Spanish Marine National Parks: Priority areas for the conservation of a vulnerable family of fishes

Background: Syngnathid fishes (Actinopterygii, Syngnathidae) are flagship species highly associated to seaweed and seagrass habitats of marine ecosystems biodiversity. Seahorses and pipefish are highly vulnerable to anthropogenic and environmental disturbances, but most species are currently Data Defficient by IUCN (IUCN, 2019), requiring more biological and ecological research. This study provides the first insights on syngnathid populations in two Spanish National Parks (PNIA –Atlantic-and PNAC –Mediterranean-). Fish were collected periodically, marked, morphologically identified, analyzed for size, weight, gender and sexual maturity, and sampled for further stable isotope and genetic identification. Due the scarcity of previous information, habitat characteristics were also assessed in PNIA. Results: Syngnathid diversity and abundances 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 NPs differed isotopically, with much lower δ15 N in PNAC. The dominant species were S. abaster in PNAC and particularly S. acus in PNIA. Syngnathids preferred less exposed sites in macroalgal assemblages in PNIA and Cymodocea meadows in PNAC. In S. acus from PNIA, the occurrence of very large specimens, the absence of small-medium sizes and the isotopic comparison with a nearby population suggest that the population is mainly founded by breeders that migrate seasonally. Novel 16S rDNA haplotypes and sequence variants were detected for Hippocampus guttulatus , N. maculatus , S. acus, and S. abaster . Our data suggest the presence of a cryptic Syngnathus 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 potential misidentifications of Syngnathus genus in PNAC, and migratory events in PNIA. We propose several preferential sites in both NPs for future monitoring of syngnathid

and stratified in summer (12-18 °C) due to the warming of upper layers. Surface water temperature typically ranges from 13.4 °C to 18.7 °C in the southern coast and from 13.4 °C to 18.0 °C in the northern coast (Puertos del Estado 2017). 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 seaweeds ranging from kelp forests, fucacean algae to algal turfs (Piñeiro et al. 2014;Fernández et al. 2020). The Western side is exposed to Atlantic open water, and extreme sea currents and waves, particularly in winter. The subtidal zone of that side is dominated by hard substrates covered by crusty, coralline and other turf-forming seaweeds, and fauna such as sponges, bryozoans or ascidians.
The Eastern side is less exposed due to its position facing the Ría de Vigo. The area is also characterized by high species richness and production creating an extraordinary commercial interest for fisheries and aquaculture (Picado et al. 2014). It is promoted by important seasonal phytoplankton blooms (Álvarez-Salgado et al. 2000;Rodil et al. 2009), and secondary production (Valdés et al. 1990; Buttay et al. 2015), with high abundances in summer and changes in community structure throughout the year. Copepods are largely predominant in winter, being accompanied in summer by other groups of fauna (Buttay et al. 2015).
The fishery system in PNIA is complex, comprising 565 boats (mostly artisanal) that use 19 fishing gears in an area of 26.6 km 2 (Ouréns et al. 2015), targeting common octopus, cuttlefish, shrimps, sardine, crabs, sea urchins, clams, razor shells or goose barnacles (Cambie et al. 2012). Some of those gears, especially active fishing ones, have negative impacts on syngnathids (by-catch and substrate degradation). Fishing ground is protected but not currently subject to a special regulation.
The pressure exerted on the resources is unknown and effective regulations cannot be implemented (Ouréns et al. 2015). Tourism has sharply increased in last decade, and nature activities (snorkel or scuba diving) are increasing, promoting the awareness for the conservation of marine ecosystems Strait. Sea surface temperature ranges between 14.6 °C in winter and 27.5 °C in summer . Coastal waters are oligotrophic (0.04-0.08 µmol phosphate l − 1 and 0.25-0.8 µmol nitrate l − 1 ) and light attenuation coefficient is extremely low (0.063 m − 1 ). Depth and hydrodynamics are the dominant abiotic factors that affect habitat distribution and vary among sites throughout the archipelago . The water is oligotrophic (Vives 1993)   The aims of this study were threefold. First, to assess distribution and habitat use of syngnathids in PNIA and PNAC, each with highly distinctive environmental characteristics and vegetal assemblages.
Second, to characterize syngnathid populations, which include the assessment of genetics identification and stable isotopes signals. 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/abundances and temporal-seasonal 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.

Results
Habitat characterization in Cíes Archipelago (PNIA) Soft bottom sediments were mostly coarse sandy (569 µm), with 90% sand, 9% bioclastic gravel and < 1% mud (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. Particle selection ranged between moderate and poor, with a prevalence of sediments with a single mode (65% of samples). Two and three mode samples in TR2, 4 and 3 (these last two near rocky outcrops) reflected a mixture of particle sizes, corresponding with greater content of bioclastic gravel (bivalves and gastropods shells) as well as maerl substrate. Skewness/asymmetry indicated a tendency towards the coarser fractions (bioclastic component).
Different sedimentary environments (wide variability of textural characteristics) were present along some transects. An example was TR3, with sectors exposed to W waves, characterized by coarse sediments and bedforms (megarripples 3D), and others protected by rocky outcrops close to the shoreline. 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 analyzed in PNIA considering data of 55 species with mediumhigh abundance (Additional files 2, 3). Diversity (H') and species richness (S) were particularly low in TR1, 2, 7 and 10, especially in spring (Additional file 2). Seaweed cover increased in summer, particularly 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 both transects (P < 0.05) and seasons (P < 0.01). Those differences were evident in the two-dimensional PCOs plot (Fig. 3). Spring (left) and summer (right) samples followed a gradient along axis 1 (20.4% of total variation). Abundances 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, 8 and 9 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 6 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 abundances (0.06-0.13 syngnathids 100 m − 2 ) were recorded in mixed (sand-rock) or rocky substrates on transects TR3 and 10 (32 and 43% of total specimens, respectively). Syngnathids were missing in the mostly exposed transects TR1, 7, 8 and 9 (northern and southern areas with rocky substrate and coarse sand patches). TR1 was facing SW waves (prevalent component during storm winter conditions), while TR7, 8 and 9 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.

Stable Isotope Signatures In Syngnathids
In PNIA, H. guttulatus and S. acus (Table 3)   were collected in C. nodosa and P. oceanica meadows, suggesting that macroalgal beds are less preferred than seagrass meadows.
In PNAC, the results showed extremely low abundances (Vincent et al. 1995). However, the highest abundances were recorded in C. nodosa meadows in Es Burri Bay (1.

Conclusions
This is the first multidisciplinary approach to the study of syngnathids in Spanish coasts, specifically in two marine NPs. It will contribute to the knowledge of syngnathid populations, leading to more informed and efficient management of both NPs. Species diversity, abundances, habitat preferences, and isotopic signatures differed in both NPs, depending on habitat characteristics. Syngnathids preferred sheltered macroalgal assemblages in PNIA and Cymodocea meadows in PNAC. It is suggested that PNIA is a breeding sanctuary for S. acus, which migrate seasonally. Genetic markers agreed with meristic characteristics, except for S. abaster in PNAC, suggesting the presence of cryptic Syngnathus species, and the need of further analyses to assess potential misidentifications in the genus.
Preferential sites for future monitoring of syngnathid populations in both NPs, some actions to undertake for conservation purposes and further research priorities are proposed. Syngnathids, particularly seahorses, are flagship species attracting the attention of citizens. Efficient further actions will enhance public engagement with marine biodiversity, resulting also in social, economic and wellbeing profits.

Study and swept areas
The study was carried out in ( PNIA is located at the northern limit of the eastern boundary upwelling system off NW Africa and SW Europe. Northerly winds induce coastal upwelling in this region during most of spring and summer (Fraga, 1981) and colder nutrient-rich subsurface water known as Eastern North Atlantic Central The depth, position and habitat type (also substrate and seaweed assemblages in PNIA) were annotated for each fish captured in both study areas. Flora and fauna nomenclature followed codes of Guiry and Guiry (2020) and WoRMS Editorial Board (2020). Swept areas were calculated according to Guerra et al. (2015), considering the effective sampling time, the net sampling distance, the distance between divers and the number of divers who participated in each census.

Fish Collection
In visual census, syngnathids were hand-caught collected or manually extracted from the fishing set capture, introduced in numbered plastic bags and transferred to a support boat. In PNIA, once on land, the fish were morphologically identified, anesthetized with Ethyl 3-aminobenzoate methane sulfonate (MS-222; 0.1 g L − 1 ; Sigma-Aldrich Co., USA) and marked subcutaneously using visible implant fluorescent elastomers (VIFE; Northwest Marine Technology Inc., USA) on the ventral surface of the trunk (pipefish) or laterally (seahorses). All anaesthetized fish were weighted (W, g) and sized for standard length (SL, cm). In PNAC, the fish were morphologically identified on board, anesthetized, sized as reported above but not weighted because it was not possible to stabilize the balance in boat conditions. A fraction of the fish collected by fishing in PNAC were sacrificed for sampling (stable isotopes and genetic analysis) due to their small size (with permission of NP authority).
Dorsal fin samples were taken by fin-clipping (Planas et al. 2008), transferred to screw-capped tubes containing 95% ethanol and conserved at 4 °C for further genetic and stable isotope analysis (SIA).
The presence of previous marks (recapture events), gender, sexual state, meristics (fin rays, body rings) and body coloration were also annotated.  (Spjotvoll and Stoline, 1973). 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 seaweeds in PNIA.
Differences between transects and seasons were analyzed using PERMANOVA for each univariate -Availability of data and material: 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.
-Competing interests: The authors declare that they have no competing interests.

Supplementary Files
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