Biodiversity of carapace epibiont diatoms in loggerhead sea turtles (Caretta caretta Linnaeus 1758) in the Aegean Sea Turkish coast

Study area

Dalyan beach is located in the province of Muğla (36°42′02′′N, 28°41′31′′E) (Fig. 1). It has one of the highest numbers of loggerhead sea turtle nests along with the beaches of Belek, Antalya, and Anamur, along the Aegean and the Mediterranean coasts of Turkey (Kaska et al., 2016). As a result, Dalyan beach was assigned as a “specially protected area” in 1988 and has “flagship beach” status for the conservation of loggerhead sea turtles (Türkozan & Yılmaz, 2008). The beach is 4.7 km long and composed of a fine-sand dune and gravel drifted from the Dalyan Delta, which is deposited to the east of the beach. Dalyan Delta is an extensive wetland with a labyrinth of reedy channels opening to Köyceğiz Lake via the Dalyan River where, during the study period (2011–2018), some foraging sea turtles were observed. The wetland complex (Dalyan Delta) opens to the sea through a channel at the northern part of the beach (Türkozan & Yılmaz, 2008).

Sampling

Samples of diatoms were collected from nesting loggerhead sea turtles, at night during the nesting season, between May–August, 2011–2014 and 2018 (Fig. 2). All sampling was carried out in accordance with the regulations of the Ministry of Environment and Urbanization (TR-15/04/2018/39). Sampling was supervised by experts from the Sea Turtle Research Rescue and Rehabilitation Centre (DEKAMER), Ref. B.32.PAU.0.AG.00.00/005. In total, 39 samples were taken. Samples were collected with toothbrushes from 20 cm² of vertebral and coastal carapace scutes of 33 turtles (curved carapace length (CCL) between 67,5–77 cm) between 2011–2014, and pieces of biofilm were scraped with a razor from six different sea turtles (according to the conservation regulations) while the turtles were laying eggs in 2018. A total of 33 samples were processed and used for light microscopy (LM) and scanning electron microscopy (SEM) (3 samples from 2011; 5 samples from 2012; 20 samples from 2013 and 5 samples from 2014). Six unprocessed fragments of biofilm (from 2014 and 2018) were used for SEM observations (Table 1).

Biofilm pieces were fixed with 70% ethanol for 4 h. Each fixed biofilm was then washed five times with distilled water, followed by washing in increasing alcohol concentration. In each concentration, the biofilm was left for 20 mins, (30 mins in absolute alcohol) at room temperature. After drying, a piece of biofilm was mounted on an aluminium stub with double-adhesive carbon tape. Untreated samples of the dried and dehydrated microbiome were sputter-coated with palladium-gold alloy and observed with a Hitachi SU8020 scanning electron microscope (Hitachi, Tokyo, Japan).

For light (LM) and scanning electron microscopy (SEM) observations, samples were cleaned to remove organic material by washing with 10% HCl, boiling in 30% H₂O₂ and rinsing with distilled water (Swift, 1967). Permanent slides were air-dried and mounted in Naphrax^®. LM observations were performed with a Zeiss Axio Imager 2 (Carl Zeiss Microscopy Gmbh, Jena, Germany) equipped with a 100 × oil immersion Plan apochromatic objective (with numerical aperture = 1.46) at the University of Szczecin (Poland), and a Nikon Eclipse Ci (Nikon Corp. Tokyo, Japan) with a Nikon DS-Fi1 camera at the Kütahya Dumlupınar University. SEM images were taken using a HITACHI S-5500 at Warsaw University of Technology (Poland). Slides and processed material are deposited at the Department of Marine and Freshwater Resources Management, Istanbul University, Istanbul (Turkey) and the diatom collection (SZCZ) of the Institute of Marine and Environmental Sciences, University of Szczecin, Szczecin (Poland).

Data analysis

The abundance of diatom species was expressed as a percentage of the total number of valves counted (relative abundances in %). The relative abundance (RA) of particular taxa and the taxa richness of the assemblages were estimated on the basis of at least 300 diatom valves counted per sample. Frequency of the most abundant taxa and their maximum RA during the four-year period (2011–2014) and for each of the years were determined.

Raw diatom counts were expressed as a relative abundance and were square-root transformed to normalize data. A resemblance matrix of the data was generated using Bray–Curtis analysis. The Bray–Curtis similarity matrix (Legendre & Legendre, 1983; Clarke & Gorley, 2006) of the relative abundance data of 457 taxa over 33 samples was constructed. Similarity percentage analysis (SIMPER, (Clarke & Warwick, 1994)) was used to identify the taxa making the most significant contribution to the similarities between epibiontic diatom assemblages. All statistical analyses were performed using the Primer v6 software (Clarke & Gorley, 2006) and Statistica 7.0 (StatSoft, Inc. 2004).

Identifications were made following Witkowski, Lange-Bertalot & Metzeltin (2000). Terminology follows Round, Crawford & Mann (1990), and nomenclature of recorded taxa follows AlgaeBase (Guiry & Guiry, 2019) and Diatombase (Kociolek et al., 2019).

Diatom composition & distribution

A total of 457 diatom taxa belonging to 86 diatom genera were identified from 33 samples (Table S1). Among them, 62, 95, 253 and 275 taxa were identified in 2011, 2012, 2013 and 2014, respectively. Among the 457 diatom taxa, 27 taxa were observed exclusively in 2011, 26 taxa in 2012, 111 taxa in 2013, and 129 taxa in 2014, while 174 taxa were found only once (sporadic).

The genera with the highest number of taxa represented were Navicula (79), Nitzschia (45), Amphora (40), Cocconeis (32), Diploneis (25), Mastogloia (23), Fallacia (14) and Achnanthes (12), followed by Halamphora (10) and Psammodictyon (10). Although Navicula and Nitzschia had the highest numbers of taxa, they occurred with an average RA of 3%. Amongst the genera which were recorded in all four sampling years, the most abundant was Achnanthes (Avg RA = 7%) (Tables 2 and 3).

The results revealed that there were 16 taxa common to all four sampling years. These taxa were Achnanthes elongata Majewska & Van de Vijver, Cocconeis sp. 8, Dimmeregramma minus var. nanum (Gregory) Van Heurck, Diplomenora cocconeiformis (Schmidt) Blazé, Diploneis bombus (Ehrenberg) Ehrenberg, Halamphora acutiuscula (Kützing) Levkov, H. tenerrima (Aleem & Hustedt) Levkov, Karayevia submarina (Hustedt) Bukhtiyarova, Meloneis mimallis Louvrou, Danielidis & Economou-Amilli, Navicula normaloides Cholnoky, N. perminuta Grunow, Nitzschia elegantula Grunow, N. liebetruthii Rabenhorst, Pinnunavis yarrensis (Grunow) Okuno, Tryblionella pararostrata (Lange-Bertalot) Lange-Bertalot, T. granulata (Grunow) Mann. Navicula perminuta and Delphineis australis (Petit) Watanabe, Tanaka, Reid, Kumada & Nagumo were recorded in 65% of samples, both with an average RA of 10% (Figs. 2 and 3, Tables S2–S5).

According to the SIMPER analysis (Tables S2–S5), samples collected from turtles in 2014 had the highest observed within-group average similarities (37.96%). As revealed by SIMPER analyses, the group of taxa contributing the most (cumulatively 50.63%) to similarity between diatom assemblages from the five samples collected in 2014 included Navicula perminuta, Nitzschia frustulum, Cocconeis placentula Ehrenberg, Navicula sp. 54, Navicula sp. 55, Nitzschia liebetruthii, Melosira moniliformis (Müller) Agardh, Tryblionella granulata and Seminavis strigosa (Hustedt) Danielidis & Economou-Amilli (Table S5).

Biofilm observations

During SEM analysis of the unprocessed biofilm samples (Figs. 4, 5) diatoms were found mixed with other microorganisms, e.g., cyanobacteria, organic detritus, broken pieces of the carapace, mineral detritus and diatomaceous detritus. However, in the carapace fragments (CAR_2018_1 and CAR_2018_5), which had sparse biofilm components, diatoms were observed as pioneer epibionts attached directly to the carapace. In the well-developed biofilm (CAR-2018_3) diatoms were abundant, well preserved and represented by epizoic forms: Achnanthes elongata, A. squaliformis (Majewska et al., 2017a), Chelonicola sp. and Tripterion spp. Another biofilm was dominated by cosmopolitan species such as Navicula perminuta and small Nitzschiae sect. Lanceolatae (N. frustulum, N. liebethrutii), with lesser participation of the above-mentioned epizoic forms. It appeared as if the layers of diatoms were bound between microlayers of a mucilage composed of unidentifiable organic matter, possibly containing microfungi. In the well-developed biofilm fragments, low occurrence of diatoms was observed. Biofilm sample CAR_2018_2 (Fig. 4D) was mostly composed of mineral and fine organic detritus along with relatively rare, usually broken, diatom frustules. Interestingly, in CAR_2018_4 (Fig. 5D), we observed organic compounds, filamentous cyanobacteria (Anabaena sp.) and fine-mineral detritus, whereas diatoms were absent. The differences on the biofilm of several loggerhead sea turtles may give ideas of the development of biofilms, also diatom composition should be taken into consideration. However there are not any data on sea turtles’ health regarding diatoms, diatom composition especially freshwater and brackish taxa may be monitored in foraging areas. Komoroske et al. (2011) observed concentrations of pollutants of carapace like metals, in further studies diatom composition and pollutants could be monitored to reveal any possible relation.

Diatom composition

In this study, we present the first detailed floristic list of epibiont diatoms observed on the carapace of loggerhead sea turtles in the Mediterranean Sea. The number of taxa (457) was higher than any floristic surveys conducted on turtles or similar biotic habitats (e. g., whales and cetaceans) in the Mediterranean and over a wider geographic area (Nemoto, 1956; Nemoto, 1958; Majewska et al., 2015b; Robinson et al., 2016). The number of diatom taxa recorded in this study was considerably larger than those recorded on the carapace of olive ridley sea turtles (21 diatom taxa) in Majewska et al. (2015b) and green sea turtles carapace (57 diatom taxa) in Rivera et al. (2018). Number of diatom taxa difference might be related with some factors such as sample numbers, sampling techniques (razoring or brushing the carapce), sea turtles’ foraging areas (Robinson et al., 2016) or the possible difficulties in marine diatom identification. However common diatom taxa of the three sea turtle species might suggest that not only the epizoic diatoms addressed to sea turtles but also other marine diatoms could occur on different sea turtle species, whereas further studies on sea turtle species may reveal the similarity or difference on diatom dispersal.

Of the 457 diatom taxa, the genus Navicula was the most diversified, with several unidentified Navicula spp. abundant in the samples analysed. However, most of the unidentified Navicula spp. were similar in morphology with only very minor differences in some characteristics. This might be a result of adaptation in the biofilm (e.g., some valves were heteropolar with a narrower valve end on one side of the valve). The second-largest group in the diatom community was Nitzschia spp., with N. frustulum as the dominant taxon overall. We observed some small-celled taxa, e.g., Nitzschia inconspicua, Halamphora tenerrima and Navicula spp. on the turtle carapaces, in accordance with previous studies from various regions (Majewska et al., 2015b; Robinson et al., 2016; Rivera et al., 2018). Occurrence on the turtle carapace might be related to the small cell size of the frustules, which may lead to rapid reproduction, as has been observed in Navicula perminuta (an opportunistic species). Mastogloia species were found in low abundance, but were represented by numerous species (e.g., M. adriatica Voigt, M. corsicana Grunow, and M. decussata Grunow). Therefore, Mastogloia species demonstrated the ability to survive under conditions in the biofilm, but were unable to reach high abundance.

Comparison with the local diatom flora

Some of the taxa observed in the biofilm on the loggerhead sea turtle carapace have been found in diatom flora on different substrata in the same region and along the Aegean Sea coast, and do not seem to have a preference either for a geographic region or for the substrate type (Kaleli, 2019; Kaleli, Kociolek & Solak, 2020). In a shallow coastal lake (Iztuzu Lake), in the same area as the beach occupied by sea turtles during the nesting season, diatoms were abundant (Kaleli, 2019), and some of the species were the same as those found associated with the C. caretta carapace (e.g., Diplomenora cocconeiformis, Diploneis bombus, Fallacia schaeferae (Hustedt) D.G. Mann, Mastogloia lanceolata, Meloneis mimallis). It is possible that diatoms were transferred by the sea turtles during the nesting season. These taxa have also been observed from different locations in the adjacent coasts and also in the Western Indian Ocean (Kaleli et al., 2018, unpublished observations).

Despite the fact that marine taxa strongly dominated the assemblages (Table S3), a few freshwater taxa were observed. The freshwater forms were usually observed as solitary valves (e.g., Encyonema minutum (Hilse) D.G. Mann and Lindavia balatonis (Pantocsek) Nakov, Guillory, Julius, Theriot & Alverson). The presence of taxa associated with fresh to brackish waters (Table S6) was not particularly surprising as Köyceğiz Lake, which is a typical freshwater lake, is located nearby, and connected through the delta, to Dalyan beach. Both male and female turtles have been observed in the shallow waters close to the banks of the channels connecting the beach to Köyceğiz Lake. Some of the turtles were also observed feeding in the lake and this could be why freshwater taxa were incorporated into the biofilm. Some taxa may be able to tolorate change in salinity (freshwater-brackish, brackish-marine) despite their apparent freshwater preference, and results also support the idea that some species could have different responses to environmental conditions, resulting in a better or worse adaptation (Underwood, Phillips & Saunders, 1998; Ribeiro et al., 2003; Miho & Witkowski, 2005; Hafner, Jasprica & Car, 2018) to variable conditions, which could be explained by the number of the freshwater forms observed on the carapace. It was also suggested by Majewska et al. (2017b) that lakes and rivers could make exclusive epibiosis where specific species could attach and grow in the biofilm and environment affects the dispersal on sea turtle carapace. The abundance of freshwater and brackish water species, presumably reveal that important amount of sea turtles access to shallow freshwaters of Dalyan and spend long periods in the surrounding areas. Nutrient enrichment in these waters provide favourable conditions for the ubiquitous taxa. Navicula perminuta and Nitzschia frustulum, which are found in marine and brackish waters, dominated the assemblages and this may indicate that species with similar ecological tolerances can settle on the carapace, but species with better adaptation (small cell size, attaching to the carapace, broad tolerance of changes in salinity and light intensity) can thrive.

Epizoic diatoms

Among the dominant taxa, Achnanthes elongata and Olifantiella seblae (Kaleli et al., 2018), recently described from the same biofilm samples, were observed, as obligately epizoic diatoms together with unidentified Chelonicola sp. and Tripterion spp. which we consider potentially new to science (Kaleli et al. in preparation). Representatives of Chelonicola and Tripterion have been described and observed as obligately epizoic forms on sea-turtle carapaces from oceanic waters (Majewska et al., 2015a; Riaux-Gobin et al., 2017a; Riaux-Gobin et al., 2017b) and have not yet been found on other substrates. Achnanthes elongata was described from samples from the Pacific coasts and with this study observed for the first time in the Mediterranean Sea, O. seblae has only been observed in the Mediterranean Sea (Kaleli et al., 2018). The epizoic taxa observed in this study (O. seblae, A. elongata) have a broad range of valve morphology in terms of outline. Morphological plasticity is common in diatom species observed on other sea turtle carapaces or whale skin (Nemoto, 1956; Nemoto, 1958; Riaux-Gobin et al., 2019). For example, Olifantiella showed high plasticity in the Mediterranean samples, and Olifantiella seblae was observed with a length range of 4.5–14.5 µm with elliptic–lanceolate valves. A recent study on Olifantiella species from the South Pacific found similar results on valve plasticity (Riaux-Gobin et al., 2019), valve outline had a wide range of polymorphisms and changes were observed also in valve structure, such as stria formation and counts and the buciniportula, though it was indicated that Olifantiella seblae and Labellicula lecohuiana Majewska, Stefano & Van de Vijver (2017) could be conspecific in Olifantiella gorandiana complex (Riaux-Gobin et al., 2019). Likewise, Achnanthes elongata and A. squaliformis valves were 20.3–70 µm and 12.3–63.1 µm long respectively and showed high plasticity in this study. These two Achnanthes species were described with quite similar lengths to our samples in Majewska et al. (2017a); 15–75 µm for A. elongata and 11.5–45 µm for A. squaliformis).

Biofilm composition

Our SEM observations of intact biofilms highlight that the biofilm is composed of microorganisms and mineral detritus along with micro-detritus from the carapace (Figs. 4 and 5). The formation of the biofilm seems to be a stochastic process, with the early colonisers (we observed diatoms) serving as a foundation for the subsequent deposition of organic and mineral detritus. A similar “messy” microstructure of the biofilm was also observed on the carapace of several species of sea turtles in Robinson et al. (2016). The biofilm observed on carapaces of olive ridley turtles from Costa Rica had a quite different spatial organisation (Majewska et al., 2015b). A relatively low diatom species number was reported (Majewska et al., 2015b), there was stable species composition with low inter-sample dissimilarities, and the epizoic microalgae were either partly immersed or entirely encapsulated within an exopolymeric coat. Here, the biofilm was formed by a massive occurrence of several diatom species with a dozen more taxa being sporadically observed. In addition, our observations on a clean carapace fragment revealed that among the micro-epibionts, diatoms might play a pioneering role as they attach with mucilage (Fuller et al., 2010) to the relatively smooth surface of the carapace.

The colonization of an existing surface by epibionts, organisms living attached to the body surface of a basibiont (host or substratum organism), constitutes one of the most substantial modifications of the basibiont’s body surface (Molino & Wetherbee, 2008; Wahl, 2008). Small epibionts, although generally ignored in the description of marine organisms, may have profound effects on the basibiont by causing a variety of either beneficial or detrimental effects. These effects should be taken into account when the ecology of the host is studied (Gillan & Cadee, 2000). Among the early settlers, microalgae play a crucial role in biofilm development and are able to settle on even the most fouling-resistant surfaces (Molino & Wetherbee, 2008).

Biogeography

Our findings of biofilms composed of epizoic diatoms (e.g., Achnanthes elongata, A. squaliformis, Olifantiella seblae, Tursiocola sp. and Tripterion sp.) showed that the carapace of loggerhead sea turtles in the Mediterranean Sea was a suitable environment for diatom growth and further distribution. It was not possible to determine from which turtle population these epizoic diatoms originated, or from which substrata diatoms were introduced to the carapace (e.g., by grazing). There have not yet been sufficient comparable observations of sea turtle epizoic diatoms and the diatom flora from coasts or coral reefs of nesting grounds in general. However, the presence of epizoic diatom taxa of sea turtles from locations such as the Pacific Coast of Costa Rica, or the Caribbean and South American coasts (Majewska et al., 2015a; Majewska et al., 2015b; Riaux-Gobin et al., 2017a; Riaux-Gobin et al., 2017b; Riaux-Gobin et al., 2019), and the Mediterranean Sea might show that populations of basibionts meet somewhere in the oceans during their foraging migrations, as in the example of Achnanthes squaliformis or the similar species, O. seblae, L. lecohuiana both observed from the Atlantic and the Mediterranean sea turtles. In the Mediterranean C. caretta were tracked and it was found that turtles spent time foraging in the Eastern Mediterranean basin (Casale et al., 2012; Casale et al., 2013). It is possible that the Mediterranean loggerhead population and the Atlantic population could exchange diatom flora (Revelles et al., 2007). Genetic data have shown that the C. caretta populations from the west Atlantic coast spend time foraging with the population from the Eastern Mediterranean (Laurent et al., 1998; Carreras et al., 2006). Species distribution comparison of green sea turtles in Costa Rica and Iran showed a remarkable difference (Majewska et al., 2017b). Different characteristics of water column in Iranian coast of the Persian Gulf and Atlantic Ocean was found as a possible effect of diatom dispersal. Water chemistry and nutrients play a role in diatom community and their growth forms, where in the Persian Gulf species numbers were lower in the challenging environment. On the contrary, the Mediterranean loggerhead sea turtles comprised high biodiversity. However, tracking of these C. caretta is challenging and more detail is needed from the coasts of the Mediterranean for a comparison. But in the oligotrophic waters of the Eastern Mediterranean Sea diatom assemblage composition was significantly richer in species with epizoic diatoms present and characterized by high frequencies. Nonetheless, our study indicated that diatoms could adapt to the sea turtle’s microbiome and form a highly diversified facultative epibiont community. The dominant taxa in RA observed here were mostly raphid diatoms (in particular Navicula spp.). Raphid diatoms are generally among the earliest and most abundant primary colonizers of natural and artificial surfaces (Hoagland, Zlotsky & Peterson, 1986).

In general, the intensity of fouling pressure varies between season, latitude, depth, and local ecological factors; however, any permanently exposed, non-defended surface will eventually become fouled (Wahl, 1989). To determine whether seasonal and spatial variability is a relevant structuring factor, observations of the epibiont diatom community structure should be conducted involving more locations (e.g., different nesting grounds) in the Mediterranean, and over a more extended time period. Due to the fact that the nesting season only happens over a few months each year, there is little opportunity to study the seasonality of the diatom assemblages on the carapaces. However, as more and more turtles are tagged and followed with remote sensing (although it is still difficult to locate the tagged sea turtle as GPS trackers could be damaged or stop sending signals, and the turtle may not come back to ashore or to the same coasts for nesting in 3–4 years’ time), it could be possible and fruitful to repeat the analysis of the microbiome composition in terms of changes in the diatom assemblages over the time on the same sea turtle.