Fast-Evolving Mitochondrial DNA in Ceriantharia: A
Reflection of Hexacorallia Paraphyly?
Sérgio N. Stampar1,2*, Maximiliano M. Maronna3, Marcelo V. Kitahara4, James D. Reimer5,

André C. Morandini2

1 Universidade Estadual Paulista ‘‘Júlio de Mesquita Filho’’, Laboratório de Biologia Aquática - LABIA, Faculdade de Ciências e Letras de Assis, Departamento de Ciências

Biológicas, Assis, São Paulo, Brazil, 2 Universidade de São Paulo, Instituto de Biociências, Departamento de Zoologia, São Paulo, São Paulo, Brazil, 3 Universidade de São

Paulo, Instituto de Biociências, Departamento de Genética e Biologia Evolutiva, São Paulo, São Paulo, Brazil, 4 Universidade de São Paulo, Centro de Biologia Marinha, São

Sebastião, São Paulo, Brazil, 5 Molecular Invertebrate Systematics and Ecology Laboratory, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa, Japan

Abstract

The low evolutionary rate of mitochondrial genes in Anthozoa has challenged their utility for phylogenetic and systematic
purposes, especially for DNA barcoding. However, the evolutionary rate of Ceriantharia, one of the most enigmatic ‘‘orders’’
within Anthozoa, has never been specifically examined. In this study, the divergence of mitochondrial DNA of Ceriantharia
was compared to members of other Anthozoa and Medusozoa groups. In addition, nuclear markers were used to check the
relative phylogenetic position of Ceriantharia in relation to other Cnidaria members. The results demonstrated a pattern of
divergence of mitochondrial DNA completely different from those estimated for other anthozoans, and phylogenetic
analyses indicate that Ceriantharia is not included within hexacorallians in most performed analyses. Thus, we propose that
the Ceriantharia should be addressed as a separate clade.

Citation: Stampar SN, Maronna MM, Kitahara MV, Reimer JD, Morandini AC (2014) Fast-Evolving Mitochondrial DNA in Ceriantharia: A Reflection of Hexacorallia
Paraphyly? PLoS ONE 9(1): e86612. doi:10.1371/journal.pone.0086612

Editor: Michael Schubert, Laboratoire de Biologie du Développement de Villefranche-sur-Mer, France

Received August 27, 2013; Accepted December 12, 2013; Published January 27, 2014

Copyright: � 2014 Stampar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 2012/01771, Coordenação de Aperfeiçoamento de
Pessoal de Nı́vel Superior (CAPES, PROEX) through the Programa de Pós-Graduação em Zoologia do Departamento de Zoologia IB-USP and Conselho Nacional de
Desenvolvimento Cientı́fico e Tecnológico - Universal 481549/2012-9 to SNS and JDR was supported by the Rising Star Program and the International Research
Hub Project for Climate Change and Coral Reef/Island Dynamics at the University of the Ryukyus. This work was partly supported by grants 2012/01771 (SNS),
2010/50174-7 (ACM), 2006/56211-6(MMM) and 2011/17537 (MVK), São Paulo Research Foundation (FAPESP). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: stampar@assis.unesp.br

Introduction

‘‘DNA barcoding’’ is the usage of a standardized DNA region

not only as a tool for fast and reliable identification of known

species, but also to assist the detection of undescribed species [1].

Researchers utilize a short DNA sequence (,700 bp) from the

mitochondrial protein-coding gene cytochrome c oxidase subunit I

(COI) that differs by several percent between even closely

metazoan related species as an adequate ‘‘barcode’’ to distinguish

species. Since then, the COI region has been widely used for DNA

barcoding in many Metazoa, including cnidarians (see more in

[2]). However, within cnidarians, the effectiveness of this marker

as a species-level barcode is purported to be limited to Medusozoa

[3] as it has levels of divergence (Kimura 2-Parameter, K2-P)

within congeners greater than 20% in Medusozoa [2], while this

divergence hardly exceeds 5% within anthozoan congeners [3–4].

Furthermore, other mitochondrial DNA markers broadly used –

such as mitochondrial 16S rDNA – also have divergence values

among anthozoan congeners significantly lower than those verified

for medusozoans [3]. As such, it appears that the anthozoan

mitochondrial molecular clock, most probably as a result of a

mismatch repair system - e.g. a MutS-like protein encoded by the

mt genomes of a number of octocorals [5–9], is slower than in

most Metazoa. This low evolutionary rate has been inferred as an

ancestral condition [3]; [10–11], although there is no evidence of

this mismatch repair system in Hexacorallia mitochondrial

genomes, or in other eukaryotes [12].

Within the ‘‘orders’’ that traditionally compose the Hexacor-

allia, only Ceriantharia does not have a representative with a

complete mitochondrial genome determined to date. Phylogenetic

studies of cerianthids based on mitochondrial and nuclear markers

have resulted in reconstructions divergent from those based on

morphology [11]; [13–15]. Thus, Ceriantharia is an ‘‘incertae sedis’’

group [13] within Anthozoa. In general, the phylogenetic

reconstruction of higher taxonomic levels (i.e. class, order)

demands molecular markers with ‘‘low’’ mutation rates (e.g. less

than 1% divergence/million years) [16]. Therefore, the long (28S)

and short (18S) nuclear ribosomal genes have been purported to

be adequate [17] if they are conserved enough to produce

unambiguous alignments and to provide an appropriate phyloge-

netic signal to define basal relationships [18]. In this study, nearly

complete sequences from 18 and 28S and partial 16S ribosomal

genes were used to reconstruct the cnidarian evolutionary history,

and specifically focus on the relative position of cerianthids within

the phylum. In addition, the genetic divergences of mitochondrial

markers (i.e. partial 16S and COI sequences) were defined, and

compared between Ceriantharia and other cnidarian groups,

indicating a ‘‘fast-evolving mtDNA profile’’ (based on medusozoan

data) in the former. In general, this study adds to the development

of a more cohesive evolutionary scenario for Ceriantharia, defines

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a phylogenetic framework for Ceriantharia systematics, and

proposes a new evolutionary scenario for the mutation history of

mtDNA in the non-bilaterian Metazoa.

Materials and Methods

DNA Extraction, Polymerase Chain Reaction and
Sequencing

Total DNA was extracted from individual cerianthid specimen

tentacles (Table 1) using InstaGeneH (Bio-Rad #732-6030) or the

DNAdvanceH kit (AgencourtH #A48705). PCR reactions and

conditions followed under predefined conditions ([13]; primers

used in the present study are listed in Table 2). Amplicons were

purified using AMPureH kit (AgencourtH #A63881) following

manufacturer’s instructions, and made ready for sequencing using

the BigDyeH Terminator v3.1 kit (Applied Biosystems #4337455;

same primers and Tm temperature conditions as in PCR

reactions). Sequencing was carried out on an ABI PRISMH3100

genetic analyzer (Hitachi), and resulting sequences were assembled

and edited using GeneiousTM 5.4.4 (Table 1). Ceriantharia

sequences from mitochondrial (COI, 16S) and nuclear (18S,

28S) molecular markers were obtained from 12 of the 50

taxonomically recognized species [28].

Mitochondrial Evolutionary Divergence between
Ceriantharia Species and Genera

Evolutionary distances of mitochondrial (16S rDNA and COI)

genes from Ceriantharia were analyzed within congeners (Isar-

achnanthus – see Table 3) and between genera in MEGA5 software

[29], using Kimura’s two-parameter model of base substitution

(K2-P) in order to calculate their respective genetic distances. In

addition, K2-P genetic distances were also estimated for other

anthozoan clades, and hydrozoans were considered as ‘‘outgroup’’

for discussion. The distances obtained for both mitochondrial

markers are compiled in four graphs in two figures, of which one

are from congeners (Figure 1), and another from species of

different genera (Figure 2). The complete datasets, including

sequences obtained from GenBank, are compiled in the supple-

mentary material (Table S1).

Phylogenetic Inferences and Hypotheses Testing
Representing all accepted cnidarian clades except Myxozoa

(sensu [11]; Text S1), 18S and 28S rDNAs sequences were used to

reconstruct the cnidarian evolutionary history. In order to avoid

the effects of an excess of missing entries on a combined analysis,

each molecular marker was analyzed with a single gene analysis

approach. In order to avoid any limitation on restricted results

from a certain method of alignment and phylogenetic method, we

analyzed our 18S and 28S datasets with several alignment

methods, masking data and all standard phylogenetic reconstruc-

tion methods: (1) ‘‘static’’ defined homology applying consistency

scores (MAFFT, [30]) and estimated secondary structure (RNA-

salsa; [31]); (2) ‘‘dynamic’’ defined homology applying probabilis-

tic approaches with indel model (Bali-Phy; [32]), gaps treated as

missing data (MAFFT; [30] and OPAL; [33] in Saté; [34]), and

Table 1. List of Ceriantharia species included in the present study.

Family Organism Obtained sequences GENBANK-DDBJ/BOLD Number

Arachnactidae Isarachnanthus bandanensis 16S/COI 16S - (JX125699) COI - (CMBIA097-11(BOLD))

Isarachnanthus maderensis 16S/COI/18S 16S - (JX125670–72/79–80/82/85–87)/COI - (JX128313–14/22–23/25/
28–33)/18S – AB859825

Isarachnanthus nocturnus 16S/COI/28S/18S 16S - (JX125669/73–78/81/83–84/88–89/91–98)/COI - (JF915196–97/
JX128315–21/24/26/27/34–42)/18S - AB859826/28S - AB859832

Cerianthidae Ceriantheomorphe brasiliensis 16S/COI/18S/28S 16S - JF915193/COI - JF915195/18S – AB859823/28S – AB859831

Ceriantheopsis americanus 16S/COI 16S – AB859834/COI – AB859839

Cerianthus membranaceus 16S/COI/18S 16S – AB859837/COI – AB859843/18S – AB859824

Cerianthus lloydii 16S/COI 16S – AB859838/COI AB859844

Pachycerianthus sp.1 16S/COI/18S/28S 16S – AB859835/COI – AB859840/18S – AB859829/28S – AB859833

Pachycerianthus borealis 16S 16S - U40288

Pachycerianthus magnus 16S/COI/18S 16S – AB859836/COI – AB859841/18S – AB859828

Pachycerianthus fimbriatus COI/18S COI – AB859842/18S –AB859827

doi:10.1371/journal.pone.0086612.t001

Table 2. List of primers used to amplify/sequence
Ceriantharia representatives in this study.

Marker Primers Tm Reference

COI LCO 1490+ HCO2198 49u [19]

LCO 1490+ HCOout 48u [20]

LCO 1490+ HCOCato 48u [21]

16S CB1+ CB2 56u [22]

CB1+ R [BR] 54u [23]

F1Mod+CB2 56u [22]

18S 18C+18Y 55u [24]

18A+18L 55u [24]

18O+18B 55u [24]

28S 5S-R635, F635–R1630,
F1379–R2077,
F2076–R2800, F2800–
R3264

63u [25] [26] [27]

doi:10.1371/journal.pone.0086612.t002

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through several transformation costs in parsimony (POY; [35]). To

consider the possible influence of gaps, and because some positions

with noise signal in molecular datasets may result in topologies

with phylogenetic artifacts, we made extra datasets for analyses: (1)

gaps coded as an additional binary datamatrix (for probabilistic

based methods; Fastgap; [36]) and (2) highly variable positions

were detected as potentially noisy positions, and were masked

(filtered) using Aliscore [37].

Considering the phylogenetic reconstructions methods, we

analyzed the datasets under Maximum Likelihood (RAxML, Saté;

[38]; [34]), Bayesian inference [MrBayes, Bali-Phy; [39], [32]) and

Maximum Parsimony (TNT, POY; [40], [35]; see Text S1 and for

Table S3 for full parameterss information on all thirteen

phylogenetic reconstructions’ approaches.) To evaluate nodal

support and to detect if support values were biased, two

parametric (aLRT and aBAYES) and two non-parametric

(Bootstrap, BS and SH-aLRT) techniques were applied in

MAFFT+RAxML results [38]; [41]). Bootstrap values were

computed on RAxML v7.3.2 (500 pseudoreplicates, same

parameters as the original phylogenetic analysis) and additional

statistical tests were performed using PhyML v3.0.1 [42]; [43])

under the same parameters as the original MAFFT+RAxML

inferences (Figure 3). As additional datasets, representing mito-

chondrial DNA we analyzed partial sequences from the mito-

chondrial ribosomal gene 16S for 63 cnidarian species with

outgroups as used in previous works (see Text S1, Table S1 and

Table S2) for final rooting, and a dataset including two extra

Ctenophora species for final outgroup consideration (both analyses

with no masking option). Sequences were aligned in MAFFT

(parameter: auto), phylogeny estimated in RAxML (GTR DNA

model, 125 replicates) and support values estimated as in 18S and

28S studies. A combined dataset was created for 18S and 16S

Figure 1. Estimates of evolutionary divergence in major Anthozoan lineages from mitochondrial molecular markers (intrageneric
level, Hydrozoa considered as an outgroup for discussion). Right graph: estimates of evolutionary divergence (Kimura 2-parameter model) of
COI among congeners of Ceriantharia (n = 9), Hydrozoa (n = 10), Actiniaria (n = 5), Scleractinia (n = 7), Zoantharia (n = 7) and Octocorallia (n = 6),
(n = number of species examined). Left graph: estimates of evolutionary divergence (Kimura 2-parameter model) of 16S ribosomal DNA among
congeners of Ceriantharia (n = 9), Hydrozoa (n = 11), Actiniaria (n = 9), Scleractinia (n = 7), Zoantharia (n = 6) and Octocorallia (n = 6), (n = number of
species examined).
doi:10.1371/journal.pone.0086612.g001

Figure 2. Estimates of evolutionary divergence in major Anthozoan lineages (intergenera level, Hydrozoa considered as outgroup
for discussion) from mitochondrial molecular markers. Right graph: estimates of evolutionary divergence (K2-P) of COI between species of
different genera in Ceriantharia (n = 8), Hydrozoa (n = 12), Scleractinia (n = 10), Zoantharia (n = 4) and Octocorallia (n = 8) (n = number of species
examined). Left graph: estimates of evolutionary divergence (Kimura 2-parameter model) of 16S ribosomal DNA between species of different genera
in Ceriantharia (n = 9), Hydrozoa (n = 13), Actiniaria (n = 12), Scleractinia (n = 11), Zoantharia (n = 5) and Octocorallia (n = 11) (n = number of species).
doi:10.1371/journal.pone.0086612.g002

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sequences, with basic similar results to single gene 16S analysis (see

Table S1 and Figure 3 and Figure S1 for sampled species and

phylogenetic results). Different data treatment and phylogenetic

analysis approaches were additionally tested on a COI dataset, but

no major recognizable cnidarian clades were recovered (these

results were not included in our main results: see Figure S2 and

Figure S3 for results and analyses’ details).

To analyze the phylogenetic signal profile along the recovered

phylogenies in figure 3, the net approach test was performed using

PhyDesign (Figure 4; [44]). Finally, to test alternative systematic

proposals and our own main results, considering the phylogenetic

position of Ceriantharia, we computed AU and other phylogenetic

hypothesis tests using consel ([45]; same input datasets and results

from the MAFFT+RAxML analysis; Table 4).

Results

Mitochondrial Genetic Diversity
While the genetic distance estimates for COI and 16S within

Ceriantharia congeners ranged from 3 to 17% and 3 to 12%,

respectively (Figures 1 and 2), values for all other anthozoan

groups excepting Octocorallia were estimated to be less than 1%

(COI) to 2% (16S). Octocorallian congeners slightly exceeded

these values, with approximately 2% and 4% divergences within

COI and 16S sequences, respectively. In general, the divergence

values estimated for ceriantharians showed a range most similar to

those from hydrozoans (medusozoan example) (intrageneric

divergence: COI/16S 3–17%/3–12% (Ceriantharia) and 4–

23%/2–14% (Hydrozoa)). Furthermore, the estimated COI

genetic divergence between genera in every major anthozoan

group (Octocorallia and Hexacorallia) was lower than the

divergence observed for ceriantharian congeners. Most anthozo-

ans had intergeneric divergences below 10% while the estimated

divergence in ceriantharians was between 14 and 22% (Figure 3).

However, the calculated 16S divergence between species of

different genera showed less difference between Ceriantharia

and other anthozoans, as observed values in Ceriantharia ranged

from 7 to 23%, while other anthozoan groups ranged from 0 to

7%.

An example of the genetic divergence of related congeneric

species is the divergence values of Isarachnanthus spp. in the Atlantic

Ocean ( = 6–9%), which are higher than levels reported from

similar comparisons in other anthozoans. The estimated diver-

gence between several specimens of I. nocturnus and I. maderensis was

based on multiple specimens and is considered reliable (Table 3).

Phylogenetic Position of Ceriantharia
The final alignments consisted of 73 (18S rDNA), 63

(mitochondrial 16S rDNA) and 25 (28S rDNA) cnidarian species,

with 1992, 1184 and 3013 positions, respectively (MAFFT results).

Instead of Porifera, Ctenophora was chosen as outgroup in all

studies, as fewer gaps were required in order to align their

sequences together with cnidarian sequences.

Results of the phylogenetic reconstructions are show in Figures 3

and 5 and summarized below.

Both 18S and 28S genes recovered monophyletic Anthozoa and

Meduzozoa with a sister relationship in almost all results (Figures 3

and 4), although the Hexacorallia monophyly suffered due to

Ceriantharia’s relative position. In most 18S reconstructions, all

anthozoan and medusozoan lineages (i.e. Octocorallia, Hexacor-

allia, Scyphozoa, Staurozoa, Cubozoa, and Hydrozoa) were

recovered as monophyletic groups. However, Ceriantharia was

recovered as a sister group to Anthozoa, or in some cases, as a

sister group of Cnidaria (Table 4). Ceriantharia, which has been

taxonomically placed within Hexacorallia, was recovered as such

only with dynamic homology with low CI and RI indexes if

compared to other MP-POY topologies from the same dataset. A

sister-like relationship between Ceriantharia and Octocorallia was

also recovered in MP topologies where gaps were treated as

missing data (TNT analysis), or in ML and MP with filtered

datasets (RAxML and POY results; Table 4; except for these cases,

Ceriantharia was recovered as a sister group to Cnidaria).

The monophyly of both Anthozoa and Medusozoa, and their

respective major component groups, were also recovered using

28S rDNA (Figure 3, Table 4). However, in general, the main

topology differed from that of 18S rDNA as Ceriantharia occupied

a sister-like position to Octocorallia, with high statistical support in

all but non-parametric bootstraps. MP topologies that recovered

Ceriantharia as sister group of Hexacorallia (traditional placing)

had several unexpected results – for example, non-monophyletic

Scyphozoa and Staurozoa as sister groups of Hydrozoa. As in 18S

rDNA, these MP topologies had lower CI and RI values compared

to results using different methods (Table S2). The 16S dataset

including two ctenophore sequences had a loss of monophyly of

the Medusozoa group and some well-recognized cnidarian clades

(e.g., Hydrozoa), with Ceriantharia grouped with the rest of the

anthozoan lineages (but not as sister group of the rest of

Hexacorallia), possibly representing a case of basal long branch

attraction for portions of the sampled Hydrozoa and Ctenophora

sequences. For the 16S dataset we rooted the 16S phylogeny

considering Medusozoa and Anthozoa as monophyletic groups,

and this analysis had highly similar results as to the 18S ML

analysis and rooted with ctenophore sequences (Figure 3). It is

important to note that the late definition of rooting does not affect

analyses in ML studies nor phylogenetic relationships between the

terminal trees. Due to better support values and more similar clade

relationships for major cnidarian lineages with 18S and 28S

results, we focused on the 16S dataset and their ML result that did

Table 3. Estimates of average evolutionary divergence over sequence pairs within Isarachnantus representatives.

Molecular Marker Species Sample Size Within Species Between Species

d (%) S.E. d (%) S.E.

16S I. maderensis 9 0.5 0.002 6.0 0.01

I. nocturnus 20 0.0 0.000

COI I. maderensis 11 0.0 0.000 8.9 0.01

I. nocturnus 21 0.1 0.000

The number of base substitutions per site from averaging over all sequence pairs within each group is shown. d – Distance in % and S.E. – Standard error estimates were
obtained by a bootstrap procedure (500 replicates).
doi:10.1371/journal.pone.0086612.t003

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not include ctenophoran sequences for Consel and PhyDesign

analysis. Consel recovered low support (negative significance value

- less than 0.05 of p-value) for Ceriantharia as sister group of

Hexacorallia compared to alternative systematic scenarios in both

18S and 16S analyses (Table 5). Based on PhyDesign outcomes, it

is believed that the 28S rDNA has more phylogenetic signal than

the 18S rDNA, and consequently is more adequate for Cnidaria

higher rank reconstruction (Figure 4). However, it is important to

note that these results may be biased due to the different number

of species included in each gene dataset (and consequently

different number of epochs in each topology; Figure 4).

Overall, our results from both nuclear and mitochondrial

ribosomal gene analysis corroborate that Ceriantharia is a

monophyletic and basal independent lineage, but indicates that

it does not have a direct relationship to other hexacorallian orders.

Thus, these results suggest that Ceriantharia should be ranked as a

separate subclass within Anthozoa.

Figure 3. ML phylogenies of Cnidaria based on ribosomal molecular markers 18S (upper left), 28S (upper right) and 16S datasets
(lower center). Major cnidarian lineages are in cladograms and phylograms for Medusuzoa clades (Hydrozoa, Scyphozoa, Cubozoa, Staurozoa) and
Anthozoa (Hexacorallia, Hexacorallia, with special emphasis with Ceriantharia (green). Divergent results in the 18S phylogeny (considering the major
clades) are shown in 16S (no Ctenophora sequences in analysis) and 28S trees (jellyfish icon for Medusozoa clades, polyp icon for Anthozoa clades).
Supports values are aBAYES and aLRT (parametric, upper branch) together with BS and SH-like (non parametric, lower branch) in clockwise fashion.
Each dataset was aligned in MAFFT and phylogeny estimated in RAxML; support values were calculated in RAxML (BS) and PhyML (aBAYES, aLRT and
SH-like; see Table S2 for details in datamatrices, software and parameters).
doi:10.1371/journal.pone.0086612.g003

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Discussion

Anthozoan Phylogenetics and Genetic Divergence
among Cnidarian Species and Genera

According to [3], ‘‘the tempo of evolution in anthozoan

mitochondrial genes appears to be at least 10–20 times lower

than the standard mitochondrial clock based on vertebrate

sequences, which averages a sequence divergence of 1–2%/Myr

[46–49]…’’. With the use of a number of statistical methods, [50]

also found that the anthozoan intra- and interspecific COI

variations were #6% or invariant (in 98% of the analyzed species).

The genetic divergence of ceriantharian mitochondrial markers

would therefore be expected to follow the slow rate of

differentiation as detected in other groups currently classified as

anthozoans [51]. However, an overview of our results suggests a

much faster rate. For both analyzed mitochondrial markers (COI

and 16S rDNA), the genetic divergence rates estimated between

Ceriantharia congeners are more similar to those from Hydrozoa

than to Anthozoa, suggesting that the mitochondrial clock in

Ceriantharia has almost the same divergence rate as in Medusozoa

and other Bilateria groups (see also [13]).

Therefore, the above statement of slow mitochondrial evolution

in Anthozoa is accurate only if Ceriantharia is not included
within Anthozoa classification. In fact, this hypothesis is not

unusual. Based on nuclear molecular markers, [15] and [11]

showed that Ceriantharia is not within Hexacorallia. Another

study also stressed that Ceriantharia could be a hexacorallian

outgroup [52], but its position was not further discussed. In

addition, analyses of complete mitochondrial genomes indicate

that the phylogenetic position of Ceriantharia is unstable and not

well defined (e.g. sister group of Hexacorallia) [53]. For discussion

on the historical context of molecular data and Ceriantharia’s

evolutionary position over the last twenty years see (Text S1).

More recently, however, a new body of data is changing the

scenario previously proposed for the origin of the rapid evolution

of mtDNA [50]. In summary, there are two most parsimonious

explanations for the slow mtDNA evolution in anthozoans (see

Figure 2 in [50]). However, both scenarios must be modified to

accommodate the Ceriantharia results presented herein. The first

scenario (Figure 5, reconstruction A) hypothesizes independent

origins for the rapid evolution of mtDNA in Medusozoa and

Bilateria. However, it is necessary to include an additional step

(origin of the rapid evolution of mitochondrial DNA) before

Ceriantharia, and consequently this reconstruction is no longer

most parsimonious. In the second scenario (Figure 5, reconstruc-

tion B) there is no necessity to add an extra step, making it the

most parsimonious scenario; namely that anthozoans (not includ-

ing Ceriantharia) decreased their mtDNA rate of evolution.

Corroborating the findings from [15], the phylogenetic

‘‘uniqueness’’ of Ceriantharia in relation to anthozoans has major

implications not only in basal metazoan mtDNA evolution but also

in cnidarian evolutionary history. The inclusion of Ceriantharia

data in molecular clock phylogenetic reconstructions may influ-

ence estimates for the appearance of Cnidaria. It may also have

effects on the stability of the position of Cnidaria in broader

Figure 4. Phydesign curves - Phylogenetic Informativeness
profiles from the 18S, 16S (no Ctenophora sequences in
analysis), and 28S datasets, computed with their original
datasets and results from the MAFFT+RAxML results. The x-
axis represents topologies considering their nodes as epoch units, the
y-axis represents the Net Phylogenetic Informativeness value for each
molecular marker along the topologies.
doi:10.1371/journal.pone.0086612.g004

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studies, where the monophyly of the group (and major lineages,

like Anthozoa and Medusozoa) are compromised by potential

incomplete taxon sampling [53–55]. From the current state of

knowledge, it is possible to infer that Ceriantharia does not belong

to Hexacorallia and thus represents a new subclass. However, the

exact phylogenetic position of Ceriantharia is still debatable. Our

study indicates that, based on a broader sampling of the group,

Ceriantharia is most likely a sister group to Anthozoa. However, a

sister-like relationship between Ceriantharia and Octocorallia

cannot be discarded and further analyses are needed to clarify this

matter.

Futures Prospects on Cnidarian mtDNA Evolutionary
Genetics

A better understanding of the mechanisms related to the shifts of

mtDNA evolutionary rates is a challenging task in current

cnidarian studies [56–58]. There are several possible historical

influences on qualitative aspects of mtDNA on this group, such as

genome linearization and fragmentation, horizontal gene transfer

from a non-bilaterian species (e.g., HGT, mtMutS gene), and gene

arrangement (e.g., [59–61]). Recombination phenomena have

been invoked to explain gene rearrangements in Octocorallia, and

plasmid insertion to explain mtDNA linearization in Medusozoa

[60]. The connection between genome components and rear-

rangements with mutation rates is not a trivial matter [62–65], as it

is proposed to have been involved as one of the main mechanisms

on evolutionary genetics of linear genomes [66]. Nonetheless, the

theory of a mtDNA mismatch repair gene in Octocorallia as the

mechanism explaining slow rates of genetic divergence does not

explain the slow rates exhibited in Hexacorallia (Figures 1–3). This

consideration reinforces the ‘‘classic’’ relationship proposed

between Octocorallia and Hexacorallia lineages, differing from

phylogenetic analyses based on mitochondrial data linking

Octocorallia as sister group of Medusozoa (Anthozoa not

monophyletic: [53]; [67]. Thus, more extensive studies on

Table 4. Phylogenetic results considering all the analyzed ribosomal datasets and methods, showing the recovery of major clades
in Cnidarian systematics proposals in our topologies and literature.

Tree Topology (phylogenetic hypotheses)

Static homology
strategy Dynamic homology strategy

ML (x4) BY (x2) MP (x30) ML (x2) BY MP(x18)

18S 28S 16S 18S 28S 18S 28S 18S 28S 18S 28S 18S 28S

Anthozoa monophyletic (1,2,3) YES YES YES YES 2/30 YES (1) YES no YES 6/18 YES

Ceriantharia sister group of rest of
Hexacorallia

no no no no_poli no no 2/30 no no no no no 4/18

Ceriantharia sister group of Octocorallia no YES no no_poli YES 1/30 19/30 no YES no YES no 9/18

Ceriantharia sister group of rest of Cnidaria ali(4) no no no_poli no 27/30 no ali(2) no YES no 7/18 no

Ceriantharia sister group of rest of Anthozoa (1,2,3) no YES no_poli no no 6/30 (1) no no no 2/18 4/18

Octocorallia monophyletic YES YES YES YES YES 29/30 YES YES YES YES YES 11/18 YES

Rest of the ‘‘Hexacorallia’’ clade monophyletic YES YES YES YES YES YES YES YES YES YES YES 14/18 15/18

Octocorallia sister group of Medusozoa ali(4) no no no no 4/30 no ali(2) no no no 4/18 no

Medusozoa monophyletic YES YES YES YES YES 28/30 26/30 YES YES YES YES 11/18 YES

Staurozoa, Scyphozoa, Cubozoa and Hydrozoa
monophyletics

YES YES YES YES YES 23/30 YES YES YES YES YES 11/18 16/18

Staurozoa sister group of rest of Medusozoa no YES no no YES 1/30 24/30 ali(2) ali(2) no YES no 6/18

Staurozoa sister group of Scyphozoa (3) & ali(4) no no no no 4/30 no no no no no no no

Staurozoa sister group of Cubozoa (1,2) no YES YES no 9/30 no (1) no YES no 13/18 no

Major results considering Ceriantharia’s position are shown in blue or black font. Results are presented from the static and dynamic homology strategies, considering
the 18S, 16S (no Ctenophora species in analysis) and 28S datasets. Static homology strategy, ML (x4) = Maximum Likelihood results considering 4 different analytical
strategies: (1) MAFFT+RAxML, (2) RNAsalsa+RAxML (with and without secondary structure info included in these analysis), (3) gap coded as binary data matrix
(RNAsalsa+FastGap+RAxML), (4): (ali: RNAsalsa+aliscore+RAxML); Static homology strategy, BY (x2) = Bayes results considering 2 different analytical strategies: (1) no gap
coded (RNAsalsa+MrBayes), (2) gap coded (RNAsalsa+FastGap+MrBayes); Static homology strategy, MP = maximum parsimony results considering 30 different analytical
strategies, being divided as 15 analyses with different cost weight regimes (3 of them treating gaps as missing data: RNAsalsa+TNT) and other 15 similar cost weight
regimes, after alignment masking with (RNAsalsa+aliscore+TNT); Dynamic Homology strategy, ML (x2) = Maximum Likelihood results considering 2 different analytical
strategies: (1) SATé (CLUSTALW+RAxML) and (2) a 18S filtered datamatrix (ali: RNAsalsa+aliscore+(SATé = MAFFT+RAxML)); Dynamic Homology strategy, BY = Bayes
results from a dynamic homology analysis (BAli-Phy); no_poli = basal politomy in Anthozoa related to major recovered clades (Ceriantharia, Octocorallia and
‘‘Hexacorallia). In cases of all result/s being the considered hypotheses: YES (black box); most of results of the considered hypotheses were equal or .50% of total
analyses: number of positive (dark grey); most of results considered the hypotheses were ,50% of total analyses: number of positive results (light grey).
doi:10.1371/journal.pone.0086612.t004

Figure 5. Graphic representation of possible evolutionary
scenarios for slow mtDNA evolution in Anthozoa, modified
from [39]. Scenario A: fast evolution originated in the Ceriantharia,
Medusozoa and Bilateria independently (adapted from [43]). Scenario B:
fast mtDNA evolved as a unique event (Cnidaria+Bilateria) but was
subsequently lost in Anthozoa (not including Ceriantharia).
doi:10.1371/journal.pone.0086612.g005

Fast-Evolving Mitochondrial DNA in Ceriantharia

PLOS ONE | www.plosone.org 7 January 2014 | Volume 9 | Issue 1 | e86612



mitochondrial genome arrangement and how it is related to

mutation/evolutionary patterns are needed in Hexacorallia.

Additional efforts should also be undertaken to increase cer-

iantharian taxon sampling, evaluation of different cnidarian

outgroups (e.g., Porifera vs Ctenophora), together with a thorough

evaluation of (1) data treatment to define homology, (2)

reconstruction parameters and methods, (3) nodal support, (4)

hypotheses testing and the (5) recovered phylogenetic signal (e.g.

[68–69]; [34]).

Recently [70] suggested that almost 90% of the Ceriantharia

species are already known. However the authors neglected to

address the existence of cryptic species, species with disjunct

distributions (e.g. Ceriantheomorphe brasiliensis, Ceriantheopsis ameri-

canus), and oceanic areas with no data (e.g. deep sea); additionally,

the evolutionary position of Ceriantharia should be clarified

because of their possible reflection of basal adaptive radiation in

cnidarians, considering their asymmetric conditions on sister

clades, based on (1) number of extinct derived species, (2) genetic

divergence and (3) morphological-ecological traits, that altogether

could represent signals of adaptative events [67] [71]. Finally, we

emphasize that the inclusion of Ceriantharia in Hexacorallia

should no longer be accepted and this group should be elevated to

subclass.

Thus the class Anthozoa should be divided into three
subclasses;

– Hexacorallia,

– Octocorallia

– Ceriantharia subclass nov.

Supporting Information

Figure S1 ML cladogram of Cnidaria based on a
combined dataset (18S+16S ribosomal molecular mark-
ers partitions). Supports values are aBAYES and aLRT

(parametric, upper branch) together with BS and SH-like (non

parametric, lower branch) in clockwise direction. Each dataset was

aligned in MAFFT and phylogeny estimated in RAxML

(independent branch length calculated for every molecular marker

partition); support values were calculated in RAxML (BS) and

PhyML. Data treatment and basic parameters (e.g., number of

replicates) were similar to individual ML analysis for 18S dataset

(MAFF+RAxML analysis; see Table S2 for details in datamatrices,

software and parameters).

(TIF)

Figure S2 ML phylograms of evolutionary relationships
presented in figure 3, considering ribosomal molecular
markers analysis (A: nuclear 18S; B: mitochondrial 16S
(no Ctenophora species in analysis); C: nuclear 28S).

(TIF)

Figure S3 ML phylograms from different analysis
datasets for cytochrome oxidase I (COI) cnidarian
sequences estimated in Garli (100 replicates; [72]).
Partition analysis with PartitionFinder [73] defined two basic

partitions as optimal to estimate gene phylogeny for COI (all

codon positions): first and second position (partition 1, model

TVM+G) and third position (partition 2, model SYM+G); then

both partitions (Figure A) and partition 1 only (Figure B) were

analyzed. Trying to overcome molecular saturation (non-phylo-

genetic related heterogeneity), the COI dataset was filtered at two

different intensity levels: treating gaps as missing data (‘‘Aliscore -

N’’ strategy; Figure C) and a more intense approach (‘‘Aliscore -N

-r -w4’’; Figure D). The COI dataset (Genbank IDs presented in

terminal’s names) was originally aligned in MAFFT (codon frame

checked); the root position was defined a posteriori (random

position; no effect on ML analysis).

(TIF)

Table S1 List of DNA sequences species used in this
study considering the 18S, 28S and 16S datasets (new
sequences remarked in blue).

(XLSX)

Table 5. Hypothesis testing results, considering Bayesian posterior probability calculated by the BIC approximation (PP) and
results based on p-values; significance level result for AU (.0.05), rejecting the Hexacorallia clade in the traditional proposal
remarked in blue).

Phylogenetic hypotheses (18S datamatrix) AU PP KH SH WKH WSH

Ceriantharia sister group of rest of Anthozoa 0.861 0.901 0.812 0.846 0.812 0.844

Ceriantharia sister group of Octocorallia 0.214 0.054 0.188 0.190 0.188 0.329

Hexacorallia ‘‘monophyletic’’ 0.151 0.045 0.169 0.173 0.169 0.306

Phylogenetic hypotheses (16S datamatrix) AU PP KH SH WKH WSH

Ceriantharia sister group of rest of Anthozoa 0.760 0.953 0.746 0.825 0.746 0.799

Ceriantharia sister group of Octocorallia 0.273 0.041 0.254 0.268 0.254 0.370

Hexacorallia ‘‘monophyletic’’ 0.035 0.06 0.101 0.114 0.101 0.192

Phylogenetic hypotheses (28S datamatrix) AU PP KH SH WKH WSH

Ceriantharia sister group of Octocorallia 0.873 1.0 0.871 0.93 0.871 0.909

Ceriantharia sister group of rest of Anthozoa 0.137 0.0 0.13 0.132 0.129 0.208

Hexacorallia ‘‘monophyletic’’ 0.003 0.0 0.021 0.022 0.021 0.045

The original phylogenetic results from these datasets and their results, as well main results from the approximately unbiased test (AU, non parametric test) and posterior
probabilities values (PP, parametric test) are shown in bold. The 18S and 28S datasets were aligned with RNAsalsa and 16S dataset (no Ctenophora species in analysis)
was aligned with MAFFT, with no data masking in all cases. Other tests: Kishino-Hasegawa test (KH); Shimodaira-Hasegawa test (SH); weighted Kishino-Hasegawa test
(WKH); weighted Shimodaira-Hasegawa test (WSH).
doi:10.1371/journal.pone.0086612.t005

Fast-Evolving Mitochondrial DNA in Ceriantharia

PLOS ONE | www.plosone.org 8 January 2014 | Volume 9 | Issue 1 | e86612



Table S2 Software and analytic details (data treatment
and parameters) for every analysis applied in this study
for 18S and 28S datasets (see Table 4 for major results
compilation).
(XLSX)

Table S3 Results from TNT analysis (18S and 28S
datasets), considering original and masked alignments
(RNAsalsa+aliscore), in all analyzed cost weight trans-
formations (indel:transitions:transversions).
(XLSX)

Text S1 Discussion on evolution and previous system-
atic considerations of Ceriantharia and Anthozoa.
(DOCX)

Acknowledgments

We are deeply indebted to Dr. Fábio L. da Silveira for providing laboratory

facilities and comments during the development of the study. We are

grateful to Drs. Peter Wirtz, Gustav Paulay, Elizabeth G. Neves, Mark

Vermeij, Alvaro E. Migotto, Kennet Lundin and Chaolun Allen Chen for

providing specimens for this study. We are also thankful to Dr. Estefania

Rodriguez for providing some DNA sequences.

Author Contributions

Conceived and designed the experiments: SNS MMM ACM. Performed

the experiments: SNS MMM. Analyzed the data: SNS MMM MVK JDR

ACM. Contributed reagents/materials/analysis tools: SNS MMM ACM.

Wrote the paper: SNS MMM MVK JDR ACM.

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