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DOI 10.1007/s12526-016-0507-0
ORIGINAL PAPER
First record of the Aeolid Anteaeolidiella fijensis (Nudibranchia,
Aeolidiidae) from India
Leila Carmona 1,3 & Juan Lucas Cervera 1 & Appukuttannair Biju Kumar 2 &
Balachandran Komalam Snehachandran 2
Received: 31 August 2015 / Revised: 9 May 2016 / Accepted: 11 May 2016
# Senckenberg Gesellschaft für Naturforschung and Springer-Verlag Berlin Heidelberg 2016
Abstract The aeolid Anteaeolidiella fijensis Carmona,
Bhave, Salunkhe, Pola, Gosliner and Cervera, 2014 is reported for the first time from India. Differences in the external
colouration compared with the original description hampered
its identification, and therefore, a molecular approach was
needed. Maximum-likelihood and Bayesian analyses of partial DNA sequences of the mitochondrial cytochrome c oxidase subunit I and 16S rRNA genes, and the nuclear gene
histone-3 were used to infer phylogenetic trees. ABGD species delimitation analyses and morphological study
complemented this contribution. This is the first record of
Anteaeolidiella fijensis outside the type locality and extends
its distribution range remarkably. Here, we also complete the
original description with new data regarding the external
colouration and internal anatomy. Finally, the instraspecific
variation may be related with an ontogenetic process or be a
populationally distinctive feature.
Keywords Mollusca . Aeolidida . India . Biodiversity . New
record
Communicated by V. Urgorri
* Leila Carmona
leila.carmona.barnosi@marine.gu.se
1
Departamento de Biología, Facultad de Ciencias del Mar y
Ambientales, Ampus de Excelencia Internacional del Mar
(CEI · MAR,) Universidad de Cádiz, Polígono Río San Pedro, s/n,
Ap.40, 11510 Puerto Real (Cádiz), Spain
2
Department of Aquatic Biology and Fisheries, University of Kerala,
Thiruvananthapuram 695581, Kerala, India
3
Present address: Department of Marine Sciences, University of
Gothenburg, Box 460, 40530 Gothenburg, Sweden
Introduction
At the beginning of this century, Miller (2001) erected the
aeolidiid genus Anteaeolidiella, which was lately reviewed
by Carmona et al. (2014a) that diagnosed the members of this
genus as aeolids with predominant orange and/or white
colouration and large oral glands covered by giant secretory
cells. Carmona et al. (2014a) pointed out the relevance of colour
patterns within Anteaeolidiella, despite the fact these markings have been considered as an intraspecific variation of the
type species A. indica Bergh 1888 (e.g. Gosliner and Griffiths
1981). In addition, Carmona et al. (2014a) stated that in terms
of biogeography, all the species of the genus Anteaeolidiella
present a quite restricted distribution, with the exception of
A. lurana (Er. Marcus and Ev. Marcus, 1967), which can be
found from the Mediterranean to Brazil as well as in the
Eastern Pacific.
On the other hand, the knowledge about the Indian
fauna of Opisthobranchia (here, used as an informal
group, since its monophyly has been rejected, e.g.
Wägele et al. 2014) is quite incomplete and irregular.
The most comprehensive study was that conducted in
the 70s by Narayanan (1969, 1970, 1971a, b), although,
in the last seven years, several contributions have increased the total knowledge of opisthobranch diversity
in India (Apte 2009; Apte et al. 2010; Ramakrishna
et al. 2010; Bhave and Apte 2011; Sankar et al. 2011;
Sreeraj et al. 2012; Apte and Bhave 2014; Poriya et al.
2015; Prasade et al. 2015).
In this contribution, we present the first record of
Anteaeolidiella fijensis Carmona, Bhave, Salunkhe, Pola,
Gosliner and Cervera, 2014 from India, the second species
of this genus reported in this country so far. In addition, we
complete the original description of this species based on the
Indian specimen.
Table 1
List of specimens used for phylogenetic analyses. Abbreviations: EA, Eastern Atlantic; EP, Eastern Pacific; GB, GenBank
Family
Tritoniidae Lamarck, 1809
Aeolidiidae Gray, 1827
Species name
Tritonia challengeriana Bergh, 1884
Anteaeolidiella cacaotica (Stimpson, 1855)
Anteaeolidiella chromosoma (Cockerell
& Eliot, 1905)
Anteaeolidiella fijensis Carmona, Bhave,
Salunkhe, Pola, Gosliner & Cervera, 2014
Anteaeolidiella ireneae Carmona, Bhave,
Salunkhe, Pola,
Gosliner & Cervera, 2014
Anteaeolidiella lurana (Marcus & Marcus,
1967)
Anteaeolidiella oliviae (MacFarland, 1966)
Anteaeolidiella poshitra Carmona, Bhave,
Salunkhe, Pola, Gosliner & Cervera, 2014
Babakinidae Roller, 1973
Anteaeolidiella saldanhensis (Barnard, 1927)
Anteaeolidiella takanosimensis (Baba, 1930)
Babakina indopacifica Gosliner, GonzálezDuarte &
Cervera, 2007
Locality
Collection dates
Voucher
GenBank accession nos.
COI
16S
H3
Bouvetoya (EA, GB)
Eastern Australia
Japan
Line Is.
Mexico (EP)
30Jun04
14Feb10
10Aug10
28Sep06
26Feb06
MNCN/ADN: 51922
MNCN/ADN: 51924
CASIZ 174212
CASIZ 173060
HM162718
JX087528
–
JQ997030
JQ997018
HM162643
JX087455
JX087457
JQ996825
JQ996812
HM162550
JX087590
–
JQ996926
JQ996911
Peru
Fiji
04Dic06
20Aug06
ZSM Mol 20090868
ZSM Mol 20070193
–
JQ997020
JQ996813
JQ996815
JQ996912
JQ996914
India
Clipperton Is.
DABF-UOK/GAS 4
1998
CASIZ 115686
KX276653
JQ997028
KX276652
JQ996823
KX276655r
JQ996924
Clipperton Is.
Eastern Australia
1998
05Aug06
CASIZ 115686
C.477129.003
JQ997029
KF273324
JQ996824
KF273321
JQ996925
KF273328
Eastern Australia
Bermuda
Brazil
Brazil
Italy
Mexico
California
India
05Aug06
20Jun09
19Dic07
14Dec08
Oct11
Aug06
26May09
11Dec09
C.477129.002
ZMBN82992
MZSP97638
MZSP97574
CASIZ 186820
CNMO 2996
CASIZ 181315
BNHS-opistho-38
KF273325
–
JQ997027
–
JQ997031
KF273326
JQ997034
KF273327
KF273322
JQ996821
JQ996822
–
JQ996826
KF273323
JQ996829
–
KF273329
JQ996922
–
JQ996923
JQ996927
–
JQ996930
–
India
South Africa (EA)
Japan
Philippines (GB)
2Jan14
06Jan08
19Mar06
20Mar08
DABF-UOK/GAS 5
CASIZ 176313
MNCN/ADN: 51923
–
KX276654
JQ997032
JX087529
HM162754
–
JQ996827
JX087456
HM162678
KX276656
JQ996928
JX087591
HM162587
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One specimen of Anteaeolidiella fijensis was studied. The
remaining species of the genus Anteaeolidiella were those
included by Carmona et al. (2014a), plus an additional specimen of A. poshitra from India (see Table 1 for a full list of
samples, localities and vouchers). Tritonia challengeriana
Bergh, 1884 and Babakina indopacifica Gosliner, GonzalezDuarte and Cervera, 2007 were chosen as outgroups based on
the contribution of Carmona et al. (2013). Tissue samples
were taken from the foot. The DNeasy blood & tissue kit of
Qiagen (Qiagen, Valencia, CA, USA; 09/2001) was used for
DNA extraction.
Partial sequences of cytochrome c oxidase subunit I (COI),
16S and H3 were amplified by polymerase chain reaction
(PCR) using the primers: LCO1490 (5′GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198
(5′-TAAACTTCAGGGTGACCAAAAATCA-3′) (Folmer
et al. 1994) for COI; 16S ar-L (5′CGCCTGTTTATCAAAAACAT-3′) and 16S br-H (5′CCGGTCTGAACTCAGATCACGT-3′) (Palumbi et al.
1991) for 16S rRNA; and H3AD5′3′ (5′ATGGCTCGTACCAAGCAGACVGC-3′) and H3BD5′3′
(5′-ATATCCTTR GGCATRATRGTGAC-3′) (Colgan et al.
1998) for H3. These three gene regions are commonly used
in systematic studies of heterobranchs (e.g. Ohnheiser and
Malaquias 2013; Ortigosa et al. 2014; Pola et al. 2014a, b;
Eilertsen and Malaquias 2015; Hallas and Gosliner 2015;
Shipman and Gosliner 2015). PCRs were conducted in
25-μl volume reactions containing 1 μl of both forward and
Fig. 1 Phylogenetic hypothesis for the genus Anteaeolidiella based on
the combined dataset (H3 + COI + 16S) inferred by Bayesian inference
(BI) analysis. Numbers above branches represent posterior probabilities
from BI. Numbers below branches represent bootstrap values from ML.
Different species delimitation methods results are also plotted. Specimens
sequenced in this study are in bold. Abbreviations: A, ABGD, based on
COI data set, with both models (Jukes-Cantor and Kimura); B, number of
species based on SplitsTree results; green rectangle = Anteaeolidiella
chromosoma; red rectangle = Anteaeolidiella oliviae; dashed
rectangle = Anteaeolidiella fijensis; orange rectangle = Anteaeolidiella
lurana; blue rectangle = Anteaeolidiella saldanhensis; yellow
rectangle = Anteaeolidiella cacaotica; grey rectangle = Anteaeolidiella
poshitra; pink rectangle = Anteaeolidiella takanosimensis; purple
rectangle = Anteaeolidiella ireneae
Material and methods
Molecular work
DNA extraction, amplification and sequencing
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reverse primers (10 μM), 2.5 μl of dNTP (2 mM), a genedependent amount of magnesium chloride (25 mM), 0.25 μl
of Qiagen DNA polymerase (5 units/μl), 5 μl of BQ-solution^
(5x), 2.5 μl of Qiagen buffer (10x; Qiagen Taq PCR core kit
cat. no. 201225) and 2 μl of genomic DNA. Magnesium chloride amounts were 3.5 μl for COI and 16S, and 2 μl for H3.
Amplification of COI was performed with an initial denaturation for 5 min at 94 °C, followed by 35 cycles of 1 min at
94 °C, 30 s at 44 °C (annealing temperature) and 1 min at
72 °C with a final extension of 7 min at 72 °C. The 16S
amplification began with an initial denaturation for 5 min at
95 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 44 °C
(annealing temperature), 1 min at 72 °C with a final extension
of 7 min at 72 °C. H3 amplification was performed with an
initial denaturation for 3 min at 95 °C, followed by 40 cycles
of 45 s at 94 °C, 45 s at 50 °C (annealing temperature), 2 min
at 72 °C, with a final extension of 10 min at 72 °C. Sequence
reactions were run on a 3730XL DNA sequencer, Applied
Biosystems. All new sequences have been deposited in
GenBank (Table 1).
Sequence alignment and phylogenetic analyses
Sequences were assembled and edited with Geneious Pro v.
4.7.6 (Drummond et al. 2009) and aligned in MAFFT (Katoh
et al. 2009). The alignments were further checked using
MacClade v. 4.06 (Maddison and Maddison 2005). Proteincoding sequences were translated into amino acids for confirmation of alignment. The most variable regions from the 16S
rRNA alignment were removed using the default settings in
Gblocks (Talavera and Castresana 2007). Excluding Bindelrich^ regions, the tree was in general very similar when
Fig. 2 Neighbour-Net graph of the COI sequences obtained with SplitsTree
including the variable regions. Therefore, final analyses were
performed including all bases. Sequences of COI, 16S and H3
were trimmed to 658, 438 and 327 base pairs, respectively.
Single gene analyses and a concatenated analysis were performed. The best-fit evolutionary model was determined in
MrModeltest v. 2.3 (Nylander 2004), using the Akaike information criterion (Akaike 1974). The GTR + G was selected
for the first and third position (H3 and COI), GTR + I for the
second codon position of H3 and COI, and HKY + I + G for
the 16S. Maximum likelihood (ML) analyses were performed
using the software RAxML v7.0.4 (Stamatakis et al. 2008).
Node support was assessed with non-parametric
bootstrapping (BS) with 5000 replicates, random starting
trees, and parameters estimated from each dataset under the
model selected for the original dataset. MrBayes v. 3.1.2
(Ronquist and Huelsenbeck 2003) was used for Bayesian inference (BI) analysis and to estimate posterior probabilities
(PP) for node support with two runs of 5,000,000 generations
each. Convergence was checked in TRACER v. 1.5
(Drummond and Rambaut 2007) with a burn-in of 25 %.
Only nodes supported by BS ≥ 70 (Hillis and Bull 1993) and
PP ≥ 0.95 (Alfaro et al. 2003) are discussed.
Species delimitation analyses
To define species, we used an integrative approach including
tree topologies, Carmona et al. (2014a) cut-off values, automatic barcode gap discovery (ABGD), the external
colouration of the living animals and the morphological data.
Pairwise uncorrected p-distance values between each taxon
were calculated for the COI gene using PAUP* 4.0b 10.0
(Swofford 2002). We also applied the ABGD method
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(Puillandre et al. 2012). This method was run for COI and for
all the specimens of the genus Anteaeolidiella included in this
study. ABGD settings were the following: Pmin = 0.001,
Pmax = 0.1, Steps = 10, X = 1.0, Nb bins = 20, and Jukes
Cantor (JC69) and Kimura (K80). Additionally, SplitsTree 4
(Huson and Bryant 2006) was used to construct the NeighborNet network (Bryant and Moulton 2004). The resulting tree
was converted to graphics in FigTree v1.4.0 and final adjustments were done in Adobe Illustrator CS5.
corresponded with each species Anteaeolidiella of the genus
included in this study. Finally, the SliptsTree network depicts a
small level of conflict (contradictory patterns) among
Anteaeolidiella species due to the presence of parallel edges
of equal lengths, especially between A. poshitra and its sister
species A. takanosimensis (Fig. 2). In addition, the SplitsTree
analysis supports the results obtained by the tree reconstructions and ABGD analysis (Figs. 1 and 2).
Systematics
Morphological study
Source of specimen and morphology
The specimen was dissected by dorsal incision. Their internal
features were examined and drawn under a stereoscopic microscope with the aid of a camera lucida. Special attention was paid
to the morphology of the reproductive system and oral and salivary glands. The buccal mass was removed and dissolved in
10 % sodium hydroxide until the radula was isolated from the
surrounding tissue. The radula was then rinsed in water, dried
and mounted for examination via scanning electron microscopy
(SEM). Voucher specimens are held at the museum collections of
the Department of Aquatic Biology and Fisheries, University of
Kerala, Thiruvananthapuram 695581, Kerala, India (Accession
numbers DABF-UOK/GAS 04 and DABF-UOK/GAS 05).
Family Aeolidiidae Gray, 1827
Genus Anteaeolidiella Miller 2001
Anteaeolidiella fijensis Carmona, Bhave, Salunkhe, Pola,
Gosliner and Cervera, 2014.
Material examined: DABF-UOK/GAS 04, one specimen
dissected, 8 mm in length preserved, Kavarathi Island (10033′
54″N 72037′34″E), India, 2 January 2014, found in
Results
Molecular results
The concatenated dataset generated an alignment of 1423 base
pairs. No saturation was observed across genes and codon
positions (not shown). Topologies of the ML trees were congruent with the results yielded by BI analysis and, thus, ML
trees are not shown. Figure 1 shows the genus Anteaeolidiella
phylogenetic hypothesis based on the combined dataset represented by BI. This figure also illustrates the results obtained
from different delimitation approaches used in this study. Our
results confirmed the monophyly of the genus Anteaeolidiella
since all the specimens of this genus included in the analyses
clustered together (PP = 1, BS = 93). The genus
Anteaeolidiella was constituted by two clades, one with A.
olivae and A. chromosoma (PP = 1, BS = 95) and a second
clade with the remaining species of this genus included in this
study (PP = 1, BS = 100). Within this larger group, both specimens of A. fijensis (one from Fiji, the type locality, and one
from India) appeared together (PP = 1, BS = 100) and with a
basal position.
The ABGD analyses recovered nine groups with P values
ranging from 0.001 to 0.06, independently from the chosen
model (Jukes and Cantor 1969 and Kimura 1980). Each group
Fig. 3 Different morphotypes of Anteaeolidiella fijensis. a–b,
Photograph of the living animals and schematic dorsal colour patterns
of the specimen from Fiji (taken from Carmona et al. 2014a); c–e,
photographs of the living animals and schematic dorsal colour patterns
of the specimen from India
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association with the bryozoan Bugula sp., at 0.80-m depth,
collected by R. Ravinesh.
Type locality: Laucala Bay, Fiji (Carmona et al. 2014a).
Geographical distribution: Known from Fiji (Carmona et
al. 2014a) and Kavarathi Island, India (present study).
External morphology (Fig. 3): Body aeolid form, elongate,
moderately slender, tapering gradually towards posterior end
of foot. Propodial tentacles stout, slightly protruded.
Background colour translucent white with bright orange pigment over dorsum (Fig. 3a–b). Head with orange mark.
Chalky white area behind rhinophores, forming a continuous
patch over dorsum, less dense over pericardial area (Fig. 3c–
e). Rhinophores translucent white, with some orange pigmentation and white tips white. Rhinophores stout. Oral tentacles
translucent white, with some orange pigmentation. Oral tentacles conical, slightly longer than rhinophores. Black eyes
visible behind rhinophores. Cerata longer than rhinophores,
moderately thick, extending from rhinophores to metapodium,
leaving bare space over dorsum. Ceratal epithelium diffusely
covered with orange pigment and chalky white pigmentation.
Ramifications of digestive gland greenish or brownish.
Subapical band translucent. Cnidosac white. Cerata in up to
13 rows, each with 2–15 cerata. Anus cleioproctic, between
second and third rows of first ceratal group of right posterior
digestive branch. Genital pore ventral to first right row.
Anatomy (Fig. 4): Masticatory border of jaws smooth
(Fig. 4a). Radular formula 18 x 0.1.0 (DABF-UOK/GAS
Fig. 4 Internal anatomy of
Anteaeolidiella fijensis. a detailed
view of the masticatory border of
the jaws, scale bar = 100 μm; b
radular teeth, scale bar = 50 μm; c
reproductive system, scale
bar = 0.5 mm. Abbreviations: am
ampulla, fgm female gland mass,
ps penial sac, rs receptaculum
seminalis, s spermoviduct, va
vagina, vd vas deferens
04). Teeth wide, bilobed. Central cusp triangular, elongate,
with 28–30 elongate and acutely pointed denticles on each
side (Fig. 4b). Outermost denticles larger than innermost.
Oral glands conspicuous, large, fusiform, dorso-laterally to
buccal bulb. Each oral gland with rows of giant secretory cells.
Salivary glands are absent.
Reproductive system diaulic (Fig. 4c). Hermaphroditic
duct widening into moderately long ampulla. Spermoviduct
dividing into oviduct and vas deferens. Vas deferens elongate,
entering wider proximal portion of penial sac with unarmed
penial papilla. Receptaculum seminis pyriform, inserting into
short oviduct, before latter forms female glands. Vagina ventral to penis.
Discussion
Carmona et al. (2014a) first described this species from the
Fiji Republic. The specimens studied by the authors presented
a less complex colouration, being predominantly orange, with
some white pigmentation (Fig. 3a–b). Therefore, the chalky
white appearance of the specimen reported herein clearly differs from the holotype of A. fijensis, but our molecular approach as well as the ABGD species delimitation analyses
support that both specimens belong to the same species.
Despite difference in the chalky white pigmentation, our specimen from India and those studied by Carmona et al. (2014a)
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have some external similarities. For instance, the colouration
of the rhinophores and oral tentacles are the same, as well as
the orange mark over the head matches. In addition, with
regard to anatomical features, the only difference was the
number of the radular denticles, more abundant in the Indian
specimen (30 against 20 denticles in the Holotype) but probably because of its larger size.
This intraspecific variation in terms of external colouration
could be ontogenetic, since those specimens studied by
Carmona et al. (2014a) were 4 mm long, while the specimen
from India reaches 8 mm. This variability due to size has been
also reported in the aeolidiid genus Spurilla, in particular
within Spurilla braziliana MacFarland, 1909 (Carmona et al.
2014b). Another hypothesis is that each morphotype is particular for each population, which was also observed in those
specimens of Spurilla braziliana from the Pacific (Carmona
et al. 2014b). However, in order to confirm any of these hypotheses more material of A. fijensis is needed.
Carmona et al. (2014a) pointed out that the diversity of the
genus Anteaeolidiella was far from being completed.
Actually, these authors suggested Bin India more than one
Anteaeolidiella species probably cohabit^, based on the pictures published on websites. This study confirms that theory,
contributes to a better knowledge of the Bopisthobranch^ diversity in India, and extends the distribution range of A.
fijensis notably.
Acknowledgments This work was supported by the research grant
(CGL2010-17187), Spanish Ministry of Economy and Competitiveness
(includes the early Ministry of Sciences and Innovation) to J. L. Cervera.
This work was supported by the Department of Biotechnology
(Government of India) as well by the Department of Science and
Technology (Government of India) with the INSPIRE fellowship. The
assistance of Mr R. Ravinesh in field collection is gratefully acknowledged. We would like to thank to Deneb Ortigosa for helping with the
SEM, and to Vanessa González Ortiz for her support with the illustrations
of this manuscript. This is CEI · MAR journal publication 130.
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