New Geographical Record of Three Cumacean Species Eudorella nana, Leucon affinis, Leucon siphonatus and One Rare Amphipod Presence Confirmation, Stenothoe bosphorana, in Adriatic Sea, Italy

Three cumacean species, Eudorella nana Sars, 1879, Leucon affinis Fage, 1951, Leucon siphonatus Calman, 1905, were recorded for the first time and one rare amphipod presence, Stenothoe bosphorana Sowinsky, 1898, was confirmed in the Adriatic basin.


Introduction
The importance of the taxonomist work in biological and ecological research as well as in biodiversity management is well known, even though most of the time it is underestimated. Misidentification of specimens may have some consequences on the accuracy of ecological works and furthermore in fields like medicine, pharmacology, agriculture, conservation biology, ecosystem management and climatology (Dubois et al. 2013).
Some species may be also widely confused by "non-specialized" taxonomists with other similar but common species and this is a possible reason for their rareness.
Among the 46 genera included within Stenothoidae the most represented is Stenothoe Dana, 1852 with 60 valid species already described, closely followed by Metopa Boeck, 1871 with 54 species (WoRMS Editorial Board 2020).
In 2006, Krapp-Schickel proposed a classification of the genus Stenothoe based on the presence of carinate body, prehensile pereopods and a naked or spinose telson. In 2015 the same author re-described the entire genus dividing the 55 valid species attributed since then in two groups: the largest one comprehending those species more similar to S. valida and the smaller one including those species similar to S. monoculoides (Krapp-Schickel 2015, 2006. The genus Stenothoe is widely distributed all over the world from boreal to temperate regions (Myers 1985;Bellan-Santini 2005;Hayward and Morley 2005;Krapp-Schickel 2006;Krapp-Schickel and Lo Brutto 2015), both as free-living and in association with sea anemones (Vader 1984;Vader and Krapp-Schickel 1996;Krapp-Schickel 2015).
Up to date, only 14 out of 60 amphipods belonging to the genus Stenothoe are known to be distributed within the Mediterranean Sea (Marin and Sinelnikov 2018) and within them only S. bosphorana Sowinsky, 1898 is still considered as an endemic species (Krapp-Schickel 2015). There was only one old record of this species along the coast of Zlarin Island, Croatia (Krapp-Schickel 1976) without any further information since then.
The contribution of this paper is to rise the knowledge on the macrozoobenthos biodiversity, adding four smalls, but not less important, crustacean species to the Adriatic Sea fauna.
It is well known the importance of the biodiversity, to the point that the first descriptor of the "Good Environmental Status (GES)" of the EU Marine Strategy Framework Directive (MSFD; Directive 2008/56/EC) is, quoting: "Biological diversity is maintained. The quality and occurrence of habitats and the distribution and abundance of species are in line with prevailing physiographic, geographic and climate conditions". In this contest, it is clear the role of taxonomists and the relevance of knowing the distribution of every single species in the biodiversity point of view; furthermore, to understand the spreading of some species (alien and/or invasive) due to climate changes and human activities is extremely crucial to have a clear picture of the native or already present species in a certain place.

Materials and Methods
Study Area The Adriatic Sea is an elongated basin in the northernmost part of the Mediterranean, between the Italian Peninsula and the Balkans countries, with the major axis in the NW-SE direction. The northern area is very shallow, gently sloping, with an average depth of about 35 m, while the central part is on average 140 m depth, with 2 pits, the Pomo pit reaching ≈ 260 m and the South Adriatic pit reaching ≈ 1200 m. The northern and central parts of the basin are affected by a great number of rivers along the Italian coast, with the Po River being the most relevant in terms of water inflow (Artegiani et al. 1997). River runoff and wind stress are the main drivers of the water circulation. West Adriatic Current (WAC) and East Adriatic Current (EAC), flowing along the coasts, are the main currents affecting the Adriatic circulation. There are three main cyclonic gyres, one in the northern part, one in the middle and one in the south (Artegiani et al. 1997).
In the southern Adriatic Sea, the cyclonic gyre is observed in all seasons and a relatively strong western coastal current and a weaker eastern current are observed during summer. A strong annual thermal variation affects the northern and central Adriatic Sea and it is more pronounced at the surface (e.g., 5-28°C) than close to the bottom (e.g., 12-17°C). The water column from the coast to the 11-12 km offshore is characterized by low temperature (5-6°C) and salinity (<37‰) in winter, while the offshore waters are warmer (10-12°C) and thicker (>38‰). A vertical thermohaline front, parallel to the coast and extended throughout the water mass, divides the coastal waters from the open sea. In summer, instead, a horizontal stratification characterizes the water column, separating the warmer surface waters with lower salinity, from the deeper, colder and more saline ones (Artegiani et al. 1997).
The area is affected by a heavy marine traffic from cargo ships, supplier vessels for offshore activities (e.g., gas platforms), ferryboats, trawl-fishing vessels, cruise and recreational crafts (Coll et al. 2007;Pranovi et al. 2016). It is also characterized by intense mussel aquaculture along the Italian coast and by fish farming along the Croatian one (Ponti et al. 2007;Fabi et al. 2009).
Samples Collection and Analysis Macrozoobenthos samples were collected during different surveys, from 2012 to 2019, within a multi-annual monitoring program aimed to assess the environmental effects of offshore gas extraction platforms.
Sampling was performed at three different square areas (A, B, C) of the central Adriatic Sea, each having a surface area of ≈ 16 km 2 ( Fig. 1; Table 1).
Twenty-four sites where randomly chosen within each area and six samples were collected at each site using a Van-Veen grab (capacity 13.0 L; surface 0.1 m 2 ). The grab samples were sieved in situ through a 0.5 mm mesh, preserved in 5% buffered formalin and later transferred to 70% ethanol. Macrozoobenthos was sorted and identified in the laboratory using a stereomicroscope Zeiss Stemi 2000C and a compound microscope Zeiss Axiolab 5 equipped with eyepiece reticles, stage micrometers and camera.
The specimens herein examined and descripted are the first of the total recorded (Table 2) during the whole surveys period. All the specimens were measured in size (Total Body Length, TL), dissected when necessary, examined under higher magnification and identified following the below-mentioned systematic keys and descriptions. Bellan-Santini et al. (1993) and Krapp-Schickel (2015) for the amphipod and Bǎcescu (1951), Fage (1951), Jones (1976) and Shalla (2011) for the cumaceans were followed for the systematic keys and description.
For some individuals of each species, pictures of body parts were taken using Zeiss Axiocam ERC5s 5mpx. The handmade draws were obtained from the pictures, using XP-PEN Graphic Tablet Deco 02 Pen 8192 and Autodesk SketchBook software.
The nomenclature herein follows the World Register of Marine Species (WoRMS Editorial Board 2020). Voucher specimens are presently kept in the reference collection of CNR-IRBIM in Ancona, Italy.

Results
During the overall sampling period, 7 E. nana and 11 L. affinis were found in A and B areas, 274 L. siphonatus in A, B and C areas and 3 S. bosphorana were only found in C area ( Material examined: Three male specimens (cod. StB-1, StB-2, StB-3) collected in C area in 2018.
Ecology and distribution: S. bosphorana is known to live on muddy bottoms between 35 and 360 m (Bellan-Santini et al. 1993).
As above mentioned, S. bosphorana is still considered an endemic species in the Mediterranean Sea (Krapp-Schickel 2015), where it is distributed in the eastern part of the basin, from the Sea of Marmara and North Aegean Sea to the Levantine Sea (Krapp-Schickel 1976; Manoudis et al. 2005;Bakir et al. 2014). Fewer records were also registered in the western part of the Mediterranean, particularly in Saint-Tropez Gulf and alongside the Tunisian coast (Chevreux and Fage 1925;Zakhama-Sraieb et al. 2009). Diagnosis: According to Bǎcescu (1951), Fage (1951), Jones (1976) and Shalla (2011).
TL: 1.2-3.8 mm. Carapace anteriorly truncate without prominent pseudorostrum (Fig. 3); efferent orifice dorsal. Anterior lateral part of carapace shaped in an evident, horizontal, elongated tip forming a unique tooth in the male and few much shorter teeth in the female; immediately above, a Vshaped excavation with a single upwards pointed tooth on top; an evident sheaf of long bristles on the posterior margin of the fifth abdominal segment.
TL: 1.8-3.6 mm. Carapace 1/4 of the total length; pseudorostrum straight largely cut off at the top; dorsal ridge with an anterior batch of 16 contiguous teeth, followed by a smooth and concave depression and by another series of 4 teeth extending up to the rear edge of the carapace; peduncle of uropods with the same length of endopod, which is a little shorter than exopod. Ecology and distribution: L. affinis is a bathyal species, living from 180 to 415 m depth (Mühlenhardt-Siegel 2009) in sandy bottoms and in deep-sea muds (Carpine 1970;Ledoyer 1983Ledoyer ,1987Corbera and Cardell 1995).

Subgenus
TL: 1.7-3.1 mm. Elongated branchial siphon developing from the front of pseudorostrum, accompanied by long bristles; dorsal and antero-lateral edge of carapace with some teeth; distal end of endopod with a long apical plumose bristle and four internal spines, exopod with internal and external bristles on margins.

Remarks
The A area bottom, with an average depth of 75 m, was characterized by sand (Shepard's classification). The    Considering the amphipods and cumaceans associated with the species recorded in this paper, in the three areas A, B and C a total of 57, 71 and 60 taxa were respectively found (Table 3). E. truncatula was the most abundant species in both areas A and B. Ampelisca diadema (Costa, 1853) was one of the dominant species in all the considered areas, together with Phtisica marina Slabber, 1769 (A area), Harpinia dellavallei Chevreux, 1910 (B area), Paraphoxus oculatus (G. O. Sars, 1879) and Orchomene grimaldii Chevreux, 1890 (C area).

Discussion
The taxonomic misidentification issue plays an important role in the marine biodiversity management, especially for very small-sized marine species. Furthermore, genetic analysis on these specimens is problematic as the small and fragile body makes difficult to collect suitable samples.
This study is aimed to contribute to the knowledge on the families Leuconidae and Stenothoidae distribution. In fact, several authors, as well as both European Register of Marine Species (Costello et al. 2020) and World Register of Marine Species (WoRMS Editorial Board 2020), report the distribution of the four species here recorded in different areas of the Mediterranean Sea (Watling 2001;Coll et al. 2010;Watling and Gerken 2014;WoRMS Editorial Board, 2020) without any (or only one old record in the case of S. bosphorana) information on their presence in the Adriatic Sea; moreover the "Italian Checklist of Marine Fauna" (Marusso 2010) does not report E. nana and L. siphonatus in the Adriatic section, and L. affinis and S. bosphorana in all Italian seas.
Only 3 specimens of S. bosphorana were found in siltysand bottom at 80 m depth in Adriatic Sea during the overall sampling period (from 2012 to 2019), highlighting the rareness of this species in this basin. Since this taxon was previously only reported living on muddy bottoms, this record adds a different bottom type where it can be found, also widening the living area.
The E. nana living habit fit with the environmental characteristics of the A and B areas where it was found, considered typical of the circalittoral zone and living in a wide range of bottom type, this record adds the Adriatic Sea to the E. nana living area, giving new information on its distribution.
In this study, L. affinis and L. siphonatus, until now considered as bathyal species living mainly on sandy and muddy deep-sea bottom, were found in shallow waters living in sand and silty-sand bottom of the Adriatic Sea, between 75 and 80 m depth, thus providing further information on their habitat and distribution.
The new record of the four species in the Adriatic Sea could be explained by the hypothesis that these species were already present in Adriatic, although not common, but had not yet been identified due to the poor knowledge about these taxa and the limited number of studies on these orders of crustacea, their small size and fragility and the difficulties to collect undamaged specimens using the common sampling techniques.
Further specific studies would be essential to better know the distribution of these taxa in this basin and therefore understand their ecological role in the benthic community.
Availability of Data and Material Voucher specimens are presently kept in the reference collection of CNR-IRBIM in Ancona, Italy. No other data sets are generated during this study.
Code Availability Not applicable.
Authors' Contributions Pierluigi Strafella main author, draft the work, participated to sampling survey, analyzed the samples and validated the taxonomic records; Vera Salvalaggio analyzed the samples, cooperated in the paper writing and the taxonomic validation, managed the sampling survey; Clara Cuicchi supported the paper writing and the taxonomic validation; Elisa Punzo contributed in the paper writing and the taxonomic validation; Angela Santelli analyzed the samples, supported the paper writing and the taxonomic validation; Alessandro Colombelli supported the paper writing, drew the images;; Gianna Fabi coordinator of the research, organized and led the scientific cruise, approved the version to be published; Alessandra Spagnolo responsible of the research, contributed in the paper writing and approved the version to be published.
Funding This study received funding from ENI S.p.A.-UPSTREAM DICS.

Declarations
Conflicts of Interest/Competing Interests The authors declare that they have no conflict of interest or competing financial and/or non-financial interests in relation to this work.
Ethics Approval This article does not contain any studies with animals performed by any of the authors.
Consent to Participate Sampling and observational field studies do not require any permits.

Consent for Publication Not applicable.
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