Morphological and molecular variability of the sea

Journal of the Marine Biological Association of the United Kingdom, page 1 of 11. doi:10.1017/S0025315414000988

# Marine Biological Association of the United Kingdom, 2014

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 1

Unidad 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

Sea anemones of the family Phymanthidae Andres, 1883 (Actiniaria: Actinoidea) are distinguished by verrucae on the distal column, no marginal sphincter muscle or a weak endodermal one, and two kinds of tentacles: marginal tentacles arranged in cycles that may have knoblike or branched protuberances, and discal tentacles arranged radially, typically very short, and vesicle-like (Carlgren, 1949; Rodrı´guez et al., 2007). Phymanthidae currently comprises two genera: Phymanthus Milne-Edwards & Haime, 1851 with eleven valid species; and Heteranthus Klunzinger, 1877 with two valid species (Fautin, 2013). These two genera are traditionally distinguished by the presence of lateral protuberances (papilliform or ramified) in the marginal tentacles and no marginal sphincter (or an indistinct one) in Phymanthus, whereas Heteranthus has smooth marginal tentacles without protuberances and a weak circumscribed marginal sphincter (Carlgren, 1949).

Corresponding author: R. Gonza´lez-Munõz Email: [email protected]

Nevertheless, morphs with and without protuberances in the marginal tentacles (as well as intermediate morphs) have been reported in specimens of Phymanthus crucifer (Le Sueur, 1817) (Duerden, 1897, 1898, 1900, 1902; Stephenson, 1922; Cairns et al., 1986). Verrill (1900, 1905) suggested that morphs with and without protuberances in the marginal tentacles should be treated as separate species that could hybridize; however Duerden (1897, 1900, 1902) argued that all forms should be treated as a single species based on the existence of forms with intermediate stages of tentacular protuberances. This morphological variability on marginal tentacles reported for P. crucifer challenges the value of this feature as a genuslevel character within Phymanthidae. Although the size of cnidae alone is not generally considered a specific taxonomic diagnostic character due to its variability within conspecific individuals (Fautin, 1988, 2009; Williams, 1996, 1998, 2000; Acunã et al., 2003, 2004; Ardelean & Fautin, 2004; Acunã & Garese, 2009), several studies have proposed quantitative analyses of the cnidae to 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 acrorhagi of the upper-shore morphotype of Actinia equina (Linnaeus, 1758), suggesting that these could help distinguish between the mid- and lower-shore morphotypes of the 1

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ricardo gonza’ lez-mun~ oz et al.

species. Other attempts to distinguish between colour morphotypes using cnidae size alone found slight differences that do not support the use of this feature to separate species (Chintiroglou & Karalis, 2000). In this study, we examined representatives of the three different marginal tentacular morphs of Phymanthus crucifer (with and without protuberances and intermediate forms) in order to identify morphological, cnidae and/or molecular distinctions that would enable separation of the morphs into different species or corroborate the broad phenotypic plasticity of P. crucifer. In addition, we tested the monophyly of Phymanthidae using three mitochondrial markers.

MATERIALS AND METHODS

Morphological and cnidae analyses We catalogued the marginal tentacular morphotypes of Phymanthus crucifer as follows: morphotype 1 (M1), specimens with protuberances in all marginal tentacles; morphotype 2 (M2), specimens completely lacking protuberances in all marginal tentacles (i.e. smooth tentacles); and morphotype 3 (M3), specimens with some smooth marginal tentacles and some marginal tentacles with protuberances. Twelve specimens (four per morphotype) were collected in La Gallega reef (19813′ 13′′ N 96807′ 37′′ W) of the Veracruz Reef System in the Gulf of Mexico in 2010; three additional specimens (one of each morphotype) were collected from Puerto Morelos reef (20855′ 50.7′′ N 86849′ 24′′ W) in the Mexican Caribbean (Figure 1). Collections were conducted by hand, snorkelling or SCUBA diving, and using a hammer and chisel. Collected specimens were transferred to the laboratory and maintained in an aquarium to register their colour while alive (Figure 2). Specimens were relaxed in a 5% 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. Measurements of column height, as well as pedal and oral

disc diameter were obtained from fixed specimens; fragments of selected specimens were dehydrated and embedded in paraffin. Histological sections 6– 10 mm thick and stained with haematoxylin –eosin (Estrada-Flores et al., 1982) were prepared to examine internal anatomy. Data on cnidae were obtained from four representatives of each of the three morphotypes (a total of 12 individuals), all collected from La Gallega reef. Seven squash preparations were obtained from the main tissue types (1 mm3) of each specimen. We analysed cnidae from the marginal tentacles tips (mtt), discal tentacles (dt), actinopharynx (ac), filaments (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 these protuberances regularly develop in morphotypes M1 and M3. From each of the seven squash preparations, the length and width of 40 undischarged capsules (replicates) of each type of cnidae were randomly measured using DIC microscopy 1000 × oil immersion (following Williams, 1996, 1998, 2000). Cnidae samples were ordered in a bi-dimensional space using principal component analysis (PCA). Differences in ordination given by morphotype, individual specimen and type of cnidae, as well as the interaction terms among these factors were analysed using a permutational MANOVA procedure (Anderson, 2001; McArdle & Anderson, 2001). Differences among cnidae were analysed for each type of tissue separately. The PERMANOVA procedure was applied on resemblance matrices based on the Euclidian distance between samples. Although length and width of the capsules were in the same measurement scale, data were standardized and normalized prior to analyses. The statistical model used was 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 of 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

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 representing morphotype (with three levels); a is the coefficient 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 of cnidae (with three or two levels, depending on tissue kind) and is orthogonal to M and I; MT and I(M)T are corresponding interactions terms; and S is the residual matrix. Permutation procedures were applied to obtain appropriate distributions for the pseudo-F statistic under the null hypothesis. All analyses were performed using permutations of residuals under the reduced model, resulting in a range from 909 to 999 unique permutations for each F-test. The experimental design was balanced in every case, and the partitioning of variation was achieved so that the test statistic (pseudo-F) represents the proportion of the variation in the bi-dimensional cloud 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 of Mexico and Mexican Caribbean Sea (Registration code: YUC – CC –254 –11) of the Unidad Multidisciplinaria de Docencia e Investigacioń en Sisal (UMDI-Sisal) at the Universidad Nacional Autońoma de Me´xico (UNAM).

Molecular analyses Acquisition of molecular data followed the protocol detailed in Lauretta et al. (2013). We obtained DNA sequences of

three mitochondrial (12S and 16S rDNA and cox3) regions for 14 specimens (11 from La Gallega reef and three from Puerto Morelos reef). Phymanthus crucifer haplotypes were compared to available GenBank sequence data for Phymanthus loligo (Hemprich & Ehrenberg in Ehrenberg, 1834) and Heteranthus sp. (for GenBank accession numbers see Rodrı´guez 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 crucifer which were added to the data matrix presented in Rodrı´guez et al. (2014) after removing all hexacoral taxa not belonging to Actiniaria (except the antiphatharian Leiopathes Haime, 1849, which was used as an outgroup) and adding Heteranthus sp.; for a complete account of taxa included in this study, we refer readers to Rodrı´guez et al. (2014). New sequences have been deposited in GenBank (Table 1). DNA sequences of each marker were separately aligned using MAFFT v.7 (online at http://mafft.cbrc.jp/alignment/ server/; Katoh et al., 2002, 2005; Katoh & Toh, 2008) using the following settings and parameters: Strategy, L-INS-i (recommended for ,200 sequences with one conserved domain and long gaps); scoring matrix, 200PAM/k ¼ 2; gap opening penalty, 1.53; offset value, 0.05; max. iterate, 1000; and retree, 1. We then concatenated the three mitochondrial markers to create a single dataset for 115 taxa and 2697 sites. The Akaike information criterion (AIC) was implemented within jModelTest v.2.1.2 (Darriba et al., 2012) to determine

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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 Investigacioń en Sisal, UNAM; AMNH, American Museum of Natural History. Family

Species

ID number

Collection locality

Voucher

Phymanthidae

Phymanthus crucifer Phymanthus crucifer Phymanthus crucifer Phymanthus crucifer Phymanthus crucifer Phymanthus crucifer Phymanthus crucifer Phymanthus crucifer Phymanthus crucifer Phymanthus crucifer Phymanthus crucifer Phymanthus crucifer Phymanthus crucifer Phymanthus crucifer

RG-128 RG-129 RG-130 RG-131 RG-133 RG-134 RG-138 RG-143 RG-182 RG-184 RG-187 RG-200 RG-219 RG-220

GoM GoM GoM GoM GoM GoM GoM GoM GoM GoM GoM MC MC MC

UMDI-Sisal UMDI-Sisal UMDI-Sisal UMDI-Sisal UMDI-Sisal UMDI-Sisal UMDI-Sisal UMDI-Sisal UMDI-Sisal UMDI-Sisal UMDI-Sisal AMNH-5312 UMDI-Sisal 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 corresponding parameters (p-inv ¼ 0.0470, gamma shape ¼ 0.3360, 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) ¼ 1.3194, (CG) ¼ 1.0000, (CT) ¼ 8.7441, (GT) ¼ 1.0000) (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 (ML) within PhyML v.3.0 (http://www.atgc-montpellier.fr/ phyml/; Guindon & Gascuel, 2003). The following parameters were implemented within PhyML: substitution model ¼ GTR + I + G (the online version of PhyML does not implement TIM2, and GTR had a DAIC of 2.3); substitution rate categories ¼ 6; p-inv ¼ 0.0470; gamma shape ¼ 0.3360; starting tree ¼ BIONJ; tree improvement ¼ SPR & NNI; optimized tree topology and branch lengths; and bootstrap replicates ¼ 350. We also conducted tree searches under maximum parsimony (results not shown) with TNT v.1.1 (random and consensus sectorial searches, tree drifting and

Table 2. Morphological analysis of all three morphotypes; all measurements are in mm. pd, pedal disc diameter; ch, column height; od, oral disc 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 M1.2 M1.3 M1.4 M2.1 M2.2 M2.3 M2.4 M3.1 M3.2 M3.3 M3.4

11 23 25 32 22 23 27 10 16 23 20 32

31 20 22 23 28 26 26 8 34 16 29 18

36 48 45 53 59 49 51 38 48 44 54 46

2–3 3–4 2–5 2–4 2–4 3–4 3–6 2–4 3–7 3–5 4–5 2–3

Male Male (?) (?) Female Female Male (?) Male (?) (?) Female

M2

M3

100 rounds of tree fusing; Goloboff et al., 2008). In all analyses, gaps ( –) were treated as missing data. Trees of minimum length were found at least five times. The concatenated data set was subjected to 1000 rounds of bootstrap resampling to assess support for clades.

RESULTS AND DISCUSSION

Morphological analyses All twelve specimens examined from La Gallega displayed external morphological diagnostic taxonomic features corresponding to Phymanthus crucifer, including verrucae in the distal column arranged in longitudinal rows, column coloration with flame-like staining pattern, discal tentacles arranged in radial rows from peristoma to margin, and marginal tentacles hexamerously arranged. The only external morphological difference among specimens, aside from coloration 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 the taxonomic diagnostic features of P. crucifer). Size of specimens (pedal and oral disc diameter and column height) and number of verrucae per longitudinal row did not exhibit a consistent pattern that could be associated with any of the three marginal tentacular morphs (Table 2). The three morphotypes contained both relatively small and larger specimens, suggesting that the development of protuberances on marginal tentacles is not related to different growth stages of these organisms in the wild. Colour patterns of the oral disc and tentacles varied among all the specimens examined but did not show a consistent pattern 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), or with endocelic radial rows marking the arrangement of the discal tentacles (Figure 2I –J). The mouth was primarily the


same colour as the oral disc or exceptionally bright green or bright orange in some specimens (Figure 2F, I and 2D, respectively). The peristoma often had a lighter tone than the rest of the oral disc (Figure 2B, G, H, K). Marginal tentacles without protuberances in representatives of morph M2 and some of M3 presented longitudinal rows of yellowish, brownish or white colorations (Figure 2E –G, I – J); and some marginal tentacles had purple shades at their tips (Figure 2I, K). Colour pattern is a controversial character to distinguish sea anemones; some species are distinguished by colour patterns while others have distinct colour morphs that are considered to be phenotypic plasticity due to local genetic adaptations (Stoletzki & Schierwater, 2005). Phymanthus crucifer is dioecious and not thought to undergo asexual reproduction (Jennison, 1981). We found

spermatic vesicles (males) in all three morphotypes (Table 2), but oocysts in only some specimens of morphs M2 and M3. Nevertheless, oocysts have been reported in specimens of morph M1 in previous studies (Gonza´lez-Munõz et al., 2012). In most dioecious species of cnidarians, males and females are macroscopically indistinguishable (Fautin, 1992), whilst sexual dimorphism has only been reported for a few hydrozoan and scyphozoan species (Fautin, 1992), and for the actiniarian Entacmaea quadricolour (Leuckart in Ru¨ppell & Leuckart, 1828) (Scott & Harrison, 2009). Crowther (2013) suggested that the symbiotic relationship with zooxanthellae is likely associated with the formation of lateral protuberances in the tentacles as it occurs in other species such as Lebrunia coralligens (Wilson, 1890) and Lebrunia danae (Duchassaing & Michelotti, 1860). However,

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

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

Cnidae analyses We found the same types of cnidae (cnidom) in all samples examined, regardless of morphotype (Figure 3). The cnidom of Phymanthus crucifer included basitrichs, microbasic p-mastigophores and spirocysts, as previously reported for the family and genus (Carlgren, 1949). We did not find any additional types of cnidae in morphotypes M1 and M3 (those with protuberances in the marginal tentacles). It is unlikely that the protuberances on the marginal tentacles could be acting as structures for competition because agonistic behaviour in actiniarians is usually associated with the presence of holotrichs, a type of nematocyst in specialized structures such as acrorhagi and catch tentacles (Bigger, 1988; Williams, 1991) commonly found in some shallow water sea anemone species (Daly, 2003; Fautin, 2009). We measured 560 cnidae capsules per specimen, separated into 14 categories of cnidae (basitrichs, microbasic p-mastigophores and spirocysts) and tissue type; this added to a total of 6720 capsules measured (Figure 3). Our results 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 individual specimens (Figure 4A –G). The PCA ordination of samples from all tissue types showed that the first principal component explained from 60 to 94.5% of the variability of the cnidae size depending on the type of tissue being analysed (Table 3). Thus, the first principal component represents the variability in cnidae length. The percentage of variation explained by the second principal component was low (from 5.5 to 21.3%) in cnidae from ac, fi, pr/mt, dt and mtt, but relatively high in cnidae from co and vp (from 35.9 to 40.0%) (Table 3). This second principal component represents cnidae width. In ac and fi the variation in cnidae width was higher for microbasic 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). Acunã et al. (2007, 2011) only considered length when comparing cnidae sizes among specimens. Although our results confirm that length was the variable that explained most 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 be considered in future studies. 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 similar regardless of the morphotype they came from (see Figure 4A– G). By contrast, differences in cnidae size among specimens within each morphotype were significant for all tissue types (Table 3: Ind(Morph) and Ind(Morph) × Type). Cnidae size (both length and width considered) also varied significantly depending on cnidae type (Table 3: Type), but differences in size between cnidae types were similar among all three morphotypes (Table 3: Morph × Type). Overall, these results suggest that individuals constitute the main source of variation when the size of cnidae are examined. Edmands & Fautin (1991) noted that the size of nematocysts does not appear to correlate with animal size in Aulactinia veratra (Drayton in Dana, 1846), and Acunã et al. (2007) suggest that there is no functional relationship between cnida length and body weight in Oulactis muscosa (Drayton in Dana, 1846). Thus, although the diameter of the pedal disc is slightly variable between examined specimens of P. crucifer (Table 2), we found it unnecessary to include the pedal 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 variation among individuals or morphotypes from the Gulf of Mexico or Mexican Caribbean. However, mitochondrial 12S (824 bp) revealed two haplotypes that were distinguished by a single substitution (K2P distance ¼ 0.1215%, see Table 4), but these haplotypes were not specific to any particular morphotype. While haplotype 1 (differentiated by a single adenine substitution) was specific to Gulf of Mexico specimens, it was shared by all three morphotypes. Haplotype 2 (differentiated by a single guanine substitution) was more broadly distributed, being shared between specimens in the Gulf of Mexico and Mexican Caribbean. Within the Gulf of Mexico, haplotype 2 was shared by M2 and M3, while in the Mexican Caribbean it was shared by all three morphotypes. Table 4 summarizes divergence estimates among sequences within morphotypes of Phymanthus crucifer and representatives of 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 similarity 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 PC2 % of variation Morph Ind(Morph) Type Morph × type Ind(Morph) × type Total number of samples

90.7 9.3 0.858 0.001 0.001 0.918 0.001 1440

64.1 35.9 0.534 0.001 – – – 480

85.9 14.1 0.912 0.001 0.001 0.873 0.001 1440

87.6 12.4 0.895 0.001 0.001 0.117 0.001 960

94.5 5.5 0.572 0.001 0.001 0.855 0.001 960

78.7 21.3 0.826 0.001 0.001 0.163 0.001 960

60.0 40.0 0.235 0.001 – – – 480


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 of sequence divergence within and among anthozoan species, finding no variation in sequences from conspecifics is not Table 4. Divergence estimates (K2P) based on sequence comparisons of the three mtDNA markers. Comparisons were made between Phymanthus crucifer and Phymanthus loligo, as well as between Phymanthus crucifer and Heteranthus sp. NA, not available. Heteranthus sp. 12S Heteranthus sp. P. loligo P. crucifer 16S Heteranthus sp. P. loligo P. crucifer cox3 Heteranthus sp. P. loligo P. crucifer

P. crucifer

P. loligo 1.93%

0.12%

unexpected, even in those from potentially isolated populations 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 higher between Phymanthus crucifer and P. loligo or Heteranthus sp. than between the two haplotypes obtained for P. crucifer. Thus, although anthozoan mtDNA is characterized by low levels of divergence, we would expect at least a similar degree of divergence among the morphotypes of P. crucifer if they were indeed distinct species. If all three P. crucifer morphotypes are indeed a single species, then mitochondrial 12S revealed, for the first time, intraspecific variation within sea anemones.

2.06% NA 1.18%

SYSTEMATICS AND TAXONOMIC STATUS OF PHYMANTHIDAE

NA 1.73% 3.06% 2.39%

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

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

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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 members of Phymanthidae grouped within Actinoidea (Rodrı´guez et al., 2014), a superfamily of mainly shallow-

water sea anemones (Rodrı´guez et al., 2012, 2014). Our results concur with those of Crowther (2013), who included a higher taxon sampling of the superfamily Actinoidea in


her study of the families Thalassianthidae Milne-Edwards & Haime, 1851 and Aliciidae Duerden, 1895. The presence of Phymanthus crucifer morphotypes without protuberances in the marginal tentacles renders Carlgren’s (1949) major distinction between the two genera of Phymanthidae invalid. The marginal sphincter muscle, the other feature used by Carlgren (1949) to distinguish between these genera, is also problematic. Heteranthus is characterized by a weak but circumscribed marginal sphincter, whereas most species of Phymanthus lack a marginal sphincter (Carlgren, 1949). However, Phymanthus muscosus (Haddon & Shackleton, 1893) has a very feeble sphincter muscle (Haddon, 1898). Carlgren (1900) initially placed Heteranthus within a different family, Heteranthidae Carlgren, 1900, but he later placed it within Phymanthidae, based on similarities with Phymanthus (Carlgren, 1943). We recovered Heteranthus as nested within Actiniidae (see Figure 5) suggesting that discal tentacles have evolved independently at least twice within Actinoidea. A comprehensive revision of the family Phymanthidae and a redefinition of its diagnostic characters are needed to establish its membership. Based on external and internal morphological features, cnidae data, and mitochondrial DNA, we conclude that all morphotypes of Phymanthus crucifer represent a single species, despite differences in the presence or absence of protuberances in the marginal tentacles. The significance and function of the protuberances in the marginal tentacles remains unknown within P. crucifer, but might be related to specific adaptations to the surrounding environment.

Acunã F.H., Excoffon A.C., Zamponi M.O. and Ricci L. (2003) Importance of nematocysts in taxonomy of acontiarian sea anemones (Cnidaria, Actiniaria): a statistical comparative study. Zoologischer Anzeiger 242, 75–81.

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 black coral taxonomy and systematics. Zoological Journal of the Linnean Society 169, 312 –361.

Dr Judith Sańchez-Rodrı´guez (ICMyL) and B.S. Alejandro Co´rdova (FES-I) helped in the field; M.S. Maribel Badillo-Alemań (UMDI-Sisal) provided access and support to histological facilities; M.S. Gemma Martıńez-Moreno and Dr Patricia Guadarrama-Cha´vez (UMDI-Sisal) helped with laboratory work and provided support in the microscopy laboratory; Dr Andrea Crowther (South Australian Museum) provided 12S and cox3 sequence data for Heteranthus sp. All specimens were collected under consent of Mexican law, collecting permit approved by Comisioń Nacional de Acuacultura y Pesca (Number 07332.250810.4060). Comments of two anonymous referees improved this manuscript.

FINANCIAL SUPPORT

This work was partially supported by the Comisioń Nacional de Ciencia y Tecnologıá (CONACyT) (R.G., grant number 35166/202677); CONACyT –SEMARNAT (N.S., grant number 108285); and DGAPA –PAPIME –UNAM (N.S., grant number PE207210).

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Correspondence should be addressed to: R. Gonza´lez-Munõz Unidad 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 Email: [email protected]

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