SHORT TERM SCIENTIFIC MISSION (STSM) SCIENTIFIC REPORT

This report is submitted for approval by the STSM applicant to the STSM coordinator

Action number: CA15219 STSM title: Filling gaps in Mediterranean biodiversity – the Okenia: species identity and species gap-analysis in DNA barcode libraries. STSM start and end date: 07/01/2019 to 01/03/2019 Grantee name: Sofía Paz Sedano

PURPOSE OF THE STSM:

The aim of this STSM was the study of the genus Okenia (Mollusca: Nudibranchia) in the Mediterranean Sea. The specific tasks were: 1) Review published records of the different Okenia species inhabiting the Mediterranean Sea; 2) Carry out morphological analyses of specimens newly sampled from the Mediterranean Sea; 3) Cross-check a-priori morphological identifications of Mediterranean specimens with barcoding tools, amplifying and sequencing COI, 16S, and H3genes; 4) Establish a new protocol for sequencing and design new effective primers for future studies; 5) Complement the study with subsequent morphological and molecular analyses of putative sister taxa sampled in both Atlantic Sea coast; 6) Possibly sequence additional taxa belonging to the family H. Adams & A. Adams, 1854; 7) Carry out an updated molecular phylogeny including the newly sequenced species in order to evaluate the evolutionary relationships of Okenia taxa; 8) Deposit all sequences obtained in GenBank as to facilitate identifications of Okenia taxa by non-specialists, as well as deposit newly sampled material in the museums of Stazione Zoologica Anton Dohrn (Naples, Italy) and National Museum of Natural History (Madrid, Spain), so that they may also constitute the starting point of additional studies by subsequent researchers.

DESCRIPTION OF WORK CARRIED OUT DURING THE STSMS

Bibliographic data Indexed and grey literature were examined for published Mediterranean records of taxa belonging to the genus Okenia, especially those accounts concerning faunistic, taxonomic, and biogeographic studies of Mollusca. Bibliographic data were critically analyzed and taxonomically updated to the latest nomenclature available, following the World Register of Marine Species. At the same time, a GenBank search was carried out to check for barcodes of Okenia material from the Mediterranean Sea, as well as other taxa belonging to the Family Goniodorididae. Taxon sampled 42 specimens from the Mediterranean Sea, including the Italian, French and Spanish coast, were collected by me and several collaborators and studied during the STSM. Those included one specimen of Okenia mediterranea (Ihering, 1886), one specimen of Okenia elegans (Leuckart, 1828), four specimens previously identified as Okenia cupella (Vogel & Schultz, 1970), and 36 specimens previously identified as Okenia zoobotryon (Smallwood, 1910). Besides, 15 additional specimens belonging to different taxa were also studied due to their similarity with the Mediterranean samples, including: eight specimens collected from Florida, four of them unidentified and four identified as O. cf. zoobotryon, two specimens from Canary Islands identified as O. cf. cupella, three specimens from Cape Verde identified as O. cf. impexa, the holotype of Okenia cupella (Virginia) and two specimens of Okenia angelensis Lance, 1966 from California. Morphological analysis The external morphology of specimens was examined from both photographs of living and laboratory observations. Internal organs were removed by a dorsal incision and drawn using a Nikon SMZ-1500 dissecting microscope, with an attached camera lucida. Radula and cuticle of the specimens were studied after dissolving the buccal mass in 10% sodium hydroxide and rinsed in water. Cuticle and penis were dried by critical point using hexamethyldisilazane. Radulae, cuticles, and penises were observed under Scanning Electron Microscope (SEM). Molecular analysis Specimens from Sabaudia Lake, Porto Ercole, and Napoli were sequenced by A. Macali, collaborator from Centro Ittiogenico Sperimentale Marino (Tuscia University, Italy). Sequences of O. angelensis were obtained by T. Gosliner, collaborator from California Academy of Science (California) (Table 1). For remaining specimens, DNA was extracted from foot tissue and performed using the DNAeasy Blood and Tissue Kit (Qiagen). Partial sequences of cytochrome c oxidase I (COI), 16S ribosomal RNA (16S rRNA), and histone H3 (H3) were amplified by PCR.

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DESCRIPTION OF THE MAIN RESULTS OBTAINED

Task 1. The literature analysis revealed records of several valid taxa. The precise number of Okenia species inhabiting the Mediterranean Sea and the identification of some specimens reported has varied over time according to different specialists. So far, eight valid species have been reported from the area: Okenia hispanica Valdés & Ortea, 1995, Okenia elegans (Leuckart, 1828), Okenia leachii (Alder & Hancock, 1854), Okenia mediterranea (Ihering, 1886), Okenia aspersa (Alder & Hancock 1845), Okenia cupella (Vogel & Schiltz, 1970), Okenia impexa (Er. Marcus, 1957), and Okenia zoobotryon (Smallwood, 1910). Task 2. Some of the newly sampled specimens of Okenia zoobotryon and Okenia cupella from the Mediterranean Sea were studied. As a result, I noticed that specimens ascribed to those taxa were originally misidentified and actually correspond to two undescribed species. Moreover, by comparing the anatomy of the specimens studied here with that of the specimens previously recorded from the Mediterranean Sea, I suggest that specimens previously recorded as O. zoobotryon belong to the first of the two new species, and that specimens ascribed to both O. cupella and O. impexa actually belong to a single entity, corresponding to the second new species. Task 3. 5, 2 and 3 sequences of COI, 16S rRNA, and 3 H3, respectively, were successfully amplified. Another 16, 16, and 9 sequences of COI, 16S rRNA, and H3, respectively, were amplified and facilitated by the collaborator A. Macali (Table 1). The sequences obtained during the present STSM are still pending of sequencing by Macrogen, Inc. However, preliminary analyses of the sequences obtained by A. Macali support morphological resuls. Task 4. Amplification and sequencing of species of is an aim sometimes difficult to achieve using the universal primers for invertebrates. Unfortunately, no time was available to design new primers. However, I expect to continue working on it in order to ease COI amplifications in future studies. Task 5. The holotype of Okenia cupella, one specimen of Okenia angelensis, eight specimens from Florida, and one specimen from Canary Island were studied. Molecularly, I successfully amplified 4, 4 and 13 COI, 16S rRNA and H3 sequences, respectively (Table 1). In this period of time I performed a complete re-description of the holotype of Okenia cupella, obtained sequences of O. angelensis, and expanded the distribution range of O. zoobotryon. I expect to find 3 potential undescribed species when the study is completed. Task 6. Three additional specimens belonging to one Okenia species from Mozambique were successfully sequenced for COI, 16S rRNA, and H3. Moreover, three specimens of the genus

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Goniodoridella were sequenced (Table 1). Both Okenia and Goniodoridella taxa are potentially undescribed. Task 7. During the present STSM, several specimens studied remained pendant of amplification. Thus, it was not possible to carry out the planned molecular phylogeny. I plan to finish this study in the next future. Task 8. Specimens will be deposited at the Stazione Zoologica Anton Dohrn (Naples, Italy), the Museum of Natural History (Madrid, Spain) and all the sequences obtained will be deposited in GenBank.

FUTURE COLLABORATIONS (if applicable)

The COST action made this collaboration possible and both parts started to work closely together in systematic and phylogeny of the eastern Atlantic/Mediterranean malacofauna. The work of the current action will result in a publication dealing with the review of the genus Okenia in the Mediterranean Sea.

Table 1. Specimens sequenced during the present STSM, including specie and locality. Genes successfully obtained marked with *****. Underlined in yellow the specimens sequenced by the present applicant. Voucher and GenBank numbers in process. Species Locality COI 16S rRNA H3 Goniodoridella sp. Australia: Mooloolah River ***** - ***** Goniodoridella sp. Australia: Mooloolah River ***** - ***** Goniodoridella sp. Australia: Mooloolah River - - ***** Okenia angelensis California, San Francisco ***** ***** ***** Okenia angelensis California, San Francisco ***** ***** ***** Okenia cupella Virginia, York River - - ***** Okenia elegans Spain, La Planassa ***** ***** ***** Okenia mediterranea Italy, Santa Maria al Bagno ***** ***** ***** Okenia sp. 1 Italy, Sabaudia Lake - - ***** Okenia sp. 1 Italy, Sabaudia Lake ***** ***** ***** Okenia sp. 1 Italy, Sabaudia Lake ***** ***** ***** Okenia sp. 1 Italy, Sabaudia Lake ***** ***** ***** Okenia sp. 1 Italy, Sabaudia Lake ***** ***** ***** Okenia sp. 1 Italy, Mar Piccolo ***** ***** ***** Okenia sp. 1 Italy, Mar Piccolo ***** - - Okenia sp. 1 Italy, Mar Piccolo ***** ***** - Okenia sp. 1 Italy, Porto Ercole ***** ***** ***** Okenia sp. 1 Italy, Porto Ercole ***** - ***** Okenia sp. 1 Italy, Porto Ercole ***** - ***** Okenia sp. 1 Italy, Porto Ercole ***** - ***** Okenia sp. 1 Italy, Porto Ercole ***** ***** ***** Okenia sp. 1 Italy, Porto Ercole ***** ***** ***** Okenia sp. 1 Italy, Porto Ercole ***** ***** ***** Okenia sp. 1 Italy, Naples ***** ***** ***** Okenia sp. 1 Italy, Naples ***** ***** ***** Okenia sp. 1 Italy, Naples ***** - ***** Okenia sp. 1 Italy, Naples ***** ***** ***** Okenia sp. 1 Italy, Naples ***** - *****

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Okenia sp. 1 Italy, Naples ***** ***** ***** Okenia sp. 1 Italy, Naples ***** - ***** Okenia sp. 1 Italy, Naples ***** - ***** Okenia sp. 1 Italy, Naples ***** ***** ***** Okenia sp. 2 Italy, Gallipoli ***** - ***** Okenia sp. 2 Italy, Gallipoli ***** - ***** Okenia zoobotryon Florida, Riviera Beach ***** ***** ***** Okenia zoobotryon Florida, Riviera Beach ***** ***** ***** Okenia sp. 3 Florida, Riviera Beach - - ***** Okenia sp. 3 Florida, Riviera Beach - - ***** Okenia sp. 4 Florida, Jupiter Lighthouse Museum ***** ***** ***** Okenia sp. 5 Florida, Jupiter Lighthouse Museum - - ***** Okenia sp. 5 Florida, Jupiter Lighthouse Museum - - ***** Okenia sp. 5 Florida, Jupiter Lighthouse Museum - ***** ***** Okenia cf. cupella Spain, Tenerife - - ***** Okenia cf. cupella Spain, Tenerife - - ***** Okenia cf. impexa Cape Verde, Mindelo - - ***** Okenia cf. impexa Cape Verde, Mindelo - - ***** Okenia cf. impexa Cape Verde, Mindelo ***** - ***** Okenia sp. 6 Mozambique, Barra Lagoon ***** ***** ***** Okenia sp. 6 Mozambique, Barra Lagoon ***** ***** ***** Okenia sp. 6 Mozambique, Barra Lagoon ***** ***** *****

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