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Fig. 3 in Description of Brooksia lacromae sp. nov. (Tunicata, Thaliacea) from the Adriatic Sea

Fig. 3. Drawing of the blastozooid of Brooksia lacromae sp. nov. (sinistral individual). A. Dorsal view. B. Ventral view. Abbreviations: ap1–3 = attachment processes; as = atrial siphon; br = branchial bar; dt = dorsal tubercle; e = embryo; en = endostyle; g = ganglion; h = heart; nu = nucleus; os = oral siphon; pb = peripharyngeal band; IL–IVL = left side muscles; IR–IIIR = right side muscles; IM1 and IM2 = intermediate muscles.

Fig. 4 in Description of Brooksia lacromae sp. nov. (Tunicata, Thaliacea) from the Adriatic Sea

Fig. 4. Brooksia lacromae sp. nov. A. Photograph of the ventral side of the dextral blastozooid. B. Photograph of the dorsal side of the sinistral blastozooid. C. Photograph of the lateral side of a blastozooid. D. Detail of the ventral posterior end of an oozooid. All indivuduals were dyed with Janus Green dye. Abbreviations: st = stolon; tp = test processus.

Fig. 2 in Description of Brooksia lacromae sp. nov. (Tunicata, Thaliacea) from the Adriatic Sea

Fig. 2. Oozooid of Brooksia lacromae sp. nov. A. Dorsal view, drawing. B. Ventral view, drawing. C. Dorsal view, photograph of an individual dyed with Janus Green, with an arrow indicating breakage in the proboscis during collection. Abbreviations: as = atrial siphon; bl = branching of the ventral longitudinal muscle; br = branchial bar; dt = dorsal tubercle; en = endostyle; g = ganglion; im = intermediate muscle; lm = longitudinal muscle; lo = openings in the longitudinal muscle; nu = nucleus; os = oral siphon; p = proboscis; pb = peripharyngeal band; st = stolon; tp = test processus; I–VII = body muscles.

Fig. 1 in Description of Brooksia lacromae sp. nov. (Tunicata, Thaliacea) from the Adriatic Sea

Fig. 1. Position of the Lokrum station in the South Adriatic, where specimens of Brooksia lacromae sp. nov. were collected.

Figure 3 in The genus Herdmania Lahille, 1888 (Tunicata, Ascidiacea) in Australian waters

Figure 3. Herdmania mentula sp. nov. (A, E, WAM Z11756; B-D, WAM Z11771;F, WAM Z11761): A, body from left side showing muscles. B, dorsal tubercule; C, portion of dorsal lamina; D, gut and gonad; E, F, distal part of gut and gonads, showing gonoducal openings and membranes, and anal border. Scale bars: A, D = 5.0 mm; B, C, E, F = 1.0 mm.

Figure 1 in The genus Herdmania Lahille, 1888 (Tunicata, Ascidiacea) in Australian waters

Figure 1. Herdmania fimbriae sp. nov. (A, C, D, I, SAM E2892; B, L, QM GH4405; E, K, QM GH 2299; F, G, J, SAM E2891; H, QM GH933). A, B, body wall from left side; C, dorsal tubercle; D, dorsal lamina; E–H, distal part of gonads showing gonoducal openings and membranes; I, gut loop and gonads; J–L, anal border. Scale bars: A, I = 5.0 mm; B = 2.0 mm; C–H, J–L = 1.0 mm.

Figure 4 in The genus Herdmania Lahille, 1888 (Tunicata, Ascidiacea) in Australian waters

Figure 4. Herdmania momus (A, C, QM G10093; B, QM GH561; D, L, QM G308151; E-G, QM G9363; H, QM GH2554; I-K, QM GH771): A, body from left side showing muscles; B, portion of dorsal lamina; C, D, dorsal tubercles; E, gut loop and gonads; F-H, distal part of gonad showing oviducal hood and male openings surrounded by testis follicles; I, testis follicles with tips of vas deferens showing amongst them; J, clumps of testis follicles with vas deferens; K, L, anal borders. Scale bars: A, E = 5.0 mm; B-D, F = 1.0 mm; H, J = 0.5 mm; K, L = 2.0 mm.

Figure 2 in The genus Herdmania Lahille, 1888 (Tunicata, Ascidiacea) in Australian waters

Figure 2. Herdmania grandis (A, C, D, F, QM GH377; B, E, QM GH2200; G, QM GH5759): A, body from left side showing muscles; B, dorsal tubercle showing early stage in development of convoluted slit; C, portion of dorsal lamina; D, gut loop; E, F, anus and gonoducal openings; G, anus. Scale bars: A, D = 5.0 mm; B, E-G = 1.0 mm; C = 0.5 mm.

Fig. 4 in Graveyards of Giant Pandas at the Bottom of the Sea? A Strange-Looking New Species of Colonial Ascidians in the Genus Clavelina (Tunicata: Ascidiacea)

Fig. 4. Clavelina ossipandae sp. nov., holotype, ICHUM 5837. A, Intestinal loop and gonads, viewed from the right side; B, larva.

Fig. 3 in Graveyards of Giant Pandas at the Bottom of the Sea? A Strange-Looking New Species of Colonial Ascidians in the Genus Clavelina (Tunicata: Ascidiacea)

Fig. 3. Clavelina ossipandae sp. nov., holotype, ICHUM 5837. A, Pharynx opened ventrally; B, enlarged view of a square in A, showing dorsal part of the peripharyngeal area.

Fig. 2 in Graveyards of Giant Pandas at the Bottom of the Sea? A Strange-Looking New Species of Colonial Ascidians in the Genus Clavelina (Tunicata: Ascidiacea)

Fig. 2. Clavelina ossipandae sp. nov., holotype, ICHUM 5837. A, In situ live colony with four zooids; B, a schematic depiction of the colony showing colony organization and zooid insertion; C, D, a single zooid detached from the colony, drawn from the right side (C) and the left side (D).

Fig. 5. A in Graveyards of Giant Pandas at the Bottom of the Sea? A Strange-Looking New Species of Colonial Ascidians in the Genus Clavelina (Tunicata: Ascidiacea)

Fig. 5. A maximum-likelihood tree based on COI sequences (690 bp), showing the phylogenetic position of Clavelina ossipandae sp. nov. among Clavelinidae. Numbers near nodes indicate UF bootstrap values (≥ 50) and posterior probabilities (≥ 0.50).

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).