INTRODUCTIONTop

The anchoveta, Engraulis ringens (Jenyns, 1842), is a coastal pelagic anchovy that is widely distributed throughout the eastern Pacific Ocean, from northern Peru (4°30′S) to Chiloé Island in southern Chile (44°14′S) (Serra 1983Serra J.R. 1983. Changes in the abundance of pelagic resources along the Chilean coast. In: Sharp G., Csirke J. (eds), Proceedings of the Expert Consultation to Examine Changes in Abundance and Species Composition of Neritic Fish Resources. FAO Fish. Rep. 291: 255-284., Pauly and Tsukayama 1987Pauly D., Tsukayama I. 1987. The Peruvian Anchoveta and its Upwelling Ecosystem: Three Decades of Change. ICLARM Studies and Reviews, 351 pp.), and is the target of one of the largest pelagic fisheries in the world. This species forms very dense shoals and is strongly influenced by biotic and abiotic environmental factors at all stages of its life cycle (Alheit and Ñiquen 2004Alheit J., Ñiquen M. 2004. Regime shift in the Humboldt Current ecosystem. Prog. Oceanogr. 60: 201-222.). A number of early life history characteristics and reproductive traits, such as egg size, larval size and yolk sac size of recently hatched larvae of the anchoveta, have been described to vary along most of its range of distribution (Llanos-Rivera and Castro 2004Llanos-Rivera A., Castro L.R. 2004. Latitudinal and seasonal egg size variations of the anchoveta Engraulis ringens off the Chilean Coast. Fish. Bull. 102: 207-212., 2006Llanos-Rivera A., Castro L.R. 2006. Inter-population differences in temperature effects on Engraulis ringens yolk-sac larvae. Mar. Ecol. Prog. Ser. 312: 245-253., Castro et al. 2009Castro L.R., Claramunt G., Krautz M.C., et al. 2009. Egg trait variations in anchoveta Engraulis ringens: A maternal effect to changing environmental conditions in contrasting spawning habitats. Mar. Ecol. Prog. Ser. 381: 237-248.). At its southern limit of distribution (41°-44°S), however, few studies of biology and ecology of adult anchoveta are available. The southern zone of the species distribution, named as the central-southern Chilean fishery zone for administrative purposes, covers a wide latitudinal range (33°S and 44°S; Fig. 1) in which several historic spawning locations have been identified along the coast (Cubillos et al. 2005Cubillos L., Castro L., Oyarzún C. 2005. Evaluación de stock desovante de anchoveta y sardina común entre la V y X Regiones, año 2004, Informe Técnico Proyecto FIP-IT/2004-03, Instituto de Fomento Pesquero, Chile, 130 pp., 2006Cubillos L., Castro L., Claramunt G., et al. 2006. Evaluación de stock desovante de anchoveta y sardina común entre la V y X Regiones, año 2005, Informe Técnico Proyecto FIP-IT/2005-02. Instituto de Fomento Pesquero, Chile, 147 pp.). In recent years, early developmental stages of anchoveta were found in the interior sea of Chiloé Island (northern Chilean Patagonia; 41°-43°S) (Landaeta and Castro 2006Landaeta M.F., Castro L.R. 2006. Seasonal variability in the spatial patterns of ichthyoplanktonic assemblages from the fjord zone of austral Chile. Cien. Tecnol. Mar. 29: 107-127., Landaeta et al. 2009Landaeta M., Muñoz M.I., Castro L.R. 2009. Seasonal and short-term variability in the vertical distribution of ichthyoplankton in a stratified fjord of southern Chile. Cien. Tecnol. Mar. 32: 27-42., Castro et al. 2015Castro L., Soto S., Llanos A., et al. 2015. Identification of spawning zones and early life stages of small pelagic fishes in inshore waters of the X and XI regions, year 2013. FIP 2013-17 Final Research Report (in Spanish). Fondo de Investigación Pesquera, Chile. 414 pp.). Asynchrony and significant differences in spawning season have been documented within the central-southern Chilean fishery zone of anchoveta, where spawning occurs from July to December, with higher gonadosomatic indices from August to November below latitude 38°S and from September to October above latitude 38°S (Cubillos et al. 2009Cubillos L., Pedraza M., Canales M., et al. 2009. Dinámica reproductiva de anchoveta y sardina común, zona centro-sur año 2006. Informe final Proyecto FIP/2006-13. Instituto de Fomento Pesquero, Chile, 141 pp., Claramunt et al. 2014Claramunt G., Cubillos L.A., Castro L., et al. 2014. Variation in the spawning periods of Engraulis ringens and Strangomera bentincki off the coasts of Chile: A quantitative analysis. Fish. Res. 160: 96-102.). Furthermore, significantly larger females have been found above latitude 38°S (Cubillos et al. 2009Cubillos L., Pedraza M., Canales M., et al. 2009. Dinámica reproductiva de anchoveta y sardina común, zona centro-sur año 2006. Informe final Proyecto FIP/2006-13. Instituto de Fomento Pesquero, Chile, 141 pp.).

Evidence suggesting some spawning stock and population connectivity in this central-southern fishery has been proposed. Soto-Mendoza et al. (2012)Soto-Mendoza S., Parada C., Castro L., et al. 2012. Modelling transport and survival of anchoveta eggs and yolk-sac larvae in the coastal zone off central-southern Chile: Assessing spatial and temporal spawning parameters. Prog. Ocean. 92: 178-191. used individually based models and concluded that a significant northward transport of eggs and larvae occurs from Corral (39°52’S) towards central Chile (36°-37°S). Other methodological approaches, including analyses of morphometrics, electrophoretic protein genetics, DNA and parasites (Ferrada et al. 2002Ferrada S., Hernández K., Montoya R., et al. 2002. Estudio poblacional del recurso anchoveta (Engraulis ringens Jenyns 1842) (Clupeiformes, Engraulidae), mediante análisis de ADN. Gayana 66: 243-248., Valdivia et al. 2007Valdivia I., Chávez A., Oliva E. 2007. Metazoan parasites of Engraulis ringens as tools for stock discrimination along the Chilean coast. J. Fish Biol. 70: 1504-1511., George-Nascimento et al. 2015George-Nascimento M., Oliva M. 2015. Fish population studies using parasites from the Southeastern Pacific Ocean: considering host population changes and species body size as sources of variability of parasite communities. Parasitology 142: 25-35.), have been used to assess spawning stock structure of anchoveta from northern to central Chile but have not been conclusive (and have sometimes been contradictory) in terms of the number of stock units reported. The stock structure of this fishery remains poorly defined. Furthermore, it is unknown whether the individuals found in the interior/exterior sea of Chiloé Island constitute a separate unit (Zúñiga and Canales 2014Zúñiga M.J., Canales C. 2014. Investigación del estatus y posibilidades de explotación biológicamente sustentables anchoveta zona centro-sur, año 2014. Cuarto Informe Proyecto 2.5. Instituto de Fomento Pesquero, Chile. 81 pp.) or are part of the central-southern fishery unit that migrates during the annual cycle.

Understanding of fish population structures and determination of discrete populations or spawning stock units is essential for sustainable management of fisheries resources (Sinclair 1988Sinclair M. 1988. Marine populations: an essay on population regulation and speciation. Washington Sea Grant Program, Seattle, Washington, 252 pp., Sinclair and Smith 2002Sinclair M.M., Smith T.D. 2002. The notion that fish species form stocks. ICES Mar. Sci. Symp. 215: 297-304., Cadrin et al. 2005Cadrin S., Friedland K., Waldman J. 2005. Stock Identification Methods: Applications in Fishery Science. Elsevier Academic Press, London, 719 pp.). Spawning stock identification is a complex task in the pelagic environment. Most common methods for distinguishing between marine populations include analyses of morphometric variation, parasitic fauna, biological markers (gonadosomatic index and sexual maturity curve) and genetic markers (Cadrin et al. 2005Cadrin S., Friedland K., Waldman J. 2005. Stock Identification Methods: Applications in Fishery Science. Elsevier Academic Press, London, 719 pp.). In recent years, trace element composition of otoliths has become a common and effective tool for identifying the spatial heterogeneity of populations and has helped identify migratory patterns and differentiate fish stocks (Hicks et al. 2010Hicks A., Closs G., Swearer S. 2010. Otolith microchemistry of two amphidromous galaxiids across an experimental salinity gradient: a multi-element approach for tracking diadromous migrations. J. Exper. Mar. Biol. Ecol. 394: 86-97., Morais et al. 2010Morais P., Babaluk J., Correia A., et al. 2010. Diversity of anchovy migration patterns in a European temperate estuary and in its adjacent coastal are: Implications for fishery management. J. Sea Res. 64: 295-303., Avigliano and Volpedo 2016Avigliano E., Volpedo A. 2016. A Review of the Application of Otolith Microchemistry Toward the Study of Latin American Fishes. Rev. Fish. Sci. Aquac. 24: 369-384.). This technique is based on the fact that otoliths originate during the embryonic stage and grow continuously throughout the life of the fish. As they grow, otoliths show characteristic morphologic and chemical composition patterns. Otolith growth that occurred during the pelagic larval stage can be distinguished from growth during the juvenile or later adult period. The ability to correlate a particular part of an otolith with a discrete point in time is one of the most valuable aspects of this structure (Campana 1999Campana S. 1999. Chemistry and composition of fish otoliths: pathways, mechanisms and applications. Mar. Ecol. Prog. Ser. 188: 263-297., Chang and Geffen 2013Chang M., Geffen A. 2013. Taxonomic and geographic influences on fish otolith microchemistry. Fish Fish. 14: 458-492., Górski et al. 2015Górski K., De Gruijter C., Tana R. 2015. Variation in habitat use along the freshwater-marine continuum by grey mullet Mugil cephalus at the southern limits of its distribution. J. Fish Biol. 87: 1059-1071.). Furthermore, otoliths do not seem to experience chemical reabsorption for the majority of elements and therefore reflect the physico-chemical characteristics of water, forming a permanent record of the past environmental conditions experienced by fish (Bath et al. 2000Bath G., Thorrold S., Jones C., et al. 2000. Strontium and barium uptake in aragonitic otoliths of marine fish. Geochim. Cosmochim. Acta 64: 1705-1714., Rooker et al. 2003Rooker J., Secor D., Zdanowicz V., et al. 2003. Identification of Atlantic bluefin tuna (Thunnus thynnus) stocks from putative nurseries using otolith chemistry. Fish. Oceanogr. 12: 75-84., Thorrold et al. 2007Thorrold S., Zacherl C., Levin L. 2007. Population Connectivity and Larval Dispersal, Using Geochemical Signatures in Calcified Structures. Oceanography 20: 80-89.).

The objective of this study was to evaluate whether otolith elemental composition could indicate changes or persistence in habitats occupied by anchovetas and thereafter help to determine whether the central-south fishery of Chile can be divided into sub-units corresponding to different areas of spawning and growth or, alternatively, the level of mixing of specimens throughout the area shows that such sub-units did not exist. To address this question, the elemental composition of otoliths from individuals caught off central-southern Chile in 2014 and 2015 was analysed.

METHODSTop

Sample locations and fish collection

The study was conducted in the central-southern Chile fishery zone (33°S-44°S) at two locations: Talcahuano (Thno., the central zone) and the Inner Sea of the Chiloé (ISCh, northern Patagonia). Mean satellite seasonal sea surface temperature was obtained along the central-southern Chilean coast, covering both sampling locations for the year of fish collections. Also, for longer term comparisons, an 11-year series (2005-2015) of mean sea surface temperature during the main spawning months (Thno, August-November; ISCh, September-October; Claramunt et al. 2014Claramunt G., Cubillos L.A., Castro L., et al. 2014. Variation in the spawning periods of Engraulis ringens and Strangomera bentincki off the coasts of Chile: A quantitative analysis. Fish. Res. 160: 96-102., Cubillos et al. 2009Cubillos L., Pedraza M., Canales M., et al. 2009. Dinámica reproductiva de anchoveta y sardina común, zona centro-sur año 2006. Informe final Proyecto FIP/2006-13. Instituto de Fomento Pesquero, Chile, 141 pp.) in the Talcahuano zone (36.48°S 72.98°W) and at the Inner Sea of Chiloé zone (42.10°S 72.94°W) was obtained from satellite data. The satellite data were obtained from MODIS Aqua Globales (4 km pixel resolution) and downloaded from OceanColor Web (http://oceancolor.gsfc.nasa.gov/), and the images were processed in SeaDas (versión 7.3.2).

At both locations, specimens of Engraulis ringens were obtained from commercial catches in autumn and spring 2014 and summer 2015; only the general catch area was reported, as the exact catch locations are commercially sensitive (Fig. 1). Fish samples were selected at random. Subsequently, sex, weight and total length (TL) were recorded for each fish that was selected for otolith analysis. The fish were sorted into two age groups following the age-length key developed by Araya et al. (2008)Araya M., Cubillos L., Peñailillo J. 2008. Validación de la edad de la anchoveta en la costa de Chile. Informe final Proyecto FIP/2004-38. Fondo de Investigación Pesquera, Chile. pp 138. and otolith ring counts. Group 1 included adult fish (11.5 and 13.4 cm TL; one year old) and Group 2 included adults larger than 13.4 cm TL (>two years old) (Table 1). Selected specimens were stored in a freezer (–20°C) until analysis at the Fisheries Oceanography and Larval Ecology Laboratory (LOPEL) of the University of Concepción, Chile. Individuals obtained from the central zone and northern Patagonia were similar in size, ranging from 13.1 to 16.1 cm in the central zone and 13.1 cm to 15.6 cm in northern Patagonia. However, during April and January only individuals two years old or older were present, with the largest specimens being observed in April in the central zone (Table 1).

Otolith preparation

After the sagittal otoliths had been extracted from each fish and any adhering tissue had been cleaned away by rinsing with Milli-Q water, the dry samples were stored in 0.5 mL polypropylene micro tubes. The left otolith of each individual was then mounted on a glass slide with epoxy resin and sonicated. The slides were stored in polyethylene bags for later microchemical analysis.

Microchemical analyses

The elemental composition of each otolith was analysed using laser ablation inductively coupled mass spectrometry (LA-ICP-MS) at the Centre for Trace Element Analysis at the University of Otago in Dunedin, New Zealand (the centre uses an ultraclean laboratory for analyses of this type). Depth profiles were obtained along ablation transects from the distal otolith surface (convex side), through the core, to the proximal surface (concave side) (Macdonald et al. 2008Macdonald J., Shelley M., Crook D. 2008. A Method for Improving the Estimation of Natal Chemical Signatures in Otoliths. Trans. Amer. Fish. Soc. 137: 1674-1682., Warburton et al. 2016Warburton M., Reid M., Stirling C., et al. 2016. Validation of depth-profiling LA-ICP-MS in otolith applications. Can. J. Fish. Aquat. Sci. 74: 572-581.). This was done using an ICP-MS Agilent 7500cs coupled to an ASI Resolution M-50 laser ablation system powered by a Coherent 193 nm ArF excimer laser.

The otoliths were mounted on a glass slide and the core was located visually through a video imaging system at 400× magnification. Each sample was ablated continuously in a cross section of the unpolished otolith using a 75 micron laser beam. The laser was operated at 5 Hz with an on-sample fluence of 2.2 J cm^–2. Ablation occurred in an atmosphere of pure helium to minimize the re-condensation of the materials and potential elemental fractioning (Eggins et al. 1998Eggins S., Kinsley L., Shelley J. 1998. Deposition and fractionation processes during atmospheric pressure laser sampling for analysis by ICP-MS. Appl. Surf. Sci. 127-129: 278-286.). For each otolith ablation, data were collected for up to 450 seconds to ensure that the transect completely traversed the core. To reduce the delay time associated with cleaning, no signal smoothing collector was used (Woodhead et al. 2008Woodhead J., Hellstrom J., Paton C., et al. 2008. A guide to depth profiling and imaging applications of LA-ICP-MS. In: Sylvester P. (ed), Laser Ablation ICP-MS in the Earth Sciences: Current Practices and Outstanding Issues; Short Course 40; Mineralogical Association of Canada: Quebec City, QC, Canada, pp. 135-145.). Count rate data were collected for the ions of the following isotopes: ²⁴Mg, ³¹P, ²⁷Al, ⁴³Ca, ⁵⁵Mn, ⁶⁰Ni, ⁶⁶Zn, ⁸⁸Sr, ⁶³Cu, ⁸⁵Rb, ¹³⁸Ba and ²⁰⁸Pb. Concentrations of these elements are usually sufficiently high in the environment (Campana 1999Campana S. 1999. Chemistry and composition of fish otoliths: pathways, mechanisms and applications. Mar. Ecol. Prog. Ser. 188: 263-297.). NIST 612 calibration glass and MACS-3 otolith reference material were run, bracketing groups of four otoliths.

Data analyses

Raw count rate data were reduced to molar ratios to calcium using the IOLITE software package (Iolitesoftware.com version 2.5). The means of the MACS-3 analyses were typically within 5% of the recommended values, with a precision generally better than 3% (relative standard deviation) for most elements. The relationship between Ca normalized concentrations of the elements Mg, Al, P, Mn, Ni, Zn, Cu, Rb, Sr, Ba and Pb was recorded. Mg:Ca and P:Ca concentration are known to decrease throughout the ontogeny of sardine, because of physiological factors influencing the deposit rate of these elements on the otolith (Javor and Dorval 2016Javor B.J., Dorval E. 2016. Stability of trace elements in otoliths of juvenile Pacific sardine Sardinops sagax. CalCOFI Rep. 57: 109-123.). This happened to also be the case in our data from anchoveta otoliths, as Mg:Ca and P:Ca were always strongly correlated over time (Pearson product-moment correlation p<00.1 for each transect). Hence, Mg:Ca and P:Ca often showed a “butterfly pattern” on the otolith transects, with a high concentration at emergence, slowly decreasing over time and mirroring on each side of the nucleus. Therefore, we used the concentration of these elements to locate the nucleus on the transects (Fig. 2).

The element:calcium ratios were averaged for core and edge regions of each of the studied otoliths. For both edges and cores, 20 s of data were averaged, corresponding to 15 micrometres. Fifteen micrometre averages were used to ensure that only the early larval period was covered in core samples (Plaza and Cerna 2015Plaza G., Cerna F. 2015. Validation of daily microincrement deposition in otoliths of juvenile and adult Peruvian anchovy Engraulis ringens. J. Fish Biol. 86: 203-216.). Mg:Ca and P:Ca were always strongly correlated and therefore only Mg:Ca was used in the statistical analyses. A permutational multivariate analysis of variance (PERMANOVA, Anderson 2001Anderson M. 2001. A new method for non-parametric multivariate analysis of variance. Austral Ecol. 26: 32-46.) was used to determine differences in the element concentrations (Mg, Al, Mn, Ni, Zn, Cu, Rb, Sr, Ba, Pb) of the otoliths between catch zones and period of capture. This provided an analysis of variance for a set of explanatory factors based on dissimilarity measures, thereby allowing differences to be tested at a multivariate level and across a wide range of distributions of empirical data. The analyses were based on Euclidean distance matrices. The tests were performed separately for microchemical concentrations of the otolith edge and core.

Principal components analysis was used to illustrate differences in the multi-elemental composition of cores and edges of otoliths among samples (Mg, Al, Mn, Ni, Zn, Cu, Rb, Sr, Ba and Pb). Subsequently, data of each element were tested for normality and homogeneity of variance using the Shapiro-Wilk and Levene tests (original and log(x+1)-transformed data). Only ratios of Sr:Ca, Mg:Ca, Ba:Ca, Zn:Ca, Rb:Ca and Mn:Ca were normally distributed (Shapiro-Wilk test, p>0.05) for both original and log-transformed data. However, only the Sr:Ca ratio met the assumption of homogeneity of variance (Levene test, p>0.05). Therefore, differences between zones were tested using a non-parametric test (Kruskal-Wallis) followed by a Wilcoxon test. The differences were tested separately for cores and edges.

RESULTSTop

Differences between locations and among seasons were observed in the mean seasonal sea surface temperature along the central-southern coast of Chile. In the Talcahuano area, temperatures were about 1° to 2°C higher than in the Inner Sea of Chiloé area in all seasons (Fig. 3A). The 11-year time series of mean sea surface temperatures in the peak spawning months of anchoveta at each location also showed that mean temperature values fluctuated inter-annually, but mean temperatures in those months were consistently higher in the Talcahuano area than in the Inner Sea of Chiloé (Thno, 11.2°C-12.5°C vs. ISCh, 10.4°C-11.1°C; χ² _0.05,1, 22.80, p<0.05) (Fig. 3B).

This study used a total of 102 adult Engraulis ringens specimens. Otoliths were extracted for laser ablation from 29 specimens that were one-year-old and from 73 that were two years old or older. In total, 56 specimens came from central Chile and 46 from northern Patagonia (Table 1). The concentrations of different trace elements at the otolith edges showed no significant differences in elemental concentrations for any of the factors (zone, month, group and sex; Table 2, Fig. 4). Furthermore, univariate tests for each of the elements separately revealed significant differences only for Mg (p<0.05), which showed higher concentrations in the central zone than in northern Patagonia (Table 3, Fig. 5).

The PERMANOVA revealed significant differences in core elemental concentrations among zones and months (PERMANOVA; pseudo-F=36.7, p<0.001, pseudo-F=6.7, p<0.001) but not among age groups and sexes (Table 2, Fig. 4). April was the only month that did not differ between zones in pairwise comparisons (p>0.05; Table 4).

Univariate analyses revealed differences between zones for some of the elements. Specifically, post-hoc comparisons indicated significant differences between zones for Ba, Mg and Pb (p<0.05) and strong trends for Rb and Zn (p<0.09) (Table 3, Fig. 5). Cores of otoliths of specimens from the central zone had higher concentrations of Ba, Mg, Pb and Zn and lower concentrations of Rb than those of specimens from northern Patagonia.

DISCUSSIONTop

The objective of this study was to evaluate whether the elemental composition of otoliths could serve as indicators of changes or of persistence in habitats occupied by the anchoveta Engraulis ringens and, thereafter, to determine whether the central-south fishery of Chile could be divided into sub-units corresponding to different areas of spawning and growth. The results showed significant differences in the elemental composition of otolith cores between the two fishing zones that form the central-south fishery, suggesting that there are two distinct spawning areas and that most individuals spent their early life stages in the area where they hatched. However, low differences in the elemental composition of otolith edges in some short periods indicated the possibility that adults from different origins mixed during the year.

In the otolith cores, the most important differences in terms of elemental composition were higher Ba, Mg, Pb and Zn concentrations in the central Chile zone than in northern Patagonia. In some fish species, magnesium has been positively associated with seawater temperature (Schuchert et al. 2010Schuchert P., Alexander I., Arkhipkin E., et al. 2010. Traveling around Cape Horn: Otolith chemistry reveals a mixed stock of Patagonian hoki with separate Atlantic and Pacific spawning grounds. Fish. Res. 102: 80-86., Javor and Dorval 2017Javor B.J., Dorval E. 2017. Composition and inter-annual variability in trace element profiles of Pacific sardine otoliths. CalCOFI Rep. 58: 95-104.), which is consistent with the sea surface latitudinal temperature gradient along the Chilean coast, where warmer waters were found off central Chile (>12°C) and colder waters occurred farther south (<11.5°C). This pattern is consistent with our results of differences in sea surface temperatures among seasons and during the 11-year time series during the months of peak spawning at both locations, and has also been reported previously in other studies (Cubillos et al. 2015Cubillos L.A., Castro L., Claramunt G., et al. 2015. Evaluación del stock desovante de anchoveta y sardina común entre la V y X Regiones, año 2014. Instituto de Fomento Pesquero, Chile, 212 pp., Lillo et al. 2015Lillo S., Lang C., Molina E., et al. 2015. Evaluación hidroacústica de pequeños pelágicos en aguas interiores de la X y XI Regiones, año 2014. Informe Final Proyecto Convenio desempeño 2014. Instituto de Fomento Pesquero, Chile, 161 pp.). Similar to patterns observed in juvenile sardines Sardinops sagax (Javor and Dorval 2017Javor B.J., Dorval E. 2017. Composition and inter-annual variability in trace element profiles of Pacific sardine otoliths. CalCOFI Rep. 58: 95-104.), here we report a strong correlation of Mg:Ca and P:Ca ratios for anchoveta Engraulis ringens. These concentrations were strong discriminants between sites with significant water temperature differences, and decreased throughout the ontogeny. Therefore, ontogenetic effects should be always considered when spatial patterns and stock structure of anchoveta are evaluated on the basis of microchemical signatures of otoliths.

We found higher concentrations of Ba in otolith cores from fish collected in central Chile (upwelling zone) than in those collected from northern Patagonia (a non-upwelling zone). Ba has a nutrient-like vertical distribution with much higher concentrations in deeper waters (Lea et al. 1989Lea D.W., Shen G.T., Boyle E.A. 1989. Coralline barium records temporal variability in Equatorial Pacific upwelling. Nature 340: 373-376., Nozaki 2001Nozaki Y. 2001. Elemental distribution. In: Steele J.H., Thorpe S.A., Turekian K.K. (eds), Encyclopedia of Ocean Sciences. Academic Press, San Diego, CA. pp. 840-845.), and its concentrations in surface waters significantly increase during upwelling events (Hsieh and Henderson 2017Hsieh Y.T., Henderson G.M. 2017. Barium stable isotopes in the global ocean: Tracer of Ba inputs and utilization. Earth Planet Sci. Lett. 473: 269-278.). Therefore, the higher Ba:Ca ratios we found in fish in the central zone are most probably an effect of upwelling. These findings are concordant with previous observations of Ba:Ca in seawater and fishes in other upwelling areas where increased Ba:Ca has been detected under upwelling conditions (Wheeler et al. 2016Wheeler S.G., Russel A.D., Fehrerenbacher J.S., et al. 2016. Evaluating chemical signatures in a costal upwelling region to reconstruct water mass associations of settlement-stage rockfishes. Mar. Ecol. Prog. Ser. 550: 191-206., Hampton et al. 2018Hampton S.L., Maloney C.L., van der Lingen C.D., et al. 2018. Spatial and temporal variability in otolith elemental signatures of juvenile sardine off South Africa. J. Mar. Sys. 188: 109-116.). A similar nutrient-like distribution might be expected for Sr (De Villiers 1999De Villiers S. 1999. Seawater strontium and Sr/Ca variability in the Atlantic and Pacific oceans. Earth Planet. Sci. Lett. 171: 623-634.), though we found no significant differences in Sr:Ca rations in otoliths between central Chile and northern Patagonia. Wheeler et al. (2016)Wheeler S.G., Russel A.D., Fehrerenbacher J.S., et al. 2016. Evaluating chemical signatures in a costal upwelling region to reconstruct water mass associations of settlement-stage rockfishes. Mar. Ecol. Prog. Ser. 550: 191-206. concluded that individual ratios of Sr:Ca in fish otoliths showed little variation in the California upwelling system and did not serve as valuable proxies of oceanic conditions.

Regarding other elements, off central Chile, Ahumada (1995)Ahumada R. 1995. Bahías: Áreas de uso múltiple un enfoque holístico del problema de contaminación. Cien. Tecnol. Mar, CONA. Número Especial, 59-68. found higher metal concentrations (Pb, Zn, Cu, Cr, Ni and Cd) from anthropogenic origins in San Vicente Bay (central Chile, 36°44′S, 73°09′W). Reports from other bays in the same area were consistent with these results. For example, Pb was the predominant trace metal found in the seawater of Concepción Bay (central Chile; 36°40′S, 73°01′W) (Carrera et al. 1993Carrera M., Rodríguez V., Ahumada R., et al. 1993. Metales trazas en la columna de agua y sedimentos blandos en Bahía de Concepción, Chile. Determinación mediante voltametría de redisolución. Rev. Biol. Mar. Oceanogr. 28: 151-163.). The greater concentrations of Pb and Zn recorded in the anchoveta otolith cores from central Chile in this study might be a result of high heavy metals recorded for this area traced to nearby iron, steel and petrochemical industries. Zinc concentrations in otoliths have previously been used to indicate particular habitats because they are influenced by fish diet (Ranaldi and Gagnon 2008Ranaldi M., Gagnon M. 2008. Zinc incorporation in the otoliths of juvenile pink snapper (Pagrus auratus Forster): The influence of dietary versus waterborne sources. J. Exper. Mar. Biol. Ecol. 360: 56-62.) and tends to be higher in urban coastal zones due to anthropogenic sources (Sturrock et al. 2012Sturrock A., Trueman C., Darnaude A., et al. 2012. Can otolith elemental chemistry retrospectively track migrations in fully marine fishes? J. Fish Biol. 81: 766-795.).

Unlike the elemental concentrations of the otolith cores of anchoveta, elemental concentrations of the otolith edges did not differ between zones. There are at least three potential explanations for these core differences and edge similarities in elemental composition. Differences among cores and similar edge concentrations might occur if the adult habitat is environmentally heterogeneous, wide in extension and covering inshore and offshore zones, but the spawning habitat where small larvae occur is restricted to the coastal zone given their limited swimming capacities. These across-shelf ontogenetic differences in elemental composition have been observed in other anchovy species in other systems (e.g. E. encrasicolus in the Gulf of Cadiz; Catalán et al. 2014Catalán I.A., Pérez-Mayol S., Alvarez I., et al. 2014. Daily otolith growth and ontogenetic geochemical signatures of age-0 anchovy (Engraulis encrasicolus) in the Gulf of Cadiz (SW Spain). Mediterr. Mar. Sci. 15: 781-789.). Under this scenario, if environmental conditions inshore in central Chile and northern Patagonia differ significantly but are similar offshore, the otolith edge elemental concentrations might not differ between individuals collected in the two zones, while their otolith core concentrations might. Heavy metal concentrations are usually reported to be several times higher in coastal than in oceanic waters due to greater continental contributions from urban areas or sites of high industrial concentration (Luoma 1990Luoma S. 1990. Processes affecting metal concentrations in estuarine and coastal sediments. In: Furnes R., Rainbow P. (eds), Heavy Metals in the Marine Environment. CRC Press. New York, pp. 51-66., Giordano et al. 1992Giordano P., Musmeci L., Ciaralli P., et al. 1992. Total content and sequential extractions of Hg, Cd and Pb in coastal sediments. Mar. Pol. Bull. 40: 1042-1050., French 1993French P. 1993. Post-industrial pollutant levels in contemporary Svern estuary intertidal sediments compared to pre-industrial levels. Mar. Poll. Bull. 26: 30-35.). In our study, besides Ba, these higher concentrations allowed us to infer the presence of two discrete spawning grounds. Prior research cruises (September-October 2009, 2010 and 2012; Cubillos et al. 2010Cubillos L.A., Castro L., Claramunt G., et al. 2010. Evaluación del Stock Desovante de Anchoveta y Sardina común entre la V y X Región, año 2009. Informe Final Proyecto FIP/2009-08. Instituto de Fomento Pesquero, Chile, 129 pp., 2011Cubillos L.A., Castro L., Claramunt G. 2011. Evaluación del Stock Desovante de anchoveta y sardina común entre la V y X Región, año 2010. Informe Final Proyecto FIP/2010-02. Instituto de Fomento Pesquero, Chile, 125 pp., 2013Cubillos L.A., Castro L., Claramunt G., et al. 2013. Evaluación del stock desovante de anchoveta y sardina común entre la V y X Regiones, año 2012. Informe Final Proyecto FIP/2012-09. Instituto de Fomento Pesquero, Chile, 151 pp.) verified that, spatially, spawning grounds occur in coastal areas not exceeding 15 nautical miles offshore. However, larger individuals could be found as far as 30 to 40 nautical miles from the coastal spawning grounds, as depicted from the current industrial fishing areas. Consequently, across-shelf movements would homogenize elemental composition differences in the otolith edges of older fishes.

A second explanation might be that the greater swimming capacity of the larger individuals might allow them to migrate north and south, could they could sometime access other spawning grounds where they would mix with specimens from local spawning and nursery areas. If this movement were frequent and involved large fractions of the populations, differences in the core elemental composition would tend to decrease significantly, and any difference in edge elemental composition (such as Mg, Table 4) indicative of different environmental temperature regimes probably associated with latitude should vanish. In this study during autumn (April), only adult fish older than one year of age were analysed (Table 1), and no differences were observed in core elemental composition between individuals from the two analysed zones. This suggests that adult individuals from both zones might mix before returning to the spawning habitats. This pattern of segregation of population units and a short period of mixing in some seasons is common in a number of fish species displaying homing behaviour associated with estuarine conditions, e.g. Engraulis encrasicolus L. in the Mediterranean Sea (Aldanondo et al. 2010Aldanondo N., Cotano U., Tiepolo M., et al. 2010. Growth and movement patterns of early juvenile European anchovy (Engraulis encrasicolus L.) in the Bay of Biscay based on otolith microstructure and chemistry. Fish. Oceanogr. 19: 196-208., Guidetti et al. 2013Guidetti P., Petrillo M., De Benedetto G., et al. 2013. The use of otolith microchemistry to investigate spawning patterns in European anchovy: a case study in the Eastern Ligurian Sea (NW Mediterranean). Fish. Res. 139: 1-4.).

A third potential explanation is that these two spawning units represent pocket populations of a broader stock that migrates and that might shift from year to year depending on annual variations in temperature, prey abundance or other undetermined factors. Unfortunately, because the length of our data series is relatively short (one year), we cannot make strong inferences on the restricted or periodical movements across several years. In other systems, elemental differentiation has been observed in cohorts of small pelagic fishes in some years but not in others (Guidetti et al. 2013Guidetti P., Petrillo M., De Benedetto G., et al. 2013. The use of otolith microchemistry to investigate spawning patterns in European anchovy: a case study in the Eastern Ligurian Sea (NW Mediterranean). Fish. Res. 139: 1-4.). While changes in the main spawning locations along the central-southern fishery zone have been reported at 5- to 10-year periods or longer (Bernal et al. 1983Bernal P.A., Robles F.L., Rojas O. 1983. Variabilidad física y biológica en la región meridional del sistema de corrientes Chile-Peru. FAO Fish Rep. 291: 683-711., Serra 1983Serra J.R. 1983. Changes in the abundance of pelagic resources along the Chilean coast. In: Sharp G., Csirke J. (eds), Proceedings of the Expert Consultation to Examine Changes in Abundance and Species Composition of Neritic Fish Resources. FAO Fish. Rep. 291: 255-284., Castro et al. 1997Castro L., Quiñones R., Arancibia H., et al. 1997. Areas de desove de anchoveta y sardina común en Chile central. Informe Técnico FIP-IT/96-11. Fondo de Investigación Pesquera. Subsecretaría de Pesca, 235 pp.), shorter-term (inter-annual) changes in migration patterns have not been reported for anchoveta units along their southern distribution range.

In summary, this study of the elemental composition of otoliths provides evidence of two discrete spawning areas of Engraulis ringens along the central-south fishery zone: one in central Chile and one in northern Patagonia, and mixing of adults during some non-spawning periods of the year. This finding agrees with the spatial and structural asynchrony in the reproductive processes in the two areas. Future studies should assess whether this pattern is consistent year to year or varies over longer time periods.

AKNOWLEDGEMENTSTop

This study was financed by the CONICYT Basal Programme COPAS Sur-Austral PIA PFB31. During the study, the first author was supported by graduate fellowships from CONICYT and COPAS Sur-Austral CONICYT PIA PFR31. LC was also supported by COPAS Sur-Austral CONICYT PIA APOYO CCTE AFB170006 and FONDECYT 1161131. The authors thank J. Betzhold, G. Vásquez, and R. Riffo for collections at the artisanal fishery port and vessels and colleagues at the Laboratorio de Oceanografía Pesquera y Ecología Larval (LOPEL) at the Universidad de Concepción for technical support.

REFERENCESTop

Ahumada R. 1995. Bahías: Áreas de uso múltiple un enfoque holístico del problema de contaminación. Cien. Tecnol. Mar, CONA. Número Especial, 59-68.

Alheit J., Ñiquen M. 2004. Regime shift in the Humboldt Current ecosystem. Prog. Oceanogr. 60: 201-222.
https://doi.org/10.1016/j.pocean.2004.02.006

Aldanondo N., Cotano U., Tiepolo M., et al. 2010. Growth and movement patterns of early juvenile European anchovy (Engraulis encrasicolus L.) in the Bay of Biscay based on otolith microstructure and chemistry. Fish. Oceanogr. 19: 196-208.
https://doi.org/10.1111/j.1365-2419.2010.00537.x

Anderson M. 2001. A new method for non-parametric multivariate analysis of variance. Austral Ecol. 26: 32-46.
https://doi.org/10.1111/j.1442-9993.2001.01070.pp.x

Araya M., Cubillos L., Peñailillo J. 2008. Validación de la edad de la anchoveta en la costa de Chile. Informe final Proyecto FIP/2004-38. Fondo de Investigación Pesquera, Chile. pp 138.

Avigliano E., Volpedo A. 2016. A Review of the Application of Otolith Microchemistry Toward the Study of Latin American Fishes. Rev. Fish. Sci. Aquac. 24: 369-384.
https://doi.org/10.1080/23308249.2016.1202189

Bath G., Thorrold S., Jones C., et al. 2000. Strontium and barium uptake in aragonitic otoliths of marine fish. Geochim. Cosmochim. Acta 64: 1705-1714.
https://doi.org/10.1016/S0016-7037(99)00419-6

Bernal P.A., Robles F.L., Rojas O. 1983. Variabilidad física y biológica en la región meridional del sistema de corrientes Chile-Peru. FAO Fish Rep. 291: 683-711.

Cadrin S., Friedland K., Waldman J. 2005. Stock Identification Methods: Applications in Fishery Science. Elsevier Academic Press, London, 719 pp.

Campana S. 1999. Chemistry and composition of fish otoliths: pathways, mechanisms and applications. Mar. Ecol. Prog. Ser. 188: 263-297.
https://doi.org/10.3354/meps188263

Castro L., Quiñones R., Arancibia H., et al. 1997. Areas de desove de anchoveta y sardina común en Chile central. Informe Técnico FIP-IT/96-11. Fondo de Investigación Pesquera. Subsecretaría de Pesca, 235 pp.

Castro L.R., Claramunt G., Krautz M.C., et al. 2009. Egg trait variations in anchoveta Engraulis ringens: A maternal effect to changing environmental conditions in contrasting spawning habitats. Mar. Ecol. Prog. Ser. 381: 237-248.
https://doi.org/10.3354/meps07922

Castro L., Soto S., Llanos A., et al. 2015. Identification of spawning zones and early life stages of small pelagic fishes in inshore waters of the X and XI regions, year 2013. FIP 2013-17 Final Research Report (in Spanish). Fondo de Investigación Pesquera, Chile. 414 pp.

Carrera M., Rodríguez V., Ahumada R., et al. 1993. Metales trazas en la columna de agua y sedimentos blandos en Bahía de Concepción, Chile. Determinación mediante voltametría de redisolución. Rev. Biol. Mar. Oceanogr. 28: 151-163.

Catalán I.A., Pérez-Mayol S., Alvarez I., et al. 2014. Daily otolith growth and ontogenetic geochemical signatures of age-0 anchovy (Engraulis encrasicolus) in the Gulf of Cadiz (SW Spain). Mediterr. Mar. Sci. 15: 781-789.
https://doi.org/10.12681/mms.819

Chang M., Geffen A. 2013. Taxonomic and geographic influences on fish otolith microchemistry. Fish Fish. 14: 458-492.
https://doi.org/10.1111/j.1467-2979.2012.00482.x

Claramunt G., Cubillos L.A., Castro L., et al. 2014. Variation in the spawning periods of Engraulis ringens and Strangomera bentincki off the coasts of Chile: A quantitative analysis. Fish. Res. 160: 96-102.
https://doi.org/10.1016/j.fishres.2013.09.010

Cubillos L., Castro L., Oyarzún C. 2005. Evaluación de stock desovante de anchoveta y sardina común entre la V y X Regiones, año 2004, Informe Técnico Proyecto FIP-IT/2004-03, Instituto de Fomento Pesquero, Chile, 130 pp.

Cubillos L., Castro L., Claramunt G., et al. 2006. Evaluación de stock desovante de anchoveta y sardina común entre la V y X Regiones, año 2005, Informe Técnico Proyecto FIP-IT/2005-02. Instituto de Fomento Pesquero, Chile, 147 pp.

Cubillos L., Pedraza M., Canales M., et al. 2009. Dinámica reproductiva de anchoveta y sardina común, zona centro-sur año 2006. Informe final Proyecto FIP/2006-13. Instituto de Fomento Pesquero, Chile, 141 pp.

Cubillos L.A., Castro L., Claramunt G., et al. 2010. Evaluación del Stock Desovante de Anchoveta y Sardina común entre la V y X Región, año 2009. Informe Final Proyecto FIP/2009-08. Instituto de Fomento Pesquero, Chile, 129 pp.

Cubillos L.A., Castro L., Claramunt G. 2011. Evaluación del Stock Desovante de anchoveta y sardina común entre la V y X Región, año 2010. Informe Final Proyecto FIP/2010-02. Instituto de Fomento Pesquero, Chile, 125 pp.

Cubillos L.A., Castro L., Claramunt G., et al. 2013. Evaluación del stock desovante de anchoveta y sardina común entre la V y X Regiones, año 2012. Informe Final Proyecto FIP/2012-09. Instituto de Fomento Pesquero, Chile, 151 pp.

Cubillos L.A., Castro L., Claramunt G., et al. 2015. Evaluación del stock desovante de anchoveta y sardina común entre la V y X Regiones, año 2014. Instituto de Fomento Pesquero, Chile, 212 pp.

De Villiers S. 1999. Seawater strontium and Sr/Ca variability in the Atlantic and Pacific oceans. Earth Planet. Sci. Lett. 171: 623-634.
https://doi.org/10.1016/S0012-821X(99)00174-0

Eggins S., Kinsley L., Shelley J. 1998. Deposition and fractionation processes during atmospheric pressure laser sampling for analysis by ICP-MS. Appl. Surf. Sci. 127-129: 278-286.
https://doi.org/10.1016/S0169-4332(97)00643-0

Ferrada S., Hernández K., Montoya R., et al. 2002. Estudio poblacional del recurso anchoveta (Engraulis ringens Jenyns 1842) (Clupeiformes, Engraulidae), mediante análisis de ADN. Gayana 66: 243-248.
https://doi.org/10.4067/S0717-65382002000200022

French P. 1993. Post-industrial pollutant levels in contemporary Svern estuary intertidal sediments compared to pre-industrial levels. Mar. Poll. Bull. 26: 30-35.
https://doi.org/10.1016/0025-326X(93)90594-A

George-Nascimento M., Oliva M. 2015. Fish population studies using parasites from the Southeastern Pacific Ocean: considering host population changes and species body size as sources of variability of parasite communities. Parasitology 142: 25-35.
https://doi.org/10.1017/S0031182014001127

Guidetti P., Petrillo M., De Benedetto G., et al. 2013. The use of otolith microchemistry to investigate spawning patterns in European anchovy: a case study in the Eastern Ligurian Sea (NW Mediterranean). Fish. Res. 139: 1-4.
https://doi.org/10.1016/j.fishres.2012.10.015

Giordano P., Musmeci L., Ciaralli P., et al. 1992. Total content and sequential extractions of Hg, Cd and Pb in coastal sediments. Mar. Pol. Bull. 40: 1042-1050.

Górski K., De Gruijter C., Tana R. 2015. Variation in habitat use along the freshwater-marine continuum by grey mullet Mugil cephalus at the southern limits of its distribution. J. Fish Biol. 87: 1059-1071.
https://doi.org/10.1111/jfb.12777

Hampton S.L., Maloney C.L., van der Lingen C.D., et al. 2018. Spatial and temporal variability in otolith elemental signatures of juvenile sardine off South Africa. J. Mar. Sys. 188: 109-116.
https://doi.org/10.1016/j.jmarsys.2018.02.001

Hicks A., Closs G., Swearer S. 2010. Otolith microchemistry of two amphidromous galaxiids across an experimental salinity gradient: a multi-element approach for tracking diadromous migrations. J. Exper. Mar. Biol. Ecol. 394: 86-97.
https://doi.org/10.1016/j.jembe.2010.07.018

Hsieh Y.T., Henderson G.M. 2017. Barium stable isotopes in the global ocean: Tracer of Ba inputs and utilization. Earth Planet Sci. Lett. 473: 269-278.
https://doi.org/10.1016/j.epsl.2017.06.024

Javor B.J., Dorval E. 2016. Stability of trace elements in otoliths of juvenile Pacific sardine Sardinops sagax. CalCOFI Rep. 57: 109-123.

Javor B.J., Dorval E. 2017. Composition and inter-annual variability in trace element profiles of Pacific sardine otoliths. CalCOFI Rep. 58: 95-104.

Landaeta M.F., Castro L.R. 2006. Seasonal variability in the spatial patterns of ichthyoplanktonic assemblages from the fjord zone of austral Chile. Cien. Tecnol. Mar. 29: 107-127.

Landaeta M., Muñoz M.I., Castro L.R. 2009. Seasonal and short-term variability in the vertical distribution of ichthyoplankton in a stratified fjord of southern Chile. Cien. Tecnol. Mar. 32: 27-42.

Lea D.W., Shen G.T., Boyle E.A. 1989. Coralline barium records temporal variability in Equatorial Pacific upwelling. Nature 340: 373-376.
https://doi.org/10.1038/340373a0

Lillo S., Lang C., Molina E., et al. 2015. Evaluación hidroacústica de pequeños pelágicos en aguas interiores de la X y XI Regiones, año 2014. Informe Final Proyecto Convenio desempeño 2014. Instituto de Fomento Pesquero, Chile, 161 pp.

Llanos-Rivera A., Castro L.R. 2004. Latitudinal and seasonal egg size variations of the anchoveta Engraulis ringens off the Chilean Coast. Fish. Bull. 102: 207-212.

Llanos-Rivera A., Castro L.R. 2006. Inter-population differences in temperature effects on Engraulis ringens yolk-sac larvae. Mar. Ecol. Prog. Ser. 312: 245-253.
https://doi.org/10.3354/meps312245

Luoma S. 1990. Processes affecting metal concentrations in estuarine and coastal sediments. In: Furnes R., Rainbow P. (eds), Heavy Metals in the Marine Environment. CRC Press. New York, pp. 51-66.

Macdonald J., Shelley M., Crook D. 2008. A Method for Improving the Estimation of Natal Chemical Signatures in Otoliths. Trans. Amer. Fish. Soc. 137: 1674-1682.
https://doi.org/10.1577/T07-249.1

Morais P., Babaluk J., Correia A., et al. 2010. Diversity of anchovy migration patterns in a European temperate estuary and in its adjacent coastal are: Implications for fishery management. J. Sea Res. 64: 295-303.
https://doi.org/10.1016/j.seares.2010.04.001

Nozaki Y. 2001. Elemental distribution. In: Steele J.H., Thorpe S.A., Turekian K.K. (eds), Encyclopedia of Ocean Sciences. Academic Press, San Diego, CA. pp. 840-845.
https://doi.org/10.1006/rwos.2001.0402

Pauly D., Tsukayama I. 1987. The Peruvian Anchoveta and its Upwelling Ecosystem: Three Decades of Change. ICLARM Studies and Reviews, 351 pp.

Plaza G., Cerna F. 2015. Validation of daily microincrement deposition in otoliths of juvenile and adult Peruvian anchovy Engraulis ringens. J. Fish Biol. 86: 203-216.
https://doi.org/10.1111/jfb.12561

Ranaldi M., Gagnon M. 2008. Zinc incorporation in the otoliths of juvenile pink snapper (Pagrus auratus Forster): The influence of dietary versus waterborne sources. J. Exper. Mar. Biol. Ecol. 360: 56-62.
https://doi.org/10.1016/j.jembe.2008.03.013

Rooker J., Secor D., Zdanowicz V., et al. 2003. Identification of Atlantic bluefin tuna (Thunnus thynnus) stocks from putative nurseries using otolith chemistry. Fish. Oceanogr. 12: 75-84.
https://doi.org/10.1046/j.1365-2419.2003.00223.x

Serra J.R. 1983. Changes in the abundance of pelagic resources along the Chilean coast. In: Sharp G., Csirke J. (eds), Proceedings of the Expert Consultation to Examine Changes in Abundance and Species Composition of Neritic Fish Resources. FAO Fish. Rep. 291: 255-284.

Sinclair M. 1988. Marine populations: an essay on population regulation and speciation. Washington Sea Grant Program, Seattle, Washington, 252 pp.

Sinclair M.M., Smith T.D. 2002. The notion that fish species form stocks. ICES Mar. Sci. Symp. 215: 297-304.

Soto-Mendoza S., Parada C., Castro L., et al. 2012. Modelling transport and survival of anchoveta eggs and yolk-sac larvae in the coastal zone off central-southern Chile: Assessing spatial and temporal spawning parameters. Prog. Ocean. 92: 178-191.
https://doi.org/10.1016/j.pocean.2011.07.001

Schuchert P., Alexander I., Arkhipkin E., et al. 2010. Traveling around Cape Horn: Otolith chemistry reveals a mixed stock of Patagonian hoki with separate Atlantic and Pacific spawning grounds. Fish. Res. 102: 80-86.
https://doi.org/10.1016/j.fishres.2009.10.012

Sturrock A., Trueman C., Darnaude A., et al. 2012. Can otolith elemental chemistry retrospectively track migrations in fully marine fishes? J. Fish Biol. 81: 766-795.
https://doi.org/10.1111/j.1095-8649.2012.03372.x

Thorrold S., Zacherl C., Levin L. 2007. Population Connectivity and Larval Dispersal, Using Geochemical Signatures in Calcified Structures. Oceanography 20: 80-89.
https://doi.org/10.5670/oceanog.2007.31

Valdivia I., Chávez A., Oliva E. 2007. Metazoan parasites of Engraulis ringens as tools for stock discrimination along the Chilean coast. J. Fish Biol. 70: 1504-1511.
https://doi.org/10.1111/j.1095-8649.2007.01429.x

Warburton M., Reid M., Stirling C., et al. 2016. Validation of depth-profiling LA-ICP-MS in otolith applications. Can. J. Fish. Aquat. Sci. 74: 572-581.
https://doi.org/10.1139/cjfas-2016-0063

Wheeler S.G., Russel A.D., Fehrerenbacher J.S., et al. 2016. Evaluating chemical signatures in a costal upwelling region to reconstruct water mass associations of settlement-stage rockfishes. Mar. Ecol. Prog. Ser. 550: 191-206.
https://doi.org/10.3354/meps11704

Woodhead J., Hellstrom J., Paton C., et al. 2008. A guide to depth profiling and imaging applications of LA-ICP-MS. In: Sylvester P. (ed), Laser Ablation ICP-MS in the Earth Sciences: Current Practices and Outstanding Issues; Short Course 40; Mineralogical Association of Canada: Quebec City, QC, Canada, pp. 135-145.

Zúñiga M.J., Canales C. 2014. Investigación del estatus y posibilidades de explotación biológicamente sustentables anchoveta zona centro-sur, año 2014. Cuarto Informe Proyecto 2.5. Instituto de Fomento Pesquero, Chile. 81 pp.