Detect coastal disturbances and climate change effects in coralligenous community through sentinel stations

RESEARCH ARTICLE Detect coastal disturbances and climate change effects in coralligenous community through sentinel stations Jose´ C. Garcı´a-Go´ mez*☯, Alexandre R. Gonza´lez☯, Manuel J. MaestreID☯, Free Espinosa☯ Laboratorio de Biologı´a Marina de la Universidad de Sevilla (LBMUS)/A´ rea de Investigacio´ n I+D+i del Acuario de Sevilla/Estacio´ n de Biologı´a Marina del Estrecho (Ceuta), Seville, Spain ☯ These authors contributed equally to this work. * jcgarcia@us.es Abstract This study was implemented to assess the Sessile Bioindicators in Permanent Quadrats (SBPQ) underwater environmental alert method. The SBPQ is a non-invasive and low-cost protocol; it uses sessile target species (indicators) to detect environmental alterations (natu- ral or anthropic) at either the local or global (i.e., climate change) scale and the intrusion of invasive species. The SBPQ focuses on the monitoring of preselected sessile and sensitive benthic species associated with rocky coralligenous habitats using permanent quadrats in underwater sentinel stations. The selected target species have been well documented as bioindicators that disappear in the absence of environmental stability. However, whether these species are good indicators of stability or, in contrast, suffer variations in long-term coverage has not been verified. The purpose of this study was to assess the part of the method based on the hypothesis that, over a long temporal series in a highly structured and biodiverse coralligenous assemblage, the cover of sensitive sessile species does not

change over time if the environmental stability characterising the habitat is not altered. Over a ten-year period (2005–2014), the sublittoral sessile biota in the Straits of Gibraltar Natural Park on the southern Iberian Peninsula was monitored at a 28 m-deep underwater sentinel stations. Analyses of the coverages of target indicator species (i.e., Paramuricea clavata and Astroides calycularis) together with other accompanying sessile organisms based on the periodic superimposition of gridded images from horizontal and vertical rocky surfaces allowed us to assess the effectiveness of the method. We conclude that no alterations occurred during the study period; only minimal fluctuations in cover were detected, and the method is reliable for detecting biological changes in ecosystems found in other geographi- cal areas containing the chosen indicator species at similar dominance levels. Introduction The long-term evaluation and quantification of changes in species are crucial for our knowl- edge of various marine ecosystems [1, 2] and constitute useful tools for the environmental PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 1 / 23 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Garcı´a-Go´mez JC, Gonza´lez AR, Maestre MJ, Espinosa F (2020) Detect coastal disturbances and climate change effects in coralligenous community through sentinel stations. PLoS ONE 15(5): e0231641. https://doi.org/10.1371/journal. pone.0231641 Editor: Carlo Nike Bianchi, Universita degli Studi di Genova, ITALY

Received: September 16, 2019 Accepted: March 27, 2020 Published: May 5, 2020 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0231641 Copyright: © 2020 Garcı´a-Go´mez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files.

monitoring, management, and conservation of coastal zones and their associated marine pro- tected areas. Historically, methodological difficulties related to the monitoring and study of subtidal communities have led to their neglect in many environmental monitoring programs [3], par- ticularly in relation to rocky habitats because of their wide heterogeneities, the fixed nature of the resident sessile organisms, the frequent difficulty of achieving access, and the fact that the sampling must be undertaken through indirect methods from boats. Warwick [4] indicated that the study of these communities on wide spatial scales involves a series of technical and implementation difficulties that are not present for work on soft bottoms, the water column, or the intertidal zone. For these reasons, studies related to the environmental monitoring, sur- veillance, and impacts of various factors on the sublittoral benthic environment have focused on endofauna (macroinvertebrates) associated with soft substrates [5–12]. Many biotic indices based on these animals have been proposed [13–15], and many of these indices have been used for or adapted to the requirements of the European Community Water Framework Directive [16–18]. Indeed, regarding monitoring methods based on indicators in the scope of the European Community Directives [19–20], the research has centred on macroalgae on hard substrates [21–28] and seagrasses [29–32], whereas macroinvertebrates associated with high-diversity rocky subtidal habitats (which have greater abundances of sensitive indicator species) have

been less well studied. However, some studies have sought to establish the ecological statuses of coralligenous assemblages [33–39]. The environmental information that can be provided by these assemblages is very powerful and reliable [40–43]; therefore, this faunal approach to rocky habitats could help to fill an important knowledge gap within the European Community Directives [19–20]. Of particular importance is the information that can be provided by pre-coralligenous and coralligenous rocky bottoms because of their high biodiversities and the abundances of sensi- tive colonial species with long life cycles. Although there are some long-term studies of species that are representative of coralligenous communities [44–49] short- and medium-term studies are more common [50,51]. In contrast, growing concern about the effects of climate change has led to more studies of epibenthic (rocky bottom) species in relation to coral bleaching [52–54], seagrass meadows [55], invasive alien species [56,57], and even the role of the marine reserve biota in climate change monitoring [58–60]. These studies have also highlighted the severe environmental impacts on benthic organisms that have arisen from abnormal temperature increase events in the western Mediterranean [61–63]. The great fragility of highly structured and mature benthic communities associated with hard substrates, such as those of pre-coralligenous and coralligenous areas, has encouraged non-invasive study methods based on video footage or photos of species that are fixed to the

substrate in random quadrats [36,51,64–75]. However, the absence of long-term series that have empirically validated these methodologies as systems for detecting changes in communi- ties has been limiting. Additionally, previous studies have focused on characterising and/or monitoring benthic communities, which implies greater complexity with respect to implementation mainly due to the high diversity of species present in these enclaves, which, in turn, entails greater effort and difficulty associated with the identification of the taxa. However, the Sessile Bioindicators in Permanent Quadrats (SBPQ) methodology, which was proposed by one of the authors of the present paper [43], differs from other methodologies, mainly in that the quadrats are perma- nently fixed to the rocky bottom and that the coverage of a previously selected target species must be at least 10% within each quadrat. Fixed quadrats allow changes in the coverage of PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 2 / 23 Funding: This research supported by the Regional Activity Centre for Special Protected Areas (RAC/ SPA) and the Mediterranean Protected Areas Network (MedPAN) projects, Consejerı´a de Medio Ambiente de la Junta de Andalucı´a, and co- financed by Autoridad Portuaria de Sevilla, Compañı´a Española de Petro´leos S.A.U. (CEPSA) Foundation and Red Ele´ctrica de España (REE). Also, research was partially supported by the University of Seville providing salaries to the authors JCG-G and, FE, and Research Foundation

of University of Seville (different projects) providing salaries to ARG and MJM. The specific roles of these authors are articulated in the ‘author contributions’ section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: This study was partly supported by Autoridad Portuaria de Sevilla, Compañı´a Española de Petro´leos S.A.U. (CEPSA) Foundation and Red Ele´ctrica de España (REE). There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

target species and the rest of the benthic community to be detected without the need for a high number of replicates, and with the certainty that these differences are not due to the character- istic spatial heterogeneity of this kind of habitat. The simplicity and relatively low cost of the method allows the monitoring of the stations to be repeated in the short term, which is essen- tial in the detection of invasive species or other anthropogenic disturbances that require rapid action. The method is not intended to assess the biodiversity or ecological status of these com- munities, but to establish an early warning system that allows the detection of changes in the coverage of target species. Based on the detection of these changes, specific studies can be designed to assess the degree and origin of the disturbance. Objectives This study aims to assess the underwater environmental SBPQ alert method, which focuses on monitoring preselected sessile and sensitive benthic species associated with rocky coralligen- ous habitats using permanent quadrats in underwater sentinel stations. The selected target spe- cies have been well documented as bioindicators that disappear in the absence of environmental stability via acute impacts. However, it has not been verified if these species are good indicators of long-term stability. The purpose of the present study was to assess the por- tion of the method based on the hypothesis that, over a long temporal series (a ten-year period in this study) in a highly structured and biodiverse coralligenous assemblage, the cover of sen-

sitive sessile species (i.e., the target species) does not change over time if the environmental sta- bility of the habitat is not altered. Materials and methods Study zone This study was performed on the benthic community of two sentry stations on a rocky bottom. The station is situated within the Strait of Gibraltar Natural Park (southern Spain) inside the Grade A protection zone (i.e., the maximum protection area within the Natural Park) charac- terized by rocky bottoms with a moderate slope that host a high biodiversity and species rich- ness, dominated by species such as Eunicella sp., Paramunicea clavata, Astroides calycularis, Pentapora sp., Crambe crambe and Salmacina sp. in the Punta Carnero locality (Algeciras), but it is proximal to zones under strong anthropic pressure (Fig 1). Despite its enormous ecological wealth and high level of protection, the SBPQ sentry station is at notable risk of anthropogenic disturbance. This is due to both to its proximity to the par- ticularly industrialised Bay of Algeciras, where many different pollution sources can be found (thermal power plant, chemical industry, petrochemicals, bunkering activities, etc.) [76,77] and the high level of marine traffic through the area. Due to the second factor, the last decade has seen polluting events related to hydrocarbon spills of varying magnitudes including serious spills from the ships Sierra Nava and New Flame, both of which occurred in 2007 [78]. Data regarding the bottom water temperature in the study area were obtained from data

base of the regional environmental authorities [79]. The implementation of the study was noti- fied to the managers of the the Strait of Gibraltar Natural Park and to the competent environ- mental authority of Andalusia Government (Regional Ministry of Environment and Territory Planning, CMAYOT). In fact, the CMAYOT has funded the publication of the spanish verison book where the methodology SBPQ is included (43). In adition the first author of the current study is part of the governing board of the Strait of Gibraltar Natural Park. No permits were required for this work. PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 3 / 23

Application of the SBPQ methodology for its validation: Target species, permanent grids, and underwater sentinel stations The SBPQ method was proposed by Garcı´a-Go´mez [43] together with an identification guide for sensitive indicator species vs. tolerant species, and was published by The Regional Activity Centre for Specially Protected Areas (RAC/SPA). The RAC/SPA was established in Tunis in 1985 and is responsible for assessing the status of the natural heritage and helping Mediterra- nean countries to implement the Protocol concerning Specially Protected Areas and Biological Diversity in the Mediterranean (SPA/BD Protocol), which came into force in 1999. This methodology has been designed as a simple, non-invasive, underwater environmental alert tool for the potential early detection of environmental impacts of anthropic origin in the sub- littoral system: in the short term (local alterations derived from pollutants from industries or emis- sions of urban origin, coastal dredging or civil engineering works on the coast, intrusion of exotic Fig 1. Location of the sampling point in the Strait of Gibraltar Natural Park. https://doi.org/10.1371/journal.pone.0231641.g001 PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 4 / 23

species with invasive potential, among other sources of alteration of coastal waters), and in the medium or long term (global warming). Even though the method is able to detect the presence of invasive species, further studies are required to test the reliability of the method for detecting other potential impacts of anthropic origin. Recently, the SBPQ methodology has already been used suc- cessfully in the early detection of exotic species with invasive potential, in particular the invasive seaweed Rugulopterix okamurae [80]. The Fig 2 shows a temporal variation of benthic mean per- cent species coverage at Tarifa Island monitoring by SBPQ method from years 2013 to 2017. It is focused on the management of sensitive indicator target species (benthic and sessile) vs. tolerant species in the Western Mediterranean and can be used in other parts of the world once the native species have been selected. The SBPQ method has been proposed for wide- spread use, not only by scientists, but by sports divers linked to diving clubs or centres that could be involved in underwater environmental monitoring of the coastal environment (Citi- zen Science and “Working with Nature” philosophies). Synthetic adaptation of the SBPQ protocol 1. Choice of vertical walls of pristine rocky bottoms, preferably between 20 and 35 metres deep, biologically structured and of high biodiversity, with the presence of adult-sized target species, sensitive, benthic and sessile indicators, that are visible underwater, preferably colo- nial, with a long life cycle, and abundant compared to the local macrobiota.

2. Selective installation of three to five permanent quadrats of 1x1 m2 (not chosen at random), located on patches of at least one previously selected target species that represents at least 10% of the total coverage per quadrat (Fig 3). The method has recently been updated with the objective of minimizing the degree of intrusion on these fragile habitats. For this pur- pose, a single hole is drilled on the rock and a small plastic bar marks off four 50 x 50 cm detachable monitoring quadrats that are fitted each time monitoring is carried out. This Fig 2. Temporal variation of benthic mean percent species coverage at Tarifa Island monitoring station fixed quadrats from years 2013 to 2017 (1, 2, 3, 4 refer to random sampling times within each year). Species with less than 10% coverage (Alcyonium sp., Aplidium sp., Asparagopsis armata, Crambe sp, Ircinia sp. and Polycitor adriaticum) were grouped under ‘Other species’. Taken from (Garcı´a-Go´mez et al., 2020). https://doi.org/10.1371/journal.pone.0231641.g002 PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 5 / 23

that are representative of the community should allow the appropriate inferences to be drawn. Sampling procedure Four fixed 1-m2 PVC quadrats were installed in two different locations. One setup had a verti- cal orientation (shady), and the other had a horizontal orientation (sunny) at a depth of 28 m Fig 3. Sentinel station showing permanent quadrats of 1x1m located on patches of target species. https://doi.org/10.1371/journal.pone.0231641.g003 PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 6 / 23

(coordinates 36˚ 4,770 N - 05˚ 25,184 W; GPS, DATUM WGS 84). These quadrats were placed on surfaces containing target species. The covers were monitored over ten consecutive years (2005–2014) in spring and summer seasons. A four video footage of 50 x 50 cms were filmed sequentially, covering a total of 1 x 1 m quadrat, in order to gain higher resolution of the subsequent photos taken from the video. A total of 320 photographs were digitally manipulated using the Adobe Photoshop 6.0 (2000) program as follows: four 50x50-cm areas were created for each quadrat (Fig 5A); the contrast and colour saturation were adjusted (Fig 5B); a complete digital frame was created (Fig 5C); and a digital grid that was adjusted to the perimeter of the monitoring area was superimposed (Fig 5D). To correct the angular deformation. To assess the degree of the cover of the target species, a system involving the determination of the presence/absence of cover using the digital grid was applied. This system aided estima- tion accuracy [89,90] and optimised the working time [64,91]. Nevertheless, it should be noted that, in multistratified communities, the system tends to underestimate the cover [64,69,89– 92] because larger species “mask” those that developed underneath them. Statistical analyses Repeated measures analysis of variance (RM-ANOVA) was applied to test whether the mean coverage of the target species significantly varied either through time (intra-subject factor ‘time’; ten levels: 2005 to 2014) or according to orientation (inter-subject factor ‘orientation’;

two levels: horizontal and vertical). Mauchly’s test of sphericity [93] was used to test the assumption that the variances in the differences between all possible pairs of groups were equal. When the sphericity condition was not verified, the F test value was corrected with the Greenhouse-Geisser epsilon index [94,95]. The factors of time and orientation were consid- ered orthogonal and fixed. The same two-factor design was considered to test for any signifi- cant differences in the mean coverages via RM-permutational analysis of variance Fig 4. The updated method with the objective of minimizing the degree of intrusion. A: Hand drill, B: Expansion anchor bolt, C: Drilling and bolt fixing sequence, D: Metal crosshead fixation, E: Sequential allocation of the detachable quadrat, F: Representation of a complete monitoring point. https://doi.org/10.1371/journal.pone.0231641.g004 PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 7 / 23

(RM-PERMANOVA). Univariate analyses were performed using SPSS.13© according to the guidelines of Pardo and Ruiz [96]. For the multivariate analyses, square root transformations were applied to the data, and the analyses were performed using the Bray-Curtis similarity. The Bray-Curtis similarity matrix was used to generate a non-metric multidimensional scaling (nMDS) analysis, and the Kruskal stress coefficient was calculated to test the ordination [97]. The multivariate analyses were performed using the PRIMER6 software (complete with the PERMANOVA+ package) [98–101]. Results The average covers for the five most abundant species (including the two pre-selected sensitive target species A. calycularis and P. clavata) over the period of 2005 to 2014 at the two locations (horizontal and vertical) are presented in Figs 6 and 7. Fig 8 illustrates the evolution of the covers of the sampled indicator species A. calycularis and P. clavata at both locations (horizontal and vertical) together with the time series of the Fig 5. Digital manipulation of the captured images prior to analysis. https://doi.org/10.1371/journal.pone.0231641.g005 PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 8 / 23

background water temperatures in the sampling zones. These series reflected no anomalous increases or decreases in temperature and only exhibited oscillations that were attributable to seasonality. The covers of both species were clearly greater in the vertical orientation than in the horizontal orientation. An increase and a decrease were observed in the covers of A. calycu- laris and P. clavata, respectively, but there were no significant differences. This decrease in the coverage of P. clavata throughout the period studied coincides with that observed in other studies [47]. Environmental factors such as climate change may be affecting certain species more slowly these trends may be analyzed when they have a longer period. RM-ANOVA of the cover of each species in each of the two orientations (Table 1) indicated significant differences (p<0.05) in the covers of Mesophyllum sp. and Salmacina incrustans in the shady (vertical) location over the time period. Regarding the sunny (horizontal) location, the covers of Mesophyllum sp. and Crambe sp. exhibited significant differences (p<0.05). No significant differences (p>0.05) were detected in the covers of the remaining species. The RM-PERMANOVA analysis indicated significant differences in the coralligenous assemblages (p<0.05) for the orientation factor (i.e., vertical vs. horizontal) but not for the time factor. The interaction of these two factors was also not significant (Table 2). The nMDS analysis (Fig 9) revealed a clear differentiation of the values between the vertical and horizontal orientations (Stress: 0.14).

Discussion Sampling procedure and analysis of the submarine images Previous studies have indicated that fixed monitoring stations constitute one of the most robust methods for detecting changes in benthic communities over time [69,102–105]. An area of at least 1 m2 is sufficient for integrating various colonies and individuals of diverse fauna in a single sample, and such diversity is a typical characteristic of benthic rocky bottom communities [69,106]. However, the definitive sampling grid size must be defined by the final Fig 6. Graphic representation of the evolution of the cover of the main species observed at the monitoring stations. H: horizontal; V: vertical. https://doi.org/10.1371/journal.pone.0231641.g006 PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 9 / 23

objective of the study [107]. In our case, the objective was not an exhaustive description of the existing community but rather the evaluation of possible changes in the covers of sessile target species within the community. Similarly, the number of quadrats or replicates used must be defined by the target objectives. In other studies of rocky bottoms or surfaces, three [36,108– 109], five [110], six [111], and eight replicates [112] have been employed. In all of these studies, the replicates had areas of significantly less than 1 m2 with the exceptions of the studies by Fraschetti et al. [109] and Piazzi et al. [36] who also used areas of 1 m2. Image analysis based on a monitoring system comprises a non-invasive method that does not interfere with the natural development or evolution of the studied community [69, 92, 103, 113, Fig 7. Photos of the same replicate taken in 2005 and 2014 for both orientations. https://doi.org/10.1371/journal.pone.0231641.g007 PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 10 / 23

114]. This method also allows for rapid data collection and permanent data record generation. The images were captured using videos rather than photographs mainly due to video’s greater speed and versatility when obtaining data [115–117]. Video was thus time-saving and conse- quently allowed for the optimisation of diving operations, which was very important given the depth at which the study was performed and the number of replicates that were monitored. Choices of high biodiversity habitats, target species, and other companion organisms In general, sublittoral marine habitats are characterised by diversity that increases with depth [66, 118, 119]. Deep communities are dominated by animals, are better structured, and exhibit Fig 8. Graphic representation of the evolution of the cover of the two main indicator species (A. calycularis and P. clavata), together with the bottom water temperature time series. The bars indicate the standard deviation. H: horizontal; V: vertical. https://doi.org/10.1371/journal.pone.0231641.g008 Table 1. Results of one-way repeated measures ANOVA on coverage of each species either in vertical and horizontal substrata. A. calycularis P. clavata Crambe sp. Eunicella sp. Mesophyllum sp. P. fascialis S. incrustans Source of variation df F P� F P� F P� F P� F P� F P� F P� Vertical Time+ 9 1.1592 0.335 1.6472 0.171 0.7932 0.557 0.6321 0.359 3.4742 0.018 2.7352 0.058 2.9312 0.021 Residuals 10 Mauchly’s test of sphericity p<0.001 p<0.001 p<0.05 p = 0.305 p<0.01 - -

Transformation None none none none none none none Horizontal Time+ 9 0.6711 0.277 0.5931 0.446 5.7202 0.002 1.7492 0.157 0.9101 0.006 1.0872 0.365 0.8902 0.426 Residuals 10 Mauchly’s test of sphericity p = 0.490 p = 0.052 p<0.01 p<0.001 p = 0.101 - - Transformation None none none none none none none � Greenhouse-Geisser correction was used when the sphericity condition was not verified. +Time correspond with years from 2005 to 2014. 1 Pillai´s trace. 2 Greenhouse-Geisser F. https://doi.org/10.1371/journal.pone.0231641.t001 PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 11 / 23

less abrupt dynamics and smaller temporal changes than shallow communities or those domi- nated by algae [66]. Additionally, as pointed out by Ballesteros [120], within the scope of marine protected areas (MPAs), the selection of a limited number of representative and/or key species from such communities is a sound strategy for aiding their understanding and management. Specific fixed benthic organisms have previously been used as indicators for the monitoring of various environmental parameters; such species have been used as indicators of global warming and climate change [63, 121–124], sea level fluctuations [125,126], and the influence of recreational diving on MPAs [62, 83, 127,128]. Table 2. Results of repeated measures PERMANOVA. Source df SS MS Pseudo-F P(perm) Unique perms Time 9 2150.5 238.95 2.4353 0.079 998 Orientation 1 30338 30338 309.2 0.001 998 Ti x Or 9 1207.9 134.21 1.3678 0.359 999 Res 60 5887.1 98.118 Total 79 39583 Transformation None https://doi.org/10.1371/journal.pone.0231641.t002 Fig 9. Graphic representation of the non-metric MDS analysis for the whole monitoring quadrats. https://doi.org/10.1371/journal.pone.0231641.g009 PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 12 / 23

Moreover, the use of representative taxa as proxies for entire communities has proven to be a reliable alternative for evaluating the state of rocky bottom communities comprising algae because the loss of information associated with the identification of only some specific taxa does not greatly alter the results compared with results obtained with complete datasets based on entire communities [3]. In hard bottom invertebrate communities, it has also been found that sampling efforts can be reduced without significant losses of information via the selection of indicator and representative species [129]. A. calycularis, P. clavata, and Eunicella sp. (which were clearly more abundant in the moni- tored vertical enclaves) play an important structural role in coralligenous assemblages. These species colonise both horizontal bottoms and vertical walls and offer ideal habitats for numer- ous other organisms [43, 130, 131]. Several previous studies of coralligenous zones in different areas of the Mediterranean, such as the coasts of Italy and France, have determined that the facies of Paramuricea and Eunicella are distinctive [87, 132]. None of these species exhibited significant cover alterations over the entire monitoring period. Although P. clavata and Eunicella sp. have similar biological and ecological characteris- tics, including similar rates of growth and production [133–135], this similarity does not extend to their turnover rates. For P. clavata, this rate oscillates between 7 and 9 years [133,135],

whereas for Eunicella sp., the rate is significantly lower with a range of 3 to 4 years [134]. For this reason, during the long-term temporal monitoring of fixed, limited points, we would expect Eunicella sp. to exhibit greater oscillations in cover percentages than those that we observed due to mortality among the colonies within the monitoring quadrats. Nevertheless, this phenome- non was not recorded in our work, possibly because the magnitude of the studied area allowed for the replacement of the dead organisms by new recruits to buffer the potential differences. Also, it is important to point out that the two main indicator species, A. calycularis and P. clavata (particularly the latter), are sensitive to changes in normal temperature conditions [122, 136–138], although they both withstand seasonal fluctuations well [120]. The differences found in the covers of certain species can also be explained by natural pro- cesses. Mesophyllum alternans is among the organisms responsible for the greatest percentage of cover (particularly in the horizontal orientation), but it is considered to be opportunistic [51], which might explain any oscillations in cover in the absence of any significant alterations to the reference species. The other two species that exhibited significant differences, i.e., Salma- cina incrustans and Crambe sp., are organisms that account for very low percentages of the overall cover. Variations in abundances in deep rocky bottom communities are usually much more noticeable among minor species than among the highly abundant or characteristic spe-

cies [139]. In any case, these two species can be classified as tolerant and relatively resistant to environmental impacts [43]. Recent studies of coralligenous areas have only used vertical enclaves [132], but, despite com- prising the same species, the biological communities that occur at the two orientations are clearly different due to the varying percentages of cover formed by those species. Encrusting algae domi- nate horizontal enclaves, whereas populations of P. clavata and A. calycularis are more abundant on vertical walls; these differences have previously been mentioned by other authors [120, 140]. Therefore, wherever possible, stations should be set up in both orientations, although vertical cor- alligenous enclaves provide more information because they have greater biodiversity, are better structured spatially and trophically, and have greater numbers of sensitive colonial species. Applications of fixed quadrats for the monitoring of rocky bottom habitats The method tested here would be particularly useful for the WFD/MSFD in relation to the environmental control of littoral zones based on biological indicators. The WFD/MSFD has PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 13 / 23

very few methodological tools for monitoring sessile benthic invertebrates associated with hard substrates, although the tools that do exist are excellent [141]. The environmental infor- mation provided by these sessile benthic species is significant because it covers a wide range of sessile organisms that are long-lived and sensitive to changes in the environment. These spe- cies can be used as reliable ecological sentries that keep watch over the quality of littoral envi- ronments because they are not able to flee or be displaced (as adults) when conditions deteriorate or significantly change. The effectiveness of this monitoring method was demonstrated over the 10-year period used to obtain a complete quantitative temporal series. As indicated by Gatti [87,142], such series are very useful for evaluating both reference conditions and environmental changes in ecosystems. Indeed, the main macrobiota species in the sublittoral community in this study were stable and remained in excellent condition from 2005 to 2014. The use of images is advantageous because, unlike the removal of physical samples by scrap- ing, it is neither destructive nor aggressive to the surroundings. The scraping approach is not effective as a monitoring method in zones in which the fauna is distributed in patches because it entails the destruction of relatively large areas [106]. In protected locations and areas of spe- cial interest, this scraping option is particularly undesirable. The present image-based monitoring method is “low cost” in addition to being “low effort”

and requires minimal maintenance (repairs or replacements of the fixed quadrats were neces- sary only twice over the 10-year monitoring period). Panayotidis [22] indicated that the use of simple monitoring programs (i.e., those involving taxonomic efforts that are limited to repre- sentative species and simple statistical treatments) and low budget methods is demonstrably effective for other rocky bottom communities. According to recent studies, the minimum replication area required for biodiversity studies of communities dominated by Paramuricea clavata [143], as well as other hard-substrate ben- thic invertebrates [92, 144,145], is less than the area used in the replicates in our present work. Additionally, the captured images remain available for subsequent, more detailed descriptions of the community. Higher quality images facilitate the visual identification of more of the macrobenthic species that are present in the community, and the system of photo-quadrats is more efficient than in situ visual census methods [92] while simultaneously avoiding the destruction of the monitored areas. As a final summary, Table 3 presents a comparison of the data from the monitoring area of the present study with the data from several other studies of coralligenous bottoms in terms of time and depth. Accounting for both area and time, the present study represents a long-term monitoring of coralligenous assemblages. Table 3. Comparison of the data of monitoring area, time and depth of several studies on coralligenous bottoms. Study Location Area (m2) Depth (m) Time (years)

[139] Peckol P (1984) North Carolina 0.132 20 2 [51] Garrabou J (2000) Medes Islands 1.085 15–19 2 [44] Garrabou J (2002) Marseilles, France 0.4 27 21 [66] Garrabou J (2002) NW Mediterranean 0.372 17–20 2 [146] De Biasi A M (2004) Aegean Sea 33.6 10–35 1 [112] Bussotti S (2006) Southern Italy 13.25 6–8 1 [147] Parravicini V (2009) Ligurian Sea 25 4–5 1 [36] Piazzi L (2017) Ligurian Sea 180 30–40 1 [92] Sant N (2017) Cabrera Archipelago 4.96 0–50 1 Present study 8 27–30 10 https://doi.org/10.1371/journal.pone.0231641.t003 PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 14 / 23

Finally, the proposed methodology could be extended to the entire geographical area over which the target species of this study are representative of the rocky bottom benthic communities that occur at similar depths and share similar biotic structures. This methodology could also be applied in other areas in which target species can be established and selected by employing the aforementioned implementation criteria. Additionally, the SBPQ method involves minimal imple- mentation difficulty both in terms of cost and installation. The a priori selection of the target spe- cies simplifies the taxonomic difficulty of visual identification. Therefore, this environmental warning and surveillance system is not only oriented toward the scientific and technical communi- ties of the relevant coastal countries but also toward environmental volunteers associated with div- ing centres and clubs. This methodology ultimately entails the aim of setting up geographical networks in regions with sufficiently high levels of homogeneity (e.g., the Alboran Sea or, on a larger scale, the western Mediterranean). Citizen science initiatives focusing on the mapping and monitoring of coralligenous assemblages have recently been implemented [148]. In the future, these networks could provide early warnings of changes to structured and unpolluted systems, par- ticularly those in littoral zones close to places subjected to strong anthropogenic pressure, which could be applied to the detection of local or general environmental alterations (i.e., climate change).

In contrast, the nature of the method, which is designed to monitor a group of taxa with respect to the total taxa of the community, implies that it is not useful as such in the ecological study of these habitats because the analysed information is biased. However, the generated databases (i.e., stored photographs) will allow for the performance of in-depth studies of the dynamics of the species within the communities over time, assuming that significant changes can be detected, and could aid parallel projects that aim to increase our knowledge of coralligenous assemblages. Conclusions The study assessed the Sessile Bioindicators in Permanent Quadrats (SBPQ) underwater environ- mental alert method in relation to the monitoring of pre-selected sensitive and sessile benthic spe- cies (P. clavata and A. calycularis) associated with rocky coralligenous habitats (which exhibit high stability and biodiversity) via the use of permanent quadrats fixed on rocky shores in under- water SBPQ sentinel stations. The obtained results have allowed for assessing the part of the method based on the initial hypothesis that, over a long temporal duration (a ten-year period in this study) and in a highly structured and biodiverse coralligenous assemblage, the cover of sensi- tive sessile species does not change over time if the environmental stability characterising the habi- tat is not altered. This stability of the cover of the sensitive sessile species is a key aspect for confirming the reliability and robustness of the SBPQ method. Given that, as in the present study,

the selected species are very sensitive to increases in temperature and deterioration of the environ- mental quality of the water column, the SBQP method is useful as an underwater environmental alert system because it should be solely sensitive to changes in the coverages of such species that result from physico-chemical changes in the system. Such changes include gradual increases in temperature due to global warming and changes due to the introduction of exotic species. A future usefulness would be to implement the SBPQ methodology in well-conserved areas so those areas can act as “SBPQ sentinel stations” in the event of possible disturbances. The method has been developed as a simple management tool for use by scientists and specialised technicians in addition to diving clubs that frequent certain areas. Supporting information S1 Table. Coverage of each species through the monitoring period. Time: 1 corresponding to 2005 and 10 to 2014. Slope: H, horizontal; V, vertical. (XLS) PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 15 / 23

Acknowledgments We want to warmly thank the Campo de Gibraltar, Centro de Investigaciones y Exploraciones Submarinas CIES Algeciras and CIES Tarifa Diving Centers, for their joint participation with the authors in a general "Citizen Science" program related to underwater sentinel stations. Also, we express our gratitude for the collaboration provided to the Seville Aquarium, Town Halls of Tarifa and La Lı´nea as well as to Puerto Deportivo La Alcaidesa, Club Marı´timo y Real Club Na´utico (RCN) de La Lı´nea. Finally, we would like to mention Academic Editor PLOS ONE and the reviewers of this paper for their valuable recommendations, which improved the contents of this work. Author Contributions Conceptualization: Jose´ C. Garcı´a-Go´mez. Data curation: Alexandre R. Gonza´lez. Formal analysis: Jose´ C. Garcı´a-Go´mez, Alexandre R. Gonza´lez. Funding acquisition: Jose´ C. Garcı´a-Go´mez. Investigation: Jose´ C. Garcı´a-Go´mez, Alexandre R. Gonza´lez, Manuel J. Maestre, Free Espinosa. Methodology: Jose´ C. Garcı´a-Go´mez. Project administration: Jose´ C. Garcı´a-Go´mez. Resources: Jose´ C. Garcı´a-Go´mez. Software: Alexandre R. Gonza´lez. Supervision: Jose´ C. Garcı´a-Go´mez. Validation: Jose´ C. Garcı´a-Go´mez, Alexandre R. Gonza´lez, Manuel J. Maestre, Free Espinosa. Visualization: Jose´ C. Garcı´a-Go´mez, Alexandre R. Gonza´lez. Writing – original draft: Jose´ C. Garcı´a-Go´mez, Alexandre R. Gonza´lez. Writing – review & editing: Jose´ C. Garcı´a-Go´mez, Alexandre R. Gonza´lez, Manuel J. Maes- tre, Free Espinosa. References

1. Gatti G, Bianchi CN, Morri C, Montefalcone M, Sartoretto S. Coralligenous reefs state along anthro- pized coasts: Application and validation of the COARSE index, based on a rapid visual assessment (RVA) approach. Ecol Indic. 2015; 52: 567–576. 2. Gatti G., Bianchi C. N., Montefalcone M., Venturini S., Diviacco G. Morri C. Observational information on a temperate reef community helps understanding the marine climate and ecosystem shift of the 1980–90s. Mar Pollut Bull. 2017; 114: 528–538. https://doi.org/10.1016/j.marpolbul.2016.10.022 PMID: 27743657 3. Puente A, Juanes J A. Testing taxonomic resolution, data transformation and selection of species for monitoring macroalgae communities. Estuar Coast Shelf Sci. 2008; 78: 327–340. 4. Warwick R M. Environmental impact studies on marine communities: pragmatical considerations. Aus- tralian Journal of Ecology. 1993; 18: 63–80. 5. Pearson T H, Rosenberg R. Macrobenthic succession in relation to organic enrichment and pollution of the marine environment. Oceanography and Marine Biology: An Annual Review. 1978; 16: 229– 311. 6. Rygg B. Distribution of species along pollution-induced diversity gradients in benthic communities in Norwegian fjords. Mar Pollut Bull. 1985; 16: 469–474. PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 16 / 23

7. Ros J D, Cardell M J. Effect on benthic communities of a major input of organic matter and other pollut- ants (coast off Barcelona, western Mediterranean). Environ Toxicol Chem. 1991; 31–32: 441–450. 8. Dauer D M. Biological criteria, environmental health and estuarine macrobenthic community structure. Mar Pollut Bull. 1993; 26: 249–257. 9. Estacio F, Garcı´a-Adiego E, Fa D, Garcı´a-Go´mez J C, Daza J L, Hortas F, et al. Ecological analysis in a polluted area of Algeciras Bay (Southern Spain): external vs. internal outfalls and environmental implications. Mar Pollut Bull. 1997; 34–10: 780–793. 10. Estacio F, Garcı´a-Adiego E M, Carballo J L, Sa´ nchez-Moyano J E, Garcı´a-Go´ mez J C. Interpreting temporal disturbances in an estuarine benthic community under combined anthropogenic and climatic effects. J Coast Res. 1999; 15–1: 155–167. 11. Sa´ nchez-Moyano J E, Garcı´a-Adiego E M, Estacio F. Garcı´a-Go´ mez J C. Effects of environmental factors on the spatial variation of the epifaunal polychaetes of the algae Halopteris scoparia in Algeci- ras Bay (Strait of Gibraltar). Hydrobiologia. 2002; 470: 133–148. 12. Guerra-Garcı´a J M, Garcı´a-Go´mez J C. Crustacean assemblages and sediment pollution in an excep- tional case study: a harbour with two opposing entrances. Crustaceana. 2004; 77–3: 353–370. 13. Majeed S A. Organic matter and biotic indices on the beaches of North Britanny. Mar Pollut Bull. 1987; 18: 490–495. 14. Grall J, Gle´ marec M. Using biotic indices to estimate macrobenthic community perturbations in the

Bay of Brest. Estuar Coast Shelf Sci. 1997; 44: 43–53. 15. de Juan S, Demestre M. A Trawl Disturbance Indicator to quantify large scale fishing impact on benthic ecosystems. Ecol Indic 2011; 18:183–190. 16. Borja A, Franco J, Pe´ rez V. A marine biotic index to establish the ecological quality of soft-bottom ben- thos within European estuarine and coastal environments. Mar Pollut Bull. 2000; 40: 1100–1114. 17. Simboura N, Zenetos A. Benthic indicators to use in ecological quality classification of Mediterranean soft bottom marine ecosystems including a new biotic index. Mediterr Mar Sci. 2002; 3: 77–111. 18. Rosenberg R, Blomqvist M, Nilsson H C, Cederwall H, Dimming A. Marine quality assessment by use of benthic species abundance distributions: a proposed new protocol within the European Union Water Framework Directive. Mar Pollut Bull. 2004; 49: 728–739. https://doi.org/10.1016/j.marpolbul.2004. 05.013 PMID: 15530516 19. EC. Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 estab- lishing a framework for Community action in the field of water policy. Official Journal of the European Union. 22/12/2000; L 327: 0001–0073. 20. EC. Directive 2008/56/EC of the European Parliament and of the Council of 17 June 2008 establishing a framework for community action in the field of marine environmental policy (Marine Strategy Frame- work Directive). Official Journal of the European Union. 25.6.2008; L 164: 19–40. 21. Orfanidis S, Panayotidis P, Stamatis N. Ecological evaluation of transitional and coastal waters: a

marine benthic macrophytes-based model. Mediterr Mar Sci. 2001; 2: 45–65. 22. Panayotidis P, Montesanto B, Orfanidis S. Use of low-budget monitoring of macroalgae to implement the European Water Framework Directive. Journal of Applied Phycology. 2004; 16: 49–59. 23. Ballesteros E, Torras X, Pinedo S, Garcı´a M, Mangialajo L, Torres M. A new methodology based on lit- toral community cartography dominated by macroalgae for the implementation of the European Water Framework Directive. Mar Pollut Bull. 2007; 55: 172–180. https://doi.org/10.1016/j.marpolbul.2006. 08.038 PMID: 17045303 24. Wells E, Wilkinson M, Wood P, Scanlan C. The use of macroalgal species richness and composition on intertidal rocky seashores in the assessment of ecological quality under the European Water Framework Directive. Mar Pollut Bull. 2007; 55: 151–161. https://doi.org/10.1016/j.marpolbul.2006. 08.031 PMID: 17074370 25. Juanes J A, Guinda X, Puente A, Revilla J A. Macroalgae, a suitable indicator of the ecological status of coastal rocky communities in the NE Atlantic. Ecol Indic. 2008; 8: 351–359. 26. Gaspar R, Pereira L, Neto J M. Ecological reference conditions and quality states of marine macroal- gae sensu Water Framework Directive: An example from the intertidal rocky shores of the Portuguese coastal waters. Ecol Indic. 2012; 19: 24–38. 27. Guinda X, Juanes J A, Puente A, Echavarri-Erasun B. Spatial distribution pattern analysis of subti- dal macroalgae assemblages by a non-destructive rapid assessment method. J Sea Res. 2012; 67: 34–43.

28. Neto J M, Gaspar R, Pereira L, Marques J C. Marine Macroalgae Assessment Tool (MarMAT) for inter- tidal rocky shores. Quality assessment under the scope of the European Water Framework Directive. Ecol Indic. 2012; 19: 39–47. PLOS ONE Detect coastal disturbances in coralligenous PLOS ONE | https://doi.org/10.1371/journal.pone.0231641 May 5, 2020 17 / 23

29. Krause-Jensen D, Greve T M, Nielsen K. Eelgrass as a bioindicator under the European Water Frame- work Directive. Water Resources Management. 2005; 19: 63–75. 30. Foden J. Assessment metrics for littoral seagrass under the European Water Framework Directive; outcomes of UK intercalibration with the Netherlands. Hydrobiologia. 2007; 579: 187–197. 31. Foden J, Brazier D P. Angiosperms (seagrass) within the EU Water Framework Directive: A UK per- spective. Mar Pollut Bull. 2007; 55: 181–195. https://doi.org/10.1016/j.marpolbul.2006.08.021 PMID: 17027036 32. Gobert S, Sartoretto S, Rico-Raimondino V, Andral B, Chery A, Lejeune P, Boissery P. Assessment of the ecological status of Mediterranean French coastal waters as required by the Water Framework Directive using the Posidonia oceanica Rapid Easy Index: PREI. Mar Pollut Bull 2009; 58(11): 1727– 1733. https://doi.org/10.1016/j.marpolbul.2009.06.012 PMID: 19700176 33. Deter J, Descamp P, Ballesta L, Boissery P, Holon F. A preliminary study toward an index based on coralligenous assemblages for the ecological status assessment of Mediterranean French coastal waters. Ecol Indic. 2012; 20: 345–352. 34. Canovas-Molina A, Montefalcone M, Bavestrello G, Cau A, Bianchi C N, Morri C, et al. A new ecologi- cal index for the status of mesophotic megabenthic assemblages in the Mediterranean based on ROV photography and video footage. Cont Shelf Res. 2016; 121: 13–20. 35. Ferrigno F, Russo G F, Sandulli R. Coralligenous Bioconstructions Quality Index (CBQI): a synthetic indi-