Two Seas for One Great Diversity: Checklist of the Marine Heterobranchia (Mollusca; Gastropoda) from the Salento Peninsula (South-East Italy)

: The Salento peninsula is a portion of the Italian mainland separating two distinct Mediterranean basins, the Ionian and the Adriatic seas. Several authors have studied the marine Heterobranchia (Mollusca, Gastropoda) fauna composition living in the Ionian Sea, but to date further knowledge regarding this interesting group of mollusks is still needed. Recent studies have corroborated the peculiarity of the Mediterranean Sea showing high levels of endemism and cryptic diversity. On the other hand, marine sea slugs have been revealed to be important indicators of the marine ecosystem’s health, due to their species-speciﬁc diet that consist of a vast variety of sessile and benthic invertebrates. A baseline study of the marine Heterobranchia diversity is therefore a necessary step to reveal the hidden diversity and to monitor the possible presence of alien species. The present study shows results from approximately 600 scientiﬁc dives carried out during a nine-year period in all of the main submarine habitats of the studied area, while accounting for the marine Heterobranchia from both the Ionian and Adriatic Seas. With this contribution, the list of marine Heterobranchia inhabiting the Salento Peninsula rises to 160. Furthermore, it also reports, for the ﬁrst time, the presence of one alien species and three new records for Italian waters. Ecological notes and geographical distribution for each added species are provided together with animal iconography, consisting mainly of in situ photographs, for species identiﬁcation. the comparison between the total species of the Salento Peninsula, clustered into the main groups.


Introduction
The Salento Peninsula (South-East Italy) in Puglia is a strip of land right on the border of the eastern Mediterranean Sea, made up of a large variety of habitats: rocky formations, open sandy beaches, marine caves, etc. The Salento Peninsula is a physically well-identified region, encompassing the innermost point of the Gulf of Taranto (40 • 05.0 E). The maximum and minimum width are 54 and 33 kilometers, respectively, and the total coastal length is 365 kilometers [1][2][3][4][5]. This peninsula protrudes between two ecoregions of the Mediterranean Sea [6], the Northern Ionian, and the Southern Adriatic seas, which are conventionally separated by the dividing line passing through Otranto (according to the

Materials and Methods
The geographical area under investigation was a stretch of more than 270 km of coastline around the Salento Peninsula in Southern Italy ( Figure 1). With 600 scuba dives in the past 9 years, different benthic habitats were surveyed, some of which were included in the European legislative context (EU WFD, EU Habitat Directive, EU MSFD): pre-coralligenous and coralligenous assemblages, soft-bottom substrates, Posidonia oceanica (Linnaeus) Delile, 1813 meadows, and algal biocoenosis on rocky substrates. All sampled sites were georeferenced (Table 1) to provide accurate data of the studied locations for future monitoring and comparison. When possible, ecological observation and in situ photographs of the individuals recorded were performed and catalogued for species identification. The Scuba dives (0-40 m depth) took place all year round, almost every week, during daylight and at night, between 2011 and 2019. Specimens between 2 and 5 mm in length were photographed alive in the laboratory, in Petri capsules illuminated by a series of low voltage LED lamps, with 6500 K bulbs. The camera used for filming was a tripod mounted Nikon D7100, with 60 mm micro Nikkor or 105 mm micro Nikkor optics, a series of extension rings and additional lenses. The underwater photographic or videography equipment (F.V.) was a Nikon D7000 body, 60 mm micro Nikkor, or 105 mm micro Nikkor optics, extension rings inside an Isotta housing, as well as additional wet lenses SubSee +10 diopter, two underwater flashes Inon z240, and two LED lamps of great luminous power. Or alternatively, (C.L.) a Canon 600D body in a Nauticam 600D housing, equipped with the following lenses-for macro photography and in some cases for micro subjects, the Canon 60 mm USM Macro, and Canon 100 mm USM Macro were used, in addition to wet lenses SubSee +5 and +10 diopter; for wide angle photography, the Tokina 10-17 mm fisheye lens was used. The light source was provided by a couple of Inon strobes z240 and a single focus light I-Torch Video Pro 3. The systematics and the validity of names were checked with the help of the Word Register of Marine Species [49]. Species identification was obtained by morphological investigation and a subsequent consultation of the existent literature ( [29,41,44] and other references cited in the present work), guide books [50,51] and websites [48,52,53]. In the case of Berthellina cf. edwardsii, the shell from an individual (Voucher RM3_1865) was extracted and used to confirm the identification as this anatomical feature is commonly considered to be diagnostic for this species. The shell was removed and dissolved in a 10% NaOH solution, then rinsed in water, dried, and mounted for examination by optical microscopy, following the same protocol described by Furfaro et al. [54]. Voucher numbers were assigned to the collected individuals that were selected for future molecular analyses; samples were preserved in 95% alcohol and stored in the Department of Science at the Roma Tre University (Rome, Italy) (Table 2). Finally, a comparison between species as recorded by previous authors (Perrone [30][31][32][33][34][35][36][37] (A), Onorato and Belmonte [38] (B), Micaroni et al. [39] (C)) and the present study (D) was carried out and reported in Table 2 with new records highlighted in bold letters. An ethical approach in this research was also carried out by complying with the restrictions in term of collected sample size, environmental survey of the collection sites, use of hand-net picking of specimens (harmless and not destructive) as well as complying with local, regional, national, and international rules, and regulations for access to biodiversity, sustainable use, and benefit sharing (Convention on Biological Diversity and its Nagoya Protocol, national regulations).  Table 1. Table 1. Number, name, geographic coordinates (latitude and longitude), and the depth range of the sampled stations. Table 2. List of species of Heterobranchia occurring around the Salento peninsula; localities are numbered according to Figure 1, with new records in column D, highlighted in bold. Column A = Perrone [30][31][32][33][34][35][36][37]; B = Onorato and Belmonte [38]; C = Micaroni et al. [39]; and D = Present study.  Figure 1, with new records in column D, highlighted in bold. Column A = Perrone [30][31][32][33][34][35][36][37]; B = Onorato and Belmonte [38]; C = Micaroni et al. [39]; and D = Present study.     Abbreviation cf. is from the Latin confer/conferatur, both meaning compare.

Results
The present checklist reports 160 marine Heterobranchia species from the Salento Peninsula (Table 2) Table 3 provides the species list with ecological remarks of all added species. Furthermore, an extensive photographic catalogue mainly consisting of pictures taken in situ is provided in Figures 2-9. Table 3. List of the newly recorded Heterobranchia from the Salento peninsula; species names, relative figure numbers, ecological notes, and phenotypical variability for each added species are reported. Abundance is indicated as number of specimens.       Observed all year long in shady pre-coralligenous habitats. Depth: 0-15 m.     Very small species 1.5-2 mm, as expected for Runcina species. Depth: 8 m. -

Discussion
This study reported 160 marine Heterobranchia species from the Salento Peninsula giving the most up to date list of marine heterobranchs inhabiting this area of the Apulian coasts. This was particularly remarkable considering that the total number of registered species belonging to this mollusks group for the whole Mediterranean Sea was approximately 550 [51]. The composition of the main groups of marine heterobranchs living in the Salento Peninsula reported in this work is shown in Figure 10. The groups with the largest numbers of species added were Cladobranchia (18) and Doridina (9), as expected, these nudibranchs belonged to the groups with the richest variety of taxa. Surprisingly, with the present contribution, 6 species of Cephalaspidea were added to the ones reported previously. Another interesting consideration was concerning the superorder Sacoglossa, which almost doubled after this study. Finally, some species were noteworthy because they are currently under studied or because the validity of the species is still in doubt. This was the case for Berthellina cf. edwardsii, Eubranchus cf. exiguus, E. cf. farrani, E. cf. linensis, Facelina fusca, Haminoea cf. orteai, Runcina cf. ferruginea and Trinchesia cf. miniostriata. In particular, in the case of Berthellina cf. edwardsii we depicted ( Figure 2B) the internal shell (4.2 mm long), since it fits the standard average length of the shell commonly used as diagnostic for this species, albeit we preferred to keep an uncertainty (cf.) before a further molecular study would allow a clear-cut identification. The studied marine area, deeply influenced by two different seas and characterized by a variety of submarine habitats, hosts a high variety of species, which makes it an important geographical area for this sea slug diversity. In fact, with this work we report that the total number of marine Heterobranchia living in the Salento Peninsula is 160, which is about a third of the total number of currently accepted species reported for the whole Mediterranean Sea. This is quite an important finding, also considering the fact that a high diversity in heterobranchs composition indirectly reflects a high structuring and diversification of the habitats involved, and consequently, of the biodiversity that they contribute to maintain [14]. Studying and monitoring the marine Heterobranchia diversity in the Salento Peninsula is valuable for highlighting the consequences of the global marine changes reported in the last decade [10][11][12]59,60], such as warming and acidification of waters or invasion of alien species. An additional detailed and constantly updated iconography is available on the website of the Salento Sommerso group (http://www.salentosommerso.it/index_opi.php), a non-profit association devoted to the preservation and documentation of the underwater biodiversity of the Salento Peninsula.

Discussion
This study reported 160 marine Heterobranchia species from the Salento Peninsula giving the most up to date list of marine heterobranchs inhabiting this area of the Apulian coasts. This was particularly remarkable considering that the total number of registered species belonging to this mollusks group for the whole Mediterranean Sea was approximately 550 [51]. The composition of the main groups of marine heterobranchs living in the Salento Peninsula reported in this work is shown in Figure 10. The groups with the largest numbers of species added were Cladobranchia (18) and Doridina (9), as expected, these nudibranchs belonged to the groups with the richest variety of taxa. Surprisingly, with the present contribution, 6 species of Cephalaspidea were added to the ones reported previously. Another interesting consideration was concerning the superorder Sacoglossa, which almost doubled after this study. Finally, some species were noteworthy because they are currently under studied or because the validity of the species is still in doubt. This was the case for Berthellina cf. edwardsii, Eubranchus cf. exiguus, E. cf. farrani, E. cf. linensis, Facelina fusca, Haminoea cf. orteai, Runcina cf. ferruginea and Trinchesia cf. miniostriata. In particular, in the case of Berthellina cf. edwardsii we depicted ( Figure 2B) the internal shell (4.2 mm long), since it fits the standard average length of the shell commonly used as diagnostic for this species, albeit we preferred to keep an uncertainty (cf.) before a further molecular study would allow a clear-cut identification. The studied marine area, deeply influenced by two different seas and characterized by a variety of submarine habitats, hosts a high variety of species, which makes it an important geographical area for this sea slug diversity. In fact, with this work we report that the total number of marine Heterobranchia living in the Salento Peninsula is 160, which is about a third of the total number of currently accepted species reported for the whole Mediterranean Sea. This is quite an important finding, also considering the fact that a high diversity in heterobranchs composition indirectly reflects a high structuring and diversification of the habitats involved, and consequently, of the biodiversity that they contribute to maintain [14]. Studying and monitoring the marine Heterobranchia diversity in the Salento Peninsula is valuable for highlighting the consequences of the global marine changes reported in the last decade [10][11][12]59,60], such as warming and acidification of waters or invasion of alien species. An additional detailed and constantly updated iconography is available on the website of the Salento Sommerso group (http://www.salentosommerso.it/index_opi.php), a non-profit association devoted to the preservation and documentation of the underwater biodiversity of the Salento Peninsula.

Conclusions
The study of marine diversity is a fundamental topic especially if focused on areas like Mediterranean Sea, which is characterised by a high rate of endemic and cryptic species. In this study we have investigated the presence of marine Heterobranchia in the Salento Peninsula (Apulia, South-Italy) considering for the first time both Ionian and Adriatic coasts. Results of a nine-year study reports 160 species inhabiting the studied area with new records from the sublittoral waters, ecological notes, local distribution and systematic remarks. This inventory of marine Heterobranchia encountered in the Salento Peninsula adds 45 species to the previously known for this area. For all the added species, we have figured live animals mostly by in situ photographs to document species identification and reported data on ecology, phenotypical variability and abundance. This inventory of marine Heterobranchia provides a baseline for future monitoring of both coastal sides and could serve as a starting point for further molecular studies aiming to unveil Mediterranean cryptic diversity.