km2 , respectively), whilst number of juveniles was highest in the Tejo estuary (29.1 × 103 individuals). Habitat quality was higher in Douro and also in Ria de Aveiro and Tejo (5.1, 4.0 and 3.9, respectively). Most adults were classified to the Tejo and Mondego estuaries as their juvenile sources (45.4% and 39.4%, respectively). Overall, the Tejo estuary contributed most to marine subpopulations and was identified as EJH, whilst the Mondego estuary as nursery due to the high contribution per unit area of its juvenile sites (88.5%). Juvenile density, habitat quantity and number of S. senegalensis were higher in Tejo and Sado (4.6 × 103 individuals km−2 and 3.1 × 103 individuals km−2 , 13.1 km2 and 8.4 km2 , 60.0 × 103 individuals and 26.5 × 103 individuals, respectively). Habitat quality was higher in Ria de Aveiro and Sado (4.7 and 4.6, respectively). The Sado estuary was identified both as EJH and nursery as it had the highest contribution to marine subpopulations overall (52.9%) and per unit area (49.2%). Densities of P. flesus were higher in the Douro (11.4 × 103 individuals km−2 ), as well as habitat quantity (2.3 km2 ) and number of juveniles (26.1 × 103 individuals). Higher habitat quality increased towards northern estuaries with highest values in Douro (4.2). The Douro estuary had the highest overall contribution to marine adult subpopulations (53.3%) and corresponded to an EJH, whilst the 1127 Mondego estuary had the highest contribution per unit area (50.8%) and was classified as a nursery. Juvenile D. labrax had higher densities in Mondego, Ria de Aveiro and Tejo (9.7 × 103 individuals km−2 , 7.3 × 103 individuals km−2 and 5.3 × 103 individuals km−2 , respectively). Habitat quantity was higher in Douro and Sado (2.8 km2 and 2.5 km2 ) whilst number of juveniles was higher in Tejo (9.6 × 103 individuals). Habitat quality was slightly higher towards southern estuaries with highest values in Mira, Tejo and Mondego (5.4, 5.3 and 5.2, respectively). The Mondego estuary had the maximum contributions to adult subpopulations overall (41.4%) and per unit area (57.9%) and was classified as both EJH and nursery. 3.2. Agreement among metrics Regarding the potential contribution of estuaries, juvenile density, habitat quantity and number of juveniles had analogous patterns of variation among estuaries, for S. senegalensis and P. flesus (Fig. 2). For the remaining species, in many cases higher densities of juveniles occurred in estuaries with smaller habitat quantity and this was particularly evident for S. solea. The combination of the first two metrics determine variations in the number of juveniles among estuaries, and for S. solea and D. labrax ranking of estuaries did not follow the pattern of either juvenile density or habitat quantity. Habitat quality related differently with the remaining potential metrics according to species, namely inversely in S. solea. Several tendencies were observed in the relationships between metrics (and hereon addressed qualitatively), albeit no significant correlations were found between potential and effective metrics (Figs. 2 and 3). Juvenile density and overall effective contribution of estuaries varied concurrently in P. flesus and D. labrax, whilst a relationship with effective contribution per unit area was observed in S. solea and D. labrax. Habitat quantity and effective contribution (overall) had a similar trend in P. flesus. The number of juveniles in an estuary and the number of adults classified to that estuary (overall) as a juvenile source showed similar trends in S. solea, P. flesus and D. labrax. However, no relationship was observed between estuarine habitat quality and the effective contribution of these systems, either overall or per unit area. Concordance among all metrics, via Kendall’s coefficient, was not significant for any of the species. 3.3. Multi-metric assessment of nursery function to multiple species Estuaries differed in the number of analysed species present. All four were only present in Ria de Aveiro and Mondego (Table 3). Overall, juvenile density of all species was higher in Mondego (34.8 × 103 individuals km−2 , respectively). Habitat quantity used by juveniles of multiple species differed among systems (higher in Tejo – 13.3 km2 and lower in Mondego – 0.6 km2 , respectively), as well as in the proportion relatively to the total estuarine area (maximum of 28.0% in Douro and 22.0% in Mira) and in the proportion relatively to the habitat quantity used by juveniles in all estuaries (higher in Tejo – 37.6% and lower in Mondego – 1.7%). Total number of juveniles was higher in Tejo (98.7 × 103 individuals, accounting for 42.5% of juveniles in all estuaries). Habitat quality for all species was higher in Douro and Sado (1 and 0.9, respectively). In the Mondego, Tejo and Mira estuaries, important sites for juveniles were dominated by multiple species simultaneously (100.0%, 98.5% and 100.0% of habitat quantity of important sites for juveniles in the estuary, respectively). Overall, the effective contribution from an estuary to adult subpopulations was highest in the Tejo and Mondego estuaries (26.6% and 26.0%, respectively) and these were identified as EJH. Habitat quantity in estuaries to which adults from these species were 1128 R.P. Vasconcelos et al. / Ecological Indicators 11 (2011) 1123–1133 Fig. 2. 1129 R.P. Vasconcelos et al. / Ecological Indicators 11 (2011) 1123–1133 Table 3 Potential and effective contribution of main estuaries along the Portuguese coast to adult marine populations of multiple species: Solea solea, Solea senegalensis, Platichthys flesus and Dicentrarchus labrax. Estuaries considered are: D – Douro, RA – Ria de Aveiro, Mo – Mondego, T – Tejo, S – Sado and Mir – Mira. Potential contribution of estuaries: number of fish species, juvenile density, habitat quantity, juvenile number and habitat quality in important sites for juveniles, considering the four fish species; and habitat quantity of important sites for juveniles used simultaneously by multiple species. Effective contribution of estuaries (assessed through otolith elemental fingerprints): habitat quantity of sites where adults spent their juvenile period; contribution of estuaries to marine adult subpopulations in percentage of marine fish identified to that estuary overall (in accordance to the Effective Juvenile Habitat definition by Dahlgren et al., 2006), and per unit area of important sites for juveniles (in accordance to the nursery definition by Beck et al., 2001). Metric Unit Estuary D RA Potential contribution: important estuarine sites for juveniles Species (n) 3 4 12.9 3.5 Juvenile density (×103 individuals km−2 ) 2 Habitat quantity (km ) 2.8 9.2 (% of estuary) 28.0 12.4 (% of juvenile sites in all 7.9 26.0 estuaries) 36.1 32.6 Juvenile number (×103 individuals) (% of juveniles in all estuaries) 15.5 14.0 Habitat quality (RNA:DNA) 1.0 0.5 Potential contribution: important estuarine sites for juveniles used simultaneously by multiple species 2.3 4.6 Habitat quantity (km2 ) (% of juvenile sites in the 82.1 50.0 estuary) Effective contribution 2.3 9.2 Habitat quantity (km2 ) (% of juvenile sites in all 6.6 26.4 estuaries) Contribution to adults (% of all adults) 15.6 16.8 (% of all adults per unit area) 11.9 3.2 assigned differed among estuaries and the contribution per unit area for all species was markedly higher in the Mondego estuary (75.9%) and the latter was identified as nursery. 4. Discussion 4.1. Multi-metric assessment of nursery function Differential nursery function of estuaries along the Portuguese coast for four commercial marine fish species (S. solea, S. senegalensis, P. flesus and D. labrax) was quantitatively explored based on estimates of the potential contribution of estuaries and effective connectivity with marine areas. For each species, results indicated that estuaries differ in their effective contribution to marine adult subpopulations and therefore in their role as EJH and/or nurseries, sensu Dahlgren et al. (2006) and Beck et al. (2001), respectively. Connectivity between juveniles in estuarine/coastal environments and adult marine subpopulations has been evaluated through otolith elemental fingerprints by quantifying contributions of different habitat types (Brown, 2006; Forrester and Swearer, 2002; Gillanders and Kingsford, 1996; Yamashita et al., 2000) and individual or groups of estuaries (Gillanders, 2002), thus providing an assessment of their importance as EJH. More recently effective connectivity has been addressed in synchrony with habitat quantity allowing measurements of nursery value (i.e. per unit area contribution) of distinct habitat types (Fodrie and Levin, 2008) or regions within an estuary (Chittaro et al., 2009). Present approach provided baseline knowledge on the importance of these estuaries as nursery and EJH, whilst exploring the relationship between the potential contribution of individual estuaries (i.e. pre-recruitment estimates) Mo T S Mir 4 34.8 0.6 6.0 1.7 3 7.4 13.3 4.2 37.6 3 3.9 8.4 4.7 23.7 2 9.6 1.1 22.0 3.1 20.9 9.0 0.4 98.7 42.5 0.4 33.5 14.4 0.9 10.6 4.6 0.5 0.6 100.0 13.1 98.5 6.7 79.8 1.1 100.0 0.6 1.7 13.3 38.1 8.4 24.1 1.1 3.2 26.0 75.9 26.6 3.5 13.2 2.8 1.7 2.7 and their effective contribution. Moreover, estuarine nursery function was viewed as a whole since the study considered multiple species that simultaneously use these estuaries during the juvenile phase. Ranking estuaries in terms of their nursery value for a single species, according to the nursery or EJH concepts, resulted in two distinct scenarios: (1) single estuaries were both nursery and EJH for a species (S. senegalensis and D. labrax) since the highest effective contributions resulted from estuaries with smaller juvenile areas; and (2) nursery and EJH were assigned to different estuaries (S. solea and P. flesus) since highest effective contributions of juveniles were from estuaries with largest juvenile distribution areas. Chittaro et al. (2009) and Fodrie and Levin (2008) also attained distinct results with EJH and nursery approaches to the importance of habitat types and sites, respectively. Estuaries with high contributions per unit area are valued with the nursery concept, whilst estuaries with smaller contributions per unit area but which overall contribute significantly to adult subpopulations are valued with the EJH concept. In general, these results support the rationale for considering both criteria to answer scientific and management objectives. Results indicated a species-specific contribution of these estuaries. No estuary was best for all species and distinct estuaries were considered important depending on species: namely Douro (EJH for P. flesus), Tejo (EJH for S. solea) or Sado (nursery and EJH for S. senegalensis) whereas the Mondego stood out as nursery for three species (S. solea, P. flesus and D. labrax) and EJH for D. labrax. This emphasized the importance of species-specific dynamics in the regulation of nursery function and the need to further develop multi-species assessments to understand the role of these areas for coastal populations and the need for their integrated management. Fig. 2. Potential and effective contributions of main estuaries of the Portuguese coast to marine adult populations of individual species: Solea solea, Solea senegalensis, Platichthys flesus and Dicentrarchus labrax. Potential contribution of analysed estuaries for each fish species: juvenile density (individuals km−2 , represented as inds. km−2 ), habitat quantity (km2 and percentage of the total area of juvenile sites in all estuaries), juvenile number (individuals, represented as inds., and percentage of the total number in all estuaries) and habitat quality (RNA:DNA) in important juvenile sites. Effective contribution of analysed estuaries for each fish species: percentage of marine adults classified to an estuarine juvenile source (overall and per unit area of important sites for juveniles in that estuary) via otolith elemental fingerprints. For each species, an estuary was identified as effective juvenile habitat (EJH), i.e. highest overall effective contribution following Dahlgren et al. (2006), and nursery, i.e. highest per unit area contribution following Beck et al. (2001). Estuaries considered are: D – Douro, RA – Ria de Aveiro, Mo – Mondego, T – Tejo, S – Sado and Mir – Mira. 1130 R.P. Vasconcelos et al. / Ecological Indicators 11 (2011) 1123–1133 Adults in marine habitat ientified to an estuary overall (% of all adults) d) Adults in marine habitat identified to an estuary per unit area (% of all adults) c) 100 80 60 40 20 0 Adults in marine habitat identified to an estuary overall (% of all adults) b) S. solea 100 80 60 40 20 0 100 80 60 40 20 0 Adults in marine habitat identified to an estuary per unit area (% of all adults) a) 100 80 60 40 20 0 h) 0 5 10 0 5 10 15 20 Juvenile density in estuaries (x 10 3 individualskm -2) D. labrax 0 5 10 15 20 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 Habitat quantity in estuaries (% of all sites) 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 Number of juveniles in estuaries (% of all juvenile s) 0 20 40 60 80 100 100 80 60 40 20 0 Adults in marine habitat identified to an estuary overall (% of all adults) g) 5 10 15 20 P. flesus 100 80 60 40 20 0 100 80 60 40 20 0 Adults in marine habitat identified to an estuary per unit area (% of all adults) f) Adults in marine habitat identified to an estuary per unit area (% of all adults) Adults in marine habitat identified to an estuary overall (% of all adults) e) 0 S. senegalensis 100 80 60 40 20 0 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 Habitat quality in estuaries (RNA:DNA) 0 2 4 6 8 10 Fig. 3. Relationship between potential and effective contributions of main estuaries of the Portuguese coast to marine adult populations of individual species: Solea solea, Solea senegalensis, Platichthys flesus and Dicentrarchus labrax. Potential contribution metrics are plotted on X-axis (juvenile density, habitat quantity, juvenile number and habitat quality) and effective contribution metrics are plotted on the Y-axis (percentage of marine adults identified to an estuary as their juvenile source overall and per unit area of important sites for juveniles). Each data point represents an estuary. Paired metrics are: (a) juvenile density vs overall effective contribution, (b) juvenile density vs effective contribution per unit area, (c) habitat quantity vs overall effective contribution, (d) habitat quantity vs contribution per unit area, (e) juvenile number vs overall contribution, (f) juvenile number vs effective contribution per unit area, (g) habitat quality vs overall contribution, (h) habitat quality vs effective contribution per unit area. Estuaries considered are Douro, Ria de Aveiro, Mondego, Tejo, Sado and Mira. R.P. Vasconcelos et al. / Ecological Indicators 11 (2011) 1123–1133 Assessing the nursery function of estuaries for multiple species, based on the sum of all species’ effective contributions, pursued a guild approach, focusing the use of estuaries by marine migrant species (Franco et al., 2008). In this assessment, Tejo was considered an EJH and Mondego a nursery. Whereas the nursery criterion attained some analogous results to those for individual species, the EJH underlined the summed importance of the Tejo for several species in terms of overall contribution, since it was the EJH for S. solea and the second best for S. senegalensis and D. labrax. The importance of estuaries successfully used by a higher number of species and with higher effective contributions to the marine subpopulations of several species is outlined. Interestingly, the Mondego and Tejo estuaries, identified as important multi-species nursery and EJH along this coast, respectively, represent contrasting cases of anthropogenic disturbances. Despite the low level of anthropogenic pressures in Mondego, this system presents a high natural vulnerability to disturbances, particularly in terms of water quality (Vasconcelos et al., 2007a). Contrarily, although Tejo has the highest level of anthropogenic pressures it is the largest estuary along the Portuguese coast and possesses a low natural vulnerability (Vasconcelos et al., 2007a). Such contrasting cases, in terms of nursery function (per unit area or overall), anthropogenic pressures and natural vulnerability imply specific management approaches. Knowledge on the use of particular estuarine areas or habitats by juveniles of single species (e.g. Cabral and Costa, 1999; Goldberg et al., 2002; Lazzari, 2008) and on enhanced growth or survival (e.g. Fonseca et al., 2006; Phelan et al., 2000; Ross, 2003) has provided strong support towards identifying essential fish habitats as the basis for healthy fisheries and efficient management strategies (Rosenberg et al., 2000). However, this knowledge may be insufficient (Beck et al., 2001). The development of integrated management plans for these areas and species should ultimately recognize sites or habitats used by juveniles and that effectively maintain adult subpopulations, in particular for multiple species simultaneously. Such identification is paramount to obtain the highest returns in terms of ecological and economical resources and functions as well as to optimize management and conservation efforts. Since nursery and EJH criteria may render converse results, an adequate definition of scientific, economic and management objectives is essential to respond to the established purposes. Despite the widely recognized value of nursery and EJH as testable hypotheses frameworks, both imply recognizing [according to Sheaves et al. (2006) and Layman et al. (2006)]: (1) the importance of habitat complexity and inter-connectivity; (2) that EJH is scale-independent, and scale may be defined in the way most relevant for the core questions, but any potential effects must be considered; and (3) the structure, function and dynamics of complex ecosystems and processes related with juveniles. By considering the main areas (and habitats they include) used by juveniles in each estuary the present approach accounts for ecosystem complexity and the study scale is objectively defined and explored. High quality nursery habitats are those where growth, survival and future reproductive potential are optimized (Gibson, 1994). Since reproductive output may also affect the overall value of a potential nursery habitat (Sheaves et al., 2006) an alternative to assess effective contribution may be to estimate population fitness, e.g. via population growth (Fodrie et al., 2009). Larval settlement in estuarine nurseries is the result of numerous processes, with larvae supply and available habitat as main promoters of differences in the densities of newly settled individuals. Links among larval sources and sinks, dispersal potential, realized transport and successful recruitment are rarely quantified (Swearer et al., 2002; Almany et al., 2007) and the extent to which these ecological strategies affect connectivity is still uncertain. 1131 Population size is considered to be regulated at larval life stages, with substantial mortality in the early post-settlement period in estuaries, and carrying capacity of nursery areas not reached (Rooker et al., 1999; van der Veer et al., 2000). Survival in estuarine environments has received little attention. Multiple biotic and abiotic factors control mortality rates and migration, therefore influencing recruitment to the habitats used by adults. However, dissociating natural mortality/survival and movement/emigration from the nurseries can be a difficult task (Nagelkerken, 2009). 4.2. Agreement among metrics Selecting efficient methodologies is key in ecological assessments. The relationship between potential (i.e. expected) and effective (i.e. realized) contributions of different juvenile sources was explored at the level of individual estuaries. Results suggest a possible relationship between juvenile density and effective contribution of estuaries, and also between number of juveniles and effective contribution. However, considering the disparities among results of the several species, the inter-dependence of indicators of potential contribution as well as the dynamics of these systems and populations further research on this relationship is advocated. Increasing the set of sampled estuaries should allow a more robust statistical analysis. Nevertheless, comprehensive data on juveniles in estuaries may be proven adequate evaluators of subsequent connectivity from nursery grounds. A match between potential and effective contributions was found by Fodrie and Levin (2008) analysing different habitat types. An agreement between potential and effective contributions is in line with current knowledge on the regulation of recruitment to adult subpopulations, particularly the minor role of juvenile stage processes in generating variability. In effect, variability in year class strength of marine estuarine-opportunist species has been assigned mostly to processes occurring during the earlier life stages, e.g. larval and post-settlement stages, whereas factors affecting juvenile, subadult and adult fish have lower influence (Le Pape et al., 2003b; Levin and Stunz, 2005). Juvenile densities are still commonly used as a sole measure of nursery function (Able et al., 2006; Cabral et al., 2007; Ross, 2003). Even though, in the present study, higher juvenile densities were associated in some species with higher nursery role or EJH that was not the general case. Likewise, little correspondence between the effective contribution of regions within an estuarine system and their mean juvenile densities has been reported (Chittaro et al., 2009). Elsewhere, juvenile densities have been a good indicator of effective unit area productivity of habitats and suggested that variability of recruitment pulses to adult stocks are not dampened by density-dependent nursery-ground processes (Fodrie and Levin, 2008). Although poorly indicative of effective contribution in the present study, as in Chittaro et al. (2009), habitat quantity has been previously applied to estimate the potential contribution of different habitat types to adult stocks, namely by modelling annual variation of juvenile distribution and densities to identify important habitats for fish juveniles (Le Pape et al., 2003a; Riou et al., 2001). On the other hand, although in some species higher total number of juveniles (estimated by combining juvenile density and the size of distribution area) seemed to indicate higher overall effective contribution of estuaries, this was not an overall trend. Even though density-dependent processes may entail depressed maximum juvenile growth in some sites, measured juvenile condition was always indicative of good nutritional status and elevated growth rates (Vasconcelos et al., 2009) and its variability among systems was poorly related with effective contribution to adult subpopulations. Overall, the relationship among juvenile density, habitat quantity and habitat quality and with effective estimates of juvenile recruitment from estuarine areas, advises 1132 R.P. Vasconcelos et al. / Ecological Indicators 11 (2011) 1123–1133 against their independent use as proxies of successful nursery function. Based on adult and juvenile distributions, densities and their inter-annual variability, recruitment levels of species which use confined nursery grounds have been related with habitat availability or quantity, as ultimately these define the carrying capacity of the nursery (Gibson, 1994; Iles and Beverton, 2000; Le Pape et al., 2003a; Manson et al., 2005; Rijnsdorp et al., 1992). Concurrently, Fodrie and Levin (2008) stated that California halibut populations could be nursery-habitat limited however, not by density-dependent growth or mortality during the juvenile phase but likely by density-dependent settlement or juvenile emigration from already occupied nurseries (Schmitt and Holbrook, 2000). Despite the effects of habitat quantity on population size, recruitment levels of juvenile flatfishes from nursery grounds are a result of the interplay between both habitat quality and quantity (Gibson, 1994) as also evidenced by higher juvenile densities and lower juvenile condition in S. solea and P. flesus (Vasconcelos et al., 2009). The variations in metrics of potential contribution among these estuaries, suggest the analysed juvenile sites and respective habitats may have a differential maximum nursery capacity determined by the combination of habitat quantity, quality and estuarine colonization processes (Brown et al., 2005; Rijnsdorp et al., 1992). Data on connectivity evidenced differential contributions between estuaries and the marine environment yet increasing adult sample size and the inclusion of a wider spatial scale with estuaries and coastal areas beyond the present study area is recommended to improve the strength of effective contribution estimates. Moreover, inter-annual changes in larval supply to estuarine nursery grounds can be reflected in larvae settlement, whilst variability in environmental conditions in estuaries may also affect juvenile growth and distribution. Therefore, knowledge on temporal consistency of estuaries/sites which act as nursery or EJH through time is of the utmost importance. In addition, future research should be directed towards the ultimate objectives of determining exchange rates and dependence levels among closely or distantly related meta-populations of these species. Acknowledgements The authors express their gratitude to all involved in fish sampling and processing. 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