Small pelagics such as anchovy support large fisheries in the Mediterranean Sea (Lleonart and Maynou 2003). Like other small pelagic species, the anchovy has a short life span, with high rates of natural mortality and fecundity in which recruitment plays a major role in setting year-to-year changes in the level of the stock (Fréon et al. 2005). Areas of high productivity generally influence the location of the spawning sites and spawning intensity, giving it a seasonal character. Sexual maturity occurs during the first year and only one or two cohorts significantly contribute to the fishery (Fréon et al. 2005). In the Spanish Mediterranean, anchovy spawning takes place from April to October, with a peak in June-July (Giráldez and Abad 1995, Palomera 1992).

Due to its high economic value, anchovy is the main target species of the purse seine fleet in the Spanish Mediterranean Sea, which covers two areas, GSA06 (northern Spain) and GSA01 (northern Alboran Sea), of the 30 stocks management areas established by the General Fisheries Commission for the Mediterranean (GFCM 2007). In the period 1990-2014, anchovy catches were highly variable, with an average of 12000 t in GSA06 and an average of 891 t in GSA01. Whereas in GSA06 the anchovy catches are spread throughout the area (from the French border until the Cape of Palos), in GSA01 they are mainly located in the Bay of Málaga, the only area where anchovy is fished throughout the year (CFCM 2015).

Fishery-independent data provided by scientific surveys play an important role in the assessment and management of fish populations (Pennington and Stromme 1998). Research surveys are used to calibrate stock assessment models based on commercial catches (i.e. fishery-dependent data) and to provide empirical, independent population checks. (Kline 1996, Pennington and Stromme 1998). In order to determinate the anchovy spawning stock biomass, the small pelagic species assessment survey (MEDIAS) is carried out every summer by the countries within the European Union using acoustic methods. The assessment survey takes place during the anchovy spawning peak when mixing between putative stocks is minimal (Cadrin et al. 2014), so the MEDIAS survey period is optimal for discriminating between anchovy stocks.

Various methods of stock discrimination in marine fishes have been widely used, especially spawning areas and seasons, abundance and distribution patterns, length-frequency distribution differences (Guerra-Sierra and Sánchez-Lizaso 1998), tagging and migration studies, parasites, serology (Cadrin et al. 2014), biochemical-genetic differences (Zarraonaindia et al. 2012, Viñas et al. 2013) and otolith morphometric parameters (Cendrero and Abaunza 2001, Carvalho and Castello 2013, Jansen et al. 2013). Otolith length is an ideal measurement for determining anchovy length, because a quasi-perfect linear relationship exists between them (Zengіn et al. 2015). Moreover, the length of the anchovy otolith first radius (R1) can be understood as an indicator of first-year growth (Hernández et al. 2013).

Otoliths, also called ear-bones, are part of the fish auditory system (Ladich and Schulz-Mirbach 2016). They are made mostly of calcium carbonate (Panfili et al. 2002), and as the fish grows, so does the otolith, by deposition of concentric layers of material according to a daily rhythm (Pannella 1971). Seasonal changes in the fish growth rate are reflected in the otolith. A year’s growth consists of a wider summer zone, reflecting a faster growth rate related to higher food availability, and a narrower winter zone, reflecting a slower growth rate (Panfili et al. 2002). Therefore, due to their special characteristics, otoliths can be a useful tool for age determination (Rodriguez Mendoza 2006). Knowledge of the population age structure of an exploited fish species is the key to understanding its basic population biology and is a necessary prerequisite for providing effective advice to fisheries managers (Cadrin et al. 2014).

During the MEDIAS survey (2012–2016), larger anchovy individuals were always collected from GSA01 (Alboran Sea). Initially, it was thought that older anchovies inhabited this area, but the interpretation of the annual rings in otoliths gave the same age structure in both areas (GSA01 and GSA06). The main question to be resolved was: Are the observed differences in anchovy growth pattern due to the fact that the GSA01 and GSA06 subareas hold different anchovy stocks?

Taking the Spanish part of the Alboran Sea and the Ebro Delta as reference sites of GSA01 and GSA06 respectively, as they are the most productive areas of each GSA (Bellido et al. 2008), the hypothesis of the existence of different anchovy stocks along the Spanish Mediterranean Sea has been tested.

In order to test the null hypothesis: "anchovy inhabiting the Spanish Mediterranean constitute a single stock" different and complementary methodologies have been applied to the MEDIAS survey time series data (2012-2016): differences in length, based on the length frequency distribution; differences in growth, based on otolith reading and otolith morphometric parameters; and differences in the condition factor, based on the length-weigh relationship parameters.

The separation of stocks is of primary importance for the sustainable management of the fisheries and has important ecological consequences. The findings highlighted in this paper have great economic and ecological relevance because the management of the anchovy population in the Mediterranean Sea as two different stocks with different fishing regulations will reduce the pressure on GSA01 anchovies and will in the future achieve the recovery of the population.

Research surveys constitute the main fisheries-independent data source and contribute to the assessment and management of fish populations (Pennington and Stromme 1998). This study is based on data collected during the MEDIAS survey, which is of added value given that (1) the data lack spatial bias because the whole anchovy distribution area is covered and not only the fishing grounds; (2) catches are not restricted by depth or mesh size, so the entire population length range is sampled; (3) fisheries acoustics is a direct method based on "ground-truth" information, through which all the anchovy echotraces are identified (McClatchie et al. 2000); and (4) the survey period coincides with the anchovy spawning peak (Giráldez and Abad 1995, Palomera 1992) and with the maximum anchovy abundance (Palomera and Sabatés 1990), so it is optimal for stock discrimination (Cadrin et al. 2014).

The results of the current study point to the existence of two separate anchovy stocks in the Spanish Mediterranean Sea (GSA06 and GSA01), based on their different growth rates. Similar results have been reported in genetic studies (Sanz et al. 2008, Zarraonaindia et al. 2012, Viñas et al. 2013) and in studies on larval growth (Quintanilla et al. 2015). Moreover, similar differences in growth by area have also been observed for sardine (Sardina pilchardus) in the Spanish Mediterranean Sea (Alemany and Álvarez 1993).

LFDs exhibited clear differences between GSA06 and GSA01, GSA01 anchovy being larger than GSA06 anchovy. But, contrary to what could be expected, GSA01 anchovy were not older than GSA06 anchovy. This was the first evidence that these anchovy might not belong to the same population. The anchovy demographic structure found in the whole Spanish Mediterranean Sea was based chiefly on two year classes (anchovy of 0 and 1 year old), given that two-year-old anchovy were very scarce in GSA06 and almost absent in GSA01. Our results are in agreement with the fisheries-dependent data collected throughout the year (GFCM 2014, 2015), which confirm the MEDIAS survey data as an excellent benchmark. Moreover, a similar anchovy demographic structure for the same time period was found in the adjacent GSAs, GSA07, Gulf of Lions (Van Beveren et al. 2014) and GSA03, southern Alboran Sea (Bacha et al. 2010, 2014, Jemaa et al. 2015). Other studies carried out in earlier years in the Mediterranean Sea reported a life expectancy of 3 to 4 years (Basilone et al. 2004, Van Beveren et al. 2014) and 5 years in the Bay of Biscay (Uriarte et al. 1996), but all concluded that only a minor fraction belongs to these oldest age classes.

Small pelagic fish are essential elements of marine ecosystems due to their significant biomass at intermediate levels of the food web, playing a considerable role in connecting the lower and upper trophic levels (Cury et al. 2000). Anchovy plays an important part in the pelagic food web in the Spanish Mediterranean for primary producers and consumers as they constitute their prey (Costalago et al. 2012), as well as for higher trophic levels, such as anchovy predators (Cardona et al. 2015). Therefore, fluctuations in small pelagic populations due to fishing or natural factors modify ecosystem structure and functioning and have a major impact on the whole ecosystem (Cury et al. 2000).

In the Mediterranean Sea the anchovy exploitation rate has been steadily increasing since 1990, with the consequence that the demographic structure of the stocks has shrunk (Vasilakopoulos et al. 2014) and the anchovy life span has decreased (GFCM 2014). In fact, anchovy life expectancy has gone down in the last few decades in the Spanish Mediterranean Sea. During studies carried out in the 1980s four-year-old anchovy were commonly found (Pertierra 1987, Morales-Nin and Pertierra 1990), whereas now, using the same ageing criteria, adopting 1 July as the arbitrary birth dates, the oldest age found is two years old.

Interpreting the otolith’s annual rings in order to age the anchovy is often a difficult task, especially given the presence of false rings and the difficulty of determining the edge type (ICES 2009, 2013). In the Spanish Mediterranean Sea two obvious distinctive otolith structures that matched the two study areas were observed during the reading process. In general, GSA01 otoliths were harder, with the anterior and posterior margin sharper, and showed less structural variability than the GSA06 otoliths, perhaps due to fewer cohorts contributing to the population in GSA01. In both areas otoliths showed a homogeneous, white, well-defined nucleus, being greater and more opaque in GSA01. GSA06 otoliths usually showed a small hyaline round area in the centre associated with the juvenile period, while in GSA01 this area was uncommon. This homogeneous nucleus was surrounded by a clear and continuous hyaline ring that corresponded to the first hyaline ring. The larger opaque centre observed in GSA01 otoliths was due to a higher anchovy growth rate during the first year, as confirmed by the R1 measures. This fact was the definitive proof of the existence of two anchovy stocks in the Spanish Mediterranean Sea, as was later corroborated by the results of the cluster analysis. The study of the degree of well-being of the anchovy population based on the condition factor index (Kn) proposed by Le Cren (1951) revealed that anchovy individuals in GSA01 were not only larger in length, but also had a greater weight for a given length than the anchovy in GSA06. Both the Ebro Delta (GSA06) and the Alboran Sea (GSA01) are essential habitats for anchovy due to their productive conditions (Bellido et al. 2008). However, the enrichment process differs: in the Alboran Sea nutrient enrichment comes mainly from coastal upwelling (Agostini and Bakun 2002, Mercado et al. 2013), whereas in the Ebro Delta area it comes via the transport of nutrients from the Gulf of Lions and the turbulent mixture occurs as a result of the discharge of nutrient-rich fresh water coming from the Ebro River (Salat 1996, Agostini and Bakun 2002). The Atlantic water enters the Mediterranean through the Strait of Gibraltar. The inflow of the Atlantic water is first directed northeastward due to the orientation of the Strait of Gibraltar, and then generally describes a clockwise gyre in the east of the Alboran Sea between Spain and Morocco. This surface flow then spreads to the African coast, mainly due to the effect of the Coriolis force (El-Geziry and Bryden 2010). The surface circulation in northern Spain (GSA06) is dominated by two well-defined, density-driven currents. In the northern part, a frontal jet flows southwestward along the slope (Castellón et al. 1990). In the south, off the Gulf of Valencia, part of the flow continues its path southward along the Spanish coast. Another branch bifurcates and recirculates cyclonically to feed the return Balearic Current along the northern Balearic Island shelf (Pinot et al. 2002).

Though both areas are productive (Bellido et al. 2008), the chlorophyll and primary production values in the Alboran Sea are higher than those in the Delta zone (Siokou-Frangou et al. 2010, Lazzari et al. 2012, Salgado and Lorenzo 2014). By contrast, the surface temperature is lower in Alboran Sea than in the Ebro Delta area, although the main trend in both areas is an increasing temperature (Shaltout and Omstedt 2014) . Therefore, it can be deduced that the environmental variables are more favourable for anchovy in GSA01. Indeed, anchovy belonging to GSA01 show better condition than those from other Mediterranean or Black Sea areas, possibly because of the influence of the Atlantic Ocean (Brosset et al. 2017).

Though the GSA management areas established by the GFCM were based on political and statistical considerations rather than on biological or economic factors (Lleonart and Maynou 2003), they are indeed appropriate for anchovy management purposes in the Spanish Mediterranean Sea, so GSA01 and GSA06 should be maintained as individual areas for assessment, as also happened with demersal stocks in GSA05 (Balearic Islands) and GSA06 (Quetglas et al. 2012).

The results obtained in this study not only have a great biological relevance in verifying the existence of different anchovy stocks in the Spanish Mediterranean, but can also have a direct application in the fields of conservation and management. Anchovy belonging to GSA01 had a greater length at the same age and were in better condition than anchovy belonging to GSA06, but the unresolved question is: Why is the anchovy biomass per area in GSA01 lower than that in GSA06? (GFCM 2015). Perhaps this fact is related to the application of the same regulation concerning minimum legal length, 9 cm (BOE 1995), for the whole Spanish Mediterranean, which could negatively affect the anchovy GSA01 population. This aspect is paramount, because anchovy is a key component of the pelagic ecosystem (Cury et al. 2000) and its conservation is crucial for the pelagic food web equilibrium. Therefore, the decline of the anchovy population in the Alboran Sea could affect other species very negatively, destabilizing the food chain. Finally, the application of different legislation for these two zones is proposed, as other authors have also suggested (Pla et al. 1996, Lleonart and Maynou 2003, Viñas et al. 2013).