Polyclinum constellatum (Tunicata, Ascidiacea), an emerging non-indigenous species of the Mediterranean Sea: integrated taxonomy and the importance of reliable DNA barcode data Abstract Author Montesanto, Federica Department of Biology, University of Bari “ Aldo Moro ”, Via E. Orabona, 4, 70125, Bari, Italy & CoNISMa, Piazzale Flaminio 9, 00197, Roma, Italy Author Chimienti, Giovanni Department of Biology, University of Bari “ Aldo Moro ”, Via E. Orabona, 4, 70125, Bari, Italy & CoNISMa, Piazzale Flaminio 9, 00197, Roma, Italy Author Gissi, Carmela CoNISMa, Piazzale Flaminio 9, 00197, Roma, Italy & Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari “ Aldo Moro ”, Via Orabona 4, 70125 Bari, Italy & Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), CNR, Via Amendola 165 / A, 70126 Bari, Italy Author Mastrototaro, Francesco Department of Biology, University of Bari “ Aldo Moro ”, Via E. Orabona, 4, 70125, Bari, Italy & CoNISMa, Piazzale Flaminio 9, 00197, Roma, Italy text Mediterranean Marine Science 2021 2022-02-07 23 1 69 83 http://dx.doi.org/10.12681/mms.28311 journal article 298603 10.12681/mms.28311 286c7f3d-51e4-499d-a05d-e0f2dfba3701 1108-393X 11989653 Polyclinum constellatum Savigny, 1816 Polyclinum constellatum Savigny, 1816:189 ; Van Name, 1945:68; Gravier, 1955:620; Millar, 1955:176 ; 1958:498 ; 1962:62; 1975:257; Tokioka, 1961:104 ; 1967:53 ; Almeida Rodrigues, 1962:194; Rodrigues Da Costa, 1969:192; Vasseur, 1970:212; Monniot C. & Monniot F., 1976:358; Monniot F., 1972:958. Material examined Taranto ( 40.478966°N , 17.225649°E ): ten colonies collected in Taranto harbour at 1 m depth, separately preserved in 4%formalin in seawater and 99% ethanol. One colony was examined from both the morphological and molecular point of view (P1), and for the others only the morphological characteristics were checked. Heraklion ( 35.343300°N , 25.136614°E ): ten colonies collected within the marina at 3-4 m depth, separately preserved both in 4%formalin and 99% in ethanol. Five colonies were examined from both the morphological and molecular point of view (K7, K11, K12, K34). The remaining five colonies were only checked for morphological characteristics. Two colonies were deposited in the collection of the Zoological Museum of the University of Bari (MUZAC): one collected in Heraklion (MUZAC: 6661 preserved in 4% formalin and MUZAC: 6662 in 99% ethanol) and the other in Taranto (MUZAC: 6663 preserved in 4% formalin and MUZAC: 6664 in 99% ethanol). Colonies The colonies collected in Taranto show brown-orange colour ( Fig. 2 A-B; colony P1), while colonies collected from Heraklion show different colours from red-orange (colony K11) to purplish black (colony K12) or greenish-blue (colony K7) ( Figs. 2 C-D, 4A-C). Some of these different coloured colonies appear to be joined or almost-completely fused together ( Fig. 4 A-C; colonies K11 and K12). Most of the colonies are massive and almost rounded in shape, up to 10 cm in height and 20 cm in width. They are characterized by a thick firm tunic without sand embedded, with zooids located only around the outer edge ( Fig. 2D ). Systems of zooids are arranged along the common cloacal openings consisting of small conical projections of the outer tunic ( Fig. 2 A-C). The red-orange colonies and the dark ones appear greyish and black, respectively, after preservation in 4% formalin. Zooids The zooids are 7-9 mm in length, reaching almost 12- 15 mm with the vascular stolon ( Fig. 3A ). The thorax is longer than the abdomen. The post-abdomen is slightly longer than the abdomen and has a very long vascular stolon at its end ( Fig. 3 A-B). The branchial siphon ends with six pointed lobes and shows four orders of oral tentacles arranged regularly ( Fig. 2B ). The atrial siphon consists of a wide aperture with a long pointed atrial languet at its edge ( Fig. 3 A-B). The pharynx is characterized by 16–18 rows of stigmata, with about 16-18 stigmata per half-row (counted at the level of the 7 th row) ( Fig. 3B ). The branchial transverse vessels show minute papillae not corresponding to the number of stigmata, but usually fewer in number ( Fig. 3E ). The dorsal lamina consists of narrow pointed languets ( Fig. 3D ). Six longitudinal muscles are visible near the branchial siphon ( Fig. 3C ) running towards the thorax on each side of the zooids. Up to 10 embryos can be incubated in the atrial cavity ( Fig. 3B ). 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). Alimentary canal The abdomen contains the alimentary canal with a funnel-shaped oesophagus, followed by a smooth stomach and a twisted gut loop which lies almost horizontally ( Fig. 3B ). The rectum is long, extending to the middle of the thorax, ending in a six-lobed anus at the level of the 9 th row of stigmata ( Fig. 3B ). Post-abdomen The sac-like post-abdomen is connected to the abdomen by a narrow stalk and contains the gonads and the heart ( Fig. 3B ). The testis consists of a cluster of follicles joined by ducts to the ovary lying among the male follicles, while the gonoducts end close to the anus ( Fig. 3B ). The heart is positioned at the end of the post-abdomen ( Fig. 3B ), followed by a long narrow vascular stolon ( Fig. 3A ). 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). Larva Several embryos (from 4 up to 15) are incubated in the peribranchial cavity. Fully developed larvae are approximately 0.5 mm in trunk length, and they have one or two calcite crystals usually placed in the middle of the body or close to the ocellus ( Fig. 5 A-B). They have three adhesive papillae on a sagittal plane and four pairs of long ectodermal ampullae. Furthermore, a group of small vesicles is usually present on the ventral side of the larvae ( Fig. 5A ). 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: 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. COI sequence comparisons The obtained COI sequences were 811-816 bp long and correspond to two haplotypes differing in only 5 nucleotides (i.e., 4 synonymous substitutions at the third codon position and one non-synonymous substitution at the first codon position). One haplotype (named P1) is shared between the Taranto and five Heraklion colonies, while the other haplotype (named K7) was only found in the blue-greenish K7 colony from Heraklion. Moreover, the COI sequences obtained from colonies with different morphotypes showed 100% identity, including the red-orange (K11) and dark blue (K12) Heraklion colonies joined together ( Fig. 4 A-E). A total of 33 Polyclinum sequences belonging to 7 different species were found in the nt-nr database by text and similarity search (NCBI, 21st Sept 2021 ). As detailed in Table 2 , many of these sequences show multiple unexpected indels, terminal regions lacking COI similarity and/or containing long homopolymers, or no significant similarity to ascidians. Six of these sequences are also described in the entry as pseudogenes or “unverified” sequences, meaning that the GenBank staff were unable to verify their accuracy and/or annotations ( Table 2 ). All these elements are clues of low-quality sequences, or possible PCR contamination. Indeed, our BlastN analyses show that three of these Polyclinum sequences belong to Lepidoptera and two others do not have significant similarity to any nr-nt sequence, so they cannot by unambiguously assigned to a species ( Table 2 ). Table 3 reports the mean inter- and intra-species pairwise uncorrected distances, calculated only for the confirmed Polyclinum sequences and excluding the above-mentioned non-homologous or low-quality terminal regions. It is noteworthy that the mean distance between P. constellatum and P. indicum sequences is only 1.34% ± 0.65% and all comparisons between P. constellatum , P. indicum , and P. madrasensis show a mean uncorrected difference <1.34%. In addition, our K7 haplotype is identical to a short P. constellatum sequence from Puerto Rico (see MT 637964 in Table 2 ), and both P1 and K7 haplotypes have a mean distance from indicum / constellatum / madrasensis ranging from 0.09 to 1.25% ( Table 3 ) Based only on these data, our specimens could be equally assigned to P. constellatum , to P. indicum , or P. madrasensis . However, our morphological results clearly show that both our haplotypes belong to P. constellatum . It should be noted that all public COI sequences, including the few published ones ( Table 2 ), lack a morphological description of the samples. Only for two P. indicum sequences (MH235542 and MH367293), do the Authors report that they were originally identified as belonging to Polyclinum saturnium through conventional morphological analyses and then re-assigned to P. indicum based on similarity to the public COI ( Khan et al ., 2020 ). A ML phylogenetic reconstruction of Polyclinidae representatives ( Fig. 6 ) shows the monophyly of the genus Polyclinum , but all nodes inside the genus remain unresolved, except for a statistically significant clade containing all P. constellatum sequences except P1 (the position of the P1 haplotype is unresolved). Moreover, our results suggest taxonomic mis-assignments of the P. indicum and P. madrasensis public COI sequences. Taxonomic remarks The Polyclinidae family includes colonial species with a lobed oral siphon, zooids arranged in systems with common cloacal openings, as well as the gonads and heart in the post-abdomen. The genus Polyclinum is characterized by a twisted gut loop, almost horizontal, and a pharynx with simple branchial papillae. Polyclinum constellatum is the type species of the genus. The only species belonging to the genus Polyclinum known thus far in the Mediterranean Sea is P. aurantium Milne-Edwards, 1841 ( Brunetti & Mastrototaro, 2017 ). This species differs from P. constellatum mainly in having one or few zooid systems per colony and the presence of sand grains in the tunic. This is also confirmed by Van Name (1945), Tokioka (1961) and Fortaleza & Lotufo (2018) who report that P. constellatum colonies are usually free of sand, and whether some sand is present on the surface or not, it does not pervade the interior of the colony. The World Register of Marine Species (WoRMS Editorial Board, 2022) reports 47 species belonging to the genus Polyclinum . Among these species, P. macrophyllum Michaelsen, 1919 , P. complanatum Herdman, 1899 , and P. hospitale Sluiter, 1909 cannot be considered valid species. In detail, P. macrophyllum cannot be separated from P. vasculosum ( sensu Kott, 1992 ). P. complanatum is not properly assigned, indeed it has a long abdomen different from that characteristic of Polyclinidae species, and it could be a species of Pseudodistoma ( Kott, 1992 ) . Finally, the correct authorship of P. hospitale should be Sluiter, 1895, and it is probably an Aplidium species ( Kott, 1992 ). Three valid species are not yet present in WoRMS: P. minutum Herdman, 1886 , P. tralaticia Sluiter, 1913 , and P. ramosum Parker-Nance, 2003 . P. tralaticia is considered valid by Kott (1992) and Parker-Nance (2003) albeit erroneously reported as P. tralatica . P. ramosum , described as a new species by Parker-Nance (2003) , has sand embedded in the tunic, branchial papillae one third of the length of the stigmata and a bilobed anus. P. minutum has a branchial sac with only 8-9 rows of stigmata. Considering the above-mentioned considerations, Table 1 reports a taxonomic key for 47 Polyclinum species , listed according to their main features and completed with authorship. Among the 47 accepted species of Polyclinum , only 9 are characterized by the absence of sand embedded in or coating the colonies. Moreover, most of the species characterized by the absence of sand outside or throughout the tunic have a lower number of rows of stigmata compared to those of P. constellatum , apart from for P. planum (Ritter & Forsyth, 1917) (13-14 rows of stigmata) and P. johnsonii Monniot & Monniot, 1989 (16-17 rows). However, P. planum can be distinguished from P. constellatum by the characteristic paddle-shaped colonies with the zooids arranged in rows only along one side of the colony, while P. johnsoni has a higher number of stigmata per half-row (23-25). Table 2. COI sequences of the nt-nr database (NCBI, 21 September 2021) analysed in this study. Incorrectly assigned sequences are reported in bold, identified as not belonging to ascidians according to BlastN analyses. *haplotypes analysed in this study.

AC Number	Notes	Species and Description	bp	Locality	Reference
AY116606	Lepidoptera from BlastN vs . nr-nt	Polyclinum aurantium , pseudogene	669	France	Stach & Turbeville, 2002
JQ408691	No significant similarity in BlastN	Polyclinum aurantium , pseudogene	380	India	Jaffar Ali et al ., 2016
KF414702	Lepidoptera from BlastN vs . nr-nt	UNVERIFIED: Polyclinum aurantium	646	India	Jaffar Ali unpub
KF414711	Lepidoptera from BlastN vs. nr-nt	UNVERIFIED: Polyclinum sp. ASP-2013	644	India	Jaffar Ali unpub
KJ944391	Terminal region non-homologous to COI	UNVERIFIED: Polyclinum indicum	507	India	Ahmed & Jaffar Ali, 2015
KP072781	Polyclinum indicum	550	India	Jaffar Ali unpub
KT276223	Polyclinum indicum	565	India	Jaffar Ali unpub
KT345963	Polyclinum indicum	550	India	Jaffar Ali unpub
KX650772	Terminal region non-homologous to COI	Polyclinum indicum	532	India	Jaffar Ali unpub
KX650773	Polyclinum indicum	576	India	Jaffar Ali unpub
KX650774	Polyclinum indicum	551	India	Jaffar Ali unpub
KX650775	Terminal region non-homologous to COI	Polyclinum indicum	682	India	Jaffar Ali unpub
KX650776	Polyclinum indicum	580	India	Jaffar Ali unpub
KX650777	Polyclinum indicum	667	India	Jaffar Ali unpub
KX650778	Polyclinum indicum	600	India	Jaffar Ali unpub
KX650779	Polyclinum indicum	589	India	Jaffar Ali unpub
KX650780	Polyclinum indicum	600	India	Jaffar Ali unpub
KX650781	Polyclinum indicum	631	India	Jaffar Ali unpub
KX650782	Terminal region non-homologous to COI and with homopolymers	Polyclinum indicum	689	India	Jaffar Ali unpub
KX650783	Multiple indels	Polyclinum indicum	609	India	Jaffar Ali unpub
KX650784	Terminal region non-homologous to COI and with homopolymers	Polyclinum indicum	593	India	Jaffar Ali unpub
KX650785	Polyclinum indicum	610	India	Jaffar Ali unpub
KX650786	Polyclinum indicum	610	India	Jaffar Ali unpub
MH235542	Multiple indels	Polyclinum indicum	508	India	Khan et al. , 2020
MH367293	Polyclinum indicum	610	India	Khan et al. , 2020
MT637964	Polyclinum constellatum	593	Puerto Rico	Nydam, unpub
MT873559*	Polyclinum constellatum , haplotype P1	816	Italy	This study
OL597608*	Polyclinum constellatum , haplotype K7	811	Grece	This study
MW858363	Polyclinum constellatum	863	Florida, USA	Nydam, unpub
MW858364	Polyclinum constellatum	859	Florida, USA	Nydam, unpub
MW990087	Polyclinum constellatum	808	Abu Dhabi Emirates	Govindharaj et al. , unpub
KX650787	Multiple indels and terminal region non-homologous to COI	Polyclinum saturnium	502	India	Jaffar Ali unpub
JN107814	No significant similarity in BlastN	UNVERIFIED: Polyclinum madrasensis	641	India	Jaffar Ali unpub
KU220954	Multiple indels	Polyclinum madrasensis	531	India	Jaffar Ali unpub
KX138512	Multiple indels and terminal region non-homologous to COI	Polyclinum sp. HAJ-2016	600	India	Jaffar Ali unpub

Most of the remaining accepted species are characterized by having sand embedded in or coating the tunic. The main morphological characters of these species are detailed and summarized in Table 1 . In particular, according to the COI comparisons, the sandy tunic species P. indicum Sebastian, 1952 and P. saturnium ( Savigny, 1816 ) is grouped with P. constellatum . P. indicum is characterized by zooids having an enlarged languet with a small protuberance at its end ( Sebastian, 1954 ) ( Erulan & Ananthan, 2009 ). The specific name P. indicum is no longer accepted, since it was revised by Brunetti (2007) , who proposed P. sebastiani as nomen novum. P. saturnium has colonies coated with sand, a denticulated atrial lip, only 8-12 rows of stigmata and a bilobed anus ( Kott, 1992 ). Lastly, the absence/presence of sand is not reported for P. hesperium Savigny, 1816 , P. meridianum Sluiter 1900, P. sibiricum Redikorzev, 1907 , or P. tralaticia ( Sluiter, 1913 ) . P. hesperium is a poorly described species collected by Savigny (1816) in the Suez gulf and then by Shenkar (2012) in the Red Sea, which is characterized by 18-20 row of stigmata. The P. meridianum description is only based on its original description ( Sluiter, 1900 ), reporting colonies with a poorly developed tunic, a trilobed atrial languet and 9 rows of stigmata. P. sibiricum has systems of 8-10 zooids with 12 rows of stigmata and 15 stigmata per half-row ( Redikorzev, 1907 ). P. tralaticia has 10 rows of stigmata with 10-12 stigmata per half-row and no branchial papillae on transverse vessels ( Sluiter, 1913 ). According to the key to species in Table 1 , P. constellatum is close to the co-generic species P. johnsoni , P. planum and P. hesperium . However, P. constellatum has a slightly different number of stigmata per half-row compared to those of P. planum and P. johnsoni and less rows of stigmata compared to those of P. hesperium . Moreover, P. johnsoni , which has only been collected at a depth of 300 m is characterized by transverse vessels bearing long papillae with an orange-yellow opaque pigment, as well as short wide dorsal languets ( Monniot & Monniot, 1989 ), while P. planum is characterized by the pedunculated shape of the colony ( Van Name, 1945).