Org Divers Evol (2014) 14:21–29 DOI 10.1007/s13127-013-0156-0

ORIGINAL ARTICLE

Integrative taxonomy of four Clathrina species of the Adriatic Sea, with the first formal description of Clathrina rubra Sarà, 1958

Mirna Imešek & Bruna Pleše & Martin Pfannkuchen & Jelena Godrijan & Daniela Marić Pfannkuchen & Michelle Klautau & Helena Ćetković

Received: 18 January 2013 /Accepted: 4 September 2013 /Published online: 29 September 2013 # Gesellschaft für Biologische Systematik 2013

Abstract The taxonomy of the class Calcarea has long relied Introduction on histological and morphological characters, with a few molecular studies done. In character-poor genera, such as The class Calcarea Bowerbank, 1864, comprises over 650 Clathrina, an integrative taxonomy, associating morphologi- exclusively marine species (van Soest et al. 2013), currently cal and molecular tools, greatly aids in the species identifica- divided into two subclasses: Calcinea Bidder, 1898, and tion. In this study, we describe four Clathrina species from the Calcaronea Bidder, 1898. According to morphological, onto- northern Adriatic Sea using morphology and DNA sequences genetic and molecular data, both subclasses represent mono- together with observations on their ecology. Clathrina phyletic groups, despite of their wide variability of forms clathrus and Clathrina blanca have been previously reported (Borojević et al. 1990, 2000; Dorhmann et al. 2006; Manuel for the Adriatic Sea; however, it is the first time that a DNA et al. 2003, 2004; Voigt et al. 2012). Unique features, such as a sequence of C. blanca has been obtained. Clathrina cf. skeleton made of calcium carbonate spicules, together with all hondurensis, a Caribbean species, is being reported in the five types of aquiferous systems—asconoid, syconoid, sole- Adriatic Sea for the first time, and we present the first descrip- noid, sylleibid and leuconoid—clearly distinguish calcareous tion and DNA sequence of Clathrina rubra,aredspecies sponges from the other poriferan classes (Cavalcanti and originally mentioned from Naples. Klautau 2011). Like many other groups of calcareans, Clathrina Gray, 1867, is a very character-poor genus. Keywords Porifera . Calcareous sponges . Clathrina . Recently, a new phylogenetic proposal of Clathrinida con- C. rubra . C. blanca . Northern Adriatic firmed that Guancha is not a valid genus (Klautau et al. 2013). In that work, new diagnoses were proposed for clades of species previously called Clathrina and Guancha.Inthe present work, we are considering the new diagnosis of š * : š : Ć ć * M. Ime ek ( ) B. Ple e H. etkovi ( ) Clathrina, which was redefined and is now characterised by Division for Molecular Biology, Ruđer Bošković Institute, Bijenička cesta 54, 10002 Zagreb, Croatia the presence of a clathroid cormus, asconoid aquiferous system e-mail: [email protected] and skeleton composed only of triactines to which diactines and e-mail: [email protected] tripods may be added (Klautau et al. 2013). To perform good taxonomical studies, an integrative approach is very important M. Pfannkuchen : J. Godrijan : D. M. Pfannkuchen Center for Marine Research, Ruđer Bošković Institute, Giordano to identify Clathrina species, which is being achieved with the Paliaga 5, 52210 Rovinj, Croatia combination of morphology and DNA sequences (Fig. 1). Some of the first sponge studies were performed in the M. Klautau Adriatic Sea (e.g. Haeckel 1870, 1872; Schmidt 1862, 1864). Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Av. Carlos Chagas Filho 373, Cidade However, species identifications are frequently doubtful, as Universitária, 21941-902 Rio de Janeiro, RJ, Brazil descriptions were poor or simply non-existent. Although the 22 M. Imešek et al.

Fig. 1 Bayesian majority-rule consensus tree based on concatenated ITS1-5.8S-ITS2 and partial 28S rDNA sequences. Bayesian posterior probabilities (when >0.80) are given above the branches and bootstrap values for maximum likelihood (ML) are given below the branches (when >70). Species obtained in this study are marked with bold

rocky coast and the deeper sandy ground of the eastern part of Materials and methods the northern Adriatic harbour more than 150 known sponge species (Brümmer et al. 2004; Gugel et al. 2006;Mülleretal. Samples 1984), only recently has a first comprehensive study of this diversity been published (Bakran-Petricioli et al. 2012) Sponge samples from the Croatian part of the northern and it covered only the classes Demospongiae and Adriatic coast attached to Cystoseira crinita Duby, 1830, Homoscleromorpha. Several calcarean species are known so specimens or on the ceilings of rocky caverns were collected far in the area (see Pansini and Longo 2008), but some by SCUBA diving and snorkelling. The whole specimens identifications must be confirmed and complete descriptions were sampled and preserved as described in Pfannkuchen must be made. Therefore, the aim of this study was to identify et al. (2010). Tissue parts designated for DNA analysis were and describe species of Clathrina from the eastern coast of the fixed in ethanol. Morphological preparations were performed northern Adriatic Sea, using morphological and molecular following standard procedures (Klautau and Valentine 2003; characters. Wörheide and Hooper 1999). Spicules were examined using Integrative taxonomy of four Clathrina species of the Adriatic 23 an Axiovert 200 microscope (Zeiss GmbH, Germany). Length Aligned sequences were imported into MEGA version 5 and thickness of 33 spicules of each category were obtained (Tamura et al. 2011), where data sets were analysed by max- using Axio Vision software V 4.8.1.0 (Zeiss GmbH, imum likelihood (ML). The model for ML analysis was Germany). The same specimen was used for both morpholog- selected using Modeltest 3.7 and the Akaike information ical and molecular analyses. criterion (AIC) (Posada and Crandall 1998), which indicated GTR+G+I (general time reversal gamma distributed with DNA isolation, amplification, cloning and sequencing invariant sites). Bayesian MCMC analysis was performed in MrBayes v. 3.1.2. (Huelsenbeck and Ronquist 2001). Total DNA was extracted from 0.3 g of tissue, using a G-spin Bootstrap tests were performed with 1,000 replicates. A phy- Genomic DNA Extraction Kit (Intron), following the manu- logenetic tree was made for 28S and ITS sequences in a facturer’s protocol. The ITS1-5.8S-ITS2 rDNA region was concatenated analysis. amplified by polymerase chain reaction (PCR) using primers 18SFow (5′-TCATTTAGAGGAAGTAAAAGTCG-3′) plus Accession numbers 5.8SRev (5′-GCGTTCAAAGACTCGATGATTC-3′)(Lôbo- Hajdu et al. 2004)andITS2F5′-CGGCTCGTGCGTCG Sequences reported in this study were submitted to GenBank, ATGAAGAAC-3′ plus ITS2R 5′-CGCCGTTACTGGG with the following accession numbers: Clathrina rubra GGAATCCCTGTTG-3′ (Harcet et al. 2010). Partial 28S (PMR-14306): 28S – KC479082; ITS1-5.8S-ITS2 – rDNA gene, covering 1,535 bp, was amplified with two pairs KC479088; 18S – KC479078. Clathrina clathrus (PMR- of primers, NL4F (5′-GACCCGAAAGATGGTGAACTA-3′) 14308): 18S – KC479079; 28S - KC479083; ITS1-5.8S- plus NL4R (5′-ACCTTGGAGACCTGATGCG-3′)(Nichols ITS2 – KC479089; 18S – KC479079. Clathrina cf. 2005) and primers CAL-28SFW (5′- GKCGGATCCGAAY hondurensis (PMR-14305): 28S – KC479084; ITS1-5.8S- GGACCG-3′)plusCAL28SRV(5′-CCTCTAATCATTCG ITS2 – KC479086; 18S – KC479080. Clathrina blanca CTTTACC-3′) designed in the Laboratory of Molecular (PMR-14307): 28S – KC479085; ITS1-5.8S-ITS2 – Genetics, RBI, based on multiple alignments with the se- KC479087; 18S – KC479081. quences available from GenBank. Partial 18S rDNA (1,611 bp) was also sequenced, but it was not used in the analyses as it was too conservative. PCR reactions were Results and discussion performed with the following conditions: 3 min/95 °C, 30 cy- cles (30 s/94 °C, 45 s/55 °C, 90 s/70 °C) and final elongation Taxonomy 10 min/72 °C. Reaction mixtures containing 2.5 μlof10×

PCR buffer, 3 μlMgCl2 (25 mM), 0.8 μlofeachprimer Systematic index (10 mM), 0.5 μl dNTPs (10 mM each), 0.2 μl Taq-DNA- Class Calcarea Bowerbank, 1864 polymerase and 100-150 ng template. PCR products were Subclass Calcinea Bidder, 1898 directly sequenced using the ABI PRISM 3100 automatic Order Clathrinida Hartman, 1958 sequencer and ABI PRISM BigDye Terminator v3.1 Ready Family Clathrinidae Minchin, 1900 Reaction Cycle Sequencing Kit (Applied Biosystems, USA). Genus Clathrina Gray, 1867 Problematic regions were ligated with the pGEM-T Vector Kit Type species: Clathrina clathrus (Schmidt, 1864) (Promega) and cloned into XL1-Blue competent cells; up to Diagnosis: Calcinea in which the cormus comprises anas- three clones were sequenced. tomosed tubes. A stalk may be present. The skeleton contains regular (equiangular and equiradiate) and/or parasagittal Sequence alignment and phylogenetic analysis triactines, to which diactines and tripods may be added. Asconoid aquiferous system (Klautau et al. 2013). Cloned sequences were assembled using Lasergene process- Clathrina blanca (Miklucho-Maclay, 1868) ing software (DNASTAR Inc., Madison, WI, USA) and Original description: Guancha blanca Miklucho-Maclay, checked visually for sequencing errors. The BLAST network 1868 service (http://www.ncbi.nlm.nih.gov/) was used for sequence Additional sources: Arnesen 1900; Borojević and Boury- homology searches. Multiple alignments were performed with Esnault 1987; Burton 1930; Haeckel 1872; Schmidt 1862;van the Q-INS-I option of the MAFFT program (Katoh and Soest 2001. Standley 2013). Alignments were done using score matrix Type locality: NE Atlantic, Canary Islands, Lanzarote 200 PAM/k=2, gap penalty 1.53 and offset value 0. (Port del Arrecife) Ambiguously aligned regions were determined and excluded Material examined: PMR-14307. Island of St. Giovanni from further analyses using the program Gblocks 0.91b under (45° 2′ 46.63″N, 13° 37′ 21.96″E), 3 m depth; collected by M. less stringent parameters (Castresana 2000). Pfannkuchen; January 2011. 24 M. Imešek et al.

Description: The specimen was attached to the rocky sub- actine. Later, Haeckel (1872) gave more details of the skeleton strate (limestone) of shallow cave ceilings. The symmetrical, of specimens from Lanzarote, saying that the actines of the globular cormus with a diameter less than a centimetre was parasagittal triactines were cylindrical and measured: 50-70/3- attached to the ceiling via a short thin stalk or peduncle (less 4 (paired actines); 80-100/3-4 (unpaired actine). than 4 mm long, less than a 1 mm wide). Colour in vivo is No morphological or molecular studies have been done to white. The specimen has a cormus composed of a peduncle compare the variation among populations of Clathrina blanca and a clathroid body (Fig. 2a). The clathroid body is com- from the Canary Islands and the Mediterranean and Adriatic posed of irregular and loosely anastomosed tubes of equal Seas, and types of C. blanca are unknown. However, studies size. Water-collecting tubes were observed (Fig. 2b). The have shown a close similarity between the fauna of the Canary skeleton of the tubes of the clathroid body is thin and Islands and the Mediterranean Sea (Maldonado and Uriz comprises regular and parasagittal triactines, which are 1995). Therefore, considering this relatedness and the small tangentially oriented around the tubes. The clathroid body difference between the morphology of the Adriatic specimens is attached to the substrate via one or several peduncles. and C. blanca from the Canary Islands (there were differences The peduncle is partially solid and its skeleton comprises only in the spicules thickness and in the partially solid pedun- only tangentially oriented parasagittal triactines with the cle in our specimen), we identified our specimens as C. unpaired, longer actine, basipetally oriented towards the blanca. Therefore, for the first time there is a DNA sequence attachment site (Fig. 2c). Reproductive structures were not of this currently considered cosmopolitan species. observed. In our tree it is possible to see that C. blanca is not only Spicules: Triactines: Equiangular and equiradiate or morphologically but also molecularly close to the Chilean parasagittal, with the unpaired actine being longer than the species Clathrina ramosa Azevedo et al., 2009 (Fig. 1). In paired ones. Actines are cylindrical, straight or slightly undu- fact, the only morphological difference between them is the lated with rounded tips (Fig. 2d). Dimensions: Regular: 51.2- external appearance of the clathroid body, which is globular in 67.6 (±7.2) -81.5 μm/4.7-5.5 (±0.5) -6.9 μm(n =33). C. blanca and coarsely branched in C. ramosa. Parasagittal: 54.7-91.5 (±17.1) -135.2 μm/4.5-5.3 (±0.4) Clathrina clathrus (Schmidt, 1864) -6.0 μm(unpaired,n =33); 29.9-58.4 (±12.8) -76.6 μm/4.5- Original description: Grantia clathrus Schmidt, 1864 5.3 (±0.4) -6.0 μm(paired,n =33). Additional sources: Bianco 1888; Bidder 1891; Borojević Remarks: Clathrina blanca was originally described by et al. 1968; Borojević and Klautau 2000; Breitfuss 1896, Miklucho-Maclay (1868) from Lanzarote (Canary Islands). 1898, 1935;Burton1935, 1963; Dendy and Row 1913; He described the external shape of the specimens and men- Ferrer-Hernandez 1916, 1918, 1922; Gray 1867; Haeckel tioned the presence of triactines only, with a longer unpaired 1870, 1872;Kirk1896; Klautau and Valentine 2003;

Fig. 2 Clathrina blanca. a. Fixed specimen, lateral view. The cormus (arrow) is clearly distinguishable from the stalk (arrowhead). Scale bar=5 mm. b.Spongein situ.Scalebar= 5mm.c. Magnification of the stalk. The arrow points towards the attachment site. Scale bar= 100 μm. d. Parasagittal spicule (arrow) and regular triactines (arrowhead). Scale bar=50 μm Integrative taxonomy of four Clathrina species of the Adriatic 25

Lackschewitsch 1886;Minchin1896; Priest 1887; Solé-Cava Description: The specimen was growing on the basal parts et al. 1991; Topsent 1894, 1934, 1936; van Soest 2001;von of a thallus of C. crinita. Colour in vivo is white to almost Lendenfeld 1891. transparent. The specimen has a clathrate cormus typical of Type locality: Lesina, Adriatic Sea the genus. The cormus is 2 cm long and 1 cm large. It is Material examined: PMR-14308. Island of St. Giovanni composed of irregular and loosely anastomosed tubes of equal (45° 2′ 46.63″N, 13° 37′ 21.96″E), 3 m depth; collected by size. Water-collecting tubes were not observed (Fig. 4a). The M. Pfannkuchen; January 2011. skeleton of the tubes is thin and comprises only one type of Description: The specimen was found in a semi-dark cave triactines, which are tangentially oriented around the tubes attached to the rocky substrate (limestone) of shallow cave and build an irregular mesh. Reproductive structures were not ceilings. Colour in vivo is yellow. The clathroid cormus is observed. composed of irregularly and loosely anastomosed tubes. Spicules: Triactines: Equiangular and equiradiate. Actines Large superficial water-collecting tubes were observed, open- are straight and conical with sharp tips. They are particularly ing to a few large oscula (Fig. 3a). The skeleton has no special wide at the base and start to get thinner half way to their tip organisation, comprising only triactines. Reproductive struc- (Fig. 4b). Dimensions: 101.8-128.0 (±9.6) -151.5 μm/13.3- tures were not observed. 15.2 (±1.3) -19.0 μm(n =33). Spicules: Triactines: Equiangular and equiradiate. Actines Remarks: Clathrina cf. hondurensis is clearly part of are cylindrical and undulated, with rounded tips (Fig. 3b). Haeckel’s species complex “Clathrina primordialis”. Dimensions: 77.9-91.1 (±4.9) -100.7 μm/6.9-7.2 (±0.2) Clathrina primordialis was first described by Haeckel in -7.7 μm(n =33). 1870 as Prosycum primordiale, a species from the Bay of Remarks: Clathrina clathrus is a common species in the Naples. However, all Haeckel’s species published in that work Mediterranean and Adriatic Seas. This species was considered of 1870 were considered to be nomina nuda and were pub- to be cosmopolitan; however, genetic and morphological lished again with new names in 1872. In Haeckel’s characters showed that Southwestern Atlantic yellow Monograph (1872), P. primordiale became Ascetta clathrinas identified as C. clathrus were a distinct species. primordialis, a so-called cosmopolitan species. Haeckel did This species was named C. aurea Solé-Cava et al., 1991 not elect any type locality, but mentioned that this species was (Solé-Cava et al. 1991; Klautau and Valentine 2003), and, first found near Nizza (1856), then in the Bay of Naples since then, other species of yellow clathrinas have been de- (1859) and in other localities, the last one being Lesina scribed (C. chrysea Borojević & Klautau, 2000, from New (1871). Analysing Haeckel’s drawings and specimens, Caledonia; C. luteoculcitella Wörheide & Hooper, 1999, from Klautau and Valentine (2003) concluded that A. primordialis Australia). As expected, morphological and molecular char- was probably a mixed group of different species and elected a acters of the specimen studied in the present work were specimen from the Bay of Naples (ZMB 1306) as lectotype of congruent with those of C. clathrus (Klautau and Valentine C. primordialis. Doing this, they defined C. primordialis as a 2003; Rossi et al. 2011)(Fig.1). species with two types of triactines (conical and slightly Clathrina cf. hondurensis Klautau & Valentine, 2003 conical) with sharp tips. Measurements given were 64.8- Original description: Clathrina hondurensis Klautau & 86.6 (±13.0) -117.6/11.3 (±0.7) (conical) and 84-91.9 (±5.8) Valentine, 2003 -103.2/9.6 (±0.5) (slightly conical). In the same work, they Type locality: Turneffe, Belize, Caribbean Sea. described a new species from this species complex, C. Material examined: PMR-14305. Beach north of the city of hondurensis. The holotype of C. hondurensis is from the Rovinj (45° 6′ 40.08″N, 13° 36′ 41.84″E), 3 m depth; collect- Caribbean Sea (near Belize, Turneffe) and is characterised ed by M. Pfannkuchen; November 2010. by a cormus formed by loosely and irregularly anastomosed

Fig. 3 Clathrina clathrus. a. Sponge in situ. Scale bar=5 mm. b. Regular triactines. The actines are cylindrical, undulated at their distal part and with rounded tips. Scale bar=50 μm 26 M. Imešek et al.

Fig. 4 Clathrina cf. hondurensis. a. Sponge in situ attached to the basal part of Cystoseira crinita thallus. Scale bar=1 cm. b. Triactine. Conical actine (arrow) with sharp tip (arrowhead). Scale bar=50 μm

tubes and a skeleton composed of triactines with conical Material examined: PMR-14306. Beach north of the city of actines and sharp tips. Even the size of the spicules matched Rovinj (45° 6′ 40.08″N, 13° 36′ 41.84″E), 3 m depth; collect- that of the specimen from the Adriatic (near Rovinj) (105.6- ed by M. Pfannkuchen; November 2010. 133.4 (±17.0) -156.0 μm/12.0-15.6 (±1.7)-19.2 μm). Description: The specimen was growing on the basal parts The presence of a calcareous species from the Caribbean in of a thallus of C. crinita.Colourin vivo is bright red to orange. the Adriatic Sea is, at least, uncommon. However, as it was The specimen has a clathrate cormus, which is 2 cm long and not possible to differentiate the present specimen from C. has a width of up to 1 cm. It is composed of irregularly and hondurensis morphologically, and as there are no DNA se- loosely anastomosed tubes of equal size. Water-collecting quences of C. hondurensis from Belize, we decided to call our tubes were not observed (Fig. 5a). The skeleton comprises specimen C. cf. hondurensis. Nonetheless, molecular and only triactines, which are tangentially oriented around the morphological studies comparing more specimens from the tubes and build an irregular mesh. Reproductive structures Caribbean and Adriatic Seas should be done to confirm this were not observed. identification. Spicules: Triactines: Equiangular and equiradiate (Fig. 5b). Comparing the ITS1-5.8S-ITS2 and 28S sequences to our Some parasagittal spicules can also be found (Fig. 5c). Actines data set, C. cf. hondurensis is most similar to Clathrina are straight and cylindrical, with blunt tips. Dimensions: 61.4- antofagastensis Azevedo et al., 2009, from the Chilean coast. 79.4 (±7.9) -93.2 μm/7.3-8.5 (±0.6) -9.8 μm(n =33). In our tree, C. cf. hondurensis and C. antofagastensis formed Remarks: This is the first time that C. rubra is being a well-supported clade (with bootstrap values of 100; Fig. 1). formally described. This species was originally mentioned as Both species are white and have only triactines forming the C. coriacea var. rubra by Sarà in 1958 in a work about the skeleton (Azevedo et al. 2009). distribution of sponges in a cave in the Bay of Naples (Italy). Clathrina rubra Sarà, 1958 In that paper he did not present a formal description of the Original description: Clathrina coriacea var. rubra Sarà, species and only mentioned that C. rubra was orange/red and 1958 very similar to C. coriacea. In 1968, Borojević et al. elevated Additional sources: Borojević et al. 1968; Pansini and this variety to the species category in a species list of sponges Longo 2008;vanSoest2001. from the English Channel near Roscoff (France); in 2008, Type locality: Bay of Naples (Grotta dei Misteri) Pansini and Longo mentioned it again in a species list of

Fig. 5 Clathrina rubra. a. Sponge in situ attached to the basal part of Cystoseira crinita thallus. Scale bar=1 cm. b. Regular triactine. Actine with cylindrical actine (arrow)and the typical wide, rounded tip (arrowhead). Scale bar=50 μm. c. Parasagittal triactine. Scale bar=50 μm Integrative taxonomy of four Clathrina species of the Adriatic 27 sponges from Italy. This is the first time that C. rubra was fellowship from the Brazilian National Research Council (CNPq) and found in the north Adriatic Sea. We compared the specimen by a research grant from the Rio de Janeiro State Research Foundation (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio from the Adriatic Sea with a slide of C. rubra prepared by de Janeiro - FAPERJ) Sarà. Although the spicules on the slide are very corroded and seem to have more rounded tips than those of our specimen, we believe that ours is C. rubra. Clathrina rubra is morphologically very similar to species References of yellow clathrinas: C. aurea, C. chrysea, C. clathrus and C. luteoculcitella (Rossi et al. 2011). However, it is more similar to C. aurea and C. clathrus as these species also have cylin- Arnesen, E. (1900). Spongier fra den norske kyst. I. Calcarea. Systematisk katalog med bemerkninger og bestemmelsestabell. drical actines. Despite this similarity, C. rubra can be differ- Bergens Museums Arbok, 5,1–44. entiated from C. aurea and C. clathrus by its colour (bright Azevedo, F., Hajdu, E., Willenz, P., & Klautau, M. (2009). New records red or orange), the presence of parasagittal spicules and mo- of Calcareous sponges (Porifera, Calcarea) from the Chilean coast. – lecular differences. Zootaxa, 2072,1 30. Bakran-Petricioli, T., Radolović, M., & Petricioli, D. (2012). How diverse As yellow clathrinas are phylogenetically very close (Rossi is sponge fauna in the Adriatic Sea? Zootaxa, 3172,20–38. et al. 2011; Klautau et al. 2013), we expected that the red C. Bianco, S. L. O. (1888). Notizie biologiche riguardanti specialmente il rubra would group with them in our molecular tree (Fig. 1). periodo di maturità sessuale degli animali del golfo di Napoli. – However, it was well separated from those species, which Mitteilungen aus der Zoologischen Station zu Neapel, 8,385 440. Bidder,G.P.(1891).Reviewof‘A Monograph of Victorian Sponges’. suggests that the red/orange colour appeared independently Quarterly Journal of Microscopical Science, 32,625–632. of the yellow colour. Borojević, R. (1967). Importance de l'étude de la répartition écologique pour la taxonomie des éponges calcaires. Helgoländer wiss. – Ecology Meeresunters, 15,116 119. Borojević, R., Cabioch, L., & Lévi, C. (1968). Inventaire de la faune marine de Roscoff: Spongiaires. 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