The species of Eucera Scopoli, subgenus Tetralonia Spinola from Sardinia (Italy) with new records and E. gennargentui sp. nov. (Hymenoptera, Apidae)

In this paper, an update of the species of the genus Eucera Scopoli, 1770, subgenus Tetralonia Spinola, 1838 from Sardinia is reported, based on data collection as well as on recent survey carried out at Gennargentu Massif. Seven species are recorded, four of which are newly added: Eucera fulvescens (Giraud, 1863), E. gennargentui sp. nov. Nobile, Catania & Bella, E. julliani (Pérez, 1879), and E. nana (Morawitz, 1873). The new species, Eucera ( Tetralonia ) gennargentui Nobile, Catania & Bella is described from the high al-titude of Gennargentu Massif. Details on distributions, host plants, and other biological aspects are given for each species treated. The taxonomic comparison of the taxa belonging to the subgenus Tetralonia from Sardinia, including E . gennargentui sp. nov. , are discussed taking into account both morphological and COI barcode sequences. An identification key to Eucera ( Tetralonia ) species from Sardinia is also provided.


Introduction
Belonging to the Eucerini tribe, Eucera Scopoli, 1770 is a genus of bees widespread in the world (Michener 2007), with a relevant number of species in the Palaearctic region and especially in the Mediterranean Basin (Tkalcu 1979(Tkalcu , 1984Risch 1997Risch , 1999Risch , 2001Risch , 2003Michener 2007;Dorchin et al. 2018). In their recent taxonomic studies Dorchin et al. (2018) treated the genus Tetraloniella Ashmead, 1899 within Tetralonia Spinola, 1838, and this last as a subgenus of Eucera.
The subgenus Tetralonia includes different oligolectic species, some of them closely associated and specialised with plants of the Asteraceae family (Müller 2008). The species of this subgenus have been revised for the Afrotropical region by Eardley (1989), for the New World by LaBerge (2001), and by Tkalcu (1979Tkalcu ( , 1984 for the Palaearctic region. Eucera (Tetralonia) includes eleven species in Italy and some endemic subspecies of the main Italian islands, such as E. dentata amseli (Alfken, 1938) described from Sardinia and E. alticincta bindai (Nobile, 1993) described from Sicily (Alfken 1938;Nobile 1993).
In this paper, we add a further four species of Eucera (Tetralonia) as new for Sardinia and describe a new endemic species E. gennargentui sp. nov. Nobile, Catania & Bella from Gennargentu Massif, based on both morphological and molecular data.

Materials and methods
The material examined belongs partly to the collection of Vittorio Nobile and partly comes from field collecting efforts carried out by the two other authors in Sardinia during the year 2020.
Observations in the field on Eucera gennargentui sp. nov. were made in the first week of August 2020 in the locality of Bruncu Spina, near the town of Fonni (40°01'48.0"N, 9°17'56.6"E), in the central area of Gennargentu Massif, at 1540 m a.s.l., characterized by several endemic high mountain species plants (Bacchetta et al. 2013) (Fig. 1); observations were carried out from 09:30 to 16:30, on the flowers of the Asteraceae family.
Gennargentu Massif is located near the centre of the island, almost directly at 40° N. Bruncu Spina, at 1829 m a.s.l., is the second highest mountain in Sardinia, after Punta La Marmora (1834 m a.s.l.), and is the northernmost summit of the main Gennargentu ridge.
In order to collect biological data on the new taxon, some specimens were marked using a dye specific to bees. The date of capture, general and Italian distribution, number of specimens observed, and the plants visited are reported for all species.
Each specimen was killed by freezing at -20 °C for use in DNA analysis and was dried and identified to species level after the observation of genital structures. The specimens were preserved in the collection of the authors, and in the collection of the Department of Agriculture, Food and Environment, sect. Applied Entomology, University of Catania (Di3A).
The specimens were studied with a stereomicroscope Leica EZ4HD, the photos taken with a Leica Application Suite vs. 4.13.
Total DNA was extracted from one posterior leg using the NucleoSpin Tissue Kit (Macherey-Nagel, Germany) following the manufacturer's instructions. Subsequently, the enzymatic amplification took place using primers LCO1490F and HC02198R (Folmer et al. 1994). PCR products were purified using ExoSAP-IT (ThermoFisher Scientific) and were sequenced at BMR genomics (Padua, Italy), and the sequences obtained were analysed using MEGA X 10.2.4 (Kumar et al. 2018).
The sequences were aligned with seven COI sequences of Eucera (Tetralonia) available in GenBank, and we also included two sequences of Habropoda tarsata (Spinola, 1838) (accession numbers MN919536, MN919537) as an outgroup (Table 1). Sequence divergences were calculated using the p-distance model (Srivathsan and Meier 2012), and a neighbour-joining (NJ) tree (Saitou and Nei 1987), as implemented in MEGA X, was used to visualize the distance matrix among taxa.

Di3A
Department of Agriculture, Food and Environment, Catania, Italy;

Detected species
Genus Eucera Scopoli, 1770 Subgenus Tetralonia Spinola, 1838 Eucera dentata amseli (Alfken, 1938) Fig. 2 Tetraloniella dentata amseli Alfken, 1938, Mem. Soc. Entomol. It., 16: 97-114. Description. Female (Fig. 3). Body length: 10 mm. Body with black integument with fine and evident punctation, grey setae. Head. Head with wrinkled punctation, finer at the edges and with white setae; clypeus black, with the lower half light yellow; this spot, extending upwards, and in the middle part, ends with a triangulated tip; labrum and mandibles black, antennae dorsally black, ventrally orange, with scape and first two flagellar articles black. Mesosoma. Mesothorax with greyish setae, median part of the metathorax with dense and large punctation. Black legs with greyish setae and the last four tarsi brown; the hind legs are equipped with a uniformly yellow-rust-coloured pollen-collection system. Slightly darkened wings, with black veins and dark brown tegulae. Metasoma. Dark brown, almost black, with dense and fine punctation. T1 with long and sparse white setae, T2-4 with basal bands of white felt, T5 with white felt on the sides and dark brown in the middle, and T6 almost completely covered with darkbrown felt. Dark-brown sternites with thick punctation and long golden-yellow fringes.  Male . Body length: 9 mm. General appearance similar to the female, with yellow clypeus and labrum, antennae are more than half of the body length long and reddish, except for a black streak on the whole dorsal part; black scape; mandibles are black, but reddish in the distal half. Mesosoma. With long silky whitish setae. Black legs with whitish setae. The hind femur, in the lower median part, has a small pointed tubercle equipped with a tuft of brownish bristles. Metasoma. As in the female, is dark brown, but all segments are covered with white felt, and T6 on the sides has 2 prominent teeth, one on each side. The last sternites are provided with a long and deep longitudinal median sulcus. Genitalia and hidden sterna. S6 without anterolateral marginal projections; posterolateral carina curved laterally at each side. S7 with medial process rounded and attached at the posterior lobes of lateral processes; the posterior lobes of lateral processes are relatively short with straight superior margins; anterior lobes of lateral processes wide with straight superior margins and rounded lateral-inferior margins. S8 with a wide concavity between rounded apical lobes; apicomedian ventral process well defined and saddle shape, with a large depressed area at the base. Genital capsule with the apex of the gonostylus remarkably arched, and inward-facing triangular protuberance placed slightly over the central part of the gonostylus. Difference with the closest related taxa. The new species Eucera gennargentui, is similar to E. alticincta (Lepeletier, 1841), including the nominate subspecies and E. alticincta bindai.
In the female of E. gennargentui, the long fringes of the sternites are yellow-gold, whereas in the closest related species E. alticincta alticincta, they are white; the scopa is yellow-rust in the new species, whitish externally and brown internally in E. alticincta alticincta.
In the male of the new species, the long silky hairs of the mesosoma are whitish, while they are brownish-yellow in E. alticincta alticincta. Genitalia of E. gennargentui is very similar to that of E. alticincta alticincta, however, in the gonostylus the inward-facing triangular protuberance, that in E. alticincta is located in the middle part, in the new species is placed slightly over half of the gonostylus, slightly closer to the apex. Further differences can be noted in the morphology of the hidden sterna, especially for the medial and lateral processes in S7, and for apical lobes and apicomedian ventral process in S8.
In both sexes of the new species, the integument and wings are considerably darker than E. alticincta alticincta, and the body length of both sexes of E. gennargentui is greater than that of the related species.
Further morphological and bio-ecological comparisons between E. alticincta alticincta, E. alticincta bindai, and E. gennargentui are reported in Tables 2 and 3.
Bio-ecological aspects. A total of 22 specimens (half captured and half marked) were observed in the sampling site: 20 females and 2 males. Specimens of E. gennargentui were observed in activity in a restricted area of about 300 m 2 , from 10:30 to 15:00; in particular, the females visited the flowers from 10:30 to 14:30, and the two males were observed from 13:30 to 15:00 on Carlina macrocephala subsp. macrocephala. The female specimens visited the same flowers at intervals of about ten minutes, with a preference for the flowers of Carlina macrocephala subsp. macrocephala.
To know better the flights period of E. gennargentui, further surveys in the same site were conducted during mid-July 2021, however no specimens were found. This confirms the relationship between the new species and its main host plant, in fact Carlina macrocephala subsp. macrocephala was at the beginning of its flowering. Sicily (Nobile, 1987;1993) (Banaszak & Ortiz-Sànchez 1993).
Range in Italy. This species is discontinuously present in the Italian peninsula, Sardinia and Sicily.

Range in Italy.
This species is discontinuously present in the Italian peninsula, and Sicily.
Distribution. Widespread in Europe, Eastern Mediterranean (Cyprus) and Western Asia (Turkey).
Range in Italy. This species is discontinuously present in the Italian peninsula, and Sicily.

Range in Italy.
This species is known in the regions of <br/> .New record for Sardinia.
Identification key to Eucera (Tetralonia) from Sardinia

Molecular evidences
High-quality DNA sequences were obtained for the two specimens analysed, one sequence of E. gennargentui sp. nov. from Sardinia (GenBank accession number MZ437082) and one of E. alticincta bindai from Sicily (1 ♀; Italy, Sicily, Mount Etna, Gurrida Lake, 25.VIII.2020 RC leg., det., and coll.) (GeneBank accession number MZ437367). Both COI sequences obtained from the samples were used as queries in BOLD-IDS tools, and the system returned a match (sequence of E. gennargentui: 97.88%; sequence of E. alticincta bindai: 98.77%) with a sequence of E. alticincta present in GenBank. Sequence divergences calculated using the p-distance model are reported in Table  4, and the neighbor-joining tree showing the relationships between the new species and other related species of Eucera (Tetralonia) are reported in Fig. 7.

Conclusion
In the current study, Eucera fulvescens, E. julliani, and E. nana are reported for the first time for Sardinia, and E. gennargentui sp. nov. is described from Gennargentu Massif. This new species shows a larger body size and a darker appearance than the closest related taxa E. alticincta alticincta and E. alticincta bindai, and other different characters reported in this work. Furthermore, following our field observations, we ascertained how the new species was linked to three Sardo-Corsican and Sardinian endemic botanical species of the flora of Gennargentu Massif.
The barcode sequence of the new species Eucera gennargentui diverged from that of E. alticincta alticincta from 2.08% and from that of E. alticincta bindai by 2.21%.
In Europe, wild bees are threatened by habitat changes resulting from human activities, e.g., urban exspansion, agricultural practices, habitat destruction or changes, and climate changing (Matheson et al., 1996;Steffan-Dewenter et al., 2005;Nieto et al. 2014;Bella et al. 2020;Fisogni et al. 2020). There was poor information for the majority of the species (56.7%) to determine their conservation status (Nieto et al. 2014).
In Italy, the availability of information on bees is particularly scarce, especially in southern and insular regions, so it is important to increase research to better understand the distribution and the population tendencies, especially for localised taxa.
More observations are necessary to understand the conservation status of this new species and of the other Eucera (Tetralonia) species present in Sardinia. However, the area of Gennargentu where Eucera gennargentui was found is rather restricted and subject to disturbing factors due to strong grazing regimes and reckless reforestation that inexorably damage the grasslands where the endemic host plants live. The frequency of numerous fires that devastate natural ecosystems and rural areas in Sardinia (Capra et al. 2018;Salis et al. 2021) represents a further important threat to the diversity of flora and consequently of wild bees.
The present study greatly improves the Sardinian bee fauna, showing that this Mediterranean island will surely deserves further new acquisitions, in order to increase our knowledge of its still underestimated pollinator fauna.