A multidisciplinary approach to identify priority areas for the monitoring of a vulnerable family of fishes in Spanish Marine National Parks

Planasetal. BMC Ecol Evo (2021) 21:4 https://doi.org/10.1186/s12862-020-01743-z RESEARCH ARTICLE A multidisciplinary approach toidentify priority areas forthemonitoring ofavulnerable family offishes inSpanish Marine National Parks Miquel Planas1* , Cristina Piñeiro‑Corbeira2, Carmen Bouza3,4, Inés Castejón‑Silvo5, Manuel Vera3,4, Marcos Regueira1, Verónica Ochoa1, Ignacio Bárbara2, Jorge Terrados5, Alexandro Chamorro1, Rodolfo Barreiro2, Jorge Hernández‑Urcera1, Irene Alejo6, Miguel Nombela6, Manuel Enrique García1, Belén G. Pardo3,4, Viviana Peña2, Pilar Díaz‑Tapia2, Javier Cremades2 and Beatriz Morales‑Nin5 Abstract Background: Syngnathid fishes (Actinopterygii, Syngnathidae) are flagship species strongly associated with sea‑ weed and seagrass habitats. Seahorses and pipefishes are highly vulnerable to anthropogenic and environmental disturbances, but most species are currently Data Deficient according to the IUCN (2019), requiring more biological and ecological research. This study provides the first insights into syngnathid populations in the two marine Spanish National Parks (PNIA—Atlantic‑ and PNAC—Mediterranean). Fishes were collected periodically, marked, morphologi‑ cally identified, analysed for size, weight, sex and sexual maturity, and sampled for stable isotope and genetic identifi‑ cation. Due the scarcity of previous information, habitat characteristics were also assessed in PNIA. Results: Syngnathid diversity and abundance were low, with two species identified in PNIA (Hippocampus guttulatus and Syngnathus acus) and four in PNAC (S. abaster, S. acus, S. typhle and Nerophis maculatus). Syngnathids from both National Parks (NP) differed isotopically, with much lower δ15N in PNAC than in PNIA. The dominant species were S. abaster in PNAC and S. acus in PNIA. Syngnathids preferred less exposed sites in macroalgal assemblages in PNIA and Cymodocea meadows in PNAC. The occurrence of very large specimens, the absence of small‑medium sizes and the isotopic comparison with a nearby population suggest that the population of Syngnathus acus (the dominant syng‑ nathid in PNIA) mainly comprised breeders that migrate seasonally. Mitochondrial cytochrome b sequence variants were detected for H. guttulatus, S. acus, and S. abaster, and a novel 16S rDNA haplotype was obtained in N. maculatus. Our data suggest the presence of a cryptic divergent mitochondrial lineage of Syngnathus abaster species in PNAC. Conclusions: This is the first multidisciplinary approach to the study of syngnathids in Spanish marine NPs. Habitat preferences and population characteristics in both NPs differed. Further studies are needed to assess the occurrence of a species complex for S. abaster, discarding potential misidentifications of genus Syngnathus in PNAC, and evalu‑ ate migratory events in PNIA. We propose several preferential sites in both NPs for future monitoring of syngnathid populations and some recommendations for their conservation. Keywords: National park, Syngnathids, Habitat, Stable isotopes, Genetic identification, Conservation © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background Syngnathidae is a singular fish family mostly inhabiting temperate and tropical sheltered, coastal marine waters [26, 47]. Seahorses and pipefishes utilize rocky, muddy, sandy, and rubble bottom habitats, generally associated Open Access BMC Ecology and Evolution *Correspondence: [email protected] 1 Department of Ecology and Marine Resources, Instituto de Investigaciones Marinas (IIM‑CSIC), Eduardo Cabello 6, 36208 Vigo, Spain Full list of author information is available at the end of the article Page 2 of 20 Planasetal. BMC Ecol Evo (2021) 21:4 with macrophytes communities [53]. Syngnathids are secondary consumers with specialized and opportunistic predatory strategies, ambushing small prey (mainly planktonic and nektonic crustaceans), showing a variety of diets, and foraging behaviours across genera and locations [53]. Seaweed and seagrass meadows promote the growth of most food sources and enhance the cryptic ability of syngnathids to avoid predators. Syngnathids are valuable flagship species for conservation programs that will simultaneously benefit other fish [83]. Many species are vulnerable and threatened by habitat loss (pollution, sedimentation and eutrophication) and disturbances through boating and shipping [97], [43]. More than half of syngnathid species (two seahorse and eleven pipefish species) inhabiting Spanish coasts are currently classified as Data Deficient, and further research is needed to understand their biology and ecology (e.g., connectivity, migrations, mortality, etc.) [43]. Misidentifications of species have been reported due to cryptic morphology and unclear diagnostic traits among species, stressed by historical reference labelling errors in particular cases (e.g., European genus Syngnathus) [37, 102]. Genetic data are useful to solve taxonomic issues and complement morphological information, as a basic step towards the characterization and conservation of species and associated habitats [102]. Different mitochondrial markers have shown strong molecular support for species identification of seahorse and pipefish to clarify population and conservation studies (e.g., [51, 86, 100, 102]. Studies on syngnathids in the Iberian Peninsula are scarce and mainly focussed on specific topics for a reduced number of species [10, 16–19, 56, 94]. The present study is the first multidisciplinary approach for the global evaluation of syngnathid populations in Spanish coasts, particularly in marine National Parks (NP). Studies conducted in NPs would be highly valuable, considering their protection status and the supposed reduced impacts of most potential disturbances. Currently, there are two marine National Parks (NPs) in Spain, differing in their characteristics and biodiversity: Atlantic Islands National Park (PNIA) (Atlantic Ocean, NW Spain) and Cabrera Archipelago National Park (PNAC) (Balearic Islands, Mediterranean Sea). NPs should be the best marine ecosystems to ensure species survival and success in biodiversity conservation. However, protection requires a deep knowledge and analysis of habitats, values and threats, particularly for exceptional species and populations. In marine protected areas, there is the risk of a negative impact for syngnathids through increased predator abundance [39]. Marine ecosystems in PNIA host complex habitats and numerous ecological niches due to the extraordinary rich biota inhabiting soft and rocky floors typical of protected, semi exposed and exposed environments. Rocky shores are covered by seaweed, whereas the Western side is dominated by hard substrates covered by crusty, coralline and other turf-forming seaweed [69]. That side is exposed to Atlantic open water and extreme sea currents and waves, mainly in winter. The Eastern side is less exposed due to its position facing the Ría de Vigo. That side is characterized by a high biodiversity and productivity, and therefore it is an area of special interest for fishing. Such high productivity is promoted by important seasonal phytoplankton blooms [3, 77], and secondary production [9, 92], with high abundance in summer and seasonal changes in community structure. Copepods are largely predominant in winter, being accompanied in summer by other groups of fauna [9]. The fisheries system in PNIA is complex [13, 63, and the use of some types of fishing impacts negatively on syngnathids (by-catch and substrate degradation). Although areas of fishing are protected, they are not subject to special regulations [63]. Increasing tourism and nature activities promote public awareness for the conservation of marine ecosystems [69]. Cabrera Archipelago National Park (PNAC) is an IUCN category II Marine Protected Area (MPA) located 10km southeast of Majorca (Balearic Islands, Mediterranean Sea), declared Spanish National Park in 1991. Algal beds, seagrass meadows and rocky bottoms dominate the subtidal zone. Three species of seagrass meadows are present: Zostera noltei (< 2m depth), Cymodocea nodosa (0–25m depth) and Posidonia oceanica (0–40m depth). Tourists visiting PNAC increases yearly, and recreational fishing and trawling in PNAC were banned in 1992. Small-scale fishing was regulated in 1995 but 80 smallscale boats from neighbouring towns continue fishing in some areas [57]. Fishing gears are regulated albeit overexploitation signs on the lobster trammel net fishery are evident [4]. The aims of this study were threefold. First, to assess distribution and habitat use of syngnathids in PNIA and PNAC (Additional file 1), each with highly distinctive environmental characteristics and vegetal assemblages. Second, to characterize syngnathid populations, which include the assessment of genetic identification and stable isotopes analyses. Finally, the unavailability of historical data for syngnathids in the Iberian Peninsula prevents the assessment of population trends. Hence, the third aim of this study was the selection of specific sites for further monitoring of distribution/abundance and temporalseasonal patterns on important biological and ecological features (e.g., diet composition, animal migration). The results achieved would be valuable for the development of further conservation actions in both NPs. Page 3 of 20 Planasetal. BMC Ecol Evo (2021) 21:4 Results Habitat characterization inCíes Archipelago (PNIA) Soft bottom sediments were mostly coarse sandy (569µm), with 90% sand and a prevalence of a single mode (Additional file2). Muddy sands, with > 20% mud (< 63µm), were only located in the deepest (17.6m to 21m) and distal areas of TR5, in the immediate vicinity of the muddy bottoms characteristics of the central part of Ría de Vigo. The presence of two or three mode samples in TR2, TR4 and TR3 reflected a mixture of particle sizes, including bioclastic gravel (bivalves and gastropods shells) and maerl elements. Different sedimentary environments (wide variability of textural characteristics) were present along some transects (e.g., TR3). Syngnathids were mostly sighted in sheltered sectors, preferring habitats with medium sands, better sorted and lacking mud (Additional file2). Similarity of seaweed assemblages was analysed considering data of 55 species with medium–high abundance (Additional file 2, Additional file 3). Diversity (H’) and species richness (S) were particularly low in TR1, TR2, TR7 and TR10, especially in spring (Additional file2). Seaweed cover increased in summer, especially in TR8 (633.8%) and TR9 (861.0%), but it was noticeable low in TR10 (42% in spring; 107% in summer) (Additional file2). PERMANOVA results showed significant differences in assemblage structures for transects (df = 10; Pseudo-F = 1.3974; P = 0.0308) and seasons (df = 1; Pseudo-F = 3.711; P = 0.0031). Those differences are reflected in the two-dimensional PCOs plot (Fig.1). Spring (left) and summer (right) samples followed a gradient along axis 1 (20.4% of total variation). Abundance increased in summer for most species, especially for Treptacantha baccata, Padina pavonica, Corallina officinalis or Codium tomentosum (strong negative correlation with PCO1; Spearman correlation > 0.65). Differences between transects were explained by axis 2 (18.1% of total variation), reflecting wave exposure. Transects TR1, TR8 and TR9 were clearly separated from the others, especially from TR10 and TR3. These results explained spatial differences between transects, with TR9, TR8 and TR7 as the most northern sites of Cíes Archipelago, and TR1 located in the west side of the southern island. The remaining transects (especially TR10) were located in areas less exposed to wave impact and current actions. Vectors overlay in PCO plot indicated that species such as T. baccata, P. pavonica or C. tomentosum were more abundant on less exposed areas, while Mesophyllum expansum, C. officinalis, Plocamium cartilagineum and Kallymenia reniformis preferred more wave-exposed sites (Spearman correlation > 0.65). Syngnathids inPNIA andPNAC In PNIA, two species of syngnathids were identified morphologically and genetically: the long-snouted seahorse Hippocampus guttulatus Cuvier, 1829, and the greater pipefish Syngnathus acus, Linnaeus 1758. A total of 28 specimens were sighted in PNIA from 4 to 15m depth (mostly at < 8m), with six transects providing at least one fish (Table1). None of the individuals marked in spring were recaptured in summer. All PCO showed a positive correlation of syngnathids with seaweed assemblages on transects TR3, TR4, TR5 and especially TR10 (Spearman correlation > 0.65) in summer (Additional file 2). The highest abundance (0.06–0.13 syngnathids 100m−2) were recorded in mixed (sand-rock) or rocky substrates on transects TR3 and TR10 (32 and 43% of total specimens, respectively). Syngnathids were missing in the more exposed transects TR1, TR7, TR8 and TR9 (northern and southern areas with rocky substrate and coarse sand patches). TR1 was facing SW waves (prevalent component during storm winter conditions), while TR7, TR8 and TR9 were facing N waves (prevalent component during storm summer conditions). The most common species was S. acus (n = 24), which comprised 86% of total fish sighted. Most collected fishes were large adults, with S. acus averaging 31.8 ± 10.0cm SL (range: 14.8–49.7cm) and H. guttulatus, 22.6 ± 2.0cm (range: 18.7–22.7cm). Mean weights were 21.9 ± 5.2g (range: 1.3–67.6g) in S. acus and 22.6 ± 2.0 (range: 14.8–25.8g) in H. guttulatus. In S. acus, meristic features were: 20 trunk rings (range: 19–20), 42 tail rings (41–44), 12 pectoral fin rays Fig. 1 PNIA—Principal coordinates ordination of samples for Transect x Season pairwise combinations in seaweed assemblages on spring (green) and summer (grey). Overlay vectors are species whose cover has a Spearman correlation > 0.65 with any axis Page 4 of 20 Planasetal. BMC Ecol Evo (2021) 21:4 (9–12), 38 dorsal fin rays (37–41), 3 anal fin rays and 10 caudal fin rays. Only four seahorses were observed (TR10; 8.0–8.5m depth). The species showed positive allometry (b = 3.32) (Additional file2), and lengths and weights in spring and summer did not differ significantly (Tukey HSD, n = 23, P = 0.519 for length, P = 0.471 for weight). Pregnant males and ovigerous females did not differ neither in length (Tukey HSD, n = 20, P = 0.464) nor weight (Tukey HSD, n = 20, P = 0.983). Abundance declined in summer (25%), when mature individuals were not observed. Contrarily, 90% of males and 70% of females collected in spring were pregnant (pouch carrying fertilized eggs/embryos) or ovigerous (full gonads with hydrated eggs), respectively. The minimum length recorded was 23.8 cm (8.4 g) in pregnant males and 25.2cm (10.7g) in ovigerous females. With regard to distribution of total syngnathids (S. acus and H. guttulatus) in Cíes Archipelago (PNIA), Maxent model achieved an AUC value of 0.98, indicating a very good degree of discrimination between the locations where the species were present and those where they were absent. Figure2 shows the probability of habitat suitability for syngnathids. The model highlights higher probabilities of occurrence in a few limited areas (red color) on the East coast of the islands. Three of those areas (TR 3, TR4-5 and TR10) were selected for further monitoring of syngnathids in PNIA (see Discussion). The Jackknife test (i.e. variable importance) showed that Table 1 PNIA—Syngnathids (Syngnathus acus andHippocampus guttulatus) captured inspring andsummer 2016 surveys TR transect, SL standard length, W wet weight *Not captured Species TR Date Depth (m) SL (cm) W (g) Sex Sexual state Substrate Spring 2016 S. acus 2 4‑may 15 23.8 8.4 Male Pregnant Gravel 5 5‑may 6 14.8 1.3 Female Sandy 6 5‑may 5.5 44.0 64.7 Female Ovigerous Sandy 6 5‑may 6 34.2 27.2 Female Ovigerous Sandy 10 20‑may 4 32.0 23.2 Male Pregnant Rocky 3 7‑jun 5.5 44.9 66.7 Male Pregnant Sandy‑Rocky 3 7‑jun 5.5 28.9 14.6 Female Ovigerous Sandy‑Rocky 3 7‑jun 6 35.0 25.1 Female Ovigerous Sandy‑Rocky 3 7‑jun 5 31.3 28.6 Male Pregnant Sandy‑Rocky 3 7‑jun 5.5 24.5 6.2 Male Pregnant Sandy‑Rocky 3 7‑jun 5.5 25.2 10.6 Female Ovigerous Sandy‑Rocky 3 7‑jun 6 34.3 25.9 Female Ovigerous Sandy‑Rocky 3 7‑jun 6 15.0 – Female Sandy‑Rocky 10 8‑jun 4 49.7 62.5 Female Ovigerous Rocky 10 8‑jun 4 25.5 16.6 Male Pregnant Rocky 10 9‑jun 7 20.5 4.1 Female Sandy 10 9‑jun 7.5 33.0 21.4 Male Pregnant Sandy 10 9‑jun 5 45.6 67.6 Male Pregnant Sandy‑Rocky H. guttulatus 10 9‑jun 8.5 22.7 25.8 Female Rocky 10 9‑jun 8.5 21.8 25.6 Male Rocky Summer 2016 S. acus 2* 1‑sep – – – – 3 6‑sep 6 40.0 50.7 Female Sandy 4 6‑sep 6.5 30.8 21.6 Male Rocky 4 6‑sep 4 17.6 3.1 Female Sandy 10 7‑sep 7.5 39.0 40.4 Female Sandy‑Rocky 10 7‑sep 7.5 42.9 58.6 Male Sandy‑Rocky H. guttulatus 10 7‑sep 8 19.5 21.3 Male Rocky 10 7‑sep 8 18.3 14.8 Female Rocky Page 5 of 20 Planasetal. BMC Ecol Evo (2021) 21:4 Fig. 2 PNIA—Maxent habitat suitability map for syngnathids (pooled specimens of S. acus and H. guttulatus) in Cíes Archipelago. Environmental suitability is depicted using a color gradient from blue (low environmental suitability) to red (high suitability). The bottom panel shows the results of the jackknife test of variable importance training data Page 6 of 20 Planasetal. BMC Ecol Evo (2021) 21:4 bathymetry and wave exposure were the most influential variables (Fig.2). In PNAC, four pipefish species were morphologically identified but seahorses were lacking. Only three specimens (one Syngnathus acus, two Nerophis maculatus Rafinesque, 1810) were sighted on 37 visual censuses and 15 specimens (10 Syngnathus abaster Risso, 1827, two Syngnathus typhle Linnaeus, 1758, two Syngnathus acus and, one Nerophis maculatus) were captured in seven fishing sets (Table2). All specimens were captured at 11–21m depth, except for two N. maculatus (< 8m depth). Occurrences in C. nodosa meadows (Es Burri) by fishing sampling and visual census were similar (1.3 and 1.2 syngnathids 100m−2, respectively), but two-folds higher than by visual censuses in P. oceanica meadows and macroalgal beds in rocky substrates (0.03 individuals per 100m−2). Genetic identification insyngnathids Genetic samples from 33 syngnathid specimens morphologically identified in PNIA (22 S. acus and 4 H. guttulatus) and PNAC (6 S. abaster and 1N. maculatus) were assayed. The marker cytochrome b (Cytb) was used to support the molecular identification of seahorse and pipefish species [100, 101]. The ribosomal mitochondrial marker 16S rDNA, which also supported molecular phylogeny in syngnathids [100], was assayed in the single sample of N. maculatus in which Cytb could not be amplified. Length for Cytb sequences was 1149 base pairs (bp) in S. acus. In PNIA, nine Cytb haplotypes (12 variable sites) were detected (Additional file2) and identified as S. acus (identity > 99.5% and e-value = 0.0), one of them (Cytb_ SA13; GenBank Accession Number: MW080699) identical to the reference used for this species (AF356040; [100]. Haplotypes Cytb_SA01 (MW080694) and Cytb_ SA02 (MW080695) were the most abundant (nine and six individuals, respectively), whereas the rest were only found in one individual (Cytb_SA07: MW080696,Cytb_ SA10: MW080697; Cytb_SA11: MW080698; Cytb_SA14: MW080700; Cytb_SA16: MW080701; Cytb_SA17: MW080702), resulting in a haplotype diversity (h) of 0.7792 in the PNIA population sampled. The four seahorse specimens studied were identified as H. guttulatus. Three Cytb haplotypes (564bp) were detected (Cytb_HG01-03), comprising two variable sites (five when the reference sequence was included) (Additional file2). Cytb_HG01, Cytb_HG02 and Cytb_HG03 were identical to H. guttulatus sequences reported across European populations [101]: KM061961 (GB10), KM061963 (GB7) and KM061980 (GB23), respectively. The most abundant H. guttulatus haplotype was Cytb_HG03 (two individuals), providing an h estimate of 0.8333. Table 2 PNAC: Syngnathids captured in2016 surveys andsampling information VC visual census, MC manual capture, TN Trawl net (gánguil), SL standard length, nm not measured Species Site Date Depth (m) SL (cm) Benthic community Sampling method Syngnathus acus Es Port 21 April 13.5–15 27.0 Cymodocea nodosa VC, MC Es Burri 6 Sept 11–13 11.5 Cymodocea nodosa TN Es Burri 8 Sept 13–15 4.4 Cymodocea nodosa TN Syngnathus abaster Es Burri 6 Sept 11–13 7.6 Cymodocea nodosa TN Es Burri 6 Sept 11–13 7.0 Cymodocea nodosa TN Es Burri 8 Sept 13–15 9.0 Cymodocea nodosa TN Es Burri 8 Sept 13–15 4.1 Cymodocea nodosa TN Es Burri 1 Dec 11–13 10.3 Cymodocea nodosa TN Es Burri 1 Dec 11–13 8.1 Cymodocea nodosa TN Es Burri 1 Dec 11–13 9.9 Cymodocea nodosa TN Es Burri 2 Dec 13–15 8.8 Cymodocea nodosa TN Es Burri 2 Dec 13–15 9.8 Cymodocea nodosa TN Es Burri 2 Dec 13–15 7.6 Cymodocea nodosa TN Syngnatus typhle Es Burri 8 Sept 13–15 6.4 Cymodocea nodosa TN Es Burri 8 Sept 13–15 6.4 Cymodocea nodosa TN Nerophis maculatus Es Burri 23 April 6–8 nm Posidonia oceanica VC, MC Es Burri 8 Sept 13–15 10.0 Cymodocea nodosa TN Es Burri 9 Sept 19–20 12.5 Cymodocea nodosa VC, MC Santa María 1 Dec 4.4–6 nm Posidonia oceanica MC (outside VC) Page 7 of 20 Planasetal. BMC Ecol Evo (2021) 21:4 For six specimens morphologically identified as S. abaster, two Cytb haplotypes were detected (Cytb_ SAb01: MW080703 and Cytb_SAb02: MW080704) in four and two fish, respectively (h = 0.5333); showing 139 variable sites respect to the S. acus sequence (AF356040; Additional file 2). These two Cytb_SAb haplotypes showed a higher sequence identity with a S. typhle reference haplotype (JX228148; identities > 98%) than with other Cytb sequences of S. abaster (JX228141; identities ≤ 95%) available at GenBank database. Thus, net genetic distances between the groups formed by the Cytb_SAb haplotypes and the S. typhle haplotypes available at the GenBank (0.0140 ± 0.0033) was lower than the distance between the groups formed by the PNAC Cytb_SAb haplotypes and the S. abaster haplotypes from GenBank (0.0478 ± 0.0061). Phylogenetic analysis also corroborated these results. Thus the two Cytb_SAb haplotypes from PNAC were grouped in a monophyletic cluster clearly differentiated from GenBank Cytb sequences of S. abaster [58] and other pipefish species distributed in Mediterranean areas, more closely related to S. typhle and S. taenionatus than to S. acus and S. rostellatus (Fig.3). Failed amplification of Cytb was observed in the single genetic sample studied of N. maculatus, but a novel 16S rDNA haplotype of 521 pb (16S_NM01: MW080705) was detected for this pipefish, with 48 variable sites respect to a related reference species (N. ophidion; AF354994), in absence of available GenBank data for N. maculatus. Stable isotope signatures insyngnathids In PNIA, H. guttulatus and S. acus (Table3) differed significantly for δ13C (ANOVA, F1,21 = 0.492, P = 0.026) but not for δ15N (F1,21 = 5.744; P = 0.491). Isotopic values in S. acus ranged from -16.6 to -14.7 ‰ for δ13C (-16.1 ± 0.4) and from 9.1 to 11.9 ‰ for δ15N (10.8 ± 0.7), being correlated with fish size (Additional file2). Season-sex comparisons showed inter-seasonal differences only for δ13C, with spring values (−16.2 ± 0.3 ‰) lower than in summer (−15.6 ± 0.5 ‰) (ANOVA, F1,15 = 9.52, P = 0.008), and lower values in males (−16.2 ± 0.2 ‰) than in females (−15.9 ± 0.6 ‰) (ANOVA, F1,15 = 6.65, P = 0.021). Sexmaturity comparisons showed similar δ13C values for mature (−16.2 ± 0.3 ‰) and immature (15.9 ± 0.6 ‰) fishes (ANOVA, F1,13 = 2.88, P = 0.104) but lower δ15N signals in the later (11.1 ± 0.5 ‰ for mature, 10.4 ± 0.8 ‰ for immature) (ANOVA, F1,13 = 2.79, P = 0.014). SIA in PNAC was only performed on a reduced number of S. abaster (n = 5; 7.6–10.3 cm length). Isotopic values were not correlated with fish size (Spearman correlation = − 0.3 and 0.1 for N15 and C13 respectively), Fig. 3 NJ tree (p‑distance) for the Syngnathus genus. “SA” and “SAb” show S. acus and S. abaster haplotypes, respectively. Numbers on branches indicate the bootstrap value for their confidence (1000 replicates). GenBank reference sequences for S. abaster (AF356060_S23; JX228141_SCA1), S. typhle (AF356042_S4; AF356059_S22; JX228148_KLU1), S. acus (AF356040), S. rostellatus (AF356041_S3), S. taenionatus (AF356061_S24; JX228146_ VEN89) and S. exilis (JF273424_S64) are also included. Following Mwale et al. [58], S. exilis was used as outgroup Page 8 of 20 Planasetal. BMC Ecol Evo (2021) 21:4 ranging from −15.2 to −19.6 ‰ for δ13C (−16.4 ± 1.8) and from 6.5 to 7.3 ‰ for δ15N (6.9 ± 0.3). Discussion Diversity, distribution andhabitat ofsyngnathids PNIA and PNAC differed in habitat characteristics and syngnathids occurrence. Sixteen syngnathid species are known in Europe [20] but only five were identified in our study. Two species were sighted in PNIA: the seahorse H. guttulatus (very low abundance) and the pipefish S. acus (Highly dominant). Most specimens sampled from PNIA were very large, lacking young or small sized fishes. In PNAC, seahorses were absent and four pipefish species (S. abaster, S. acus, S. typhle, N. maculatus) were recorded, comprising mostly small specimens. Syngnathids were considered uncommon in PNAC, though occurrences of S. acus, S. typhle, H. guttulatus and H. hippocampus were known [76]. Our results indicate low pipefish occurrences, with higher abundance in Es Burri Bay, particularly for S. abaster. This species is also the most common in other nearby areas (Mar Menor, SW Spain) [21]. H. guttulatus and N. maculatus are classified as Data Deficient, whereas the others are considered Least Concern [42]. In Balearic Islands, S. abaster is Vulnerable, S. typhle is Near Threatened, and S. acus and N. maculatus are Least Concern [31]. High congruence between genetic and morphological data for species identification was observed, except for S. abaster in PNAC respect to previous mitochondrial sequences for this species. In PNIA, eight novel Cytb haplotypes were found for S. acus, but also common sequence variants respect to Northern and Southern European populations of S. acus (1 haplotype) and H. guttulatus (3 haplotypes), respectively [100, 101]. Available genetic sampling in PNAC allowed detecting novel haplotypes for a small number of pipefish morphologically identified as N. maculatus (one 16S rDNA haplotype in a single specimen) and S. abaster (two Cytb haplotypes for six individuals). These two new S. abaster Cytb haplotypes detected in PNAC clustered in a single monophyletic group, supporting the morphological identification, but separately from previous Cytb sequences available for voucher samples of the same species from Italian coasts [58], and also from other congeneric pipefish distributed in the Mediterranean Sea (S. acus, S. rostellatus, S. taenionotus, S. typhle). Morphological discrimination from other possible species like S. schmidti and S. phlegon was also stablished based on non-overlapping ranges for meristic traits (http://speci es-ident ifica tion.org/index .php,[37], in the absence of available Cytb data to be compared in these species. Some sample misidentification during insitu surveys could be possible, according to confuse discriminations previously reported for some Mediterranean Syngnathus species, like S. rostellatus [37]. However, the combined morphological and genetic results in our study are congruent with previous data based on different mitochondrial markers, which support that S. abaster does not constitute a monophyletic taxon [81]. Indeed, highly divergent S. abaster mitochondrial lineages were described in the westernmost Mediterranean Sea respect to more eastern Italian coasts, which may be acknowledged as distinct related species [81]. The Table 3 PNIA—Mean (± sd) δ13C andδ15N values inH. guttulatus andS. acus sampled inspring andsummer 2016 inCíes Archipelago δ13C δ15N Species Season Mean ± sd Max Min n Mean ± sd Max Min n H. guttulatus Pooled − 16.6 ± 0.2 − 16.3 − 16.8 4 11.0 ± 0.4 11.6 10.6 4 Spring − 16.5 ± 0.2 − 16.3 − 16.6 2 11.2 ± 0.5 11.6 10.9 2 Summer − 16.7 ± 0.2 − 16.5 − 16.8 2 10.8 ± 0.4 11.1 10.6 2 ♂ Spring − 16.3 ± 0.0 − 16.3 − 16.3 1 11.6 ± 0.0 11.6 11.6 1 ♂ Summer − 16.8 ± 0.0 − 16.8 − 16.8 1 11.1 ± 0.0 11.1 11.1 1 ♀ Spring − 16.6 ± 0.0 − 16.6 − 16.6 1 10.9 ± 0.0 10.9 10.9 1 ♀ Summer − 16.5 ± 0.0 − 16.5 − 16.5 1 10.6 ± 0.0 10.6 10.6 1 S. acus Pooled − 16.1 ± 0.4 − 14.7 − 16.6 21 10.8 ± 0.7 11.9 9.1 21 Spring − 16.2 ± 0.3 − 15.8 − 16.6 16 11.0 ± 0.7 11.9 9.8 16 Summer − 15.6 ± 0.5 − 14.7 − 16.1 5 10.3 ± 0.8 11.2 9.1 5 ♂ Spring − 16.2 ± 0.3 − 15.9 − 16.6 8 11.1 ± 0.6 11.9 10.0 8 ♂ Summer − 16.0 ± 0.1 − 16.0 − 16.1 2 10.1 ± 1.5 11.2 9.1 2 ♀ Spring − 16.2 ± 0.3 − 15.8 − 16.6 8 10.9 ± 0.5 11.7 9.8 8 ♀ Summer − 15.3 ± 0.5 − 14.7 − 15.7 3 10.3 ± 0.2 10.5 10.2 3 Page 9 of 20 Planasetal. BMC Ecol Evo (2021) 21:4 Cytb haplotypes in this study confirmed a strong differentiation between the Italian S. abaster lineage, represented by reference samples reported by Mwale etal. [58] and the S. abaster haplotypes from PNAC, in Balearic Islands, which has been proposed to be part of the westernmost group along with adjacent sectors in Sardinian Sea [81]. Genetically divergent populations in species associated with long term isolation and restricted potential for dispersal has been pointed in different species inhabiting marine coastal habitats, including syngnathids, such as reported for the north-western Pacific messmate pipefish [81, 86]. Differences in diversity, distribution and abundance of syngnathids are related to habitat characteristics [62, 96, 101]. Many species are algae and seagrass residents closely associated with specific habitats that best enable camouflage [26, 45, 54, 82, 102]. In PNIA, seaweed communities are structurally complex and patchily distributed on mixed or rocky substrates [66, 69]. Most syngnathids in PNIA were located in semi-exposed or sheltered habitats on areas that showed the highest similarity regarding seaweed communities. Those areas were clearly identified in the estimated distribution map, and include rocky and sandy-gravel substrates, maerl beds as well as seaweed communities enhancing protection and habitat suitability for syngnathids. Transect TR10 was particularly interesting since it was located in the most sheltered area and the unique site with seahorse occurrences. As for S. acus in PNIA, dominant pipefish species form monospecific populations [54, 96] but many European pipefish species may vary their habitat occupancy and overlap a great deal [96], as shown in PNAC. Seagrass meadows are lacking in PNIA [29, 30] but PNAC seabed was partially covered by large extensions of seagrass meadows (P. oceanica and C. nodosa), which is a typical cover enhancing the occurrence of syngnathids in some Mediterranean areas [99]. That is the case of S. typhle, a pipefish that preferentially displays an upright position in seagrasses with narrow leaves (e.g. Zostera) [84, 96]. Its absence in PNIA could rely on the lack of seagrass meadows, even though this species may adapt to different types of habitats [88]. Appropriate habitats for syngnathids may not be determined simply by the presence or absence of vegetation but also by the prevalence of seaweed communities that best enable them to remain inconspicuous to predators [45]. All pipefish in PNAC were collected in C. nodosa and P. oceanica meadows, suggesting that macroalgal beds are less preferred than seagrass meadows. In PNAC, the results showed unexpected low pipefish abundance, which agrees with previous observations in similar habitats [96]. The highest abundance was recorded in C. nodosa meadows in Es Burri Bay (1.2–1.3 syngnathids 100 m−2). Visual censuses of syngnathids in dense meadows are difficult due to fish crypsis. Captures with the first visual censuses from 2.8 to 21.5m depth resulted substantially improved with gánguil gear operating at 11–16.5 m depth. However, global species richness and abundance in PNAC could have been underestimated. European syngnathids usually inhabit brackish areas (< 10m depth) but C. nodosa meadows are present at deeper depths (11–13m depth) in Es Burri Bay. The dominant pipefish S. abaster in PNAC commonly inhabits at 0.5–5m depth [20, 21], which is clearly above the depths imposed by gear, site and fishing permissions in gánguil sampling. Changes in macroalgal assemblages in PNIA are occurring since 2012. Abundance of Treptacantha baccata, T. usneoides and Saccorhiza polyschides decrease, while turf (Halopteris scoparia, Chondria coerulescens or Corallina spp.), and non-native (Codium fragile, Asparagopsis armata) species increase [12]. The progressive habitat loss and the increase in less optimal seaweed species can also cause dramatic changes in resident fauna and community composition [89, 90]. Most syngnathids from PNIA were captured in shallow waters (< 10m depth) on sandy substrates with low proportions of gravel, some mud and preferably nearby rocky outcrops that provides better refuge and protection (TR3, TR10). Coastal sheltered areas protected from SW (TR1) and N waves (TR7–TR9) were preferred but areas with high bottom mobility (sand waves and megarriples 3D) were avoided (TR2 and TR4). Some syngnathid species appear to be generalist considering distribution patterns and algal community characteristics whereas others prefer certain seaweed forms and feed on specific sources [54, 71]. Distribution patterns can be partially explained by the exposure to waves and open sea [54, 85] which has a great impact on seaweed cover. However, S. acus was also able to inhabit shallow and rocky areas (TR3) near the shore wave-breaking zone submitted to a certain degree of water agitation. Due to the high dominance of S. acus in PNIA, the species deserves special consideration. The length–weight relationship was similar to that in the western Black Sea [105]. The large specimens (31.8 ± 10.0cm SL) in PNIA was noteworthy compared to PNAC and other Mediterranean populations but did not differ from others in eelgrass meadows from Northern Europe [20, 35, 36, 96, 105]. However, the absence of small-medium sized specimens in Cíes Archipelago raises the questions of whether there is a resident population of adults (with dispersal of small individuals towards other areas) and/or whether the fishes migrate seasonally to Cíes from nearby areas only for breeding. None of the specimens marked in spring were recaptured in summer, suggesting that they Page 16 of 20 Planasetal. BMC Ecol Evo (2021) 21:4 whenever possible. The sexual status was recorded considering pregnancy in males and trunk shape (holding of hydrated eggs) in females. Species identification was evaluated genetically using DNA extracted from dorsal fin samples available from PNIA and PNAC surveys. In PNIA, all fishes from visual censuses were released at the capture site within 2–3h after sampling. For SL measurement, the fishes were placed on a plate including a measurement scale and photographed laterally (seahorses) or measured directly (pipefish). Seahorse images were analysed in the laboratory to determine length using image-processing software (NIS Elements Nikon and ImageJ2). Seahorses were measured as head + trunk + tail length (curved measurement in seahorses) [52]. Allometry in fishes was assessed using the following equation: where, TL is total length, a is an empirical coefficient, W is body weight and b is the allometric exponent. Sediment analysis The analysis of sediments was only carried out in PNIA. For compositional analysis, the content of organic carbon and inorganic carbon (calcium carbonate content is equivalent to bioclastic component for this regional setting) was determined by a LECO CNS-2000 Macro Elemental Analyser at CACTI (University of Vigo). Those analyses were performed on the fractions < 2 mm, in order to avoid distortional results due to gravel components (> 2mm, maerl, bivalve and gastropod shells). For textural analysis, the bulk grain size distribution was performed by dry sieving. Previously to grain size analysis, the organic matter was removed using 30% H2O2 for several days and salts were removed with further washings with distilled water. Afterwards, the samples were dried at 50°C and dry sieved between 4mm and 63μm (sieve size intervals of 1/2 ø). The resulting grain size distribution was treated with the GRADISTAT program [7]. For statistical parameters (mean, selection, asymmetry and kurtosis or pointing of the grain size curve), the nomenclature of Folk and Ward [25] classification was used. DNA sequence analysis DNA was extracted from dorsal fin tissue collected from the following morphologically identified specimens: (i) twenty-two wild greater pipefish (Syngnathus acus) and four long-snouted seahorses (Hippocampus guttulatus) from PNIA; and (ii) six black-striped pipefish (S. abaster) and one spotted pipefish (Nerophis maculatus) from PNAC. Genomic DNA was isolated using NucleoSpin TL =aWb Tissue XS kit (Macherey–Nagel Inc., Germany) and for extremely small tissue samples further amplified using GenomiPhi V2 kit (Healthcare, USA). The mitochondrial marker cytochrome b (Cytb) used for phylogenetic analysis in the Family Syngnathidae [100] was assayed for the molecular identification of all specimens studied from two divergent phylogenetic groups (Syngnathinae and Nerophinae subfamilies,[38]. Universal primers L14275F [64] and H15926R [100] were used to amplify Cytb in the pipefish species, while the specific primers SHORSE5.3L [13] and GUT CYT BR [101] in seahorses. To overcome the poor Cytb amplification success in N. maculatus, the universal primers 16SaL2510 and 16Sb-H3080 [65] were used in this species to amplify 16S rDNA, an informative marker also used for phylogenetic analyses in Syngnathidae [100]. PCR reactions in 50 μL included 100ng of template DNA, 1X PCR Gold Buffer (Applied Biosystems), 2.5 mM of MgCl2, 400µM of dNTPs, 0.2µM of each primer and 1 and 1.25 units of Amplitaq Gold™ DNA polymerase (Applied Biosystems) for pipefish and seahorse, respectively. Specific PCR programs were used for pipefish (95°C for 10min, 33 cycles of 93°C for 1min, 50°C for 1min and 72°C for 3min, plus final extension at 72°C for 10min) and seahorses (94°C for 10min, 35 cycles of 94°C for 30s, 50°C for 30s and 72°C for 1min, plus final extension at 72°C for 2min). Sequences were obtained using the ABI PRISM BigDye™ Terminator v3.1 Cycle Sequencing Kit on an ABI PRISM® 3730xl Genetic Analyzer (Applied Biosystems, Foster City, CA). Variable sites were checked with SEQSCAPE 2.5 (Applied Biosystems), using Genbank sequences AF356040, AF354994 (from Sweden; [100] and AF192664 (from UK [13], as reference for S. acus, N. ophidion and H. guttulatus, respectively. Variable positions, haplotypes and genetic distances (estimated with the p-distance method) were obtained using MEGA 7.0 [48] while haplotype diversity [59] for the different species was calculated using DnaSP 5.0 [50]. Species identification of sampled haplotypes was performed using BLASTn tool with default parameters within NCBI database. Evolutionary relationships among S. abaster haplotypes from PNAC and GenBank sequences of Mediterranean-distributed Syngnathus species were inferred using the Neighbor-Joining method based on p-distance implemented in MEGA with S. exilis (JF273424) as outgroup [58], and clustering support evaluated using bootstrap test (1000 replicates). Stable isotopes analysis (SIA) For δ13C and δ15N analysis in syngnathids, the samples were rinsed with distilled water, transferred to tin capsules, dried in oven at 60°C for 24h and weighted (± 1 μg). Due to the low lipid content in fin samples Page 17 of 20 Planasetal. BMC Ecol Evo (2021) 21:4 conserved in ethanol (< 5% lipids, C/N < 3.56) [74], further full defatting was not necessary [72, 93]. Samples were analysed at SAI (University of A Coruña) by continuous flow isotope ratio mass spectrometry using a FlashEA1112 elemental analyser (Thermo Finnigan, Italy) coupled to a Delta Plus mass spectrometer (FinniganMat, Germany) through a Conflo II interface. Isotopic values are expressed as permil (‰) in conventional delta relative to VPDB (Vienna Pee Dee Belemnite) and Atmospheric Air. The precision (standard deviation) for SIA of the laboratory standard (acetanilide) was ± 0.15‰ (1-sigma, n = 10). Geographic information GIS was managed with ArcGIS v.10.5 software to represent the maps. Layers of bionomic maps for both NPs (OAPN, unpublished observations) were incorporated. Sampled sites/transects and syngnathid capture locations were recorded and added to a geodatabase. Biological information of the specimens (species, sex, size, weight and sexual stage) was joined to each register. Available abiotic information (topographic and bathymetric layers), as well as bionomic information, were also added to geodatabase. Cartographic data were projectedin UTM 29N/UTM 31N reference system (for PNIA and PNAC, respectively) using ETRS89 Datum. Species distribution estimates Modeling distribution of syngnathids was only assessed in PNIA as the number of specimens collected in PNAC was insufficient. For that, Maxent (Maximum Entropy model) was implemented [67, 68], [24], using MaxEnt v.3.4.1 program (https ://biodi versi tyinf ormat ics.amnh. org/open_sourc e/maxen t/). For modelling, bathymetric, substrate and oceanographic variables were used as predictors of species habitat suitability. Bathymetry (BM) and seabed slope were used as bathymetric variables. BM data were obtained from the PNIA cartographic database (PNIACD; unpublished), which was provided for the managers of PNIA. Slope was derived from the bathymetric layer using the Spatial Analyst tool from ArcMap (ArcGis 10.5). Slope describes the proportion of change in elevation over distance. Low values of slope are associated with flat sea bottoms, while higher values indicate potential rocky bottoms. Sediment Texture (ST) was used as substrate variable and it was introduced in the models to define the sediment substrata. ST data were obtained from PNIA cartographic database. The ST map was constructed according to the Krumbein’s Phi Scale [46], using the following ST classes as a function of the diameter of the particle: very fine sand, fine sand, medium sand, coarse sand, very coarse sand, gravel, cobble and boulder. Waves Exposure (WE) was used as oceanographic variable. WE values were extracted from the Model of Waves of Galicia (Meteogalicia database, www.meteo galic ia.gal). WE describes the annual mean power per meter wave front. Low values of WE are associated with sheltered areas, while higher values suggest high influence to wave force. Data analysis All means are reported with standard deviation. The data were checked for normality and homogeneity of variances (Shapiro–Wilk and Levene’s tests). Analyses of variance (ANOVA) were used to examine the effects of season, sex, reproductive status, length, weight and isotopic values in syngnathids. Tukey’s HSD test adjusted for unequal sample sizes were performed for post hoc comparisons [87]. Statistical analyses were performed using R packages, with significance set at P = 0.05. Diversity, species richness and total number of species were estimated for seaweed in PNIA. Differences between transects and seasons were analysed using PERMANOVA for each univariate variable. P-values were estimated with an asymptotic permutation distribution generated by the Monte Carlo method. PERMANOVA was also used for seaweed assemblage comparisons across transects and seasons using Bray–Curtis pairwise similarities. Patterns in the structure of assemblages were visualized with principal coordinates (PCO) plots of samples and centroids of each combination of Transect × Time in the Bray–Curtis space. Data and statistical analysis were performed with R (Glht and Factoextra packages) and PRIMER-e v6 and PERMANOVA + for PRIMER (Massey University, New Zealand). Supplementary Information The online version contains supplementary material available at https ://doi. org/10.1186/s1286 2‑020‑01743 ‑z. Additional file1: 3D video showing surveyed sites and locations of syngnathid captures in Cíes Archipelago (PNIA). The final sites for further monitoring are also shown. Additional file2: Textural and compositional parameters of sediment (PNIA), general characteristics of macroalgal species and communities (PNIA), haplotypes detected in syngnathids (PNIA and PNAC), PCO Prin‑ cipal coordinates ordination for seaweed assemblages and syngnathids (PNIA), and length–weight relationships in syngnathids (PNIA). Additional file3: Illustrations of main species (medium–high abundance) of macroalgae and bathymetric zonation of seaweed assemblages in transects surveyed on Cíes Archipelago (PNIA). Abbreviations C: Total carbon; GIS: Geographic information system; OAPN: Organismo Autónomo de Parques Nacionales españoles; IUCN: International Union for the Conservation of Nature and Natural Resources; ANOVA: Analysis of variance; N: Total nitrogen; NCBI: National Center for Biotechnology Information; NP: National Park; PCO: Principal coordinates analysis; PERMANOVA: Permutational analysis of variance; PNAC: Cabrera Archipelago National Park; PNIA: Atlantic Islands National Park; SCUBA: Self‑contained underwater breathing apparatus; Page 18 of 20 Planasetal. BMC Ecol Evo (2021) 21:4 SD: Standard deviation; SIA: Stable isotope analysis; SL: Standard length; TR: Transect; UTM: Universal Transverse Mercator system; W: Weight; WORMS: World Register of Marine Species. Acknowledgements We acknowledge I. Ferreiro, D. Costas, R. Chamorro, L. Pereda, L. Royo, I. Zabarte, P. Arechavala, L. Ínsua, J. Pintado, the staff and keepers of PNIA and PNAC, and the Regional Government Xunta de Galicia for support in the project. Special thanks to Ledicia Prieto (University of the Basque Country, Spain) for excellent illustrations on seaweed assemblages in Cíes Archipelago. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). Authors’ contributions MP: Project coordinator, site selection and field sampling, laboratory and data analysis, SIA, manuscript writing and review. CPC: Site selection and field sam‑ pling, macroalgal assemblages, data analysis, writing contribution, manuscript review. CB: Genetic analyses coordinator, data analysis, writing contribution, manuscript review. IC: Field sampling, laboratory and data analysis, SIA, writing contribution, manuscript review. MV: Mitochondrial DNA analyses, data analy‑ sis, writing contribution, manuscript review. MR: GIS mapping, data analysis, supplementary materials (video). VO: Macroalgal assemblages, data analysis. IB: Site selection and field sampling, macroalgal assemblages, data analysis, writing contribution, manuscript review. JT: Site selection, sampling design and field sampling, manuscript review. AC: Sites selection, field sampling. RB: Macroalgal assemblages, data analysis, manuscript review. JHU: Field sampling, data analysis, writing contribution, manuscript review. IA: Sediment analysis, writing contribution, manuscript review. MN: Sediment analysis, writing contribution, manuscript review. MEG: Sites selection, field sampling in PNIA, video. BGP: DNA extraction and sequencing analysis, data analysis, writing contribution, manuscript review. MEG: Sites selection, field sampling, supplementary materials (video). VP: Field sampling, manuscript review. PD: Field sampling, manuscript review. JC: Field sampling design, manuscript review. BMN: Field sampling design, manuscript review. All authors have read and approved the manuscript. Funding Study funded by Proyecto Hippoparques (1541S/2015 and 1580S/2015; Organismo Autónomo de Parques Nacionales—OAPN, Ministerio de Agri‑ cultura, Alimentación y Medio Ambiente, Spain). Project 1580S/2015 was in charge of genetics. Availability of data and materials As we are working on a long‑term project, the datasets used and analysed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Fish capture, handling and sampling were approved by the Regional Govern‑ ment Xunta de Galicia (Reference number: REGA ES360570202001/16/FUN/ BIOL.AN/MPO02), and conducted in compliance with all bioethics standards on animal experimentation of the Spanish Government (R.D. 1201/2005, 10th October) and All the procedures were approved by the Bioethics Commit‑ tee of CSIC. The corresponding author declares that all listed co‑authors agreed to participate in the study and consent to being involved in the publication.Consent for publicationWritten informed consent/permissions for publication were obtained by OAPN for images/maps (Figs. 6 and 7), which will be freely available on the internet and may be seen by the general public. Competing interests The authors declare that they have no competing interests. Author details 1 Department of Ecology and Marine Resources, Instituto de Investigaciones Marinas (IIM‑CSIC), Eduardo Cabello 6, 36208 Vigo, Spain. 2 BioCost Research Group, Facultad de Ciencias and CICA, Universidade da Coruña, 15071 Coruña, Spain. 3 Department of Zoology, Genetics and Physical Anthropology, Facultade de Veterinaria, Universidade de Santiago de Compostela, Campus de Lugo, Avenida Carballo Calero S/N, 27002 Lugo, Spain. 4 Instituto de Acuicultura, Universidade de Santiago de Compostela, Campus Vida s/n, 15782 Santiago de Compostela, Spain. 5 Mediterranean Institute for Advanced Studies (CSIC‑UIB), 07190 Esporles, Spain. 6 Department of Marine Geo‑ sciences and Territorial Planning, Marine Sciences Faculty, University of Vigo, 36310 Vigo, Spain. Received: 8 October 2020 Accepted: 29 December 2020 References 1. Álvarez I, de Castro M, Gómez‑Gesteira M, Prego R. 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