INTRODUCTIONTop

The Mediterranean Sea is a global hotspot for marine traffic under strong bioinvasion pressure (Ulman et al. 2017Ulman A., Ferrario J., Occhpinti-Ambrogi A., et al. 2017. A massive update of non-indigenous species records in Mediterranean marinas. PeerJ 5: e3954.). A total of 821 marine non-indigenous species (NIS) have already been recorded (Zenetos et al. 2017Zenetos A., Çinar M.E., Crocetta F., et al. 2017. Uncertainties and validation of alien species catalogues: The Mediterranean as an example. Estuar. Coast. Shelf Sci. 191: 171-187.), accounting for approximately 4.8% of its total marine biodiversity (López and Richter 2017López E., Richter A. 2017. Non-indigenous species (NIS) of polychaetes (Annelida: Polychaeta) from the Atlantic and Mediterranean coasts of the Iberian Peninsula: an annotated checklist. Helgol. Mar. Res. 71: 19.). Shipping is the most common introduction pathway for NIS, either through hull fouling or ballast water (Çinar 2013Çinar M.E. 2013. Alien polychaete species worldwide: Current status and their impacts. J. Mar. Biol. Assoc. UK 93: 1257-1278.). Marinas play a major role as invasion hubs for dispersal (Ferrario et al. 2017Ferrario J., Caronni S., Occhipinti-Ambrogi A., et al. 2017. Role of commercial harbours and recreational marinas in the spread of non-indigenous fouling species. Biofouling 33: 651-660.), and NIS appear to be more successful on artificial substrates than native species (Glasby et al. 2007Glasby T.M., Connell S.D., Holloway M.G., et al. 2007. Nonindigenous biota on artificial structures: could habitat creation facilitate biological invasions? Mar. Biol. 151: 887-895., Tyrrel and Byers 2007Tyrrel M.C., Byers J. 2007. Do artificial substrates favor nonindigenous fouling species over native species? J. Exp. Mar. Biol. Ecol. 342: 54-60., Megina et al. 2016Megina C., González-Duarte M.M., López-González P.J. 2016. Benthic assemblages, biodiversity and invasiveness in marinas and commercial harbours: an investigation using a bioindicator group. Biofouling 32: 465-475.). Harbour walls and floating pontoons provide ideal substrates for settlement of invasive encrusting biota (Mineur et al. 2012Mineur F., Cook E.J., Minchin D., et al. 2012. Changing coasts: marine aliens and artificial structures. Oceanogr. Mar. Biol. 50: 189-234., Megina et al. 2013Megina C., González-Duarte M.M., López-González P.J., et al. 2013. Harbours as marine habitats: Hydroid assemblages on sea-walls compared with natural habitats. Mar. Biol. 160: 371-381.), most likely due to the enclosed nature of these specialized habitats. Sedentary tube worms belonging to the family Serpulidae are commonly found within these fouling communities along Mediterranean marinas. Invasive serpulids are of particular concern because they cause an economic burden in fuel consumption due to extra friction and professional cleaning required to remove them from hulls (Rouse 2000Rouse G.W. 2000. Family Serpulidae. In: Beesley P.L., Ross G.L.B., Glasby C.J. (ed.), Polychaetes and Allies: The Southern Synthesis, CSIRO. Melbourne. pp. 184-189.).

The Mediterranean Sea has shown the highest increase in NIS records (41%) since 2012 (Zenetos et al. 2017Zenetos A., Çinar M.E., Crocetta F., et al. 2017. Uncertainties and validation of alien species catalogues: The Mediterranean as an example. Estuar. Coast. Shelf Sci. 191: 171-187.) and hosts nearly half (63/134) of the total number of polychaete NIS in the world (Çinar 2013Çinar M.E. 2013. Alien polychaete species worldwide: Current status and their impacts. J. Mar. Biol. Assoc. UK 93: 1257-1278.). Polychaetes constitute up to one third of hard-bottom assemblages in both abundance and species richness in the Mediterranean (Antoniadou et al. 2004Antoniadou C., Nicolaidou A., Chintiroglou C. 2004. Polychaetes associated with the sciaphilic alga community in the northern Aegean Sea: Spatial and temporal variability. Helgol. Mar. Res. 58: 168-182., Giangrande et al. 2004Giangrande A., Delos A.L., Musco L., et al. 2004. Polychaete assemblages of rocky shore along the South Adriatic coast (Mediterranean Sea). Cah. Biol. Mar. 45: 85-95. ) and represent 12% of the total NIS (Zenetos et al. 2010Zenetos A., Gofas S., Verlaque M., et al. 2010. Alien species in the Mediterranean Sea by 2010. A contribution to the application of European Union’s Marine Strategy Framework Directive (MSFD). Part I. Spatial distribution. Mediterr. Mar. Sci. 11: 381-493. ). Artificial substrates in Mediterranean harbours are usually dominated by species of Hydroides Gunnerus, 1768Gunnerus J.E. 1768. Om Nogle Norske Coraller. Kongel. Norske Vidensk. Selsk. Skr. 4: 38-73. (e.g. Çinar 2006Çinar M.E. 2006. Serpulid species (Polychaeta: Serpulidae) from the Levantine coast of Turkey (eastern Mediterranean), with special emphasis on alien species. Aquat. Inv. 1: 223-240.), but other alien calcareous tubeworms are becoming increasingly common. For example, a recent study across 50 marinas showed Hydroides elegans (Haswell, 1883Haswell W.A. 1883. On some new Australian tubicolous annelids. Proc. Linn. Soc. N.S.W. 7: 633-638. ) to be present in 66%, Hydroides dirampha Mörch, 1863Mörch O.A.L. 1863. Revisio critica Serpulidarum. Et Bidrag til Rørormenes Naturhistorie. Naturh. Tidsskr. København, Ser. 3, 1: 347-470. in 32% and Ficopomatus enigmaticus (Fauvel, 1923Fauvel P. 1923. Un nouveau serpulien d’eau saumatre Mercierella n. g., enigmatica n. sp. Bull. Soc. Zool. Fr. 47: 424-430.) in 14% of them (Ulman et al. 2019aUlman A., Ferrario J., Forcada A., et al. 2019a. A hitchhiker’s guide to alien species settlement in Mediterranean marinas. J. Environ. Manage. 241: 328-339.). Serpulids were the most common family in boat hull biofouling communities, with H. elegans found on 71% (N=418) of the hulls and all serpulids combined accounting for over one-third of NIS records in relative abundance (Ulman et al. 2019bUlman A., Ferrario J., Forcada A., et al. 2019b. Alien species spreading via biofouling on recreational vessels in the Mediterranean Sea. J. Appl. Ecol. 56: 2620-2629. ).

The serpulid genus Spirobranchus Blainville, 1818Blainville H.M.D. de. 1818. Mémoire sur la classe des Sétipodes, partie des Vers à sang rouge de M. Cuvier, et des Annélides de M. de Lamarck. Bull. Sci. Soc. Philom. Paris 1818: 78-85. currently includes 34 nominal species (Read and Fauchald 2019Read G., Fauchald K. (ed.). 2019. World Polychaeta database. Accessed on 19/10/2019. ), 1 subspecies and 3 taxa inquirenda; five of these species have been reported from the Mediterranean: S. lima (Grube, 1862Grube A.E. 1862. Mittheilungen über die Serpulen, mit besonderer Berücksichtigung ihrer Deckel. Jahresber. Abh. Schles. Ges. Breslau 39: 53-69. ), S. polytrema (Philippi, 1844Philippi A. 1844. Einige Bemerkungen über die Gattung Serpula, nebst Aufzählung der von mir im Mittelmeer mit dem Thier beobachteten Arten. Arch. Naturgesch. 10: 186-198.), S. triqueter (Linnaeus, 1758Linnaeus C. von. 1758. Systema Naturae, 10th edition, Vol. 1. L. Salvius, Holmiae, 823 pp. ), S. lamarcki (Quatrefages, 1866Quatrefages A. de. 1866. Histoire naturelle des Annelés marins et d’eau douce. Annélides et Géphyriens. Librarie Encyclopédique de Roret. Paris. Vol. 1. 1-588) and S. tetraceros (Schmarda, 1861Schmarda L.K. 1861. Neue Wirbellose Thiere: Beobachted und Gesammelt auf einer Reise um die Erdr 1853 bis 1857. In: Turbellarien, Rotatorien und Anneliden. Leipzig, Verlag von Wilhelm Engelmann. Erster Band, Zweite Hälfte. ). Previous reports of S. kraussii (Baird, 1865Baird W. 1865. Description of several new species and varieties of tubicolous annelides = Tribe LIMIVORA of Grube, in the collection of the British Museum. J. Linn. Soc. Lond. Zool. 8: 10-22.) in the Mediterranean should be assigned to a different species, S. cf. kraussii, apparently undescribed (Simon et al. 2019Simon C.A., van Niekerk H.H., Burghardt I., et al. 2019. Not out of Africa: Spirobranchus kraussii (Baird, 1865) is not a global fouling and invasive serpulid of Indo-Pacific origin. Aquat. Inv. 14: 221-249.). Spirobranchus tetraceros is considered an NIS of Indo-Pacific origin (Çinar 2013Çinar M.E. 2013. Alien polychaete species worldwide: Current status and their impacts. J. Mar. Biol. Assoc. UK 93: 1257-1278.), with its type locality being New South Wales, Australia (ten Hove and Kupriyanova 2009ten Hove H.A., Kupriyanova E.K. 2009. Taxonomy of Serpulidae (Annelida, Polychaeta): The state of affairs. Zootaxa 2036: 1-126.). The distribution of S. tetraceros has been subject to debate in recent decades due to its wide range and invasive capabilities (ten Hove and Kupriyanova 2009ten Hove H.A., Kupriyanova E.K. 2009. Taxonomy of Serpulidae (Annelida, Polychaeta): The state of affairs. Zootaxa 2036: 1-126., Ben-Eliahu and ten Hove 2011Ben-Eliahu M.N., ten Hove H.A. 2011. Serpulidae (Annelida: Polychaeta) from the Suez Canal from a Lessepsian migration perspective (a monograph). Zootaxa 2848: 1-147.). Spirobranchus tetraceros is ranked among the 100 worst invasive species in the Mediterranean (Streftaris and Zenetos 2006Streftaris N., Zenetos A. 2006. Alien marine species in the Mediterranean - the 100 “worst invasives” and their impact. Mediterr. Mar. Sci. 7: 87-118.) and has been historically considered a Lessepsian invader entering through the Suez Canal (Çinar 2013Çinar M.E. 2013. Alien polychaete species worldwide: Current status and their impacts. J. Mar. Biol. Assoc. UK 93: 1257-1278.). Its first Mediterranean record is from the Lebanese coast (Laubier 1966Laubier L. 1966. Sur quelques Annélides Polychètes de la région de Beyrouth. Am. Univ. Beirut Misc. Pap. 5: 9-22.), and it has been repeatedly collected along the eastern Mediterranean coasts since then (Ben-Eliahu 1991Ben-Eliahu M.N. 1991. Red Sea serpulids (Polychaeta) in the eastern Mediterranean. Ophelia Suppl. 5: 515-528., Ben-Eliahu and ten Hove 1992Ben-Eliahu M.N., ten Hove H.A. 1992. Serpulid tubeworms (Annelida: Polychaeta) a recent expedition along the Mediterranean coast of Israel finds new populations buildups of Lessepsian migrant species. Isr. J. Zool. 38: 35-53. , Ulman et al. 2017Ulman A., Ferrario J., Occhpinti-Ambrogi A., et al. 2017. A massive update of non-indigenous species records in Mediterranean marinas. PeerJ 5: e3954.; see Fig. 1). Reported in 2016 from Siracusa (Sicily), S. tetraceros is considered to be undergoing a westward expansion (Ulman et al. 2017Ulman A., Ferrario J., Occhpinti-Ambrogi A., et al. 2017. A massive update of non-indigenous species records in Mediterranean marinas. PeerJ 5: e3954.). The only previous record from western Mediterranean waters is that of six S. tetraceros specimens found (1979) in the biofouling community of the French aircraft carrier Foch arriving via the Suez Canal in Toulon after a stay of seven months in the Indian Ocean (Zibrowius 1979Zibrowius H. 1979. Serpulidae (Annélida Polychaeta) de l’Océan Indien arrivés sur le côques de bateaux à Toulon (France, Méditerranée). Rapp. P.-V. Reun. Comm. Int. Explor. Sci. Mer Mediterr. 25-26: 133-134. ), but no establishment ever ensued in the area.

The first established population of S. tetraceros in the western Mediterranean is reported here, with specimens collected during 2015-2017 representing the first country record for Spain and the first regional record for the Marina Real (Valencia Port). Molecular evidence using cytochrome b (cytb) sequence data suggests that the nominal taxon S. tetraceros comprises in fact multiple species. Specimens from the S. tetraceros type locality (New South Wales, Australia) were genetically distinct from both Red Sea and Mediterranean material. An illustrated morphological account of the Valencia and Heraklion specimens and an updated taxonomic key for Spirobranchus taxa in the Mediterranean Sea are provided.

MATERIALS AND METHODS Top

Sampling

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. 2017Ulman A., Ferrario J., Occhpinti-Ambrogi A., et al. 2017. A massive update of non-indigenous species records in Mediterranean marinas. PeerJ 5: e3954.). Sequences of S. tetraceros specimens from the Red Sea (Eilat, Israel), already available in GenBank (Perry et al. 2018Perry O., Bronstein O., Simon-Blecher N., et al. 2018. On the genus Spirobranchus (Annelida, Serpulidae) from the northern Red Sea, and a description of a new species. Invertebr. Syst. 32: 605-625.), were also included in the molecular analyses (Table 1).

Morphological analyses

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)Bastida-Zavala J.R., ten Hove H.A. 2002. Revision of Hydroides Gunnerus, 1768 (Polychaeta: Serpulidae) from the western Atlantic region. Beaufortia 52: 103-178. : 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.

DNA analyses

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. 2010Palero F., Hall S., Clark P.F., et al. 2010. DNA extraction from formalin-fixed tissue: new light from the Deep-Sea. Sci. Mar. 74: 465-470.) 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)Boore J.L., Brown W.M. 2000. Mitochondrial genomes of Galathealinum, Helobdella, and Platynereis: sequence and gene arrangement comparisons indicate that Pogonophora is not a phylum and Annelida and Arthropoda are not sister taxa. Mol. Biol. Evol. 17: 87-106.) 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, 1766Pallas P.S. 1766. Miscellanea Zoologica. Petrum van Cleef. Hagae Comitum, The Hague, 224 pp) from Brazil (NC032055); S. latiscapus (Marenzeller, 1885Marenzeller E. von. 1885. Südjapanische Anneliden. II. Ampharetea, Terebellacea, Sabellacea, Serpulacea. Denkschriften der Akademie der Wissenschaften, Wien 49: 197-224. ) from New Zealand (JX144879), S. corniculatus (Grube, 1862Grube A.E. 1862. Mittheilungen über die Serpulen, mit besonderer Berücksichtigung ihrer Deckel. Jahresber. Abh. Schles. Ges. Breslau 39: 53-69. ) from the Red Sea and S. cariniferus (Gray, 1843Gray E. 1843. Fauna of New Zealand. In: Dieffenbach E., Travels in New Zealand. vol. 2: 30-138, John Murray, London.) from New Zealand (e.g. JX144873, JX144875) (Fig. 2). Sequences were aligned using Muscle ver. 3.6 (Edgar 2004Edgar R.C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32: 1792-1797.) and conserved (ungapped) blocks of sequence were extracted using the Gblocks server with default parameters (Castresana 2000Castresana J. 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol. 17: 540-552., Talavera and Castresana 2007Talavera G., Castresana J. 2007. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst. Biol. 56: 564-577.). 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. 2018Kumar S., Stecher G., Li M., et al. 2018. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 35: 1547-1549.). 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. 2018Kumar S., Stecher G., Li M., et al. 2018. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 35: 1547-1549.). The aligned sequences and selected evolutionary model were then used to estimate the maximum likelihood phylogenetic tree in RAxML (Stamatakis 2014Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312-1313.). Node support was evaluated with 1000 bootstrap replicates.

RESULTSTop

Molecular identification and genetic distances

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.

Morphological analyses and systematic account

Genus Spirobranchus Blainville, 1818Blainville H.M.D. de. 1818. Mémoire sur la classe des Sétipodes, partie des Vers à sang rouge de M. Cuvier, et des Annélides de M. de Lamarck. Bull. Sci. Soc. Philom. Paris 1818: 78-85.

Spirobranchus cf. tetraceros

Spirobranchus tetraceros Ben-Eliahu and ten Hove 2011Ben-Eliahu M.N., ten Hove H.A. 2011. Serpulidae (Annelida: Polychaeta) from the Suez Canal from a Lessepsian migration perspective (a monograph). Zootaxa 2848: 1-147.: 88-95, Fig. 34, Table 5
Spirobranchus tetraceros sensu lato Ulman et al. 2017Ulman A., Ferrario J., Occhpinti-Ambrogi A., et al. 2017. A massive update of non-indigenous species records in Mediterranean marinas. PeerJ 5: e3954.: 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)Schmarda L.K. 1861. Neue Wirbellose Thiere: Beobachted und Gesammelt auf einer Reise um die Erdr 1853 bis 1857. In: Turbellarien, Rotatorien und Anneliden. Leipzig, Verlag von Wilhelm Engelmann. Erster Band, Zweite Hälfte. , 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 (*)

1	Collar chaetae absent	Spirobranchus cf. kraussii (*)
–	Collar chaetae present	2
2	Collar chaetae few, fine and capillary	3
–	Collar chaetae numerous, large and Spirobranchus-type	4
3	Opercular ampulla cup-shaped, higher than distal calcareous cap, which may be flat, concave or slightly convex, with or without distal projections (in this case, with a central cylindrical protuberance from which project 1-3 short tips); anterior margin of lateral-dorsal lobes of collar not fringed	Spirobranchus lamarcki
–	Opercular ampulla flat as an empty balloon, thinner than the distal convex, often conical calcareous cap, with or without projections (often with three teeth); anterior margin of lateral-dorsal collar lobes finely fringed	Spirobranchus triqueter
4	Opercular plate typically with three groups of dichotomously branched spines, sometimes conical cap only; interradiolar membrane and anterior margin of peduncular wing with finger-like processes	S. cf. tetraceros (*)
–	Opercular plate without spines; interradiolar membrane without finger-like processes	5
5	Opercular plate flat or concave; opercular peduncle with wide wings fringed at the tip; tube rose, with about five longitudinal serrated keels	S. lima
–	Opercular plate convex, often with two dorsal tubercles; opercular peduncle with narrow, rarely bifid wings; tube white, with three keels and a series of lateral alveoli	S. polytrema

DISCUSSIONTop

Several specimens of S. cf. tetraceros were collected from the Marina Real of Valencia Port during both winter and summer surveys in three consecutive years (2015-2017). Consequently, an established population of S. cf. tetraceros from the western Mediterranean is reported here for the first time, also representing a first record for the Iberian Peninsula. Spirobranchus tetraceros was first reported from the western Mediterranean 40 years ago, fouling the aircraft carrier “Foch” in Toulon (Zibrowius 1979Zibrowius H. 1979. Serpulidae (Annélida Polychaeta) de l’Océan Indien arrivés sur le côques de bateaux à Toulon (France, Méditerranée). Rapp. P.-V. Reun. Comm. Int. Explor. Sci. Mer Mediterr. 25-26: 133-134. , and see above), but did not establish then. Despite a recent intensive survey of dozens of recreational marinas along the Mediterranean coast, Ulman et al. (2017)Ulman A., Ferrario J., Occhpinti-Ambrogi A., et al. 2017. A massive update of non-indigenous species records in Mediterranean marinas. PeerJ 5: e3954. did not find S. tetraceros sensu lato in the Marina of Alicante (Spain) or the OneOcean Port Vell Marina in Barcelona. The fact that this invader was present in Valencia during the same time period, but apparently not in Alicante or Barcelona, underscores the necessity of a comprehensive study of Mediterranean ports and marinas in order to identify established invasive species.

Morphology and molecular sequence analyses confirm that the specimens of S. cf. tetraceros from Valencia are identical to those found in the eastern Mediterranean (Heraklion, Crete). Most importantly, Mediterranean specimens are shown to be genetically different from specimens of S. tetraceros sensu stricto collected from the type locality (New South Wales, Australia). This result directly supports the hypothesis (in Perry et al. 2018Perry O., Bronstein O., Simon-Blecher N., et al. 2018. On the genus Spirobranchus (Annelida, Serpulidae) from the northern Red Sea, and a description of a new species. Invertebr. Syst. 32: 605-625.), that S. tetraceros is not a single widely distributed invader of Australian origin, but rather a complex of cryptic species. The second implication is that the Mediterranean specimens examined herein may belong to a yet undescribed species of the S. tetraceros complex. The identity and origin of the Mediterranean population remains uncertain, because the widely accepted hypothesis of Ben-Eliahu (1991)Ben-Eliahu M.N. 1991. Red Sea serpulids (Polychaeta) in the eastern Mediterranean. Ophelia Suppl. 5: 515-528. that S. cf. tetraceros is a Lessepsian migrant passively crossing the Suez Canal to the Mediterranean is not conclusively supported by our results. Genetic distances between Red Sea (Gulf of Eilat) specimens and Mediterranean S. cf. tetraceros seem large enough to be considered as belonging to distinct taxa. Nevertheless, a more comprehensive worldwide revision, including more populations and genetic markers, should be carried out before drawing a final conclusion on the taxonomic status of these populations.

Morphological species delimitation is particularly difficult in Spirobranchus because of their high intraspecific variability opercular structures, considered one of the major taxonomic characters of the genus. Several taxa were initially synonymized by ten Hove (1970)ten Hove H.A. 1970. Serpulinae (Polychaeta) from the Caribbean: I - The genus Spirobranchus. Stud. Fauna Curaçao Caribb. Isl. 32: 1-57. under S. tetraceros and a cosmopolitan distribution was hypothesized for this taxon, among other reasons because of its high opercular variation (see also Perry et al. 2018Perry O., Bronstein O., Simon-Blecher N., et al. 2018. On the genus Spirobranchus (Annelida, Serpulidae) from the northern Red Sea, and a description of a new species. Invertebr. Syst. 32: 605-625.). Ben-Eliahu and ten Hove (2011)Ben-Eliahu M.N., ten Hove H.A. 2011. Serpulidae (Annelida: Polychaeta) from the Suez Canal from a Lessepsian migration perspective (a monograph). Zootaxa 2848: 1-147. and Willette et al. (2015)Willette D.A., Iñiguez A.R., Kupriyanova E.K., et al. 2015. Christmas tree worms of Indo-Pacific coral reefs: untangling the Spirobranchus corniculatus (Grube, 1862) complex. Coral Reefs 34: 899-904. also reported highly variable opercula for S. tetraceros specimens from the Suez Canal and the Indo-Pacific S. corniculatus, respectively. The molecular characterization of Valencia Port and Heraklion specimens carried out here, including specimens with either conical or fully-branched opercula, confirms that this morphological variation simply corresponds to intraspecific plasticity. This result highlights the importance of using molecular data for species delimitation and the need to further analyse the morphology of the species of Spirobranchus. Other characters should be used to discriminate species within the S. tetraceros complex, such as the shape and distribution of multilobed processes from the interradiolar membrane.

Worldwide distributed cryptic invaders are particularly difficult to track because they are often assumed to be native species or wrongly assigned to other invasive species (Morais and Reichard 2018Morais P., Reichard M. 2018. Cryptic invasions: a review. Sci. Total Environ. 613-614: 1438-1448.). Our results are relevant for the management of Mediterranean NIS, showing that S. tetraceros represents a species complex rather than a single widely distributed species. Mediterranean specimens differ genetically from S. tetraceros sensu stricto from the type locality and may have a different ecology, so management practices should be planned taking this into account. Further sampling and ecological studies across both temperate and tropical areas, including populations from West Africa and the Caribbean Sea, are necessary to complete a worldwide revision of the S. tetraceros complex. Reliable species delimitation within this complex will require a complete re-evaluation of morphological characters and ecological and biogeographical considerations, as well as the analysis of both mitochondrial and nuclear markers (e.g. microsatellites or single nucleotide polymorphisms). The combined use of morphological and molecular data, as carried out here, should be considered of paramount importance for the study of widely distributed invasive species.

ACKNOWLEDGEMENTSTop

Thanks are due to two anonymous reviewers whose comments and suggestions helped us to improve the manuscript. We thank Eunice Wong (formerly Australian Museum, Sydney) and Carol Simon (Stellenbosch University, South Africa) for their permission to use photos of Spirobranchus spp. for Figure 2.

REFERENCESTop

Antoniadou C., Nicolaidou A., Chintiroglou C. 2004. Polychaetes associated with the sciaphilic alga community in the northern Aegean Sea: Spatial and temporal variability. Helgol. Mar. Res. 58: 168-182.
https://doi.org/10.1007/s10152-004-0182-6

Baird W. 1865. Description of several new species and varieties of tubicolous annelides = Tribe LIMIVORA of Grube, in the collection of the British Museum. J. Linn. Soc. Lond. Zool. 8: 10-22.
https://doi.org/10.1111/j.1096-3642.1864.tb02002.x

Bastida-Zavala J.R., ten Hove H.A. 2002. Revision of Hydroides Gunnerus, 1768 (Polychaeta: Serpulidae) from the western Atlantic region. Beaufortia 52: 103-178.

Ben-Eliahu M.N. 1991. Red Sea serpulids (Polychaeta) in the eastern Mediterranean. Ophelia Suppl. 5: 515-528.

Ben-Eliahu M.N., ten Hove H.A. 1992. Serpulid tubeworms (Annelida: Polychaeta) a recent expedition along the Mediterranean coast of Israel finds new populations buildups of Lessepsian migrant species. Isr. J. Zool. 38: 35-53.

Ben-Eliahu M.N., ten Hove H.A. 2011. Serpulidae (Annelida: Polychaeta) from the Suez Canal from a Lessepsian migration perspective (a monograph). Zootaxa 2848: 1-147.
https://doi.org/10.11646/zootaxa.2848.1.1

Blainville H.M.D. de. 1818. Mémoire sur la classe des Sétipodes, partie des Vers à sang rouge de M. Cuvier, et des Annélides de M. de Lamarck. Bull. Sci. Soc. Philom. Paris 1818: 78-85.

Boore J.L., Brown W.M. 2000. Mitochondrial genomes of Galathealinum, Helobdella, and Platynereis: sequence and gene arrangement comparisons indicate that Pogonophora is not a phylum and Annelida and Arthropoda are not sister taxa. Mol. Biol. Evol. 17: 87-106.
https://doi.org/10.1093/oxfordjournals.molbev.a026241

Castresana J. 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol. 17: 540-552.
https://doi.org/10.1093/oxfordjournals.molbev.a026334

Çinar M.E. 2006. Serpulid species (Polychaeta: Serpulidae) from the Levantine coast of Turkey (eastern Mediterranean), with special emphasis on alien species. Aquat. Inv. 1: 223-240.
https://doi.org/10.3391/ai.2006.1.4.6

Çinar M.E. 2013. Alien polychaete species worldwide: Current status and their impacts. J. Mar. Biol. Assoc. UK 93: 1257-1278.
https://doi.org/10.1017/S0025315412001646

Edgar R.C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32: 1792-1797.
https://doi.org/10.1093/nar/gkh340

Fauvel P. 1923. Un nouveau serpulien d’eau saumatre Mercierella n. g., enigmatica n. sp. Bull. Soc. Zool. Fr. 47: 424-430.

Ferrario J., Caronni S., Occhipinti-Ambrogi A., et al. 2017. Role of commercial harbours and recreational marinas in the spread of non-indigenous fouling species. Biofouling 33: 651-660.
https://doi.org/10.1080/08927014.2017.1351958

Giangrande A., Delos A.L., Musco L., et al. 2004. Polychaete assemblages of rocky shore along the South Adriatic coast (Mediterranean Sea). Cah. Biol. Mar. 45: 85-95.

Glasby T.M., Connell S.D., Holloway M.G., et al. 2007. Nonindigenous biota on artificial structures: could habitat creation facilitate biological invasions? Mar. Biol. 151: 887-895.
https://doi.org/10.1007/s00227-006-0552-5

Gray E. 1843. Fauna of New Zealand. In: Dieffenbach E., Travels in New Zealand. vol. 2: 30-138, John Murray, London.

Grube A.E. 1862. Mittheilungen über die Serpulen, mit besonderer Berücksichtigung ihrer Deckel. Jahresber. Abh. Schles. Ges. Breslau 39: 53-69.

Gunnerus J.E. 1768. Om Nogle Norske Coraller. Kongel. Norske Vidensk. Selsk. Skr. 4: 38-73.

Haswell W.A. 1883. On some new Australian tubicolous annelids. Proc. Linn. Soc. N.S.W. 7: 633-638.

Kumar S., Stecher G., Li M., et al. 2018. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 35: 1547-1549.
https://doi.org/10.1093/molbev/msy096

Laubier L. 1966. Sur quelques Annélides Polychètes de la région de Beyrouth. Am. Univ. Beirut Misc. Pap. 5: 9-22.

Linnaeus C. von. 1758. Systema Naturae, 10th edition, Vol. 1. L. Salvius, Holmiae, 823 pp.

López E., Richter A. 2017. Non-indigenous species (NIS) of polychaetes (Annelida: Polychaeta) from the Atlantic and Mediterranean coasts of the Iberian Peninsula: an annotated checklist. Helgol. Mar. Res. 71: 19.
https://doi.org/10.1186/s10152-017-0499-6

Marenzeller E. von. 1885. Südjapanische Anneliden. II. Ampharetea, Terebellacea, Sabellacea, Serpulacea. Denkschriften der Akademie der Wissenschaften, Wien 49: 197-224.

Megina C., González-Duarte M.M., López-González P.J., et al. 2013. Harbours as marine habitats: Hydroid assemblages on sea-walls compared with natural habitats. Mar. Biol. 160: 371-381.
https://doi.org/10.1007/s00227-012-2094-3

Megina C., González-Duarte M.M., López-González P.J. 2016. Benthic assemblages, biodiversity and invasiveness in marinas and commercial harbours: an investigation using a bioindicator group. Biofouling 32: 465-475.
https://doi.org/10.1080/08927014.2016.1151500

Mineur F., Cook E.J., Minchin D., et al. 2012. Changing coasts: marine aliens and artificial structures. Oceanogr. Mar. Biol. 50: 189-234.

Morais P., Reichard M. 2018. Cryptic invasions: a review. Sci. Total Environ. 613-614: 1438-1448.
https://doi.org/10.1016/j.scitotenv.2017.06.133

Mörch O.A.L. 1863. Revisio critica Serpulidarum. Et Bidrag til Rørormenes Naturhistorie. Naturh. Tidsskr. København, Ser. 3, 1: 347-470.

Pallas P.S. 1766. Miscellanea Zoologica. Petrum van Cleef. Hagae Comitum, The Hague, 224 pp.

Palero F., Hall S., Clark P.F., et al. 2010. DNA extraction from formalin-fixed tissue: new light from the Deep-Sea. Sci. Mar. 74: 465-470.
https://doi.org/10.3989/scimar.2010.74n3465

Perry O., Bronstein O., Simon-Blecher N., et al. 2018. On the genus Spirobranchus (Annelida, Serpulidae) from the northern Red Sea, and a description of a new species. Invertebr. Syst. 32: 605-625.
https://doi.org/10.1071/IS17061

Philippi A. 1844. Einige Bemerkungen über die Gattung Serpula, nebst Aufzählung der von mir im Mittelmeer mit dem Thier beobachteten Arten. Arch. Naturgesch. 10: 186-198.
https://doi.org/10.5962/bhl.part.29558

Quatrefages A. de. 1866. Histoire naturelle des Annelés marins et d’eau douce. Annélides et Géphyriens. Librarie Encyclopédique de Roret. Paris. Vol. 1. 1-588
https://doi.org/10.5962/bhl.title.122818

Read G., Fauchald K. (ed.). 2019. World Polychaeta database. Accessed on 19/10/2019.
http://www.marinespecies.org/polychaeta

Rouse G.W. 2000. Family Serpulidae. In: Beesley P.L., Ross G.L.B., Glasby C.J. (ed.), Polychaetes and Allies: The Southern Synthesis, CSIRO. Melbourne. pp. 184-189.

Schmarda L.K. 1861. Neue Wirbellose Thiere: Beobachted und Gesammelt auf einer Reise um die Erdr 1853 bis 1857. In: Turbellarien, Rotatorien und Anneliden. Leipzig, Verlag von Wilhelm Engelmann. Erster Band, Zweite Hälfte.

Simon C.A., van Niekerk H.H., Burghardt I., et al. 2019. Not out of Africa: Spirobranchus kraussii (Baird, 1865) is not a global fouling and invasive serpulid of Indo-Pacific origin. Aquat. Inv. 14: 221-249.
https://doi.org/10.3391/ai.2019.14.2.05

Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312-1313.
https://doi.org/10.1093/bioinformatics/btu033

Streftaris N., Zenetos A. 2006. Alien marine species in the Mediterranean - the 100 “worst invasives” and their impact. Mediterr. Mar. Sci. 7: 87-118.
https://doi.org/10.12681/mms.180

Talavera G., Castresana J. 2007. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst. Biol. 56: 564-577.
https://doi.org/10.1080/10635150701472164

ten Hove H.A. 1970. Serpulinae (Polychaeta) from the Caribbean: I - The genus Spirobranchus. Stud. Fauna Curaçao Caribb. Isl. 32: 1-57.

ten Hove H.A., Kupriyanova E.K. 2009. Taxonomy of Serpulidae (Annelida, Polychaeta): The state of affairs. Zootaxa 2036: 1-126.
https://doi.org/10.11646/zootaxa.2036.1.1

Tyrrel M.C., Byers J. 2007. Do artificial substrates favor nonindigenous fouling species over native species? J. Exp. Mar. Biol. Ecol. 342: 54-60.
https://doi.org/10.1016/j.jembe.2006.10.014

Ulman A., Ferrario J., Occhpinti-Ambrogi A., et al. 2017. A massive update of non-indigenous species records in Mediterranean marinas. PeerJ 5: e3954.
https://doi.org/10.7717/peerj.3954

Ulman A., Ferrario J., Forcada A., et al. 2019a. A hitchhiker’s guide to alien species settlement in Mediterranean marinas. J. Environ. Manage. 241: 328-339.
https://doi.org/10.1016/j.jenvman.2019.04.011

Ulman A., Ferrario J., Forcada A., et al. 2019b. Alien species spreading via biofouling on recreational vessels in the Mediterranean Sea. J. Appl. Ecol. 56: 2620-2629.
https://doi.org/10.1111/1365-2664.13502

Willette D.A., Iñiguez A.R., Kupriyanova E.K., et al. 2015. Christmas tree worms of Indo-Pacific coral reefs: untangling the Spirobranchus corniculatus (Grube, 1862) complex. Coral Reefs 34: 899-904.
https://doi.org/10.1007/s00338-015-1294-y

Zenetos A., Gofas S., Verlaque M., et al. 2010. Alien species in the Mediterranean Sea by 2010. A contribution to the application of European Union’s Marine Strategy Framework Directive (MSFD). Part I. Spatial distribution. Mediterr. Mar. Sci. 11: 381-493.

Zenetos A., Çinar M.E., Crocetta F., et al. 2017. Uncertainties and validation of alien species catalogues: The Mediterranean as an example. Estuar. Coast. Shelf Sci. 191: 171-187.
https://doi.org/10.1016/j.ecss.2017.03.031

Zibrowius H. 1979. Serpulidae (Annélida Polychaeta) de l’Océan Indien arrivés sur le côques de bateaux à Toulon (France, Méditerranée). Rapp. P.-V. Reun. Comm. Int. Explor. Sci. Mer Mediterr. 25-26: 133-134.