Turkish Journal of Zoology Turk J Zool (2015) 39: 998-1003 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1310-4

Morphology and phylogeny of the (: : Actiniaria) from Chabahar Bay, Iran

Gilan ATTARAN-FARIMAN*, Pegah JAVID, Arash SHAKOURI Department of Marine Biology, Faculty of Marine Sciences, Chabahar Maritime University, Chabahar, Iran

Received: 06.10.2013 Accepted/Published Online: 07.07.2015 Printed: 30.11.2015

Abstract: Carpet anemones of the genus Stichodactyla are characterized by having no calcium carbonate skeleton, being flattened with rather short tentacles, being exclusively tropical, and being hosts for clown fish species. Most of the species belonging to this genus, however, are often very similar morphologically; thus, identification by external features is rather difficult. In this study, we have described the morphology and phylogenetic affinities of a carpet anemone species collected from Chabahar Bay. Although the Iranian species showed different coloration patterns within the same geographical area, they strongly resemble Stichodactyla haddoni Saville- Kent, 1893 in having short tentacles densely covering the undulated oral disc and white, pointed, recognizable exocoelic tentacles. Comparison of 18S rDNA with other actiniarians revealed that the 2 Iranian species are closely related to Stichodactyla haddoni, , and . This is the first record of Stichodactyla haddoni from the southeastern coast of Iran and the northern part of the Sea of Oman.

Key words: Stichodactyla haddoni, Actiniaria, 18S rDNA, Iran’s southeastern coast

1. Introduction morphologic features among these clades is not completely Sea anemones are present in all water depths and well supported (Fukami et al., 2004; Sinniger et al., 2005; marine habitats (Rodriguez and Daly, 2010). Few Daly et al., 2008; Rodriguez and Daly, 2010). Members of morphological characters are useful in the of the order Actiniaria show the most variation in biology, anthozoans because they do not have many distinguishing anatomy, and life history among hexacorallians, so the characteristics; this makes taxonomic classification accurate determination of Actiniaria is unresolved yet difficult (Bernston et al., 1999). Thus, because there is no and is very problematic (Daly et al., 2008). There are some consensus about the phylogeny of Actiniaria based on factors that may be important in the sea anemone’s external morphologic characteristics, it is important to examine morphology and identification: habitat preferences and the DNA (Daly et al., 2010). Many biologists agree that coloration pattern (Daly et al., 2004, Fautin et al., 2008), phylogenetic analyses should be the basis of research in size of specimen and diameter of oral disc, type and size of different fields of biology (Soltis and Soltis, 2003). The tentacles and their arrangement on the oral disc, and kind molecular evolution among anthozoans, however, is of verrucae on the column (Fautin et al., 2008). slower compared with that of other marine invertebrates Several studies have been done on molecular aspects (Daly et al., 2010). of the class Anthozoa, and different regions of their The genus Stichodactyla has a well-developed adherent ribosomal gene have already been studied: 16S rDNA pedal disc, the column is broader than tall, and it has a (France et al., 1996), 18S rDNA (Song and Won, 1997), broad flat or undulating oral disc packed with many 28S rDNA (Chen et al., 1995), and the combination of short, hollow tentacles (Fautin et al., 2008), features that 16S rDNA and 18S rDNA (Bridge et al., 1995). Among distinguish it from other sea anemones. Many features in them, 18S rDNA acts more effectively in revealing the this genus (e.g., coloration patterns, tentacles) are shared relationships between lower-level taxa; longer markers between species, making their recognition in the field may have not valuable data in phylogenetic studies at the very difficult (Fautin et al., 2008). Although orders in genus level (Daly et al., 2010). The main purpose of this the subclass are presumably monophyletic study was the assessment and identification of 2 specimens (Daly et al., 2008), taxonomic information based only on of Stichodactyla haddoni species with different color * Correspondence: [email protected] 998 ATTARAN-FARIMAN et al. / Turk J Zool patterns through morphological and molecular analysis. leidyi Agassiz, 1865 (Ctenophora; Cyclocoela; Lobata; The specimens were collected from Chabahar Bay located Bolinopsidae) as the outgroup. on the southeastern coast of Iran.

2. Materials and methods 3. Results 2.1. Sampling, DNA extraction, and PCR conditions Stichodactyla haddoni [both CHIAS1 (Figures 1A–1C) Two specimens of Stichodactyla haddoni were collected and CHIAS2 (Figures 2A–2C)] has 2 kinds of tentacles. by hand from the intertidal zone of Chabahar Bay, on Exocoelic tentacles: pointed white tentacles that are the southeastern coast of Iran (25°35’N–36’N, 60°60’E). longer than endocoelic tentacles; these are present only Since there were 2 distinguishable morphs between the at the edge of the oral disc, and each of these alternates specimens, we named them CHIAS1 (CH: Chabahar, I: with many rows of small spherical tentacles on the oral Invertebrate, A: Anthozoa, S: Stichodactyla, 1: species 1) disc. Endocoelic tentacles: internal; each group between and CHIAS2 (CH: Chabahar, I: Invertebrate, A: Anthozoa, the white tentacles arises simultaneously and are the S: Stichodactyla, 1: species 2) according to their coloration same length or shorter than exocoelic tentacles; they patterns. Some specimens were transferred to the are in all parts of the oral disc with various colorations. laboratory and kept in aquaria for detailed morphological Each exocoelic tentacle of approximately 5 mm in length examination and measurements. The noted morphological alternates with rows of small spherical endocoelic tentacles features were compared and analyzed for the 2 specimens on the oral disc. The number of tentacles is lower near the with different colorations. mouth (an area of approximately 10–15 mm around the Tissue from the pedal disc or column was scalped mouth lacks tentacles). Oral disc is undulated and wider from the specimens and then frozen at –20 °C. DNA than pedal disc. was extracted by modified phenol-chloroform isoamyl The diameter of CHIAS1 is approximately 35–40 alcohol method and was suspended in 50–100 µL of cm when expanded; oral disc densely covered by

TE or D2W. The quality and quantity of extracted DNA short tentacles with bulbous sticky tips (Figure 1A). was determined via 1% agarose gel electrophoresis and Column yellow to light green, fully covered with rows of ethidium bromide staining. The extracted DNA was loaded nonadhesive verrucae. The mouth is orange, and the pedal onto electrophoresis gel. The amount of loaded DNA in disc is light brown to cream (Figure 1B). The tentacles of electrophoresis was approximately 15 ng/µL. Extracted the oral disc are brown and cream with white streaks, and DNA was used as the template in PCR amplification. PCR white exocoelic tentacles are visible only at the edge of the primers were as follows: 18Sa (forward) (5’-ACC CTG oral disc (Figure 1C). GTT GAT CCT GCC AGT-3’) (Medlin et al., 1988) and CHIAS2 is approximately 33–35 cm. Exocoelic tentacles 18Sb (reverse) (5’-GAT CCT TCT GCA GGT TCA CCT are long and visible at the edge of the oral disc (Figure 2A). AC-3’) (Medlin et al., 1988). Reactions were carried out in Endocoelic tentacles are dark brown and opaque green 50 µL of master mix, with 10X PCR buffer (Biofluxbiotech), with gray and purple coloration in some parts, and the –1 25 mM MgCl2, 10 mM dNTPs, 10 µmol µL of each mouth is red (Figure 2B). Rows of nonadhesive verrucae primer, 5 U of Taq DNA polymerase, and about 15 ng µL–1 are visible on the column (Figure 2C). of template. The 18S rDNA was amplified using a DNA Totals of 970 bp for CHIAS1 and 820 bp for CHIAS2 thermal cycler with the following profile: 95 °C for 4 min; were analyzed in the final alignment. The Iranian 38 cycles of 94 °C for 1 min, 56.5 °C for 1 min, and 72 °C specimens (CHIAS1 and CHIAS2) are within the family for 2 min and 30 s; and a final cycle of 72 °C for 10 min. (Figure 3). There were 48 base pairs of PCR products were visualized through 1% agarose gel, and nucleotide differences among the total 823 bases. In the the PCR products were then sequenced. survey using the 18S segment of DNA as the marker, 2.2. Phylogenetic analyses CHIAS1, Stichodactyla gigantea, and Heteractis magnifica DNA sequence base-calling errors were verified and have virtually identical sequences, unlike CHIAS2. The manipulated by manual inspection using BioEdit 7.1.11 morphological evidence and NJ analyses showed that (Hall, 1999). A BLAST search was performed in GenBank, CHIAS1 and CHIAS2 correspond to Haddon’s carpet and the matching homologous actiniarian sequences anemone Stichodactyla haddoni. The similarity of were retained for subsequent alignment. DNA sequence CHIAS1, S. gigantea, and Heteractis magnifica is supported alignments were carried out in Clustal-X 1.83 (Jeanmougin with 69% bootstrap support. CHIAS2 is also supported et al., 1998). Phylogenetic reconstruction was performed with 62% bootstrap support. Both specimens of CHIAS1 through the neighbor-joining (NJ) method (Saitou and and CHIAS2 are closely related to S. haddoni, with Nei, 1987) using a p-distance matrix. All phylogenetic 100% bootstrap support. Furthermore, morphological analyses were carried out with Mega 5.1 software (Tamura examination indicates that Iranian samples CHIAS1 and et al., 2011). The NJ tree was rooted using Mnemiopsis CHIAS2 belong to S. haddoni.

999 ATTARAN-FARIMAN et al. / Turk J Zool

Figure 1. Stichodactyla haddoni. A) CHIAS1 in situ, Chabahar Bay. B) CHIAS1 in aquaria. C) Specimen exposing exocoelic tentacles (Ex) and endocoelic tentacles (En). Scale bars: 10 cm for A and B, 5 mm for C. Photos A and B: Mohammad Bahman; Photo C: Pegah Javid.

Figure 2. Stichodactyla haddoni. A) CHIAS2 in situ (Ex: exocoelic tentacles are seen at the edge of the oral disc; En: endocoelic tentacles). B) CHIAS2 in aquaria. Note the coloration, tentacles, and red oral disc. The density of tentacles is lower near the mouth. C) Aboral view of CHIAS2: arrow shows the row of nonadhesive verrucae (V). The diameter of the oral disc is much broader than that of the pedal disc. Scale bars: 8 cm for A and B, 6 cm for C. Photo A: Mohammad Bahman; Photos B and C: Pegah Javid.

1000 ATTARAN-FARIMAN et al. / Turk J Zool

Figure 3. Neighbor-joining consensus tree for 23 species, including Iranian sea anemone species (CHIAS1 and CHIAS2), based on 18S rDNA sequences. Cl1: Stichodactylidae; Cl2: Actiniidae; Cl3: Hormathiidae; Cl4: Aiptasiidae; Cl5: Actinostolidae. The numbers beside the branches are bootstrap values with 1000 replications. Bootstrap supports under 50% are not shown in this analysis.

4. Discussion species are their anatomy, habitat, patterns of coloration, Phylogenetic frames can disclose the evolutionary patterns the approximate length of tentacles and their types, of many morphological characteristics (Soltis and Soltis, verrucae, and cnidae present on the column (Daly, 2004). 2003). Independent analyses of life history, anatomy, and As was mentioned before, some characteristics are used to molecular sequence cannot provide a full framework for distinguish different species of sea anemones: the habitat, understanding the phylogenetic relationships among as sea anemones may be found on substrates with dead hexacorallians, especially among sea anemones (Daly et coral species and sandy clay (Sen Gupta et al., 2003), or al., 2003). Due to lack of light, specimens frequently expel clay and silt with patches of fine coralline sand (Hashimi their zooxanthellae, making them lose their coloration et al, 1978), on rocks (Fautin et al., 2008), or in sea grass after a short time (Figure 1C). Other differences in how beds and muddy sand flats (Fautin et al., 2009); patterns they look in situ and in aquaria make identification of coloration, which are diverse among sea anemones difficult (Collingwood, 1868; Fautin, 2013). (Daly, 2004); the length of tentacles, which can be diverse Although different in color and size, CHIAS1 and in different species of sea anemones or among different CHIAS2 share many other characteristics such as number species of a genus (Fautin et al., 2008) (this factor was the and type of tentacles, verrucae, and form of oral disc. most important in our species’ identification); verrucae The characteristics that may vary among sea anemone that are not similar in all species of sea anemones, nor even

1001 ATTARAN-FARIMAN et al. / Turk J Zool in all species of genus Stichodactyla (Fautin et al., 2008); Recent studies have shown that fragments of 12S and and cnidae (Daly, 2004; Fautin et al., 2008). Fautin et al. 18S rDNA are more effective in recovering well-supported (2008) reported S. haddoni with an undulating oral disc nodes than 16S and 28S (Daly et al., 2010). Although with a diameter greater than that of the pedal disc, which molecular evolution is slow in Actiniaria (Daly et al., is covered with sticky tentacles, and exocoelic tentacles 2008), the nuclear gene evolution rate is higher than the that are more robust than endocoelic ones. These features rate of evolution in mitochondria (Hellberg, 2007; Daly et were the factors that we observed in conjunction with al., 2008); thus, it is better to study 18S rDNA instead of Stichodactyla haddoni in our study, too. mitochondrial genes. Different studies have been carried In comparison to Stichodactyla, which burrows in out on 18S rDNA among cnidarians (Bridge et al., 1995; sediments, has a wide mouth, and has longitudinal rows of Won, 1997; Bernston et al., 1999; Bernston et al., 2001; nonadhesive verrucae on the column (Fautin et al., 2008), Daly et al., 2002, 2003, 2008; Worthington Wilmer and Mitchell, 2008). Phylogenetic studies and relations among Heteractis Allman, 1864 rarely burrows in sediments or orders of hexacorallians are difficult and are independent sands. The tentacles are long and densely cover the oral of anatomy, life cycle, and molecular sequence analyses; disc, and can reach 40 mm in length and 3 mm in width thus, there are no clear relationships among hexacorallians when expanded. The mouth is small and hardly tractable; (Daly et al., 2003; Medina et al., 2006; Brugler and France, it is the center of the oral disc. The most distinctive feature 2007). that separates S. haddoni from S. gigantea is their exocoelic In our analyses, Stichodactyla and Heteractis are tentacles being more robust than their endocoelic supported with 100% bootstrap support. According to all tentacles (Fautin et al., 2008; Fautin, 2013). In this regard, the morphological and phylogenetic evidence, we concluded the morphology of CHIAS1 and CHIAS2 did not concur that Iranian specimens CHIAS1 and CHIAS2 belong to with the diagnosis of the genus Heteractis or Stichodactyla Stichodactyla haddoni. This is the first report of this species gigantea. for the southeastern coast of Iran, in Chabahar Bay.

References

Bernston EA, Bayer FM, McArthur AG, France SC (2001). Daly M, Chaudhuri A, Gusmao LC, Rodriguez E (2008). Phylogenetic Phylogenetic relationships within the Octocorallia (Cnidaria: relationships among sea anemones (Cnidaria: Anthozoa: Anthozoa) based on nuclear 18S rRNA sequences. Mar Biol Actiniaria). Mol Phylogenet Evol 48: 292–301. 138: 235–246. Daly M, Fautin DG, Cappola VA (2003). Systematics of the Bernston EA, France SC, Mullineaux LS (1999). Phylogenetic Hexacorallia (Cnidaria: Anthozoa). Zool J Linn Soc-LOND relationships within the class Anthozoa (Phylum Cnidaria) 139: 419–437. based on nuclear 18S rDNA sequences. Mol Phylogenet Evol Daly M, Gusmao LC, Reft AJ, Rodriguez E (2010). Phylogenetic 13: 417–433. signal in mitochondrial and nuclear markers in sea anemones Bridge D, Cunningham CW, DeSalle R, Buss LW (1995). Class- (Cnidaria, Actiniaria). Integr Comp Biol 50: 371–388. level relationships in the phylum Cnidaria: molecular and Daly M, Lipscomb DL, Allard MW (2002). A simple test: evaluating morphological evidence. Mol Biol Evol 12: 679–689. explanations for the relative simplicity of the Edwardsiidae Brugler MR, France SC (2007). The complete mitochondrial genome (Cnidaria: Anthozoa). Evolution 56: 502–510. of the black coral Chrysopathes formosa (Cnidaria: Anthozoa: Fautin DG (2013). Hexacorallians of the World. Lawrence, KS, USA: Antipatharia) supports classification of antipatharians within University of Kansas. Available online at http://hercules.kgs. the subclass Hexacorallia. Mol Phylogenet Evol 42: 776–788. ku.edu/hexacoral/anemone2/index.cfm. Chen CA, Odorico DM, ten Lohuis M, Veron JEN, Miller DJ (1995). Fautin DG, Crowther AL, Wallace CC (2008). Sea anemones Systematic relationships within the Anthozoa (Cnidaria: (Cnidaria: Anthozoa: Actiniaria) of Moreton Bay. Mem Anthozoa) using the 5’-end of the 28S rDNA. Mol Phylogenet Queensl Mus Nature 54: 35–64. Evol 4: 175–183. Fautin DG, Tan SH, Tan R (2009). Sea anemones (Cnidaria: Collingwood C (1868). Note on the existence of gigantic sea- Actiniaria) of Singapore: abundant and well-known shallow- anemones in the China Sea, containing within them quasi- water species. Raffles B Zool 22: 121–143. parasitic fish. Ann Mag Nat Hist 1: 31–33. France SC, Rosel PE, Agenbroad JE, Mullineaux LS, Kocher TD Daly M (2004). Anatomy and taxonomy of 3 species of sea anemones (1996). DNA sequence variation of mitochondrial large- (Cnidaria: Anthozoa: Actiniidae) from the Gulf of California, subunit rRNA provides support for a two-subclass organization including Isoaulactinia hespervolita Daly, n. sp. Pac Sci 58: of the Anthozoa (Cnidaria). Mol Mar Biol Biotechnol 5: 15–28. 377–390.

1002 ATTARAN-FARIMAN et al. / Turk J Zool

Fukami H, Budd AF, Levitan DR, Jara J, Kersanach R, Knowlton N Saitou N, Nei M (1987). The neighbor-joining method: a new (2004). Geographic differences in species boundaries among method for reconstructing phylogenetic trees. Mol Biol Evol members of the Montastraea annularis complex based on 4: 406–425. molecular and morphological markers. Evolution 58: 324–337. Sen Gupta R, Patel MI, Ramamoorthy K, Deshmukhe G (2003). Hall TA (1999). BioEdit: a user-friendly biological alignment editor Coral Reefs of the Gulf of Kachchh: A Subtidal Videography. and analysis program for Windows 95/98/NT. Nucleic Acids Gujarat, India: Gujarat Ecological Society. Symp Series 41: 95–98. Shearer TL, Van Oppen MJH, Roman SL, Worheid G (2002). Slow Hashimi NH, Nair RR, Kidwai RM (1978). Sediments of the Gulf mitochondrial DNA sequence evolution in the Anthozoa of Kutch: a high-energy tide dominated environment. Indian (Cnidaria). Mol Ecol 11: 2475–2487. J Mar Sci 7: 1–7. Sinniger F, Montoya-Burgos JI, Chevaldonne P, Pawlowski J (2005). Hellberg ME (2007). Footprints on water: the genetic wake of Phylogeny of the order Zoantharia (Anthozoa, Hexacorallia) dispersal among reefs. Coral Reefs 26: 463–473. based on the mitochondrial ribosomal genes. Mar Biol 147: 1121–1128. Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ (1998). Multiple sequence alignment with Clustal X. Trends Soltis DE, Soltis PS (2003). The role of phylogenetics in comparative Biochem Sci 23: 403–500. genetics. Plant Physiol 132: 1790–1800. Medina M, Collins AG, Takaoka TL, Kuehl JV, Boore JL (2006). Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S Naked corals: skeleton loss in Scleractinia. P Natl Acad Sci 103: (2011). MEGA5: Molecular evolutionary genetics analysis 9096–9100. using maximum likelihood, evolutionary distance, and maximum parsimony method. Mol Biol Evol 28: 2731–2739. Medlin L, Elwood HJ, Stickel S, Sogin ML (1988). The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding Won JH (1997). Systematic relationship of the anthozoan orders regions. Gene 71: 491–499. based on the partial nuclear 18S rDNA sequences. Korean J Biol Sci 1: 43–52. Rodriguez E, Daly M (2010). Phylogenetic relationships among deep sea and chemosynthetic sea anemones: Actinoscyphiidae and Actinostolidae (Actiniaria: Mesomyaria). PLoS One 5: 1–8.

1003