# Searching for Mediterranean bath sponges
(Demospongiae: Dictyoceratida: Spongiidae) in the
Northeast Atlantic reveals a new species: an integrative
taxonomic approach

Marie Grenier, Charlotte Simmler, Pierre Chevaldonné, Noëlle Callizot, Thierry Pérez

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## To cite this version:

Marie Grenier, Charlotte Simmler, Pierre Chevaldonné, Noëlle Callizot, Thierry Pérez. Searching for Mediterranean bath sponges (Demospongiae: Dictyoceratida: Spongiidae) in the Northeast Atlantic reveals a new species: an integrative taxonomic approach. Zoological Journal of the Linnean Society, 2024, 202 (1), pp.1-23/zlad166. 10.1093/zoolinnean/zlad166. hal-04318081

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Original Article

# Searching for Mediterranean bath sponges
(Demospongiae: Dictyoceratida: Spongiidae) in the
Northeast Atlantic reveals a new species: an integrative
taxonomic approach

Marie Grenier¹,², Charlotte Simmler¹, Pierre Chevaldonné¹, Noëlle Callizot², Thierry Pérez¹,*

¹Institut Méditerranéen de Biodiversité et d'Ecologie marine et continentale (IMBE), UMR CNRS 7263, IRD 237 – Aix Marseille Université – Avignon Université, Station Marine d'Endoume, Chemin de la Batterie des Lions, 13007, Marseille, France
²Neuralia SAS, 410 Chemin Départemental 60, 13120, Gardanne, France

[Version of Record, published on 14 November 2023; http://zoobank.org/urn:lsid:zoobank.org:pub:3ED45D8A-954B-4723-9D0F-714357A7DAB5]

*Corresponding author. Institut Méditerranéen de Biodiversité et d'Ecologie marine et continentale (IMBE), UMR CNRS 7263, IRD 237 – Aix Marseille Université – Avignon Université, Station Marine d'Endoume, Chemin de la Batterie des Lions, 13007, Marseille, France. Tel: +33(0)413945044. E-mail: thierry.perez@imbe.fr

ABSTRACT

Bath sponges, included in the Spongiidae (Demospongiae: Dictyoceratida), are distributed across the oceans of the world, with a greater abundance in temperate, subtropical, and tropical zones. Their harvest started during ancient times in the Mediterranean Sea, which shed light on the whole family. Most of the Mediterranean Spongiidae have been reported repeatedly from the Northeast Atlantic, notably along the Iberian peninsula. The aim of this study was to clarify the taxonomic status of these sponges after a recent sampling effort in the French Pays Basque and the Spanish Cantabria, complemented by several Mediterranean Sea stations, including the Strait of Gibraltar. An integrative taxonomic approach was adopted for the first time on a set of Spongiidae samples from the Northeast Atlantic, assessing the congruence of analyses of: (i) the morphology of the spongin skeleton; (ii) two different parts of the genome (mitochondrial and nuclear); and (iii) metabolomic fingerprints, to delimit the frontiers between species better. A new bath sponge species is described herein, and significant advances and changes are proposed for the systematics of the family.

**Keywords:** metabolomic fingerprints; mitochondrial marker; molecular taxonomy; nuclear marker; Porifera; *Spongia*; spongin fibres; systematics

INTRODUCTION

Bath sponges, included in the Spongiidae (Demospongiae: Dictyoceratida), are distributed across the oceans of the world, with a greater abundance in temperate, subtropical, and tropical zones (de Voogd et al. 2023). Their harvest started during ancient times in the Mediterranean Sea (Fourt 2019), and nowadays bath sponge fisheries exist in the Caribbean region (Cuba, Florida, and the Bahamas), in the Mediterranean Sea (Tunisia, Croatia, and Greece) and in the South Pacific Ocean (Josupeit 1990, Fourt 2019).

Their use has diversified over time, from being basic tool in medicine during the Roman Empire to being personal hygiene care object during the late 19th century (Pronzato and Manconi 2008, Faget 2017). Currently, the value of natural bath sponges

is based on the sole exploitation of the cleaned spongin skeleton of these species in order to supply the cosmetics industry. However, various studies have also highlighted the pharmacological potential of some Spongiidae that produce chemical compounds with cytotoxic, antibacterial, neurotropic, anticvulsant, or even analgesic properties (Tokue et al. 2006, Dellai et al. 2012, Máximo et al. 2016). Sponge fishermen are aware of this high potential (Fourt 2019). After two golden ages, in the late 19th century and after the Second World War, bath sponge availability and fishery production has gradually decreased owing to multiple factors, including overfishing and climate change (Pronzato and Manconi 2008, Voultsiadou et al. 2011, Fourt et al. 2020). By the end of the 20th century, the increasing frequency of marine heat waves multiplied fatal blows, bringing
Mediterranean populations to the brink of extinction (Pérez and Vacelet 2014, Grenier et al. 2023). In this context, the description of any new bath sponge represents a significant event for a vast population of putative end-users.

Today, Spongiidae include 149 valid species, distributed among six genera: *Spongia* Linnaeus, 1759 (72 species), *Hyattella* Lendenfeld, 1888 (28 species), *Hippospongia* Schulze, 1879 (24 species), *Coscinoderma* Carter, 1883 (16 species), *Leiosella* Lendenfeld, 1888 (eight species) and *Rhopaloeides* Thompson, Murphy, Bergquist & Evans, 1987 (one species) (de Voogd et al. 2023). The current taxonomy of this group is based mainly on the presence or absence of an armouring surface, which is an organized dermal crust made of various mineral debris and foreign spicules, sub-dermal or internal lacunae, and on the architecture of a skeleton made of a dense network of spongin fibres arranged in different levels of hierarchical reticulation. This spongin network is usually composed of fibres of various thickness, which may contain foreign inclusions, such as broken or entire spicules of syntopic sponge species, sand debris, and/or a pith (Vacelet 1959, Cook and Bergquist 2002). Thus, the organization, the density, the structure, and the thickness of these fibres vary between genera and species, with their cross-linking conferring variable levels of elasticity, resistance, and absorption capacity that contribute to their economic value (Castritsi-Catharios et al. 2007).

Despite the significant progress recently achieved for overall sponge taxonomy and systematics, bath sponge classification remains a matter of debate. The substantial intraspecific morphological plasticity has triggered several taxonomic changes that remain questionable (Cook and Bergquist 2002, Castritsi-Catharios et al. 2011). Therefore, this difficult morphology-based taxonomy has led to incomplete and/or ambiguous identifications, explaining the unresolved status of most phylogenies at the family level and at the level of the order Dictyoceratida (Cook 2007, Erpenbeck et al. 2012, Redmond et al. 2013, Thacker et al. 2013). For instance, the first comprehensive molecular approach based on the partial mitochondrial cytochrome c oxidase subunit 1 gene (*CO1*) and on the D3-D5 partition of the nuclear large ribosomal subunit gene (28S) failed to resolve the phylogeny of Spongiidae (Erpenbeck et al. 2012). Likewise, Redmond et al. (2013) and Thacker et al. (2013) could not resolve robustly the relationships between Dictyoceratida taxa, including Spongiidae. Consequently, Spongiidae represent one of the very few cases of challenging Porifera taxonomy and systematics that has not yet been improved by molecular biology; despite several attempts, no significant advance has been realized within the last 20 years (for a review, see Cárdenas et al. 2012).

Undoubtedly, the various traditional molecular markers, always used alone so far, provide insufficient resolution to answer all Dictyoceratida-related phylogenetic questions (Erpenbeck et al. 2020). Also, the great number of incomplete or erroneous identifications of sequenced individuals, owing to the difficulty to assess morphological traits adequately, explains the incongruency between molecular genetic phylogenies and higher-level morphological classification (Agne et al. 2022).

Sponge species with a very large geographical distribution are nowadays most often split after an accurate examination of specimens from different parts of the distribution area (Cárdenas et al. 2012). The history of the 'elephant ear' sponge is one of the best

examples. This bath sponge, described first as *Spongia agaricina* by Pallas (1766), who did not indicate any type locality, is well known owing to its vernacular name related to its shape. A century later, Schulze (1879) described *Spongia lamella* from the Adriatic Sea, but this species was long considered as a synonym of *S. agaricina*, like the Indo-Pacific *Spongia thienemanni* described by Arndt (1943). During more than two centuries, *S. agaricina* was thus considered to occur from the Mediterranean Sea to the Pacific Ocean, and it is only recently that the morphology, the organization of the skeleton, and the tensile strength of two sets of elephant ear sponge samples, from the Mediterranean Sea and the Indian Ocean, respectively, revealed *S. agaricina* and *S. lamella* to be two distinct species (Castritsi-Catharios et al. 2007). Two neotypes were designated: one for the Indian Ocean, *S. agaricina* Pallas, 1766, and the other one for the Mediterranean Sea, *S. lamella* Schulze, 1879 (Pronzato and Manconi 2008). The latter species is also reported from the Atlantic Ocean by a number of studies that rarely present true taxonomic analyses (Ferrer Hernández 1914, Lopes and Boury-Esnault 1981, Carballo and Garcia-Gómez 1994, Boury-Esnault et al. 2005, Noyer et al. 2009, 2011, Noyer and Becerro 2012), hence its status deserves to be checked.

The type species of the genus, *Spongia officinalis* Linnaeus, 1759, offers an even more puzzling history since the very first description. Schmidt (1862) described *Spongia adriatica* exclusively from the Adriatic Sea, specifying that this sponge could be observed along the cliffs and rocky bottoms from Kvarner, the Dalmatian coasts, and islands, to the Ionian islands. This sponge long remained a variety of *S. officinalis* (var. adriatica), well recognized by fishermen and sponge traders, and nowadays formally accepted as *S. officinalis* in the World Porifera Database (de Voogd et al. 2023). The story of this species could thus be very simple if other authors of the 19th and 20th centuries had not introduced a bit of chaos. Burton (1934) designated as neotype of *S. officinalis* one specimen identified by Schulze (1879) as *S. officinalis* var. adriatica. But Burton did not pay attention to the geographical origin of this specimen, which came indeed from the Adriatic region, from Lesina which might correspond to two locations presenting totally different environmental conditions: the current Croatian Island of Hvar (rocky bottoms, open sea) or an Italian coastal lagoon (muddy bottoms, brackish waters). Therefore, although a sequence of this neotype could be obtained recently (Agne et al. 2022), this sponge cannot be considered as a reference for *S. officinalis*.

Finally, the validity of another species is open to debate. Schmidt (1862) described *Spongia mollissima* from the Levantine Sea. Then, Schulze (1879) added a few localities in the Aegean Sea, until Arndt (1937) indicated that its distribution should be restricted to the eastern Mediterranean Sea. Long considered as a variety of *S. officinalis* (var. mollissima), this sponge has been accepted formally by Pronzato and Manconi (2008), who designated a specimen collected along the Syrian coast as neotype. However, a study conducted by Dailianis et al. (2011) did not find notable genetic differences between *S. officinalis* and *S. mollissima* collected across the Mediterranean Sea. Although no difference was found between these two species, these authors could highlight a significant divergence between them and specimens of *S. officinalis* sampled in the Strait of Gibraltar. In
this last study, only the external morphology was investigated on
several specimens, which represents only the starting point of an
accurate taxonomic description.

Most of the Mediterranean Spongiidae have been reported re-
peatedly from the Northeast Atlantic, notably along the Iberian
peninsula (Ramirez 2002). The aim of the present study was to
clarify the taxonomic status of these sponges after a recent sam-
pling effort in the French Pays Basque, in the Spanish Cantabria,
and in different places in the Mediterranean Sea, including the
Strait of Gibraltar. For the first time with Spongiidae, we applied
an integrative taxonomic approach, assessing the congruence of
analyses of the morphology of the skeleton with genetics and
metabolomic fingerprints to decipher the frontiers between species
clearly. Incidentally, a new bath sponge species is described, and a
significant turnover in the systematics of the family is warranted.

MATERIALS AND METHODS

Sampling

In this study, sponges from eight different localities were inves-
tigated, three of them in the Northeast Atlantic Ocean (Fig. 1).
Specimens from the Northeast Atlantic Ocean were collected
in June 2016, June and August 2020, and June and July 2021,
along the French Pays-Basque coast and along the Asturian and
Cantabrian shores of Spain. In the French Pays Basque, sampling
was performed in two reef flat stations off Hendaye and then off
Ciboure in a semi-dark tunnel. In Spain, sampling was conducted
off Gijon on the rocky bottoms of Las Gemelas island, and then
in two stations, a chaos of rocks, and semi-dark and dark tunnels
around Mouro island, off Santander.

Specimens from the Strait of Gibraltar were collected in 2007
and 2009 in Ceuta during the sampling of the ECIMAR project.

These specimens belonged to the set of samples considered by
Dailianis *et al.* (2011) but were subjected to new analyses in the
present work.

In the Mediterranean Sea, the specimens considered in this
work were sampled between 2020 and 2022 off Marseille and
La Ciotat (France), mostly in semi-dark caves; Porto Cesareo
(Apulia, Italy), mostly on a wreck; Tel Aviv (Israel), on shallow
and mesophotic rocky bottoms; and Djerba (Tunisia), in sea-
grass meadows. All these samples were collected by SCUBA
diving except for the sample from Tunisia, which was provided
by a fisherman. Apart from that specimen, underwater pictures
and general information of the external morphology, such as the
size, the growth form, the colour, and the consistency, were re-
corded for each individual. One part of each sample was fixed
in 95% ethanol for morphological and molecular analyses, and
the remaining part was frozen at -20°C for further metabolomic
analyses. Only the samples from Ceuta were not available frozen
for metabolomics.

Morphological analysis

The presence of a more or less dense network of fibres can make it difficult to cut the spongin skeleton. The sections, mostly perpendicular to the surface, must be as thin as possible and as large as possible, in order to observe all types of spongin fibres (primary, secondary, and even tertiary in some case), which could be located in different areas of the sponge body (from the surface to the core of the specimen). For this reason, two types of complementary techniques were used in this study.

The first technique involves the preparation of thin histo-
logical slides and requires several steps to prepare the biological
material. A piece of sponge was embedded in araldite following a
succession of baths, according to a gradient from 100% acetone

<figure><img src="image_3.png" /><figcaption>Figure 1. Study area. Black stars indicate the sampling locations of Spongiidae from this work.</figcaption></figure>
<table><caption>Table 1. Species names, voucher codes and GenBank accession numbers of Spongiidae used in this study.</caption><thead><tr><th>Species</th><th>Voucher code</th><th>CO1<br>dgLCO1490-dgHCO2198</th><th>28S<br>C2-D2</th></tr></thead><tbody><tr><td rowspan="20">Spongia maitasuna</td><td>20210726-CIB-BTP6</td><td rowspan="20">OR297953</td><td>OR291312</td></tr><tr><td>20210726-CIB-BTP11</td><td>OR291313</td></tr><tr><td>20210726-CIB-BTP12</td><td>OR291314</td></tr><tr><td>20210630-CIB-BPC2</td><td>OR291315</td></tr><tr><td>20210729-ESP-MTP1</td><td>OR291316</td></tr><tr><td>20210729-ESP-MTP2</td><td>OR291317</td></tr><tr><td>20210729-ESP-MTP3</td><td>OR291318</td></tr><tr><td>20210729-ESP-MTP4</td><td>OR291319</td></tr><tr><td>20210729-ESP-MTP6</td><td>OR291320</td></tr><tr><td>20210729-ESP-MTP7</td><td>OR291321</td></tr><tr><td>20210729-ESP-MTP8</td><td>OR291322</td></tr><tr><td>20210730-ESP-M2TP1</td><td>OR291323</td></tr><tr><td>20210730-ESP-M2TP7</td><td>OR291324</td></tr><tr><td>20210730-ESP-M2TP8</td><td>OR291325</td></tr><tr><td>20210730-ESP-M2TP9</td><td>OR291326</td></tr><tr><td>20210730-ESP-M2TP10</td><td>OR291327</td></tr><tr><td>20210730-ESP-M2TP13</td><td>OR291328</td></tr><tr><td>20210730-ESP-M2TP14</td><td>OR291329</td></tr><tr><td rowspan="20">Spongia lamella</td><td>20200602-MRS-MTP3</td><td>OR297956</td><td>OR291330</td></tr><tr><td>20200602-MRS-MTP4</td><td>OR297957</td><td>OR291331</td></tr><tr><td>20200602-MRS-MTP5</td><td>OR297958</td><td>OR291332</td></tr><tr><td>20200602-MRS-MTP6</td><td>OR297959</td><td>OR291333</td></tr><tr><td>20200602-MRS-MTP7</td><td></td><td>OR291334</td></tr><tr><td>20200602-MRS-MTP8</td><td></td><td>OR291335</td></tr><tr><td>20210726-CIB-BTP1</td><td></td><td>OR291336</td></tr><tr><td>20210726-CIB-BTP2</td><td>OR297960</td><td>OR291337</td></tr><tr><td>20210726-CIB-BTP3</td><td>OR297961</td><td>OR291338</td></tr><tr><td>20210726-CIB-BTP4</td><td></td><td>OR291339</td></tr><tr><td>20210726-CIB-BTP7</td><td>OR297962</td><td>OR291340</td></tr><tr><td>20210726-CIB-BTP10</td><td></td><td>OR291341</td></tr><tr><td>20210630-CIB-BPC1</td><td>OR297963</td><td>OR291342</td></tr><tr><td>20210630-CIB-BPC3</td><td>OR297964</td><td>OR291343</td></tr><tr><td>20200814-HEN-AMNC3</td><td>OR297965</td><td>OR291344</td></tr><tr><td>20200624-HEN-BMNC2</td><td></td><td>OR291345</td></tr><tr><td>20160607-GIJ-JC1</td><td></td><td>OR291346</td></tr><tr><td>20210729-ESP-MTP5</td><td>OR297966</td><td>OR291347</td></tr><tr><td>20210730-ESP-M2TP2</td><td>OR297967</td><td>OR291348</td></tr><tr><td>20210730-ESP-M2TP3</td><td>OR297968</td><td>OR291349</td></tr><tr><td>20210730-ESP-M2TP12</td><td>OR297969</td><td>OR291350</td></tr><tr><td rowspan="10">Spongia officinalis</td><td>20200519-MRS-ITP3</td><td>OR297970</td><td>OR291351</td></tr><tr><td>20200519-MRS-ITP5</td><td>OR297971</td><td></td></tr><tr><td>20200522-MRS-FTP2</td><td></td><td>OR291352</td></tr><tr><td>20200522-MRS-FTP3</td><td></td><td>OR291353</td></tr><tr><td>20200602-MRS-MTP9</td><td></td><td>OR291354</td></tr><tr><td>20200617-MRS-NTP1</td><td></td><td>OR291355</td></tr><tr><td>20220909-MRS2-RTP4</td><td></td><td>OR291356</td></tr><tr><td>20220909-MRS2-RTP5</td><td></td><td>OR291357</td></tr><tr><td>20220909-MRS2-RTP6</td><td>OR297972</td><td>OR291358</td></tr><tr><td>20220923-MRS2-ITP25</td><td></td><td>OR291359</td></tr><tr><td>20220923-MRS2-ITP26</td><td>OR297973</td><td>OR291360</td></tr></tbody></table>
<table><caption>Table 1. Continued</caption><thead><tr><th>Species</th><th>Voucher code</th><th>CO1<br>dgLCO1490-dgHCO2198</th><th>28S<br>C2-D2</th></tr></thead><tbody><tr><td rowspan="4">Spongia cf. officinalis</td><td>20220923-MRS2-RTP27</td><td rowspan="4">OR297974</td><td>OR291361</td></tr><tr><td>20090617-CEU-ECITP1</td><td>OR291362</td></tr><tr><td>20070701-CEU-ECITP2</td><td>OR291363</td></tr><tr><td>20070705-CEU-ECITP3</td><td>OR291364</td></tr><tr><td rowspan="10">Spongia mollissima</td><td>20200809-IT-NTP1</td><td rowspan="5">OR297975</td><td>OR291365</td></tr><tr><td>20200809-IT-NTP2</td><td>OR291366</td></tr><tr><td>20200809-IT-NTP3</td><td>OR291367</td></tr><tr><td>20200809-IT-NTP4</td><td>OR291368</td></tr><tr><td>20200809-IT-NTP5</td><td>OR291369</td></tr><tr><td>20200809-IT-NTP8</td><td>OR291370</td></tr><tr><td>20200810-IT-CTP1</td><td>OR291371</td></tr><tr><td>20200810-IT-StTP2</td><td>OR291372</td></tr><tr><td rowspan="2">Spongia nitens</td><td>Po.25493</td><td rowspan="2">KX866744<sup>a</sup></td><td>KX688726<sup>a</sup></td></tr><tr><td>Po.25665</td><td>KX688730<sup>a</sup></td></tr><tr><td>Spongia zimocca</td><td>Po.25742</td><td></td><td>OR291373</td></tr><tr><td>Spongia virgultosa</td><td>20070718-MRS-ECITP5</td><td>OR297977</td><td>OR291374</td></tr><tr><td rowspan="4">Hippospongia communis</td><td>20200519-MRS-JTP1</td><td>OR297978</td><td>OR291375</td></tr><tr><td>20200522-MRS-FTP4</td><td>OR297979</td><td>OR291376</td></tr><tr><td>20200522-MRS-FTP6</td><td>OR297980</td><td>OR291377</td></tr><tr><td>20201225-TUN-Ep8</td><td></td><td>OR291378</td></tr><tr><td>Coscinoderma sporadense</td><td>Po.25932</td><td>KX866774<sup>a</sup></td><td>OR291379</td></tr><tr><td rowspan="2">Ircinia oros</td><td>Po.25670</td><td></td><td>KX688731<sup>a</sup></td></tr><tr><td>PO10-3-7</td><td>JN655189<sup>a</sup></td><td></td></tr></tbody></table>

to 25% acetone and 75% araldite. Then, sections of ~5 mm thickness were cut with a low-speed saw and wet-ground with abrasive paper or a polishing disc to obtain thinner sections (<1 mm thick). This relatively tedious but precise method provides a clean cut from the surface to the base of the sponge.

The second method was to prepare skeleton sections by hand. The cut was made using a razor blade, directly on the piece of sponge preserved in ethanol. The section was then stained with Acid Fuchsin and soaked in absolute ethanol. A final bath of a few minutes in acetone was necessary to promote araldite penetration inside fibres before mounting the slide. This is a much faster technique, requiring very few steps, but more difficult to perform depending on the consistency of the sponge skeleton. These two types of sections were mounted between a glass slide and a coverslip in a drop of araldite, put in the oven at 60°C for a minimum of 24 h, and observed under a light microscope. Measurements of fibres and the network are expressed as the minimal and maximal size (in micrometres).

## Molecular analysis

For the molecular work, we chose CO1 and ribosomal 28S as molecular markers to access very different parts of the genome (mitochondrial and nuclear). CO1 is universally used for sponge barcoding. Most of the time, it exhibits adequate diversity to distinguish effectively between taxa of the same genus and/or family described using morphological traits (Heim et al. 2007, Cárdenas et al. 2010) or implied by ecological traits (Duran and

Rützler 2006, Wulff 2006) and helps in tracing cryptic speciation (Xavier et al. 2010). The C2–D2 region of 28S DNA is suitable for all sponge classes and often provides a higher resolution and easier amplification than CO1 (Chombard et al. 1998).

DNA extractions were performed from small sponge fragments (0.5 cm³) using a NucleoSpin Tissue DNA Mini kit (Macherey Nagel, Hoerdt, France). Two sets of primers were used respectively for the CO1 and 28S: (i) the degenerated primers dgLCO1490 and dgHCO2198 to amplify an 800 bp fragment of the CO1 mitochondrial gene (Folmer et al. 1994, Meyer et al. 2005); and (ii) the universal primers C2 and D2 to amplify a 450 bp fragment of the ribosomal 28S gene (Chombard et al. 1998). Amplifications were done in a total reaction volume of 40 µL comprising 2 µL of each primer (0.5 µM), 0.8 µL of dNTPs (0.2 mM), 4 µL of DreamTaq buffer, which contains 2 mM of MgCl₂, 0.2 µL of DreamTaq (Thermo Fisher Scientific; 1 U µL⁻¹), 2 µL of extracted DNA, and 29 µL of ultrapure water (HyPure™ molecular biology grade water). The PCRs were performed on an Eppendorf AG Mastercycler Pro S vapo protect thermocycler, with an initial step of 5 min at 95°C, followed by one amplification cycle (denaturation at 94°C for 30 s, annealing at 50°C for CO1 for 1 min (57°C for 28S), and extension at 72°C for 1 min), followed by a further 34 amplification cycles (denaturation at 94°C for 30 s, annealing at 50°C for CO1 for 30 s (57°C for 28S), and extension at 72°C for 1 min), and a final extension step at 72°C for 10 min. The PCR products were directly sequenced in each primer direction by Eurofins (Ebersberg, Germany).

<footnote><sup>a</sup>Sequences downloaded from GenBank.</footnote>
A total of 28 sequences of CO1 and 68 of 28S were generated. We also downloaded two sequences of CO1 and two of 28S from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank). Voucher numbers and GenBank accession numbers of all DNA sequences used in this work are listed in Table 1.

Sequences were aligned using BIOEDIT v.7.2.5 (Hall 1999). First, phylogenetic trees were constructed using both the neighbour-joining (NJ) method (1000 bootstrap replicates) with CLUSTAL X v.2.1 (Larkin et al. 2007) and maximum-likelihood (ML) methods (1000 bootstrap replicates) with the online plateform PHYML (Guindon et al. 2010). The substitution model chosen for ML analysis was TN93+G according to the smart model selection (SMS) in PhyML, using the Bayesian information criterion (BIC) (Lefort et al. 2017). Then, a maximum-likelihood concatenated tree of CO1 + 28S was generated using RAXMLGUI v.2.0 (Edler et al. 2021). Bootstrap supports were obtained with the rapid bootstrap using 1000 replicates. The substitution model chosen for the phylogenetic analyses was HKY+G for both molecular markers, according to the software JMODELTEST previously used to evaluate separately the best evolutionary model for each marker (Darriba et al. 2012). Finally, Bayesian inference (BI) was performed in MRBAYES v.3.2.7 (Ronquist et al. 2012) in order to generate another concatenated tree. We have used the same evolutionary model parameters as selected for the ML analyses, with two runs and four independent chains for 10⁶ generations and a 2% burn-in, resulting in a majority consensus tree (of the sampled trees). In all analyses, the Dictyoceratida *Ircinia oros* (Schmidt, 1862), was used as the outgroup. The p-distance values were calculated using the software MEGA v.11.0.13 (Tamura et al. 2021) to estimate the interspecific nucleotide variability.

## Metabolomic analysis

Soon after collection, all sponges were transported in sealed jars of seawater without exposure to air and then frozen at -20°C. In the laboratory, frozen samples were freeze-dried for ≥48 h. For each sample, the final dry sponge biomass was reduced to a thin powder with a knife mill (M20, IKA-WERKE). One gram of each sponge powder was mixed with 10 mL MeOH/CH₂Cl₂ LC-MS grade (50:50 v/v), sonicated for 3 min (J.P SELECTA, 50/60 Hz) and the full operation was repeated three times. The pooled supernatants containing extracted metabolites, collected using a syringe, were then mixed with 500 mg of C-18 powder (Polygoprep 60-50, Macherey-Nagel) and concentrated to dryness under vacuum (SpeedVac®, Savant SPD111V). Each C-18 mixed extract was processed further on Strata C18-E Giga SPE cartridges (55 µm, 70 Å, 2 g/12 mL, Phenomenex) to remove residual sea salts. Extracts were washed with 36 mL of LC-MS-grade water, then eluted with 24 mL of LC-MS-grade MeOH, leading to the production of desalted sponge crude extract.

All resulting desalted extracts were concentrated to dryness under vacuum, then prepared at 0.2 mg/mL in LC-MS-grade MeOH and filtered on PTFE luer-lock filters (0.22 µm, catalogue no. 26142, Restek) before injection. A total of 63 extracts were prepared, with seven of them corresponding to pooled extracts based on taxonomic identification.

Chromatographic separations were achieved on a Luna® Omega Polar C-18 UHPLC column (150 mm × 2.1 mm, 1.6 µm,

Phenomenex), maintained at 42°C, with an elution gradient composed of water (A) and acetonitrile (B) both with 0.1% formic acid, in the following conditions: from 40% B to 95% B in 20 min (flow rate of 0.45 mL/min, injection volume of 2 µL).

Mass spectrometry detection parameters using ionization by electrospray (ESI-Q-ToF, Bruker Impact II) in positive mode were set as follows: nebulizer gas N₂ at 3.5 bar, dry gas at 12 L/min, capillary temperature of 200°C and voltage at 4500 V. The MS/MS acquisition mode was set with a scan rate of 4 Hz (full scan from 50 to 1200 m/z) and a mixed collision energy of 20–40 eV (50% time at each collision energy, stepping mode). A sodium formate/acetate solution forming clusters in the studied mass range was used as calibrant and injected automatically before each sample for internal mass calibration, ensuring a precision of m/z of <2 ppm in the mass range. Extracts were injected randomly to integrate any memory effect on the column and time-dependent MS drift. Pooled samples, corresponding to a mixture of all the samples in the sequence, injected every six samples from the beginning to the end of the series, were used for further ion filtering and data processing.

Following their calibration, the acquired MS data were converted to the open format *.mzXML using MSCONVERT (Chambers et al. 2012) and processed further in MZMINE v.3.5.0 (Schmid et al. 2023) for feature detection as follows: (i) mass detection (centroid, MS1 noise level 5E3 and MS2 noise level 0); (ii) ADAP Chromatogram Builder (minimum consecutive scans 2, minimum intensity for consecutive scans 8E3, minimum absolute height 1E4, m/z tolerance 10 ppm); (iii) chromatogram resolving using ADAP resolver algorithm (Myers et al. 2017) (S/N threshold 10, minimum feature height 1E4, coefficient area threshold 10, peak duration range 0.0–2 min, RT wavelet range 0.0–0.1 min, RT range for MS2 scan pairing 0.03 min); (iv) isotope grouper using ¹³C isotope filter (m/z tolerance 10 ppm, RT tolerance 0.1 min, maximum charge 2, representative isotope most intense); (v) join aligner (m/z tolerance 10 ppm, weight for m/z 75%, RT tolerance 0.1 min, weight for RT 25 %); (vi) feature list filtering (features detected in MeOH and SPE blank were deleted using feature list blank subtraction, minimum of detection in blanks one); (vii) gap filling peak finder [intensity tolerance 10%, m/z tolerance 10 ppm, RT tolerance 0.1 min, minimum scans (data points) three]; (viii) feature list filtering using feature list rows filter [minimum aligned features (samples) maximum of three samples or 1%, RT 0.5–18 min, keep feature with MS2 scans, never remove feature with MS2, reset the feature number ID] and feature filter (area 1E3–1E7, height 1.5E4–1E7, duration 0–1, keep only features with MS2 scan). The first MS2 aligned feature list contained 915 signals. Using the same data processing, a second aligned feature list containing 240 signals was generated with MS data from extracts that had been pooled based on taxonomic identification.

Exported as a .csv file, the first aligned feature list was processed on the METABOANALYST v.5.0 web application (https://www.metaboanalyst.ca/home.xhtml) as follows: data integrity check was set as default, log-transformed, and normalized by Pareto scaling. Hierarchical clustering analyses were performed using Euclidean distance calculations with the Ward algorithm as linkage criterion. The results are represented as a dendrogram and as a heatmap to assess the
similarities and differences between metabolomic finger-
prints of each investigated Spongiidae. The second aligned
feature list, also exported as a .csv file, was used to generate
Venn diagrams using the web platform Bioinformatics and
Evolutionary Genomics (https://bioinformatics.psb.ugent.
be/webtools/Venn/).

RESULTS
SYSTEMATICS

Phylum Porifera Grant, 1836

Class Demospongiae Sollas, 1885

Order Dictyoceratida Minchin, 1900

Family Spongiidae Gray, 1867

*Definition:* Dictyoceratida with homogeneous skeletal fibres, without distinct laminations, typically dominated by sub-primary skeletal fibres, and with diplodal choanocyte chambers (from *Cook and Bergquist 2002*).

Genus *Spongia* Linnaeus, 1759

*Type species:* *Spongia officinalis* Linnaeus, 1759.

*Definition:* Unarmoured spongiids, not heavily lacunose, with simple primary fibres (from *Cook and Bergquist 2002*).

*Spongia maitasuna* Grenier & Pérez, 2023 sp. nov.

(Fig. 2A–D)

Material examined

*Holotype:* MNHN-IP-2018-419, Baloo tunnel at 14 m depth, Ciboure, France (43°24′59″N, 1°39′52″W). Collector: T. Pérez, 26 July 2021. Voucher code: 20210726-CIB-BTP6. GenBank accession numbers OR297953 and OR291312 (CO1 and 28S, respectively).

*Paratype 1:* MNHN-IP-2018-420, Western part of Mouro island, semi-dark to dark cave, between 3 and 12 m depth, Santander, Spain (43°28′22″N, 3°45′22″W). Collector: T. Pérez, 29 July 2021. Voucher code: 20210729-ESP-MTP1. GenBank accession number OR291316 (28S).

*Paratype 2:* MNHN-IP-2018-421, Eastern part of Mouro island, shallow water cliffs and chaos of rocks and crevices, between 3 and 12 m depth, Santander, Spain (43°28′21″N, 3°45′18″W). Collector: T. Pérez, 30 July 2021. Voucher code: 20210730-ESP-M2TP1. GenBank accession numbers OR297955 and OR291323 (CO1 and 28S, respectively).

Other specimens examined

20210630-CIB-BPC2, Baloo tunnel at 14 m depth, Ciboure, France (43°24′59″N, 1°39′52″W). Collector: P. Chevaldonné, 30 June 2021.

20210726-CIB-BTP11, Baloo tunnel at 14 m depth, Ciboure, France (43°24′59″N, 1°39′52″W). Collector: T. Pérez, 26 July 2021.

20210726-CIB-BTP12, Baloo tunnel at 14 m depth, Ciboure, France (43°24′59″N, 1°39′52″W). Collector: T. Pérez, 26 July 2021.

20210729-ESP-MTP2, Western part of Mouro island, semi-
dark to dark cave, between 3 and 12 m depth, Santander, Spain
(43°28′22″N, 3°45′22″W). Collector: T. Pérez, 29 July 2021.

20210729-ESP-MTP3, Western part of Mouro island, semi-
dark to dark cave, between 3 and 12 m depth, Santander, Spain
(43°28′22″N, 3°45′22″W). Collector: T. Pérez, 29 July 2021.

20210729-ESP-MTP4, Western part of Mouro island, semi-
dark to dark cave, between 3 and 12 m depth, Santander, Spain
(43°28′22″N, 3°45′22″W). Collector: T. Pérez, 29 July 2021.

20210729-ESP-MTP6, Western part of Mouro island, semi-
dark to dark cave, between 3 and 12 m depth, Santander, Spain
(43°28′22″N, 3°45′22″W). Collector: T. Pérez, 29 July 2021.

20210729-ESP-MTP7, Western part of Mouro island, semi-
dark to dark cave, between 3 and 12 m depth, Santander, Spain
(43°28′22″N, 3°45′22″W). Collector: T. Pérez, 29 July 2021.

20210729-ESP-MTP8, Western part of Mouro island, semi-
dark to dark cave, between 3 and 12 m depth, Santander, Spain
(43°28′22″N, 3°45′22″W). Collector: T. Pérez, 29 July 2021.

20210730-ESP-M2TP7, Eastern part of Mouro island, shallow water cliffs and chaos of rocks and crevices, between 3 and 12 m depth, Santander, Spain (43°28′21″N, 3°45′18″W). Collector: T. Pérez, 30 July 2021.

20210730-ESP-M2TP8, Eastern part of Mouro island, shallow water cliffs and chaos of rocks and crevices, between 3 and 12 m depth, Santander, Spain (43°28′21″N, 3°45′18″W). Collector: T. Pérez, 30 July 2021.

20210730-ESP-M2TP9, Eastern part of Mouro island, shallow water cliffs and chaos of rocks and crevices, between 3 and 12 m depth, Santander, Spain (43°28′21″N, 3°45′18″W). Collector: T. Pérez, 30 July 2021.

20210730-ESP-M2TP10, Eastern part of Mouro island, shallow water cliffs and chaos of rocks and crevices, between 3 and 12 m depth, Santander, Spain (43°28′21″N, 3°45′18″W). Collector: T. Pérez, 30 July 2021.

20210730-ESP-M2TP11, Eastern part of Mouro island, shallow water cliffs and chaos of rocks and crevices, between 3 and 12 m depth, Santander, Spain (43°28′21″N, 3°45′18″W). Collector: T. Pérez, 30 July 2021.

20210730-ESP-M2TP13, Eastern part of Mouro island, shallow water cliffs and chaos of rocks and crevices, between 3 and 12 m depth, Santander, Spain (43°28′21″N, 3°45′18″W). Collector: T. Pérez, 30 July 2021.

20210730-ESP-M2TP14, Eastern part of Mouro island, shallow water cliffs and chaos of rocks and crevices, between 3 and 12 m depth, Santander, Spain (43°28′21″N, 3°45′18″W). Collector: T. Pérez, 30 July 2021.

Comparative material examined
*Spongia officinalis* Linnaeus, 1759

(Fig. 2E)

20200519-MRS-ITP3, Impérial de Terre, semi-dark cave, between 10 and 20 m depth, Marseille, France (43°10′22″N, 5°23′35″E). Collector: T. Pérez, 19 May 2020.

20200519-MRS-ITP4, Impérial de Terre, semi-dark cave, between 10 and 20 m depth, Marseille, France (43°10′22″N, 5°23′35″E). Collector: T. Pérez, 19 May 2020.
<figure><img src="image_4.png" /><figcaption>**Figure 2.** A–D, *in situ* pictures of *Spongia maitasuna* in Baloo tunnel, Ciboure, France (A, B) and Mouro Island, Santander, Spain (C, D). E, *Spongia officinalis* from Marseille, France. F, *Spongia cf. officinalis* from Ceuta, Strait of Gibraltar, Spain.</figcaption></figure>

20200519-MRS-ITP5, Impérial de Terre, semi-dark cave, between 10 and 20 m depth, Marseille, France (43°10′22″N, 5°23′35″E). Collector: T. Pérez, 19 May 2020.

20200522-MRS-FTP2, Fauconnière, semi-dark tunnel, between 5 and 10 m depth, Saint-Cyr Les Lecques, France (43°09′16″N, 5°40′59″E). Collector: T. Pérez, 22 May 2020.

20200522-MRS-FTP3, Fauconnière, semi-dark tunnel, between 5 and 10 m depth, Saint-Cyr Les Lecques, France (43°09′16″N, 5°40′59″E). Collector: T. Pérez, 22 May 2020.

20200602-MRS-MTP9, Pharillons de Maire, cliff at 20 m depth, Marseille, France (43°12′27″N, 5°20′17″E). Collector: T. Pérez, 2 June 2020.
20200617-MRS-NTP1, Niolon, semi-dark cave, between 8 and 10 m depth, Marseille, France (43°20'15"N, 5°15'22"E). Collector: T. Pérez, 17 June 2020.

20220909-MRS2-RTP4, Riou island, overhang at 28 m depth, Marseille, France (43°10'23"N, 5°23'15"E). Collector: T. Pérez, 9 September 2022.

20220909-MRS2-RTP5, Riou island, overhang at 28 m depth, Marseille, France (43°10'23"N, 5°23'15"E). Collector: T. Pérez, 9 September 2022.

20220909-MRS2-RTP6, Riou island, overhang at 28 m depth, Marseille, France (43°10'23"N, 5°23'15"E). Collector: T. Pérez, 9 September 2022.

20220923-MRS2-ITP25, Impérial de Terre, semi-dark cave at 20 m depth, Marseille, France (43°10'22"N, 5°23'35"E). Collector: T. Pérez, 23 September 2022.

20220923-MRS2-ITP26, Impérial de Terre, semi-dark cave at 20 m depth, Marseille, France (43°10'22"N, 5°23'35"E). Collector: T. Pérez, 23 September 2022.

20220923-MRS2-RTP27, Riou island, overhang at 25 m depth, Marseille, France (43°10'23"N, 5°23'15"E). Collector: T. Pérez, 23 September 2022.

### Spongia cf. officinalis
(Fig. 2F)

20090617-CEU-ECITP1, ECIMAR campaign, between 3 and 30 m depth, Ceuta, Spain (35°54'25"N, 5°18'00"W). Collector: T. Pérez, 17 June 2009.

20070701-CEU-ECITP2, ECIMAR campaign, between 3 and 30 m depth, Ceuta, Spain (35°52'38"N, 5°18'20"W). Collector: T. Pérez, 1 July 2007.

20070705-CEU-ECITP3, ECIMAR campaign, between 3 and 30 m depth, Ceuta, Spain ((35°52'49"N, 5°18'45"W). Collector: T. Pérez, 5 July 2007.

### Spongia mollissima Schmidt, 1862

20200809-IT-NTP1, *Neuralia* wreck, between 30 and 35 m depth, Porto Cesareo, Italy (40°10'27"N, 17°54'19"E). Collector: T. Pérez, 9 August 2020.

20200809-IT-NTP2, *Neuralia* wreck, between 30 and 35 m depth, Porto Cesareo, Italy (40°10'27"N, 17°54'19"E). Collector: T. Pérez, 9 August 2020.

20200809-IT-NTP3, *Neuralia* wreck, between 30 and 35 m depth, Porto Cesareo, Italy (40°10'27"N, 17°54'19"E). Collector: T. Pérez, 9 August 2020.

20200809-IT-NTP4, *Neuralia* wreck, between 30 and 35 m depth, Porto Cesareo, Italy (40°10'27"N, 17°54'19"E). Collector: T. Pérez, 9 August 2020.

20200809-IT-NTP5, *Neuralia* wreck, between 30 and 35 m depth, Porto Cesareo, Italy (40°10'27"N, 17°54'19"E). Collector: T. Pérez, 9 August 2020.

20200809-IT-NTP6, *Neuralia* wreck, between 30 and 35 m depth, Porto Cesareo, Italy (40°10'27"N, 17°54'19"E). Collector: T. Pérez, 9 August 2020.

20200809-IT-NTP8, *Neuralia* wreck, between 30 and 35 m depth, Porto Cesareo, Italy (40°10'27"N, 17°54'19"E). Collector: T. Pérez, 9 August 2020.

20200810-IT-CTP1, El Camino, photophilic rocky bottoms at 17 m depth, Porto Cesareo, Italy (40°16'12"N, 17°51'15"E). Collector: T. Pérez, 10 August 2020.

20200810-IT-StTP2, La Strea, photophilic rocky bottoms at 17 m depth, Porto Cesareo, Italy (40°14'54"N, 17°53'38"E). Collector: T. Pérez, 10 August 2020.

### Spongia nitens (Schmidt, 1862)

Po.25493, Tel Aviv, Israel (Idan et al. 2018).
Po.25665 Tel Aviv, Israel (Idan et al. 2018).

### Spongia zimocca Schmidt, 1862

Po.25742 Tel Aviv, Israel (Idan et al. 2018)

### Hippospongia communis (Lamarck, 1814)

20200519-MRS-JTP1, Jarre, cave in the semi-dark part at 15 m depth, Marseille, France (43°11'46"N, 5°21'55"E). Collector: T. Pérez, 19 May 2020.

20200522-MRS-FTP4, Fauconnière, semi-dark tunnel, between 5 and 10 m depth, Saint-Cyr Les Lecques, France (43°09'16"N, 5°40'59"E). Collector: T. Pérez, 22 May 2020.

20200522-MRS-FTP6, Fauconnière, semi-dark tunnel, between 5 and 10 m depth, Saint-Cyr Les Lecques, France (43°09'16"N, 5°40'59"E). Collector: T. Pérez, 22 May 2020.

20201225-TUN-Ep8, harvested by a fisherman in a seagrass meadow, Djerba, Tunisia, 25 December 2020.

### Etymology

The species name 'maitasuna' comes from 'maitasun', which means 'love' in Euskara, the Basque Country language. Collected first in a site of the French Pays Basque, the new sponge is thus dedicated to all beloved members of our families, with special thoughts to those recently departed, especially to Marguerite Grenier, century-old grandmother, who passed away while this manuscript was in preparation.

### Diagnosis

Massive sponge with unarmoured and non-lacunose surface. Surface covered with evenly disposed conules, sometimes with traces of epibiosis or foreign bodies. Soft and compressible consistency. Skeleton made of primary, secondary, and tertiary fibres. Primary fibres are anastomosed and denser at the surface, piercing the surface, forming conules, and presenting an open space variable in size between each anastomosed primary fibre. Primary fibres harbouring inclusions such as foreign spicules and/or a pith. Secondary fibres containing a pith inclusion and forming a clearly and regular honeycomb network. Tertiary fibres present both at the surface and within the sponge body.

### Description

The new sponge is massive and irregular, sometimes slightly branched, with small lobes or protuberances (Fig. 2A–D). The sponge can measure ≤25 cm in its largest diameter. Its surface is white, beige, light to dark grey, whereas the internal tissue is usually tawny yellow. No change in colour was detected after fixation in 95% ethanol. The sponge surface is sometimes covered by dirty foreign bodies or epibiotic organisms. The surface is very conulose. Oscules can be grouped or not and are often found on the top of each lobe or protuberance. They are bordered by a thin and rather translucid membrane (Fig. 2A–D). The sponge consistency is rather soft and tearable.
The ectosomal skeleton presents a very thin epidermal skin, which has a star-like appearance, is easily detachable, and is made of collagen fibrils, from 60 to 350 µm in thickness (Fig. 3A). Scattered foreign spicules, debris, or remains of associated invertebrates, such as cirripeds, can be observed.

The choanosomal skeleton presents primary fibres, which are irregular and sinuous, sometimes difficult to observe and to distinguish because they appear anastomosed with secondary fibres, and in some places, they can also divide and join again (Fig. 3B-D). They measure 30-70 µm in diameter and are cored

with inclusions of foreign spicules and/or debris of various abundance. They also include a black or translucid pith. These primary fibres with a pith can be observed near the surface into conule formation and/or across the sponge body (Fig. 3A, C). However, when foreign debris and spicules are widely present, the pith in the primary fibres can be difficult to observe. Near the surface, close to the conule formations, the network of primary fibres is denser in comparison to the rest of the sponge body. The space between two primary fibres, thus between conules, seems to be empty, with very few tertiary fibres and foreign spicules

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Sections of the spongin skeleton of *Spongia maitasuna*. A, general view of the surface and the skeleton, showing the epiderm made of collagen fibrils and the primary fibres with a pith (arrows). B, secondary fibres organized in a regular honeycomb network. C, in the primary fibres, the pith can be black (black arrow) or translucent (white arrow). D, perforated plates close to the primary fibres. E, secondary fibres with a pith (arrows). F, spicules and debris in the ectoderm. G, tertiary fibres. H, detail of a junction between secondary and tertiary fibres (arrow).</figcaption></figure>
and debris. Perforated plates are inconsistently present close to the primary fibres (Fig. 3D). The secondary fibres form a dense network, clearly showing a honeycomb shape of variable mesh size (Fig. 3B), from 70 µm × 120 µm to 300 µm × 500 µm. These fibres measure 20–50 µm in diameter and they can contain an irregular pith, black or translucid (Fig. 3E). Tertiary fibres, ~10 µm in diameter, are found throughout the sponge body (Fig. 3F, G). Sometimes they form a thinner network that also has a clear honeycomb shape with variable mesh size (50–130 µm in diameter). In some places, these fibres appear to be linked to the thicker secondary fibres (Fig. 3H), but in some places they look rather disconnected from the rest of the skeletal architecture.

## Distribution

South European Atlantic Shelf (Spalding et al. 2007), Bay of Biscay, French Pays Basque, and Spanish Cantabria (this work).

## Ecology

*Spongia maitasuna* is found on rocky substrate in shallow water cliffs, boulders, or semi-dark and dark cavities, habitats usually with high wave energy. This sponge presents a patchy distribution and lives in syntopy with *S. lamella*. In some cases, both Spongiidae present contact interactions.

Some specimens may have epibiotic organisms, mostly encrusting sponges and zoanthids. No sign of predation was observed. In the specimens collected between late June and late July, various stages of embryogenesis were observed, including early fertilized eggs, various morula stages, and pre-larvae. No spermatic cysts were observed.

## Taxonomic remarks

The new species is assigned to *Spongia* because its body is not lacunose, as in *Hippospongia* or *Hyattella*, and it does not present an armoured ectosomal skeleton, as in *Coscinoderma* or *Leiosella*. Unlike *Rhopaloeides*, which presents long and simple fascicles of primary fibres, the primary fibres of the new species are simple and sometimes anastomosed. In *Spongia*, the new species should belong to the subgenus *Spongia*, because it presents a simple primary skeleton with regular and polygonal meshes. However, considering the present status of the genus, we prefer not to use this infrageneric, lower rank classification (see the Discussion section).

*Spongia maitasuna* shares with the type species of *Spongia* the very thin collagenic epidermal skin and the fact that primary fibres are never forming fascicles, in other words they are never fused. In *S. officinalis*, the primary fibres are thicker than in the new species, ranging from 50 to 100 µm. The network of secondary fibres is much denser in *S. officinalis*, and the meshes do not have the typical honeycomb shape of the new species. This structure of the secondary skeleton is found in *S. cf. officinalis* from Ceuta, Strait of Gibraltar. The main difference in this case lies in the primary fibres, which are slightly larger than in the new species (70–110 µm), and in the overall growth form (Fig. 2). *Spongia maitasuna* has a morphology similar to *S. nitens*, and both species display a pith within their primary fibres. But *S. nitens* has a denser secondary skeleton, and the secondary fibres are devoid of pith. Moreover, tertiary fibres were present in the two studied specimens of *S. nitens*, but according

to the literature they can be absent (Vacelet 1959, Manconi et al. 2013). They were recorded in all specimens of the new species. Finally, the new species shares with the studied specimen of *S. zimocca* the anastomosed primary fibres and the thickness of the secondary fibres, although they can be reported thicker (50–80 µm) in the literature (Vacelet 1959, Manconi et al. 2013). In the studied specimen of *S. zimocca*, the primary fibres are much thicker, 50–100 µm (100–200 µm in the literature) than in the new species. These fibres also present a pith, although this trait has not been reported previously (Vacelet 1959, Castritsi-Catharios et al. 2011, Manconi et al. 2013), and rare foreign spicules. Finally, the spongin network is overall irregular and denser in *S. zimocca* than in the new species.

## *Spongia lamella* (Schulze, 1879)

(Fig. 4)

### Material examined

20200602-MRS-MTP3, Pharillons de Maire, cliff at 20 m depth, Marseille, France (43°12′27″N, 5°20′17″E). Collector: T. Pérez, 2 June 2020.

20200602-MRS-MTP4, Pharillons de Maire, cliff at 20 m depth, Marseille, France (43°12′27″N, 5°20′17″E). Collector: T. Pérez, 2 June 2020.

20200602-MRS-MTP5, Pharillons de Maire, cliff at 20 m depth, Marseille, France (43°12′27″N, 5°20′17″E). Collector: T. Pérez, 2 June 2020.

20200602-MRS-MTP6, Pharillons de Maire, cliff at 20 m depth, Marseille, France (43°12′27″N, 5°20′17″E). Collector: T. Pérez, 2 June 2020.

20200602-MRS-MTP7, Pharillons de Maire, cliff at 20 m depth, Marseille, France (43°12′27″N, 5°20′17″E). Collector: T. Pérez, 2 June 2020.

20200602-MRS-MTP8, Pharillons de Maire, cliff at 20 m depth, Marseille, France (43°12′27″N, 5°20′17″E). Collector: T. Pérez, 2 June 2020.

20160607-GIJ-JC1, Las Gemelas island, rock substrate at 12 m depth, Gijón, Spain (43°33′22″N, 5°36′31″W). Collector: J. Cristobo, 7 June 2016.

20200624-HEN-BMNC2, Belhara, on reef flat at 22 m depth, Hendaye, France (43°24′00″N, 1°43′03″W). Collector: M. N. de Casamajor, 24 June 2020.

20200814-HEN-AMNC3, Abbadia, on reef flat at 13 m depth, Hendaye, France (43°23′56″N, 1°45′28″W). Collector: M. N. de Casamajor, 14 August 2020.

20210630-CIB-BPC1, Baloo tunnel at 14 m depth, Ciboure, France (43°24′59.0″N, 1°39′52.2″W). Collector: P. Chevaldonné, 30 June 2021.

20210630-CIB-BPC3, Baloo tunnel at 14 m depth, Ciboure, France (43°24′59.0″N, 1°39′52.2″W). Collector: P. Chevaldonné, 30 June 2021.

20210726-CIB-BTP1, Baloo tunnel at 14 m depth, Ciboure, France (43°24′59.0″N, 1°39′52.2″W). Collector: T. Pérez, 26 July 2021.

20210726-CIB-BTP2, Baloo tunnel at 14 m depth, Ciboure, France (43°24′59.0″N, 1°39′52.2″W). Collector: T. Pérez, 26 July 2021.
<figure><img src="image_6.png" /><figcaption>**Figure 4.** *In situ* pictures of *Spongia lamella* in the Northeast Atlantic Ocean and the Mediterranean Sea, A, Baloo tunnel, Ciboure, France. B, C, Mouro Island, Santander, Spain. D, Marseille, France.</figcaption></figure>

20210726-CIB-BTP3, Baloo tunnel at 14 m depth, Ciboure, France (43°24′59.0″N, 1°39′52.2″W). Collector: T. Pérez, 26 July 2021.

20210726-CIB-BTP4, Baloo tunnel at 14 m depth, Ciboure, France (43°24′59.0″N, 1°39′52.2″W). Collector: T. Pérez, 26 July 2021.

20210726-CIB-BTP7, Baloo tunnel at 14 m depth, Ciboure, France (43°24′59.0″N, 1°39′52.2″W). Collector: T. Pérez, 26 July 2021.

20210726-CIB-BTP10, Baloo tunnel at 14 m depth, Ciboure, France (43°24′59.0″N, 1°39′52.2″W). Collector: T. Pérez, 26 July 2021.

20210729-ESP-MTP5, western part of Mouro island, semi-
dark to dark cave, between 3 and 12 m depth, Santander, Spain
(43°28′22.0″N, 3°45′21.7″W). Collector: T. Pérez, 29 July 2021.

20210730-ESP-M2TP2, eastern part of Mouro island, shallow water cliffs and chaos of rocks and crevices, between 3 and 12 m depth, Santander, Spain (43°28′21.1″N, 3°45′18.3″W). Collector: T. Pérez, 30 July 2021.

20210730-ESP-M2TP3, eastern part of Mouro island,
shallow water cliffs and chaos of rocks and crevices, between 3

and 12 m depth, Santander, Spain (43°28′21.1″N, 3°45′18.3″W).
Collector: T. Pérez, 30 July 2021.

20210730-ESP-M2TP12, eastern part of Mouro island,
shallow water cliffs and chaos of rocks and crevices, between 3
and 12 m depth, Santander, Spain (43°28′21.1″N, 3°45′18.3″W).
Collector: T. Pérez, 30 July 2021.

Comparative material examined

Spongia virgultosa (Schmidt, 1868)

20070718-MRS-ECITPS, Jarre, cave in the semi-dark part at 15 m depth, Marseille, France (43°11′46″N, 5°21′55″E). Collector: T. Pérez, 18 July 2007.

Coscinoderma sporadense Voultsiadou-Koukoura, van Soest & Koukouras, 1991

Po.25932, Tel-Aviv, Israel (Idan et al. 2018).

Description

This sponge can be massive and irregular, with lobes or protuber-
ances, fan- or vase-shaped when it becomes very big (Fig. 4A–D).
The largest studied specimens measured 50 cm in their largest diameter, but this dimension can be >1 m in the Mediterranean Sea (Fig. 4D). No true vase- or fan-shaped specimen was observed among our Atlantic specimens. This sponge presents a light to dark grey external colour, and the internal tissue is usually light or tawny yellow. The colour does not change upon fixation in 95% ethanol. The sponge consistency is flexible and hardly tearable. The surface is finely covered with small, evenly distributed conules. Oscules can be grouped in large individuals, or more randomly distributed in smaller ones, always on the outer sides in large individuals or on the top of each lobe or protuberance in smaller ones. They measure 1–3 mm in diameter and are slightly raised, with a small whitish membrane delimiting the opening.

The ectosomal skeleton harbours an epidermal skin, 150–600 µm thick, easily detachable, with a star-like appearance. It is made of a network of abundant foreign spicules and debris (Fig. 5A–C). Small canals can be observed under the surface. In the choanosome, debris and foreign spicules are still abundant at the periphery of canals, and thus throughout the sponge body (Fig. 5D). The primary fibres, 30–120 µm in diameter, are common, simple, irregular, and cored with numerous inclusions of foreign spicules and debris (Fig. 5A, B). Near the surface, they can be ramified. Secondary fibres, 20–40 µm in diameter, without inclusions or pith, form a dense ramified and irregular network (Fig. 5B, E). Tertiary fibres are present or absent. When they exist, their thickness is ≤10 µm in diameter.

### Distribution

Mediterranean Sea and South European Atlantic Shelf (e.g. Noyer and Becerro 2012). This study expands the distribution of *S. lamella* northwards.

### Ecology

In the Mediterranean Sea, *S. lamella* is well known to be distributed mainly along coralligenous cliffs or on deep mesophotic bottoms (Vacelet 1959, Pronzato and Manconi 2008). In the Northeast Atlantic, *S. lamella* has mostly been found in semi-dark and dark cavities, sometimes in crevices found along shallow water cliffs or under rocks. All specimens reported here were found in habitats with very high wave energy, which might explain why no large or fan-shaped individuals were observed. This sponge has a patchy distribution, and it lives in syntopy with *S. maitasuna*. In caves, this species is commonly associated with numerous other sponges, solitary scleractinians, such as *Leptopsammia pruvoti* Lacaze-Duthiers, 1897?, or zoanthids, such as *Parazoanthus axinellae* (Schmidt, 1862). No sign of epibiosis or predation was observed. No reproductive element was recorded in the processed specimens.

### Taxonomic remarks

The 15 specimens examined from the Northeast Atlantic differ slightly in their growth form from *S. lamella* in the Mediterranean Sea, but their skeletons are identical. Openings of the oscula are also twice as large in the Atlantic specimens. They all present in their ectosome a rather thick layer of foreign spicules and debris, which is an unusual structure among *Spongia* that was not observed in *S. maitasuna*. Moreover, the overall network of fibres cannot be confounded with the specific honeycomb-shaped

skeleton of the new species. A rather organized ectosomal skeleton made of foreign spicules and debris is known only in *Coscinoderma* and *Leiosella*, but in these cases, this structure is much thicker and the organization of the choanosome skeleton very different (e.g. intertwined secondary fibres in the Mediterranean *C. sporadense*). Atlantic and Mediterranean specimens of *S. lamella* share the same common simple and irregular primary fibres, with inclusions of foreign spicules and debris, easily observable from the inner part of the sponge to the surface. They remain simple until the formation of conules, whereas in other *Spongia* species they become anastomosed at this level. The size of the primary fibres appears a little more variable in the Atlantic specimens (30–120 µm in diameter) than in the Mediterranean specimens (50–100 µm in diameter). Foreign spicules and debris seem to be more abundant in Atlantic specimens. In comparison, the primary fibres of *S. virgultosa*, which has an unusual encrusting papillate growth form for *Spongia*, are also full of mineral debris, but they are rare and significantly thinner (40–50 µm in diameter) than in *S. lamella*.

Therefore, the slight morphological differences recorded between Atlantic and Mediterranean specimens (growth form, abundance of debris, and variation in the size of primary fibres) do not seem to us to be sufficient to consider the Atlantic specimens as a different species and are thus interpreted as variations attributable to the different environmental contexts.

### DNA analysis of studied species

GenBank accession numbers for all sequences generated or downloaded in this study are presented in Table 1. For the 28S (C2–D2 fragment) and COI (dgLCO1490–dgHCO2198 fragment) molecular markers, we obtained, respectively, 18 and 3 sequences of *S. maitasuna*, 21 and 14 sequences of *S. lamella*, 14 and 5 sequences for *S. officinalis*, 8 and 2 sequences of *S. mollissima*, 4 and 3 sequences of *H. communis*, 1 and 1 sequence of *S. virgultosa*, and only 1 sequence of *S. zimocca* (28S) and 1 sequence of *C. sporadense* (28S).

Molecular markers allowed us to build highly congruent trees, although the phylogenetic reconstruction with 28S gave a better resolution (Fig. 6) than COI (Supporting Information, Fig. S1). Both methods (NJ and ML) of phylogenetic reconstruction using 28S produced the same topology: 70 Spongiidae specimens divided in two, with only one sequence of *C. sporadense* on one side and 69 specimens of *Spongia* and *Hippospongia* on the other side (Fig. 6). This set of specimens can be divided into three different groups. The first group is well supported by both methods of phylogenetic constructions (NJ and ML). It is composed by the proposed new species *S. maitasuna*, individuals from the two sampled localities presenting the same sequence of 28S, and by a well-supported group made of one sequence of *S. zimocca*, two downloaded sequences of *S. nitens*, and three sequences obtained in the present study of *S. cf. officinalis* from Ceuta. *Spongia maitasuna* thus appears closely related to this group, with the p-distance being very low between the proposed new species and *S. zimocca* (1.05%, four substitutions), *S. nitens*, and *S. cf. officinalis* (0.79%, the substitutions). The second group is weakly supported, whatever the method of construction. It includes *S. virgultosa* and a well-supported group of all *S. lamella* from the Mediterranean and Northeast Atlantic. With only one exception, the sequenced individuals are grouped by geographical
<figure><img src="image_7.png" /><figcaption>**Figure 5.** Sections of the spongin skeleton of *Spongia lamella*. A, general view of the surface and the ectosome, showing the crust made of foreign spicules and debris at the surface, and the simple primary fibres. B, primary fibres including foreign spicules and debris, and secondary fibres forming an irregular and dense network. C, tangential section of the surface crust. D, foreign spicules and debris around canals. E, details of the secondary fibres.</figcaption></figure>
origin. The Mediterranean sequence (20200602-MRS-MTP8) mixed with the Atlantic presents a p-distance of 0.26% (one substitution) from the rest of the Mediterranean *S. lamella*. In comparison, *S. virgultosa* presents a p-distance of 13.4% (51 substitutions) from all *S. lamella*. The third group is composed by a weakly supported set of sponges, including all *S. mollissima*, *S. officinalis*, and *H. communis*. The genetic distances between these three species are low, with a minimal p-distance of 0.26% between *S. mollissima* and *H. communis* from Tunisia, and 0.79–1.57% (three to six substitutions) between *S. mollissima* and *S. officinalis* or *H. communis*.

A phylogenetic tree was made by concatenating all sequences available for both 28S and CO1 (Fig. 7), with the exception of two samples of *S. zimocca* and *H. communis* for which only 28S could be amplified. The topology of the concatenated tree is congruent with the 28S tree. Both phylogenetic reconstruction methods used here (BI and ML) presented the same pattern. Thirty specimens are grouped into two different groups representing the Spongiidae family. Group 1 is composed by one specimen of the genus *Coscinoderma*, whereas group 2 is composed by 29 specimens of *Spongia* and *Hippospongia*. The proposed new species, *S. maitasuna*, forms a well-supported group in both phylogenetic analyses and still separate but relatively close to *S. zimocca*, *S. nitens*, and *S. cf. officinalis* from Ceuta. All *S. lamella* form a well-supported group, which still appears separate by geographical origin. In this representation, *S. officinalis* and *H. communis* appear mixed in a subgroup also containing *S. mollissima*.

## Chemical fingerprints of the studied species

We evaluated whether Spongiidae could be organized and grouped based on their chemical fingerprints and whether this chemical-based classification was congruent with the molecular phylogeny. Using the first aligned feature list, containing 915 chemical signals, we performed hierarchical clustering analysis to group sponge extracts according to their chemical similarities.

The dendrogram (Fig. 8) shows distinct groups of extracts all attributed to three distinct groups of species. The first group (group 1) includes all specimens of the proposed new species, *S. maitasuna*, in a subgroup, and *S. nitens* and *S. zimocca* in another subgroup. No sample of *S. cf. officinalis* from Ceuta (Strait of Gibraltar) was available for this analysis. The second group (group 2) includes all specimens of *S. lamella*, still separated into two subgroups according to their geographical origin. The Mediterranean exception (20200602-MRS-MTP8) that was grouping in the 28S phylogenetic with Atlantic specimens is grouped here with all other Mediterranean samples of the same species. The third main group (group 3) includes *S. officinalis*, *S. mollissima*, and *H. communis*, but in the case of this metabolomic analysis, all species formed well-separated subgroups. Thus, although its typology cannot be compared with those of the phylogenetic trees, the groupings presented in the dendrogram resulting from the metabolomic analysis are well congruent with those permitted by the morphological and genetic analyses.

The heatmap (Fig. 9) complements these results by depicting the whole set of chemical features that participated in the sample classification. This representation illustrates clearly that extracts from *S. maitasuna* contain a distinct set of chemical features

compared with other Spongiidae samples. Next, we assessed the distribution of features within the three identified groups of species to evaluate the chemical diversity and number of unique chemical signals characterizing each species.

To that end, a second aligned feature list was generated from pooled extracts from each identified taxonomic unit and contained 240 features. Those features represent the most abundant and redundant chemical signals in each taxonomic unit. Venn diagrams were constructed to depict the number of chemical features shared between species as opposed to those that were detected in one or two species (Fig. 10).

The lowest number of features was detected for group 1, for which 45 chemical signals were shared between all three species (*S. maitasuna*, *S. nitens*, and *S. zimocca*). A total of 16 unique signals were detected for *S. maitasuna*, illustrating a much higher chemical diversity and uniqueness in extracts than in *S. nitens* (2) and *S. zimocca* (1). At this stage, none of these features has been identified formally, because the purpose of this analysis was not to assign the associated molecular structure to each feature. However, it is certain that none of the well-known and referenced compounds from groups 2 and 3 (as shown later) were detected among the signals.

The highest number of features was associated with group 2, represented by *S. lamella* from different locations (Mediterranean and Atlantic). There were between 80% and 85% similarities of detected signals (111 in total) in all extracts. Among the detected chemical features, nitenin is one of the most abundant and well-known furanoterpenoids from this species (Noyer et al. 2011). Extracts from the Mediterranean specimens present a greater number of unique detected signals (13) than those from the Atlantic specimens (5 and 4).

Group 3, represented by *S. officinalis*, *S. mollissima*, and *H. communis*, also contains a relatively high number of detected features compared with the proposed new species. Extracts from these three species have 98 chemical signals in common. Some other well-known and characteristic furanoterpenoid compounds could be identified among these signals, such as furospongin-1, furospongin-4, and demethylfurospongin-4 (Bauvais et al. 2017). Extracts from *S. mollissima* have the highest number of unique detected signals (23), compared with *S. officinalis* (3) and *H. communis* (9). Among all Spongiidae, *S. lamella* is the species presenting the highest chemical diversity, with a total of 170 chemical signals (including its unique chemical signals and others shared with other Spongiidae). In comparison, extracts from the proposed new species contain 151 chemical features, and >10% of them are unique and thus have unknown molecular structures. After undergoing purification and determination of their chemical/molecular structure, they have the potential to become chemotaxonomic markers.

Therefore, the hierarchical classification of species, based on their chemical fingerprints, is congruent with the phylogenetic trees previously presented. Each species can be differentiated by its chemical fingerprints associated with a unique set of chemical features that might serve as chemotaxonomic markers. These putative new phenotypic traits would need to be annotated or identified after isolation, purification, and structural analysis by means of additional spectroscopic (e.g. nuclear magnetic resonance) and spectrometric methods.
<figure><img src="image_8.png" /><figcaption>**Figure 6.** Phylogenetic reconstruction of Northeast Atlantic and Mediterranean Spongiidae using sequences of the C2–D2 region of 28S, and both the maximum-likelihood (ML) and neighbour-joining (NJ) methods. Bootstrap values are given for both analyses (1000 replicates). The NJ values are in parentheses. Each voucher code is associated with a species name. The ML bootstrap values are given only if >500.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Figure 7.** Phylogenetic reconstruction of Northeast Atlantic and Mediterranean Spongiidae concatenating the C2–D2 region of 28S and the Folmer fragment of *COI* using both Bayesian inference (BI) and maximum likelihood (ML) methods. Posterior probabilities and bootstraps are given for both analyses (1000 replicates). The ML values (as percentages) are in parentheses. \*Sequences obtained only in 28S in this study.</figcaption></figure>

DISCUSSION

The two Northeast Atlantic *Spongia* studied here were highly abun-
dant in the sampling sites. Only four dives, in France and Spain,

allowed us to record several individuals per square metre. It is very
likely that these two species are much more widely distributed, in
relatively shaded rocky habitats often dominated by sponges (La
<figure><img src="image_10.png" /><figcaption>**Figure 8.** Metabolomic analysis of Northeast Atlantic and Mediterranean Spongiidae. Dendrogram from hierarchical clustering analysis using Euclidean distance calculations under the Ward algorithm. *Individuals for which no genetic data could be obtained.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Figure 9.** Metabolomic analysis of Northeast Atlantic and Mediterranean Spongiidae. Heatmap from hierarchical clustering analysis using Euclidean distance calculations under the Ward algorithm, grouping sponge extracts based on their chemical similarities. Chemical features or signals are depicted as horizontal lines, with their colour indicating the normalized level of detection intensity on a scale from -2 to +2, from dark blue to red. The heatmap shows that each species is characterized by a unique set of chemical features (dark red), collectively defining their chemical signature.</figcaption></figure>

Rivière *et al.* 2022), around the Iberian Peninsula, where several previous reports of *S. officinalis* (Lopes and Boury-Esnault 1981, Carballo and Garcia-Gómez 1994, Ramirez 2002; J. Cristobo and J. Xavier pers. comm.) might correspond to *S. maitasuna* sp. nov.

However, we can wonder how far north along the French coast Spongiidae could be found, because these sponges are typically more prevalent in warm-temperate to tropical waters (de Voogd *et al.* 2023). Along the Atlantic coast, the nearest area that has seen the largest effort to study sponge biodiversity is French Brittany (Topsent 1890, 1892, 1928, Lévi 1950, Lévi and Vacelet 1958, Cabioch 1968, Descatoire 1969), and no members of Spongiidae have ever been recorded there.

The relative abundance of these two *Spongia* could have motivated their exploitation, but there is no historical source

reporting local bath sponge fisheries. We believe that even if there were temporary occurrences of such a practice around the Iberian Peninsula by foreign fishermen, sponge taxonomists would have become aware of it eventually. The absence of very large individuals cannot be attributed to fishing pressure, as is the case for many other marine resources suffering overexploitation (Pauly 2008). Thus, the slight morphological differences between *S. lamella* from the Mediterranean Sea and the Atlantic Ocean, and especially the absence of the so-called elephant ear shape in our Atlantic sampling sites, is likely to be linked to contrasting environmental conditions. Although metabolomics grouped Mediterranean and Atlantic individuals separately, we were able to demonstrate that all individuals grouped together and shared a very high number of chemical features. As a result,
<figure><img src="image_12.png" /><figcaption>**Figure 10.** Venn diagrams representing the distribution of chemical features between species of the three main hierarchical groups of Spongiidae of the Mediterranean Sea and Northeast Atlantic Ocean. Black numbers indicate a unique set of features for each species. White numbers represent shared chemical signals between species. The number of features shared between all species of a given group are in white bold.</figcaption></figure>

this alternative taxonomic approach did not lead us to consider
the existence of two different species.

No single ideal molecular marker for all sponges exists, and cases
of low genetic distances between species exhibiting obviously di-
vergent phenotypical traits are common (Yang et al. 2017). This is
the reason why sponge integrative taxonomy, using multiple and
complementary sources of data, has developed well (Cárdenas et
al. 2012). The congruence of all datasets used in this study led us
to propose S. maitasuna as a Spongiidae well separated from all
Mediterranean and Northeast Atlantic species. None of the sampled
individuals in the French Pays Basque or Spanish Cantabria could be
assigned to S. officinalis because of their specific morphological, gen-
etic, and chemical traits, hence all past records of S. officinalis in the
Northeast Atlantic (Lopes and Boury-Esnault 1981, Carballo and

Garcia-Gómez 1994, Ramirez 2002, Xavier et al. 2010, J. Cristobo
pers. comm.) are likely to correspond to incorrect identifications.
Among the species closest to S. maitasuna, we could not determine
the exact status of S. cf. officinalis from Ceuta, Strait of Gibraltar,
which was previously reported to be genetically different from
S. officinalis and S. mollissima (Dailianis et al. 2011). The new species
presents a rather low genetic distance from this sponge from Ceuta
found in a transitional area between the Mediterranean Sea and the
Atlantic Ocean, but in the same range of values as that observed
between two Mediterranean species currently belonging to distinct
genera, S. officinalis and H. communis. This Spongia from Ceuta is
certainly also a new species that deserves a description based on
the size of its primary fibres and the shape of its secondary skel-
eton. However, because we miss the proper material to apply our
entire integrative approach (i.e. frozen samples for metabolomics), we must postpone this description to another work that will revise the status of all Mediterranean *Spongia* and *H. communis*.

Small genetic distances between sponge species (e.g. see Boury-Esnault *et al.* 2013) have often been observed using CO1 as molecular marker. This is the case for the two Homoscleromorpha *Oscarella lobularis* (Schmidt, 1862) and *Oscarella tuberculata* (Schmidt, 1868), which present a p-distance of 0.02% (Ruiz *et al.* 2022). In that case, cytology and metabolomics came to the rescue of a barcoding approach that is frequently reaching its limits in sponge taxonomy. However, our study is the first report of such low genetic distances between genera (here, *Hippospongia* and *Spongia*) having distinct morphological traits, and consequently, *Spongia* clearly appears non-monophyletic (Figs 6, 7) as current taxonomy stands. For the time being, the most parsimonious assignment of the new species, considering its morphological traits, was to *Spongia*. Nevertheless, we believe that the morphological traits separating *Spongia* and *Hippospongia*, a lacunous ectosome and a certain rarity of primary fibres in the latter genus, would deserve to be reconsidered in an extensive revision of the Spongiidae. This revision will require a good molecular reference for *Spongia* and the whole family, because the sequence of Schulze's neotype recently obtained by Agne *et al.* (2022) cannot be considered. Indeed, as mentioned earlier, its origin is too uncertain, and moreover, the sequence is, at present, so inaccurate (11 ambiguities) that it introduces too much noise for the building of solid phylogenetic trees (unpublished data by Grenier M.). Given that *Linnaeus* (1759)'s specimen has disappeared, such a revision will also require examination of some of Schmidt's specimens of the former *S. adriatica*, and ideally, obtain their sequences to position them in our phylogenetic trees. Considering the tremendous history of the Spongiidae, it is likely that several new genera will have to be created or resurrected. For example, *H. communis* (Lamarck, 1814), went through different generic names: *Spongia*, *Euspongia*, and *Hippospongia*. Considering the distance between *S. lamella* or *S. virgultosa* and most of the remaining Mediterranean *Spongia* species, transfers to other genera are likely to be needed. Vacelet (1959) already debated about the epidermal crust found in *S. lamella*, which is highly unusual for *Spongia*. Unfortunately, we could not check the occurrence of this crust in *S. agaricina*, and it has not been documented in the work that formally separated the two elephant ear bath sponges (Pronzato and Manconi 2008). The revision of the Spongiidae will also have to include many other representatives of the main genera that are debated here, with an accurate analysis of the three subgenera currently acknowledged among *Spongia*. Finally, this revision cannot be completed without a major clean-up of the sequences currently available in databases. Only a very low percentage of GenBank sequences are reliable (32% for the Mediterranean Sea). Several sequences correspond to fast, preliminary, inaccurate identifications (e.g. 38 of 121 CO1 or 28S sequences of Spongiidae sp. or *Spongia* sp.), and sometimes simply to incorrect identifications. For instance, GenBank presently hosts a sequence of the Mediterranean *S. officinalis* sampled from India (sequence available under the voucher code NIO:GoM-D but unpublished) and of *S. zimocca* from Papua New Guinea (Thacker *et al.* 2013).

This study, applying an integrative taxonomic approach to the Spongiidae (i.e. a precise analysis of the spongin skeleton, the combination of two molecular markers, and metabolomics) for

first time, has shown that it is possible to make systematic decisions where each trait taken separately displays only subtle differences. Given that Spongiidae are the first sponges described 4000 years ago by Greek philosophers (Voultsiadou 2007, Pronzato and Manconi 2008) and a sponge family that remains exploited by fishermen throughout the world, a significant effort needs to be made to propose new taxonomic references. Given the serious threats to these species, which frequently suffer disease outbreaks and mass mortality events (Pérez and Vacelet 2014, Grenier et al. 2023), this work should be carried out before some of these species become extinct because of climate change.

## SUPPLEMENTARY DATA

Supplementary data is available at *Zoological Journal of the Linnean Society* online.

## ACKNOWLEDGEMENTS

The authors acknowledge financial supports by the ANRT for Marie GRENIER PhD fellowship (CIFRE n°2019/1375). This work was performed in the framework of the French-Brazilian Associated International Laboratory 'LIA MARRIO' funded by the CNRS, and the Biodiversa+ SpongeBiodiv project funded by ANR-22-EBIP-0004-06. CNRS INEE BIODIVMEX program also contributed to the funding of a field trip in Italy. Authors are grateful to Frine Cardone, Stazione Zoologica Anton Dohrn (Italy), Javier Cristobo and Pilar Rios, Instituto Espanol de Oceanografia (Spain), Tal Idan and Sigal Shefer, Tel-Aviv University, Marie-Noëlle de Casamajor, IFREMER (France), Dorian Guillemain, Institut Pytheas (France), Sacha Molinari, IMBE (France), the Punta Campanella Diving Club, Tech-Océan centre de plongée de Ciboure (France), Patrick Astruch (GIS Posidonie, France), Christophe Lejeusne (IMBE, France) and Josiane Popovsky (CAPENA, France) for their involvement in the field works. We also thank Christian Marschal IMBE (France) for his help preparing part of the skeleton sections, Cécile Chemin, Caroline Rocher and the SCBM (IMBE, France) for their help during the molecular biology works in the laboratory. Finally, we are also much obliged to all the participants of the last sponge conference (Leiden, October 2022) who enthusiastically received the preliminary presentation of this work, and in the same way, we are definitely grateful to Nicole Boury-Esnault and Jean Vacelet for the great source of inspiration they represent, and for their always positive criticisms on our work.

## AUTHOR CONTRIBUTION

MG and TP designed the research experiment. MG, TP and PC sampled the specimens. MG performed all the laboratory analysis. MG, PC, CS, NC and TP analysed the data. MG and TP wrote the species descriptions. All authors contributed to the manuscript and approved its revised and final version.

## DATA AVAILABILITY STATEMENT

All data generated in this study are available upon request.

## CONFLICT OF INTEREST

The authors declare no conflict of interest.

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