Crust Biometry of Hydrolithon Boreale (Rhodophyta: Hydrolithaceae) on Leave of Posidonia Oceanica Along a Mediterranean Coast Fed by Siliciclastic-Carbonate

A study was conducted to estimate biometry of an epiphytic micro-calcareous red algae, Hydrolithon boreale found on leaves of a Mediterranean meadow, Posidonia oceanica along the entire Turkish coast of the Mediterranean Sea in time (winter and summer 2019) and space (regions, and bottom type and depth). Percent occurrence of the epiphyte was lower in winter (25%), particularly found in a small bay in the west than summer (44%), entirely along the coast. The epiphyte grew up to 5 mm in diameter, 0.35 mm in thickness of the crust size, and was populated up to 1006 ind/m 2 in summer owing to the increased utilization of the carbonate by the epiphyte with the increased water temperature. The size was contrasted to the density (abundance and biomass) in space. The biometry was signicantly dependent on the siliciclastic-carbonate deposition as inferred from SiO 2 of the water in relation the leaf area index (LAI) of P. oceanica. Therefore, this deposition induced specimens growing in size, followed by the reduced density in relation to N-based nutrient of the water. Further major environmental parameters which negatively affected the biometry were pH and total suspended matter of the water, analogous to the turbidity. Of the trace elements, Ni was negatively correlated with the biometry whereas the LAI was however positively correlated with all the anthropogenic-sourced trace elements (V, Cu, Zn, As, Cd, and Pb) in the leaves. Of the bottom types, the calcite rock had higher density than the other soft bottoms in contrast to the size of the epiphyte.


Introduction
The epiphytes on the seagrass suggest ecological indications and status for the marine environments. As occurring at every taxon, the epiphytes have sensitive and tolerant species to the levels of the disturbance in the marine environments (Sfriso et al. 2007(Sfriso et al. , 2009). Therefore, their density (abundance and biomass) and species composition showed direction and levels of the ecological conditions.
One of the sensitive seagrass in the Mediterranean Sea is a meadow, Posidonia oceanica (Linnaeus) Delile, 1813 and covered by diverse epiphytes hosted on its rhizomes and leaves (Nesti et al. 2009). The One of the epiphyte taxa which are noticed and paid attention are the calcareous species which are acted as processors in the sediments (Walker and Woelkerling 1988). The most dominant encrusting species were Hydrolithon and Pneophyllum species as epiphytes on the seagrass in the Mediterranean Sea (Jacquemart and Demoulin 2008; Piazzi et al. 2000). Only these two genera species predicted reasonably most of the ecological indications ( (Martin et al. 2008). In the Mediterranean Sea, overall the decomposition of thionitrophilous taxa reduced pH values which slowed down the growth of small calcareous species such as Pneophyllum fragile Kützing, 1843 and Hydrolithon spp which cannot grow in poor-bad conditions (Sfriso et al. 2007(Sfriso et al. , 2009(Sfriso et al. , 2014. Inherently, crustose coralline algae that settled in the extremely low pH (mean pH 6.9), however, were signi cantly smaller and exhibited altered skeletal mineralogy (high Mg calcite to gypsum, hydrated calcium sulfate, Kamenos et al. 2016). In an undisturbed site where the NO 3 and NO 2 were lower and Si(OH) 4 was higher, some species (Hydrolithon farinosum (J.V.Lamouroux) Penrose & Y.M.Chamberlain, 1993, Fosleilla spp. and P. fragile) of epiphytic red algae were dominant (Mabrouk et al. 2013).
A species of the present study, Hydrolithon boreale (Foslie) Y.M.Chamberlain, 1994 is one of the larger specimens of the microcalcareous red algae in term of size of its crust (up to 2-5 mm in diameter; Reyes and Afonso-Carrillo 1995; Bradassi 2011), and has been reported here for the Turkish Mediterranean Sea.
Researches on invasion success and mechanisms can guide scientists to understand regulation by a complex set of factors, including biotic and abiotic features of recipient systems, disturbance regimes, and invader life traits (Sol et al. 2012). Therefore, this brings the following present study. Regarding to the ecological importance of the calcareous seaweed in the marine environment and the historical lack of comprehensive information on their distribution and ecology in the Turkish waters, the aim of this study is to provide baseline information on bathymetric and seasonal distribution and biometrical patterns (density and plant traits) of the H. boreale recently appeared on the leaves of P. oceanica in the infralittoral of the Turkish pristine coast, a region of the most ultra-oligotrophic waters (Sisma-Ventura et al. 2017) of the Mediterranean Sea, and to determine biometrical dynamics and the species-environment relation of the biggest microcalcareous species emphasizing the siliciclastic-carbonate deposition.

Study area
The study area was located in infralittoral zone regionally limited by a bottom depth of 30 m extending occasionally to 40-43 m hereby along the southern coast of Turkey (Mutlu et al. 2020a). The area was between Taşuçu Bay, Mersin through Antalya and Datça Bay, Muğla, along three provinces ( Figure 1). The study area was restricted by Taşucu Bay in the eastern Turkish coast since there are no further locations of a meadow, P. oceanica beyond Taşucu Bay to eastern end of the eastern Mediterranean Sea (Ergün Taşkın who researches presence of the meadows and macrophytes by SCUBA, pers. comm.). The study area has a coastline length of about 1100 km (Figure 1a).
Eastern half of Mersin Bay is nutritionally fed by three major (Seyhan, Ceyhan and Göksu) rivers and partially under in uence of the Nile River (Figure 1b). Owing to few brooks having moderate ow rate, Antalya Bay is oligotrophic compared to the Mersin Bay. Muğla's bays and coves are devoid of rivers and brooks but, partially fed with local creeks and have undergone the effect of the Aegean Sea from east to west ( Figure 1).
Overall, sea surface temperature is at minima in December-February (14-17 to 21 o C), at maxima in July (19-28 o C)-August (28-30, and to 31 o C), and decreased from eastern to western coasts of the present study area. Overall, the salinity varied seasonally between 38 and 39 PSU (~40 PSU in August-early September) (Mutlu et al. 2020a).
The prevailing surface rim current of the Turkish Mediterranean coast was cyclonically circulated from İskenderun Bay (the easternmost Mediterranean Sea), owed off Mersin Bay reaching Taşucu Bay, and crossed off Antalya Gulf (cape to cape, Figure 1f) then to Rhode Island where the current is bifurcated northerly and southerly (El-Geziry and Bryden 2010). The current velocity was faster than 20 cm s -1 typical for the entire Mediterranean Sea, but was generally about 50 cm s -1 on average along the Turkish Mediterranean coast (Poulain et al. 2013).
Anthropogenic effects were present with a variety of the sources as follows: Regarding to the marine litter presumably shading the canopy of the meadows has increased from east to west of the study area by the tourism, shery and maritime activities (Gündoğdu and 2021) studied anthropogenic effect of trace elements in Posidonia blades and its sediments in the present study area; the highest bioaccumulation factor in P. oceanica was calculated for Cd. Coastal waters was heavily exposed to trace elements and signi cant positive correlations were detected between the anthropogenic trace element pollutants (As, Cd, Cu, Zn, Pb, and V) and natural sourced trace element (Ni and Cr). The Mediterranean Sea coast of Turkey did not have signi cant levels of Zd, Cd, Cu, and Pb pollution, whereas 65% of the stations were heavily polluted with As. The study area, particularly Muğla's coasts which have high angle of bottom trend has been visited by thousands of the pleasure crafts affecting the population of the meadows by their anchorages, especially in summer (Mutlu et al. 2020b). Furthermore, the study area is under in uence of intensive coastal agricultural activities, particularly throughout the coast of Mersin and Antalya providences (Figure 1f).
There are three main rivers with high ow rates in the easternmost of the Turkish Mediterranean coasts, which in uence coastal zone till Anamur Cape from the east by diluting the rate westward. There are many small scale brooks along the Antalya's coast, and some creeks along Muğla's coasts (Figure 1b). Springs of the streams were sourced through the Taurus mountain range parallel to the Turkish Mediterranean coast (Figure 1b). The study area has been in uenced by the geological structure of the mountain. The geological chronology showed that the mountain had mixed siliciclastic-carbonate during the Palaeozoic, carbonate deposition during Jurassic-Cretaceous and then shallow-water carbonates inducing deeper-water deposits during Palaeocene-Eocene (Dumant et al. 2017). Therefore, the streams and groundwater feed and enhance the study area with the siliciclastic-carbonate and drains of anthropogenic sources around beds of the streams as well (Figure 1). The ow rates of the streams and groundwater have decreased in time due to climate change and global warming.  Figure 1c).

Materials And Methods
Some environmental parameters of water columns were measured and sampled from the surface (pre x S) and near-bottom (pre x N) waters of the stations on the board. These parameters were physical (T; temperature in o C, pH; pH, S; salinity in PSU, and O; oxygen in mg/l) using a multi-parameter probes (YSI, HiTech), chemical (nutrients; NO 2 +NO 3 , NH 4 , and PO 4 , SiO 2 , chl a and TSM; total suspended matter). The oxygen was measured only in winter since the oxygen probe was malfunctioned in summer and the chemical parameters were sampled only in summer due to low occurrence of the epiphyte in winter (Figures 2-3). One litter of the water was ltered through CF/C for each, of the nutrients, and total suspended matter and through CF/F lters for chl a, and then all was kept at freezer having -20 o C on board during the eld survey.

Bottom types
Bottom types were determined since the type can differentiate the biometry of the meadows (Giovannetti Harmonizing data obtained by SCUBA, and the grab, nal bottom types were classi ed as pure white sand, sand, muddy sand, mud, mostly biogenic materials; molluscan fragment originated-gravel, rocks and matte (about 2 m high) of P. oceanica along the study area ( Figure 1). White sand was kept distinguished from the gray sand and mud (mud) because presumably of different origin of the sand source and total organic carbon content in the sediment. Gravelly bottom was not encountered in summer survey.

Sample studies
Onboard, samples of the meadow were preserved in borax-buffered formaldehyde (3%) in plastic jars on deck of the R/V "Akdeniz Su".
Total suspended solids (material was dried in an oven at 60 o C for 24 h, and then weighed before the weight of the dried membrane was subtracted from the total dry weight), and chlorphyll a (chl-a) using a method described by Lorenzen (1967). Secchi disk depth was recorded once at each station.
A total of 45 and 100 stations were analyzed for the biometry of a calcareous epiphyte, H. boreale in winter and summer, respectively. A total of 2928 and 5028 shoots, and 11662 and 24509 leave of the meadows were measured for scanning the epiphyte in winter and summer, respectively. Of the epiphytes, H. boreale was majorly recorded on the leave of P. oceanica (Appendices 2-3), regardless of very tiny epiphytes at a scale of ultra/net size (Appendix 2). The leaves were generally clear of the other epiphyte owing to the study area being at oligotrophic level (Appendix 1). Crust diameter (D) and thickness (T, height of about cylindrical crust) of H. boreale were measured using an ocular micrometer scale under binocular microscope. All measurements were converted to mm. The crust weight was estimated from the crust volume (cylinder) and density of calcium carbonate (2.711 kg/m³). Abundance (TN, ind/m 2 ) and biomass (B, µg/m 2 ) of H. boreale were calculated to be values per square meter. The biomass was estimated because it depended on the size and abundance together. Furthermore, one-sided Leaf Surface Area (LAI, m 2 /m 2 ) of P. oceanica was calculated to relate to the biometry of the epiphyte.

Statistical analyses
Of the univariate analyses, Spearman rank correlation and partial correlation were subjected to data between biometry of the epiphyte and the environmental data to estimate the hidden environmental variables. The Spearman correlation was applied between trace elements (mg/kg) in blades of P. oceanica and its sediments (Öztürk et al. 2021), the biometrics of the epiphyte and LAI of P. oceanica to estimate effect of anthropogenic trace elements on the biometrics. The Generalized Additive Model (GAM) was used to estimate the effect of the predicted environmental parameters (all of physics, chemists and optics, and each of physics, and chemists separately) to the response biometrical variables of the epiphyte for summer survey. The univariate statistical analyses were performed using the statistical tools of the MatLab (vers. 2021a, Mathworks inc).
The following statistical analyses designed according to the results of the univariate statistical analyses. Of the multivariate analyses, the multiple regression was used to test the relationship and correlation between the biometrics of the epiphyte and LAI of P. oceanica & concentration of sea surface SiO 2 using the MatLab statistical tool. Principal Coordinate Analyses (PCO) was applied to a triangular Euclidean distance matrix of the biometry to gure out the best variable with the variation on the component and then the PCO con guration on the axes was correlated with the environmental variables using the Spearman rank correlation for summer survey. The multivariate analyses were performed using PRIMER 6 (vers. 6.1.13) + PERMANOVA + (1.0.3.).

Results
Understanding the biometrical dynamic of an encrusting red algae, P. fragile on a seagrass, C. nodosa in time, data of a previous unpublished study (Mutlu et al. 2014) were examined since they were sampled at ve months a year ( Figure 2). This guides at lling the gap of their temporal dynamic between winter and summer samplings conducted in the present study. The abundance and biomass increased slightly from January to April, and then peaked in August, followed by occurrence of decrement in November ( Figure  2). The size was distributed in contrast to the temporal density of the epiphyte, and the LAI of C. nodosa did the same distribution as well ( Figure 2). However, any individual of calcareous red algae on leave of P. oceanica in the Gulf of Antalya was not encountered during 2011-2012 (Mutlu et al. 2014). Coverage of P. fragile was 6.4% of leaf surface of C. nodosa on average and varied between 1.6% in March and 16.0% in August.

Distribution of Hydrolithon boreale
Hydrolithon boreale on leave of P. oceanica occurred at 25% of the total stations in winter, at 44% in summer during the present study ( Figure 3). An average coverage of H. boreale was 0.12% (max: 0.75%) of leaf surface of P. oceanica. Most occurrences took place along the westernmost coast of the study area, Muğla's coast in winter, and all coasts of three provinces in summer (Figures 3-4).
An average abundance of 41 and 49 ind/m 2 on the study area (163 and 112 ind/m 2 on stations where the epiphyte occurred) was estimated in winter and summer, respectively (Table 1). In winter, maximum abundance and biomass was recorded in a particular area of the Muğla's coast where there used to be sh farms in 2000s (c.a. [2000][2001][2002][2003][2004][2005]. In summer, the abundance and biomass were maximized in the easternmost location opposed to the location in winter, and were highly variable with the regions along the coast of the study area (Figures 3-4). The lowest biomass and abundance occurred along the Antalya's coast with a few exceptions of the locations, and a moderate value was along the Muğla's coasts.
Diameter of H. boreale's crust was less in winter than that in winter on average, almost similar around 2.5-2.9 mm in all stations in winter while the diameter was very different in the regions of the study area ( Table 1). The maximum diameter almost doubled the winter values in summer in contrast to the abundance and biomass in summer with exception of Antalya's Gulf where the meadows were found only on the rocks (Figures 1-4). This contrast seemed to be more pronounced on soft bottom (sand and mud) and dead matte in about 2 m high.
Similar to the diameter, thicker crust of H. boreale was observed in Muğla's coast in both winter and summer. This trend was more apparent in summer. The thickness was in similar range between winter and summer (Table 1). Of the environmental parameters, sea surface SiO 2 concentration was however straight-proportionally overlapped on the size of the epiphyte, but reversely on the density (abundance and biomass) depending on the size of epiphyte and LAI of P. oceanica in summer ( Figure 3). The larger epiphyte carried the less abundance on a given LAI.

Epiphyte-environment relation
Biometrics of the epiphyte was correlated negatively with sea surface temperature, and positively dissolved oxygen concentrations of the near-bottom water in winter at p<0.05. However, there was a signi cant correlation between the biometrics and bottom type ( Table 2). Sea surface temperature was negatively correlated with the diameter and oxygen positively with density variables of the epiphyte in winter. Table 2 Spearman correlation between the biometrics of epiphyte and physical environmental parameters (see Table 6  In summer, near-bottom temperature correlated positive-signi cantly the density variables (abundance and biomass) of the epiphytes in contrast to that in winter. Interestingly, the biometrics was negativesigni cantly correlated with sea surface concentration of the SiO 2 , but positively with salinity of the nearbottom water ( Table 3). The PO 4 of the near-bottom water was positively correlated with only crust diameter at p < 0.05 (Table 3). Table 3 Spearman correlation between the biometrics of epiphyte and physical and chemical environmental parameters (see Table 6 for the abbreviations), and bottom depth (De) and type (BT) in summer. There was an obvious relationship between density and diameter of the epiphyte and the sea surface silicate (SiO 2 ) and LAI of the meadow in summer (Figure 4). However, biometrics of the epiphyte was not signi cantly correlated with the LAI of P. oceanica at p < 0.05, which has brought about the multipleregression between the variables aforementioned.
Statistics of the linear multiple-regression was given in Table 4 for the relationship between biometrics of the epiphyte and the sea surface silicate (SiO 2 ) and LAI of the meadow in summer. To extract out the hidden variables of the LAI and SiO 2 in linear relation to the biometrics, Spearman partial correlations showed that none of the variables was correlated with the biometrics at p<0.05. Multiple-linear regression showed that there was however a signi cant correlation only between the thickness and the LAI and SiO 2 at p< 0.05 in Table 4. Regarding to the b value, the LAI affected biometrics positively, but the SiO 2 negatively (Table 4). Based on all environmental variables, the abundance of the epiphyte was not partial-correlated with any of the environmental parameters. The nutrients were not partial-correlated with any of the biometrics (Table 5). Diameter, thickness and biomass were positively partial-correlated with six of the environmental parameters at p < 0.05 ( Table 5). The LAI of P. oceanica was highly correlated with the biometrics of the epiphyte (Table 5). Table 5 Partial correlation coe cients between biometrics and the environmental parameters (see Table 6 for the abbreviations) for summer samplings. Values are only correlation coe cients which were signi cantly partial-correlated at p < 0.05.  With respect to only physical environmental parameters, the abundance was positively affected by the ST, followed negatively by the SS and slightly Sec. The diameter and thickness were mostly in uenced by the pH and S; the pH affected positively the size and biomass as a function of the diameter and thickness, but the salinity negatively did (Figure 6).
In terms of effect of only chemical parameters, the TN was under negative effect of the NTSM and NNO 3 , and followed by that under positive effect of the SPO 4 and SSi. Overall, N-based nutrients affected negatively the abundance (Figure 7). The NNO 2 +NO 3 , SSi and NNO 2 increased the diameter of epiphyte, and the size was reduced by the NNO 3 and SNO 2 ( Figure 7). As occurred in the abundance, NTSM decreased the biomass, but the SSi and SNH 4 increased the biomass of the epiphyte (Figure 7).
Signi cant part (97.5%) of the total explained variance occurred on the PCO1 to estimate the rst component based and launched on the biometrics well-correlated with SSi (Table 6 and Figure 8). The density (B and TN) of the epiphyte increased with the NS and ST, and decreased with the SSi (Table 6 and Figure 8a, b, d). The distribution of the crust diameter (D) was exactly contrasted to the density of the epiphytes on the PCO (Figure 8a-c). The best descriptive trait of the biometric in direct correlation with the SSi was the diameter of the epiphyte (Figure 8c, d) since the other biometrics changed dependently on LAI of its host, P. oceanica and thickness. The enlargement of the diameter was accelerated after a threshold of about 80 µM of the SSi to about 210 µM which inhibited occurrence of the epiphyte (Figure 8c, d*). This threshold could be due to action as limiting factor for SiO 2 on the growth of the epiphyte.
Of the trace elements on the blades of the meadow, the Ni originated by the nature was negatively correlated with the crust biometrics of the epiphyte and LAI of P. oceanica at p < 0.05 (Table 7). Of the anthropogenic-sourced trace elements (V, Cu, Zn, As, Cd, and Pb) in the blades, Zn was negatively correlated with only diameter of the epiphyte. In the sediments, the Ni affected negatively with the TN, T, and B of the epiphyte. However, the LAI was positively correlated with all the anthropogenic-sourced trace elements. The As was positively correlated with diameter of the crust (Table 7).

Discussion
Of the epiphytes on P. oceanica, encrusting algae were signi cantly different in their distribution between leaves and rhizomes of P. oceanica, and species belonging to genus Pneophyllum and Hydrolithon spp were found generally on the leaves (Nesti et al. 2009). The epiphytic assemblages were indicators of various types of natural and anthropogenic disturbances, and occurred to grow detecting the moderate nutrient level (Balata et al. 2008). Hydrolithon spp and P. fragile are sensitive species for undisturbed areas (Sfriso et al. 2007(Sfriso et al. , 2009). and status which were then recovered to healthy conditions (Sfriso et al. 2020). High N-based (particularly NO 3 ) nutrientphilic epiphytes produced the high biomass contrasted to moderate nutrientphilic epiphytes assigned as sensitive species (Balata et al. 2008). In summer, the epiphytes were increased on seagrass owing to low-concentration of the nutrient induced by the leaf turnover of the seagrass which was minima in winter (Peterson et al. 2007). This brought about a difference of 20% in the occurrence of epiphyte between winter and summer (Peterson et al. 2007) similar to the difference estimated in the present study.
On the leaves of the seagrass, biomass and number of epiphytic species were high at agricultural drainage, but was low at the references sites in regard to the different nutrient levels and LAI of the seagrass as well (Prado 2018 (Boudouresque et al. 1984), and rapid absorption of nutrients (Lepoint et al. 2007  Con ict of interest The authors have no con icts of interest to declare that are relevant to the content of this article. On behalf of all authors, the corresponding author states that there is no con ict of interest.
Availability of data and material The data are not shared but the data will be available if requested by the journal.
Code availability all software used in the present study was used with the license of each code.
Ethical approval The authors declare that all applicable guidelines for sampling, care and experimental use of animals in the study have been followed.
Consent to participate All authors declare their participation in the study and the development of the manuscript herein. Declaration of Competing Interest The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.   Biometrical distribution of Hydrolithon boreale; abundance (TN; ind/m2), biomass (B; µg/m2), crust diameter (D; mm), and thickness (T; mm) in winter. Maximum values were maximum values of the winter survey, and scaling value for the circles in both winter, and summer in Figure 4, respectively.

Figure 5
The GAM solution to estimate effective variables from all environmental parameters (see Table 6 for the abbreviations) on the biometrics (Abundance; TN, D; crust diameter, and biomass, B) of the epiphyte in summer.

Figure 6
The GAM solution to estimate effective variables from only physical environmental parameters (see Table 6 for the abbreviations) on the biometrics (Abundance; TN, D; crust diameter, and biomass, B) of the epiphyte in summer.

Figure 7
The GAM solution to estimate effective variables from only chemical environmental parameters (see Table 6 for the abbreviations) on the biometrics (Abundance; TN, D; crust diameter, and biomass, B) of the epiphyte in summer.