Morphological and molecular variability ofthe sea anemone Phymanthus crucifer(Cnidaria, Anthozoa, Actiniaria, Actinoidea)

ricardo gonza’ lez-mun~oz1,2

, nuno simo~es1

, maite mascaro’1

, jose’ luis tello-musi3

,

mercer r. brugler4,5

and estefani’a rodri’guez4

1Unidad Multidisciplinaria de Docencia e Investigacion en Sisal (UMDI-Sisal), Facultad de Ciencias, Universidad NacionalAutonoma de Mexico (UNAM), Puerto de Abrigo, Sisal, C.P. 97356 Yucatan, Mexico, 2Posgrado en Ciencias del Mar y Limnologıa(PCMyL), UNAM, Instituto de Ciencias del Mar y Limnologıa (ICMyL), Circuito Exterior, Ciudad Universitaria, C.P. 04510,Mexico, 3Laboratorio de Zoologıa, Facultad de Estudios Superiores Iztacala (FES-I), UNAM, Avenida de los Barrios 1, Los ReyesIztacala, C.P. 54090 Estado de Mexico, Mexico, 4Division of Invertebrate Zoology, Sackler Institute for Comparative Genomics,American Museum of Natural History, Central Park West at 79th Street, New York, NY 10024, USA, 5Biological SciencesDepartment, NYC College of Technology (CUNY), 300 Jay Street, Brooklyn, NY 11201, USA

The shallow water sea anemone Phymanthus crucifer exhibits three distinct morphotypes, characterized by the presence orabsence of protuberances on the marginal tentacles, as well as intermediate forms. The taxonomic status of the different mor-photypes and the diagnostic value of protuberances on the tentacles have been debated for this species and the familyPhymanthidae. We analysed the external and internal anatomy, cnidae and three mitochondrial molecular markers forrepresentatives of each of the three morphotypes. In addition, we address the putative monophyly of the familyPhymanthidae based on molecular data. With the exception of the protuberances, our morphological and molecularresults show no differences among the three morphotypes; thus, we consider this feature to be intraspecific variabilitywithin P. crucifer. Furthermore, molecular data reveal that the family Phymanthidae is not monophyletic. In addition,we discuss several diagnostic morphological features of the family Phymanthidae.

Keywords: Phymanthidae, mitochondrial DNA, marginal tentacles, cnidocysts, morphotypes, coral reefs

Submitted 20 April 2014; accepted 21 June 2014; first published online 31 July 2014

I N T R O D U C T I O N

Sea anemones of the family Phymanthidae Andres, 1883(Actiniaria: Actinoidea) are distinguished by verrucae on thedistal column, no marginal sphincter muscle or a weak endo-dermal one, and two kinds of tentacles: marginal tentaclesarranged in cycles that may have knoblike or branched pro-tuberances, and discal tentacles arranged radially, typicallyvery short, and vesicle-like (Carlgren, 1949; Rodrıguez et al.,2007).

Phymanthidae currently comprises two genera:Phymanthus Milne-Edwards & Haime, 1851 with elevenvalid species; and Heteranthus Klunzinger, 1877 with twovalid species (Fautin, 2013). These two genera are traditionallydistinguished by the presence of lateral protuberances (papilli-form or ramified) in the marginal tentacles and no marginalsphincter (or an indistinct one) in Phymanthus, whereasHeteranthus has smooth marginal tentacles without protuber-ances and a weak circumscribed marginal sphincter (Carlgren,1949).

Nevertheless, morphs with and without protuberances inthe marginal tentacles (as well as intermediate morphs) havebeen reported in specimens of Phymanthus crucifer (LeSueur, 1817) (Duerden, 1897, 1898, 1900, 1902; Stephenson,1922; Cairns et al., 1986). Verrill (1900, 1905) suggested thatmorphs with and without protuberances in the marginal ten-tacles should be treated as separate species that could hybrid-ize; however Duerden (1897, 1900, 1902) argued that all formsshould be treated as a single species based on the existence offorms with intermediate stages of tentacular protuberances.This morphological variability on marginal tentacles reportedfor P. crucifer challenges the value of this feature as a genus-level character within Phymanthidae.

Although the size of cnidae alone is not generally consid-ered a specific taxonomic diagnostic character due to its vari-ability within conspecific individuals (Fautin, 1988, 2009;Williams, 1996, 1998, 2000; Acuna et al., 2003, 2004;Ardelean & Fautin, 2004; Acuna & Garese, 2009), severalstudies have proposed quantitative analyses of the cnidae tohelp distinguish among colour morphs in some species(Allcock et al., 1998; Watts & Thorpe, 1998; Manchenkoet al., 2000; Watts et al., 2000). Watts & Thorpe (1998)found significant differences in the size of holotrichs in theacrorhagi of the upper-shore morphotype of Actinia equina(Linnaeus, 1758), suggesting that these could help distinguishbetween the mid- and lower-shore morphotypes of the

Corresponding author:R. Gonzalez-MunozEmail: ricordea.gonzalez@gmail.com

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Journal of the Marine Biological Association of the United Kingdom, 2015, 95(1), 69–79. # Marine Biological Association of the United Kingdom, 2014doi:10.1017/S0025315414000988

species. Other attempts to distinguish between colour mor-photypes using cnidae size alone found slight differencesthat do not support the use of this feature to separatespecies (Chintiroglou & Karalis, 2000).

In this study, we examined representatives of the three dif-ferent marginal tentacular morphs of Phymanthus crucifer(with and without protuberances and intermediate forms) inorder to identify morphological, cnidae and/or molecular dis-tinctions that would enable separation of the morphs into dif-ferent species or corroborate the broad phenotypic plasticityof P. crucifer. In addition, we tested the monophyly ofPhymanthidae using three mitochondrial markers.

M A T E R I A L S A N D M E T H O D S

Morphological and cnidae analysesWe catalogued the marginal tentacular morphotypes ofPhymanthus crucifer as follows: morphotype 1 (M1), speci-mens with protuberances in all marginal tentacles; morpho-type 2 (M2), specimens completely lacking protuberances inall marginal tentacles (i.e. smooth tentacles); and morphotype3 (M3), specimens with some smooth marginal tentacles andsome marginal tentacles with protuberances.

Twelve specimens (four per morphotype) were collected inLa Gallega reef (19813′13′′N 96807′37′′W) of the VeracruzReef System in the Gulf of Mexico in 2010; three additionalspecimens (one of each morphotype) were collected fromPuerto Morelos reef (20855′50.7′′N 86849′24′′W) in theMexican Caribbean (Figure 1). Collections were conductedby hand, snorkelling or SCUBA diving, and using a hammerand chisel. Collected specimens were transferred to the labora-tory and maintained in an aquarium to register their colourwhile alive (Figure 2). Specimens were relaxed in a 5%MgSO4 seawater solution and fixed in 10% seawater–bufferedformalin. Additionally, small samples of tissue were obtainedfrom the pedal disc and preserved in 96% ethanol.Measurements of column height, as well as pedal and oral

disc diameter were obtained from fixed specimens; fragmentsof selected specimens were dehydrated and embedded in par-affin. Histological sections 6–10 mm thick and stained withhaematoxylin–eosin (Estrada-Flores et al., 1982) were pre-pared to examine internal anatomy.

Data on cnidae were obtained from four representatives ofeach of the three morphotypes (a total of 12 individuals), allcollected from La Gallega reef. Seven squash preparationswere obtained from the main tissue types (�1 mm3) of eachspecimen. We analysed cnidae from the marginal tentaclestips (mtt), discal tentacles (dt), actinopharynx (ac), filaments(fi), column (co), vesicle-like marginal projections (vp), andprotuberances on the marginal tentacles (pr/mt). For speci-mens of M2 (lacking protuberances), cnidae preparations ofthe marginal tentacles were obtained from regions wherethese protuberances regularly develop in morphotypes M1and M3. From each of the seven squash preparations, thelength and width of 40 undischarged capsules (replicates) ofeach type of cnidae were randomly measured using DICmicroscopy 1000 × oil immersion (following Williams,1996, 1998, 2000).

Cnidae samples were ordered in a bi-dimensional spaceusing principal component analysis (PCA). Differences inordination given by morphotype, individual specimen andtype of cnidae, as well as the interaction terms among thesefactors were analysed using a permutational MANOVA pro-cedure (Anderson, 2001; McArdle & Anderson, 2001).Differences among cnidae were analysed for each type oftissue separately. The PERMANOVA procedure was appliedon resemblance matrices based on the Euclidian distancebetween samples. Although length and width of the capsuleswere in the same measurement scale, data were standardizedand normalized prior to analyses. The statistical model usedwas given by:

Yijkl = a+ Mi + I(M) j(i) + Tk + MTik + I(M)T j(i)k + Sijkl

where Y is the response matrix with n samples (number ofrows depending on tissue type; Table 2) ∗ P ¼ 2 variables

Fig. 1. Map of the southern Gulf of Mexico and Mexican Caribbean indicating the localities sampled in this study.

70 ricardo gonza’ lez-mun~ oz et al.

(number of columns: length and width); M is a fixed factorrepresenting morphotype (with three levels); a is the coeffi-cient representing the intercept of the multivariate regression;I is a random factor representing individuals nested in M(with four levels); T is the fixed factor representing type ofcnidae (with three or two levels, depending on tissue kind)and is orthogonal to M and I; MT and I(M)T are correspond-ing interactions terms; and S is the residual matrix.Permutation procedures were applied to obtain appropriatedistributions for the pseudo-F statistic under the null hypoth-esis. All analyses were performed using permutations of resi-duals under the reduced model, resulting in a range from 909to 999 unique permutations for each F-test. The experimentaldesign was balanced in every case, and the partitioning of vari-ation was achieved so that the test statistic (pseudo-F) repre-sents the proportion of the variation in the bi-dimensionalcloud that is explained by the source of variation being tested.

Specimens, as well as histological and cnidae preparations,were deposited in the Collection of Cnidarians of the Gulf ofMexico and Mexican Caribbean Sea (Registration code:YUC–CC–254–11) of the Unidad Multidisciplinaria deDocencia e Investigacion en Sisal (UMDI-Sisal) at theUniversidad Nacional Autonoma de Mexico (UNAM).

Molecular analysesAcquisition of molecular data followed the protocol detailedin Lauretta et al. (2013). We obtained DNA sequences of

three mitochondrial (12S and 16S rDNA and cox3) regionsfor 14 specimens (11 from La Gallega reef and three fromPuerto Morelos reef). Phymanthus crucifer haplotypes werecompared to available GenBank sequence data forPhymanthus loligo (Hemprich & Ehrenberg in Ehrenberg,1834) and Heteranthus sp. (for GenBank accession numberssee Rodrıguez et al. (2014) and Crowther (2013), respectively).Divergence estimates (based on the Kimura 2-parameter(K2P)) were obtained using Mega v.5.05 (Tamura et al., 2013).

Herein, we provide new sequences for Phymanthus cruciferwhich were added to the data matrix presented in Rodrıguezet al. (2014) after removing all hexacoral taxa not belongingto Actiniaria (except the antiphatharian Leiopathes Haime,1849, which was used as an outgroup) and addingHeteranthus sp.; for a complete account of taxa included inthis study, we refer readers to Rodrıguez et al. (2014). Newsequences have been deposited in GenBank (Table 1).

DNA sequences of each marker were separately alignedusing MAFFT v.7 (online at http://mafft.cbrc.jp/alignment/server/; Katoh et al., 2002, 2005; Katoh & Toh, 2008) usingthe following settings and parameters: Strategy, L-INS-i(recommended for ,200 sequences with one conserveddomain and long gaps); scoring matrix, 200PAM/k ¼ 2; gapopening penalty, 1.53; offset value, 0.05; max. iterate, 1000;and retree, 1. We then concatenated the three mitochondrialmarkers to create a single dataset for 115 taxa and 2697 sites.

The Akaike information criterion (AIC) was implementedwithin jModelTest v.2.1.2 (Darriba et al., 2012) to determine

Fig. 2. Images of specimens examined: (A–D) morphotype 1 (M1); (E–G) morphotype 2 (M2); (H–K) morphotype 3 (M3). Scale bars: 10 mm.

characterizing variability within phymanthus crucifer 71

the appropriate evolutionary model (TIM2 + I + G) and cor-responding parameters (p-inv ¼ 0.0470, gamma shape ¼0.3360, freqA ¼ 0.3034, freqC ¼ 0.1821, freqG ¼ 0.2212,freqT ¼ 0.2933, (AC) ¼ 1.3194, (AG) ¼ 5.0386, (AT) ¼1.3194, (CG) ¼ 1.0000, (CT) ¼ 8.7441, (GT) ¼ 1.0000)(number of candidate models: 88; number of substitutionschemes: 11; base tree likelihood calculations: BIONJ usingPhyML v3.0 (Guindon et al., 2010)).

We searched for optimal trees using maximum likelihood(ML) within PhyML v.3.0 (http://www.atgc-montpellier.fr/phyml/; Guindon & Gascuel, 2003). The following parameterswere implemented within PhyML: substitution model ¼GTR + I + G (the online version of PhyML does not imple-ment TIM2, and GTR had a DAIC of 2.3); substitution ratecategories ¼ 6; p-inv ¼ 0.0470; gamma shape ¼ 0.3360; start-ing tree ¼ BIONJ; tree improvement ¼ SPR & NNI; opti-mized tree topology and branch lengths; and bootstrapreplicates ¼ 350. We also conducted tree searches undermaximum parsimony (results not shown) with TNT v.1.1(random and consensus sectorial searches, tree drifting and

100 rounds of tree fusing; Goloboff et al., 2008). In all ana-lyses, gaps (–) were treated as missing data. Trees ofminimum length were found at least five times. The concate-nated data set was subjected to 1000 rounds of bootstrapresampling to assess support for clades.

R E S U L T S A N D D I S C U S S I O N

Morphological analysesAll twelve specimens examined from La Gallega displayedexternal morphological diagnostic taxonomic features corre-sponding to Phymanthus crucifer, including verrucae in thedistal column arranged in longitudinal rows, column color-ation with flame-like staining pattern, discal tentaclesarranged in radial rows from peristoma to margin, and mar-ginal tentacles hexamerously arranged. The only externalmorphological difference among specimens, aside from color-ation patterns, was the marginal tentacular protuberances.Internal anatomy was also similar in all the specimens (seeGonzalez-Munoz et al., 2012 for a complete description ofthe taxonomic diagnostic features of P. crucifer).

Size of specimens (pedal and oral disc diameter andcolumn height) and number of verrucae per longitudinalrow did not exhibit a consistent pattern that could be asso-ciated with any of the three marginal tentacular morphs(Table 2). The three morphotypes contained both relativelysmall and larger specimens, suggesting that the developmentof protuberances on marginal tentacles is not related to differ-ent growth stages of these organisms in the wild.

Colour patterns of the oral disc and tentacles varied amongall the specimens examined but did not show a consistentpattern characterizing a particular morph (Figure 2A–K).The oral disc is mainly green, but presented a distinct tone,from olive green (Figure 2A, C–E, G) to dark green(Figure 2B, F); it could also be brown (Figure 2H, K), orwith endocelic radial rows marking the arrangement of thediscal tentacles (Figure 2I–J). The mouth was primarily the

Table 1. Voucher specimen location and GenBank accession numbers for new sequences provided in this study. See Rodrıguez et al. (2014) for a com-plete list of taxa and data included in the analysis and Crowther (2013) for data regarding Heteranthus sp. UMDI-Sisal, Unidad Multidisciplinaria de

Docencia e Investigacion en Sisal, UNAM; AMNH, American Museum of Natural History.

Family Species ID number Collection locality Voucher

Phymanthidae Phymanthus crucifer RG-128 GoM UMDI-SisalPhymanthus crucifer RG-129 GoM UMDI-SisalPhymanthus crucifer RG-130 GoM UMDI-SisalPhymanthus crucifer RG-131 GoM UMDI-SisalPhymanthus crucifer RG-133 GoM UMDI-SisalPhymanthus crucifer RG-134 GoM UMDI-SisalPhymanthus crucifer RG-138 GoM UMDI-SisalPhymanthus crucifer RG-143 GoM UMDI-SisalPhymanthus crucifer RG-182 GoM UMDI-SisalPhymanthus crucifer RG-184 GoM UMDI-SisalPhymanthus crucifer RG-187 GoM UMDI-SisalPhymanthus crucifer RG-200 MC AMNH-5312Phymanthus crucifer RG-219 MC UMDI-SisalPhymanthus crucifer RG-220 MC AMNH-5316

Because all sequences were identical across 16S and cox3, only a single sequence for each gene was uploaded to GenBank (16S: KJ910345; cox3: KJ910346).Two haplotypes were recovered for 12S; thus a single sequence representing each haplotype was submitted to GenBank (haplotype 1: KJ910343; haplotype2: KJ910344).

Table 2. Morphological analysis of all three morphotypes; all measure-ments are in mm. pd, pedal disc diameter; ch, column height; od, oraldisc diameter; nv, range of the number of verrucae per longitudinal

row; sx, sex; (?), no gametogenic tissue present.

Morph Specimen code pd ch od nv sx

M1 M1.1 11 31 36 2–3 MaleM1.2 23 20 48 3–4 MaleM1.3 25 22 45 2–5 (?)M1.4 32 23 53 2–4 (?)

M2 M2.1 22 28 59 2–4 FemaleM2.2 23 26 49 3–4 FemaleM2.3 27 26 51 3–6 MaleM2.4 10 8 38 2–4 (?)

M3 M3.1 16 34 48 3–7 MaleM3.2 23 16 44 3–5 (?)M3.3 20 29 54 4–5 (?)M3.4 32 18 46 2–3 Female

72 ricardo gonza’ lez-mun~ oz et al.

same colour as the oral disc or exceptionally bright green orbright orange in some specimens (Figure 2F, I and 2D,respectively). The peristoma often had a lighter tone thanthe rest of the oral disc (Figure 2B, G, H, K). Marginal tenta-cles without protuberances in representatives of morph M2and some of M3 presented longitudinal rows of yellowish,brownish or white colorations (Figure 2E–G, I–J); andsome marginal tentacles had purple shades at their tips(Figure 2I, K). Colour pattern is a controversial character todistinguish sea anemones; some species are distinguished bycolour patterns while others have distinct colour morphsthat are considered to be phenotypic plasticity due to localgenetic adaptations (Stoletzki & Schierwater, 2005).

Phymanthus crucifer is dioecious and not thought toundergo asexual reproduction (Jennison, 1981). We found

spermatic vesicles (males) in all three morphotypes(Table 2), but oocysts in only some specimens of morphsM2 and M3. Nevertheless, oocysts have been reported in spe-cimens of morph M1 in previous studies (Gonzalez-Munozet al., 2012). In most dioecious species of cnidarians, malesand females are macroscopically indistinguishable (Fautin,1992), whilst sexual dimorphism has only been reported fora few hydrozoan and scyphozoan species (Fautin, 1992), andfor the actiniarian Entacmaea quadricolour (Leuckart inRuppell & Leuckart, 1828) (Scott & Harrison, 2009).

Crowther (2013) suggested that the symbiotic relationshipwith zooxanthellae is likely associated with the formation oflateral protuberances in the tentacles as it occurs in otherspecies such as Lebrunia coralligens (Wilson, 1890) andLebrunia danae (Duchassaing & Michelotti, 1860). However,

Fig. 3. Cnida types and their distribution among tissues per morphotype (M1, M2, M3). Scale bars: 25 mm.

characterizing variability within phymanthus crucifer 73

we found zooxanthellae in all specimens examined, includingthose without protuberances (M2). Quantitative comparisons ofthe densities of zooxanthellae within the different morphotypesmay offer some insight about the feasibility of this hypothesis.

Cnidae analysesWe found the same types of cnidae (cnidom) in all samplesexamined, regardless of morphotype (Figure 3). The cnidomof Phymanthus crucifer included basitrichs, microbasicp-mastigophores and spirocysts, as previously reported forthe family and genus (Carlgren, 1949). We did not find anyadditional types of cnidae in morphotypes M1 and M3(those with protuberances in the marginal tentacles). It isunlikely that the protuberances on the marginal tentaclescould be acting as structures for competition because agonisticbehaviour in actiniarians is usually associated with the pres-ence of holotrichs, a type of nematocyst in specialized struc-tures such as acrorhagi and catch tentacles (Bigger, 1988;Williams, 1991) commonly found in some shallow water seaanemone species (Daly, 2003; Fautin, 2009).

We measured 560 cnidae capsules per specimen, separatedinto 14 categories of cnidae (basitrichs, microbasicp-mastigophores and spirocysts) and tissue type; this addedto a total of 6720 capsules measured (Figure 3). Our resultsshowed no significant variation in the size of cnidaebetween morphotypes (Table 3), whereas cnidae varied insize within each morphotype depending on cnidae type andindividual specimens (Figure 4A–G).

The PCA ordination of samples from all tissue typesshowed that the first principal component explained from60 to 94.5% of the variability of the cnidae size dependingon the type of tissue being analysed (Table 3). Thus, the firstprincipal component represents the variability in cnidaelength. The percentage of variation explained by the secondprincipal component was low (from 5.5 to 21.3%) in cnidaefrom ac, fi, pr/mt, dt and mtt, but relatively high in cnidaefrom co and vp (from 35.9 to 40.0%) (Table 3). This secondprincipal component represents cnidae width.

In ac and fi the variation in cnidae width was higher formicrobasic p-mastigophores than for basitrichs (Figure 4A–B). This was not the case in co, pr/mt, dt, mtt and vp tissues,in which cnidae width was similar among all types examined(Figure 4C–G).

Acuna et al. (2007, 2011) only considered length whencomparing cnidae sizes among specimens. Although ourresults confirm that length was the variable that explainedmost of the variation between samples (60–94.5%), we

found slight differences in the width of some types of cnidae(e.g. microbasic p-mastigophores), a feature that should beconsidered in future studies.

The different morphotypes did not explain the variation ofcnidae size in any of the tissues examined (Table 3: Morph).The ordination of samples from all types of tissue wassimilar regardless of the morphotype they came from (seeFigure 4A–G). By contrast, differences in cnidae size amongspecimens within each morphotype were significant for alltissue types (Table 3: Ind(Morph) and Ind(Morph) × Type).Cnidae size (both length and width considered) also variedsignificantly depending on cnidae type (Table 3: Type), butdifferences in size between cnidae types were similar amongall three morphotypes (Table 3: Morph × Type). Overall,these results suggest that individuals constitute the mainsource of variation when the size of cnidae are examined.

Edmands & Fautin (1991) noted that the size of nemato-cysts does not appear to correlate with animal size inAulactinia veratra (Drayton in Dana, 1846), and Acunaet al. (2007) suggest that there is no functional relationshipbetween cnida length and body weight in Oulactis muscosa(Drayton in Dana, 1846). Thus, although the diameter ofthe pedal disc is slightly variable between examined specimensof P. crucifer (Table 2), we found it unnecessary to include thepedal disc as a covariable in the analyses.

Molecular analyses

variation within phymanthus crucifer

Comparison of aligned sequences for cox3 (663 base pairs (bp)in length) and 16S (428 bp) did not reveal any variationamong individuals or morphotypes from the Gulf of Mexicoor Mexican Caribbean. However, mitochondrial 12S(824 bp) revealed two haplotypes that were distinguished bya single substitution (K2P distance ¼ 0.1215%, see Table 4),but these haplotypes were not specific to any particular mor-photype. While haplotype 1 (differentiated by a single adeninesubstitution) was specific to Gulf of Mexico specimens, it wasshared by all three morphotypes. Haplotype 2 (differentiatedby a single guanine substitution) was more broadly distribu-ted, being shared between specimens in the Gulf of Mexicoand Mexican Caribbean. Within the Gulf of Mexico, haplo-type 2 was shared by M2 and M3, while in the MexicanCaribbean it was shared by all three morphotypes. Table 4summarizes divergence estimates among sequences withinmorphotypes of Phymanthus crucifer and representatives ofthe family Phymanthidae (P. loligo and Heteranthus sp.).

Table 3. Probability associated with pseudo-F values obtained through restricted permutations of the residuals of MANOVA models applied to the simi-larity matrices (Euclidian distance) calculated from cnidae data sizes (length and width). ac, actinopharynx; co, column; fi, filaments; pr/mt, protuberances

or middle part of the tentacle; dt, discal tentacle; mtt, marginal tentacle tip; vm, vesicle-like marginal projections.

Source ac co fi pr/mt dt mtt vp

PC1 % of variation 90.7 64.1 85.9 87.6 94.5 78.7 60.0PC2 % of variation 9.3 35.9 14.1 12.4 5.5 21.3 40.0Morph 0.858 0.534 0.912 0.895 0.572 0.826 0.235Ind(Morph) 0.001 0.001 0.001 0.001 0.001 0.001 0.001Type 0.001 – 0.001 0.001 0.001 0.001 –Morph × type 0.918 – 0.873 0.117 0.855 0.163 –Ind(Morph) × type 0.001 – 0.001 0.001 0.001 0.001 –Total number of samples 1440 480 1440 960 960 960 480

74 ricardo gonza’ lez-mun~ oz et al.

Because mitochondrial DNA (mtDNA) exhibits low levelsof sequence divergence within and among anthozoan species,finding no variation in sequences from conspecifics is not

unexpected, even in those from potentially isolated popula-tions that are geographically distant from each other(Shearer et al., 2002; Hellberg 2006; Brugler et al., 2013).Sequence divergence based on 12S was 15–17 times higherbetween Phymanthus crucifer and P. loligo or Heteranthussp. than between the two haplotypes obtained for P. crucifer.Thus, although anthozoan mtDNA is characterized by lowlevels of divergence, we would expect at least a similardegree of divergence among the morphotypes of P. cruciferif they were indeed distinct species. If all three P. crucifer mor-photypes are indeed a single species, then mitochondrial 12Srevealed, for the first time, intraspecific variation withinsea anemones.

S Y S T E M A T I C S A N D T A X O N O M I CS T A T U S O F P H Y M A N T H I D A E

A phylogenetic reconstruction based on the three concate-nated mitochondrial genes recovered the two 12S-basedPhymanthus crucifer haplotypes as sister taxa, and these assister to P. loligo (Figure 5). However, Heteranthus sp. isrecovered as sister to the actiniid genus Anemonia Risso,

Fig. 4. Principal component analyses of cnidae data (length/width) of all types of cnidae in each type of tissue; data from all specimens examined. Green dots,cnidae of M1; dark blue dots, cnidae of M2; light blue dots, cnidae of M3. Cnidae from: (A) actinopharynx; (B) filaments; (C) column; (D) marginal vesicles; (E)marginal tentacles; (F) discal tentacles; (G) protuberances midtentacle.

Table 4. Divergence estimates (K2P) based on sequence comparisons ofthe three mtDNA markers. Comparisons were made betweenPhymanthus crucifer and Phymanthus loligo, as well as between

Phymanthus crucifer and Heteranthus sp. NA, not available.

Heteranthus sp. P. crucifer P. loligo

12SHeteranthus sp. 1.93%P. loligo 0.12%P. crucifer 2.06%

16SHeteranthus sp. NAP. loligo 1.18%P. crucifer NA

cox3Heteranthus sp. 1.73%P. loligo 3.06%P. crucifer 2.39%

12S, 792 base pairs (bp) compared; 16S, 428 bp compared; cox3, 513 bpcompared.

characterizing variability within phymanthus crucifer 75

1826, thus rendering Phymanthidae polyphyletic. All studiedmembers of Phymanthidae grouped within Actinoidea(Rodrıguez et al., 2014), a superfamily of mainly shallow-

water sea anemones (Rodrıguez et al., 2012, 2014). Ourresults concur with those of Crowther (2013), who includeda higher taxon sampling of the superfamily Actinoidea in

Fig. 5. Phylogenetic reconstruction of the Actiniaria. Tree resulting from PhyML analysis of concatenated 12S, 16S and cox3. Grey boxes indicate superfamilieswithin the order; the name of each superfamily is inside or next to the coloured box. Species epithets are given only for genera represented by more than one species;for a complete list of taxa, see Rodrıguez et al. (2014). Numbers above the branches are bootstrap resampling values expressed as a percentage; values ,50 notindicated; filled-in circles indicate nodes with support of 100%. Taxa in bold belong to Phymanthidae.

76 ricardo gonza’ lez-mun~ oz et al.

her study of the families Thalassianthidae Milne-Edwards &Haime, 1851 and Aliciidae Duerden, 1895.

The presence of Phymanthus crucifer morphotypes withoutprotuberances in the marginal tentacles renders Carlgren’s(1949) major distinction between the two genera ofPhymanthidae invalid. The marginal sphincter muscle, theother feature used by Carlgren (1949) to distinguishbetween these genera, is also problematic. Heteranthus is char-acterized by a weak but circumscribed marginal sphincter,whereas most species of Phymanthus lack a marginal sphinc-ter (Carlgren, 1949). However, Phymanthus muscosus(Haddon & Shackleton, 1893) has a very feeble sphinctermuscle (Haddon, 1898). Carlgren (1900) initially placedHeteranthus within a different family, HeteranthidaeCarlgren, 1900, but he later placed it within Phymanthidae,based on similarities with Phymanthus (Carlgren, 1943). Werecovered Heteranthus as nested within Actiniidae (seeFigure 5) suggesting that discal tentacles have evolved inde-pendently at least twice within Actinoidea. A comprehensiverevision of the family Phymanthidae and a redefinition of itsdiagnostic characters are needed to establish its membership.

Based on external and internal morphological features,cnidae data, and mitochondrial DNA, we conclude that allmorphotypes of Phymanthus crucifer represent a singlespecies, despite differences in the presence or absence of pro-tuberances in the marginal tentacles. The significance andfunction of the protuberances in the marginal tentaclesremains unknown within P. crucifer, but might be related tospecific adaptations to the surrounding environment.

A C K N O W L E D G E M E N T S

Dr Judith Sanchez-Rodrıguez (ICMyL) and B.S. AlejandroCordova (FES-I) helped in the field; M.S. MaribelBadillo-Aleman (UMDI-Sisal) provided access and supportto histological facilities; M.S. Gemma Martınez-Moreno andDr Patricia Guadarrama-Chavez (UMDI-Sisal) helped withlaboratory work and provided support in the microscopylaboratory; Dr Andrea Crowther (South AustralianMuseum) provided 12S and cox3 sequence data forHeteranthus sp. All specimens were collected under consentof Mexican law, collecting permit approved by ComisionNacional de Acuacultura y Pesca (Number07332.250810.4060). Comments of two anonymous refereesimproved this manuscript.

F I N A N C I A L S U P P O R T

This work was partially supported by the Comision Nacionalde Ciencia y Tecnologıa (CONACyT) (R.G., grant number35166/202677); CONACyT–SEMARNAT (N.S., grantnumber 108285); and DGAPA–PAPIME–UNAM (N.S.,grant number PE207210).

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Correspondence should be addressed to:R. Gonzalez-MunozUnidad Multidisciplinaria de Docencia e Investigacion en Sisal(UMDI-Sisal), Facultad de Ciencias, Universidad NacionalAutonoma de Mexico (UNAM), Puerto de Abrigo, SisalC.P. 97356 Yucatan, MexicoEmail: ricordea.gonzalez@gmail.com

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