Multidisciplinary Analysis of Cystoseira sensu lato (SE Spain) Suggest a Complex Colonization of the Mediterranean

: Cystoseira sensu lato (sl) are three genera widely recognized as bioindicators for their restricted habitat in a sub-coastal zone with low tolerance to pollution. Their ecological, morphological and taxonomic features are still little known due to their singular characteristics. We studied seven species of Cystoseira sl spp. in Cabo de las Huertas (Alicante, SE Spain) and analyzed their distribution using Permutational Analysis of Variance (PERMANOVA) and Principal Component Ordination plots (PCO). A morphological cladogram has been constructed using ﬁfteen phenotypic taxonomic relevant characters. We have also developed an optimized Cystoseira sl DNA extraction protocol. We have tested it to obtain amplicons from mt23S, tRNA-Lys and psbA genes. With these sequence data, we have built a phylogenetic supertree avoiding threatened Cystoseira sl species. Cartography and distribution analysis show that the response to hydrodynamism predicts perennial or seasonal behaviors. Morphological cladogram detects inter-speciﬁcal variability between our species and reference studies. Our DNA phylogenetic tree supports actual classiﬁcation, including for the ﬁrst-time Treptacantha sauvageauana and Treptacantha algeriensis species. These data support a complex distribution and speciation of Cystoseira sl spp. in the Mediterranean, perhaps involving Atlantic clades. The high ecological value of our area of study merits a future protection status as a Special Conservation Area.


Introduction
The family Sargassaceae Kützing (Phaeophyceae) inhabits all oceans, from polar waters to the warmest tropical seas [1]. The Cystoseira sensu lato (sl) species have their maximum diversity in the Mediterranean Sea, with two thirds of all species described found there [2][3][4]. Some of them have habitats as reduced as coastal platforms [5]. Outside the Mediterranean Sea, they are mainly found in the Northeast Atlantic. This may explain why they were reintroduced six million years ago into the Mediterranean Sea. During the Zanclean deluge, after the Messinian desiccation crisis, they may have been dragged by the currents [2,6,7], starting a new colonization process. Recent research corroborates the polyphyletic nature of these algae, distinguishing three genera [8][9][10]. They comprise Cystoseira sensu stricto, Carpodesmia and Treptacantha.
As an engineering species they form dense meadows on rocky substrates up to 100 m deep [2,5,11,12]. They are widely recognized as bioindicators for their restricted habitat in the The lowest value (1) corresponds to isolated individuals. Several individuals forming no patches score 2. For isolated patches, the abundance value is 3. For patches forming a discontinuous horizon, the value is 4. A continuous horizon of the same species of Cystoseira sl scores 5. Sixty-one linear transects (30-80m) were performed along the coastline of study (2018, May-July), recording the abundance of Cystoseira sl communities per horizon.
Trails were georeferenced using GPSies + (Klaus Bechtold, ©2017) and distribution maps drawn using QGIS v 2.18 with the WGS84 (EPSG:4326) coordinate system. An orthophoto of the region provided by the National Plan for Aerial Orthophotography (PNOA) of the National Geographic Institute [43] was used as model to draw a map according to the real geography.

Abiotic Factors and Spatial Variability of Cystoseira Spp.
Platform width, wave exposure and the sublittoral horizons were recorded as the main hydrodynamism variables (Table S1). Geomorphological characteristics of the substrate (lithology, slope, presence of pools or rifts) were also scored. Records have been analysed with Primer software (v.6.1) [44]. A three fixed factor PERMANOVA analysis has also been carried out with the Bray Curtis similarity matrix to study the influence of the main variables related to hydrodynamism on the abundance of Cystoseira sl species. A reduced model with 4999 permutations has been chosen. The distribution patterns of the samples have been analysed with a Principal Component Ordination Analysis (PCO), built with the same Bray-Curtis matrix.

Morphological Characterization
Specimens were collected in spring (2018) using chisel and hammer, avoiding damaging the basal structure and kept in 10% alcohol in seawater until analysis. A qualitative matrix using Primer 7 software [45] with 15 phenotypic characters of 37 Cystoseira sl was constructed (Table S2) [9] were recorded as reference algal groups. A cladogram was constructed using a dissimilarity Bray-Curtis matrix with the 'simple matching' method and a SIMPROF test carried out to distinguish statistical differences between individuals, adding a 'dummy variable' to improve the robustness of the cladogram.

DNA Extraction
Individuals collected were kept in cold seawater. They were cleaned of epiphytes and lyophilized in less than 24 h. Fine lyophilized thallus ground in liquid nitrogen was used for DNA extraction. DNA extractions were performed using a protocol based on the pre-treatment of Lane et al. (2006) [47] in combination with the cleaning steps of Rogers and Bendich (1989) [48]. All treatments were performed on ice to avoid DNA degradation. Lyophilized thallus is added to Buffer A (1.65 M sorbitol, 50 mM MES (sulphonic acid) pH 6.1, 10 mM EDTA, 2% (w/v) PVP-40, 0.1% (w/v) BSA (Bovine Serum Albumin) and 5 mM ß-mercaptoethanol) while stirring for 2-3 min. The mixture is filtered with Miracloth ® and centrifuged at 3000× g for 2 min. Buffer A is removed, the pellet is resuspended in Buffer B (Buffer A without PVP) and centrifuged at 3000× g for 2 min. Buffer B is discarded, and the pellet is resuspended in DNA extraction buffer [48]. Samples were incubated on ice for 1 h then centrifugated at 15,000× g for 10 min. Aqueous phase is transferred into new tubes for phenol, chloroform and isoamyl alcohol extraction for 5 min and then centrifugation. This step was performed again without phenol for 2 min. DNA is treated with −20 • C isopropyl alcohol on ice for 20 min. After centrifugation for 10 min, the supernatant is discarded and the pellet washed with ethanol at −20 • C. Pellets containing DNA are resuspended in nuclease free water and stored at −80 • C.

Gene Amplification, Sequencing and Phylogenetic Analysis
Six sets of primers from previous work were used (Table S3). Some of them (psbA-FR2 and psbA-F2R) can be used in three different combinations [2]. PCR reactions included a preheating stage of 2 min at 92 • C, nine cycles of 30 s at 94 • C, 1 min at 45 • C with a final extension of 1 min at 72 • C plus 29 cycles of 30 s at 94 • C, 30 s at 50 • C with a final extension of 1 min at 72 • C. With the psbA-FR primer, final extensions were increased from 1 to 2 min. PCR reactions were repeated when required to supply enough DNA for Sanger sequencing. Amplicons were run in 2% agarose gels for quality testing, then treated with a DNA purification kit (Qiagen). Sequencing was carried out by Macrogen Inc. (Korea). Consensus sequences were determined using Bioinformatics 'Reverse complement' [49] and Omega Cluster tools [50]. A sequence database has been set up based on previous studies [2,9]. Phylogeny analyses were performed using Mega X software [51], through a Muscle type alignment [52]. Both supertree and isolate trees for individual genes (mt23S, tRNA-Lys and psbA) have been built using the 'Neighbor-Joining' method [53,54] and phylogenetic test 'Bootstrap' with 1500 interactions [55]. All taxa used in the phylogenetic analyses are listed in Supplementary Table S4

Cartography of Communities Associated with the Coastal Fringe
Cystoseira sl species were found over more than 4 km of the 14 km sampled (Figure 1a,b), in nearly uninterrupted communities at Cabo de las Huertas (Cape area) and separate individuals or patches on La Almadraba beach, Paseo Marítimo and La Playita bay ( Figure 1c). Cystoseira compressa, C. humilis, C. foeniculacea, Carpodesmia amentacea var. stricta, C. brachycarpa var. balearica, Treptacantha algeriensis and T. sauvageauana were the species found in the area of study.

Distribution of Cystoseira Sensu Lato
Cystoseira sl communities are frequent in the area of study, in rocky shores with high hydrodynamism (Figure 2a). They usually disappear abruptly when the rock substrate changes from platform to boulders (Figure 2b). Cystoseira sl communities on narrow platforms and medium wave exposure are different from those under low or high wave exposure ( Tables 1 and 2 and S5). In wide platforms, the degree of wave exposure conditions Cystoseira sl communities. For instance, Treptacantha sauvageauana likes low wave exposure because it is the third most abundant species under these conditions. On the contrary, other species such as Carpodesmia amentacea var. stricta, Treptacantha algeriensis or Cystoseira compressa prefer large wave exposure. The abundance of Cystoseira sl species depends on their location at the platform, since populations from proximal and distal horizons also differ significantly. A PCO of Cystoseira sl abundance with environmental factors was analysed using 183 samples from 61 transects recorded ( Figure 3). Axes explain 79.9% of the total variability and vectors with high correlation are represented (cor > 0.2). Main hydrodynamism variables vectors are located to the lower right quadrant indicating the direction of higher values of hydrodynamism. Two geomorphologic variables have close correlation with hydrodynamism, 'Inclination' negatively and 'Rifts' positively. Canopy height is also directly linked. The lowest heights of Cystoseira sl have been located in La Calita, which is a cove with high anthropogenic impact ( Figure S1). Thus, Cystoseira sl general abundance cannot be explained by a single environmental factor. When Cystoseira sl individual species are considered, different patterns of distribution appear (Figure 4). For instance, C. compressa has a wide distribution, since this species is the most environmentally tolerant of all found. With the largest ecological range, it is the most abundant and is nearly homogeneously distributed in all horizons of El Cabo de las Huertas (Figure 4a). C. amentacea var. stricta and T. algeriensis are distributed positively with vectors, therefore to hydrodynamism. They have a slightly smaller ecological range, with irregular distribution (Figure 4b,c). Meanwhile, C. humilis, T. sauvageauana and C. foeniculacea have less correlation with hydrodynamism, with reduced distribution usually located at proximal horizon (low wave exposure) (Figure 4d-f). The proximal horizon has a discontinuous presence of C. compressa, C. amentacea var. stricta, T. algeriensis, T. sauvageauana and C. humilis while more exposed horizons have a continuous community of these species ( Figure S2).

Morphological Analysis
Our morphological cladogram includes eight clades (Cystoseira I-VIII) ( Figure 5). Cystoseira VIII is the clade with the least similarity, followed by Cystoseira VII. From Cystoseira VI to I, branches can be differentiated with increasing similarity values. Similarities of more than 85% prevent species assignments. Most of our samples lay close to their counterparts included in previous reference studies [9,45,46]. However, several species (T. sauvageauana, C. foeniculacea, C. compressa) display more variability within a given clade or are located in different clades.

Phylogenetic Analysis
Our DNA protocol completely removed Cystoseira sl secondary metabolites and allowed template amplification with all primers used ( Figures S3-S6). In this study, 30 Cystoseira sl DNA samples have been sequenced, corresponding to 3 genes. We have built a concatenated tree (supertree) with a total of 98 Fucaceae sequences from 41 species; 22 of these are Cystoseira sl species (Figure 6). The supertree is more robust (higher node stats values) than those trees built using single gene sequences (Figures S7-S9).
Three clades (I-III) have been obtained. Cystoseira-I comprises C. foeniculacea, in a separate subclade, C. compressa and C. humilis together in a further subclade. Cystoseira-II includes four differentiated branches with Treptacantha spp. species (T. abies-marina, T. baccata, T. usneoides, T. susanensis and T. barbata). The latter two species clustered in a separate subclade. Cystoseira-II includes sequences of T. sauvageauana and T. algeriensis for the first time. Both sequences appear separated from the rest. Cystoseira-III includes C. amentacea, C. brachycarpa, C. crinita, C. funkii, C. mediterranea, C. tamariscifolia, and C. zosteroides, this last species as an outlier. Within clade III, C. crinita and C. brachycarpa can be clearly differentiated from the rest. Cystoseira-I and Cystoseira-II appear closer phylogenetically, unlike Cystoseira-III.

Discussion
Hydrodynamism plays a key role in the Cystoseira sl population distribution from the SE Spanish coast. This has also been found in previous works [13,31,34,56]. In our study, Cystoseira sl, in general terms, is most abundant and widely distributed in zones with large exposure to waves. For instance, C. amentacea var. stricta, T. algeriensis and C. compressa, with large productive potential, require light saturation [57] and high hydrodynamism [13,27,31,37,58,59]. Consequently, they are frequently found in rifts and other exposed places. There are seasonal species which have their optimal development during spring [13]. The widespread distribution of C. compressa may reflect its high ecological tolerance, even to anthropogenic impact. This is perhaps the reason why it is the only Cystoseira sl species found in urban areas such as La Almadraba beach [13,19,60]. Other species (e.g., T. sauvageauana, C. foeniculacea) are distributed preferentially in more sheltered environments [61,62] where hydrodynamism is less relevant. They have a perennial behavior, maintaining their thalluses all year [30,63]. Although C. brachycarpa inhabits sheltered environments [45,64], in our study it was found in an abrasion platform. The orientation on the coast, facing south, receives less impact from the dominant eastern waves [65] allowing C. brachycarpa to survive under these conditions. A coastline slope does not favor the presence of Cystoseira sl populations, as found in the Thyrrenian islands [66]. Slope degree has a close inverse relationship with those populations, since above 60 • can highly reduce the probability of settlement of these communities. Human trampling is to be considered because it takes place on coastal platforms during the summer season. Those coves with a high tourist influx have lower values of abundance and canopy of Cystoseira sl, indicating that they are very sensitive to this anthropogenic disturbance. Even low intensities of trampling can highly affect the spatial distribution of algal communities [60,67], causing the simplification of the platform communities [12]. Therefore, future efforts could be applied studying this relation in order to apply management plans for Cystoseira sl protection.
Recent studies show that natural hybridization is operating in seaweed forests of Cystoseira sl [2,8,16]. The high variability of T. sauvageauana, C. foeniculacea, C. compressa in our morphological cladogram would agree with this. Each Cystoseira sl gender has morphological characters with various phenotypes [9], usually one of them more common than the rest. So, perhaps species that exhibit uncommon morphotypes could not be classified appropriately in the cladogram. Numerical taxonomy techniques are useful to tackle this intraspecific variability. For instance, the lack of spines in secondary and higher order branches of C. crinita would lead the transfer of this species from Cystoseira-III into Cystoseira-I, in which all species are characterized by the absence of this type of appendages. The latest studies indicate that there are many exceptions to this character, reinforcing the need to combine morphological with phylogenetical studies [9]. Like any other method of morphological identification, it needs to rely on molecular taxonomy to clarify the classification. Therefore, morphological data has been completed with phylogenetic studies [2,10].
Our molecular supertree of sequences of psbA, mt23S and tRNA-Lys spacer gene from Cystoseira sl from SE of Spain mostly agrees with previous studies [2,9,10]. In agreement with Orellana et al. [9], our clades Cystoseira-II (Treptacantha) and Cystoseira-I (Cystoseira sensu stricto) are closer phylogenetically and with other genera such as Polycladia, Stephanocystys, Sargassum and Sirophysalis, and far from Cystoseira-III (Carpodesmia). This supports the lack of a common ancestor of these genera [2,9,10]. Cystoseira-III (Carpodesmia) is the most chemically complex genus [68,69] and most members are Mediterranean [2,70], suggesting that the seasonality of this sea [71] may be driving a pressure on these algae enhancing the production of their metabolites to deal with stressors such as UV and temperature [56]. Neither Bruno de Sousa et al. [9] nor this study have succeeded in the resolution on C. tamariscifolia/C. amentacea clade. Therefore, future studies should be aimed to resolve these phylogenetic relationships. We have generated for the first-time sequence data from T. algeriensis and T. sauvageauana which support that both species are well differentiated and separated from the rest. This would also agree with their different morphology and ecological preferences [45,46]. The challenge of understanding their taxonomy may reside in the recent and continuous speciation of these genera in Mediterranean Sea [2,61,72], their adaptative convergence or possible genetic constraints [10]. There is an urgent need to keep investigating to understand their evolution and ecology in order to enhance their protection and maintain conservation efforts that help to preserve marine ecosystems for future generations.
This study provides the first bionomic cartography and evaluation of conservation of Cystoseira sl populations in SE Spain. Out of the 14 Cystoseira sl species cited in this area [73], half have been found in this study in a coastal fringe of just 4 km. In view of the diversity and abundance found, this region displays a relevant ecological quality [74]. The relative continuous algal canopy would also imply a good water status, as reported [75]. Therefore, because of its environmental characteristics and high algal diversity in the shores, this place should be a Special Conservation Area (SCA), integrated in the Natura 2000 Network (EU Directive 92/43/EEC). The inclusion of Cystoseira, Treptacantha and Carpodesmia genera in El Cabo de las Huertas Community Importance Site would also be a great achievement for the conservation of marine natural habitats of the Spanish Levant coast. The site may be useful for future extraction of specimens for transplants to former habitats which have undergone local extinctions [23]. There is also a pressing need to develop a management plan in view of the increasing disappearance of the prairies of eastern Mediterranean Fucaceae [19][20][21][22]. Long-term monitoring of these biocenosis would be desirable, which could be carried out using the CARLIT methodology, that employs Cystoseira sl populations as a key species establishing the health of the water body [13]. Cystoseira sl evolution data would generate time series to know the state of these communities, thus evaluating the health of our coast. This type of study is already being carried out in different areas of the Mediterranean Sea [20,31,59,76].

Conclusions
The bionomic cartography of Cystoseira sl assesses the good ecological quality and good water body status of El Cabo de las Huertas. Distribution analysis shows two levels of relation to hydrodynamism which predicts perennial or seasonal behaviors. Morphological cladogram detects inter-specifical variability between our species and previous reference studies of these genera. Our phylogenetic tree supports actual classification. Previously unpublished sequences of psbA, mt23S and tRNA-Lys of T. algeriensis and T. sauvageauana confirm those are included in Bruno de Sousa's Cystoseira II group (2019), in agreement with morphological studies.
Supplementary Materials: The following are available online at: http://www.mdpi.com/2077-1312/8/12/961/s1. Table S1: Geomorphological characteristics of the substrate and its levels selected to PERMANOVA (*) and Principal Component Analysis (PCO) analyses. Table S2: List of qualitative phenotypical characters used for the building of Bray-Curtis dissimilarity matrix. Table S3: List of primers used for phylogenetic analysis. Table S4: GenBank accession numbers used in this study. Table S5: Abundance of Cystoseira sl dominant species attending on Wave exposure, Width of the platform and Horizons of the platform. Percentage of contribution of each species to the dissimilarity. Figure S1: Canopy height of Cystoseira sl recorded in the area of study. Figure Figure S7: Maximum likelihood phylogenetic tree obtained with Nearest Neighbour Interaction on mt23S sequences of samples from Sargassaceae family. Values of branches represent maximum likelihood bootstrap support values (>50%). Cystoseira sl samples sequenced in this study labelled with 1 or 2. Figure S8: Maximum likelihood phylogenetic tree obtained with Nearest Neighbour Interaction on tRNA-Lys spacer sequences of samples from Sargassaceae family. Values of branches represent maximum likelihood bootstrap support values (>50%). Samples sequenced in this study labelled with 1. Figure S9: Maximum likelihood phylogenetic tree obtained with Nearest Neighbour Interaction on psbA sequences of samples from Sargassaceae family. Values of branches represent maximum likelihood bootstrap support values (>50%). Samples sequenced in this study labelled with 1, 2 or 3.