Abstract
Symbiotic dinoflagellates of the genus Symbiodinium, also called zooxanthellae, are found in association with a wide diversity of shallow-water anthozoans. The Symbiodinium genus includes numerous lineages, also referred to as clades or phylotypes, as well as a wide diversity of genetic sub-clades and sub-phylotypes. There are few studies characterizing the genetic diversity of zooxanthellae in Mediterranean anthozoans. In this study, we included anthozoans from the Western Mediterranean Sea and by means of internal transcriber (ITS) and large sub-unit (LSU) rRNA markers we corroborate what has been previously identified, demonstrating that phylotype “Temperate A” is very common among host Cnidaria in this basin. Our finding of fixed differences in ITS and LSU markers that correspond to different host taxa, indicate that this clade may comprise several closely-related species. Previous studies have reported the occurrence of Symbiodinium psygmophilum (formerly sub-clade B2) associated with Oculina patagonica and Cladocora caespitosa in the Eastern Mediterranean. Here, we identify this association in O. patagonica from the Western Mediterranean but not in C. caespitosa, suggesting some differences in symbiotic combinations between the Western and Eastern Mediterranean Basins.
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1 Introduction
Many members of the Class Anthozoa have symbiotic relationships with photosynthetic dinoflagellates belonging to the Symbiodinium genus (Trench 1993). These organisms, also known as zooxanthellae, contribute to host nutrition providing fixed carbon, while the host provides inorganic nutrients, a well lit environment environment, and refuge from herbivory (Weis et al. 2001). Most of the anthozoans serving as symbiotic hosts to algal cells show reduced survivorship in the absence of symbiosis (see Furla et al. 2005 for a review).
The genus Symbiodinium includes numerous evolutionary lineages, also referred to as phylotypes or clades (A, B, C, D, etc.) (Baker 2003; Coffroth and Santos 2005). Genetic differentiation of these lineages has usually been supported by a variety of genetic markers (Sampayo et al. 2009; Thornhill et al. 2013; Pochon et al. 2014). In general, each clade or phylotype includes a diversity of genetic sub-clades or sub-phylotypes, which exhibit distinctive biogeographical, ecological and host-specific patterns. However, symbiont distributions in scleractinian corals may differ over large geographic ranges (Baker and Rowan 1997; LaJeunesse et al. 2003; LaJeunesse et al. 2010). Some symbiont taxa are widely distributed, both among different hosts and across geographic regions (Loh et al. 2001; Rodriguez-Lanetty and Hoegh-Guldberg 2003), whereas other taxa show high host specificity or appear to be regionally endemic (Baker 1999; Baillie et al. 2000; Santos et al. 2002; LaJeunesse et al. 2003; LaJeunesse et al. 2004; LaJeunesse et al. 2010). However, in some temperate regions, such as the Mediterranean Sea, it is difficult to assess the specificity of Symbiodinium sp. phylotypes and host taxa, mainly because the study of zooxanthellae diversity in that region is still in the early stages in comparison with studies available from tropical regions. Currently, there are only few studies characterizing the diversity of zooxanthellae from Western Mediterranean anthozoans (Savage et al. 2002; Visram et al. 2006; Forcioli et al. Forcioli et al. 2011), with all of them indicating the “Temperate A” clade as dominant in this basin.
The Mediterranean is considered a biodiversity hotspot with a high level of endemism, as well as an assortment of temperate and subtropical elements (Coll et al. 2010). This is mainly related to its narrow connection with the Atlantic Ocean, to its east–west orientation and its geological history (Boudouresque 2004). Thus, the current biological diversity is due to the interaction between ecological factors, as well as historical processes that shaped the Mediterranean Basin throughout the course of history (Templado 2014).
This study lends new insight on the diversity of zooxanthellae hosted by Western Mediterranean anthozoans using: the large subunit of the ribosomal RNA (LSU) and the Internal Transcriber Spacers (ITS: ITS1-5.8S-ITS2).
2 Material and methods
Samples from two groups of symbiotic anthozoans, Actiniaria (Anemonia viridis, Bunodeopsis strumosa and Paranemonia cinerea) and Scleractinia (Cladocora caespitosa and Oculina patagonica), were collected at different localities in the Western Mediterranean region (Table 1). Samples were fixed in absolute ethanol and stored until genetic analysis. Sequences from LSU and ITS regions from other zooxanthellate species across different global regions were obtained from GenBank (http://www.ncbi.nlm.nih.gov/genbank/).
Symbiont DNA was isolated from individuals preserved in ethanol following a modified protocol from Coffroth et al. (1992). Molecular variation was detected by polymerase chain reaction (PCR) amplification of the LSU with primers 24D15F1 and 24D2R1 (Baker et al. 1997) and the ITS region using primers ZITSUPM13 and ZITSDNM13 (Santos et al. 2001), and with their corresponding reaction conditions. After PCR amplification, products were purified by ethanol/sodium acetate precipitation or by excising bands. Samples were cycle-sequenced using the ABI Prism BigDye Terminator, and subsequently running them on an ABI 3730 Genetic Analyzer (Applied Biosystems).
2.1 Phylogenetic analysis
DNA sequences were edited using SEQUENCHER 4.6 (Gene Codes), aligned using SeaView 4.4.2. (Gouy et al. 2010) and further revised by eye.
Phylogenetic reconstructions were obtained using the Bayesian inference (BI) and Maximum Likelihood (ML) methods. The evolutionary molecular model that best fit the data sets was selected using jModelTest v3.7 (Posada and Crandall 1998) under Bayesian criterion (BIC). Bayesian analyses were performed using MRBAYES v3.1.2 (Huelsenbeck and Ronquist 2001), with two independent runs of four Metropolis-coupled chains with 5 000 000 generations each, to estimate the posterior probability distribution. Maximum-likelihood (ML) analyses were conducted in PHYML v2.4.4 (Guindon and Gascuel 2003) using the evolutionary model selected by jModelTest. The robustness of the ML-inferred trees was tested by nonparametric bootstrapping (Bs) (Felsenstein 1985), with 1 000 pseudoreplicates in each case. Bayesian posterior probabilities (BPPs) were used as a measure of the robustness of Bayesian trees.
3 Results and discussion
With the use of ITS and LSU markers, we provide additional information on the genetic diversity of Symbiodinium sp. hosted by Mediterranean anthozoans. Both markers show similar levels of resolution (Sampayo et al. 2009), nevertheless, the LSU region has been commonly used to assign Symbiodinium isolates to clades or phylotypes and inferring the relationships between them (Coffroth and Santos 2005; Barbrook et al. 2006) and ITS markers are more commonly used to obtain phylogenetic resolution at the sub-phylotype or sub-clade level (LaJeunesse 2001; Rodriguez-Lanetty 2003).
By means of different phylogenetic analyses with ITS and LSU regions, it is determined that the dominant Symbiodinium phylotype for our anthozoan species in the Western Mediterranean Sea is “Temperate A” (Fig. 1). This corroborates previous results published by other authors (Savage et al. 2002; Barbrook et al. 2006; Visram et al. 2006). This clade was previously described by Savage et al. (2002) as a phylotype only detected in the NE Atlantic and Western Mediterranean and an ancestral lineage of clade A. Although only moderately supported, our analyses also revealed that the “Temperate A” clade includes some nucleotide sequence diversity, structured in different sub-phylotypes, supported in the ITS phylogeny by a range of 0.80–0.90 B.P. and 70–85 % Bs under the Bayesian and ML analyses respectively (Fig. 2).
From our study, the only Mediterranean species that showed a different Symbiodinium sp. phylotype was the scleractinian coral Oculina patagonica. The LSU gene phylogeny identifies the clade hosted by this species as clade B with 1 B.P. and 100 % Bs under the Bayesian and ML analyses, respectively (Fig. 1). The ITS analyses defined the sub-phylotype hosted by O. patagonica as close to sub-clade B2, with less than 0.90 B.P. under the Bayesian analyses and less than 85 % Bs under the ML analyses (Fig. 2). Symbiodinium phylotype B has also been described by Visram et al. (2006) in the sea anemone Bunodeopsis strumosa from the NW Mediterranean (Banyuls, Gulf of Lyon), suggesting that the Western Mediterranean Basin might be richer in terms of Symbiodinium diversity than previously established. The Symbiodinium ITS sub-phylotype B2, also called Symbiodinium psygmophilum (LaJeunesse et al. 2012), has been previously reported only in northerly coral reef habitats in the Western Atlantic, including the Florida Keys and Bermuda (LaJeunesse 2001; Santos et al. 2001; Savage et al. 2002). More recently, LaJeunesse et al. (2012) found phylotype B2 in three species of scleractinians from the coast of Israel in the Eastern Mediterranean Basin: Oculina patagonica, Cladocora caespitosa and Madracis pharensis. In addition, Meron et al. (2012) found this same phylotype in Balanophyllia europaea and C. caespitosa in the Central Mediterranean (Iscchia Island, Southern Tyrrhenian Sea). However, these authors also found clade “Temperate A” in some individuals of B. europaea from the same locality. Cladocora caespitosa was also included in our study considering samples from the Western Mediterranean (Castellón and Tarragona, Balearic Sea); however, they harbor the Symbiodinium sp. “Temperate A” clade, which differs from that found by LaJeunesse et al. (2012) and Meron et al. (2012). This regional variation in Symbiodinium associations within the same host species has been previously reported in the Western Atlantic as well as the Indo-Pacific Oceans, suggesting that those changes might be dependent on regional environmental conditions (Rodriguez-Lanetty et al. 2001; Savage et al. 2002).
Clade B symbionts are particularly common in temperate Western Atlantic regions (Finney et al. 2010), as has been found in anthozoans from temperate regions in the Indo-Pacific (Loh et al. 1998; Rodriguez-Lanetty et al. 2001). However, this clade is not restricted to temperate regions, and has been found in scleractinian corals and other invertebrates in tropical areas (Loh et al. 1998). Symbiodinium psygmophilumrepresents a cold-tolerant lineage able to survive conditions inhospitable to most other Symbiodinium species (Thornhill et al. 2008). An experimental study conducted by Thornhill et al. (2008) concluded that S. psygmophiulm (formerly sub-phylotype B2) is capable of quickly recovering photosynthetic function upon the return of normal conditions after long periods of cold temperatures. Therefore, assuming the same behavioral pattern in hospite, this symbiont may remain photosynthetically inactive throughout colder periods, persisting without major contribution to the nutrition of the host; while during the warm seasons, the species likely increases its photosynthetic function and therefore would contribute more actively to its host’s calcification and growth, thus playing a key role shaping temperate coral communities (Dimond and Carrington 2007).
In this study, we showed that the diversity of zooxanthellae phylotypes in the Western Mediterranean comprises at least two species. Including samples from different Mediterranean anthozoans, we have corroborated the occurrence of Symbiodinium sp. phylotype “Temperate A” in the scleractinian coral Cladocora caespitosa and the actiniaria Anemonia viridis. As well we show the presence of this clade in an anthozoan species that has not been previously study, the actiniarian Paranemonia cinerea. It is noteworthy to mention that our study differs from Visram et al. 2006, in that we found “Temperate A” clade in the actiniarian Bunodeopsis strumosa, that suggest that the Symbiodinium diversity within the same host taxa at the Western Mediterranean Basin might be richer than previously assessed. Moreover, we assessed that the diversity within phylotype “Temperate A” may include two or more closely related species that deserve further genetic and morphologic characterization.. We also verified the occurrence of Symbiodinium psygmophilum in the scleractinian coral Oculina patagonica in the Western Mediterranean Basin.
Given the importance of zooxanthellae for the survival of host Cnidaria, in deep studies on the characterization and ecology of this species are highly recommended. These kinds of studies are on its beginnings in the Mediterranean Sea. Provided the differences we have found, studies characterizing the genetic diversity of the endemic Symbiodinium clade “Temperate A” are needed. As well, the geographical differences we found on symbionts hosted by the scleractinian coral Cladocora caespitosa, suggests the need of a complete characterization of the Symbiodinium species present in the Mediterranean Basin along latitudinal and longitudinal gradients. All together these studies are of importance to better understand the ecology and evolution of these symbionts and host taxa at the Mediterranean Basin.
References
Baillie BK, Belda-Baillie CA, Maruyama T (2000) Conspecificity and Indo-Pacific distribution of Symbiodinium genotypes (Dinophyceae) from giant clams. J Phycol 36:1153–1161
Baker AC, Rowan R, Knowlton N (1997) Symbiosis ecology of two Caribbean Acroporid corals. In: Lessios HA, Macintyre IG (eds) Proceedings 8th international coral reef symposium, Vol. 2. Smithsonian Tropical Research Institute, Balboa, Panama, pp 1295–1300
Baker AC, Rowan R (1997) Diversity of symbiotic dinoflagellates (zooxanthellae) in scleractinian corals of the Caribbean and eastern pacific. In: Lessios HA, Macintyre IG (eds) Proceedings 8th international coral reef symposium, Vol. 2. Smithsonian Tropical Research Institute, Balboa, Panama, pp 1301–1306
Baker AC (1999) The symbiosis ecology of reef-building corals. Ph.D. dissertation. University of Miami, Miami, FL, 120 pp
Baker AC (2003) Flexibility and specificity in coral-algal symbiosis: diversity, ecology and biogeography of Symbiodinium. Annu Rev Ecol Evol Syst 34:661–689
Barbrook AC, Visram S, Douglas AE, Christopher JH (2006) Molecular diversity of dinoflagellate symbionts of Cnidaria: the psbA minicircle of Symbiodinium. Protist 157:159–171
Boudouresque CF (2004) Marine biodiversity in the Mediterranean: status of species, populations and communities. Scientific Reports Port-Cross Natural Park 20:97–146
Coll M, Piroddi C, Steenbeek J, Kaschner K, Lasram FBR, Aguzzi J, Ballesteros E, Bianchi CN, Cobera J, Dailianis T et al (2010) The biodiversity of the Mediterranean Sea: estimates, patterns, and threats. PLoS One 5:e11842
Coffroth MA, Lasker HR, Diamond ME, Bruenn JA, Birmingham E (1992) DNA fingerprints of a gorgonian coral: a method for detecting clonal structure in a vegetative species. Mar Biol 114:317–325
Coffroth MA, Santos SR (2005) Genetic diversity of symbiotic dinoflagellates in the genus Symbiodinium. Protist 156:19–34
Dimond J, Carrington E (2007) Temporal variation in the symbiosis and growth of the temperate scleractinian coral Astrangia poculata. Mar Ecol Prog Ser 348:161–172
Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783–791
Finney JC, Pettay DT, Sampayo EM, Warner ME, Oxendorf HA, LaJeunesse TC (2010) The relative significance of host-habitat, depth, and geography on the ecology, endemism, and peciation of coral endosymbionts in the genus Symbiodinium. Microb Ecol 60:250–263
Forcioli D, Merle P-L, Caligara C, Ciosi M, Muti C, Francour P, Cerrano C, Allemand D (2011) Symbiont diversity is not involved in depth acclimation in the Mediterranean sea whip Eunicella singularis. Mar Ecol Prog Ser 439:57–71
Furla P, Allemand D, Shick JM, Ferrier- Pagès C, Richier S, Plantivaux A, Merle PL, Tambutté (2005) The symbiotic anthozoan: a physiological chimera between algal and animal. Integr Comp Biol 45:595–604
Gouy M, Guindon S, Gascuel O (2010) SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 27:221–224
Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 5:696–704
Huelsenbeck JP, Ronquist F (2001) MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17:754–755
LaJeunesse TC (2001) Investigating the biodiversity, ecology and phylogeny of endosymbiotic dinoflagellates in the genus Symbiodinium using the ITS region: in search of a “species” level marker. J Phycol 37:866–880
LaJeunesse TC, Loh WKW, vanWoesik R, Hoegh-Guldberg O, Schmidt GW, Fitt WK (2003) Low symbiont diversity in southern great barrier reef corals relative to those of the Caribbean. Limnol Oceanogr 48:2046–2054
LaJeunesse TC, Thornhill DJ, Cox EF, Stnton FG, Fitt wK, Schmidt W (2004) High diversity and host specificity observed among symbiotic dinoflagellates in reef coral communities from Hawaii. Coral Reefs 23:596–603
LaJeunesse TC, Pettay T, Sampayo EM, Phongsuwan N, Brown B, Obura D, Hoegh-Guldberg O, Fitt WK (2010) Special Paper: Long-standing environmental conditions, geographic isolation and host–symbiont specificity influence the relative ecological dominance and genetic diversification of coral endosymbionts in the genus Symbiodinium. J Biogeogr 37:785–800
LaJeunesse TC, Parkinson J, Reimer JD (2012) A genetic-based description of Symbiodinium minutum sp. nov. and S. psygmophilum sp. nov. (Dinophyceae), two dinoflagellates symbiotic with cnidarian. J Phycol 48:1380–1391
Loh WKW, Carter D, Hoegh-Guldberg O (1998) Diversity of zooxanthellae from scleractinian corals of One tree island (The great barrier reef). In: Greenwood JG, Hall NJ (eds) Proceedings of the Australian coral reef society, 75th annual conference. University of Queensland, Brisbane, pp 141–150
Loh WKW, Loi T, Carter D, Hoegh-Guldberg O (2001) Genetic variability of the symbiotic dinoflagellates from the wide ranging coral species Seriatopora hystrix and Acropora longicyathus in the Indo-West Pacific. Mar Ecol Prog Ser 222:97–107
Meron D, Rodolfo-Metalpa R, Cunning R, Baker AC, Fine M, Banin E (2012) Changes in microbial communities in response to a natural pH gradient. ISME J 6:1775–1785
Pochon X, Putnam HM, Gates RD (2014) Multi-gene analysis of Symbiodinium dinoflagellates: a perspective on rarity, symbiosis and evolution. Peerj, e394.
Posada D, Crandall KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14:817–818
Rodriguez-Lanetty M, Loh W, Carter D, Hoegh-Gulberg O (2001) Latitudinal variability in symbiont specificity within the widespread scleractinian coral Plesiastrea versipora. Mar Biol 138:1175–1181
Rodriguez-Lanetty M (2003) Evolving lineages of Symbiodinium-like dinoflagellates based on ITS1 rDNA. Mol Phylogenet Evol 28:152–168
Rodriguez-Lanetty M, Hoegh-Guldberg O (2003) Symbiont diversity within the widespread scleractinian coral Plesiastrea versipora, across the northwestern Pacific. Mar Biol 143:501–509
Sampayo AM, Dove S, LaJeunesse (2009) Cohesive molecular genetic data delineate species diversity in the dinoflagellate genus Symbiodinium. Mol Ecol 18:500–519
Santos SR, Taylor DJ, Coffroth MA (2001) Genetic comparisons of freshly isolated vs. cultured symbiotic dinoflagellates: Implications for extrapolating to the intact symbiosis. J Phycol 37:866–880
Santos SR, Taylor DJ, Kinzie RA, Hidaka M, Sakai K, Coffroth AM (2002) Molecular phylogeny of symbiotic dinoflagellates inferred from partial chloroplast large subunit (23S)-rDNA sequences. Mol Phylogenet Evol 23:97–11
Savage AM, Goodson MS, Visram S, Trapido-Rosenthal H, Wiedenmann J, Douglas AE (2002) Molecular diversity of symbiotic algae at the latitudinal margins of their distribution: dinoflagellates of the genus Symbiodinium in corals and sea anemones. Mar Ecol Prog Ser 244:17–26
Templado J (2014) Future trends of Mediterranean biodiversity. In: Goffredo S, Dubinsky Z (eds) The Mediterranean Sea: Its history and present challenges, Chapter: 28. Springer, New York
Thornhill DJ, Kemp DW, Bruns BU, Fitt WK, Schmidt GW (2008) Correspondence between cold tolerance and temperate biogeography in a western Atlantic Symbiodinium (Dinophyta) lineage. J Phycol 44:1126–1135
Thornhill DJ, Yiang X, Pettay DT, Zhong SSR (2013) Population genetic dataof a model symbiotic cnidarian system reveal remarkable symbiotic specificity and vectored introductions across ocean basins. Mol Ecol 2:4499–4515
Trench RK (1993) Microalgal-invertebrate symbioses- a review. Endocytobiosis and Cell Res 9:135–175
Visram S, Wiedenmann J, Douglas AE (2006) Molecular diversity of symbiotic algae of the genus Symbiodinium (Zooxanthellae) in cnidarians of the Mediterranean Sea. J Mar Biol Assoc UK 86:1281–1283
Weis VM, Reynolds WS, deBoer MD, Krupp DA (2001) Host-symbiont specificity during onset of symbiosis between the dinoflagellates Symbiodinium spp. and planula larvae of the scleractinian coral Fungia scutaria. Coral Reefs 20:301–308
Acknowledgments
The authors want to acknowledge F. Canovas, J. Martinez-Garrido, A. Lerida and A. Addamo for their help in the field with sample collection; R. Garcia-Jimenez and S. Valente for their help and assistance in the molecular laboratory. Dr. Ester Serrão from CCMAR allowed us to carry out the genetic work in her laboratory. M.A. Coffroth for her help with new ideas. We would like also to thanks T. LaJeunesse for his revision on the manuscript and suggestions for improving it. S. Young and K. Nielsen did help us to improve the English grammar and edition. This study was in part performed within the Association of Marine Biology Laboratories Program (ASSEMBLE) under grant agreement no. 227799, carried out at the Center of Marine Sciences (CCMAR) at Algarve University; as well as supported by CUMFISH projects (PTDC/MAR/119363/2010; http://www.ccmar.ualg.pt/cumfish/) funded by Fundacão para Ciência e Tecnologia (Portugal), CGL2011-23306 funded by the Spanish Ministry of Science and Innovation. P. Casado-Amezua is currently supported by an internship from the Alfred Wegener Institute (AWI), Helmholtz Centre for Polar and Marine Research. M. González-Wangüemert is supported by a FCT postdoctoral fellowship (SFRH/BPD/70689/2010).
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Casado-Amezúa, P., Machordom, A., Bernardo, J. et al. New insights into the genetic diversity of zooxanthellae in Mediterranean anthozoans. Symbiosis 63, 41–46 (2014). https://doi.org/10.1007/s13199-014-0286-y
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DOI: https://doi.org/10.1007/s13199-014-0286-y