Doctoral Programme on Marine Ecosystem Health and Conservation
 PhD Subject Catalogue Fifth Edition - 2014
Assessing the connectivity, fish movement patterns and population structure of sparid species in SW Portugal using otolith fingerprints as a tool for a rational management of fisheries resources.
PhD Code: MARES_14_17:
  • Host institute 1: P14 - Interdisciplinary Centre for Marine and Environmental Research (CIIMAR)
  • Host institute 2: P4 - Galway Mayo Institute of Technology (GMIT)
  • Host institute 3: P5 - University of Algarve (UAlg)
Research fields:
  • T4 - Natural Resources: overexploitation, fisheries and aquaculture
  • Jorge Gonçalves
  • Deirdre Brophy
  • Aberto Correia (CIIMAR)
Contact Person and email: Alberto Correia -

Subject description
Scientific Background
Spondyliosoma cantharus, Diplodus vulgaris and Diplodus sargus are important sparid species found in the NE Atlantic, from Great Britain to Senegal, including Madeira, Azores and Canaries Archipelagos, and also in the Mediterranean [1]. These coastal fish live on rocky and sandy substrates and in seagrass meadows, at depths ranging between 50 and 160 m [2]. Adults spawn offshore in deep waters, during winter and spring, with reproductive strategies varying between species [3,4,5]. During spring/summer, and after 1-2 months of pelagic life, the larvae are transported by oceanographic currents towards inshore shallow nursery areas, such as coastal lagoons, estuaries and artificial reefs, where they settle and remain for several months [6]. After reaching 4.5 to 5.5 cm in length, the juveniles disperse outside the nursery area to join the adult population [7,8,9,10]. Sparids are a valuable resource in the south of Portugal [11], being a target for gill nets and longlines used by artisanal fisheries [9]. Landings of sea breams in general and for most Sparidae species have shown a steady decline over the past ten years. However, no regulations are currently imposed on this fishery, other than minimum landing sizes and catch quotas [12]. Despite its fishery value and ecological importance, the existing studies regarding the population structure, fish movement patterns and connectivity are scarce in SW Portugal [13,14,15]. Recent studies show the existence of spatially distinct elemental tags in otoliths of D. vulgaris juveniles (age 0+) collected in several Portuguese estuaries [16]. However the attempt to assess the contribution of these nurseries to the adult recruitment failed, probably because it did not take into account some unidentified nursery areas [17]. Otolith signatures of young adults (age 2+) suggest that D. vulgaris captured on the Portuguese SW coast is resident in the regional fishing areas during the adult stage. However, it could not be conclusively determined whether or not recruitment to these grounds was derived from different spawning and/or nursery areas [14]. For the above-mentioned species, it is unclear, at present, whether young juveniles that recruit to different inshore nursery areas are derived from different spawning sources and which inshore nursery areas are important sources of recruits to the adult spawning populations and fishery grounds in different coastal regions [14, 15]. Although genetic techniques are useful for studying fish population structure, they are often inconclusive if gene flow is sufficient to maintain genetic homogeneity across populations that are widely separated and potentially characterised by different local recruitment, growth and mortality rates [19]. This may often be the case in meta-population complexes, where sub-populations may have similar genetics but for management purposes should be treated independently. Resolving meta-population structures is a problem in fisheries management because of the difficulty of quantifying connectivity rates or interdependency of replenishment among populations in different areas. Otolith chemistry and shape are non-genetic methods to investigate population structure for fish, since it provides one alternative solution for addressing such questions of connectivity and separation between spawning and juvenile sources and adult stocks [20, 21, 22, 23]. The purpose of this work will be to assess the utility of chemical signatures and shape analysis of otoliths to provide information on the population structure, movement patterns and connectivity of some sparid fish (Diplodus vulgaris, Diplodus sargus and Spondyliosoma cantharus) along the Portuguese southwest coast.
  • 1. To record the otolith fingerprints of young juveniles collected in the main known nurseries areas (estuaries and coastal lagoons), including some artificial reefs, to construct an atlas of baseline signatures. 
  • 2. To use natal signatures to identify juvenile origin of 2 year old adult fish from the same cohort and assess connectivity of nursery grounds and adult habitats (including the contribution of the artificial reefs as alternative nurseries for sea breams);
  •  3. To compare habitat quality between nursery areas and its influence on growth, survival and recruitment rate success, using biochemical (RNA:DNA and TAG:ST), morphometric (Fulton’s K) and growth (marginal otolith increment width) based fish condition indices; 
  • 4. To use otolith composition and shape of whole otoliths (i.e. entire life history prior to capture) to distinguish between adult fish from different fishing grounds and evaluate underlying stock structure; 
  • 5. To formulate and recommend appropriate measures for fisheries conservation and rational management of sparids.
Biological Sampling: juveniles (age 0+) will be collected in spring 2016 using beach seine and beam trawl in the estuaries and coastal lagoons known as nurseries grounds for sea breams [8] (Mondego, Tejo, Sado, Mira, Ria Formosa, Alvor, Arade, Guadiana, Odiel and Guadalquivir). Additional samples obtained by fish traps from artificial reefs off Algarve will be included [10]. Adults (age 2+) will be collected in the SW fisheries grounds (Mira, Sesimbra, Sines, Sagres, Portimão, Olhão and Vila Real Santo António) [9] in spring 2018 through the artisanal fleets using trammel nets, gillnets and longlines. Information about the location of capture will be requested from fishermen. If available, some adults specimens from Madeira, Azores, Canaries and Mediteranean will be included as outgroups. Individuals from the same age (cohort) will be used. The ages will be later confirmed by absence of annual ring (0+) or by counting the annual growth increments (2+) [14,15]. A sub-sample of thirty individuals (0+ and 2+) will be selected from each nursery and fishery ground for further analyses. A few specimens from 2017 (juveniles or adults) will be used to assess the temporal homogeneity of the otolith (edge) signatures. Fish will be measured, weighed and frozen for later analyses. Otoliths will be carefully extracted, differentiated to left and right sagitta and stored. A portion of muscle will be extracted and preserved in ethanol for RNA/DNA analysis.
Age Determination: The annual growth increments will be counted using, preferentially, non-destructive methodologies. Otoliths will be immersed in a clearing mixture and annuli counted using a stereoscopic microscope under reflected light against a dark background at 25X magnification. If needed, otoliths will be mounted in epoxy resin and slight sections will be made using a low speed saw. The age will be estimated by counting the translucent increments, taking into consideration the assumed date of birth (1st January) and the date of capture. Three readers will age each otolith independently. Only fish with 100% concordance between age readings will be used [14,15].
Otolith Microchemistry: Otolith fingerprint analysis will focus on the core region of the otoliths (natal origin) from both young juveniles (0+: 2014) and adults (2+: 2016) using laser ablation inductively coupled plasma mass spectrometry (ICP-MS-LA). Sagittae will be cleaned of organic tissues, rinsed with Milli-Q water, air dried and mounted in epoxy. Transverse sections will be taken through the primordium with a diamond saw. Otolith sections will be grounded with silicon carbide papers and further polished with diamond pastes until the core became visible. The polished sections will be fixed on microscope slides with epoxy resin, sonicated in Milli-Q water, further rinsed and dried in a laminar flow cabinet. Data will be collected for a range of isotopes already known to be informative for these species (88Sr, 138Ba, 55Mn, 25Mg, 7Li, 65Cu, Pb208 and 66Zn) along with 43Ca which is used as the internal standard [14,15]. The laser spot diameter will be of 200 µm which corresponds to the otolith section in age 0+ juveniles [17]. Calibration will be achieved with the National Institute of Standards (NIST) and accuracy assessed using a standard reference otolith material (FEBS-1). The core isotopic composition (16O/18O and 13C/12C) of the otoliths will be also performed by Isotope Ratio Mass Spectrometry (IRMS) using a high-resolution computer-controlled micromill sampling device. Edge analysis (ICP-MS-LA) of a few juveniles or adults collected in two consecutive years will be also assessed (temporal stability of the fingerprint). Whole otolith analysis of adults will be made through ICP-MS-SB (solution based) and IRMS using standard protocols [14,15]. 
Otolith Shape Analysis: Otoliths will be photographed for shape analysis of the outline using fourier descriptors and shape indices (circularity, aspect ratio, roundness and solidity). Morphometric measurements and elliptical fourier coefficients will be obtained using ImageJ V.1.4.3 and Shape V.1.3 softwares, respectively. Data will be analysed multivariate statistical procedures after removing the possible confounding effect related with different fish sizes [21,22,23].
Fish Condition Indices: The use of general well being proxies for juveniles (0+), such as biochemical (RNA:DNA and TAG:ST) morphometric (Fulton’s Condition, K) and growth (Otolith Marginal Increment Width index, OG) will allow the habitat quality of the nurseries to be assessed. K will be calculated by the formula K=100 (W/L3) where W is the eviscerated body mass (mg) and L is the standard length (mm). RNA:DNA concentrations for each individual will be obtained using ethidium bromide through the fluorimetric method. ATG/ST (triacylglycerols:sterols) ratio will be quantified following an extraction of the lipids using standard protocols. OG is the mean otolith daily increments width during the last 10 days prior to capture [18]. 
Statistics: Data will be checked for normality and homogeneity of variances prior to statistical analysis. If needed, data will be transformed as appropriate to conform to distributional assumptions. Univariate and multivariate tests will be made in accordance with the specific objective of each study. One-Way ANOVA, followed by the Tukey pos-hoc test, if needed, will be used to discriminate mean differences against several groups (e.g. variables: µg element g-1 of calcium; Factor: location). MANOVA will be used to test for spatial and temporal differences in otolith elemental signatures. Locations and year will be treated as fixed factors. For the MANOVA, we will report the approximate F-ratio statistic for the most robust test of multivariate statistics (Pillai’s trace). Post-hoc multivariate pairwise comparisons between locations will be performed using the Hotelling T-square test. Linear or Quadratic Discriminant Function Analyses (LDFA or QDFA) will be use to visualize spatial differences and to examine the re-classification accuracy success of fishes to this original location. Cross validations will be performed by using jackknife (‘‘leave one out’’) procedures [19]. Additionally, Maximum Likelihood Analysis (MLA) of the juvenile core otolith chemistry data (natal baseline) will be used to determine the contributions of the nurseries areas to the adults stocks using the HISEA programme. For each cohort, the simulation mode with 1000 simulations will be used to estimate the variability of the estimator (i.e. baseline data). Bootstrapping (1000 re-samplings) of the baseline and mixed sample data will be used to estimate the mean and standard deviations of the proportions of adults originating from each nursery area.
Consortium and Supervision team
The 3-year PhD proposal has been designed to provide an integrated and multidisciplinary doctoral training across three leading European marine research institutes (CIIMAR, GMIT and UAlg). The three institutes will provide complementary expertise, equipment and facilities to the candidate. Furthermore partners have within national and European ongoing research projects that will support field trips, laboratorial and analytical costs. All the supervisors (Alberto Correia, Deirdre Brophy and Jorge Gonçalves from CIIMAR, GMIT and UAlg, respectively) are independent senior researchers holding a PhD in Biological Sciences. They worked in the last 10-15 years in Marine Biology and Fisheries Sciences with a broad experience in the use of otolith chemical signatures to study fish population structure, to track migratory patterns, to assess connectivity issues and to discriminate fish stock, including sea breams. They have very good track record in high rated journals, participate regularly in international conferences and have a large post-graduate teaching experience. All have/had several national and international funded projects in the Marine Sciences area, including as principal investigator.
  • [1] Bauchot ML, Hureau JC. 1986. Sparidae. In: Whitehead PJP, Bauchot ML, Hureau JC, Nielsen J, Tortonese E. Eds. Fishes of the North-eastern Atlantic and the Mediterranean, vol. II. UNESCO, Paris, pp. 883–907.
  • [2] Vigliola L, Harmelin-Vivien ML, Biagi F, Galzin R, Garcia-Rubies A, Harmelin JG, Jouvenel J Y, Le Direach-Boursier L, Macpherson E, Tunesi L (1998). Spatial and temporal patterns of settlement among sparid fishes of the genus Diplodus in the northwestern Mediterranean. Mar Ecol Prog Ser 168: 45-56.
  • [3] Divanach P. 1985. Contribution à la connaissance de la biologie et de l’élevage de 6 Sparidés méditerranéens: Sparus aurata, Diplodus sargus, Diplodus vulgaris, Diplodus annularis, Lithognathus mormyrus, Puntazzo puntazzo (Poissons Téléostéens). Thése d’Etat. Univ Sci Tech Languedoc
  • [4] Gonçalves JMS, Erzini K. 2000. The reproductive biology of Spondyliosoma cantharus (L.) from the SW Coast of Portuga. Sci Mar 64: 403-411.
  • [5] Buxton CD, Garratt PA. 1990. Alternative reproductive styles in seabreams (Pisces: Sparidae). Environ Biol Fish 28: 113-124.
  • [6] Macpherson E. 1998. Ontogenetic shifts in habitat use and aggregation in juvenile sparid fishes. J Exp Mar Biol Ecol 220: 127-150.
  • [7] Gonçalves JMS, Bentes L, Coelho R, Correia C, LinoPG, Monteiro CC, RibeiroJ, Erzini K .2003. Age and growth, maturity, mortality and yield-per-recruit for two banded bream (Diplodus vulgaris Geoffr.) from the south coast of Portugal. Fish Res 62: 349–359.
  • [8] Vinagre C, Cabral HN, Costa MJ. 2010. Relative importance of estuarine nurseries for species of the genus Diplodus (Sparidae) along the Portuguese coast. Estuar Coast Shelf Sci 86: 197-202.
  • [9] Erzini K, Gonçalves JM S, Bentes L, Lino PG, Cruz J. 1996. Species and size selectivity in a Portuguese multispecies artisanal long-line fishery. ICES J Mar Sci 53: 811–819.
  • [10] Santos MN, Leitão F, Moura A, Cerqueira M, Monteiro CC. 2011. Diplodus spp. on artificial reefs of different ages: influence of the associated macrobenthic community . ICES J Mar Sci 68: 87-97.
  • [11] INE. 2009. Estatísticas da Pesca 2008. Instituto Nacional De Estatística, Lisboa.
  • [12] FAO. 2005. Fishery and Aquaculture Country Profiles. Portugal. Available in: Accessed in 22th of September 2013.
  • [13] Abecasis D, Bentes L, Erzini K. 2009. Home range, residency and movements of Diplodus sargus and Diplodus vulgaris in a coastal lagoon: Connectivity between nursery and adult habitats. Estuar Coast Shelf Sci 85: 525-529.
  • [14] Correia AT, Pipa T, Gonçalves JMS, Erzini K, Hamer PA, 2011. Insights into population structure of Diplodus vulgaris along the SW Portuguese coast from otolith elemental signatures. Fish Res 111: 82– 91.
  • [15] Correia AT, Gomes P, Gonçalves JMS, Erzini K, Hamer PA. 2012. Population structure of the black seabream Spondyliosoma cantharus along the south-west Portuguese coast inferred from otoliths chemistry. J Fish Biol 80: 427-443.
  • [16] Reis-Santos P, Vasconcelos RP, Ruano M, Latkoczy C, Gunther D, Costa MJ, Cabral H. 2008. Interspecific variations of otolith chemistry in estuarine fish nurseries. J Fish Biol 72: 2595–2614.
  • [17] Vasconcelos RP, Reis-Santos P, Tanner S, Maia A, Latkoczy C, Günther D, Costa MJ, Cabral H. 2008. Evidence of estuarine nursery origin of five coastal fish species along the Portuguese coast through otolith elemental fingerprints. Estuar Coast Shelf Sci 79: 317-327.
  • [18] Vasconcelos RP, Reis-Santos P, Fonseca V, Ruano M, Tanner S, Costa MJ, Cabral HN. 2009. Juvenile fish condition in estuarine nurseries along the Portuguese coast. . Estuar Coast Shelf Sci 82:128-138.
  • [19] Correia AT, Ramos AA, Barros F, Silva G, Hamer P, Morais P, Cunha RL, astilho R (2012) Population structure and connectivity of the European conger eel (Conger conger) across the Northeastern-Atlantic and Western-Mediterranean: integrating molecular and otolith elemental approaches. Mar Biol 159:1509-1525
  • [20] Campana SE, Chouinard GA, Hanson JM, Fréchet A, Brattey J. 2000. Otolith elemental fingerprints as biological tracers of fish stocks. Fish Res 46: 343–357.
  • [21] Paul K, Oeberst R, Hammer C. 2013. Evaluation of otolith shape analysis as a tool for discriminating adults of Baltic cod stocks. J Appl Ichth 29:743–750.
  • [22] Stransky C. 2013. Morphometric Outlines. In Stock Identification Methods: Applications in Fishery Science Second Edition. Editors Steven X. Cadrin, ‎Lisa A. Kerr, ‎Stefano Mariani Published by Elsevier.
  • [23] Keating JP, Brophy D, Officer RA, Mullins E. 2014. Otolith shape analysis of blue whiting suggests a complex stock structure at their spawning grounds in the Northeast Atlantic. Fish Res 157: 1–6

Expected outcomes
The candidate is expected to produce, at least, three scientific publications in international peer-reviewed journals with scope in Marine Biology and Fisheries. Participation in two international specialized conferences (e.g. ICES Annual Science Conferences) is also expected. In terms of outreach to the general public, any successful candidate would be expected to report findings through public lectures and seminars, as well as to target exposure via the mass media. Finally the candidate will formulate scientific advice to government agencies and local stakeholders to support fisheries assessment and management of sparids.

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