Fig. 1 in Polyclinum constellatum (Tunicata, Ascidiacea), an emerging non-indigenous species of the Mediterranean Sea: integrated taxonomy and the importance of reliable DNA barcode data Abstract

Fig. 1: Map of the Mediterranean Sea showing the literature records (black rhombuses) of P. constellatum and the new findings (red dots). performed in a final reaction volume of 25 μl contain- nus was reconstructed with the online software PHYML ing: 1X reaction buffer with 1 mM final concentration of v3.0 (http://www.atgc-montpellier.fr/phyml-sms/) (Guin- MgCl 2 (Takara Bio Inc.), 0.2 mM of each dNTP, 0.3 μM don & Gascuel, 2003), which also includes the automatof each primer and 1.25 Units of PrimeStar HS (Takara ic model selection algorithm SMS (Smart Model Selec- Bio Inc.). Amplification conditions were: 30 cycles with tion). The best-fit substitution model was selected using denaturation for 10 s at 98°C, annealing for 15 s at 46°C the Akaike Information Criterion (AIC). Bootstrap val- or 50°C, extension for 1 min 30 s at 72°C; a final elonga- ues, indicating node reliability, were based on 100 reption step of 5 min at 72°C. licates. The sequence dataset used for this phylogenetic PCRs with the DreamTaq polymerase were performed reconstruction is reported in Supplementary Table S1 and in a final volume of 25 μl containing: 1X reaction buffer was extracted from the phylogenetic dataset published in with 2 mM final concentration of MgCl 2 (Thermo Fish- Tabudravu et al. (2019). It includes representative species er Scientific), 0.2 mM of each dNTP, 0.4 μM of each of of the Polyclinidae family plus Eudistoma and Pseudodithe two primers, and 1.25 Units of DreamTaq polymerase stoma species chosen as outgroups for their morphologi- (Thermo Fisher Scientific). The amplification conditions cal similarities with Polyclinidae. were as follows: an initial denaturation for 3 min at 95°C, then 34 amplification cycles (denaturation for 30 s at 95°C; annealing for 30 s at 46-50°C; extension for 1 min Results 30 s at 72°C) followed by a final elongation step of 5 min at 72°C. Morphological analyses The obtained amplicons were purified with the DNA Clean&Concentrator kit (Zymo Research) and directly The colonies collected in Taranto harbour and Hersequenced according to the Sanger method by Microsynth aklion marina were all morphologically identified as P. AG (Switzerland). The sequence quality check, compar- constellatum based on the following features: colonies isons and alignment were carried out with Geneious ver. without sand in/outside, zooids arranged in systems, 5.5.7.2 (Kearse et al., 2012). The sequences obtained post-abdomen (without vascular stolon) shorter than the were deposited in the GenBank database (see Accession thorax and abdomen combined, pharynx with 16-18 rows numbers MT873559 and OL597608). For comparative of stigmata, more than 15 stigmata per row, and a 6-lobed analyses, homologous sequences of the genus Polycli- anus. These characteristics are in accordance with the key num were searched for in the non-redundant nucleotide of Polyclinum species edited by Kott (1963) and they are database (nr-nt db, on 21st September 2021) of the NCBI also reported in the description of the species made by (National Center for Biotechnology Information) by En- Van Name (1945). trez text search, and by BLASTn (Altschul et al., 1990) using our P. constellatum sequences as the query. Uncorrected pairwise distances were calculated with PAUP 4.0a (Swofford, 2002), while a Maximum Likelihood (ML) phylogenetic tree of the genus Polyclinum ge-