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 1983, Pauly and Tsukayama 1987), 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 2004). 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 2004, 2006, Castro et al. 2009). 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. 2005, 2006). 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 2006, Landaeta et al. 2009, Castro et al. 2015). 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. 2009, Claramunt et al. 2014). Furthermore, significantly larger females have been found above latitude 38°S (Cubillos et al. 2009).

Evidence suggesting some spawning stock and population connectivity in this central-southern fishery has been proposed. Soto-Mendoza et al. (2012) 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. 2002, Valdivia et al. 2007, George-Nascimento et al. 2015), 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 2014) 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 1988, Sinclair and Smith 2002, Cadrin et al. 2005). 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. 2005). 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. 2010, Morais et al. 2010, Avigliano and Volpedo 2016). 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 1999, Chang and Geffen 2013, Górski et al. 2015). 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. 2000, Rooker et al. 2003, Thorrold et al. 2007).

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.

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.

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. 2010, Javor and Dorval 2017), 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. 2015, Lillo et al. 2015). Similar to patterns observed in juvenile sardines Sardinops sagax (Javor and Dorval 2017), 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. 1989, Nozaki 2001), and its concentrations in surface waters significantly increase during upwelling events (Hsieh and Henderson 2017). 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. 2016, Hampton et al. 2018). A similar nutrient-like distribution might be expected for Sr (De Villiers 1999), though we found no significant differences in Sr:Ca rations in otoliths between central Chile and northern Patagonia. Wheeler et al. (2016) 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) 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. 1993). 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 2008) and tends to be higher in urban coastal zones due to anthropogenic sources (Sturrock et al. 2012).

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. 2014). 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 1990, Giordano et al. 1992, French 1993). 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. 2010, 2011, 2013) 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. 2010, Guidetti et al. 2013).

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. 2013). 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. 1983, Serra 1983, Castro et al. 1997), 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.