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 Investigacio´n en Sisal (UMDI-Sisal), Facultad de Ciencias, Universidad Nacional Auto´noma de Me´xico (UNAM), Puerto de Abrigo, Sisal, C.P. 97356 Yucata´n, Me´xico, 2Posgrado en Ciencias del Mar y Limnologı´a (PCMyL), UNAM, Instituto de Ciencias del Mar y Limnologı´a (ICMyL), Circuito Exterior, Ciudad Universitaria, C.P. 04510, Me´xico, 3Laboratorio de Zoologı´a, 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 mor- photypes 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; Acun˜a et al., 2003, 2004; Heteranthus has smooth marginal tentacles without protuber- Ardelean & Fautin, 2004; Acun˜a & 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 speci- mens 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;