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Fig. 6 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. 6: ML phylogenetic tree of the genus Polyclinum (sequences abbreviation: Pln) based on COI nucleotide sequences (1560 aligned nucleotide sites; best-fit substitution model GTR+I+G; bootstrap on 100 replicates). Eudistoma and Pseudodistoma species were used as outgroups. The sequence list and species abbreviations are reported in Supplementary table S1. Black dots: bootstrap values ≥ 70 %; red: P. constellatum sequences; blue: P. indicum sequences; yellow background: our sequences.

Fig. 4 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. 4: A, C) Colonies of Polyclinum constellatum with different colours photographed and collected in the Heraklion marina (Crete) (A: colony K11 and C: colony K12); B) Transversal section of the colonies, joined only at the surface layer (upper white arrow); D) Zooid extracted from the red-orange colony (K11), with magnification of the 6-lobed anus; E) Zooid extracted from the dark blue colony (K12) with magnification of the 6-lobed anus. Both K11 and K12 have the same COI haplotype (sequence AC number: MT873559).

Fig. 5 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. 5: A) Larva of P. constellatum, showing the ocellus, four long narrow ampullae, three adhesive papillae and a group of a few small ventral vesicles (red arrow). am, ampullae; ap, adhesive papillae; oc, ocellus; B) Larva of P. constellatum, red arrow pointing out the calcite crystal in the middle of the body.

Fig. 2 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. 2: A) Orange colony of Polyclinum constellatum from Taranto harbour (colony P1); B) Magnification of the oral (arrow pointing put the oral tentacles of different size) and cloacal aperture (asterisk); C) P. constellatum collected in Heraklion (colony K19) with zooids arranged in systems around the cloacal apertures; D) Section of the colony showing the zooids located only around the outer edge (arrow).

Fig. 3 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. 3: A) Whole zooid of Polyclinum constellatum, showing a clear division into thorax, abdomen and post-abdomen with a long vascular stolon. ab, abdomen; pa, post-abdomen; th, thorax; vs, vascular stolon; B) Zooid with evident pharynx, rectum, anus and four embryos incubated in the atrial cavity. The funnel-shaped oesophagus, the smooth stomach and the twisted gut loop are visible in the abdomen. The post-abdomen shows the heart at its terminal end, as well as several rounded testicular follicles and the ovary, with the gonoducts running parallel to the rectum. an, anus; e, embryos; gd, gonoducts; gl, gut loop; oe, oesophagus; ov, ovary; h, heart; r, rectum; st, stomach; tf, testicular follicles; C) Magnification of the oral siphon with six pointed lobes (arrows) and six longitudinal muscle bands (indicated with numbers 1-6); D) Branchial sac with 18 rows of stigmata and narrow languets of the dorsal lamina (arrows); E) Magnification of the pharynx, with minute papillae (arrows) at the level of the transverse vessels; F) Magnification of the six-lobed anus (lobes indicated with numbers 1-6).

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-

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).

Fig. 14 in Revision of the genus complex Gibbula: an integrative approach to delineating the Eastern Mediterranean genera Gibbula Risso, 1826, Steromphala Gray, 1847, and Phorcus Risso, 1826 using DNA-barcoding and geometric morphometrics (Vetigastropoda, Trochoidea)

Fig. 14 Phorcus turbinatus (Born, 1778). a PCA plot of PC1 vs. PC2 for genus Phorcus. Phorcus turbinatus (petrol) separates from all other species. b Representative specimen of Ph. turbinatus from this study. c One syntype of Ph. turbinatus (NHMW 14002). Scale bars 5 mm

Fig. 12 in Revision of the genus complex Gibbula: an integrative approach to delineating the Eastern Mediterranean genera Gibbula Risso, 1826, Steromphala Gray, 1847, and Phorcus Risso, 1826 using DNA-barcoding and geometric morphometrics (Vetigastropoda, Trochoidea)

Fig. 12 Phorcus richardi (Payraudeau 1826). a PCA plot of PC1 vs. PC2 of genus Phorcus. Phorcus richardi (maroon) is the most abundant species in the current sample. It separates well from Ph. articulatus and Ph. turbinatus. One group of Ph. mutabilis is not distinguishable from Ph.

Fig. 11 in Revision of the genus complex Gibbula: an integrative approach to delineating the Eastern Mediterranean genera Gibbula Risso, 1826, Steromphala Gray, 1847, and Phorcus Risso, 1826 using DNA-barcoding and geometric morphometrics (Vetigastropoda, Trochoidea)

Fig. 11 Steromphala divaricata (Linnaeus, 1758). a PCA plot of PC1 vs. PC2 from genus Steromphala. Steromphala divaricata (pink) cannot be recovered as a completely separated group as it shows a small overlap with St. varia. Type material and material from this study do not overlap. Three individuals (Linné 41–43) of the type material (pink group on the

Fig. 10 in Revision of the genus complex Gibbula: an integrative approach to delineating the Eastern Mediterranean genera Gibbula Risso, 1826, Steromphala Gray, 1847, and Phorcus Risso, 1826 using DNA-barcoding and geometric morphometrics (Vetigastropoda, Trochoidea)

Fig. 10 Steromphala varia (Linnaeus, 1758). a PCA plot of PC1 vs. PC3 of genus Steromphala. Steromphala varia (blue) separates from all other species. Specimens from this study overlap with the type material. b

Fig. 4 in Revision of the genus complex Gibbula: an integrative approach to delineating the Eastern Mediterranean genera Gibbula Risso, 1826, Steromphala Gray, 1847, and Phorcus Risso, 1826 using DNA-barcoding and geometric morphometrics (Vetigastropoda, Trochoidea)

Fig. 4 Plot of first and third principal component of combined lateral and ventral landmark data from Phorcus and Steromphala individuals. A morphological separation of the two genera becomes apparent

Fig. 5 in Revision of the genus complex Gibbula: an integrative approach to delineating the Eastern Mediterranean genera Gibbula Risso, 1826, Steromphala Gray, 1847, and Phorcus Risso, 1826 using DNA-barcoding and geometric morphometrics (Vetigastropoda, Trochoidea)

Fig. 5 Lectotype of Steromphala cineraria (Linnaeus, 1758). Located at LSL (LSL.502). Scale bar 5 mm

Fig. 2 Landmark templates for representative a lateral and b in Revision of the genus complex Gibbula: an integrative approach to delineating the Eastern Mediterranean genera Gibbula Risso, 1826, Steromphala Gray, 1847, and Phorcus Risso, 1826 using DNA-barcoding and geometric morphometrics (Vetigastropoda, Trochoidea)

Fig. 2 Landmark templates for representative a lateral and b ventral standardised views. Filled circles show fixed landmarks. Empty circles show semilandmarks, processed as sliding landmarks

FIGURE 4 in Integrative taxonomy reveals disjunct distribution and first record of Hoplias misionera (Characiformes: Erythrinidae) in the Amazon River basin: morphological, DNA barcoding and cytogenetic considerations

FIGURE 4 | Neighbor joining (NJ) tree of Hoplias inferred from partial COI (Cytochrome c Oxidase Subunit I gene) sequences using the Kimura 2-parameter model. The lateral bar indicates the partitions of species delimitation performed by the GMYC, ABGD and BIN analysis. The clade Hoplias misionera nested individuals from the La Plata and Amazon basins (blue tips).

FIGURE 7 in Integrative taxonomy reveals disjunct distribution and first record of Hoplias misionera (Characiformes: Erythrinidae) in the Amazon River basin: morphological, DNA barcoding and cytogenetic considerations

FIGURE 7 | Updated distribution map of Hoplias misionera showing former known localities in Argentina and southern Brazil (Rosso et al., 2016) and the new records from Amazon basin (triangles). Star = type locality.

FIGURE 1 in Integrative taxonomy reveals disjunct distribution and first record of Hoplias misionera (Characiformes: Erythrinidae) in the Amazon River basin: morphological, DNA barcoding and cytogenetic considerations

FIGURE 1 | Hoplias misionera, UFOPA AMTRA131-19, 237 mm SL, Amazonas River, Alenquer, Pará, Brazil. Lateral view. Scale bar = 1 cm. Photo by L.R.R. Rodrigues.

FIGURE 2 in Integrative taxonomy reveals disjunct distribution and first record of Hoplias misionera (Characiformes: Erythrinidae) in the Amazon River basin: morphological, DNA barcoding and cytogenetic considerations

FIGURE 2 | Configuration of the medial margins of the dentary in Hoplias misionera. A. Y-shaped, UFOPA AMTRA126-19, 214 mm SL. B. V-shaped, UFOPA AMTRA127-19, 232 mm SL. Scale bars = 1 cm. Photos by L. R. R. Rodrigues. Illustration by T. M. A. Lima.

FIGURE 6 in Integrative taxonomy reveals disjunct distribution and first record of Hoplias misionera (Characiformes: Erythrinidae) in the Amazon River basin: morphological, DNA barcoding and cytogenetic considerations

FIGURE 6 | Partial idiogram of the four largest chromosome pairs of Hoplias malabaricus (karyomorphs C and F) and H. misionera showing marked size reduction from the first to second metacentric pair only in the karyomorph F.

FIGURE 3 in Integrative taxonomy reveals disjunct distribution and first record of Hoplias misionera (Characiformes: Erythrinidae) in the Amazon River basin: morphological, DNA barcoding and cytogenetic considerations

FIGURE 3 | Last vertical series of scales on the base of the caudal-fin rays. Comparison between Hoplias misionera (A), UFOPA AMTRA131-19, 237 mm SL and Hoplias cf. malabaricus (B), UFOPA AMTRA110, 201 mm SL. Scale bars = 1 cm. Photos by L. R. R. Rodrigues. Illustration by T. M. A. Lima.