ORIGINAL ARTICLE
Integrative taxonomic description of Plakina kanaky, a newpolychromatic sponge species from New Caledonia(Porifera: Homoscleromorpha)C�esar Ruiz1, Julijana Ivani�sevi�c2, Pierre Chevaldonn�e1, Alexander V. Ereskovsky1,Nicole Boury-Esnault1, Jean Vacelet1, Olivier P. Thomas3 & Thierry P�erez1
1 Institut M�editerran�een de Biodiversit�e et d0Ecologie marine et continentale (IMBE), CNRS – IRD – Aix Marseille Universit�e – Universit�e
d’Avignon, Marseille, France
2 Scripps Center for Metabolomics and Mass Spectrometry, The Scripps Research Institute, La Jolla, CA, USA
3 Institut de Chimie de Nice (ICN), PCRE, CNRS – Universit�e de Nice-Sophia-Antipolis, Nice, France
Keywords
Homoscleromorpha; integrative taxonomy;
metabolomic fingerprinting; phenotypic
variation; Porifera.
Correspondence
Thierry P�erez, Institut M�editerran�een de
Biodiversit�e et d0Ecologie marine et
continentale (IMBE), UMR 7263 CNRS –
IRD – Aix Marseille Universit�e – Universit�e
d’Avignon, Station Marine d0Endoume, Rue
de la Batterie des Lions, 13007 Marseille,
France.
E-mail: [email protected]
Accepted: 24 June 2014
doi: 10.1111/maec.12209
Abstract
Four similar sponges of different colors, all unknown to science, were collected
in submarine caves of New Caledonia. We aimed at determining whether the
four chromotypes represented different species or phenotypic variations of a
unique new species. We used an integrative taxonomic approach combining
morphologic, molecular and metabolomic analyses. The main traits that define
these specimens are a skeleton made of monolophose, trilophose and tetralo-
phose calthrops only, high chemical diversity and a high abundance and diver-
sity of prokaryotic symbionts. The symbiotic community includes two unique
prokaryote morphotypes, which are described for the first time in Homosclero-
morpha, and appeared to be vertically transmitted. Although several features
slightly differ among chromotypes, the most parsimonious conclusion was to
propose a single new species Plakina kanaky sp. nov. Our phylogenetic analysis
indicated the paraphyly of the Plakina genus, with P. kanaky sp. nov. belonging
to a clade that includes Plakina jani and Plakina trilopha. The present work
demonstrates that integrative taxonomy should be used in order to revise the
entire Plakinidae family and especially the non-monophyletic genus Plakina.
Introduction
Sponge taxonomists have mainly relied on morphological
characters such as external anatomy (e.g. color, texture
and growth form) and skeletal features (e.g. morphology
and arrangement of spicules and spongin fibers) to
accurately recognize and delineate species boundaries.
However, natural populations of marine invertebrates,
especially sponges, often exhibit morphological variability
(L�opez-Legentil et al. 2010; Freckelton et al. 2012; Loh
et al. 2012), that can be due to environmental conditions
(Maldonado & Young 1998; Mercurio et al. 2000; Meroz-
Fine et al. 2005; C�ardenas & Rapp 2013), to biotic factors
such as predation (Loh & Pawlik 2009) or to the occur-
rence of photosynthetic symbionts, such as cyanobacteria
for instance. In some cases, individuals of a given species
can appear less similar than those of different species
(Ackers et al. 1992). This intra-specific variation has been
a long-standing source of taxonomic difficulties, espe-
cially when the main taxonomic characters are lacking or
when they are too polymorphic. This has important
implications in the related fields of ecology, biodiversity
management and the research of new pharmaceutical
compounds (Miller et al. 2001).
In recent years, sponge taxonomists have been adopt-
ing new concepts and methods to gain access to addi-
tional sources of data. For example, the sponge DNA
barcoding initiative, using mitochondrial gene fragments,
provides an informative tool to assist in the identification
of sponge species (Erpenbeck et al. 2006). However, there
Marine Ecology (2014) 1–15 ª 2014 Blackwell Verlag GmbH 1
Marine Ecology. ISSN 0173-9565
is no consensus on a universally relevant barcoding frag-
ment for sponges, and DNA amplification and PCR can
be highly challenging in some families (Vargas et al.
2012).
Sponges are among the most important sources of sec-
ondary metabolites in the sea, with a great diversity of
structures and biological activities (Blunt et al. 2013). The
comparison of the diversity of these organic compounds
in order to classify and identify organisms is called
chemotaxonomy (Bergquist & Wells 1983). One of the
main limitations of this approach is the targeted search of
original molecules by natural product chemists that has
underestimated the taxonomic value of the highly diversi-
fied pool of sponge metabolites. Another approach, the
metabolomic fingerprinting, is an untargeted, comprehen-
sive assessment of the metabolite pool within an organ-
ism. This holistic approach allows the comparison of
multiparametric patterns (or fingerprints) as dynamic
metabolomic phenotypes of a high number of samples
(Wolfender et al. 2009). These comparisons can lead to
the proposal of or support hypothetic classifications, or
highlight potential chemotaxonomical markers. Such
markers are particularly useful for species delimitation in
sponges with highly polymorphic characters (Ereskovsky
et al. 2009; Ivani�sevi�c et al. 2011; C�ardenas et al. 2012).
Integrative taxonomy proposes the use of multiple and
complementary sources of data to evaluate the status of a
species (Dayrat 2005). This approach combines data sets
of different origin, such as morphology, reproduction,
DNA sequences, and ecological and chemical characteris-
tics, and thus appears to be the most reliable way to
evaluate the specific status of specimens (Dayrat 2005;
Padial et al. 2010). Today, this approach is generally
accepted as the best way to answer the challenges of
sponge systematics (C�ardenas et al. 2012). Homosclero-
morpha is a sponge group with difficult systematics.
Phylogenetic relationships within this taxon remain
unsettled and are subject to frequent controversies
(Borchiellini et al. 2004; Gazave et al. 2012). The system-
atics of Homoscleromorpha is rather complex because
several representatives lack a skeleton, which is the funda-
mental character used in sponge systematics. Conse-
quently, Homoscleromorpha was one of the first sponge
clades studied with integrative taxonomy, an approach
that has encouraged the exploration of the species diver-
sity within this clade and allowed the description of 42%
of the overall Homoscleromorpha diversity during the
last 20 years (C�ardenas et al. 2012; Boury-Esnault et al.
2013). Formerly included in Demospongiae, Homosclero-
morpha are accepted today as the fourth Porifera class
(Gazave et al. 2012). They presently include two families,
Oscarellidae Lendelfeld, 1887, and Plakinidae Schulze
(1880); with seven genera and 98 species described so far
(Van Soest et al. 2014). Today, the Oscarellidae are
mainly characterized by the absence of a skeleton,
whereas the classification of Plakinidae is mainly based
on the shape and the organization of spicules in the skel-
eton. There are three main types of spicules: diods, triods
and calthrops, which may be non-lophose (rays without
any ramification) or lophose (rays presenting multiple
branching). Lophose spicules may be homolophose, with
homogenous ramifications in size and ornamentation, or
heterolophose, with an heterogeneous pattern of ramifica-
tion. The type of ramification in the actines of calthrops
is diagnostic, with at least three distinct general spicular
morphologies recognized among the plakinid genera with
lophose calthrops: homolophose calthrops with one, two,
three or four lophate rays for Plakina, large lophodiods,
lophotriods and lophocalthrops for Placinolopha, and
small, heterolophose calthrops for Corticium (Muricy &
Diaz 2002). In addition to the skeleton, cytological and
genetic characters have also been useful for species
description and for solving species complexes (Muricy
et al. 1996a,b, 1999; Gazave et al. 2010; Ereskovsky et al.
2014). Nevertheless, the phylogenetic relationships inside
the Plakinidae family and especially the monophyly of the
Plakina genus are still debated because of the wide vari-
ability in morphological characters previously observed
and the lack of diagnostic characters to clearly define the
genus (Muricy & Diaz 2002; Gazave et al. 2010, 2012;
C�ardenas et al. 2012).
In the present study, we used an integrative approach
based on morphologic description, histologic and cyto-
logic characteristics, DNA barcoding and metabolomic
fingerprinting in order to demonstrate that four sponge
chromotypes discovered in the Pacific Ocean belong to
the same new Plakina species, and to position this species
within the above-mentioned complex Homoscleromorpha
systematics.
Material and Methods
Sampling
Sampling was conducted in New Caledonia (Tropical
Southwestern Pacific Ocean). A total of nine specimens
was collected in November 2010 and February 2013 by
SCUBA diving at Bourail (21°41.3030 S, 165°27.8110 E),Hiengh�ene (20°36.7560 S, 164°56.9580 E) and Touho
(20°45.5030 S, 165°16.9640 E) (Fig. 1). They were all
found in small dark cavities or caves (15–25 m depth)
on the outer slope of coral reefs. The specimens’ general
morphology (color, size, consistency and shape) was reg-
istered in situ, with underwater photographs. Samples
were preserved by the following methods: in 95% etha-
nol for morphologic and DNA analyses, fixed in 2.5%
2 Marine Ecology (2014) 1–15 ª 2014 Blackwell Verlag GmbH
Plakina kanaky, a new polychromatic sponge Ruiz, Ivani�sevi�c, Chevaldonn�e, Ereskovsky, Boury-Esnault, Vacelet, Thomas & P�erez
glutaraldehyde for cytological observations and frozen
for metabolomic analyses.
Skeletal composition
For spicule preparation, a small fragment of each speci-
men was boiled with 5 ml nitric acid for organic degra-
dation. After several washes with distilled water, the
final suspension containing the siliceous spicules was
placed on slides for light microscopy in order to con-
duct observation and measurement of spicules. A mini-
mum of 30 measurements was carried out for each type
of spicule, and the size range is provided in the descrip-
tion. For scanning electron microscopy (SEM), slides
containing spicules were coated with gold-palladium and
observed under a Hitachi S-570 microscope. The skele-
ton architecture was studied by light microscopy on pol-
ished sections. These sections were obtained by cutting a
piece of non-desilicified sponge embedded in AralditeTM
with a low-speed saw using a diamond-wafering blade.
The sections, about 5 mm thick, were then wet-ground
with abrasive paper or polishing discs to obtain thinner
sections <1 mm thick, mounted on glass slides and
observed under a light microscope (Boury-Esnault et al.
2002).
Cytology
A small fragment of each specimen was fixed in 2.5%
glutaraldehyde in 2 M phosphate buffer and filtered sea-
water (1 vol.: 4 vol.: 5 vol.), then post-fixed in 2% OsO4
in seawater (Boury-Esnault et al. 1984). Siliceous spicules
were dissolved with 5% hydrofluoric acid for 2 h. Each
fragment was then embedded in AralditeTM for semi-thin
and ultra-thin sections. Semi-thin sections were stained
with toluidine blue and observed under a Leica DMLB
light microscope (LM). Digital pictures were taken using
an Evolution LC camera. Ultra-thin sections were per-
formed with an RCM ultramicrotome PTXL. The cuts
were placed on a copper grid (3.05 mm in diameter, 300
meshes) and stained with 2% uranyl acetate for 15 min.
Observations were carried out with a JEOL JEM-1400
transmission electron microscope. The cytological compo-
sition of each sample was described, including their pro-
karyotic composition. Different prokaryotic morphotypes
were determined, taking into account the cells’ morphol-
ogy, dimensions and cytoplasm characteristics.
DNA analysis
DNA extraction from a small (2 ml) fragment of sponge
was realized using QIAamp DNA Mini Kit (QIAGEN)
extraction columns. The universal primers LCO1490 and
HCO2198 were used to amplify a 658-bp portion of the
cytochrome oxidase I (COI) mitochondrial gene (Folmer
et al. 1994). Amplification was performed in a 20 ll totalreaction volume with: 2 ll of each primer (10 lM), 3.2 lldNTPs (10 mM), 4 ll polymerase buffer, 2.5 ll MgCl2(25 mM), 0.1 ll Taq polymerase (5 U�ll�1) and 1.2 llextracted DNA. PCR was performed on a Mastercycler
gradient PCR-S Eppendorf thermocycler with an initial
step of 5 min at 94 °C followed by 40 amplification
cycles (denaturation at 94 °C for 1 min; annealing at
42 °C for 1 min; and extension at 72 °C for 1 min), and
a final extension step at 72 °C for 5 min. PCR products
were directly sequenced in each primer direction by the
Genoscreen laboratory (Lille, France). Eleven sequences
were obtained from GenBank and aligned with our new
sequences with BIOEDIT 7.0.5.3 (Hall 1999). Phyloge-
netic trees were constructed using the neighbor-joining
(NJ) method (1000 bootstrap replicates) with CLUSTAL
X 2.0 (Larkin et al. 2007).
Fig. 1. Location of the collection sites of Plakina kanaky sp. nov. in New Caledonia (Tropical Southwestern Pacific).
Marine Ecology (2014) 1–15 ª 2014 Blackwell Verlag GmbH 3
Ruiz, Ivani�sevi�c, Chevaldonn�e, Ereskovsky, Boury-Esnault, Vacelet, Thomas & P�erez Plakina kanaky, a new polychromatic sponge
Metabolomics
For metabolomic fingerprinting, we applied an extraction
protocol that allowed the extraction of compounds with a
wide range of chemical diversity, from polar to very apolar,
and including rather hydrosoluble compounds (Blunt et al.
1987; Regalado et al. 2010; Ivani�sevi�c et al. 2011;. Speci-
mens were kept at �20 °C after collection, then lyophilized
and ground to obtain a homogenous powder. 0.1 g of
each individual was extracted using 1 ml MeOH/CH2Cl21:1 in an ultrasonic bath. The suspension was filtered
on standard filter paper, the filtrates were then evaporated
to dryness using 0.1 g silica powder and then loaded
onto C18 solid phase extraction cartridges (Phenomenex
Strata). The conditioned cartridges were first washed with
5 ml distilled water for desalting and eluted with 3 ml
MeOH/CH2Cl2 1:1 in a 5 ml volumetric flask. Sponge
extracts were analysed using an Ultra Performance Liquid
Chromatography (UPLC) system (1200 series, Agilent
Technologies) coupled to a 6538 Accurate Mass Spectro-
meter (MS) Q-TOF (Agilent Technologies). A Zorbax 300
SB-C18, 5 lm, 150 9 0.5 mm ID column (Agilent) was
used for UPLC/MS analysis in positive electrospray ioniza-
tion (ESI) mode. The standard mobile phase, A = 0.1%
formic acid in water and B = 0.1% formic acid in acetoni-
trile was used, applying a linear gradient elution from
100% A (0–5 min) to 100% B (50–55 min). The flow rate
was 30 ll�min�1 and the sample injection volume was
5 ll. Every sample (together with blank injections) was run
in triplicate. ESI source conditions were set as follows: gas
temperature 325 °C, drying gas 5 l�min�1, nebulizer
15 psi, fragmenter 120 V, skimmer 65 V and capillary volt-
age 4000 V. The instrument was set to acquire over the m/
z range 60–1000, with the MS acquisition rate of 1.67 spec-
tra per s. Raw HPLC/MS data were converted to mzXML
files using PROTEOWIZARD MS CONVERT version
3.0.4146. The mzXML files were processed using XCMS for
peak detection, alignment and isotope annotation (Smith
et al. 2006; Tautenhahn et al. 2012). The parameters in
XCMS were set as follows: centWave settings for feature
detection (Dm/z = 15 ppm, minimum peak width = 10 s
and maximum peak width = 60 s); obiwarp settings for
retention time correction (profStep = 1); and other
parameters including mzwid = 0.015, minfrac = 0.1 and
bw = 5 for chromatogram alignment. The entire metabolic
profiles (total ion chromatograms in ESI positive mode),
encompassing all aligned metabolite features detected by
XCMS, were used for sample comparison and classifica-
tion. Each metabolite feature (including isotopes, adducts,
multiple charged features and in-source fragments) repre-
sented a variable with a peak area as a relative quantifica-
tion measure. Data were normalized by scaling the peak
areas to the total profile area observed in each sample.
Hierarchical cluster analysis (HCA) of the metabolomic
fingerprints was performed with STATISTICA software
(StatSoft Inc.) using Euclidean distance and Ward0s linkagemethod (Ward 1963).
Results
Systematics
Phylum Porifera Grant 1836.
Class Homoscleromorpha Bergquist 1978.
Order Homosclerophorida Dendy 1905.
Family Plakinidae Schulze (1880).
Genus Plakina Schulze (1880).
Type species
Plakina monolopha Schulze (1880).
Definition
Plakinidae with a spiculation of diods, triods and calthr-
ops in a single size class, and with homolophose calthrops
with one, two, three or four lophate rays (Muricy & Diaz
2002).
Plakina kanaky sp. nov. Ruiz & P�erez (this paper)
Holotype: Museum National d0Histoire Naturelle de
Paris, MNHN DJV 174, brick-red chromotype, depth
18 m, outer slope of the reef in a cave. Bourail, New Cal-
edonia (21°41.3030 S, 165°27.8110 E). Collector T. P�erez.Paratype 1: Museum National d0Histoire Naturelle de
Paris, MNHN DJV 175, yellow chromotype, depth 22 m,
outer slope of the reef in a cave, Hiengh�ene, New Caledo-
nia (20°36.7560 S, 164°56.9580 E). Collector T. P�erez.Paratype 2: Museum National d0Histoire Naturelle de
Paris, MNHN DJV 176, blue chromotype, same location
as the holotype. Collector T. P�erez.
Specimens examined for comparison: Plakina corticolo-
pha (paratype) DCL2970 MNHN. New Caledonia.
Etymology
The species name refers to the four chromotypes, which
harbor the colors of the Kanaky flag (red, blue, green and
yellow), and honors the people of Kanaky (New Caledo-
nia), their history and their culture. The new species can
thus be considered as a new part of their natural heritage.
4 Marine Ecology (2014) 1–15 ª 2014 Blackwell Verlag GmbH
Plakina kanaky, a new polychromatic sponge Ruiz, Ivani�sevi�c, Chevaldonn�e, Ereskovsky, Boury-Esnault, Vacelet, Thomas & P�erez
Diagnosis
Plakina species inhabiting shaded cavities and caves in
coral reefs. Yellow, brick-red, green or blue color. Lobate
surface and fleshy consistency. Monolophose, trilophose
and tetralophose calthrops. Neither diods nor triods.
Well-developed mesohyl with sub-ectosomal cavities.
High abundance and diversity of prokaryotic symbionts,
including a filamentous morphotype and an irregular
globular morphotype.
Description
Morphology
Small, yellow, brick-red, blue or green sponge, about 2–5 9 5–15 cm in size and 0.5–1 cm thick (Fig. 2). Its sur-
face is smooth with irregularly rounded lobes. Each lobe
often harbors a tiny osculum, about 1 mm in diameter,
bordered by a thin protruding membrane. The sponge is
firmly attached to the substratum and its consistency is
fleshy.
Skeleton
The skeleton, arranged around the choanosomal cavi-
ties, is dense (Fig. 3A) in all the color morphs except
the yellow one whose spicule density displayed a 10-
fold decrease compared with the other color morphs.
Three types of spicules are present within the different
color morphs. Monolophose calthrops are mainly con-
centrated in the ectosome, while trilophose and tetralo-
phose calthrops are randomly arranged in the mesohyl.
Neither diods nor triods were observed. Monolophose
calthrops are abundant in all color morphs, except the
yellow one, in which the most abundant spicules at the
surface are tetralophose calthrops. The non-lophose
actins have one or two spines at their basis, and the
lophose actines have one or two distal ramifications.
Each actin is 20–30 lm long (Fig. 3B; Table 1). Trilo-
phose calthrops are present in all specimens, but rare
in the yellow one. The principal actin (non-lophose) is
15–27.5 lm long (Fig. 3C) and can have a distal rami-
fication. The three lophose actins, 2.5–5 lm long from
the basis to the ramification tip, often present some
ramifications at the distal part of the actin. Tetralo-
phose calthrops are the most abundant type in the yel-
low morphotype, and are less abundant in the other
color morphs. The ramification pattern is variable, usu-
ally divided four times. Each actin measures 2.5–5 lmfrom the basis to the beginning of the ramification
(Fig. 3D).
Soft tissue organization
The ectosome is 30–75 lm thick and is separated from
the choanosome by sub-ectosomal cavities that are 10–45 lm thick (Fig. 4A, B). The leuconoid aquiferous sys-
tem has well-developed inhalant and exhalant canals.
Choanocyte chambers (20–50 lm in diameter) are dipl-
odal, spherical to ovoid (Figs 4A and 5A).
Fig. 2. Four chromotypes of Plakina kanaky
sp. nov. collected in New Caledonia.
Marine Ecology (2014) 1–15 ª 2014 Blackwell Verlag GmbH 5
Ruiz, Ivani�sevi�c, Chevaldonn�e, Ereskovsky, Boury-Esnault, Vacelet, Thomas & P�erez Plakina kanaky, a new polychromatic sponge
Cytology
Choanocytes are ovoid to pyramidal (Fig. 5A, B),
2.5–7 lm wide and 2.4–7 lm high. Their collar is
about 3–4 lm in diameter and composed of about 28–35microvilli. Their nucleus (1.3–2.5 lm in diameter) has an
apical to central position. The cytoplasm usually contains
one or two phagosomes of 1 lm in diameter, and smaller
digestive vacuoles, osmiophilic and lipidic inclusions,
may be present. Choanocytes are underlined by a base-
ment membrane. Apopylar cells are flat or rounded, 8–12 lm long and 2–3.2 lm high, with an anucleolated
nucleus 2 lm in diameter, occupying almost all the cyto-
plasmic area. No vacuoles or inclusions were observed
(Fig. 5A). Exo- and endopinacocytes are flat or ovoid, 8–15 lm long and 1–2 lm high, with an anucleolated
nucleus of 1.2–2 lm in diameter. Both cell types are
Table 1. Morphologic characteristics of the four chromotypes of Plakina kanaky sp. nov. in comparison with Plakina jani and Plakina trilopha.
Characters Plakina kanaky Plakina jani
Plakina
monolopha
Color Yellow Brick-red Blue Green Yellow White/cream
Growth form Irregular-lobate Irregular-lobate Irregular-lobate Irregular-lobate Irregular-lobate Discoidal
Aquiferous system Leuconoid Leuconoid Leuconoid Leuconoid Intermediary Syllebid
Diameter of chambers 24–39 lm 20–35 lm 26–40 lm 22–38 lm 30–50 lm 35–55 lm
Type of chamber Aphodal Aphodal Aphodal Aphodal Eurypylous/aphodal Eurypylous
Diameter of
choanocytes
2–7 lm 2–6 lm 4–6 lm 2–6 lm 3–8 lm No data
Diods – – – – a, 43–100 lm c, 52–93 lm
Triods – – – – a, 16–37 lm a, 16–37 lm
Monolophose
calthrops (long, actine)
r, 20–25 lm (1d) a, 15–30 lm
(1d)
a, 25–30 lm (1d) a, 20–30 lm (1d) c, 16–38 lm (1d) c, 8–31 lm
(1 m, ts)
Trilophose calthrops
(long, non-lophate
actine)
r, 15–25 lm
(1 m, 2d, ts)
c, 15–25 lm
(1 m, 2d, ts)
c, 17–28 lm
(1 m, 2d, ts)
c, 15–20 lm
(1 m, 2d, ts)
a, 30–35 lm
(1 m, 2d, ts)
–
Tetralophose calthrops
(long, actine)
a, 5–10 lm
(1 m, 2d, ts)
r, 5–10 lm
(1 m, 2d, ts)
r, 5 lm
(1 m, 2d, ts)
r, 25 lm
(1 m, 2d, ts)
c, 10–25 lm
(1 m, 2d, ts)
–
Prokaryote type 1, 2, 3, 4, 5, 6 1, 2, 3, 4, 5, 6 2, 3, 4, 5, 6 1, 2, 3, 4, 5, 6 3, 6 –
For spicules: –, absent; a, abundant; c, common; r, rare.
For their pattern of ramification: 1, first ramification; 2, second ramification; d, distal; m, middle; ts, terminal spines.
Prokaryote types are indicated according to their morphologic description (Muricy et al. 1998, 1999; Muricy & Diaz 2002).
A B
C
D
Fig. 3. Skeleton components of Plakina kanaky sp. nov. (A) Transverse cut showing spicule density and their arrangement. This density is found
in the brick-red, green and the blue chromotypes (scale bar = 50 lm). Scanning electron micrographs of spicules: (B) monolophose calthrops
with and without spines, scale bar = 8 lm; (C) trilophose calthrops; (D) tetralophose calthrops (scale bars C and D = 3.7 lm).
6 Marine Ecology (2014) 1–15 ª 2014 Blackwell Verlag GmbH
Plakina kanaky, a new polychromatic sponge Ruiz, Ivani�sevi�c, Chevaldonn�e, Ereskovsky, Boury-Esnault, Vacelet, Thomas & P�erez
flagellated, with small vacuoles and dark inclusions
(Fig. 5C, D). Both endopinacocytes and exopinacocytes
are underlined by a basement membrane. Collagen fibrils
are present in the adjacent mesohyl. Archaeocytes were
the only cells found in the sponge mesohyl. They have an
irregular form; their size is variable, ranging from 5 to
9 lm long with a spherical anucleolated nucleus of 0.9–2 lm. Their cytoplasm contains abundant glycogen parti-
cles. They were always found engulfing some of the prok-
aryotes described below (Fig. 5E, F). No cells with
vacuoles or inclusions were recorded.
Prokaryotic symbionts
This sponge can be considered as a high microbial abun-
dance (HMA) sponge. All prokaryotes were found to be
extra-cellular, randomly dispersed in the mesohyl
(Fig. 6A). Neither bacteriocytes nor intra-nuclear microbes
were observed. Among the different morphological types
found in the mesohyl, two dominant morphotypes are
here recorded for the first time in Homoscleromorpha.
The first morphotype corresponds to encapsulated pro-
karyotic cells that are often in groups of 2–6 cells that
form irregular globular aggregates of about 10 lm in
diameter. Each globular cell is 2 9 1.6 lm large, has a
clear membrane 0.2–0.5 lm thick and an inner and outer
thin dark membrane. Their dense cytoplasm can contain
between 8 and 25 small clear vacuoles of 0.1 lm in diame-
ter (Fig. 6B), or between 10 and 25 small dark inclusions
of about 0.1–0.2 lm in diameter (Fig. 6C). This prokary-
otic type was recorded in all color morphs except for the
blue one. The second morphotype is a filamentous micro-
organism (Fig. 6D, E) measuring up to 20 lm long, and
composed of 2–15 cells. Each cell has a dimension of
about 3–5 9 1.6 lm, with a cell membrane 0.1 lm thick.
Their cytoplasm is dense, but with no vacuoles or inclu-
sions. Another smaller and ovoid microorganism was usu-
ally observed in close interaction with this filamentous
type (see arrows in Fig. 6D, E). This filamentous microor-
ganism was recorded in all color morphs. Other morpho-
logical types, 0.5–2 lm large, were found to be present in
all sponge color morphs. These prokaryotes were more
difficult to differentiate because of their similar forms and
cytoplasmic components (Fig. 6F, G and H). These mor-
photypes are very similar to those found in other HMA
sponges, with numerous division figures.
Reproduction
No reproductive elements were recorded in specimens col-
lected in November 2010. Gametogenesis was never
observed, but coeloblastula pre-larvae were found in blue,
brick-red and green specimens collected in February 2013.
Prokaryotic transfer from the adult sponge to the larvae
was observed inside the pre-larval cavity of the main pro-
karyote morphotypes found in the adult mesohyl (Fig. 7).
DNA analysis
A fragment of 658 bp of the mitochondrial COI gene was
obtained for all color morphs except the green, for which
PCR was unsuccessful. All sequenced specimens share
exactly the same nucleotide composition (EMBL acces-
sion number HG799484). This single sequence was
aligned with 11 additional sequences from closely related
Homoscleromorpha. The NJ phylogenetic reconstruction
shows two clades with Plakina species, the first one being
formed by Plakina trilopha Schulze (1880); Plakina jani
Muricy et al. (1998) and Plakina kanaky sp. nov. Plakina
A B
Fig. 4. Internal organization in Plakina
kanaky sp. nov. Semi-thin section (light
microscope) (A) showing the general
organization of the mesohyl and the
ectosome (scale bar = 50 lm). (B) Detailed
view of the ectosome region (scale
bar = 10 lm). Cc, choanocyte chamber; E,
ectosome; Ec, exhalant canal; Ex,
exopinacocyte; M, mesohyl; Pk, prokaryote;
Sc, subectosomal cavity.
Marine Ecology (2014) 1–15 ª 2014 Blackwell Verlag GmbH 7
Ruiz, Ivani�sevi�c, Chevaldonn�e, Ereskovsky, Boury-Esnault, Vacelet, Thomas & P�erez Plakina kanaky, a new polychromatic sponge
kanaky sp. nov. differs by 18 nucleotide substitutions
(2.7%) from the closest sponge, P. jani. A second group
is formed by Plakina crypta Schulze (1880) and the type
species Plakina monolopha Schulze (1880). The nucleotide
divergence between P. kanaky sp. nov. and P. monolopha
is 8.8% (58 substitutions; Fig. 8).
Metabolomics
The metabolomic fingerprint of Plakina kanaky sp. nov.
(taking into consideration all four different morphotypes)
consists of 8030 abundant metabolite features with inten-
sities higher than 10,000 ion counts (10e+04). Base peak
chromatograms of the four color morphs display similar
patterns with the most abundant peaks eluted in reten-
tion time ranging from 24 to 26 min (Fig. 9). Metabolic
fingerprints of the four chromotypes and Plakina jani
display specific features, as well as shared metabolite fea-
tures primarily eluted in the retention time range from
24 to 30 min [e.g. Mass. (M+H) = 288.287 and 316.317).
Plakina jani can be clearly distinguished by the presence
of major, specific features annotated as (M+H)
A
B C
D
E F
Fig. 5. Transmission electron micrographs of (A) choanocyte
chamber (scale bar = 10 lm); (B) choanocytes (scale bar = 2.5 lm);
(C, D) pinacocytes (scale bars: C = 2.6 lm; D = 2 lm); (E, F)
archaeocytes (scale bars = 2 lm). Ap, apopylar cells; Bm, basal
membrane; Cc, choanocyte chamber; Co, choanocyte; En,
endopinacocyte; f, flagellum; N, nucleus; Pi, Pinacocyte; Pk,
prokaryotes.
A
B
C
D
E F
G H
Fig. 6. Transmission electron micrographs of the prokaryotic
community in Plakina kanaky sp. nov., each number indicating a given
type of prokaryotic cells. (A) General view of prokaryotic density (scale
bar = 5 lm). (B, C) Detailed view of the globular morphotype. (D, E)
Detailed view of the filamentous morphotype. The arrows show a small
microorganism that was commonly associated with the filamentous
prokaryote. (F, G, H) Detailed view of different morphotypes associated
with all chromotypes of P. kanaky sp. nov. (scale bars B to H = 2 lm).
8 Marine Ecology (2014) 1–15 ª 2014 Blackwell Verlag GmbH
Plakina kanaky, a new polychromatic sponge Ruiz, Ivani�sevi�c, Chevaldonn�e, Ereskovsky, Boury-Esnault, Vacelet, Thomas & P�erez
= 212.174, 244.199 and 341.255, and eluted in the reten-
tion time ranging from 16 to 20 min. In addition to the
absence of these latter features, P. kanaky (comprising all
four morphotypes) can also be distinguished from P. jani
by the presence of specific, although minor, features [e.g.
(M+H) = 468.303, 520.333, 409.340, 423.319] mainly
eluted after 30 min. Moreover, the blue morphotype of
P. kanaky seems to be distinctly characterized by the
presence of a highly abundant feature annotated as
(M+H) = 324.161, also eluted in the second part of the
gradient, after 30 min.
Ecology
Plakina kanaky sp. nov. is a sciaphilous species dwelling
in dark cavities, caves and tunnels in the coral reef. It
was found between 15 and 25 m depth, generally growing
on rocks. No sign of epibiosis or predation were
observed, which can easily be explained by the high
chemical diversity recorded, possibly including metabo-
lites with allelopathic activities. Some of the chromotypes
were observed in the same square meter. Blue, brick-red
and yellow types were found in Bourail in two different
caves, green and yellow types were found in Hiengh�ene
in the same cave, and blue and brick-red types were
found in Touho, also in the same cave.
Discussion
Twenty-seven Plakina species have been described so far,
most of them on the basis of their skeleton and spicule
A B
CFig. 7. Transmission electron micrographs of
(A) coeloblastula of Plakina kanaky sp. nov.
(scale bar = 50 lm); (B) detailed view of
flagellated cells and prokaryote morphotype 1
inside sponge larvae and sponge mesohyle
(scale bar = 10 lm); (C) detailed view of a
coeloblastula flagellated cell (scale
bar = 5 lm). C, central cavity; Flc, flagellated
cells; N, nucleous; Oi, osmiophilic inclusion;
Pk, prokaryotes.
Fig. 8. Phylogenetic relationships between
Plakina kanaky sp. nov. and nine other
Homoscleromorpha sequences using
neighbor-joining reconstruction from a 658-
bp fragment of the mitochondrial cytochrome
oxidase I gene. Bootstrap values (1000
replicates) are shown at nodes.
Marine Ecology (2014) 1–15 ª 2014 Blackwell Verlag GmbH 9
Ruiz, Ivani�sevi�c, Chevaldonn�e, Ereskovsky, Boury-Esnault, Vacelet, Thomas & P�erez Plakina kanaky, a new polychromatic sponge
A
B
C
D
E
Fig. 9. Metabolomic fingerprint of the four color morphs of Plakina kanaky sp. nov. (A–D) and Plakina jani (E). Base peak chromatograms (mass
spectrometry positive electrospray ionization) show the metabolic features of the principal compounds according to their retention time (x-axis)
and intensity in ion counts (y-axis).
10 Marine Ecology (2014) 1–15 ª 2014 Blackwell Verlag GmbH
Plakina kanaky, a new polychromatic sponge Ruiz, Ivani�sevi�c, Chevaldonn�e, Ereskovsky, Boury-Esnault, Vacelet, Thomas & P�erez
morphology. The presence of lophose diods, triods or
calthrops remains the key character in order to classify
homoscleromorph species within the Plakina genus (Mur-
icy & Diaz 2002), although sometimes the presence or
absence of one of these diagnostic characters may lead to
taxonomic errors in this group (Muricy et al. 1996b).
One of the best examples is the Mediterranean Plakina
trilopha, which has long been considered a single species
with several morphotypes on the basis of its skeletal com-
position only, whereas the development of genetic tools
(allozymes) and the use of a new set of morphological
characters allowed the description of several new cryptic
species (Muricy et al. 1998). Today, several non-Mediter-
ranean records of P. trilopha (Van Soest et al. 2014)
remain to be carefully revised.
One of the main characteristics of Plakina kanaky sp.
nov. is the absence of diods, present in almost all Plakina
species described so far (Table 1). Nevertheless, the pres-
ence or absence of a spicule type could be also the result
of bio availability of silica in the surrounding waters
(Maldonado et al. 1999; C�ardenas & Rapp 2013). Only
two other Plakina species described from the Pacific
Ocean, Plakina australis (Gray 1867) and Plakina corticol-
opha L�evi & L�evi 1983; lack diods and triods, but they
differ from P. kanaky sp. nov. in the shape of their cal-
throps, which are strongly spined (L�evi & L�evi 1983). We
did not have much information on P. australis, but its
type locality is rather far from New Caledonia. Plakina
corticolopha has been collected in New Caledonia at
400 m depth, and the difference in silica bio availability,
which is higher in deep waters, might affect the shape of
spicules (Maldonado et al. 1999; C�ardenas & Rapp 2013).
However P. corticolopha still differs from the new species
in its external morphology as this sponge was reported to
be grey, thin encrusting without lobes and with few tiny
oscules (L�evi & L�evi 1983).
Up to now, only four Mediterranean species of Plakina
have been described using characters other than morphol-
ogy, such as cytology, symbiotic composition and genetics
(Muricy et al. 1996a,b, 1998). Our cytological analysis of
P. kanaky sp. nov. reveals some diagnostic characters in
common with the Mediterranean species, such as the
absence of cells with vacuoles or inclusions and a high
abundance of prokaryotic symbionts. As pointed out by
Muricy et al. (1999) for the Mediterranean Plakina species,
a unique combination of symbiotic prokaryote morpho-
types is associated with each species, and their number
and composition can be used to discriminate among spe-
cies. The new Plakina species harbors an original composi-
tion of prokaryotes, such as the globular and filamentous
morphotypes, which are two morphotypes never recorded
in Homoscleromorpha before. Some prokaryotic morpho-
types were seen inside the coeloblastula pre-larvae, which
corroborates the vertical transmission of symbionts in this
group (Ereskovsky & Boury-Esnault 2002; Maldonado
2007; Ereskovsky et al. 2010) and a likely co-evolutionary
process. For the filamentous prokaryote recorded here, this
finding is particularly puzzling as this morphotype may
belong to the genus Entotheonella, which is otherwise
known to be an environmental bacterial taxon (Wilson
et al. 2014). Besides a rather high diversity and density of
prokaryotic cells, P. kanaky sp. nov. harbors very few types
of sponge cells in the mesohyl. Only archaeocytes were
observed. Neither vacuolar nor spherulous cells, present in
numerous Homoscleromorpha, were found, nor sclero-
cytes, reported in other Plakina species (Donadey 1979;
Muricy et al. 1999). In Corticium candelabrum, Maldona-
do & Riesgo (2007) indicated that spicules could be pro-
duced both by sclerocytes and pinacocytes, but this is not
likely to be the case in this new species, making the bio-
synthesis of its skeleton enigmatic. The present analysis
showed that the COI mitochondrial gene fragment can
provide molecular classifications of Homoscleromorpha
that are congruent with phylogenies obtained with several
other molecular markers (Ivani�sevi�c et al. 2011). The Plak-
ina genus appeared once again as paraphyletic, which is in
line with the non-monophyly hypothesis proposed by pre-
vious authors (Muricy et al. 1996a, 1998; Muricy & Diaz
2002; Gazave et al. 2010, 2012; and for review see Boury-
Esnault et al. 2013). The COI sequence of P. kanaky sp.
nov. is most closely related to P. jani and P. trilopha. This
relationship was also evidenced by comparing the meta-
bolomic fingerprints (Fig. 10). The metabolomic analysis
with the high definition mass detector revealed that
P. kanaky sp. nov. produces a great number of secondary
metabolites (more than 8000 metabolite features detected
with intensity >10,000 ion counts). This preliminary meta-
bolomic study did not allow the identification of second-
ary metabolites, although it is known from the literature
that sponge species of the Plakina genus produce steroidal
alkaloids, the so-called plakinamines (Rosser & Faulkner
1984). However, we can confirm that the first plakinam-
ines described (A and B) were not detected in any of the
four chromotypes of P. kanaky or in P. jani. Further
research will also be needed to identify the compounds
that explain the color of the new species. Pigments can be
polar or apolar compounds (Blunt et al. 1987; Freckelton
et al. 2012). Considering our extraction protocol, there
is a good probability that they were contained within
our crude extracts, and likely within the 4% of chemical
diversity that differs among the chromotypes.
Sponges are well known not only for their phenotypic
plasticity (e.g. L�opez-Legentil et al. 2010; Freckelton et al.
2012; Loh et al. 2012) but also for the high probability of
obtaining species complexes among taxonomic groups in
which fundamental taxonomic characters are lacking, or
Marine Ecology (2014) 1–15 ª 2014 Blackwell Verlag GmbH 11
Ruiz, Ivani�sevi�c, Chevaldonn�e, Ereskovsky, Boury-Esnault, Vacelet, Thomas & P�erez Plakina kanaky, a new polychromatic sponge
are present but not diagnostic (C�ardenas et al. 2012).
Homoscleromorpha taxonomy is particularly challenging
and several chromotypes have already been found in this
group, not only in well-defined species (e.g. Oscarella tu-
berculata), but also in several cryptic species complexes
(e.g. Oscarella spp., Plakina trilopha) (Boury-Esnault et al.
1992; Ivani�sevi�c et al. 2011; C�ardenas et al. 2012). There-
fore, an integrative approach was needed in order to
define the status of our specimens from New Caledonia
and to verify whether the four chromotypes belonged to
several or to a single Plakina species.
Besides the color differences, the spicule and skeleton
analysis showed a marked variation in density between
the yellow and the other three chromotypes. Although
the internal organization was the same in all investigated
individuals, subtle differences were observed in prokary-
otic composition, in particular a lack of the ‘globulous’
prokaryote morphotype in the blue specimens, which is
puzzling as this prokaryote seems to be spread by vertical
transmission in the other chromotypes. The chemical fin-
gerprints showed only minimal differences in the metabo-
lome composition among all four chromotypes, about
4% of divergence, which might be explained by the
occurrence of different pigments. The DNA analysis did
not show any differences in nucleotide composition
among the three chromotypes successfully sequenced (yel-
low, brick-red and blue). It is well known that COI can-
not always efficiently distinguish sponge individuals at
the intra-specific level (e.g. for population genetics or
phylogeographic purposes); however, we have previously
demonstrated that this marker can be used to distinguish
sister species and describe cryptic species complexes
(P�erez et al. 2011; Reveillaud et al. 2012).
The color variability of P. kanaky sp. nov. will require
further investigation, such as new sampling of a greater
number of individuals, which might even reveal new
chromotypes. In sponges, environmental factors such as
depth or light intensity may influence the color (Sar�a &
Vacelet 1973; Maldonado & Young 1998). This may be
explained by the high abundance of pigments, which are
key molecular components of sponges’ photoprotection
and/or photosensitivity (see Wicksten 1989; Loh et al.
2012; De Bruyn & Gosselin 2014). It is also often related
to the variability in abundance of photosynthetic micros-
ymbionts. Plakina kanaky sp. nov. inhabits reef caves or
dark cavities and all chromotypes were found in adjacent
locations; thus, in this case, there is no relationship
between the degree of exposure to light and the different
chromotypes.
Thus, although several features (prokaryotic associa-
tions, spicular composition or density, metabolites)
slightly differ among chromotypes, the most parsimoni-
ous conclusion of our data analysis is that we recorded
only one new species, P. kanaky sp. nov. Its close rela-
tionship with P. jani and P. trilopha fits well with the
clade Tetralophosa recently proposed by Gazave et al.
(2012), a clade that should gather all Plakina sponges that
have a well-developed mesohyl, a well-differentiated ecto-
some and that present sub-ectosomal cavities and tetralo-
phose calthrops. We believe that such an integrative
approach will be needed to review the entire Plakinidae
family and especially the genus Plakina. This approach
has already demonstrated its relevance for solving species
complexes and for supporting phylogenetic hypotheses,
but it can also significantly improve our knowledge of
the mechanisms at the origin of diversity of taxonomic
characters in order to propose new evolutionary scenarios
within sponge lineages. Particular attention is needed in
order to better understand the ecological role of prokaryotic
symbionts. Taking into account that Homoscleromorpha
Fig. 10. Hierarchical cluster analysis of
Homoscleromorpha sponge species based on
UPLC/MS metabolomic fingerprints. The four
chromotypes are represented by the letters b,
blue; br, brick-red; g, green; y, yellow; D,
Distance.
12 Marine Ecology (2014) 1–15 ª 2014 Blackwell Verlag GmbH
Plakina kanaky, a new polychromatic sponge Ruiz, Ivani�sevi�c, Chevaldonn�e, Ereskovsky, Boury-Esnault, Vacelet, Thomas & P�erez
are widely distributed in marine caves, one might wonder
if their prokaryotic and chemical heritage could explain
their ecological success in this particular, sometimes con-
sidered extreme, marine habitat.
Acknowledgements
This work was partly supported by the Associated Interna-
tional Laboratory ‘MARRIO’. We are grateful to Sandrine
Chenesseau, J€oel Courageot and Caroline Rocher for their
technical assistance, to Guilherme Muricy, Manuel Maldo-
nado and Maria Cristina Diaz for their helpful advice and
to the Institut de Recherche pour le D�eveloppement
(IRD) for providing diving facilities in New Caledonia.
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