Spatial and temporal dynamics of larval fish communities in relation to environmental variability in the NW Mediterranean

PhD program in Marine Sciences Spatial and temporal dynamics of larval fish communities in relation to environmental variability in the NW Mediterranean Doctoral thesis by: Vanesa María Raya Rodrigálvarez Thesis advisor: Ana María Sabatés Freijo Institut de Ciències del Mar, CSIC Barcelona, May 2025 This thesis was conducted in the Institut de Ciències del Mar (ICM – CSIC) and was made possible thanks to the projects CACO (REN 2002-01339/MAR), FISHJELLY (MAR-CTM201018874) and WINFISH (CTM2015-68543-R). This work acknowledges the Severo Ochoa Centre of Excellence accreditation (CEX2019000928-S). A mi familia, por su apoyo incondicional. A todas las personas que he encontrado en el camino, porque cada paso me ha llevado a donde estoy y a que esta tesis sea posible. i Table of contents Abstract iii Keywords iv Preface. Prefacio v Acknowledgements. Agradecimientos ix List of tables xiii List of figures xiv 1. Introduction 1 1.1. Introduction 3 1.1.1. General features of the NW Mediterranean 3 1.1.2. The Mediterranean Sea as a biodiversity hotspot 6 1.1.3. The Mediterranean Sea as a climate change hotspot 6 1.1.4. The relevance of ichthyoplankton studies 8 1.1.5. Larval fish assemblages 10 1.1.6. Evolution of fish larvae studies in the Mediterranean Sea 11 1.1.7. Larval fish communities and environmental conditions in the NW Mediterranean 13 1.2. Objectives 15 1.3. General methods 17 1.3.1. Field sampling and oceanographic data 17 1.3.2. Fish larvae identification 19 1.3.3. Statistical analysis of fish larvae data 20 2. Results 21 Chapter 1. Variability of larval fish assemblages under contrasting winter environmental conditions in the NW Mediterranean 23 Abstract 25 2.1.1. Introduction 26 2.1.2. Materials and methods 28 2.1.3. Results 32 2.1.4. Discussion 43 References 49 Chapter 2. Recurrence of the spatial structure of summer larval fish assemblages linked to hydrodynamics in the NW Mediterranean 61 Abstract 63 2.2.1. Introduction 64 2.2.2. Materials and methods 65 2.2.3. Results 68 2.2.4. Discussion 81 References 85 Chapter 3. Long-term variability of larval fish community in the NW Mediterranean in summer 95 Abstract 97 2.3.1. Introduction 98 2.3.2. Materials and methods 99 2.3.3. Results 101 2.3.4. Discussion 113 References 118 ii Chapter 4. The Box-Balance Model: A new tool to assess fish larval survival, applied to field data on two small pelagic fish 131 Abstract 133 2.4.1. Introduction 134 2.4.2. Materials and methods 136 2.4.3. Results 141 2.4.4. Discussion 150 2.4.5. Conclusions 155 References 155 Appendix. Applying the Box-Balance Model (BBM) to field data 163 3. General discussion 165 3.1. Seasonality in larval fish communities 168 3.1.1. Diversity of larval fish communities 168 3.1.2. Influence of environmental variables on larval fish communities 169 3.2. Larval fish communities in the context of climate change 173 3.2.1. Long-term changes in summer larval fish communities 174 3.3. Comparative analysis of larval survival 177 3.4. Future directions 179 4. Conclusions 181 5. General bibliography 185 6. Annex. Published work 209 iii Abstract The early developmental stages of fish, eggs and larvae, found in the planktonic environment are subject to a high mortality. Thus, the study of larval survival has been a key issue in fisheries science since the early 20th century. Spatial patterns in the larval fish communities are influenced by a complex array of environmental processes that interacts with fish biology at different temporal and spatial scales. These processes include those of large scale, such as climate patterns and seasonal and interannual environmental variability, which determine adults’ distribution and their spawning strategies. At local and short time scale, larval fish communities are shaped by the hydrodynamics that influence fish larval dispersal and retention, and by biologic factors, such as food concentration and predation, that ultimately determine their survival. This thesis characterises the structure of the larval fish community in summer and winter in the Catalan coast (NW Mediterranean), an area with a wide array of environmental conditions and high hydrodynamic activity. The aim is to understand its spatial and interannual variability in response to changes in environmental conditions, including oceanographic variables and hydrodynamic processes. Within the context of climate change, the thesis describes long-term changes in the structure of the summer larval fish communities and aims to understand the interactions between larvae of established species and species that are expanding northwards in the area. To investigate the influence of winter environmental conditions on the structure of fish larval communities, two winters, 2017 and 2018, with contrasting environmental conditions were compared. 2017 was mild, while 2018 was more severe, with intense vertical mixing and deep-water formation and cascading events that enhanced shelf-slope water exchanges. Differences in the structure of larval fish community were found in relation to shelf-slope water exchange processes. A high spatial heterogeneity in larval fish communities was detected in the summers of 2003, 2004 and 2012, related to environmental factors, such as the continental shelf structure, latitudinal difference in surface temperature, primary productivity, and stratification level. Hydrodynamic structures such as instabilities of the Northern Current and anticyclonic eddies, also played an important role in the configuration of these communities. In summer, over three decades, 1980s, 2000s and 2010s, an increase in sea water temperature and a decrease in chlorophyll were detected. Changes in the composition and abundance of the larval fish community were also detected. These were mainly due to the presence of warm water species in the area for the first time, or to their increase in abundance, in the 2000s in relation to the northward expansion of the adults' range. Other species showed a decline in abundance over time, probably due to the decrease in chlorophyll. iv This work quantitatively compared the survival chances for larvae of E. encrasicolus (an established species) and S. aurita (a species expanding northwards). To this aim, a new method, the Box-Balance Model, was developed to evaluate the role of hydrodynamic structures in their mortality. The model revealed that despite the warming trend would contribute to the expansion of S. aurita, it has not yet developed an adaptation strategy as successful as that of E. encrasicolus, a well-established species in the area. Keywords Biological oceanography, larval fish distribution, shelf-slope exchanges, deep-water formation, environmental conditions, hydrodynamic structures, fish spawning habits, interannual variability, larval fish growth, larval fish survival. v Preface. Prefacio Desde el punto de vista científico, el Mar Mediterráneo es un sistema único, por su situación entre continentes, por su circulación termohalina a pequeña escala y por albergar ecosistemas complejos, de gran biodiversidad y con un gran número de especies endémicas. Es además uno de los mares donde los impactos antrópicos son más numerosos y llevan actuando desde tiempos remotos. Los ecosistemas del Mar Mediterráneo son muy vulnerables, y los peces suponen un elemento importante de todos estos ecosistemas. Además, los estadios iniciales de la vida de los peces son las etapas que están sometidas a una mayor presión. Por su alta vulnerabilidad, las larvas de peces suponen una pieza única para averiguar cómo afectan los cambios inducidos por las actividades humanas a los ecosistemas marinos. Esta tesis pretende contribuir a la comprensión del papel que desempeñan estas piezas clave en el sistema del Mar Mediterráneo. Pero esta tesis no surge solamente con la intención de contribuir al conocimiento científico. Del amor al mar nace esta tesis, porque el Mediterráneo es mucho más que una inmensa masa de agua entre continentes y mucho más que un ecosistema único. Nace también la certeza de que la conservación de los océanos y en particular, del mar Mediterráneo es necesaria. Y para la conservación es necesario primero el conocimiento. Por eso esta tesis para mí significa mucho más que un título académico. xii xiii List of tables Table 2.1.1. Mean abundance and standard deviation (No. larvae 10 m - 2 ), relative abundance (RA, in %) and frequency of occurrence (FO, in %) of all identified larval fish taxa in 2017 and 2018. The taxa are listed according to adult habitat (shelf and oceanic). 35 Table 2.1.2. Summary of the similarity percentage routine (SIMPER): P-value obtained in the pairwise comparisons, the five species that most contribute to the cluster differences (ordered from highest to lowest contribution), and the cumulative percentage contribution to these differences. 38 Table 2.1.3. Summary of the canonical correspondence analysis (CCA) results. The individual variables are ordered by the percentage of variance explained (Exp. Var.), the significance of each variable (P) together with its test statistic (F-value), and the inter-set correlations between the environmental variables and the first two axes of the CCA. 40 Table 2.1.4. Weighted mean depth (WMD, in m) and standard deviation, during day and night, for the most abundant larval fish taxa, in 2017 and 2018. 44 Table 2.2.1. Mean abundance and standard deviation (No. larvae 10 m - 2 ), relative abundance (RA, in %) and frequency of occurrence (FO, in %) of all identified larval fish taxa in July 2003, 2004 and 2012. 71 - 72 Table 2.2.2. Summary of the canonical correspondence analysis (CCA) results. Individual variables are ordered by the percentage of variance explained (Exp. Var.), in relation to the total and constrained variance. The significance of each variable (P) together with its test statistic (F-value), and the interset correlations of environmental variables with the three significant axes of the CCA are provided. 76 Table 2.3.1. Mean abundance and standard deviation (No. larvae 10m - 2 ) of all identified larval fish taxa in the 1980s, 2000s and 2010s surveys. 103 -105 Table 2.3.2. Summary of the redundancy analysis (RDA) results. The individual variables are ordered by the percentage of variance explained (Exp. Var.), the significance of each variable (P) together with its test statistic (F-value), and the inter-set correlations between the environmental variables and the first two axes of the RDA. 108 Table 2.4. 1. Summary of the regression analyses for: 1) the correction of the fish larvae SL after shrinkage by preservation; 2) the age-length relationship; and 3) the catchability factor (q) for day/night samples comparisons (see text for more details). SE(a) and SE(b) are the standard error of the coefficients and SE is the standard error of the estimate. 141 Table 2.4. 2. Summary of the Wilcoxon rank-sum test for the differences between day and night abundances at length for Engraulis encrasicolus and Sardinella aurita larvae. 142 Table 2.4. 3. Linear regression analyses of larval log-abundances at age for the mortality estimation of Sardinella aurita and Engraulis encrasicolus by survey, specifying the age ranges (S, in days) used. SE is the standard error of the estimate. Comparisons among surveys are shown by the ANCOVA for both species. 144 Table 2.4. 4 . Linear regression of corrected larval log-abundances at age for the mortality estimations, as applied to the BBM for S. aurita and E. encrasicolus, specifying the age ranges used for each survey (S, in days). SE is the standard error of the estimate. 145 xiv List of figures Fig. 1. 1. Map of the Mediterranean Sea showing the circulation of the Atlantic Water and areas of deep water formation. 3 Fig. 1. 2. Spatial trends (°C per decade) over 1981–2020, and total change multi-model mean projected changes from 1995–2014 to 2081–2100 (From IPCC, 2021). 6 Fig. 1. 3. Projected water cycle changes. Long-term (2081–2100) projected annual mean changes (%) relative to present-day (1995–2014) for (a) precipitation and (b) total runoff. (From IPCC, 2021). 7 Fig. 1.4. Diagram of the different factors that affect the formation, maintenance and disruption of larval fish assemblages, acting at different temporal and spatial scales. Adapted from Doyle (1993). 10 Fig. 1.5. Peer reviewed SCI publications between 1970 and 2024 containing the words ichthyoplankton, ‘fish larv*’ or ‘larval fish’ and ‘larval fish assemblage*/communit*’ (LFA: Larval fish assemblages) worldwide and in the Mediterranean. The ordinate axis on the left indicates the number of larval fish publications and the ordinate axis on the right the number of publications of larval fish assemblages. Source: Scopus. 12 Fig.1.6. Sampling area covered by the different surveys conducted in the Catalan coast. The area marked with the blue stripe pattern corresponds to the winter surveys, the yellow area to the summer surveys conducted in 2000s and 2010 and the salmon area to the study performed over the three decades. 17 Fig. 1.7. Anatomical, morphological and some specialised characters of post-flexion larvae used for the larval identification. 19 Fig. 2. 1. 1. Study area in the NW Mediterranean, south of the Gulf of Lion. Crosses indicate sampling stations (CTD and Bongo net) during 2017 (orange) and 2018 (dark blue) surveys. Triangles indicate stations where only CTD casts were performed. Orange circles correspond to MOCNESS net samplings. Stars correspond to depth-stratified sampling at fixed stations in 2017 (orange) and 2018 (blue) surveys. Open ellipses (green, Coast, Shelf, Slope and Palamós canyon) indicate the vertical profiles of oceanographic variables shown in figure 4. The isobaths shown are 50, 200, 1000 and 2000 m. 28 Fig. 2.1.2. Spatial distribution of surface (10 m) temperature (SST, a and b), salinity (SSS, c and d), chlorophyll-a (SSChla, e and f) and dynamic height relative to 500 m (DH) overlaid on density (, g and h) in winter 2017 (left panels) and 2018 (right panels). Arrows indicate the flow direction and intrusions of the Northern Current and the extrusions of coastal waters. The isobaths shown are 50, 200, 1000 and 2000 m. 31 Fig 2.1.3. Vertical profiles of density (  ), mean (circle) and standard deviation (horizontal lines) of CTD stations sampled in 2017 and 2018. 32 Fig. 2.1.4. Vertical profiles of temperature (red), salinity (blue), density (  , black), and chlorophyll-a (green), for four selected stations for 2017 and 2018, at different locations: coast (a and b), shelf (c and d), slope (e and f) and the Palamós Canyon (g and h) (for station locations see figure 1). 34 Fig. 2.1.5. Spatial distribution of total fish larvae (a and b), larvae of shelf (c and d) and oceanic (e and f) fish taxa, according to the classification in Table 1, overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m). 36 xv Fig. 2.1.6. (a) Ward’s hierarchical clustering of stations in winter 2017 and 2018, and heatmap showing the abundance of the fish larvae (colour gradation from white to dark red represents from absence to the highest abundance), the six clusters are indicated by coloured boxes labelled with numbers. (b) Spatial distribution of sampling stations showing the groups identified in the cluster analysis. (c) Non-metric multidimensional scaling (nMDS) ordination plot. Symbols of the same color indicate clusters of stations with similar species composition. Circles correspond to the stations sampled in 2017 and triangles to the stations sampled in 2018. 39 Fig. 2.1.7. Ordination plot of the canonical correspondence analysis (CCA) showing the relationships between the fish larval taxa, the sampling stations, and the explanatory environmental variables (vectors) for the winter of 2017 and 2018. Symbols of the same color indicate clusters of stations with similar species composition. Circles correspond to the stations sampled in 2017 and triangles to the stations sampled in 2018. Abbreviations of the taxa names are detailed in Table 2.1.1. 40 Fig. 2.1.8. Spatial distribution of score values of stations for the axis 1 (a and b) and axis 2 (c and d) that resulted from the canonical correspondence analysis (CCA) for 2017 (a and c) and 2018 (b and d). Colour symbols denote the stations belonging to the group identified in the cluster analysis. (a) and (b): Scores ordination in relation to the first ordination axis. (c) and (d): Scores ordination in relation to the second axis. Black line separates positive and negative values. 41 Fig. 2.1.9. Spatial distribution of fish larvae of some representative oceanic species overlaid on sea surface temperature (SST) and bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in 2017 and 2018. 42 Fig. 2.1.10. Spatial distribution of fish larvae of some representative shelf and upper slope species overlaid on sea surface temperature (SST) and bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in 2017 and 2018. Note that S. pilchardus and Arnoglossus spp. are represented on a different size scale to M. poutassou and G. macrophthalmus. 43 Fig. 2.1.11. Violin plot showing the vertical distribution of larvae of Arctozenus risso, Myctophum punctatum, Sardina pilchardus and Micromessistius poutassou, during day and night, for 2017 and 2018. The width of the violin represents the proportion of larvae at each depth stratum. 45 Fig. 2.2.1. Study area off the Catalan coast, in the NW Mediterranean. Blue circles indicate sampling stations (CTD and Bongo net) during the July 2003 and 2004 surveys. Crosses indicate sampling stations (CTD and Bongo net) during the July 2012 survey. 66 Fig. 2.2.2. Spatial distribution of surface (5 m) temperature (SST; a, b and c), salinity (SSS; d, e and f), geostrophic current field (arrows) and dynamic height (DH) indicted by the colour grading (g, h and i), chlorophyll-a (SSChla; j, k and l) and pynocline depth (PD; black lines) overlaid on the maximum density gradient (Gmax; m, n and o), in July 2003 (left panels), 2004 (central panels) and 2012 (right panels). Dark arrows indicate the location of the Blanes and Palamós canyons. 69 Fig. 2.2.3. Spatial distribution of abundance of larvae of shelf (upper panels, a, b and c) and oceanic (central panels, d, e and f) fish taxa, according to the classification in Table 1, and species richness (bottom panels, g, h and i), overlaid on bathymetry. Isobaths shown: 50, 200 and 1000 m. 73 Fig. 2.2.4. (a) Dendrogram showing the classification of the station assemblages using a Ward’s clustering method based on the Bray–Curtis similarity matrix of the abundance of larval fish off the Catalan coast in 2003, 2004 and 2012. The six significant clusters are identified by the colour squares. The scale (height) corresponds to the squared distances. (b) Spatial distribution of sampling stations showing the groups identified in the cluster analysis. Isobaths shown: 50, 200 and 1000 m. 73 Fig. 2.2.5. Plot of the mean abundance (No. per 10m 2 ) of taxa for each cluster. 74 xvi Fig 2.2.6. Non-metric multidimensional scaling (nMDS) ordination plot. Symbols of the same colour indicate clusters of stations with similar species composition. Squares correspond to the stations sampled in 2003, circles to the stations sampled in 2004 and triangles to the stations sampled in 2012. 75 Fig. 2.2.7. Venn diagram of the variation partitioning analysis showing the percentage of variation explained in the larval fish assemblages by each term, spatial, environmental, and inter-annual. 76 Fig. 2.2.8. Ordination plots of the environmental canonical correspondence analysis (CCA) (left panels, a and c) and of the spatial CCA (right panels, b and d). Upper panels, a and b: Ordination of the sampling stations in the CCA plots, showing the relationships between the sampling stations and the explanatory environmental variables (blue vectors), for July of 2003, 2004 and 2012. Symbols of the same colour indicate clusters of stations with similar species composition. Squares correspond to the stations sampled in 2003, circles to the stations sampled in 2004 and triangles to the stations sampled in 2012. Lower panels, c and d: Ordination of the larval fish taxa in the CCA plots, showing the relationships between the larval fish taxa and the explanatory environmental variables (blue vectors), for July of 2003, 2004 and 2012. The abbreviations of larval fish names are indicated in Table 2.2.1. 77 Fig. 2.2.9. Spatial distribution of score values of stations for the axis 1 (upper panels, a, b and c) and axis 2 (lower panels, d, e and f) that resulted from the environmental canonical correspondence analysis (CCA) for 2003 (left panels, a and c), 2004 (central panels, b and e) and 2012 (right panels, c and f). Symbols of the same colour denote stations belonging to the same group identified in the cluster analysis. Isobaths shown: 50, 200 and 1000 m. 78 Fig. 2.2.10. Spatial distribution of the fitted score values of stations for the axis 1 (colour gradient) that resulted from the spatial canonical correspondence analysis (CCA) on the detrended larval fish matrix for 2003 (left panel), 2004 (central panel) and 2012 (right panel). Geostrophic current field (arrows) and bathymetry have been overlaid. Isobaths shown: 50, 200 and 1000m. 78 Fig. 2.2.11. Spatial distribution of fish larvae of representative shelf and oceanic species in 2003, 2004 and 2012. Seranus hepatus overlaid on SSChla (a, b and c), Sardinella aurita on SST (d, e and f), Engraulis encrasicolus (g, h and i), Trachinus draco (j, k and l), and Ceratoscopelus maderensis (p, q and r) on the geostrophic current field, and Argyropelecus hemigymnus on bathymetry (m, n and o). Isobaths shown: 50, 200 and 1000 m. 80 Fig. 2.3.1. Study area off the Catalan coast, in the NW Mediterranean. Open circles indicate sampling stations (CTD and Bongo net) during 1983 surveys, blue circles in 2003 and 2004 surveys and crosses in 2011 and 2012 surveys. 100 Fig. 2.3.2. Box plots of the environmental variables of each survey. (a) Sea surface temperature, (b) Sea surface salinity and (c) Sea surface chlorophyll-a. In each box plot, the darkened line indicates the median; boxes show the interquartile range and whiskers indicate the expected extent of 99% of the data for a Gaussian distribution. The letters at the bottom indicate the groups identified by the Tukey's post hoc test. 102 Fig. 2.3.3. Box plots of (a) total larval abundance (N), (b) species richness (S, number of species, (c) Shannon-Wiener diversity index (H’, using a natural logarithm) and (d) Pielou’s measure of evenness (J’) for each survey. In each box plot, the darkened line indicates the median; boxes show the interquartile range and whiskers indicate the expected extent of 99% of the data for a Gaussian distribution. The letters at the bottom indicate the groups identified by the Tukey's post hoc test. 106 Fig. 2.3.4. Two-way hierarchical clustering of surveys and heatmap showing the abundance of fish larvae (colour gradation from white to dark red represents from absence to highest abundance). Groups of species are indicated by coloured boxes labelled with a cod of letters and numbers, and survey groups are indicated by a different colour. 107 xvii Fig. 2.3.5. Ordination plot of the redundancy analysis (RDA) showing the relationships between the larval fish taxa, the sampling stations, and the explanatory environmental variables (vectors) for the summer 1980s, 2000s and 2010s. Blue symbols correspond to the stations sampled in the 1980s, red symbols to those in the 2000s and yellow symbols to those in the 2010s. Abbreviations of the taxa names are detailed in Table 2.3.1. 109 Fig. 2.3.6. Mean abundance and spatial distribution of Engraulis encrasicolus larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. 110 Fig. 2.3.7. Mean abundance and spatial distribution of Sardinella aurita larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. 110 Fig. 2.3.8. Mean abundance and spatial distribution of Pomatomus saltatrix larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. 111 Fig. 2.3.9. Mean abundance and spatial distribution of Caranx rhonchus larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. Note that this species was absent in the 1980s. 111 Fig. 2.3.10. Mean abundance and spatial distribution of Coris julis larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. 112 Fig. 2.3.11. Mean abundance and spatial distribution of Thalassoma pavo larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. Note that this species was absent in the 1980s. 112 Fig. 2.3.12. Mean abundance and spatial distribution of Serranus cabrilla larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. 112 Fig. 2.3.13. Mean abundance and spatial distribution of Myctophum punctatum larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. 113 Fig 2.4.1. Map of the study region, R, in the NW Mediterranean, showing the sampling stations. Grey arrows indicate the typical path of the Northern Current along the continental slope. 136 Fig 2.4.2. Age-length relationships of E. encrasicolus and S.aurita larvae. 141 Fig 2.4.3. Abundances at length for E. encrasicolus and S. aurita larvae (bars, left axis), showing day/night differences. Ratio day/night (circles) for the adjustment of the curve to obtain the catchability factor (q, right axis) (See text for details). 143 Fig 2.4.4. Larval mortality rates for E. encrasicolus and S. aurita in each survey, estimated from log-abundances at age, without applying the day-night correction (upper panels), and applying the correction (lower panels) used in the BBM. 143 Fig 2.4.5. Maps of the surface circulation overlaid on the balances (see methods) estimated at each sampling station, showing the structures of larval import/retention and export/dispersion areas in each survey, for E. encrasicolus (left) and S. aurita (right). Eddies observed in Sabatés et al (2013) are identified. The white areas indicate the sampling stations where the balance determination was not possible. 146 xviii Fig 2.4.6 . Box-balances in areas of net larval import: The north import area (July 2003 and July 2004; upper rows) and the south import area (June 2004; lower row) for E. encrasicolus and S. aurita. The reference maps (left column) indicate their respective position in the region, A is the area analysed. N indicates the number of sampling stations pooled. Solid line in bar graphs indicates the larval abundance at age predicted by the mortality estimate. The bars indicate the abundances at age: in white those that do not contribute to the mortality estimates, in blue the portion that exceed the predicted value (gain), and in red the abundance that would be required to reach the predicted value (loss). 147 Fig 2.4.7 . Box-balances in one of the retention areas, the eddy A3 in July 2004 (upper panels), its neighbourhood (central panels) and the total central area (eddy A3 and its neighbourhood) (lower panels). Descriptions of panels as in figure 6. 148 Fig 2.4.8 . Box-balances in one of the dipolar structures, the eddy C2 in June 2004. Descriptions of panels as in figure 6. 149 Fig 2.4.9 . Box-balances in the south area of net larval export in June 2004 (upper panels) and July 2004 (lower panels). Descriptions of panels as in figure 6. 149 Fig. 3.1. Schematic of some potential effects of climate-related and other anthropogenically induced changes on fish early life history. The complexity and potential for interactions of effects are evident from the many arrows from the potential stressors (dark red boxes) shared by each influential ecosystem component (i.e., predators and prey of early life stages, dark green boxes) and early life stage process (dark blue boxes). Dark red lines indicate direct effects on early life stages, while yellow lines are effects on predators and prey of early stages that can, in turn, impact early life survival. This diagram is not exhaustive in the number of potential effects or stressors and the stressors are broad (e.g., “habitat degradation” could range from chemical pollution to sound pollution, pelagic environments to benthic) and are not mutually exclusive. To reduce complexity, interactions among early life stage process boxes are not included. Adapted from Llopiz et al (2014). 173 1. INTRODUCTION 1. Introduction 3 1.1. Introduction 1.1.1. General features of the NW Mediterranean The Mediterranean Sea is a unique marine region, characterized by being surrounded almost completely by land between continents (Fig. 1.1). It is connected to the Atlantic Ocean through the narrow Strait of Gibraltar (width~13 km, sill depth~300 m) and to the Black Sea through the Dardanelles - Marmara Sea - Bosphorus system. The Mediterranean is also connected to the Red Sea through the Suez Canal, although in an artificial manner. The Strait of Sicily divides the Mediterranean Sea into two deep basins, and connects the Western Mediterranean with the Ionian Sea (Robinson and Golnaraghi, 1994). The Mediterranean is relatively small (covers a total area of 2.5 million km2) and is a deep basin (average depth of 1460 m, maximum 5267 m). Continental shelves are typically narrow and account for less than 20% of the total sea surface. The net evaporation exceeds precipitation and river inputs functioning as a concentration basin with high salinity (37.5 – 39.5). Through the Strait of Gibraltar, the Mediterranean imports nutrient poor Atlantic water and exports remineralised nutrient rich water, resulting in its oligotrophy and low primary productivity (Tanhua et al., 2013). Fig. 1.1. Map of the Mediterranean Sea showing the circulation of the Atlantic Water and areas of deep water formation. The general circulation of the Mediterranean is thermohaline and current patterns are cyclonic (Millot, 1999) (Fig. 1.1). The inflow of Atlantic water, of relatively low salinity (~36.2), through the Strait of Gibraltar, follows two main paths. One part follows the North African coast showing intense mesoscale activity and enters the eastern basin through the Strait of Sicily (Millot and Taupier-Letage, 2005). The other part flows northwards following the northern side of the Balearic Islands to the west coast of Corsica (García et al., 1994). On the eastern side of the Ligurian Sea the Atlantic water flow from the 1. Introduction 10 activity (Pepin 1991). Also very important are physical processes, such as currents, which define larval transport pathways of larvae to favourable or unfavourable areas, and fronts and eddies that favour the concentration/retention of fish larvae in areas suitable for their survival (Bakun, 2006; Govoni, 2005). 1.1.5. Larval fish assemblages A larval fish assemblage, or larval fish community, is a group of species whose larvae are collected in the same area at the same time, and should be considered a static snapshot that does not necessarily imply or require evolutionary convergence or biological interactions (e.g., competition, predation). It is transient by definition, and the juvenile and adult phases of these species may not concur in time or space (Miller 2002). Co-occurrence of individual species in an assemblage suggests that they share common requirements during their early life. An alteration in the ecosystem that negatively affects one species is likely to negatively affect all species in the assemblage. Thus, by studying the assemblage as a whole, it is possible to have a better understanding of the processes that regulate larval survival to solve the 'recruitment problem'. To do this, it is crucial to know the processes involved in the formation of larval fish assemblages, their maintenance and their disruption (Miller, 2002). These processes include a complex array of physical and biological factors that interacts with the biology of fish populations at different temporal and spatial scales (Doyle et al., 1993) (Fig. 1.4). Fig. 1.4. Diagram of the different factors that affect the formation, maintenance and disruption of larval fish assemblages, acting at different temporal and spatial scales. Adapted from Doyle (1993). The main factors involved in the formation of larval fish assemblages are the distribution range of adult fish populations and their reproductive strategies, which have evolved to adapt to the dominant oceanographic characteristics of the region in which they are found (Parrish et al., 1981). Thus, the specific composition, distribution, and abundance of fish larvae within the complex and variable marine ecosystems are the result of a process of co-adaptation of species' spawning strategies (e.g., timing and location of spawning, duration and frequency of spawning) (Doyle et al., 1993). As a result of these 1. Introduction 11 strategies, offspring are placed where larvae can find the best conditions for successful feeding, growth and survival (Cowen and Sponaugle, 2009; Cushing, 1969; Siddon et al., 2011). Annual or larger-scale variability in environmental factors, such as climate change, can lead to variations in the distribution range of adult fish and in their reproductive strategies (Fig. 1.4). These variations are reflected in the distribution patterns and abundance of their larvae (Doyle et al. 1993). Thus, changes in the larval fish community provide early indicators of variations in fish community dynamics (Doyle et al., 2009) and in oceanographic and climatic conditions (Auth et al., 2011; McClatchie et al., 2018). Spatial mesoscale and seasonal changes in environmental conditions may cause fish spawning to occur at a place and time not suitable for successful feeding, growth and survival of larvae (Cushing 1969; Llopiz et al 2014). Therefore, the seasonality of the spawning period of fish also plays a role in the formation of larval fish communities, especially in mid and high latitudes, where seasonal variability of environmental conditions is greater than in low latitudes (Miller 2002). Some physical processes can generate macroscale structures (e.g. water mass separation fronts, large currents) and mesoscale structures (e.g. convergent fronts, eddies), which can concentrate eggs and larvae, thus contributing to the formation of the assemblages (Bakun 2006; Govoni 2005). Physical processes can also contribute to the maintenance of larval fish communities by transporting larvae to favourable areas or retaining them in mesoscale structures, or lead to disruption by transporting them to unfavourable areas (Miller 2002). The main biological factors that contribute to the maintenance of larval fish communities or lead to their disruption are those that influence larval growth and survival, such as feeding and predation, as mentioned above. Competition for food occurs mainly among larvae in advanced stages and is important when preys are at low concentration. In this case, competition could lead to a reduction of food to levels that would result in lower survival of larvae of all species in the community (Miller 2002). To avoid competition for food and space in the early stages, fish species (often belonging to the same genus or family) that occupy the same area and require similar habitats spawn in different time periods and, in most cases, not spatially overlapping (Sabatés et al., 2007a; Tsikliras et al., 2010). 1.1.6. Evolution of fish larvae studies in the Mediterranean Sea Since the 1970s, there has been a steady increase in the number of published articles on ichthyoplankton worldwide (Fig. 1.5). Since the mid-1990s, this type of studies has become increasingly relevant, showing that this is an active topic in science. In the Mediterranean, the number of published works on fish larvae has increased significantly since the 2010s. Most ichthyoplankton studies focus on a limited number of species, mainly those of commercial interest. Research on larval fish assemblages started in the mid1990s, and represent a small percentage (<5%) of the overall number of fish larvae studies. A significant proportion of this work on larval assemblages has been carried out in the Mediterranean. 1. Introduction 12 Fig. 1.5. Peer reviewed SCI publications between 1970 and 2024 containing the words ichthyoplankton, ‘fish larv*’ or ‘larval fish’ and ‘larval fish assemblage*/communit*’ (LFA: Larval fish assemblages) worldwide and in the Mediterranean. The ordinate axis on the left indicates the number of larval fish publications and the ordinate axis on the right the number of publications of larval fish assemblages. Source: Scopus. In the Mediterranean, early research on fish larvae was aimed to describe the larval development of fish species for identification purposes, as well as to provide information on locations and periods of spawning areas (e.g. Demir, 1961; Marinaro, 1991). Among them, it is worth mentioning the excellent work of Lo Bianco et al. (1956), which compiled a high number of descriptions for the identification of fish larvae specimens of the Mediterranean fish species. This pioneering study constitutes the basis for ichthyoplankton research in the Mediterranean region, and to this day, these descriptions serve as reference guide for the identification of specimens from field samples. Subsequently, research on fundamental aspects of larval biology was initiated in some species, mainly the European anchovy (Engraulis encrasicolus) and the European sardine (Sardina pilchardus). Improvements in otolith reading and analysis techniques made it possible to address larval growth (e.g. Dulčić, 1995; Palomera et al., 1988; Regner, 1980) and to quantify the larval mortality rates (Palomera and Lleonart, 1989; Regner, 1985). A crucial topic is the analysis of larval feeding of numerous species, the small pelagics (Borme et al., 2013; Catalán et al., 2010), tunas (Morote et al., 2008a; Uriarte et al., 2019), red mullet (Sabatés et al., 2015), sparids (Sánchez-Velasco and Norbis, 1997) and mesopelagic fish (Contreras et al., 2015; Sabatés and Saiz, 2000). Studies have provided valuable information on marine food webs, and great advances have been possible thanks to the introduction of novel techniques such as stable isotope (Mir-Arguimbau et al., 2021; Quintanilla et al., 2020), fatty acids (Costalago et al., 2011; Rossi et al., 2006) and molecular analyses (Uriarte et al., 2019; Yebra et al., 2019). Research on the relationship between larval growth and condition and its influence on larval survival began in the 2000s (Catalán et al., 2006; Costalago et al., 2011; Ramírez et al., 2004). As in all scientific fields, research on fish larvae has been linked to different technological advances. The use of multiple nets and other sampling systems that allow the collection of samples at different depth levels of the water column has allowed the study of the vertical distribution of fish larvae, along with that 1. Introduction 13 of their prey. These studies have shown that the larvae of most species are located near the surface above the thermocline (e. g. Granata et al., 2011; Olivar and Sabatés, 1997; Sabatés, 2004), while the larvae of oceanic species have preference for subsurface layers (Olivar et al., 2010). Some studies have shown that certain species perform daily vertical migrations for feeding (Olivar et al., 2001; Sabatés et al., 2008; Somarakis and Nikolioudakis, 2007). Advances in oceanographic data collection and remote sensing systems have allowed progress in the interaction between environmental variables, e.g. temperature, salinity and chlorophyll-a, as well as hydrodynamic structures in the spatial and temporal distribution of fish larvae. Improvements in computing have made it possible to perform complex statistical analyses efficiently and implement individual based models to study connectivity between areas (Mariani et al., 2010; Ospina-Alvarez et al., 2015) and habitat models to forecast larval fish distributions of pelagic species under climate change scenarios (Macías et al., 2014; Maynou et al., 2020a). Despite all these advances, there are still gaps in our knowledge about the biology and ecology of fish larvae. It is important to further progress in topics such as the taxonomy of fish larvae, and fundamental aspects of their biology (e.g. diet, growth rates, swimming ability, etc.) since all these aspects are crucial for their survival. Other important aspects to consider are larval predation by numerous taxonomic groups (jellyfish, ctenofors, adult fish, etc.) as well as competition for resources. 1.1.7. Larval fish communities and environmental conditions in the NW Mediterranean The larval fish community in the NW Mediterranean was first described by Sabatés (1990). In this work, the spatio-temporal distribution of fish larvae was analysed and a marked seasonality in the presence of larvae in the plankton was evidenced in relation to the spawning period of the adults. This seasonality has also been described in subsequent works in the area (Alemany, 1997; Álvarez et al., 2012; Olivar et al., 2010) and in other Mediterranean regions (Koutrakis et al., 2004; Zarrad et al., 2020). Most Mediterranean fish species spawn during late spring and summer, under conditions of oligotrophy and stratification (Tsikliras et al., 2010). Summer ichthyoplankton is characterised by high species diversity and comprises an extensive assemblage of neritic species (e.g. Sparidae, Labridae, Mullidae, Serranidae, Scombridae), small and medium pelagics (e.g. Engraulidae, Clupeidae, Carangidae) and oceanic species (e.g. Myctophidae, Gonostomatidae). In winter, when the water column is well mixed, species from relatively cold water reproduce, such as Sardina pilchardus, and other species belonging to the families Ammodytidae, Gadidae and Pleuronectidae (Sabatés et al., 2007a). In addition to the spawning period, the determining factor in the distribution of fish larvae, and therefore in fish larval assemblages, is the habitat of the adults and the location where they spawn. In the NW Mediterranen, as in other geographic regions, depth explains the distribution of fish larvae, usually separating neritic from oceanic species (Isari et al., 2008; Sabatés, 1990; Somarakis et al., 2011). Sabatés and Olivar (1996) demonstrated that the density front associated with the Northern Current limited the distribution of larvae of coastal species towards the open sea, separating the larvae of neritic and 1. Introduction 14 mesopelagic species. Mesoscale structures associated with the Northern Current, such as anticyclonic eddies, have demonstrated to retain and concentrate anchovy larvae that are transported by the current (Sabatés et al., 2013, 2007b). Most of the studies carried out along the Catalan coast dealing with the role of environmental factors (e.g. bathymetry, temperature and chlorophyll-a) and hydrodynamic structures (e.g. currents, anticyclonic eddies) on the distribution, concentration and dispersal of fish larvae have been carried out on pelagic species, such as Engraulis encrasicolus and Sardinella aurita (Sabatés et al., 2013, 2009, 2004). The studies addressing the effect of environmental factors on the fish larval community as a whole are scarce and restricted to a limited area (Olivar et al., 2010; Sabatés and Olivar, 1996). In addition, these studies have been conducted in the spring-summer period and the information available on this subject for the winter period is scarce and restricted to a few species (sardine, blue whiting and mesopelagic fish) (Olivar et al., 2003; Sabatés et al., 2024). In the global warming context experienced by the Mediterranean, coldwater fish species that reproduce in winter would be adversely affected (Lloret, et al. 2015). The Catalan coast, located south of the Gulf of Lion, is one of the coldest areas in the Mediterranean, where deep convection takes place in winter, and cold-water fish species, the most threatened by the climate change, are particularly abundant. As mentioned above, a northward expansion of fish and larvae of warm water species has been documented along the Catalan coast in relation to sea warming (Raya and Sabatés, 2015; Sabatés et al., 2012, 2006). On a global scale, studies on the influence of climate variability on fish larvae community are very scarce (e.g. Auth et al., 2017; Koslow et al., 2013; Nielsen et al., 2020) and in the Mediterranean, although they have been addressed at the level of a particular species (Maynou et al., 2020b; Sabatés et al., 2015, 2006), they have not been conducted in the entire larval fish community. Some questions remain unknown, such as whether the coexistence of larvae of indigenous species with those of newly arrived species implies possible competition for resources or space. The influence of rising sea temperature on the larvae of established fish species in the area also remains to be investigated. This thesis addresses these questions by analysing the spatial and interannual variability of the larval fish communities on the Catalan coast (NW Mediterranean) and explores its long-term variability in a climate change context. This region, with its high hydrodynamic activity and wide range of environmental conditions (Saiz et al., 2014; Salat, 1996) provides an ideal location to analyse the influence of environmental factors on the spatial structure of the larval fish community. To elucidate whether the coexistence between larvae of well established species (E. encrasicolus) and a species that is expanding northwards in relation to sea warming (Sardinella aurita), which occupy a similar trophic niche, can be detrimental to the larvae of E.encrasicolus, the survival of both species is analysed. To this end, a new tool (Box-Balance Model) has been developed to quantify the dispersal and retention of larvae of both species and to evaluate the role of hydrodynamic structures in their survival along the Catalan coast. 1. Introduction 15 1.2. Objectives The present thesis characterizes the structure of the larval fish community, i.e. its composition, abundance and spatial distribution of the species in summer and winter, in the NW Mediterranean. The objective is to understand its spatial and interannual variability in response to changes in environmental conditions, including oceanographic variables and hydrodynamic processes. Within the current context of climate change in the Mediterranean, the thesis aims to describe long-term changes in the structure of the summer larval fish communities and to understand the interactions between larvae of well established species and species that are expanding northwards in the area. The general aim of the thesis is divided into several specific objectives, which are addressed in the different chapters: Chapter 1. Variability of larval fish assemblages under contrasting winter environmental conditions in the NW Mediterranean This chapter provides the first characterisation of larval fish communities in two consecutive winters of contrasting environmental conditions in the Catalan coast. The Catalan coast is located south of the Gulf of Lion, one of the coldest areas in the Mediterranean, where deep convection takes place in winter, and cold water fish species, the most threatened by the climate change, are particularly abundant. In this context, the objectives are: (a) to describe the species composition, abundance and diversity of the fish larval community in winter, (b) to analyse the influence of hydrodynamic processes on the structure of the larval fish assemblages, and (c) to ascertain the extent to which interannual shifts in the larval fish assemblages are indicators of changes in environmental conditions. Chapter 2. Recurrence of the spatial structure of summer larval fish assemblages linked to hydrodynamics in the NW Mediterranean The objective of this chapter is to assess the role of different sources of variability, including environmental, spatial and interannual, on the conformation of larval fish assemblages in summer along the Catalan coast. In particular, the influence of environmental factors (e.g. bathymetry, temperature and primary productivity) and mesoscale hydrodynamic structures (e.g. currents, anticyclonic eddies) on the spatial structure of the larval fish community is investigated Chapter 3. Long-term variability of larval fish community in the NW Mediterranean in summer This chapter investigates the main changes experienced by the summer larval fish community over three decades along the Catalan coast. The objectives are (a) to identify the long-term changes in the species composition, abundance and distribution of the larval fish community from 1980s to 2010s, and (b) to explore the links between environmental variables and the larval fish community trends within the current context of climate change in the Mediterranean 1. Introduction 16 Chapter 4. The Box-Balance Model: A new tool to assess fish larval survival, applied to field data on two small pelagic fish In the light of the northward expansion and increasing abundance of warm water fish species in the NW Mediterranean, the objective of this chapter is to quantitatively compare the survival of larvae of Engraulis encrasicolus (well established species) and Sardinella aurita (species that is expanding northwards) and to identify suitable (or unsuitable) areas for their development. To address these issues, a new tool, the Box-Balance Model, has been developed, which allows the quantification of larval dispersion and retention by mesoscale dynamics in combination with growth and mortality rates of the two species. 1. Introduction 17 1.3. General methods 1.3.1. Field sampling and oceanographic data Ichthyoplankton data used in this thesis come from biological-oceanographic research programs conducted by the Institute of Marine Sciences (CSIC) in Barcelona (Fig. 1.6): ARECES 1980s (June, July and September 1983), CACO 2000s (July and September 2003; June and July 2004), FISHJELLY 2010s (June 2011 and July 2012) and WINFISH (February 2017 and 2018). In all summer surveys carried out off the Catalan coast (NW Mediterranean) the same area was covered (from 40.15˚N to 42˚N and from 0.5˚E to 3.5˚E), between the coast and the continental shelf break, and the same sampling methodology was applied. The winter surveys were conducted in the northern part of the Catalan coast (from 41.2˚N to 42.3˚N and from 2.6˚E to 3.8˚E), using the same sampling methodology. In the summer surveys carried out in the 2000s and 2010s and in the winter surveys the sampling area was extended towards the open sea. All surveys were carried out with the R/V Garcia del Cid. Fig.1.6. Sampling area covered by the different surveys conducted in the Catalan coast. The area marked with the blue stripe pattern corresponds to the winter surveys, the yellow area to the summer surveys conducted in 2000s and 2010 and the salmon area to the study performed over the three decades. At each station, vertical profiles of the basic hydrographic variables (temperature and salinity) were obtained with a conductivity-temperature-depth (CTD, Neil Brown, WOCE standard), equipped with a Sea-Tech in situ fluorometer. To calibrate the fluorometer, water samples were collected at different levels of the water column at each station to determine chlorophyll-a concentration. Water samples were filtered through Whatman GF/F filters and frozen. In the laboratory, the chlorophyll-a concentration (μg l-1) was determined fluorometrically following the method of Yentsch and Menzel (1963). Chlorophyll-a was extracted from filters immersed in 6 mL of 90% acetone (24 h at 4 ºC in darkness), 1. Introduction 18 and the extract was analysed with a Turner Designs fluorometer calibrated with pure chlorophyll-a. The vertical profiles obtained by the CTD were interpolated to 1-m depth intervals. Contour maps of environmental variables (mainly temperature, salinity, density and chlorophyll-a) were drawn using SURFER 13 (Golden Software, 2015). Fish larvae were collected at each station during day and night, using a Bongo net with a mouth opening of 60 cm of diameter and a mesh size of 300 μm. The net was towed at 2 knots, obliquely, from a maximum depth that depends on the survey, to the surface. The volume of filtered water by the nets was estimated by means of a flowmeter placed in the centre of each net mouth. In addition, in the winter surveys depth-stratified samplings were performed using a MOCNESS net of 1 m2 mouth opening and 300 μm mesh size. Zooplankton samples were fixed in 5% formaldehyde buffered with sodium tetraborate. 1.3.2. Fish larvae identification In the laboratory, all fish larvae were sorted from the preserved samples under a stereomicroscope. Larvae were counted and identified to the lowest taxonomic level possible by the same group of experts, enabling direct comparison between all the surveys of the same spawning season. The identification of larvae cannot be done by dichotomous keys due to the important changes that larvae undergo during their development. The assignment of a specimen to a particular species is based on the descriptions of larval development existing in the literature. For this thesis, numerous guides and other publications were used for the identification of fish larvae. Some of them, specific to the Mediterranean, such as Lo Bianco et al. (1956), Sabatés (1988), Alemany (1997) and Demir (1961), and other works such as Moser et al. (1984) Olivar and Fortuño (1991), Aboussouan (1975, 1967), Fahay (2007), and Richards (2005). Usually, the process of larval identification is carried out in several steps. The first step is to identify unknown larvae by order or family, based on shape and general appearance, followed by meristic values, pigment, and morphology (Miller and Kendall, 2009). Some characters vary with larval development, thus size and stage of development should be considered when comparing the shape of an unknown larva with illustrations and descriptions of known specimens. The characters used in the identification of fish larvae can be grouped into four types: (1) Morphological characters, such as shape and length of the body, head and gut and relative position of the fins. These characters were used for differentiation between families and between genera. (2) Meristic characters, such as number of spines and fin rays or the number of myomeres. Fin elements develop gradually during larval development, thus state of development must be considered. The number of myomeres correspond to the number of vertebrae in the adult stage. This character was especially useful to distinguish some very similar species, such as Sardina pilchardus and Sprattus sprattus. (3) Pigmentation pattern as taxonomic character on larvae is limited to melanophores, since in formalin-fixed larvae, melanophores 1. Introduction 19 are the only pigments that remain after fixation. Melanophores are characteristic of each species and were used to differentiate between species of the same family or genus, for example Trachurus trachurus from Trachurus mediterraneus or Serranus hepatus from Serranus cabrilla. (4) Specialised larval characters are those that develop exclusively during the larval stage and lost in the juvenile stage (Fig. 1.7). These include spines and cranial armour head spines, used for the distinction, for example of Caranx rhonchus from T. mediterraneus larvae. Other specialised characters, such as barbel, characteristic of Mullus spp., and pedunculated eyes for some myctophid species were also useful in the distinction of different species. Fig. 1.7. Anatomical, morphological and some specialised characters of postflexion larvae used for the larval identification (From Miller and Kendall, 2009) 1.3.3. Statistical analysis of fish larvae data The number of larvae of each species for each sampling station was standardized to number per 10 m2 of the sea surface and ln(x + 1) transformed to reduce the weight of the dominant species. These transformed larval abundances were used to perform statistical analysis and to draw maps to obtain the geographical distributions of the larvae of the different species using SURFER 13 (Golden Software, 2015). Multivariate statistical analyses were performed using the R statistical software (R Core Team, 2023) to study the larval fish community. The aim was to identify patterns in the distribution of species by identifying groups of objects (sampling stations or surveys) with similar characteristics in terms of specific composition and abundance of larvae of each species. For this purpose, clustering analyses, such as the Ward's (1963) minimum variance method and ordination techniques, in this case non-metric multidimensional scaling (nMDS), were used. To investigate the influence of environmental variables on the specific composition, abundance and distribution of fish larvae canonical ordination methods such as canonical correspondence analysis (CCA) and redundancy analysis (RDA) were used. 2.1. Variability under contrasting environmental conditions 26 2.1.1. Introduction Spatial patterns in the larval fish assemblages (LFA), i. e. occurrence, distribution, and abundance of fish larvae, are influenced by a complex array of environmental processes that interacts with the biology of fish at different temporal and spatial scales (Doyle et al., 1993). These processes include those of large scale, as climate patterns (Di Pane et al., 2020; Guan et al., 2015), as well as environmental variability at seasonal and interannual time scales, that determine adults distribution and their spawning strategies (Auth, 2008; Doyle et al., 2009). At a local and short time scale, LFA will be shaped by the hydrodynamics (e.g. circulation, fronts, instabilities) that influence fish larval dispersal and retention (Moser and Smith, 1993; Suthers et al., 2023) and by biologic factors, such as food concentration and predation (Houde, 2008; Miller and Kendall, 2009), that ultimately determine fish recruitment (Cushing, 1990; Houde, 2008). From a global approach, changes in ichthyoplankton assemblages provide early indicators of shifts in fish community dynamics (Doyle et al., 2009) and oceanographic and climatic conditions (Auth et al., 2011; Ciannelli et al., 2022; McClatchie et al., 2018). The Mediterranean is a relatively small, semi-enclosed sea between continents. As a result, it is very sensitive, and responds rapidly to atmospheric forcing and anthropogenic impacts, as compared to oceanic time scales (Giorgi, 2006). In this context, it can be considered as an excellent site to study the effects of climate variability on marine communities. The basin is in the temperate zone of the Northern Hemisphere within a relatively narrow latitudinal range (30–45ºN). This involves a marked seasonal cycle, with the alternation of stratified and mixing periods that results in strong seasonality in primary production, with its annual maximum in late winter/early spring and minimum in late summer (Ribera d’Alcalà et al., 2004). Recurrent late winter/early spring phytoplankton blooms are only regularly observed in the northwestern region, and intermittently in a few other areas (D’Ortenzio and Ribera D’Alcalà, 2009). The seasonal bloom in the NW Mediterranean is triggered by deep convection episodes that occur offshore in the Gulf of Lion in winter. These episodes are driven by evaporation caused by strong, cold and dry northerly winds (MEDOC GROUP, 1970; Schott et al., 1996). Occasionally, intense cooling and evaporation extend over the shelf waters, which become denser than those offshore, and cascade down-slope to the depth where the same density is found (Fieux, 1974; Salat and Font, 1987). All these winter convection episodes, responsible for the deep-water formation, are the major contributors to overall primary production in the whole western Mediterranean. Interannual variability in primary production is highly dependent on the extension, intensity and duration of deep-water formation episodes, which increase in colder and drier years (Latasa et al., 2022; Marty and Chiavérini, 2010). Over the shelves and upper slope, where the bottom is shallower, convection can sustain a high productivity during winter (Salat et al., 2002). The study area, off the Catalan coast, is located in the NW Mediterranean, south of the Gulf of Lion, one of the coldest areas in the Mediterranean (Theocharis and Georgopoulos, 1993). The continental shelf is generally narrow (typically less than 20 km wide), furrowed by a series of deep canyons whose heads are 2.1. Variability under contrasting environmental conditions 27 not far from the coastline. The oceanographic dynamics is largely controlled by the Northern Current (NC), a slope current associated to a shelf-slope density front, detectable down to about 400 m depth, which separates the relatively low salinity shelf waters from the more saline open sea waters (Font et al., 1988). The NC displays high mesoscale variability that causes oscillations, meandering and eddy generation (Rubio et al., 2005). The NC may interact with the canyons, strongly modifying the local circulation and generating shelf-slope exchanges (Flexas et al., 2008) that play a key role in the distribution and abundance of planktonic organisms (Guerrero et al., 2016; Sabatés et al., 2004). In the NW Mediterranean, the presence of fish larvae in the plankton shows a marked seasonality related to the spawning cycles of the adults (Palomera and Olivar, 1996; Sabatés et al., 2007). Most fish species spawn during spring and summer (Tsikliras et al., 2010), when oligotrophy prevails in the upper water column. In winter, a period of high primary productivity and maximum phytoplankton biomass (Morán and Estrada, 2005; Ribera d’Alcalà et al., 2004), few fish species reproduce (Olivar et al., 2003b). These are species characteristic of relatively cold waters (of North Atlantic biogeographic affinity), which are of important commercial interest, including the European sardine, Sardina pilchardus, the Atlantic mackerel, Scomber scombrus, the European seabass, Dicentrarchus labrax and various species of Gadiformes (hake, Merluccius merluccius and blue whiting, Micromessistius poutassou) and Pleuronectiformes, such as the four-spot megrim, Lepidorhombus boscii (Sabatés et al., 2007). In the Catalan coast, most ichthyoplankton studies, conducted in the spring-summer months, showed that LFA basically responded to the bathymetry, including the submarine canyons, the presence of continental outflows, and the circulation patterns, such as the anticyclonic eddies and the shelf-slope density front (Álvarez et al., 2015; Sabatés and Olivar, 1996). However, the information available on this subject for the winter period is scarce and restricted to a few species (sardine, blue whiting and mesopelagic fish) (Olivar et al., 2003b; Sabatés et al., 2024). The impact of climate change on the structure of ichthyoplankton assemblages has been studied worldwide (Auth et al., 2018; Hsieh et al., 2009). In the global warming context, in the Mediterranean (Salat et al., 2019; Vargas-Yáñez et al., 2017), cold-water fish species could be adversely affected (Lloret et al., 2015), and it has been reported that survival of early life stages of these species is lower in mild winters than in severe winters (Mir-Arguimbau et al., 2022; Moyano et al., 2023). The present study provides the first characterisation of LFA in two consecutive winters of contrasting environmental conditions in the NW Mediterranean Sea. Our study area was the northern Catalan coast, south of the Gulf of Lion, one of the coldest areas in the Mediterranean, where deep convection takes place in winter, and cold water fish species, the most threatened by the climate change, are particularly abundant. In this context, the objectives are: (a) to describe the species composition, abundance and diversity of the fish larval community in winter, (b) to analyse the influence of hydrodynamic processes 2.1. Variability under contrasting environmental conditions 28 on the structure of the LFA, and (c) to ascertain the extent to which interannual shifts in the LFA are indicators of changes in environmental conditions. 2.1.2. Materials and methods Sample collection and data processing Two oceanographic surveys were conducted in two consecutive winters (from 18 February to 3 March 2017 and from 14 to 26 February 2018) in the Catalan Sea (NW Mediterranean Sea; Fig. 2.1.1) on board the R/V García del Cid. Sampling stations, 33 in 2017 and 29 in 2018, were located approximately 7−14 km apart, and placed on transects perpendicular to the shoreline, from near the coast to the slope (Fig. 2.1.1). At each station, vertical profiles of basic hydrographic variables (temperature, salinity and fluorescence) were acquired with a conductivity-temperature-depth (CTD) profiler equipped with a fluorometer. In order to calibrate the fluorometer, water samples for chlorophyll-a determination were collected at selected stations, evenly distributed over the area, with a rosette system at different depth levels down to 70 m, throughout day and night. To get geostrophic circulation patterns, as well as vertically integrated information on the dynamic structures, dynamic heights (Asch and Checkley, 2013) were calculated with a reference depth level of 500 m. Where station depth was lower, dynamic heights were extrapolated using the continuity equation applied to the deepest level of three-station clusters (cf. Hidaka, 1940). Fig. 2.1.1. Study area in the NW Mediterranean, south of the Gulf of Lion. Crosses indicate sampling stations (CTD and Bongo net) during 2017 (orange) and 2018 (dark blue) surveys. Triangles indicate stations where only CTD casts were performed. Orange circles correspond to MOCNESS net samplings. Stars correspond to depth-stratified sampling at fixed stations in 2017 (orange) and 2018 (blue) surveys. Open ellipses (green, Coast, Shelf, Slope and Palamós canyon) indicate the vertical profiles of oceanographic variables shown in figure 4. The isobaths shown are 50, 200, 1000 and 2000 m. 2.1. Variability under contrasting environmental conditions 29 Chlorophyll-a concentration (µg l–1) was determined fluorometrically (Yentsch and Menzel, 1963). Water samples of 100 to 200 ml were filtered through Whatman GF/F filters. Chlorophyll-a was extracted from filters immersed in 6 ml of 90% acetone (24 h at 4°C in darkness). The extract was analysed using a Turner Designs fluorometer calibrated with pure chlorophyll-a. The relationship between chlorophyll-a concentration and fluorescence obtained on each cruise was used to convert the continuous CTD fluorescence register to chlorophyll-a concentration. Fish larvae were collected at each station using a Bongo net with a mouth opening of 60 cm diameter and a mesh size of 300 μm. The net was towed obliquely from a maximum depth of 500 m to the surface, at a vessel speed of 2 knots. In the 2017 survey, some of the stations over the slope were sampled using a MOCNESS net of 1 m2 mouth opening and 300 μm mesh size to perform depth-stratified zooplankton samplings (Fig. 2.1.1). In addition, to analyse the day-night vertical distribution of larvae, depth-stratified samples were obtained at several fixed stations, one in 2017 and three in 2018, during 24 - 48 h, avoiding sunset and sunrise hours (see Fig. 2.1.1). As the net enabled sampling 8 depth strata, these were defined according to the bottom depth. The MOCNESS net was deployed to collect the samples obliquely, moving from deep to shallow layers, at a ship speed of 2 - 2.5 knots. The volume of filtered water by the different nets was estimated by means of calibrated flowmeters. All zooplankton samples were immediately fixed in 5% formaldehyde buffered with sodium tetraborate. In the laboratory, all fish larvae were sorted and identified to the lowest possible taxonomic level based on existing descriptive literature (Lo Bianco et al., 1956). The number of larvae collected with the Bongo net was standardized to number per 10 m2 of the sea surface. The larvae collected by the MOCNESS net at each depth stratum were standardized to 1000 m3 of filtered water. Statistical analyses and spatial patterns of species For each species and survey, the mean abundance values, frequency of occurrence (FO, percentage of stations where a taxon occurred) and the relative abundance (RA, percentage contribution of a taxon to the total abundance of individuals) were calculated. To detect significant differences in environmental variables (SST, SSS, SSSig, SSChla and DH) between years, t-tests were performed. The threshold of statistical significance for all analyses was set at α = 0.05. To analyse the spatial and temporal patterns in LFA, multivariate analyses were performed using the “cluster” (Maechler et al., 2022) and “vegan” (Oksanen et al., 2022) packages in the statistical software R v. 4.2.1 (R Core Team, 2022). Data of fish larval abundance were ln(x + 1) transformed to reduce the weight of the dominant species, and taxa with FO < 5% were not included in the analyses. To assess the existence of groups of stations with similar taxa composition and abundance, a non-metric multidimensional scaling (nMDS) (Clarke, 1993), and a cluster analysis were performed based on the Bray-Curtis dissimilarity index (Bray and Curtis, 1957). The dendrogram was constructed using the Ward’s hierarchical agglomeration method (Ward, 1963). The groups of stations were plotted on a map of 2.1. Variability under contrasting environmental conditions 30 the sampled region to identify geographical patterns if they exist. Permutational multivariate analysis of variance (Adonis test, 999 permutations) (Anderson, 2001) and pairwise tests were used to test for significant differences in fish larval abundance and composition between years and between clusters. To quantify the contribution of the species to the dissimilarity between years and clusters, similarity percentage routine (SIMPER) was performed (Clarke, 1993). To study the influence of environmental conditions on the fish larval community canonical correspondence analysis (CCA) was used. The environmental data matrix included: the oceanographic variables at the surface (10 m), temperature (SST), salinity (SSS), density (SSSig) and chlorophyll-a (SSChla); mean chlorophyll-a in the upper 50 m of the water column (MChla_50); dynamic height (DH); bottom depth (log-transformed; Depth); distance from a sampling station to the coast (Dist_Coast). This last variable was considered to compensate the high bottom depths reached close to the coast in the submarine canyons. DH was included because it can be used as an indicator that integrates the effects of multiple oceanic processes (Asch and Checkley, 2013). It is a proxy for the integrated temperature and salinity of the upper water column. Thus, areas with fresh, warm seawater correspond to higher dynamic heights than areas with salty, cool seawater, which have lower dynamic heights. Moreover, DH can be used to identify hydrodynamic structures, such as eddies and meanders (Asch and Checkley, 2013). As the DH values in the Mediterranean are negative, they were transformed into positive values in the analysis for convenience by adding a constant. All these factors influence the spawning habitat of fishes. Collinearity between pairs of variables was assessed using Pearson’s correlation coefficients (cut-off value |0.6|). As a result, SSSig and MChla_50 were discarded. The CCA analysis was performed using the forward selection method to rank the variables in their order of importance and select only those that contributed significantly to explaining the variance. All maps were drawn using SURFER 13 (Golden Software, 2015). Contour maps of environmental variables were obtained using the minimum curvature gridding method. Contour maps of the CCA scores were obtained using the kriging interpolation method, introducing the variogram fitted by the least squares criterion previously calculated. To visualise the variation in larval abundance with depth, violin plots were performed using a Gaussian density function. Weighted mean depth (WMD) for each species was calculated as the mean depth sampled by each net (MOCNESS data, standardised to 1000m3) weighted by the proportion of larvae caught in the net (Hawes et al., 2020). 2.1. Variability under contrasting environmental conditions 31 Fig. 2.1.2. Spatial distribution of surface (10 m) temperature (SST, a and b), salinity (SSS, c and d), chlorophyll-a (SSChla, e and f) and dynamic height relative to 500 m (DH) overlaid on density (, g and h) in winter 2017 (left panels) and 2018 (right panels). Arrows indicate the flow direction and intrusions of the Northern Current and the extrusions of coastal waters. The isobaths shown are 50, 200, 1000 and 2000 m. 2.1. Variability under contrasting environmental conditions 32 2.1.3. Results Hydrographic conditions The most striking feature that appeared when comparing SST and, in particular, SSS distributions between years was the presence of a water mass of low temperature (< 12.8 ºC) and salinity (< 37.5) along the coast in winter 2017. This band of coastal waters covered a significant part of the continental shelf (Fig. 2.1.2 a and c). SST and SSS increased towards the open sea, with values ~13.6 ºC and ~38.4, respectively, offshore. In 2018, SST and SSS values showed a smoother transition between coastal and offshore waters (Fig. 2.1.2 b and d). SST was slightly lower than in 2017, both near the coast, ~12.6 ºC, and offshore (~13.4 ºC; Fig. 2.1.2 a and b). The range of SSS was much lower than in 2017, with ~38.1 near the coast and ~38.5 offshore (Fig. 2.1.2 c and d). Overall, SST was not statistically different between years (p = 0.9802), but significant differences were found in SSS (p < 0.05). SSSig followed the same pattern as SSS, with the highest values over the slope (Fig. 2.1.2 e and f). In 2017, a marked gradient related to the low salinity coastal water mass was evident over the shelf (Fig. 2.1.2 e). As observed in SSS, significant differences in SSSig were detected between years (p < 0.05). The dynamic height overlaid on density clearly showed the presence of the Northern Current, associated with the shelf-slope density front, flowing southwestward along the continental slope with significantly higher DH values in 2017 than in 2018 (p < 0.05). In both years, an anticyclonic eddy was detected over the Palamós Canyon, more marked in 2018 (Fig. 2.1.2 e and f). Two intrusions of offshore waters to the coast were detected, one in the north of the area (~ 42.2°N) and the other following the southern side of Fig 2.1.3. Vertical profiles of density (), mean (circle) and standard deviation (horizontal lines) of CTD stations sampled in 2017 and 2017 2.1. Variability under contrasting environmental conditions 33 the anticyclonic eddy, over the Palamós canyon. These intrusions were also evident in the SST and SSS distributions (Fig. 2.1.2 a, b, c and d), although in 2017 they were masked by the presence of the low salinity coastal waters. In the Blanes canyon in 2017, an intrusion of offshore waters towards the coast was detected (Fig. 2.1.2 e), while in 2018 there was an offshore spreading of coastal waters towards the southeast through the canyon, as also shown by the low temperature and salinity values (Fig. 2.1.2 b and d). High chlorophyll-a concentrations were associated to the low salinity surface layer in 2017, although relatively high surface chlorophyll-a patches (> 1 µg l-1) were also found beyond the low salinity coastal band (Fig. 2.1.2 g). In 2018, surface chlorophyll-a was, in general slightly lower than in 2017, except in the vicinity of the Blanes canyon where high values (nearly 2 µg l-1) extended offshore, in relation to the offshore spreading of coastal waters (Fig. 2.1.2 h). No significant differences were observed in chlorophyll-a between years (t-test, p = 0.4742). In general, the water column presented slight stratification in 2017, due to the coastal water mass of low salinity, while it was more homogeneous in 2018 (Fig. 2.1.3). The vertical profiles of hydrographic parameters of the different stations representative of each zone (coast, shelf, slope and the Palamós canyon; see figure 2.1.1 for station locations) showed clear differences between both years (Fig. 2.1.4). In 2017, the coastal water mass of low temperature and salinity was detected at surface to 20 m depth, with a clear pycnocline, while on the rest of the shelf the vertical density gradient was weaker (Fig. 2.1.4 a and c). Over the slope, the temperature profile showed a layer of lower values between 70 and 250 m, suggesting subduction of shelf and upper slope waters (Fig. 2.1.4 e). In the Palamós Canyon, the signature of coastal waters sinking was detected between 100 and 350 m, as shown by the lower temperature and salinity at this depth interval (Fig. 2.1.4 g). In 2018 the water column was more homogeneous everywhere (coast, shelf and slope) and the temperature, salinity and density profiles showed a slight increase with depth (Fig. 2.1.4 b, d and f). The anticyclonic eddy identified in the horizontal distributions over the Palamós Canyon could be observed down to a depth of 100 m with a clear pycnocline (Fig. 2.1.4 h). Below this depth, traces of sinking or cascading events were detected at 400 m and below 1000 m, as shown by the temperature and salinity inversions, much deeper than in the previous year (Fig. 2.1.4 h). The vertical distribution of chlorophyll-a presented differences between years in relation to the stratification of the upper layer, although within each year the patterns were similar across zones. In 2017 high chlorophyll-a values were detected in surface waters down to 40 m depth, mainly in the coast, while in 2018, the chlorophyll-a distribution was more homogeneous down to ~150 m (Fig. 2.1.4). 2.1. Variability under contrasting environmental conditions 34 Fig. 2.1.4. Vertical profiles of temperature (red), salinity (blue), density (, black), and chlorophyll-a (green), for four selected stations for 2017 and 2018, at different locations: coast (a and b), shelf (c and d), slope (e and f) and the Palamós Canyon (g and h) (for station locations see figure 2.1.1). 2.1. Variability under contrasting environmental conditions 35 Table 2.1.1. Mean abundance and standard deviation (No. larvae 10m-2), relative abundance (RA, in %) and frequency of occurrence (FO, in %) of all identified larval fish taxa in 2017 and 2018. The taxa are listed according to adult habitat (shelf and oceanic). 2.1. Variability under contrasting environmental conditions 42 Fig. 2.1.9. Spatial distribution of fish larvae of some representative oceanic species overlaid on sea surface temperature (SST) and bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in 2017 and 2018. Fish larval vertical distribution Most of the taxa showed a deeper larval distribution in 2018 than in 2017 (Table 2.1.4). Larvae of oceanic species, inhabiting over the slope and submarine canyons, showed a deep and relatively wide vertical distribution in the water column, being shallower during the day than at night (Table 2.1.4, Fig 2.1.11 a and b). For instance, larvae of A. risso were located in the first ~120 m during the day and deeper at night, between 40 m and 350 m (Fig. 2.1.11 a). The vertical distribution of M. punctatum larvae extended into the first 100 m of the water column, except during the night in 2018, which were found down to ~250 m (Fig. 2.1.11 b). The vertical distribution of larvae of shelf species was shallower than that of oceanic species and deeper in 2018 than in 2017 (Table 2.1.4). They were closer to the surface during the day than at night. For instance, the vertical distribution of S. pilchardus larvae extended to the first 100 m, being particularly abundant in the upper 40 m during the day. At night in 2018, their distribution was deeper reaching ~250 m depth (Fig. 2.1.11 c). As observed for the shelf species, the vertical distribution of larvae of species that inhabits the shelf break and upper slope, e.g. M. poutassou, showed a relatively superficial distribution during the day and deeper at night (Table 2.1.4). Larvae of these species were located in the first 100 m during the day, with the highest abundances at ~50 m. At night they showed a wide vertical distribution, extending to ~400 m in 2017 (Fig. 2.1.11 d). 2.1. Variability under contrasting environmental conditions 43 Fig. 2.1.10. Spatial distribution of fish larvae of some representative shelf and upper slope species overlaid on sea surface temperature (SST) and bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in 2017 and 2018. Note that S. pilchardus and Arnoglossus spp. are represented on a different size scale to M. poutassou and G. macrophthalmus. 2.1.4. Discussion Environmental setting The main oceanographic conditions found during the study were those expected for the season, with a coastal-offshore gradient of SST and SSS and the presence of the Northern Current associated with the shelf-slope density front along the slope (Font et al., 1988). However, in 2017 the coastal zone was occupied by cold and low-saline waters at the surface, due to the occurrence of heavy rain episodes a few days before the cruise (Sabatés et al., 2024). Although the sea surface temperatures detected in both cruises were quite similar, winter 2017 was mild, with a short period of cold temperatures and limited vertical mixing (Mir-Arguimbau et al., 2022). The stratification detected in the coastal area was due to the low salinity surface waters (Fig. 2.1.4 a), but also offshore, where subduction of coastal waters into middepth layers was observed, as evidenced by their lower temperature and salinity values (Fig 2.1.4 e and g). The stratification and mild winter conditions that year would restrict vertical motions and hinder deep convection (Margirier et al., 2020; Parras-Berrocal et al., 2022). In contrast, winter 2018 was more severe, temperatures reached lower values and persisted for a longer period than in 2017 (Mir-Arguimbau et al., 2022). These conditions would favour the mixing of the water column and dense water formation 2.1. Variability under contrasting environmental conditions 44 with subduction and cascading events down the slope to deeper layers, as shown by the temperature and salinity inversions detected at ~400 m and below 1000 m in the Palamós Canyon (Fig. 2.1.4 h). In the Gulf of Lion, the intense convection episodes reported that year reached at least 1900 m (Fourrier et al., 2022; Margirier et al., 2020). Such processes of deep-water formation, convection and cascading, are particularly important, as they are responsible for shelf-slope water exchanges in the region (Ivanov et al., 2004). Thus, the conditions found in 2018 would enhance the shelf-slope exchanges while the hydrodynamic conditions of 2017 would restrict these processes. The distinct situations observed between the two years would also be reflected in the differences in DH values, higher in 2017 than in 2018, as DH vertically integrates the effects of multiple oceanic processes (Asch and Checkley, 2013). Table 2.1.4. Weighted mean depth (WMD, in m) and standard deviation, during day and night, for the most abundant larval fish taxa, in 2017 and 2018. 2.1. Variability under contrasting environmental conditions 45 Fig. 2.1.11. Violin plot showing the vertical distribution of larvae of Arctozenus risso, Myctophum punctatum, Sardina pilchardus and Micromessistius poutassou, during day and night, for 2017 and 2018. The width of the violin represents the proportion of larvae at each depth stratum. Environmental influence on the structure of larval fish assemblages In both years, the values of the species richness were comparable to those usually found during the mixing period in the Mediterranean (Koutrakis et al., 2004; Olivar et al., 2010, 2014; Zarrad et al., 2020). However, these values were lower than those found in spring-summer (e.g. Álvarez et al., 2015; Sabatés et al., 2007), since most Mediterranean fish species reproduce during the spring–summer period (Tsikliras et al., 2010). The predominance of clupeid larvae, S. pilchardus and S. sprattus, found in the two studied years (Table 2.1.1) is a common feature of winter ichthyoplankton in the Mediterranean (Koutrakis et al., 2004; Olivar et al., 2003a; Sabatés et al., 2004) and in other areas around the world (Di Pane et al., 2020; Moyano and Hernández-León, 2009; Rodriguez, 2008). These species, and larvae of shelf species in general, were more abundant in 2017 (~70% of the total larval abundance) in the coastal band of low salinity and high chlorophyll-a waters. In 2018, oceanic fish larvae were more dispersed throughout the study area in relation to the shelf-slope exchange processes of that year. The values of relative abundances of oceanic species were similar to those reported for many LFA during periods of vertical mixing in the western Mediterranean (Olivar et 2.1. Variability under contrasting environmental conditions 46 al., 2014), as well as in other geographical areas (Moyano and Hernández-León, 2009; Osorio-Zúñiga et al., 2018; Sassa and Konishi, 2015). The high larval concentrations of both shelf and oceanic species detected in the Blanes Canyon would support the role of submarine canyons as a conduit for larvae from deep-water spawning sites to coastal grounds, highlighting their crucial role in shaping LFA (Mordy et al., 2019). Cluster analysis separated the LFA into two branches (Fig. 2.1.6 a), with a gradation from larvae of shelf to oceanic fish species. In 2017, the coastal and offshore assemblages were well delimited, with the coastal group 6, associated to the low salinity and high SSChla waters, bounded by the marked density gradient that would retain these larvae in the productive coastal waters. The assemblage of oceanic species was located offshore of this density gradient and clearly separated from the costal group. This oceanic assemblage appeared on the inner part of the shelf-slope front and the associated Northern Current. The presence of larvae of oceanic fish species in the inner part of the shelf-slope front has been observed in previous occasions in the area and has been related with the position of the front far from the coast (Masó et al., 1998; Sabatés and Olivar, 1996), in our study over the 1000 m isobath. In 2018, the assemblages were not so geographically delimited. In addition, the absence of a strictly coastal group and the occurrence of transitional assemblages (3, 4 and 5) would evidence the role of the shelf-slope water exchange processes in the transport of fish larvae across the shelf this year. The CCA results showed the relationship of oceanic clusters (1 and 2) with high SST values, characteristic of offshore waters. This relationship is confirmed by the geographical representation of the scores and the spatial distribution of the oceanic fish taxa (Fig. 2.1.8). The relationship of cluster 1 (mainly in 2017) and cluster 2 (2018) with high and low DH values, respectively, reflects the distinct hydrodynamic conditions found each year, with important shelf-slope water exchange processes in 2018. In that year, the high abundances of larvae of oceanic species and their wide distribution all over the area, including the shelf stations, revealed their transport from offshore areas towards the shelf (Fig. 2.1.5), where vertical mixing can sustain a high productivity (Salat et al., 2002). It should be noted that larvae of oceanic species with a deeper vertical distribution, such as A. risso and A. hemigymnus, did not undergo this coastal transport as was observed for M. muelleri and M. punctatum (Fig. 2.1.9). These differences in the transport of oceanic fish larvae in relation to their vertical distribution depth were previously reported (Olivar et al., 2010; Vargas-Yáñez and Sabatés, 2007). The cascading and vertical mixing processes detected in 2018 would disperse larvae in the water column, which would result in a deeper vertical distribution of larvae than in 2017, while favouring an homogeneous vertical distribution of chlorophyll-a in the first 150 m of the water column (Fig. 2.1.4 f and h). These strong vertical mixing processes have been reported to lead to high primary productivity and subsequent zooplankton abundance (Estrada et al., 2014; Vandromme et al., 2011). In addition to the shelf-slope exchanges induced by these hydrodynamic conditions in 2018, the intrusions of the Northern Current detected in both years would also favour the transport of these larvae towards the coast, as previously reported in the area (Masó et al., 1998; Sabatés 2.1. Variability under contrasting environmental conditions 47 et al., 2018). The presence of larvae of oceanic species over the shelf, associated with current instabilities, is a common process in different geographical regions (Cuttitta et al., 2018; Govoni and Spach, 1999; Sassa and Konishi, 2015). Concerning larvae of shelf species, this study evidences the association of the coastal group, 6, with the band of low SSS, SST and high SSChla values found in 2017. This stratified productive area would offer favourable conditions for feeding and development of larvae of these species. These results agree with the observations of Catalán et al., (2006), who found that in the mixed season, the distribution of shelf fish larvae was associated with the high concentrations of zooplanktonic organisms. In that year, the shallow vertical distribution of these species, such as S. pilchardus, would be related to the vertical density gradient that would restrict their diel vertical migration or even their passive night sinking (Davis et al., 1990; Olivar et al., 2001). In 2018, the horizontal distribution of shelf species was wider across the shelf and slope, as their offshore distribution was limited by the location of the shelf-slope front. This suggests, as observed in the area, that the front would act as a barrier preventing the dispersal of larvae, and other planktonic organisms, towards the open sea (Guerrero et al., 2016; Sabatés and Olivar, 1996; Saiz et al., 2014). In this year, the vertical distribution of these species, such as S. pilchardus (Fig. 2.1.11), was deeper than that found in other studies (John, 1985; Olivar et al., 2001; Sabatés, 2004), suggesting that the homogeneity of the water column would facilitate the vertical movements of larvae. Moreover, the cascading events detected in 2018 may overwhelm the depth-related behavioural patterns of larvae, causing them not to follow their usual vertical distribution patterns as observed in other areas (Gray and Miskiewicz, 2000). Among the species that appeared in the central part of the CCA, without an obvious relationship with any environmental variable, it is worth mentioning M. poutassou and G. macrophthalmus whose adults inhabit the shelf break and the upper slope (Barros-García et al., 2018; Mir-Arguimbau et al., 2022). Larvae of M. poutassou showed a wide distribution over the shelf and slope in both years, being limited offshore by the shelf-slope front. They showed a wide vertical distribution in the water column, which agrees with previous studies conducted in other areas (Ådlandsvik et al., 2001; Hillgruber and Kloppmann, 2000). Small larvae of this species would be located in deep waters, where the adults dwell, and larvae gradually rise towards the surface reaching the illuminated and more productive surface layers for feeding (Sabatés et al., 2024). The wide horizontal and vertical distribution of this species would allow it to take advantage of the high surface productivity in the coastal band in 2017, as well as the relatively high chlorophyll-a concentrations, associated with the vertical mixing, detected down to about 150 – 200 m depth outside the shelf. Larvae of G. macrophthalmus were found on the oceanic side of the coastal band in 2017, but were absent in these productive waters, suggesting that the surface density gradient would prevent their distribution towards the coast. In addition, the shallower vertical distribution of this species with respect to M. poutassou, particularly during the day, would further limit their reaching 2.1. Variability under contrasting environmental conditions 48 the coast. In 2018, the scattered distribution of larvae over the whole area could be related to the high hydrodynamics that year. In 2018, the absence of a strictly coastal group and the occurrence of transitional assemblages (3, 4 and 5), would evidence the role of the shelf-slope water exchange processes in the transport of fish larvae across the shelf in that year. These processes were more evident over the canyons, where all groups were detected (Fig. 2.1.6 b). Previous studies already reported that canyons interact with the Northern Current and enhance the shelf-slope water exchanges, favouring the transport of fish larvae and other planktonic organisms across the shelf and increasing primary and secondary productivity (Alvarez et al., 1996; Flexas et al., 2008; Masó et al., 1998). Both the high abundance of shelf species in the transition group 5 (Fig. 2.1.6 a), related to high CCA scores of axis 1, and the spatial distribution of scores far from the coast over the Blanes canyon in 2018 (Fig. 2.1.8 b), suggest that the extrusion of coastal waters mixed with offshore waters, resulting in a very productive area (Fig. 2.1.2 h). The Palamós Canyon would be a particular case, where a clear deep cascading was detected that year, which would channel water and particles from coastal areas to the deep ocean (Canals et al., 2013; Romano et al., 2017). The cascading events would enhance the transport of oceanic larvae towards the coast at the surface as reflected in the presence of clusters 1, 2 and 3 over the canyon and in the distribution of negative values of axis 1 scores very close to the coast (Fig. 2.1.8 b). The responsible for this surface transport would be the eddy detected in the canyon (Sabatés et al., 2024, 2013), as occurs in other areas (Cuttitta et al., 2016). The differences in the structure of the LFA in the two winters studied are closely linked to the environmental conditions observed each year, mainly to the transport processes associated with the deepwater formation episodes. Thus, LFA are good indicators of the variability of the environmental processes. Larval fish assemblages under a climate change scenario In the northwestern Mediterranean, climate trends show an increase in air temperature along with a decrease in precipitation and in the frequency of northerly winds (e.g. Alpert et al., 2002; Jordà et al., 2017; Vicente-Serrano and Rodríguez-Camino, 2017). At sea, an increasing trend in temperature and salinity has been detected in surface and intermediate waters (e.g. Piñeiro et al., 2019; Salat et al., 2019; Vargas-Yáñez et al., 2017). This scenario involves an intensification of the water column stratification and, consequently, a decrease in the nutrient injection into the photic layer and the primary production (Calvo et al., 2011). In the open sea, as stratification in the upper layer becomes more persistent, favourable conditions for the deep convection become less frequent (Grignon et al., 2010) and since winter 2014 deep-water formation episodes did not reach the deepest levels of the basin (Margirier et al., 2020; Salat et al., 2019). In addition, long-term predictions point to a decrease in the occurrence of deep convection events in future climate scenarios (Josey and Schroeder, 2023; Macias et al., 2018). In this context, the present study provides an outline of the effects that these trends may have on fish larvae by 2.1. Variability under contrasting environmental conditions 49 comparing two environmentally contrasting years. 2017 could be considered as an example of a future mild winter, in which deep convection and the consequent shelf-slope exchange processes were inhibited, while winter 2018 was closer to a current “typical” winter, with quite intense vertical mixing and dense water cascading that enhanced the transport of larvae of oceanic species towards the coast. In addition, the deep convection and mixing process results in high primary production and zooplankton abundance (Estrada et al., 2014; Vandromme et al., 2011) that would enhance the survival of fish larvae (MirArguimbau et al., 2022). In winter 2017, stratified conditions and limited vertical mixing would be expected to decrease primary production in the surface layers, suggesting that environmental conditions were not so suitable for larval development. The high abundance of larvae of shelf fish species (i.e S. pilchardus and Arnoglossus sp.), and M. poutassou, found in the coastal band would apparently contradict the above statements. However, it must be considered that these coastal waters, associated to heavy rain episodes, were highly productive. In any case, in the Mediterranean, such coastal fertilisation mechanisms are subject to great variability and are of short duration (1 – 3 weeks), which would not be time enough to maintain zooplankton populations (Salgado-Hernanz et al., 2019). Moreover, long term declining trends in the surface chlorophyll concentration have been reported in coastal areas in the Mediterranean (Gómez-Jakobsen et al., 2022). This would imply that in successive winters the larvae of these species would face progressively less favourable conditions for their development and survival. 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Abstr. 10, 221–231. https://doi.org/10.1016/0011-7471(63)90358-9 Zarrad, R., Rodríguez, J.M., Alemany, F., Charef, A., Jarboui, O., Missaoui, H., 2020. Larval fish community composition and distribution of the central-southern mediterranean under summer and winter conditions. Acta Ichthyol. Piscat. 50, 313–324. https://doi.org/10.3750/AIEP/02957 Chapter 2 Recurrence of the spatial structure of summer larval fish assemblages linked to hydrodynamics in the NW Mediterranean 2.2. Recurrence of the spatial structure in summer 63 Recurrence of the spatial structure of summer larval fish assemblages linked to hydrodynamics in the NW Mediterranean Vanesa Raya, Jordi Salat, Ana Sabatés (submitted) Abstract In this study we aim to investigate the contribution of various sources of variability, environmental, spatial, and interannual on the larval fish assemblages (LFA) in summer off the Catalan coast, an area with a wide array of environmental conditions and high hydrodynamic activity. The study was based on three ichthyoplankton surveys carried out in July 2003, 2004 and 2012. The number of identified taxa, belonging to shelf and oceanic fish species, was quite similar among cruises, around 80, with the small pelagics Engraulis encrasicolus and Sardinella aurita being the dominant species. A high spatial heterogeneity in LFA was detected throughout the studied area. Most of this heterogeneity was related to the spatial variability of environmental factors, such as the continental shelf structure, latitudinal difference in surface temperature, chlorophyll-a concentration, and stratification level. Hydrodynamic structures, mainly the thermal front across the shelf, instabilities of the Northern Current and anticyclonic eddies, also played an important role in the configuration of the LFA. Although these showed certain interannual variability, the recurrence of hydrodynamic structures, together with the spawning habits of adults, means that the spatial structure of LFA is maintained in different years. 2.2. Recurrence of the spatial structure in summer 64 2.2.1. Introduction Larval fish assemblages (LFA), i. e. occurrence, distribution, and abundance of fish larvae within the complex and variable marine ecosystems, are the outcome of co-adaptation process of species spawning strategies (e. g. spawning time and location, spawning duration and frequency) (Doyle et al., 1993). These strategies have evolved to adapt to specific features of the environments that fish inhabit, ensuring that the offspring is placed where larvae can find the best conditions that allow successful feeding, growth and survival (Cowen and Sponaugle, 2009; Peck et al., 2012). Spatial patterns in LFA are ultimately determined by environmental processes that operate at different temporal and spatial scales (Doyle et al., 1993). These processes include those of large scale, as climate patterns (Di Pane et al., 2020; Koslow et al., 2013), as well as environmental variability at seasonal and interannual time scales, which determine adult distribution and their spawning strategies (Auth, 2008; Doyle et al., 2009). At a local and short time scale, LFA will be determined by hydrodynamic structures (e.g. currents, fronts, eddies) that influence fish larval dispersal and retention (Moser and Smith, 1993; Suthers et al., 2023) and by biologic factors, such as food concentration and predation (Houde, 2008; Miller and Kendall, 2009), that ultimately determine fish larvae survival (Cushing, 1990; Houde, 2008). The Mediterranean fish fauna is characterised by high diversity (Bianchi and Morri, 2000; Quignard and Tomasini, 2000). Species of subtropical origin are mainly found in the eastern basin and the southern Mediterranean, where water temperature is higher than average (Theocharis and Georgopoulos, 1993). Cold-temperate species inhabit the northern areas (Gulf of Lion, Ligurian Sea and northern Adriatic: Bianchi and Morri, 2000) where the water is colder with a seasonal variation at the surface ranging from 13 to 25ºC (Salat, 1996). In the Mediterranean, the presence of fish larvae in the plankton shows a marked seasonality related to the spawning cycles of the adults (Sabatés et al., 2007a). Most fish species spawn during spring and summer (Tsikliras et al., 2010), when oligotrophy prevails in the upper water column. The higher temperatures of the summer period favour faster larval growth, and the stability of the water column could contribute to maintain food patches (Sabatés et al., 2007a). The summer period is characterized by a stratified water column, with a marked thermocline that limits vertical mixing. Consequently, primary production remains concentrated at the deep chlorophyll maximum, a thin layer at the deepest levels of the photic zone (Estrada, 1985). Surface productivity is restricted to some coastal zones (Atienza et al., 2016) and to areas under the influence of runoff waters, mainly the Rhône and the Ebro (Salat, 1996). The study area, off the Catalan coast, in the NW Mediterranean Sea, is characterized by a marked latitudinal gradient of surface temperature. The northern sector, which is more directly influenced by strong northerly winds, is colder than central and southern parts and a thermal front develops at surface, perpendicular to the coast, associated with the southern limit of the strong northerlies (Sabatés et al., 2009). The continental shelf is generally narrow, widening in the southernmost part, in the vicinity of the 2.2. Recurrence of the spatial structure in summer 65 Ebro river delta, and in the north between the two major submarine canyons (Blanes and Palamós canyons). The oceanographic dynamics is largely controlled by the Northern Current (NC), which typically flows southwestwards along the continental slope at approximately 30−50 cm s−1, at the surface (Millot, 1990; Salat, 1995). The NC is in geostrophic equilibrium with a shelf-slope density front, detectable down to about 400 m depth, and leaves fresher and cooler waters on its coastal side (Font et al., 1988; Millot, 1990). The NC displays high mesoscale variability that causes oscillations, meandering and eddy generation (Rubio et al., 2005). These mesoscale structures are triggered by the complex bathymetry (canyons, abrupt changes in the direction of the continental slope, widening or narrowing shelves) that strongly modify the local circulation and generate shelf-slope exchanges (Flexas et al., 2008; Masó et al., 1998). Most of the studies carried out along the Catalan coast dealing with the role of environmental factors (e.g. bathymetry, temperature and chlorophyll-a), as well as hydrodynamic structures (e.g. currents, anticyclonic eddies), on the distribution, concentration and dispersal of fish larvae have been carried out on pelagic species, such as Engraulis encrasicolus and Sardinella aurita (Sabatés et al., 2013, 2009, 2004). The studies addressing the effect of environmental factors on the fish larval community as a whole are scarce and restricted to a limited area (Olivar et al., 2010; Sabatés and Olivar, 1996). In the present study, we investigate the species assemblages of the fish larval community along the Catalan coast. This area, with its high mesoscale activity and wide array of environmental conditions (Saiz et al., 2014; Salat, 1996) provides an ideal location to analyse the effect of the environmental factors on the spatial structure of LFA. The study is based on three ichthyoplankton surveys carried out in summer 2003, 2004 and 2012. The aim was to assess the role of different sources of variability, including environmental, spatial, and interannual on the conformation of LFA in summer along the Catalan coast. 2.2.2. Materials and methods Oceanographic data and ichthyoplankton sampling Three oceanographic surveys were conducted along the Catalan coast, NW Mediterranean, in July: 18– 25, 2003; 21–29, 2004 and 12–23, 2012 (Fig. 2.2.1). In 2003 and 2004, samplings were conducted at 66 stations located along transects perpendicular to the shoreline, from near the coast to the slope. In 2012, the sampling was extended offshore but, due to the bad weather conditions, the two northern transects could not be carried out and a total of 73 stations were sampled. In all surveys, the stations were placed ~14 km apart, and the distance between transects was ~18.5 km (Fig. 2.2.1). At each station, vertical profiles of the basic hydrographic variables (temperature, salinity and fluorescence) were obtained with a Neil Brown Mark III-CTD (WOCE standard) equipped with a SeaTech in situ fluorometer. The vertical profiles were interpolated to 1-m depth intervals. The dynamic heights were calculated with a reference level of 600 m and, where depth was lower, were extrapolated 2.2. Recurrence of the spatial structure in summer 66 using the continuity equation applied to the deepest level of the three-station clusters (cf. Hidaka, 1940). Geostrophic velocity components at the stations were derived from a stream function obtained by interpolating dynamic heights over the whole area (Stewart, 2008). The maximum vertical density gradient was calculated by centred differences at 1-m intervals, and the mean depth of the pycnocline was established where the maximum of the vertical density gradient was observed. To calibrate the fluorometer, water samples for chlorophyll-a determination were collected with a rosette system at three depths down to 80 m, throughout the day and night (see Sabatés et al., 2009 for methodological details). Fig. 2.2.1. Study area off the Catalan coast, in the NW Mediterranean. Blue circles indicate sampling stations (CTD and Bongo net) during the July 2003 and 2004 surveys. Crosses indicate sampling stations (CTD and Bongo net) during the July 2012 survey. Fish larvae were collected at each station, during day and night, using a Bongo net with a mouth opening of 60 cm of diameter and a mesh size of 300 μm. The net was towed at 2 knots, obliquely from a maximum depth of 200 m to the surface. The volume of filtered water by the nets was estimated by means of a flowmeter placed in the centre of each net mouth. Zooplankton samples were fixed in 5% formaldehyde buffered with sodium tetraborate. In the laboratory, all fish larvae were sorted from the preserved samples. Larvae were identified to the lowest taxonomic level possible. Data analysis The number of larvae of each taxon collected at each station was standardized to number per 10 m2 of the sea surface. For each species and survey, the mean abundance values, frequency of occurrence (FO, 2.2. Recurrence of the spatial structure in summer 67 percentage of stations where a taxon occurred) and the relative abundance (RA, percentage contribution of a taxon to the total abundance of individuals) were calculated. To analyse the spatial and interannual patterns in LFA, multivariate analyses were performed using the “cluster” (Maechler et al., 2022) and “vegan” (Oksanen et al., 2022) packages in the software R v. 4.3.1 (R Core Team, 2022). Data of larval abundance were ln(x + 1)-transformed to reduce the weighting of the dominant species. Analyses were performed considering all three surveys together, and only taxa that were present in at least 5% of all the stations sampled (a total of 59 taxa) were included. The threshold of statistical significance for all analyses was set at α = 0.05. To assess the existence of groups of stations with similar taxa composition and abundance, a cluster analysis was carried out using the Bray-Curtis index as the distance measure (Bray and Curtis, 1957) and the Ward’s hierarchical agglomeration method (Ward, 1963). Groups of stations were plotted on a map of the sampled region to identify possible geographical patterns. Permutational multivariate analysis of variance (Adonis test, 999 permutations; Anderson, 2001) was used to test for significant differences in larval fish abundance and taxa composition between clusters. The mean larval abundance of different taxa in each cluster was calculated to visually represent these differences. In addition, a non-metric multi-dimensional scaling (nMDS), was performed to provide a two-dimensional representation of the assemblage structure. The influence of environment, space and year on the variability in the composition and abundance of the different taxa in the LFA was analysed using the variation partitioning method (Borcard et al., 1992; Mood, 1971; Peres-Neto et al., 2006). This method considers different sets of explanatory variables (environmental, spatial and interannual) and estimates the amount of variation in the fish larvae matrix that can be attributed exclusively to one or the other set of explanatory variables, after removing the effect of the other sets of variables. It also allows to estimate the amount of variation explained jointly by two explanatory data sets. Hereafter, we will use ‘term’ to refer to a dataset of explanatory variables. The variation partitioning method was performed in two steps (Borcard et al., 2011; Legendre and Legendre, 2012). First, canonical correspondence analyses (CCA) were used to explain the variation in the fish larval community, one analysis for each term. The second step was to perform partial canonical analyses to determine the relative contribution of each term. The significance of the variation explained by each term was assessed by a Monte Carlo permutation test (999 permutations). Finally, variation partitioning was performed. The environmental data set used in the CCA to analyse the influence of environmental factors on the fish larval community included: bottom depth (log, Depth); the surface (5 m) temperature (SST), salinity (SSS) and chlorophyll-a (SSChla); maximum density gradient (Gmax); pycnocline depth (PD); geostrophic velocity components; and surface (10 m) dynamic height (DH). Gmax and PD were considered to analyse the effect of the strength of the stratification. The geostrophic velocity components and DH were included to analyse the influence of hydrodynamic structures (Asch and Checkley, 2013). As the DH values in the Mediterranean are negative, they were transformed into positive values in the 2.2. Recurrence of the spatial structure in summer 74 Fig. 2.2.5. Plot of the mean abundance (No. per 10m2) of taxa for each cluster. Cluster analysis identified six groups of stations with significant differences between them (p < 0.05; Fig. 2.2.4 a). The spatial distribution of these groups showed a gradation in the fish larval community composition, from shelf to oceanic stations, mainly in 2003 and 2012 (Fig. 2.2.4 b and Fig. 2.2.5). Group A was the most coastal assemblage in the three sampled years, and was mainly located in the southern half of the area. It was characterised by the lowest number of taxa, almost exclusively shelf species, such as E. encrasicolus, S. aurita, Sparidae and Gobiidae (Fig. 2.2.4 b and Fig. 2.2.5). Groups B, C and D were located on the shelf. Group B in 2012 and 2004 and Group C in 2003 and 2004 were mainly found in the 2.2. Recurrence of the spatial structure in summer 75 Ebro shelf, and Group D in the northern half in the three sampled years (Fig. 2.2.4 b). Cluster B showed a relatively high abundance of some shelf benthic species, such as Serranus hepatus, Trachinus draco, Arnoglossus spp. and Diplodus annularis, but the oceanic species C. maderensis and C. braueri, were also abundant (Fig. 2.2.5). The species composition of group C was similar to that of group B. The distinction between them was a higher abundance of larvae of some thermophilic species in the latter, such as S. aurita, Pomatomus saltatrix and Caranx rhonchus (Fig. 2.2.5). Group D was characterised by the presence of both, shelf (e. g. E. encrasicolus, S. aurita, S. hepatus, Arnoglossus spp. and Auxis rochei) and oceanic taxa (e.g. C. braueri, Ceratoscopelus maderensis, Lampanyctus crocodilus and Argyropelecus hemigymmnus) (Fig. 2.2.5). Groups E and F were found offshore in the three years. Cluster E assembled stations located on the shelf break and on the slope, and cluster F the most offshore stations (Fig. 2.2.4 b). These oceanic groups exhibited the highest abundances of the oceanic taxa, while cluster E also had elevated abundances of the shelf species (Fig. 2.2.5). Fig 2.2.6. Non-metric multidimensional scaling (nMDS) ordination plot. Symbols of the same colour indicate clusters of stations with similar species composition. Squares correspond to the stations sampled in 2003, circles to the stations sampled in 2004 and triangles to the stations sampled in 2012. The nMDS ordination analysis (2D stress = 0.178) showed some segregation between the six clusters (Fig. 2.2.6). The stations of the oceanic groups (E, F) were located on the right side of the plot, being cluster E stations mainly located on the negative side of the second axis and those of the group F on its positive side. The other four groups were situated on the left side of the plot. Clusters A and B were found on the positive side of the second axis and clusters C and D on its negative side (Fig. 2.2.6). Influence of environment on the spatial and interannual variability of LFA All terms (environmental, spatial and interannual) included in the variation partitioning analysis were significant (p < 0.05) and explained 33.3% of the variation in the larval fish community (Fig. 2.2.7). The highest percentage of variation was explained by the environmental variables (21.4%), and the 2.2. Recurrence of the spatial structure in summer 76 contribution of spatial and interannual terms was 14.4% and 7.6%, respectively. These values include the shared variation between the three terms (Fig. 2.2.7). Considering exclusively the effect of each term, environment explained 11.7%, space 7.1% and year 4.9% of the total variation. Fig. 2.2.7. Venn diagram of the variation partitioning analysis showing the percentage of variation explained in the larval fish assemblages by each term, spatial, environmental, and inter-annual. The environmental CCA identified seven significant variables: Depth, SST, SSS, SSChla, DH (hydrodynamics), PD and Gmax (stratification) (Table 2.2.2). The first ordination axis explained 60.03% of the constrained variance and was negatively correlated with Depth, and positively with SSChla. The second axis explained 16.94% of the constrained variance and was positively correlated with SST and negatively with PD. The spatial CCA of the detrended matrix of fish larvae was significant (p < 0.05). The first axis explained 46.3% and the second 20.7% of the constrained variance. Table 2.2.2. Summary of the canonical correspondence analysis (CCA) results. Individual variables are ordered by the percentage of variance explained (Exp. Var.), in relation to the total and constrained variance. The significance of each variable (P) together with its test statistic (F-value), and the interset correlations of environmental variables with the three significant axes of the CCA are provided. The ordination plot of the first two axes of environmental CCA showed the segregation of shelf and oceanic clusters, as observed in the nMDS (Fig. 8 a). The shelf groups were placed on the right side of the plot. Group A was associated with the lowest Depth and the highest SSChla and Gmax values (Fig. 2.2.8 a). Cluster B was related to high SST and Gmax and low PD and SSS. Group C was associated with the 2.2. Recurrence of the spatial structure in summer 77 highest values of SST, SSChla and Gmax and low values of PD, SSS, and Depth. Cluster D was associated with high DH, SSChla and Gmax, and low Depth values. The oceanic groups, E and F were located on the left side of the plot, being cluster F associated with higher Depth, SSS and PD and lower SSChla than cluster E (Fig. 2.2.8 a). The geographical representation of the scores of each station on axis 1 from the environmental CCA, showed their relationship with Depth, with positive values in coastal locations and negative offshore (Fig. 2.2.9 a, b and c). The scores on axis 2 exhibited the latitudinal gradient observed for SST, with positive values in the south and negative in the north (Fig. 2.2.9 d, e and f). These results evidence the relationship between environmental and spatial terms. Fig. 2.2.8. Ordination plots of the environmental canonical correspondence analysis (CCA) (left panels, a and c) and of the spatial CCA (right panels, b and d). Upper panels, a and b: Ordination of the sampling stations in the CCA plots, showing the relationships between the sampling stations and the explanatory environmental variables (blue vectors), for July of 2003, 2004 and 2012. Symbols of the same colour indicate clusters of stations with similar species composition. Squares correspond to the stations sampled in 2003, circles to the stations sampled in 2004 and triangles to the stations sampled in 2012. Lower panels, c and d: Ordination of the larval fish taxa in the CCA plots, showing the relationships between the larval fish taxa and the explanatory environmental variables (blue vectors), for July of 2003, 2004 and 2012. The abbreviations of larval fish names are indicated in Table 2.2.1. 2.2. Recurrence of the spatial structure in summer 78 Fig. 2.2.9. Spatial distribution of score values of stations for the axis 1 (upper panels, a, b and c) and axis 2 (lower panels, d, e and f) that resulted from the environmental canonical correspondence analysis (CCA) for 2003 (left panels, a and c), 2004 (central panels, b and e) and 2012 (right panels, c and f). Symbols of the same colour denote stations belonging to the same group identified in the cluster analysis. Isobaths shown: 50, 200 and 1000 m. Fig. 2.2.10. Spatial distribution of the fitted score values of stations for the axis 1 (colour gradient) that resulted from the spatial canonical correspondence analysis (CCA) on the detrended larval fish matrix for 2003 (left panel), 2004 (central panel) and 2012 (right panel). Geostrophic current field (arrows) and bathymetry have been overlaid. Isobaths shown: 50, 200 and 1000m. 2.2. Recurrence of the spatial structure in summer 79 The spatial analysis identified the different clusters although they were not as clearly segregated as in the environmental analysis (Fig. 2.2.8 b). The geographical representation of the fitted site scores of each station on axis 1 resulting from the spatial CCA showed similar spatial structures in the three studied years (Fig. 2.2.10). Positive values were observed in the southern shelf and in the shelf, between the Palamós and Blanes canyons, at the northern side of the anticyclonic eddies. Negative values were found offshore in the northernmost part of the area, associated with the intrusion of the NC, and in the central part related to instabilities of the NC (Fig. 2.2.10). The species ordination, both in the environmental and in the spatial CCA plots, showed segregation between shelf and oceanic species (Fig. 2.2.8 c and d). The shelf species were mainly located on the positive side of axis 1, associated with high values of SSChla and Gmax in the environmental CCA, and low depth and SSS. Larvae of these species, such as S. hepatus, were located all over the shelf, being especially abundant in the southern area, related to the highest concentrations of SSChla and low salinity waters from the Ebro river runoff (Fig. 2.2.8 c and Fig. 2.2.11 a, b and c). The thermophilic species e.g. Sardinella aurita, Caranx rhonchus and Pomatomus saltatrix were associated with high SST and low PD values (Fig. 2.2.8 c). Larvae of these species, illustrated by S. aurita, were located along the continental shelf, their distribution towards the north being limited by the position of the thermal front (Fig. 2.2.11 d, e and f). It should be noted that the abundance of these species was particularly low in 2012. Larvae of E. encrasicolus and Trachinus draco were located on the central part of the environmental CCA plot and showed no clear relationship with any environmental variable (Fig. 2.2.8 c). E. encrasicolus, the most abundant species, was present in all assemblages. They were located along the entire shelf in the three studied years with highest abundances in the northern half of the area, in coincidence with the intrusion of the NC on the northern boundary and the presence of anticyclonic eddies (Fig 2.2.11 g, h and i). Larvae of T. draco, placed rightmost on the spatial CCA plot, were particularly abundant in two areas: over the Ebro shelf and in the shelf between the Palamós and Blanes canyons, at the northern side of the anticyclonic eddies (Fig. 2.2.8 d and Fig. 11 j, k and l). In both CCAs, the oceanic species were located on the negative side of the axis 1, associated with high Depth and SSS and low SSChla and Gmax values in the environmental analysis (Fig. 8 c and d). Larvae of A. hemigymnus, placed leftmost on the spatial CCA plot, were located offshore, all along the slope, being especially abundant in 2012 (Fig. 2.2.11 m, n and o). The maximum abundances of C. maderensis were also located over the slope, although its distribution extended to the continental shelf, particularly in 2012 in the north of the Ebro shelf where an intrusion of the NC was detected (Fig. 2.2.11 p, q and r). 2.2. Recurrence of the spatial structure in summer 80 Fig. 2.2.11. Spatial distribution of fish larvae of representative shelf and oceanic species in 2003, 2004 and 2012. Seranus hepatus overlaid on SSChla (a, b and c), Sardinella aurita on SST (d, e and f), Engraulis encrasicolus (g, h and i), Trachinus draco (j, k and l), and Ceratoscopelus maderensis (p, q and r) on the geostrophic current field, and Argyropelecus hemigymnus on bathymetry (m, n and o). Isobaths shown: 50, 200 and 1000 m. 2.2. Recurrence of the spatial structure in summer 81 2.2.4. Discussion This study analyses the contribution of various sources of variation, including environmental, spatial, and interannual, on the LFA in summer off the Catalan coast, an area with a wide array of environmental conditions and high hydrodynamic activity. Our findings show that environmental conditions were the main driver of the observed variation, with their contribution to spatial variability being higher than their influence on interannual variability. Environmental setting In the NW Mediterranean, the circulation pattern is dominated by the Northern Current, with its meandering behaviour and some intrusions towards the coast. The meandering behaviour of the NC leads to some anticyclonic eddies on its coastal side that have been previously reported in the north-western Mediterranean coasts (Flexas et al., 2002; Sabatés et al., 2004). In the three studied years, a recurrent eddy was located at nearly the same position, over the Blanes canyon, that would be favoured by the interaction between the NC and the canyon (Flexas et al., 2008; Rubio et al., 2005) (Fig. 2.2.2 g, h and i). The presence of cold waters in the northern region contributes to the formation of the persistent thermal front, north of the Blanes canyon, in the three cruises. However, in 2012, the thermal front was blurred by the strong intrusion of cold, low salinity water advected by the NC along the continental slope and trapped by the anticyclonic eddy (Fig. 2.2.2 c). These waters probably come from the Rhône river runoff in the Gulf of Lion, as previous studies have described that they can be advected by the NC southwards along the continental slope, being detected to the south of the Blanes canyon mainly in spring and summer (Sabatés et al., 2007b; Salat, 1996). In July 2003 the surface temperature was around 2°C higher than usual in the whole region, reaching extremely high values in the southern part. These warm waters were the result of the successive heat waves that affected south-western Europe during spring and summer of 2003 (Black et al., 2004; Luterbacher et al., 2004). The anomalous warming was limited to the upper levels of the water column, contributing to an increase in the stratification intensity (Sparnocchia et al., 2006), in accordance with the high Gmax and low PD values observed in the southern part of the study area this year. In the southern area, the mesoscale dynamics was in general dominated by the Ebro river runoff and the relatively wide continental shelf. High SSChla values were associated with the low-salinity water patches from the Ebro runoff, as previously reported in that area (Arin et al., 2005; Sabatés et al., 2008). The change in the orientation of the shelf break in the north of the Ebro shelf affects the stability of the NC, enhancing the intrusion of slope waters in that area (Salat et al., 2002). The circulation patterns and the SSS distributions suggest that both processes, intrusions and river runoff, interact dynamically against each other leading to a fragmented structure of small patches, clearly evident in 2003 and 2004 (Sabatés et al., 2013). Thus, the Ebro shelf is usually characterized by high productivity and has been described as 2.2. Recurrence of the spatial structure in summer 82 an important spawning area for shelf dwelling fish species (Palomera et al., 2007; Sabatés et al., 2007a). In 2012, a decline in the Ebro river runoff (Cozzi et al., 2018), would allow the intrusion of slope waters to reach the coast. In this year, a decrease in the concentration of nutrients from the runoff of the Ebro and Rhône rivers (Cozzi et al., 2018), probably caused the reduction in SSChla concentration detected this year in the whole area. LFA and interannual changes The high species richness values (79 taxa) were quite consistent among years. These values are similar to those usually found in summer in the Mediterranean Sea (e. g. Álvarez et al., 2015; Sabatés, 1990; Somarakis et al., 2011a), and only lower to those reported for tropical and subtropical areas (e.g. Beckley et al., 2018; Keane and Neira, 2008; Meinert et al., 2020). This species richness is considerably higher than that observed in winter and is obviously related to the spawning cycles of the adults, since most Mediterranean fish species (e.g. Sparidae, Labridae, Blennidae, Mullidae, Serranidae, Carangidae) reproduce during the spring–summer period (Tsikliras et al., 2010). The predominance of clupeiformes, Engraulis encrasicolus and Sardinella aurita (Table 2.2.1) is a common feature of Mediterranean LFA in summer, being the former the most abundant species (Olivar et al., 2010; Tsikliras et al., 2009; Zarrad et al., 2013). The highest abundances of larvae of shelf species were found in the northern half of the study area, suggesting that hydrodynamic structures, as the NC, eddies and the temperature front, play a role in shaping their spatial distribution (Bakun, 2006; Goldstein et al., 2019; Sabatés et al., 2007b). Overall, larvae of oceanic species were abundant over the shelf break, particularly in 2012, when sampling was carried out further offshore. However, this year, larvae of oceanic species were also detected north of the Ebro shelf and in the central region, which would be related to the intrusion of slope waters towards the coast (Atienza et al., 2016; Olivar et al., 2010). The spatial distribution of species richness showed the highest values in the northern half of the area, in agreement with the LFA identified. In this region, where the shelf is narrow and furrowed by submarine canyons, the assemblages included a high number of species, both coastal and oceanic. In the wider Ebro shelf, the assemblages contained a lower number of species, except at the shelf break, due to the coexistence of larvae of shelf and oceanic taxa (Doyle et al., 1993; Isari et al., 2008; Olivar et al., 2010). While several studies have shown high interannual variability in LFA in relation to environmental factors (e.g. Álvarez et al., 2015; Auth et al., 2018; Hsieh et al., 2009), in the present study the variation partitioning method evidenced that the interannual differences in the LFA were lower than the spatial variation. Of this interannual variability, a small part would be due to changes in environmental factors, such as SST and SSChla. Most of this variability would not be related to any of the variables considered in this study, suggesting that LFA depend on other factors, for example, those that may affect larval 2.2. Recurrence of the spatial structure in summer 83 survival, such as food abundance (zooplankton) and the presence of predators (Houde, 2008; Peck et al., 2012; Pepin et al., 2003). It may also reflect changes in ‘adult related processes’ (e.g, spawning time, duration and fecundity; Isari et al., 2008) that may affect the occurrence, distribution and abundance of their larvae (Doyle et al., 1993). Environmental factors controlling LFA The variation partitioning method revealed that the environmental factors play the most important role in structuring the LFA. According to the CCA results, depth is the primary environmental variable shaping the LFA, mainly reflecting the spawning habitat of adults, as widely reported in different systems (Álvarez et al., 2015; Richardson et al., 1980; Somarakis et al., 2011b). The relationship between depth and LFA was evidenced in the coastal-offshore ordination of assemblages, particularly in the southern region, where the continental shelf is wider. Some few species were related to low depths, mainly those found in the coastal assemblage (e. g. Buglossidium luteum, Gobiidae) while most of the shelf species, associated to intermediate depths, were found in the transitional clusters, over shelf to the shelf break. Larvae of oceanic species, such as A. hemigymnus, were abundant over the slope in the oceanic assemblages, in relation to the highest depths. Other oceanic species, such as the myctophid C. maderensis, also showed high abundances over the shelf, belonging to the transitional and oceanic assemblages. The presence of C. maderensis larvae on the continental shelf is common in other parts of the Mediterranean and in the Atlantic (Isari et al., 2008; John and Zelck, 1997). The larvae of this species are located in the shallower levels of the water column, unlike A. hemigymnus larvae which have a deeper vertical distribution (Olivar et al., 2014), thus, they are more susceptible to being transported by mesoscale structures from the open sea (Sabatés and Olivar, 1996; Vargas-Yáñez and Sabatés, 2007). SSS, a variable that increases towards the open sea, also played a role in shaping the LFA. Coastal waters, influenced by river runoff showed lower salinity than slope waters, separated by the shelf-slope density front (Font et al., 1988). Larvae of oceanic species, such as A. risso and the myctophids (e. g. 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Hierarchical Grouping to Optimize an Objective Function. J. Am. Stat. Assoc. 58, 236– 244. https://doi.org/https://doi.org/10.1080/01621459.1963.10500845 Zarrad, R., Alemany, F., Rodriguez, J.M., Jarboui, O., Lopez-Jurado, J.L., Balbin, R., 2013. Influence of summer conditions on the larval fish assemblage in the eastern coast of Tunisia (Ionian Sea, Southern Mediterranean). J. Sea Res. 76, 114–125. https://doi.org/10.1016/j.seares.2012.08.001 Chapter 3 Long-term variability of larval fish community in the NW Mediterranean in summer 2.3. Long-term variability 97 Long-term variability of larval fish community in the NW Mediterranean in summer Vanesa Raya, Ana Sabatés Abstract This work investigates the main changes experienced by the summer larval fish community over three decades along the Catalan coast, an area characterized by a wide array of environmental conditions. The study was based on nine ichthyoplankton surveys carried out in June, July and September in three decades, 1980s, 2000s and 2010s, covering the same area and applying the same sampling methodology. Changes in the environmental variables were detected throughout the study period. An increase in sea surface temperature, particularly marked in June, and a decrease in surface chlorophyll-a associated with a decrease in runoff of the Ebro and Rhône rivers was observed. Marked changes in the composition and abundance of the larval fish community were also detected between June and July in the 1980s and the following decades. These changes were mainly due to the presence for the first time in the area of warm water species, e.g. Thalassoma pavo and Caranx rhonchus, or to their increase in abundance, e.g. Sardinella aurita and Pomatomus saltatrix, in the 2000s in relation to the northward expansion of the adults' range. The presence of larvae of warm water species in the 2000s and 2010s contributed to an increase in specific richness in the larval fish community compared to values obtained in the 1980s. Other species showed a decline in abundance over time, probably due to a decrease in surface chlorophyll-a, e.g. Engraulis encrasicolus although overexploitation is also an important factor to consider. Larvae of other species, such as mesopelagic fish, did not show changes in abundance over the three decades. In a future scenario of increasing sea water temperature and marine heatwaves events, enhanced stratification, and decreasing surface primary production, the changes that may occur in the fish larvae community will largely depend on the adaptive responses of individual species to the new environmental conditions. 2.3. Long-term variability 98 2.3.1. Introduction The effects of global warming on the dynamics of marine ecosystems are widespread worldwide. High temperatures alter oceanographic conditions which can influence fish life-history traits such as reproduction (e.g. Petitgas et al., 2013) and growth (e.g. Pörtner and Peck, 2010). It is well documented that in relation to increasing sea water temperature, fish communities have undergone poleward shifts in latitudinal distribution (Costa et al., 2021; Di Pane et al., 2020; Walsh et al., 2015). However, for a species to colonise a new region, it must successfully reproduce and its offspring must survive (Bates et al., 2014; Sabatés et al., 2006). The spawning strategies of marine fishes (e. g. timing and location, duration and frequency of spawning) are adapted to the specific features of the habitats, with the result that offspring is placed where larvae can find the best conditions that allow them to successful feed, growth and survival (Cowen and Sponaugle, 2009; Cushing, 1969; Siddon et al., 2011). Changes in fish distribution would result in changes in spawning grounds and in the times and environments in which fish eggs and larvae occur (Hsieh et al., 2009; Llopiz et al., 2014). The larval fish communities, i.e. occurrence, distribution, and abundance of fish larvae are influenced by a complex array of environmental processes that interacts with the biology of fish at different temporal and spatial scales (Doyle et al., 1993). From a global approach, changes in the larval fish community provide early indicators of shifts in fish community dynamics (Doyle et al., 2009) and oceanographic and climatic conditions (Auth et al., 2011; Ciannelli et al., 2022; McClatchie et al., 2018). The Mediterranean Sea is one of the most exposed areas worldwide to the numerous impacts of climate change, including one of the highest warming rates, and increasing frequency of extreme weather events (Darmaraki et al., 2019; Jordà et al., 2012; Macias et al., 2018). The sea warming is known to affect primary production at global and regional scales by increasing stratification of the sea, reducing the input of nutrients into surface waters from mixing (Barale et al., 2008; Macias et al., 2018). The Mediterranean is also a biodiversity hotspot (Bianchi and Morri, 2000; Coll et al., 2010), threatened by many anthropogenic impacts, such as overfishing, habitat degradation, and invasive species (e.g. Lejeusne et al., 2009). These threats interact synergistically and can severely alter the balance of highly vulnerable Mediterranean ecosystems, thereby affecting marine biodiversity and fish populations (Lejeusne et al., 2009; Stergiou et al., 2016). The Mediterranean fish fauna is characterised by an unusually high biodiversity for a temperate sea (Bianchi and Morri, 2000; Quignard and Tomasini, 2000). Species of subtropical origin are mainly found in the eastern basin and the southern Mediterranean, where water temperature is higher than average (Theocharis and Georgopoulos, 1993). Cold-temperate species inhabit the northern coldest areas (Gulf of Lion, Ligurian Sea and northern Adriatic: Bianchi and Morri, 2000). The distribution of fish species in the Mediterranean has undergone significant changes in recent decades in relation to the increase in seawater temperature. Species characteristic of the warm waters of the southernmost parts of the Mediterranean are expanding their range towards the northernmost and coldest areas, a process known as meridionalization 2.3. Long-term variability 99 (e. g. Azzurro, 2008; Bianchi and Morri, 2003; Francour et al., 1994; Lejeusne et al., 2009). These distribution changes do not occur exclusively among fish, but have also been described in other taxonomic groups, such as algae, crustacea and cnidaria (Guerrero et al., 2018; Parravicini et al., 2015). In the NW Mediterranean, the presence of fish larvae in the plankton shows a marked seasonality related to the spawning cycles of the adults (Sabatés et al., 2007a). Most fish species spawn during spring and summer (Tsikliras et al., 2010), among them neritic species (e.g. Sparidae, Labridae, Mullidae, Serranidae, Scombridae), small and medium pelagics (e.g. Engraulidae, Clupeidae, Carangidae) and oceanic species (e.g. Myctophidae, Gonostomatidae). The summer ichthyoplankton community, thus, is characterised by high specific diversity in a period when oligotrophy prevails in the upper water column. The higher temperatures of the summer period favour faster larval growth and hence a high probability of survival (Anderson, 1988; Houde, 1987). The summer period in the Mediterranean is characterized by a stratified water column, with a marked thermocline that limits vertical mixing. Consequently, primary production remains concentrated at the deep chlorophyll maximum, a thin layer at the deepest levels of the photic zone (Estrada, 1985). Surface productivity is restricted to some coastal zones and to areas under the influence of runoff waters, mainly the Rhône and the Ebro (Salat, 1996). The study area, off the Catalan coast, in the NW Mediterranean, is characterized by a marked latitudinal gradient of surface temperature. The northern sector, which is more directly influenced by strong and cold northerly winds, is colder than central and southern parts and a thermal front develops at surface, perpendicular to the coast, associated with the southern limit of the strong northerlies (Sabatés et al., 2009). In the light of the northward expansion and increasing abundance of warm water fish species in the NW Mediterranean, the Catalan coast, with a clear gradient of sea surface temperature and wide array of environmental conditions (Saiz et al., 2014; Salat, 1996), provides an ideal location to analyse possible changes in the fish larvae community in summer. The objectives were i) to identify the long-term changes in the species composition, abundance and distribution of the summer larval fish community from 1980s to 2010s, and ii) to explore the links between environmental variables and the larval fish community trends within the current context of climate change in the Mediterranean. 2.3.2. Materials and methods Data sources Ichthyoplankton data came from biological-oceanographic research programs conducted by the Institute of Marine Sciences (CSIC) in Barcelona (http://www.icm.csic.es) in summer along the Catalan coast (NW Mediterranean) (Fig. 2.3.1): ARECES 1980s (June, July and September 1983), CACO 2000s (July and September 2003; June and July 2004), and FISHJELLY 2010s (June 2011 and July 2012). All surveys were performed with the R/V Garcia del Cid, the same area was covered (from 40.15˚N to 42˚N and from 0.5˚E to 3.5˚E), and the same sampling methodology was applied. While 2–3 surveys per decade over a thirty-year period may seem low, the sampling surveys encompass a wide mesoscale area, 2.3. Long-term variability 106 The analysis of the different metrics used to detect possible changes in the larval fish community, showed significant differences (p < 0.05) between the surveys. In general, the highest larval fish abundances (N) were found in June and July in all three decades, with those in September being sensibly lower (Fig. 2.3.3 a). It is worth noting, however, the low abundances of larvae detected in July 2012. The specific richness (S) showed the lowest values in the 1980s, being significantly different from later years, and the highest values were detected in June 2004 (Fig. 2.3.3 b). Both, the Shannon-Wiener biodiversity index (H') and the Pielou's evenness index (J') were significantly lower in June and July 1983 (Fig. 2.3.3 c and d). Fig. 2.3.3. Box plots of (a) total larval abundance (N), (b) species richness (S, number of species, (c) Shannon-Wiener diversity index (H’, using a natural logarithm) and (d) Pielou’s measure of evenness (J’) for each survey. In each box plot, the darkened line indicates the median; boxes show the interquartile range and whiskers indicate the expected extent of 99% of the data for a Gaussian distribution. The letters at the bottom indicate the groups identified by the Tukey's post hoc test. Temporal variability The two-way cluster analysis classified the surveys into five clusters (p < 0.05) separated into two main branches, with September surveys in one branch and those of June and July in the other (Fig. 2.3.4). The analysis also divided the species into two branches, which in turn included four major groups (A to D) according to their occurrence and abundance. The upper branch (groups A and B) comprised taxa that occurred in few surveys and were present in relatively low abundance (Fig. 2.3.4). Group A included species, such as Citharus linguatula, Pagellus acarne and Scorpaena porcus, which were almost exclusively found in September months (Fig 2.3.4, table 2.3.1). The species that occurred in different months but with low abundance, i.e. the coastal fish Chromis chromis and Anthias anthias and the myctophyd Lampanyctus pusillus, formed group B.1. Group B.2 was formed by species slightly more abundant, such as Oblada melanura and Labridae, that were found exclusively, or were particularly 2.3. Long-term variability 107 abundant in June 83, and Diplodus sp. and the mesopelagic fish Arctozenus risso in July 2012 (Fig 2.3.4, Fig. 2.3.4. Two-way hierarchical clustering of surveys and heatmap showing the abundance of fish larvae (colour gradation from white to dark red represents from absence to highest abundance). Groups of species are indicated by coloured boxes labelled with a cod of letters and numbers, and survey groups are indicated by a different colour. 2.3. Long-term variability 108 abundant in June 83, and Diplodus sp. and the mesopelagic fish Arctozenus risso in July 2012 (Fig 2.3.4, table 2.3.1). The lower branch (groups C and D) comprised relatively abundant taxa. Warm water species such as Caranx rhonchus, Thalassoma pavo and Sphyraena sphyraena formed group C.1 and were mainly present in the cluster assembling June 2004 and 2011 and July 2003 and 2004, being absent in all months of 1983 (Fig. 2.3.4, table 2.3.1). Group C.2.1 included species such as Trachinus draco, Pomatomus saltatrix and Symphurus nigescens, which were present in all surveys, with the exception of June 1983. Group C.2.2 were composed by species such as Trachurus trachurus, Serranus cabrilla, Coris julis, and the myctophid fish Myctophum punctatum (Fig. 2.3.4, table 2.3.1), which were found in June and July of all decades, being practically absent in September surveys. Group D included the 12 most common and abundant species. Group D.1 was formed by species such as Auxis rochei, Trachurus mediterraneus and the myctophids Ceratoscopelus maderensis and Lampanyctus crocodilus, which showed low abundances in September 83 and 2003. Species such as the small pelagics, Engraulis encrasicolus and Sardinella aurita, and the mesopelagic fish Cyclothone braueri formed group D.2 and were present and abundant in all surveys, although S. aurita larvae were absent in June 1983. Table 2.3.2. Summary of the redundancy analysis (RDA) results. The individual variables are ordered by the percentage of variance explained (Exp. Var.), the significance of each variable (P) together with its test statistic (F-value), and the inter-set correlations between the environmental variables and the first two axes of the RDA. Regarding the RDA in relation to environmental conditions, the first ordination axis explained 46.9% of the constrained variance and was negatively correlated with depth (Table 2.3.2). The second axis explained 30.83% of the constrained variance and was negatively correlated with temperature (Table 2.3.2). The ordination of the sampling stations in the RDA plot showed that stations sampled in the 1980s were mainly located in the upper part of the plot, associated with low temperature and high chlorophyll-a values, and those of the 2000s in the lower part, related to high temperature and low chlorophyll-a (Fig. 2.3.5). The stations sampled in the 2010s were located in the central part of the plot. Species ordination showed that most of them were placed in the central part of the plot (Fig. 2.3.5). However, some species exhibited a relationship with specific environmental variables. Thus, the mesopelagic fish Cyclothone braueri and the myctophids (e.g. Lampanyctus crocodilus, Myctophum punctatum) were located in the left part of the plot, associated with high depth values. Another group of species, such as Oblada melanura and Trachurus trachurus placed in the upper part of the plot, showed a relationship with low 2.3. Long-term variability 109 temperature and high chlorophyll-a values. Warm water species such as S. aurita, Trachurus mediterraneus and Pomatomus saltatrix were located in the lower part of the plot, associated with higher temperatures. Fig. 2.3.5. Ordination plot of the redundancy analysis (RDA) showing the relationships between the larval fish taxa, the sampling stations, and the explanatory environmental variables (vectors) for the summer 1980s, 2000s and 2010s. Blue symbols correspond to the stations sampled in the 1980s, red symbols to those in the 2000s and yellow symbols to those in the 2010s. Abbreviations of the taxa names are detailed in Table 2.3.1. Distribution trends across decades A marked decline in the abundance of E. encrasicolus larvae was detected from the 1980s to the 2010s (Fig. 2.3.6). Throughout the whole studied period, larvae of this species were found all along the coast, with two main centres of abundance, at the northern half of the area and to the south, on the Ebro River shelf. It is worth noting the high abundance of larvae detected in the northern part in June and July of the 1980s. S. aurita larvae were absent in June 1983, their abundance in July 1983 was extremely low and their distribution was limited to the southern half of the area (Fig. 2.3.7). The abundance of this species increased markedly in the 2000s, its distribution extended along the entire shelf, reaching the northern limit of the study area. In the 2010s however, its abundance notably declined, showing a very coastal distribution extending all along the study area. The temporal distribution of P. saltatrix larvae over the three decades was quite similar to that of S. aurita larvae (Fig. 2.3.8). That is, larvae were absent in June 1983, in July 1983 their distribution was limited to the Ebro shelf. In the 2000s, their distribution extended to the whole study area, while in the 2010s, their abundance decreased notably, showing a more coastal distribution. 2.3. Long-term variability 110 Fig. 2.3.6. Mean abundance and spatial distribution of Engraulis encrasicolus larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. Fig. 2.3.7. Mean abundance and spatial distribution of Sardinella aurita larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. Caranx rhonchus larvae were absent in the 1980s (Fig. 2.3.9). In 2000s, larvae were relatively abundant, particularly in June 2004, with the highest abundances mainly in the south, on the Ebro shelf. They showed a relatively coastal distribution extending close to the northern limit of the study area. Their abundance declined in the 2010s, similar to the pattern detected in S. aurita and P. saltatrix. Coris julis larvae were relatively abundant in the 1980s, particularly in July (Fig. 2.3.10). Their abundance declined slightly in the 2000s and increased again in the 2010s, in contrast to the pattern shown by the species mentioned above. In all the studied years, their distribution extended along the entire coast. 2.3. Long-term variability 111 Fig. 2.3.8. Mean abundance and spatial distribution of Pomatomus saltatrix larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. Fig. 2.3.9. Mean abundance and spatial distribution of Caranx rhonchus larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. Note that this species was absent in the 1980s. Thalassoma pavo larvae, like those of C. rhonchus, were absent in the 1980s (Fig. 2.3.11). In the following two decades, their abundance was very similar, being always more common in June than in July, showing a scattered distribution throughout the study area. The abundance of Serranus cabrilla larvae showed similar abundances in all three decades, although always higher in June than in July (Fig. 2.3.12). Their distribution was widespread throughout the area. M. punctatum larvae did not show significant changes in abundance or distribution over the three decades. Larvae were located at the shelf break throughout the study area (Fig. 2.3.13). 2.3. Long-term variability 112 Fig. 2.3.10. Mean abundance and spatial distribution of Coris julis larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. Fig. 2.3.11. Mean abundance and spatial distribution of Thalassoma pavo larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. Note that this species was absent in the 1980s. Fig. 2.3.12. Mean abundance and spatial distribution of Serranus cabrilla larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. 2.3. Long-term variability 113 Fig. 2.3.13. Mean abundance and spatial distribution of Myctophum punctatum larvae overlaid on bathymetry (isobaths shown: 50, 200, 1000 and 2000 m) in June and July of the 1980s, 2000s and 2010s. 2.3.4. Discussion In the present study, important changes in the ichthyoplankton community have been detected over the three decades of the study period, with a major shift occurring between the 1980s and the 2000s. While these changes may be the result of the interaction of multiple factors, both abiotic and biotic, temperature has been identified as the key driver of these changes, influencing both adult fish populations and larval stages (e.g. Houde, 1989; Peck et al., 2012; Vary et al., 2023). The results of the different analyses performed showed that larvae of some warm water fish species that were not present in the 1980s were detected from the 2000s onwards. Other warm water species increased in abundance and their distribution spread northwards. Changes in the values of the environmental variables were detected along the three decades of the study period. There was a temperature increase, particularly marked for the June surveys. This is in accordance with the warming trend documented in the Mediterranean (Skliris et al., 2012; Vargas-Yáñez et al., 2009), with the most significant temperature increase recorded in the spring and summer months (Salat et al., 2019; Skliris et al., 2012; Yan and Tang, 2021). Extremely high temperatures were detected in July 2003 as a result of the successive heat waves that affected south-western Europe during spring and summer of that year (Black et al., 2004; Luterbacher et al., 2004). Climatic models predict rapid warming in the Mediterranean along with a greater occurrence of marine heatwave events (Darmaraki et al., 2019; Oliver et al., 2019; Pastor and Khodayar, 2023; Smale et al., 2019). Surface salinity and chlorophyll-a showed opposite trends, increasing and decreasing respectively, along the three decades. The low salinity values detected in the 1980s would be related to the high river runoff waters, mainly the Ebro and the Rhône (Sabatés, 1990) that show a decreasing trend over time (Cozzi et al., 2018). In the NW Mediterranean, during the stratification period, river runoff is the main mechanism 2.3. Long-term variability 114 that can supply nutrients to the surface, and a good correspondence between low salinity and high chlorophyll-a at surface is characteristic of this period (Masó et al., 1998; Sabatés et al., 2013, 2009). A decrease in chlorophyll-a concentration has also been documented in relation to the decrease in river runoff waters, accompanied by a decrease in nutrients concentration (Colella et al., 2016; Cozzi et al., 2018; Salgado-Hernanz et al., 2022) and an intensification of water column stratification due to sea temperature increase (Coma et al., 2009; Durrieu de Madron et al., 2011; Somot et al., 2006). The cluster analysis showed marked changes in the composition and abundance of the larval fish community between June and July in the 1980s and the following decades. The RDA indicated that temperature was the main environmental variable contributing to these changes. This analysis also showed a clear relationship of warm water species with temperature, which were detected for the first time, or exhibited a marked increase in abundance in the 2000s, during the warmest years of the studied period. The presence of larvae of warm water species in the 2000s and 2010s, not detected in the 1980s, e.g. the carangids, Trachinotus ovatus, Caranx rhonchus and the coastal benthic fish, Thalassoma pavo, is in agreement with the records of southern fish species in the colder areas of the NW Mediterranean from the late 1980s (Bianchi et al., 2019; Francour et al., 1994; Lejeusne et al., 2009). Although sea warming may be responsible for these distribution changes, species’ response to climate change involves interactions with various biotic and abiotic conditions (Bates et al., 2014) and therefore other factors, such as the establishment of marine reserves and the subsequent reduction or prohibition of fishing in such areas, may have favoured an increase in the populations of some thermophilic species (Bodilis et al., 2003). In the case of carangids, the northward expansion of warm water species (i.e. Trachurus mediterraneus, C. rhonchus, Caranx crysos, T. ovatus and Seriola dumerili) could also be related to the increase of floating objects and jellyfish in the Mediterranean, as their juvenile stages have been frequently found associated with fish aggregating devices, drift algae and jellyfish (Psomadakis et al., 2011; Raya and Sabatés, 2015; Tilves et al., 2018). T. pavo is a coastal warm water fish confined before 1980s to the southern parts of the Mediterranean coasts. In the 1980s, larvae of this species were absent in the study area, their abundance increasing in the following decades. As commented above, this species was detected in the mid-1990s in the northern sector of the Mediterranean (Bianchi, 2007; Milazzo et al., 2011). T. pavo coexists with the sympatric species C. julis that tolerates cooler waters and is found along the northern coast of the Mediterranean (Milazzo et al., 2011). Larvae of C. julis were abundant in the 1980s and declined slightly in the 2000s. This decline would suggest a greater adaptive advantage of T. pavo, which ‘won’ the competition for space over C. julis (Milazzo et al., 2013). While sea warming can modify interspecific interactions between similar ecological species (Milazzo et al., 2013), our results show that larvae of both species coexist in space and time while it remains to be confirmed how the interaction between the two species will evolve in the near future. Larvae of other species, such as S. aurita and P. saltatrix, increased markedly in abundance between the 1980s and the 2000s, and their distribution extended further north (where temperatures are cooler). This is 2.3. Long-term variability 115 in accordance with the increasing abundance and gradual northward expansion of adults of these species along western Mediterranean coasts in relation to sea warming (Sabatés et al., 2012, 2006). The expansion, colonization, and increase in abundance of native warm water species into the colder north Mediterranean regions is an expected outcome of meridionalization (Azzurro et al., 2019; Lloret et al., 2015). The presence of larvae of warm water species in the study area from the 2000s onwards indicates that they reproduce successfully and confirms the establishment of these species in their new distribution ranges (Sabatés et al., 2006). However, it is important to highlight the low abundance of larvae of most species detected in July 2003 (Table 2.3.1), during the marine heat wave. This suggests that larvae would be negatively affected by high temperatures reached (~27ºC), probably being above the physiological optimum for their growth and survival (Maynou et al., 2014; Takasuka et al., 2008). Rising temperatures, particularly during heat waves, can have different impacts on ecosystems and, consequently, in the structure larval fish assemblages (Nielsen, 2020). It is expected that in the near future, in a scenario of increased marine heatwaves (Darmaraki et al., 2019; Oliver et al., 2019; Pastor and Khodayar, 2023; Smale et al., 2019) fish larvae of most species will not find favourable conditions for their growth and survival. Larvae of some species, present in the 1980s, do not appear to have experienced major changes in abundance and distribution between the three decades (Table 2.3.1; Fig. 2.3.10 and Fig. 2.3.12). This is the case of coastal benthic species such as Serranus cabrilla, Serranus hepatus, and Coris julis. These species are well established in the area, reproduce in spring-summer and are not subject to fishing exploitation. The larvae of mesopelagic species, e.g. Myctophum punctatum, Cyclothone braueri, and Arctozenus risso also did not show these changes between the three decades (Table 2.3.1; Fig. 2.3.13). The oceanic habitat of these species, as indicated the RDA, would mean that they would be less influenced by the observed environmental trends, increasing temperature and decreasing river runoff, which are more pronounced on the continental shelf than in the open sea (Salgado-Hernanz et al., 2022) (Fig. 2.3.11). The presence of larvae of warm-water species in the 2000s and 2010s contributed to an increase of the specific richness (S) of the larval fish community compared to the richness values obtained in 1980s (Fig. 2.3.3). In this context, some authors have already suggested that the increase of warm water species would lead to a biotic homogenization of fish communities in the NW Mediterranean (Ben Rais Lasram and Mouillot, 2008). The Shannon-Wiener (H’) and Pielou’s (J) biodiversity indices showed low values in the 1980s compared to the following decades. These low biodiversity values would be related to the dominance of E. encrasicolus in the 1980s. The marked decline in the abundance of E. encrasicolus larvae observed from the 1980s to the 2000s would be related to the excessive fishing pressure on this species (FAO, 2024). A decreasing trend in anchovy population has been reported in the NW Mediterranean since the 1980s (FAO, 2023). Fishing exploitation results in unbalanced demographic structure of the small pelagic stocks by preferentially removing larger individuals (e.g. Brosset et al., 2.3. Long-term variability 122 Doyle, M.J., Picquelle, S.J., Mier, K.L., Spillane, M.C., Bond, N.A., 2009. Larval fish abundance and physical forcing in the Gulf of Alaska, 1981-2003. Prog. 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