Journal of the Marine Poorly known sponges in the Mediterranean Biological Association of the United Kingdom with the detection of some taxonomic inconsistencies cambridge.org/mbi Julio A. Díaz1,4 , Sergio Ramírez-Amaro1,2, Francesc Ordines1 , Paco Cárdenas3 , Pere Ferriol4, Bàrbara Terrasa2 and Enric Massutí1 Original Article 1Instituto Español de Oceanografía, Centre Oceanogràfic de les Balears, Moll de Ponent s/n, 07015 Palma, Spain; 2Laboratori de Genètica, Biology Department, University of the Balearic Islands, Carretera de Valldemossa km 7.5, Cite this article: Díaz JA, Ramírez-Amaro S, 07122 Palma de Mallorca, Spain; 3Pharmacognosy, Department of Medicinal Chemistry, BioMedical Centre, Ordines F, Cárdenas P, Ferriol P, Terrasa B, 4 Massutí E (2020). Poorly known sponges in the Husargatan 3. Uppsala University, 751 23 Uppsala, Sweden and Interdisciplinary Ecology Group, Biology Mediterranean with the detection of some Department, University of the Balearic Islands, Carretera de Valldemossa km 7.5, 07122 Palma de Mallorca, Spain taxonomic inconsistencies. Journal of the Marine Biological Association of the United Abstract Kingdom 100, 1247–1260. https://doi.org/ 10.1017/S0025315420001071 The poorly known sponge species Axinella vellerea (Topsent, 1904), Acarnus levii (Vacelet, 1960) and Haliclona poecillastroides (Vacelet, 1969) are reported from bottom-trawl samples Received: 16 July 2020 off the Balearic Islands, Western Mediterranean. A re-description is provided for all three spe- Revised: 7 September 2020 Accepted: 21 October 2020 cies and the Folmer fragment of cytochrome oxidase subunit I (COI) obtained for A. levii and First published online: 10 December 2020 H. poecillastroides. This is the second report of A. vellerea in the Mediterranean, the first time that A. levii is reported outside Corsica and the first time that H. poecillastroides is documented Key words: outside the Gulf of Lion, France. The systematic allocation of A. levii and H. poecillastroides is Acarnidae; Balearic Islands; barcoding; Mediterranean Sea; new records; Petrosiidae; discussed based on a COI phylogenetic analysis and morphology. The poorly understood phyl- Porifera ogeny of the Haplosclerida does not permit us to find a proper allocation for H. poecillastroides, although its current position in the genus Haliclona or the family Chalinidae is not defensible. Author for correspondence: On the other hand, A. levii currently fits best in the family Microcionidae, and seems related to Julio A. Díaz, E-mail: [email protected] some Clathria species with mixed features between Clathria and Acarnus. Considering that the species of the genus Acarnus shares a strong synapomorphy (the possession of Cladotylotes), it is plausible for all Acarnus species to be Microcionids. We conclude that H. poecillastroides needs to be reallocated to a new genus: Xestospongia poecillastroides comb. nov. (Petrosiidae). However, a reallocation of A. levii is not advisable for the moment, as this would imply major systematic changes such as the reallocation of the whole genus Acarnus to Microcionidae, and the redescription of Microcionidae and Acarnidae. Introduction The Mediterranean is considered a hotspot of sponge diversity, with about 680 species reported including ∼265 endemic species (Pansini & Longo, 2003; Voultsiadou, 2009;Van Soest et al., 2012; Xavier & Van Soest, 2012). Moreover, new species and new geographic records are periodically reported in this sea (e.g. Vacelet et al., 2007; Bertolino et al., 2013, 2015; Sitjà & Maldonado, 2014; Corriero et al., 2015; Melis et al., 2016). Knowledge on Mediterranean sponge fauna comes mainly from species living in shallow habitats accessible through scuba diving. Less is known about the circalittoral and bathyal domains (Danovaro et al., 2010), although these deep ecosystems may harbour important communities of filter feeding animals, such as corals or sponges, that can also function as habitat engineers (Maldonado et al., 2015). Improving the scientific knowledge of this fauna contributes to the management of these fragile ecosystems and their protection. The Balearic Promontory, in the Western Mediterranean, is an area of high ecological interest because of the high oligotrophy of its waters, a consequence of the lack of rivers and upwelling zones, the scarcity of rain and the karstic nature of its rocks (Estrada, 1996; Acosta et al., 2002). Moreover, the oceanographic fronts and currents of the Balearic Archipelago and between the Islands and the Iberian Peninsula may act as genetic barriers to the dispersal of sponges and other benthic organisms, contributing to its isolation © Marine Biological Association of the United Kingdom 2020 (Duran et al., 2004; Galarza et al., 2009; Pérez-Portela et al., 2015; Pascual et al., 2016). The first taxonomic studies of the sponge fauna in the Balearic Islands date back to the end of the 19th and the beginning of the 20th centuries and focus on samples collected from Maó in Menorca, Palma Bay in Mallorca and Cabrera Island (Lackschewitz, 1886; Ferrer-Hernández, 1916, 1921). Then, there is a gap in the literature until the 1980s, when sev- eral authors relaunched sponge research, mainly from shallow waters (Bibiloni & Gili, 1892; Bibiloni et al., 1989; Bibiloni, 1990; Vacelet & Uriz, 1991; Martí et al., 2004; Gràcia et al., 2005, 2014; Guzzetti et al., 2019), although with some exceptions from deeper habitats (Uriz & Rosell, 1990; Maldonado et al., 2015). Recently, Santín et al.(2018) have analysed the sponges of the Menorca Channel from a community-level perspective. Within the ecosystem approach to fisheries, the MEDITS surveys programme provides data and samples of benthic and demersal species on the sedimentary bottoms of the continental Downloaded from https://www.cambridge.org/core. University of Athens, on 27 Sep 2021 at 09:23:06, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0025315420001071 1248 Julio A. Díaz et al. shelf and upper slopes along the northern Mediterranean (Spedicato et al., 2019). The aim of this work is to re-describe three poorly known deep-sea sponge species, recorded for the first time from the circalittoral soft bottoms of the Balearic Promontory using an integrative taxonomy approach (combining morphological descriptions and molecular sequences). This is taken as an opportunity to revisit and question the phylogenetic relationships of these species. Material and methods Samples The specimens were collected during the MEDITS surveys devel- oped off the Balearic Islands (western Mediterranean), using the experimental bottom trawl gear GOC-73 (Bertrand et al., 2002) Fig. 1. Map of the studied area showing the stations (numbers) where sponges were (see Figure 1 and Table 1 for sampling station details). collected. Coloured symbols represent the distribution of Axinella vellerea, Haliclona Additional sampling with a Jennings’ type beam trawl (Reiss poecillastroides and Acarnus levii. The small map shows the previous records of the species in the Mediterranean for A. vellerea (Sitjà & Maldonado, 2014; Sitjà et al., et al., 2006) has also been carried out during these surveys in 2019) and A. levii (Vacelet, 1960, 1961), and previous documented records for H. poe- order to improve the sampling of benthic species. Sponges ana- cillastroides (Vacelet, 1969). lysed in this study were collected with both sampling methods during the MEDITS surveys carried out in 2016, 2017 and 2018 around Mallorca and Menorca. μ μ μ 1.75 MgCl2 0.5 l of each primer, 0.5 l BSA, 0.05 l TAQ and Once on deck, sponges were separated from the rest of the 1 μl DNA). The PCR thermal profile applied was: initial stage catch and photographed with a Nikon DSLR D300 digital camera of 94°C for 5 min, then 37 cycles at 94°C for 15 s, 46°C for 15 s on a graph chart. Whole samples were preserved in absolute etha- and 72°C for 15 s, followed by a final extension at 72°C for 7 nol (EtOH). Sponge specimens were deposited in the Marine min. PCR amplification of A. vellerea did not work, including Fauna Collection (http://www.ma.ieo.es/cfm/; CFM-IEOMA) another set of primers (LCO and Tetract-minibarR1) designed based at the Centro Oceanográfico de Málaga (Instituto Español to amplify the COI mini-barcode (the first 130 bp of the Folmer de Oceanografía) with the following identification reference num- fragment) (Cárdenas & Moore, 2019). PCR products were puri- – bers: CFM-IEOMA-6390 6399. fied using the QIAquickR PCR Purification Kit (QIAGEN). Both heavy and light strands were sequenced on an ABI 3130 Morphological descriptions sequencer using the ABI Prism Terminator BigDyeR Terminator Cycle Sequencing Reaction Kit (Applied Biosystems). External morphology, colour and texture were annotated prior to Sequences were imported into BioEdit 7.0.5.2. (Hall, 1999) and the sample conservation. Spicules preparations and histological checked for quality and accuracy with nucleotide base assignment. sections were made according to the standard methods described Multiple sequence alignments (MSA) were obtained with by Hooper (2003). ClustalW (Thompson et al., 1994). The DNA sequences obtained Spicules were observed with a Nikon S-Ke optical microscope were deposited in the GenBank database (http://www.ncbi.nlm. and photographed with a CMOS digital camera. Images were pro- nih.gov/genbank/) under the following accession numbers: cessed with the Fiji software (Schindelin et al., 2012). For each sam- MN508968, MN508969 and MN508967. Sequences were vali- ple, 30 spicules per spicule class or category were counted. Spicular dated using the BLAST function from the GenBank database sizes are provided as minimum-mean-maximum in chord length (Altschul et al., 1990). The sequences were also used to recon- and minimum-mean-maximum in width and expressed in microns struct the phylogenetic relationships between species, through a (μm). Thick sections of both tangential-surface and transverse- phylogenetic tree based on Bayesian inference (BI) and surface sections were made with a scalpel and, when necessary, Maximum likelihood (ML). To build our alignments, we made dehydrated with alcohol and cleared with xylene.