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: thierry.perez@imbe.fr

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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