Morphological and Molecular Variability of the Sea

Journal of the Marine Biological Association of the United Kingdom, page 1 of 11. # Marine Biological Association of the United Kingdom, 2014 doi:10.1017/S0025315414000988 Morphological and molecular variability of the 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 Investigacioń en Sisal (UMDI-Sisal), Facultad de Ciencias, Universidad Nacional Autońoma de Me´xico (UNAM), Puerto de Abrigo, Sisal, C.P. 97356 Yucatań, Me´xico, 2Posgrado en Ciencias del Mar y Limnologıá (PCMyL), UNAM, Instituto de Ciencias del Mar y Limnologıá (ICMyL), Circuito Exterior, Ciudad Universitaria, C.P. 04510, Me´xico, 3Laboratorio de Zoologıá, Facultad de Estudios Superiores Iztacala (FES-I), UNAM, Avenida de los Barrios 1, Los Reyes Iztacala, C.P. 54090 Estado de Me´xico, Me´xico, 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 Sciences Department, 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 or absence of protuberances on the marginal tentacles, as well as intermediate forms. The taxonomic status of the different morphotypes and the diagnostic value of protuberances on the tentacles have been debated for this species and the family Phymanthidae. We analysed the external and internal anatomy, cnidae and three mitochondrial molecular markers for representatives of each of the three morphotypes. In addition, we address the putative monophyly of the family Phymanthidae based on molecular data. With the exception of the protuberances, our morphological and molecular results show no differences among the three morphotypes; thus, we consider this feature to be intraspeciﬁc variability within 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

INTRODUCTION Nevertheless, morphs with and without protuberances in the marginal tentacles (as well as intermediate morphs) have Sea anemones of the family Phymanthidae Andres, 1883 been reported in specimens of Phymanthus crucifer (Le (Actiniaria: Actinoidea) are distinguished by verrucae on the Sueur, 1817) (Duerden, 1897, 1898, 1900, 1902; Stephenson, distal column, no marginal sphincter muscle or a weak endo- 1922; Cairns et al., 1986). Verrill (1900, 1905) suggested that dermal one, and two kinds of tentacles: marginal tentacles morphs with and without protuberances in the marginal ten- arranged in cycles that may have knoblike or branched pro- tacles should be treated as separate species that could hybrid- tuberances, and discal tentacles arranged radially, typically ize; however Duerden (1897, 1900, 1902) argued that all forms very short, and vesicle-like (Carlgren, 1949; Rodrı´guez et al., should be treated as a single species based on the existence of 2007). forms with intermediate stages of tentacular protuberances. Phymanthidae currently comprises two genera: This morphological variability on marginal tentacles reported Phymanthus Milne-Edwards & Haime, 1851 with eleven for P. crucifer challenges the value of this feature as a genus- valid species; and Heteranthus Klunzinger, 1877 with two level character within Phymanthidae. valid species (Fautin, 2013). These two genera are traditionally Although the size of cnidae alone is not generally consid- distinguished by the presence of lateral protuberances (papilli- ered a specific taxonomic diagnostic character due to its vari- form or ramified) in the marginal tentacles and no marginal ability within conspecific individuals (Fautin, 1988, 2009; sphincter (or an indistinct one) in Phymanthus, whereas Williams, 1996, 1998, 2000; Acunã et al., 2003, 2004; Heteranthus has smooth marginal tentacles without protuber- Ardelean & Fautin, 2004; Acunã & Garese, 2009), several ances and a weak circumscribed marginal sphincter (Carlgren, studies have proposed quantitative analyses of the cnidae to 1949). help distinguish among colour morphs in some species (Allcock et al., 1998; Watts & Thorpe, 1998; Manchenko et al., 2000; Watts et al., 2000). Watts & Thorpe (1998) found significant differences in the size of holotrichs in the

Corresponding author: acrorhagi of the upper-shore morphotype of Actinia equina R. Gonza´lez-Mun˜oz (Linnaeus, 1758), suggesting that these could help distinguish Email: [email protected] between the mid- and lower-shore morphotypes of the

1 2ricardogonza’ lez-mun~ oz et al.

species. Other attempts to distinguish between colour mor- disc diameter were obtained from fixed specimens; fragments photypes using cnidae size alone found slight differences of selected specimens were dehydrated and embedded in par- that do not support the use of this feature to separate affin. Histological sections 6–10 mm thick and stained with species (Chintiroglou & Karalis, 2000). haematoxylin–eosin (Estrada-Flores et al., 1982) were pre- In this study, we examined representatives of the three dif- pared to examine internal anatomy. ferent marginal tentacular morphs of Phymanthus crucifer Data on cnidae were obtained from four representatives of (with and without protuberances and intermediate forms) in each of the three morphotypes (a total of 12 individuals), all order to identify morphological, cnidae and/or molecular dis- collected from La Gallega reef. Seven squash preparations tinctions that would enable separation of the morphs into dif- were obtained from the main tissue types (1mm3) of each ferent species or corroborate the broad phenotypic plasticity specimen. We analysed cnidae from the marginal tentacles of P. crucifer. In addition, we tested the monophyly of tips (mtt), discal tentacles (dt), actinopharynx (ac), filaments Phymanthidae using three mitochondrial markers. (fi), column (co), vesicle-like marginal projections (vp), and protuberances on the marginal tentacles (pr/mt). For specimens of M2 (lacking protuberances), cnidae preparations of the marginal tentacles were obtained from regions where MATERIALS AND METHODS these protuberances regularly develop in morphotypes M1 and M3. From each of the seven squash preparations, the Morphological and cnidae analyses length and width of 40 undischarged capsules (replicates) of each type of cnidae were randomly measured using DIC We catalogued the marginal tentacular morphotypes of microscopy 1000 × oil immersion (following Williams, Phymanthus crucifer as follows: morphotype 1 (M1), speci- 1996, 1998, 2000). mens with protuberances in all marginal tentacles; morpho- Cnidae samples were ordered in a bi-dimensional space type 2 (M2), specimens completely lacking protuberances in using principal component analysis (PCA). Differences in all marginal tentacles (i.e. smooth tentacles); and morphotype ordination given by morphotype, individual specimen and 3 (M3), specimens with some smooth marginal tentacles and type of cnidae, as well as the interaction terms among these some marginal tentacles with protuberances. factors were analysed using a permutational MANOVA pro- Twelve specimens (four per morphotype) were collected in cedure (Anderson, 2001; McArdle & Anderson, 2001). ′ ′′ ′ ′′ La Gallega reef (19813 13 N96807 37 W) of the Veracruz Differences among cnidae were analysed for each type of Reef System in the Gulf of Mexico in 2010; three additional tissue separately. The PERMANOVA procedure was applied specimens (one of each morphotype) were collected from on resemblance matrices based on the Euclidian distance ′ ′′ ′ ′′ Puerto Morelos reef (20855 50.7 N86849 24 W) in the between samples. Although length and width of the capsules Mexican Caribbean (Figure 1). Collections were conducted were in the same measurement scale, data were standardized by hand, snorkelling or SCUBA diving, and using a hammer and normalized prior to analyses. The statistical model used and chisel. Collected specimens were transferred to the labora- was given by: tory and maintained in an aquarium to register their colour while alive (Figure 2). Specimens were relaxed in a 5% = + + + + + + Yijkl a Mi I(M) j(i) Tk MTik I(M)T j(i)k Sijkl MgSO4 seawater solution and fixed in 10% seawater–buffered formalin. Additionally, small samples of tissue were obtained from the pedal disc and preserved in 96% ethanol. where Y is the response matrix with n samples (number of Measurements of column height, as well as pedal and oral rows 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. characterizing variability within phymanthus crucifer 3

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.

(number of columns: length and width); M is a fixed factor three mitochondrial (12S and 16S rDNA and cox3) regions representing morphotype (with three levels); a is the coeffi- for 14 specimens (11 from La Gallega reef and three from cient representing the intercept of the multivariate regression; Puerto Morelos reef). Phymanthus crucifer haplotypes were I is a random factor representing individuals nested in M compared to available GenBank sequence data for (with four levels); T is the fixed factor representing type of Phymanthus loligo (Hemprich & Ehrenberg in Ehrenberg, cnidae (with three or two levels, depending on tissue kind) 1834) and Heteranthus sp. (for GenBank accession numbers and is orthogonal to M and I; MT and I(M)T are correspond- see Rodrı´guez et al. (2014) and Crowther (2013), respectively). ing interactions terms; and S is the residual matrix. Divergence estimates (based on the Kimura 2-parameter Permutation procedures were applied to obtain appropriate (K2P)) were obtained using Mega v.5.05 (Tamura et al., 2013). distributions for the pseudo-F statistic under the null hypoth- Herein, we provide new sequences for Phymanthus crucifer esis. All analyses were performed using permutations of resi- which were added to the data matrix presented in Rodrı´guez duals under the reduced model, resulting in a range from 909 et al. (2014) after removing all hexacoral taxa not belonging to 999 unique permutations for each F-test. The experimental to Actiniaria (except the antiphatharian Leiopathes Haime, design was balanced in every case, and the partitioning of vari- 1849, which was used as an outgroup) and adding ation was achieved so that the test statistic (pseudo-F) repre- Heteranthus sp.; for a complete account of taxa included in sents the proportion of the variation in the bi-dimensional this study, we refer readers to Rodrı´guez et al. (2014). New cloud that is explained by the source of variation being tested. sequences have been deposited in GenBank (Table 1). Specimens, as well as histological and cnidae preparations, DNA sequences of each marker were separately aligned were deposited in the Collection of Cnidarians of the Gulf of using MAFFT v.7 (online at http://mafft.cbrc.jp/alignment/ Mexico and Mexican Caribbean Sea (Registration code: server/; Katoh et al., 2002, 2005; Katoh & Toh, 2008) using YUC–CC–254–11) of the Unidad Multidisciplinaria de the following settings and parameters: Strategy, L-INS-i Docencia e Investigacioń en Sisal (UMDI-Sisal) at the (recommended for ,200 sequences with one conserved Universidad Nacional Autońoma de Me´xico (UNAM). domain and long gaps); scoring matrix, 200PAM/k ¼ 2; gap opening penalty, 1.53; offset value, 0.05; max. iterate, 1000; Molecular analyses and retree, 1. We then concatenated the three mitochondrial markers to create a single dataset for 115 taxa and 2697 sites. Acquisition of molecular data followed the protocol detailed The Akaike information criterion (AIC) was implemented in Lauretta et al. (2013). We obtained DNA sequences of within jModelTest v.2.1.2 (Darriba et al., 2012) to determine 4ricardogonza’ lez-mun~ oz et al.

Table 1. Voucher specimen location and GenBank accession numbers for new sequences provided in this study. See Rodrı´guez et al. (2014) for a complete list of taxa and data included in the analysis and Crowther (2013) for data regarding Heteranthus sp. UMDI-Sisal, Unidad Multidisciplinaria de Docencia e Investigacio´n en Sisal, UNAM; AMNH, American Museum of Natural History.

Family Species ID number Collection locality Voucher

Phymanthidae Phymanthus crucifer RG-128 GoM UMDI-Sisal Phymanthus crucifer RG-129 GoM UMDI-Sisal Phymanthus crucifer RG-130 GoM UMDI-Sisal Phymanthus crucifer RG-131 GoM UMDI-Sisal Phymanthus crucifer RG-133 GoM UMDI-Sisal Phymanthus crucifer RG-134 GoM UMDI-Sisal Phymanthus crucifer RG-138 GoM UMDI-Sisal Phymanthus crucifer RG-143 GoM UMDI-Sisal Phymanthus crucifer RG-182 GoM UMDI-Sisal Phymanthus crucifer RG-184 GoM UMDI-Sisal Phymanthus crucifer RG-187 GoM UMDI-Sisal Phymanthus crucifer RG-200 MC AMNH-5312 Phymanthus crucifer RG-219 MC UMDI-Sisal Phymanthus 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; haplotype 2: KJ910344).

the appropriate evolutionary model (TIM2 + I + G) and cor- 100 rounds of tree fusing; Goloboff et al., 2008). In all ana- responding parameters (p-inv ¼ 0.0470, gamma shape ¼ lyses, gaps (–) were treated as missing data. Trees of 0.3360, freqA ¼ 0.3034, freqC ¼ 0.1821, freqG ¼ 0.2212, minimum length were found at least five times. The concate- freqT ¼ 0.2933, (AC) ¼ 1.3194, (AG) ¼ 5.0386, (AT) ¼ nated data set was subjected to 1000 rounds of bootstrap 1.3194, (CG) ¼ 1.0000, (CT) ¼ 8.7441, (GT) ¼ 1.0000) resampling to assess support for clades. (number of candidate models: 88; number of substitution schemes: 11; base tree likelihood calculations: BIONJ using PhyML v3.0 (Guindon et al., 2010)). We searched for optimal trees using maximum likelihood RESULTS AND DISCUSSION (ML) within PhyML v.3.0 (http://www.atgc-montpellier.fr/ phyml/; Guindon & Gascuel, 2003). The following parameters Morphological analyses were implemented within PhyML: substitution model ¼ GTR + I + G (the online version of PhyML does not imple- All twelve specimens examined from La Gallega displayed ment TIM2, and GTR had a DAIC of 2.3); substitution rate external morphological diagnostic taxonomic features corre- categories ¼ 6; p-inv ¼ 0.0470; gamma shape ¼ 0.3360; start- sponding to Phymanthus crucifer, including verrucae in the ing tree ¼ BIONJ; tree improvement ¼ SPR & NNI; opti- distal column arranged in longitudinal rows, column color- mized tree topology and branch lengths; and bootstrap ation with flame-like staining pattern, discal tentacles replicates ¼ 350. We also conducted tree searches under arranged in radial rows from peristoma to margin, and mar- maximum parsimony (results not shown) with TNT v.1.1 ginal tentacles hexamerously arranged. The only external (random and consensus sectorial searches, tree drifting and morphological difference among specimens, aside from color- ation patterns, was the marginal tentacular protuberances. Internal anatomy was also similar in all the specimens (see Gonza´lez-Munõz et al., 2012 for a complete description of Table 2. Morphological analysis of all three morphotypes; all measure- the taxonomic diagnostic features of P. crucifer). ments are in mm. pd, pedal disc diameter; ch, column height; od, oral Size of specimens (pedal and oral disc diameter and disc diameter; nv, range of the number of verrucae per longitudinal row; sx, sex; (?), no gametogenic tissue present. column height) and number of verrucae per longitudinal row did not exhibit a consistent pattern that could be asso- Morph Specimen code pd ch od nv sx ciated with any of the three marginal tentacular morphs M1 M1.1 11 31 36 2–3 Male (Table 2). The three morphotypes contained both relatively M1.2 23 20 48 3–4 Male small and larger specimens, suggesting that the development M1.3 25 22 45 2–5 (?) of protuberances on marginal tentacles is not related to differ- M1.4 32 23 53 2–4 (?) ent growth stages of these organisms in the wild. M2 M2.1 22 28 59 2–4 Female Colour patterns of the oral disc and tentacles varied among M2.2 23 26 49 3–4 Female all the specimens examined but did not show a consistent M2.3 27 26 51 3–6 Male pattern characterizing a particular morph (Figure 2A–K). M2.4 10 8 38 2–4 (?) The oral disc is mainly green, but presented a distinct tone, M3 M3.1 16 34 48 3–7 Male from olive green (Figure 2A, C–E, G) to dark green M3.2 23 16 44 3–5 (?) (Figure 2B, F); it could also be brown (Figure 2H, K), or M3.3 20 29 54 4–5 (?) M3.4 32 18 46 2–3 Female with endocelic radial rows marking the arrangement of the discal tentacles (Figure 2I–J). The mouth was primarily the characterizing variability within phymanthus crucifer 5 same colour as the oral disc or exceptionally bright green or spermatic vesicles (males) in all three morphotypes bright orange in some specimens (Figure 2F, I and 2D, (Table 2), but oocysts in only some specimens of morphs respectively). The peristoma often had a lighter tone than M2 and M3. Nevertheless, oocysts have been reported in spe- the rest of the oral disc (Figure 2B, G, H, K). Marginal tenta- cimens of morph M1 in previous studies (Gonza´lez-Munõz cles without protuberances in representatives of morph M2 et al., 2012). In most dioecious species of cnidarians, males and some of M3 presented longitudinal rows of yellowish, and females are macroscopically indistinguishable (Fautin, brownish or white colorations (Figure 2E–G, I–J); and 1992), whilst sexual dimorphism has only been reported for some marginal tentacles had purple shades at their tips a few hydrozoan and scyphozoan species (Fautin, 1992), and (Figure 2I, K). Colour pattern is a controversial character to for the actiniarian Entacmaea quadricolour (Leuckart in distinguish sea anemones; some species are distinguished by Ru¨ppell & Leuckart, 1828) (Scott & Harrison, 2009). colour patterns while others have distinct colour morphs Crowther (2013) suggested that the symbiotic relationship that are considered to be phenotypic plasticity due to local with zooxanthellae is likely associated with the formation of genetic adaptations (Stoletzki & Schierwater, 2005). lateral protuberances in the tentacles as it occurs in other Phymanthus crucifer is dioecious and not thought to species such as Lebrunia coralligens (Wilson, 1890)and undergo asexual reproduction (Jennison, 1981). We found Lebrunia danae (Duchassaing & Michelotti, 1860). However,

Fig. 3. Cnida types and their distribution among tissues per morphotype (M1, M2, M3). Scale bars: 25 mm. 6ricardogonza’ lez-mun~ oz et al.

we found zooxanthellae in all specimens examined, including found slight differences in the width of some types of cnidae those without protuberances (M2). Quantitative comparisons of (e.g. microbasic p-mastigophores), a feature that should be the densities of zooxanthellae within the different morphotypes considered in future studies. may offer some insight about the feasibility of this hypothesis. The different morphotypes did not explain the variation of cnidae size in any of the tissues examined (Table 3: Morph). The ordination of samples from all types of tissue was Cnidae analyses similar regardless of the morphotype they came from (see We found the same types of cnidae (cnidom) in all samples Figure 4A–G). By contrast, differences in cnidae size among specimens within each morphotype were significant for all examined, regardless of morphotype (Figure 3). The cnidom × of Phymanthus crucifer included basitrichs, microbasic tissue types (Table 3: Ind(Morph) and Ind(Morph) Type). p-mastigophores and spirocysts, as previously reported for Cnidae size (both length and width considered) also varied the family and genus (Carlgren, 1949). We did not find any significantly depending on cnidae type (Table 3: Type), but differences in size between cnidae types were similar among additional types of cnidae in morphotypes M1 and M3 × (those with protuberances in the marginal tentacles). It is all three morphotypes (Table 3: Morph Type). Overall, unlikely that the protuberances on the marginal tentacles these results suggest that individuals constitute the main could be acting as structures for competition because agonistic source of variation when the size of cnidae are examined. behaviour in actiniarians is usually associated with the pres- Edmands & Fautin (1991) noted that the size of nemato- ence of holotrichs, a type of nematocyst in specialized struc- cysts does not appear to correlate with animal size in tures such as acrorhagi and catch tentacles (Bigger, 1988; Aulactinia veratra (Drayton in Dana, 1846), and Acunã Williams, 1991) commonly found in some shallow water sea et al. (2007) suggest that there is no functional relationship anemone species (Daly, 2003; Fautin, 2009). between cnida length and body weight in Oulactis muscosa We measured 560 cnidae capsules per specimen, separated (Drayton in Dana, 1846). Thus, although the diameter of into 14 categories of cnidae (basitrichs, microbasic the pedal disc is slightly variable between examined specimens p-mastigophores and spirocysts) and tissue type; this added of P. crucifer (Table 2), we found it unnecessary to include the to a total of 6720 capsules measured (Figure 3). Our results pedal disc as a covariable in the analyses. showed no significant variation in the size of cnidae between morphotypes (Table 3), whereas cnidae varied in size within each morphotype depending on cnidae type and Molecular analyses individual specimens (Figure 4A–G). The PCA ordination of samples from all tissue types variation within phymanthus crucifer showed that the first principal component explained from Comparison of aligned sequences for cox3 (663 base pairs (bp) 60 to 94.5% of the variability of the cnidae size depending in length) and 16S (428 bp) did not reveal any variation on the type of tissue being analysed (Table 3). Thus, the first among individuals or morphotypes from the Gulf of Mexico principal component represents the variability in cnidae or Mexican Caribbean. However, mitochondrial 12S length. The percentage of variation explained by the second (824 bp) revealed two haplotypes that were distinguished by principal component was low (from 5.5 to 21.3%) in cnidae a single substitution (K2P distance ¼ 0.1215%, see Table 4), from ac, fi, pr/mt, dt and mtt, but relatively high in cnidae but these haplotypes were not specific to any particular mor- from co and vp (from 35.9 to 40.0%) (Table 3). This second photype. While haplotype 1 (differentiated by a single adenine principal component represents cnidae width. substitution) was specific to Gulf of Mexico specimens, it was In ac and fi the variation in cnidae width was higher for shared by all three morphotypes. Haplotype 2 (differentiated microbasic p-mastigophores than for basitrichs (Figure 4A– by a single guanine substitution) was more broadly distribu- B). This was not the case in co, pr/mt, dt, mtt and vp tissues, ted, being shared between specimens in the Gulf of Mexico in which cnidae width was similar among all types examined and Mexican Caribbean. Within the Gulf of Mexico, haplo- (Figure 4C–G). type 2 was shared by M2 and M3, while in the Mexican Acunã et al. (2007, 2011) only considered length when Caribbean it was shared by all three morphotypes. Table 4 comparing cnidae sizes among specimens. Although our summarizes divergence estimates among sequences within results confirm that length was the variable that explained morphotypes of Phymanthus crucifer and representatives of most of the variation between samples (60–94.5%), we the 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 60.0 PC2 % of variation 9.3 35.9 14.1 12.4 5.5 21.3 40.0 Morph 0.858 0.534 0.912 0.895 0.572 0.826 0.235 Ind(Morph) 0.001 0.001 0.001 0.001 0.001 0.001 0.001 Type 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 characterizing variability within phymanthus crucifer 7

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.

Because mitochondrial DNA (mtDNA) exhibits low levels unexpected, even in those from potentially isolated popula- of sequence divergence within and among anthozoan species, tions that are geographically distant from each other finding no variation in sequences from conspecifics is not (Shearer et al., 2002; Hellberg 2006; Brugler et al., 2013). Sequence divergence based on 12S was 15–17 times higher Table 4. Divergence estimates (K2P) based on sequence comparisons of between Phymanthus crucifer and P. loligo or Heteranthus the three mtDNA markers. Comparisons were made between sp. than between the two haplotypes obtained for P. crucifer. Phymanthus crucifer and Phymanthus loligo, as well as between Thus, although anthozoan mtDNA is characterized by low Phymanthus crucifer and Heteranthus sp. NA, not available. levels of divergence, we would expect at least a similar degree of divergence among the morphotypes of P. crucifer Heteranthus sp. P. crucifer P. loligo if they were indeed distinct species. If all three P. crucifer mor- 12S photypes are indeed a single species, then mitochondrial 12S Heteranthus sp. 1.93% revealed, for the first time, intraspecific variation within P. loligo 0.12% sea anemones. P. crucifer 2.06% 16S Heteranthus sp. NA P. loligo 1.18% SYSTEMATICS AND TAXONOMIC P. crucifer NA STATUS OF PHYMANTHIDAE cox3 Heteranthus sp. 1.73% A phylogenetic reconstruction based on the three concate- P. loligo 3.06% nated mitochondrial genes recovered the two 12S-based P. crucifer 2.39% Phymanthus crucifer haplotypes as sister taxa, and these as 12S, 792 base pairs (bp) compared; 16S, 428 bp compared; cox3, 513 bp sister to P. loligo (Figure 5). However, Heteranthus sp. is compared. recovered as sister to the actiniid genus Anemonia Risso, 8ricardogonza’ lez-mun~ oz et al.

Fig. 5. Phylogenetic reconstruction of the Actiniaria. Tree resulting from PhyML analysis of concatenated 12S, 16S and cox3. Grey boxes indicate superfamilies within 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 Rodrı´guez et al. (2014). Numbers above the branches are bootstrap resampling values expressed as a percentage; values ,50 not indicated; ﬁlled-in circles indicate nodes with support of 100%. Taxa in bold belong to Phymanthidae.

1826, thus rendering Phymanthidae polyphyletic. All studied water sea anemones (Rodrı´guez et al., 2012, 2014). Our members of Phymanthidae grouped within Actinoidea results concur with those of Crowther (2013), who included (Rodrı´guez et al., 2014), a superfamily of mainly shallow- a higher taxon sampling of the superfamily Actinoidea in characterizing variability within phymanthus crucifer 9 her study of the families Thalassianthidae Milne-Edwards & Acunã F.H., Excoffon A.C., Zamponi M.O. and Ricci L. (2003) Haime, 1851 and Aliciidae Duerden, 1895. Importance of nematocysts in taxonomy of acontiarian sea anemones The presence of Phymanthus crucifer morphotypes without (Cnidaria, Actiniaria): a statistical comparative study. Zoologischer protuberances in the marginal tentacles renders Carlgren’s Anzeiger 242, 75–81. (1949) major distinction between the two genera of Acunã F.H. and Garese A. (2009) The cnidae of the acrospheres of the Phymanthidae invalid. The marginal sphincter muscle, the corallimorpharian Corynactis carnea (Studer, 1878) (Cnidaria, other feature used by Carlgren (1949) to distinguish Corallimorpharia, Corallimorphidae): composition, abundance and between these genera, is also problematic. Heteranthus is char- biometry. Belgian Journal of Zoology 139, 50–57. acterized by a weak but circumscribed marginal sphincter, Acunã F.H., Ricci L. and Excoffon A.C. (2011) Statistical relationships of whereas most species of Phymanthus lack a marginal sphinc- cnidocyst sizes in the sea anemone Oulactis muscosa (Actiniaria: ter (Carlgren, 1949). However, Phymanthus muscosus Actiniidae). Belgian Journal of Zoology 141, 32–37. (Haddon & Shackleton, 1893) has a very feeble sphincter muscle (Haddon, 1898). Carlgren (1900) initially placed Acunã F.H., Ricci L., Excoffon A.C. and Zamponi M.O. (2004) A novel Heteranthus within a different family, Heteranthidae statistical analysis of cnidocysts in acontiarian sea anemones Carlgren, 1900, but he later placed it within Phymanthidae, (Cnidaria, Actiniaria) using generalized linear models with gamma errors. Zoologischer Anzeiger 243, 47–52. based on similarities with Phymanthus (Carlgren, 1943). We recovered Heteranthus as nested within Actiniidae (see Allcock A.L., Watts P.C. and Thorpe J.P. (1998) Divergence of nemato- Figure 5) suggesting that discal tentacles have evolved inde- cysts of two colour morphs of the intertidal beadlet anemone Actinia pendently at least twice within Actinoidea. A comprehensive equina. Journal of the Marine Biological Association of the United revision of the family Phymanthidae and a redefinition of its Kingdom 78, 821–828. diagnostic characters are needed to establish its membership. Anderson M.J. (2001) A new method for non-parametric multivariate Based on external and internal morphological features, analysis of variance. Austral Ecology 26, 32–46. cnidae data, and mitochondrial DNA, we conclude that all morphotypes of Phymanthus crucifer represent a single Andres A. (1883) Le Attinie. Roma: Coi Tipi der Salviucci, 460 pp. species, despite differences in the presence or absence of pro- Ardelean A. and Fautin D.G. (2004) Variability in nematocysts from a tuberances in the marginal tentacles. The significance and single individual of the sea anemone Actinodendron arboreum function of the protuberances in the marginal tentacles (Cnidaria: Anthozoa: Actiniaria). Hydrobiologia 530/531, 187–197. remains unknown within P. crucifer, but might be related to Bigger C.H. (1988) The role of nematocysts in anthozoan aggression. In specific adaptations to the surrounding environment. Hessinger D.A. and Lenhoff H.M. (eds) The biology of nematocysts. San Diego, CA: Academic Press, pp. 295–308.

ACKNOWLEDGEMENTS Brugler M.R., France S.C. and Opresko D.M. (2013) The evolutionary history of the order Antipatharia (Cnidaria: Anthozoa: Hexacorallia) as inferred from mitochondrial and nuclear DNA: implications for Dr Judith Sańchez-Rodrı´guez (ICMyL) and B.S. Alejandro black coral taxonomy and systematics. Zoological Journal of the Co´rdova (FES-I) helped in the field; M.S. Maribel Linnean Society 169, 312–361. Badillo-Alemań (UMDI-Sisal) provided access and support to histological facilities; M.S. Gemma Martıńez-Moreno and Cairns S., den Hartog J.C. and Arneson C. (1986) Class Anthozoa Dr Patricia Guadarrama-Cha´vez (UMDI-Sisal) helped with (Corals, Anemones). In Sterrer W. and Schoepfer-Sterrer C. (eds) laboratory work and provided support in the microscopy Marine fauna and flora of Bermuda. New York: John Wiley & Sons, laboratory; Dr Andrea Crowther (South Australian pp. 164–194. Museum) provided 12S and cox3 sequence data for Carlgren O. (1900) Ostafrikanische actinien. Mittheilungen aus dem Heteranthus sp. All specimens were collected under consent Naturhistorischen Museum 17, 21–144. of Mexican law, collecting permit approved by Comisioń Carlgren O. (1943) East-asiatic corallimorpharia and actiniaria. Kungliga Nacional de Acuacultura y Pesca (Number Svenska Vetenskapsakademiens Handlingar 20, 1–43. 07332.250810.4060). Comments of two anonymous referees improved this manuscript. Carlgren O. (1949) A survey of the Ptychodactiaria, Corallimorpharia and Actiniaria. Kunglia Svenska Vetenskaps—Akademiens Handlingar 1, 1–121.

FINANCIAL SUPPORT Chintiroglou C.C. and Karalis P. (2000) Biometric investigations on the cnidae of the Aegean colour morphs of Anemonia viridis. Journal of This work was partially supported by the Comisio´n Nacional the Marine Biological Association of the United Kingdom 80, 543–544. de Ciencia y Tecnologı´a (CONACyT) (R.G., grant number Crowther A.L. (2013) Character evolution in light of phylogenetic analysis 35166/202677); CONACyT–SEMARNAT (N.S., grant and taxonomic revision of the zooxanthellate sea anemone families number 108285); and DGAPA–PAPIME–UNAM (N.S., Thalassianthidae and Aliciidae. PhD thesis. University of Kansas, grant number PE207210). USA.

Daly M. (2003) The anatomy, terminology, and homology of acrorhagi and pseudoacrorhagi in sea anemones. Zoologische Verhandenlinhen REFERENCES Leiden 345, 89–101.

Acun˜a F.H., Excoffon A.C. and Ricci L. (2007) Composition, biometry Dana J.D. (1846) Zoophytes. Volume VII of the United States exploring and statistical relationships between the cnidom and body size in the expedition. During the years 1838, 1839, 1840, 1841, 1842. Under the sea anemone Oulactis muscosa (Cnidaria: Actiniaria). Journal of the command of Charles Wilkes, U.S.N. Philadelphia, PA: Lea and Marine Biological Association of the United Kingdom 87, 415–419. Blanchard, 740 pp. 10 ricardo gonza’ lez-mun~ oz et al.

Darriba D., Taboada G.L., Doallo R. and Posada D. (2012) jModelTest Haime J. (1849) Sur le polypieroide d’un Leiopathes glaberrima. Annales 2: more models, new heuristics and parallel computing. Nature des Sciences Naturelles 12, 224–226. Methods 9, 772. Hellberg M.E. (2006) No variation and low synonymous substitution Duchassaing P. and Michelotti G. (1860) Me´moire sur les Coralliaires des rates in coral mtDNA despite high nuclear variation. BMC Antilles. Turin: Imprimerie Royale, 89 pp. doi: 10.5962/bhl.title.11388. Evolutionary Biology 6, 24. Duerden J.E. (1895) On the genus Alicia (Cladactis), with an anatomical Jennison B.L. (1981) Reproduction in three species of sea anemones from description of A. costae, Panc. Annals and Magazine of Natural Key West, Florida. Canadian Journal of Zoology 59, 1708–1719. History 15, 213–218. Katoh K., Misawa K., Kuma K.I. and Miyata T. (2002) MAFFT: a novel Duerden J.E. (1897) The actiniarian family Aliciidae. Annals and method for rapid multiple sequence alignment based on fast Fourier Magazine of Natural History 20, 1–15. transform. Nucleic Acids Research 30, 3059–3066. Duerden J.E. (1898) The Actiniaria around Jamaica. Journal of the Katoh K., Kuma K.I., Toh H. and Miyata T. (2005) MAFFT version 5: Institute of Jamaica 2, 449–465. improvement in accuracy of multiple sequence alignment. Nucleic Acids Research 33, 511–518. Duerden J.E. (1900) Jamaican Actiniaria. Part II. Stichodactylinæ and Katoh K. and Toh H. Zoantheæ. Scientific Transactions of the Royal Dublin Society 7, (2008) Improved accuracy of multiple ncRNA 133–208. alignment by incorporating structural information into a MAFFT-based framework. BMC Bioinformatics 9, 212. Duerden J.E. (1902) Report of the Actinians of Porto Rico (Investigations Klunzinger C.B. (1877) Die Korallthiere des Rothen Meeres. 1: Die of the aquatic resources and fisheries of Porto Rico by the U. S. Fish Alcyonarien und Malacodermen. Berlin: Gutmann’schenBuchhandlung, Commission Steamer Fish Hawk in 1899). Bulletin of the US Fish 98 pp. Commission 20, 323–374. Lauretta D., Haüssermann V., Brugler M.R. and Rodrı´guez E. (2014) Edmands S. and Fautin D.G. (1991) Redescription of Aulactinia veratra Isoparactis fionae sp. nov. (Cnidaria: Anthozoa: Actiniaria) from n. comb. (¼ Cnidopus veratra) (Coelenterata: Actiniaria) from Southern Patagonia with a discussion of the family Isanthidae. Australia. Records of the Western Australian Museum 15, 59–68. Organisms Diversity and Evolution 14, 31–42. Ehrenberg C.G. (1834) Beitra¨ge zur physiologischen Kenntniss der Le Sueur C.A. (1817) Observations on several species of the genus Actinia; Corallenthiere im allgemeinen, und besonders des rothen Meeres, illustrated by figures. Journal of the Academy of Natural Sciences of nebst einem Versuche zur physiologischen Systematik derselben. Philadelphia 1, 149–154, 169–189. Abhandlungen der Ko¨niglichen Akademie der Wissenschaften zu Berlin 1, 225–380. Linnaeus C. (1758) Systema Naturae. Regnum Animale. Lipsiae: Facsimile produced by the Cura Societatis Zoologiae Germanie Ed. 10. Estrada-Flores E., Peralta L. and Rivas P. (1982) Manual de Tećnicas Histolo´gicas.Me´xico: AGT, pp. 32–65. Manchenko G.P., Dautova T.N. and Latypov Y.Y. (2000) High level of genetic divergence between sympatric colour morphs of the littoral Fautin D.G. (1988) Importance of nematocysts to actinian taxonomy. In sea anemone Anthopleura orientalis (Anthozoa: Actiniaria). Hessinger D.A. and Lenhoff H.M. (eds) The biology of nematocysts. Biochemical Systematics and Ecology 28, 737–750. San Diego, CA: Academic Press, Inc., pp. 487–500. McArdle B.H. and Anderson M.J. (2001) Fitting multivariate models to Fautin D.G. (1992) Cnidaria. In Adiyodi K.G. and Rita G. (eds) community data: a comment on distance-based redundancy analysis. Reproductive biology of invertebrates, Volume 5 (sexual differentiation Ecology 82, 290–297. and behaviour). New Delhi: Oxford and IBH Publishing Company, pp. 31–52. Milne-Edwards H. and Haime J. (1851) Archives du Museúm d’Historie Naturelle. 5: Monographie des polypiers fossils des terrains palfozoo- Fautin D.G. (2009) Structural diversity, systematics, and evolution of ques, pricidie d’un tableau general de la classification des polypes. cnidae. Toxicon 54, 1054–1064. Paris: Gide et J. Baudry, 502 pp. Fautin D.G. (2013) Hexacorallians of the world. Available at: http://geo- Risso A. (1826) Histoire naturelle des principales de l’Europe Me´ridionale. portal.kgs.ku.edu/hexacoral/anemone2/index.cfm (accessed 1 July Volume 5. Paris: F. Chez, G. Levrault, Libraire, Rue de la Harpe, 2014). pp. 284–290. Goloboff P.A., Farris J.S. and Nixon K. C. (2008) TNT, a free program Rodrı´guez E., Barbeitos M.S., Brugler M.R., Crowley L.M., Grajales A., for phylogenetic analysis. Cladistics 24, 774–786. Gusmaõ L., Haüssermann V., Reft A. and Daly M. (2014) Hidden among sea anemones: The first comprehensive phylogenetic recon- Gonza´lez-Munõz R., Simo˜es N., Sańchez-Rodrı´guez J., Rodrı´guez E. struction of the the order Actiniaria (Cnidaria, Anthozoa, and Segura-Puertas L. (2012) First inventory of sea anemones Hexacorallia) reveals a novel group of hexacorals. Plos One 9, (Cnidaria: Actiniaria) of the Mexican Caribbean. Zootaxa 3556, 1–38. e96998. doi: i10.1371/journal.pone.0096998. Guindon S., Dufayard J.F., Lefort V., Anisimova M., Hordijk W. and Rodrı´guez E., Barbeitos M., Daly M., Gusmaõ L.C. and Haüssermann Gascuel O. (2010) New algorithms and methods to estimate V. (2012) Toward a natural classification: phylogeny of acontiate sea maximum-likelihood phylogenies: assessing the performance of anemones (Cnidaria, Anthozoa, Actiniaria). Cladistics 1, 1–18. PhyML 3.0. Systematic Biology 59, 307–321. Rodrı´guez E., Daly M. and Fautin D.G. (2007) Order actiniaria. In Guindon S. and Gascuel O. (2003). A simple, fast, and accurate algorithm Zhang Z.-Q. and Shear W.A. (eds) Linnaeus Tercentenary: Progress to estimate large phylogenies by maximum likelihood. Systematic in Invertebrate Taxonomy. Zootaxa 1668, 131–136. Biology 52, 696–704. Ru¨ppell E. and Leuckart F.S. (1828) Atlas zu der Reise im No¨rdlicehn Haddon A.C. (1898) The actiniaria of Torres Straits. Scientific Afrika von Eduard Ru¨ppell, Neue Wirbellose Thiere des Rothen Transactions of the Royal Dublin Society 6, 393–520. Meers. Frankfurt am Main: Heinrich Ludwig Brvnner, 47 pp. Haddon A.C. and Shackleton A.M. (1893) Description of some new Scott A. and Harrison P.L. (2009) Gametogenic and reproductive cycles species of actiniaria from Torres Straits. Scientific Proceedings of the of the sea anemone, Entacmaea quadricolour. Marine Biology 156, Royal Dublin Society 8, 116–131. 1659–1671. characterizing variability within phymanthus crucifer 11

Shearer T.L., Van Oppen J.H., Romano S.L. and Wo¨rheide G. (2002) Williams R.B. (1991) Acrorhagi, catch tentacles and sweeper tentacles: a Slow mitochondrial DNA sequence evolution in the Anthozoa synopsis of “aggression” of actiniarian and scleractinian Cnidaria. (Cnidaria). Molecular Ecology 11, 2475–2487. Hydrobiologia 216/217, 539–545. Stephenson T.A. (1922) On the classification of Actiniaria. Part III. Williams R.B. (1996) Measurements of cnidae from sea anemones Definitions connected with the forms dealt with in Part II. Quarterly (Cnidaria: Actiniaria): statistical parameters and taxonomic relevance. Journal of Microscopical Science 66, 247–319. Scientia Marina 60, 339–351. Stoletzki N. and Schierwater B. (2005) Genetic and colour morph differ- Williams R.B. (1998) Measurements of cnidae from sea anemones entiation in the Caribbean sea anemone Condylactis gigantea. Marine (Cnidaria: Actiniaria), II: further studies of differences amongst Biology 147, 747–754. sample means and their taxonomic relevance. Scientia Marina 62, 361–372. Tamura K., Stecher G., Peterson D., Filipski A. and Kumar S. (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Williams R.B. (2000) Measurements of cnidae from sea anemones Molecular Biology and Evolution 30, 2725–2729. (Cnidaria: Actiniaria), III: ranges and other measures of statistical dis- Verrill A.E. (1900) Additions to the Anthozoa and Hydrozoa of the persion, their interrelations and taxonomic relevance. Scientia Marina 64, 49–68. Bermudas. Anthozoa. Transactions of the Connecticut Academy of Arts and Sciences 10, 551–572. and Verrill A.E. (1905) The Bermuda Islands. Part IV. Geology and paleon- Wilson H.V. (1890) On a new actinia, Hoplophoria coralligens. Studies at tology, and Part V. An account of the coral reefs. Transactions of the the Biological Laboratory of the John Hopkins University (Baltimore) 6, Connecticut Academy of Arts and Sciences 12, 45–348. 379–387. Watts P.C., Allcock A.L., Lynch S.M. and Thorpe J.P. (2000) An analysis of the nematocysts of the beadlet anemone Actinia equina and the Correspondence should be addressed to: green sea anemone Actinia prasina. Journal of the Marine Biological R. Gonza´lez-Munõz Association of the United Kingdom 80, 719–724. Unidad Multidisciplinaria de Docencia e Investigacioń en Sisal Watts P.C. and Thorpe J.P. (1998) Phenotypic identification of three gen- (UMDI-Sisal), Facultad de Ciencias, Universidad Nacional etically differentiated morphs of the intertidal beadlet anemone Autońoma de Me´xico (UNAM), Puerto de Abrigo, Sisal Actinia equina (Anthozoa: Cnidaria). Journal of the Marine C.P. 97356 Yucatań, Me´xico Biological Association of the United Kingdom 78, 1365–1368. Email: [email protected]