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Journal of the Marine Biological Association of the United Kingdom, 2015, 95(1), 69 79.

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 gonzalez-mun~oz1,2, nuno simo~es1, maite mascaro1, jose luis tello-musi3,
mercer r. brugler4,5 and estefania rodriguez4
1

Unidad Multidisciplinaria de Docencia e Investigacion en Sisal (UMDI-Sisal), Facultad de Ciencias, Universidad Nacional
Autonoma de Mexico (UNAM), Puerto de Abrigo, Sisal, C.P. 97356 Yucatan, Mexico, 2Posgrado en Ciencias del Mar y Limnologa
(PCMyL), UNAM, Instituto de Ciencias del Mar y Limnologa (ICMyL), Circuito Exterior, Ciudad Universitaria, C.P. 04510,
Mexico, 3Laboratorio de Zoologa, Facultad de Estudios Superiores Iztacala (FES-I), UNAM, Avenida de los Barrios 1, Los Reyes
Iztacala, 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 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 intraspecic 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; rst published online 31 July 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; Rodrguez 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 ramied) 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. Gonzalez-Munoz
Email: ricordea.gonzalez@gmail.com

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 specic taxonomic diagnostic character due to its variability within conspecic individuals (Fautin, 1988, 2009;
Williams, 1996, 1998, 2000; Acuna et al., 2003, 2004;
Ardelean & Fautin, 2004; Acuna & 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 signicant 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
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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 xed 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 xed specimens; fragments


of selected specimens were dehydrated and embedded in parafn. 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), laments
(), 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 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 xed factor


representing morphotype (with three levels); a is the coefcient representing the intercept of the multivariate regression;
I is a random factor representing individuals nested in M
(with four levels); T is the xed 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 Investigacion en Sisal (UMDI-Sisal) at the
Universidad Nacional Autonoma de Mexico (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 Rodrguez 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 Rodrguez
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 Rodrguez 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 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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ricardo gonza lez-mun~ oz et al.

Table 1. Voucher specimen location and GenBank accession numbers for new sequences provided in this study. See Rodrguez 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 Investigacion 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 0.0470, gamma shape
0.3360, freqA 0.3034, freqC 0.1821, freqG 0.2212,
freqT 0.2933, (AC) 1.3194, (AG) 5.0386, (AT)
1.3194, (CG) 1.0000, (CT) 8.7441, (GT) 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
GTR + I + G (the online version of PhyML does not implement TIM2, and GTR had a DAIC of 2.3); substitution rate
categories 6; p-inv 0.0470; gamma shape 0.3360; starting tree BIONJ; tree improvement SPR & NNI; optimized tree topology and branch lengths; and bootstrap
replicates 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

23
34
25
24
24
34
36
24
37
35
45
23

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 ve 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 ame-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
Gonzalez-Munoz 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

characterizing variability within phymanthus crucifer

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 (Gonzalez-Munoz
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
Ruppell & 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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ricardo gonza lez-mun~ oz et al.

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 nd 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 signicant 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 rst 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 rst
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, , 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 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).
Acuna et al. (2007, 2011) only considered length when
comparing cnidae sizes among specimens. Although our
results conrm 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 signicant for all
tissue types (Table 3: Ind(Morph) and Ind(Morph) Type).
Cnidae size (both length and width considered) also varied
signicantly 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 Acuna
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 0.1215%, see Table 4),
but these haplotypes were not specic to any particular morphotype. While haplotype 1 (differentiated by a single adenine
substitution) was specic 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; , laments; pr/mt, protuberances
or middle part of the tentacle; dt, discal tentacle; mtt, marginal tentacle tip; vm, vesicle-like marginal projections.
Source

ac

co

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

characterizing variability within phymanthus crucifer

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) laments; (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,
nding no variation in sequences from conspecics 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 rst time, intraspecic 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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ricardo gonza 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 Rodrguez et al. (2014). Numbers above the branches are bootstrap resampling values expressed as a percentage; values ,50 not
indicated; lled-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
(Rodrguez et al., 2014), a superfamily of mainly shallow-

water sea anemones (Rodrguez et al., 2012, 2014). Our


results concur with those of Crowther (2013), who included
a higher taxon sampling of the superfamily Actinoidea in

characterizing variability within phymanthus crucifer

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 Carlgrens
(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 redenition 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 signicance and
function of the protuberances in the marginal tentacles
remains unknown within P. crucifer, but might be related to
specic adaptations to the surrounding environment.

Acuna 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, 7581.

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 Sanchez-Rodrguez (ICMyL) and B.S. Alejandro


Cordova (FES-I) helped in the eld; M.S. Maribel
Badillo-Aleman (UMDI-Sisal) provided access and support
to histological facilities; M.S. Gemma Martnez-Moreno and
Dr Patricia Guadarrama-Chavez (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 Comision
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 Comision Nacional


de Ciencia y Tecnologa (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. Gonzalez-Munoz
Unidad Multidisciplinaria de Docencia e Investigacion en Sisal
(UMDI-Sisal), Facultad de Ciencias, Universidad Nacional
Autonoma de Mexico (UNAM), Puerto de Abrigo, Sisal
C.P. 97356 Yucatan, Mexico
Email: ricordea.gonzalez@gmail.com

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