The Port of Valencia, Spain in the western Mediterranean Sea consists of three boathouses, the “Marina Real” and an outer harbour. Malvarrosa Beach, north of the port, is a highly-anthropized fine sand beach with several artificial concrete reefs installed in 2014 at 4 m depth, less than 200 m from the coast (Station M in Fig. 1). Sampling was carried out at three stations of the Marina Real of Valencia Port (39°26.9′N, 0°18.1′W) and one on the artificial reef (station M: 39°28′39.2″N, 0°19′13.4″W) located at Malvarrosa Beach (Fig. 1; Table 1). The Marina Real sampling was carried out at surface level (0-0.3 m) at two stations, the sailing school (V: 39°27′41.5″N, 0°19′06.5″W) and the gas station (G: 39°27′40.2″N, 0°18′45.8″W), by manual scraping using a 25×25 cm square on biological concretions located in the submerged areas of the pontoons and internal walls of the Marina. Outside the Marina, at the north breakwater (Station E: 39°27′46.1″N, 0°18′50.2″W) samples were obtained by SCUBA divers from 2-3 m depth. Samples from the Marina Old Venetian Harbour of Heraklion (35°20′51.0″N, 25°08′27.4″E) were obtained in a similar way, scraping 25×20 cm at 1.5 m depth. All biological samples were obtained from artificial substrates.

Spirobranchus cf. tetraceros specimens were collected in summer 2015 and in summer and winter 2016 at all three Valencia Port stations (V, sailing school; G, gas station; E, north breakwater), but not from the Malvarrosa Beach artificial reefs (see Fig. 1). Additional specimens from Valencia Port (sailing school station) were found in August and October 2016 and July 2017. Specimens were anaesthetized with 7.5% magnesium chloride in seawater and sieved in the laboratory using a 1 mm mesh. Some individuals were removed from their tubes and fixed in 4% formaldehyde for 24 h, rinsed in seawater and transferred to 70% ethanol, while other specimens were directly preserved in 100% ethanol for later molecular analysis. Sequences were obtained for two specimens from the Valencia Port with different operculum types (simple conical and flat fully branched). To ensure a proper comparison with S. tetraceros, we also sequenced material collected from the type locality (New South Wales, Australia) and a previously reported population from Heraklion, Crete, Greece in the eastern Mediterranean Sea (Ulman et al. 2017). Sequences of S. tetraceros specimens from the Red Sea (Eilat, Israel), already available in GenBank (Perry et al. 2018), were also included in the molecular analyses (Table 1).

In order to identify and document morphological features, the specimens were examined using two Leica dissecting microscopes (models M165C and DMS 1000) and photographed using a Leica DFC420 digital camera. Chaetae and uncini were mounted under a Leica DM3000 microscope and photographed using a Leica DFC450 digital camera. Measurements were taken using the Leica Application Suite software and following Bastida-Zavala and ten Hove (2002): total length from the tip of radioles to end of pygidium; thoracic length in ventral view from the posterior edge of the apron to the anterior edge of collar; thoracic width measured over the ventral side of the collar region across the fifth unciniger; radiolar length from the base of the radiolar crown to the tip; abdominal length from the posterior edge of the apron to the end of the pygidium in lateral view; opercular diameter; number of abdominal chaetigers; and number of radioles in each half of the crown.

Total genomic DNA was extracted from samples of Spirobranchus collected from Heraklion, Valencia and NSW (Australia) (see Table 1 for details) using a QIAamp DNA Mini Kit (QIAGEN Inc) and following the manufacturer’s instructions. DNA quality was assessed by gel electrophoresis (1% agarose) (Palero et al. 2010) and quantified using a Qubit 3.0 fluorometer (Life Technologies). A fragment (~400 bp) of the mitochondrial cytochrome b gene was amplified with ~30 ng of genomic DNA in a reaction containing 1 U of Taq polymerase (Amersham), 1 × buffer (Amersham), 0.2 mM of each primer (Cytb 424F = GGWTAYGTWYTWCCWTGRGGWCARAT and Cytb 876R = GCRTAWGCRAAWARRAARTAYCAYTCWGG; Boore and Brown (2000)) and 0.12 mM dNTPs. The polymerase chain reaction (PCR) thermal profile was 94°C for 4 min for initial denaturation, followed by 30 cycles of 94°C for 30 s, 54°C for 30 s, 72°C for 30 s and a final extension at 72°C for 4 min. Amplified PCR products were purified using QIAquick PCR Purification Kit (QIAGEN Inc.) before direct sequencing of the product. The sequences were obtained using the BigDye v3.1 (Applied Biosystems) kit on an ABI Prism 3770. Chromatograms for each PCR amplicon were checked visually and ambiguous positions were left as such using IUPAC codes. Primer sequences and flanking regions were removed from the consensus sequences created from forward and reverse strands using BioEdit ver. 7.2.5.

Sequences of several species of Spirobranchus were obtained from GenBank, including S. tetraceros from the Red Sea (MF319330, MF319331), S. giganteus (Pallas, 1766) from Brazil (NC032055); S. latiscapus (Marenzeller, 1885) from New Zealand (JX144879), S. corniculatus (Grube, 1862) from the Red Sea and S. cariniferus (Gray, 1843) from New Zealand (e.g. JX144873, JX144875) (Fig. 2). Sequences were aligned using Muscle ver. 3.6 (Edgar 2004) and conserved (ungapped) blocks of sequence were extracted using the Gblocks server with default parameters (Castresana 2000, Talavera and Castresana 2007). Estimates of p-distances (proportion of genetic differences) and Kimura 2-Parameter (K2P) evolutionary divergence between groups were obtained from the aligned cytb dataset using MEGA X (Kumar et al. 2018). Before running molecular phylogenetic analyses, the most suitable nucleotide substitution model was selected according to the BIC criterion as implemented in MEGA X (Kumar et al. 2018). The aligned sequences and selected evolutionary model were then used to estimate the maximum likelihood phylogenetic tree in RAxML (Stamatakis 2014). Node support was evaluated with 1000 bootstrap replicates.

After adding GenBank data and Gblocks trimming, the final cytb alignment included 317 bp positions (from the original 400 bp). The selected DNA substitution model was the Hasegawa-Kishino-Yano model (HKY+G+I) with invariant positions (34% of the sites invariable) and heterogeneity across sites (G=1.10). The phylogenetic tree obtained by maximum likelihood (Ln=–2999.90) provides further support for the separation of the Australian S. tetraceros from the Mediterranean specimens, showing that these two populations are not monophyletic. Therefore, Mediterranean Spirobranchus are here referred to as S. cf. tetraceros and considered to belong to a different species, most likely undescribed, rather than to S. tetraceros sensu stricto from Australia. Red Sea samples clustered (with high bootstrap support) with samples from the Mediterranean (Fig. 2).

Both p-distances and K2P distances showed a similar pattern, with intraspecific genetic distances (not shown) being much lower (<0.02) than inter-specific distances (>0.14) (Table 2). Observed values for the K2P genetic distances between S. tetraceros sensu stricto from the type locality (NSW) and S. cf. tetraceros from Mediterranean (0.424±0.045) or Red Sea (0.385±0.042) were larger than distances between S. cf. tetraceros from Mediterranean and Red Sea (0.274±0.034). For comparison, K2P distances between those two groups of S. cf. tetraceros were larger than distances observed between other pairs of valid species such as S. aloni and S. corniculatus (0.197±0.028) or S. aloni and S. gardineri (0.252±0.033). Several non-synonymous changes could be observed between the S. cf. tetraceros from Mediterranean and Red Sea when translating the DNA sequences into protein, which suggests that these two populations may correspond in fact to valid (most likely undescribed) taxa. Nevertheless, a more comprehensive revision, including more populations and genetic markers should be carried out before drawing a final conclusion on the taxonomic status of these two groups.

Genus Spirobranchus Blainville, 1818

Spirobranchus cf. tetraceros

Spirobranchus tetraceros Ben-Eliahu and ten Hove 2011: 88-95, Fig. 34, Table 5
Spirobranchus tetraceros sensu lato Ulman et al. 2017: 34-35, table 2

Material examined. Western Mediterranean Sea, Marina Real (Valencia Harbour, Spain): 14 specimens, Stations:V (7), E (6), G (1); eastern Mediterranean Sea, Heraklion (Crete, Greece) at the Marina Old Venetian Harbour: six specimens. Measurements and detailed morphological descriptions are based on the largest complete specimens from Valencia Port (Station E; Fig. 1) and Heraklion, and complemented with details on structures (e.g. tubes) from other specimens collected at the same localities. The specimens are deposited at Department of Zoology, School of Biological Sciences, University of Valencia (Spain).

Tube. Attached to artificial substrates such as plastic pontoons, vertical cement walls, buoys, metal ladders and cement blocks. Tube outside and inside predominantly white (though occasionally slightly pinkish internally near opening), triangular to circular, with a tooth over entrance and one high, irregular longitudinal ridge, a pair of low lateral keels and many transversal ridges (Fig. 3A, B).

Total length of the largest specimen. 51.3 mm (Valencia) and 11.9 mm (Heraklion). Note that differences in size of the largest specimen depends on sampling. The number of complete specimens examined from the Valencia Port area (N=14) is larger than those found in Heraklion (N=6). Moreover, specimens from Valencia were collected both in summer and winter and therefore it is reasonable that they show a greater variation in size.

Radiolar crown. Composed of two circular lobes each with 25 (Valencia) or 18 (Heraklion) radioles (Fig. 4A-C). Lenght of radiolar crown 7.1 mm (Valencia) and 2.2 mm (Heraklion). Colour blue/white.

Interradiolar membrane. Usually with unilobed, bilobed or multilobed processes (Fig. 4C-E), the shape of which may vary from complex dorsally to simple ventrally.

Peduncle. Inserted on the left of median line, pigmented with white/blue colours (Fig. 4B). Lateral distal wings clearly protruding left and right of opercular plate with pointed tips and crenulated on their inner and outer margins (doubly fringed) (Fig. 5A).

Operculum. Peduncle joining operculum in dorsal position. Diameter 3.8 mm (Valencia) and 2.2 mm (Heraklion). Operculum with circular calcareous endplate, which may be flat, concave, convex or even conical; endplate bearing three groups of dichotomously branched (antler-like) spines, sometimes appearing as three spines only (particularly in the conical operculum) (Fig. 5B-E); position of spines always the same: one (or one group) medio-ventrally and two (or two groups) latero-dorsally. The most complex opercula showing one medio-ventral spine split thrice and two latero-dorsal spines split twice to thrice, with medial spinules irregularly placed.

Polymorphism of opercula. Simple conical (Fig. 5D, E), flat and flatened fully branched (Fig. 5C) as well as intermediate forms (Fig. 5B) were found within the Valencia Port samples.

Collar and thoracic membranes. Collar divided into one ventral and two lateral lobes. Latero-dorsal lobes continuing into thoracic membranes (Fig. 4A) producing a short ventral apron with shallow midventral indent (Fig. 5E). Collar chaetae of two types: special Spirobranchus-type covered with minute denticles (Fig. 6A) and limbate-striated (not shown).

Thorax. Seven thoracic chaetigers, including six uncinigerous. Collar fascicle (CC in Fig. 3C) situated at some distance anterior to remaining thoracic chaetae (Fig. 3C). Thoracic chaetae limbate. Uncini saw-shaped with peg (anterior-most tooth) gouge-shaped (Fig. 6B). Length of thorax: 8.2 mm (Valencia) or 3.4 mm (Heraklion). Thoracic width at 5th row of uncini: 5.9 mm (Valencia) or 1.6 mm (Heraklion). Ventral ends of thoracic uncinigerous tori widely separated anteriorly, gradually approaching one another towards the end of thorax, thus leaving a triangular depression (Fig. 4A).

Abdomen. 87 (Valencia) or 37 (Heraklion) chaetigers. Length 36.2 mm (Valencia) or 7.5 mm (Heraklion). True trumpet-shaped chaetae (Fig. 6C) in a single fascicle, becoming increasingly longer posteriorly. Saw-shaped uncini with gouge-shaped pegs.

Colouration of preserved specimens. Anterior end of thorax, radioles, peduncle and operculum dark blue (Fig. 4B).

Remarks. The original description of the species by Schmarda (1861), based on material from NSW (Australia), does not follow current standards and prevents a suitable comparison with Mediterranean material. A re-description of S. tetraceros from the type locality could not be included here. An accurate and comprehensive morphological revision of material from multiple localities is needed before pointing out useful characters to discriminate between putative taxa.

Key to Mediterranean Spirobranchus, including alien species (*)