The findings address a notable gap in Mediterranean bee research. Although bees pollinate most wild and cultivated plant species, the conservation status of most bee species remains unknown because distributions, taxonomy and relationships with flowering plants are inadequately documented. The IUCN European Red List of Bees reported that 9.2% of assessed species were threatened with extinction and 5.2% were considered Near Threatened, while the status of 56.7% remained unknown because of insufficient information. Sardinia is no exception: a checklist of Italian wild bees reported 316 species from the island, yet additional island records have continued to appear. The Culuccia Peninsula, an area of roughly 3 square kilometres, had previously lacked a dedicated survey of its bee fauna.

The peninsula lies on the edge of the Maddalena Archipelago, between Santa Teresa di Gallura and Palau, at elevations from sea level to 93 metres. It is protected as a Permanent Wildlife Protection and Capture Reserve and contains a mosaic of habitats. Mediterranean maquis is the dominant land-cover class, followed by transitional woodland and shrub, while smaller areas include wetlands, herbaceous dune vegetation, water bodies, bare rock, artificial surfaces, pasture, garigue, beach and vineyards. The vegetation includes Mediterranean scrub, thermophilous, psammophilous and xerophilous plants, coastal species and halophytes. Two small vineyards, roughly 100 beehives and some pastures add areas of relatively low human disturbance to the landscape.

To assemble the inventory, the team sampled bees from April to October across seven sessions in spring, summer and autumn in 2022 and 2023. Seven fixed transects, each 200 metres long and one metre wide, were located in the main land-cover classes and separated by at least 450 metres. Each transect was walked once per session by two people for 50 minutes of regular walking, at different times between 10:00 and 16:00 to capture species with different periods of activity. Sampling occurred only when temperatures were above 15°C, wind was light, there was no rain and vegetation was dry. Additional random sampling with nets was carried out across the peninsula, while two Malaise traps, separated by 1,000 metres, operated continuously from May to October 2022 and were emptied every two weeks.

Each collected specimen was assigned a unique identification code, date, WGS84 coordinates and Corine Land Cover class. For specimens collected on flowers, the researchers also recorded the associated plant taxa; the evidence does not indicate that every specimen had a flowering-plant association. Plants were photographed in the field and identified with published identification works and assistance from botanical specialists. Bees were prepared, stored dry in entomological boxes and preserved at the Museum of Zoology and Comparative Anatomy in the Department of Science at Roma Tre University in Rome. The distinction between specimen-level collection data and flower-visit observations is important because the latter form a separate ecological dataset used in the plant–bee network.

The two collection methods captured complementary portions of the fauna. Nets collected 383 bees and Malaise traps collected 86. Among the 76 species, 43.4% were sampled exclusively with nets, 42.1% with both methods and 14.5% only with Malaise traps. The survey recovered five taxa described as Sardo-Corsican endemics: Bombus terrestris sassaricus, B. xanthopus, Hoplitis legoffi, Panurgus corsicus and Megachile sicula corsica. Some of the six new Sardinian records may be related to the peninsula’s proximity to Corsica, where H. tridentata, H. praestans and B. xanthopus are known, but the study could not establish whether the species are restricted to northeastern Sardinia or occur elsewhere on the island.

For molecular analysis, the researchers extracted genomic DNA from one leg using a salting-out protocol. They amplified the mitochondrial COI barcode fragment with established primer sets, applying a nested protocol and, for some difficult taxa, an alternative pair previously used for Andrena, Hylaeus and Colletes. Amplification success was checked on agarose gels, and purified products were sequenced by Macrogen in Madrid and Milan. The resulting sequences were deposited in BOLD and GenBank under accession numbers PQ472941–PQ473152. Species assignments were initially based on Kimura two-parameter-corrected genetic distances with a 3% cut-off in the BOLD identification system. Sequences were aligned with MUSCLE, and distance-based neighbour-joining trees were constructed in MEGA 7 under the K2P model with 500 bootstrap replicates.

The final assignments combined molecular and morphological evidence rather than relying on one method alone. The researchers consulted dichotomous keys, taxonomic literature, the Meloni collection of Sardinian wild bees in Genoa, distribution references and online identification resources. They also sought specialist support for uncertain material, including specialists in Tetralonia, Andrena, Colletes, Hylaeus, Lasioglossum and Hoplitis. Final names were assigned on the basis of morphological identification, BOLD similarity and, when necessary, the position of the analysed specimen in phylogenetic trees. This integrative approach allowed every sample to be identified to the level the evidence supported, although three samples remained unnamed: two Andrena species and one Anthophora species.

Most genetic clusters corresponded to the species recognized morphologically, but several cases exposed unresolved taxonomic questions. The Sardinian B. terrestris sassaricus and B. xanthopus specimens clustered together and showed 99.83–100% similarity to BOLD sequences labelled B. terrestris xanthopus. Their treatment remains part of a wider debate: molecular and ecochemical evidence supported raising xanthopus to species status in 2015, while other analyses and reports of possible hybridization have questioned that status. The team therefore followed the morphological classification pending further work. Sequences assigned to Andrena afzeliella also grouped with sequences labelled A. ovatula, with 99.81–100% similarity, apparently because some older database identifications predated a revision that distinguished the two taxa using morphological and molecular evidence.

Other examples remained uncertain for different reasons. Two Andrena specimens provisionally assigned to A. clypella fell into distinct genetic clades, suggesting that morphologically similar individuals could represent different species; without public A. clypella reference sequences, the study could not establish their relationship. Another Andrena sequence grouped with nominal A. niveata specimens, although the support was below the researchers’ threshold. Colletes maidli showed 96.95% similarity to reference specimens of the same species but occupied an isolated position in the tree. Several Hoplitis sequences had relatively low similarity to public conspecifics, and the recently described H. legoffi matched H. adunca at 93.78% while forming a distinct lineage. In Lasioglossum, L. leucozonium appeared paraphyletic in four clades, while L. prasinum showed 93.50% similarity to a nominal conspecific. The authors also detected distinct Barcode Index Numbers within L. leucozonium and L. villosulum, indicating possible cryptic diversity in species complexes that recent integrative studies have divided into multiple candidate lineages.

Bee visitors formed a network involving 63 species and 20 flowering-plant families. Asteraceae was visited by the largest percentage of recorded bee species, at 46%, followed by Plumbaginaceae at 23.8%, Cistaceae at 20.6%, Fabaceae at 19%, Asphodelaceae at 17.4%, and Brassicaceae and Lamiaceae at 15.9% each. Apiaceae, Primulaceae and Scrophulariaceae were each visited by only one recorded bee species. The analysis grouped plants at family rather than species or genus level to simplify the visualization and mitigate bias associated with small sample sizes for some bees. It documented previously unreported floral associations, including Andrena antonellae on Cistaceae, A. morio on Plumbaginaceae, H. praestans on Asteraceae and A. afzeliella on Asphodelaceae and Caryophyllaceae. It also provided the first observed flower visits by the Sardo-Corsican endemic Panurgus corsicus, which was recorded only on Asteraceae in this survey.

The records connect local occurrence with several taxa of conservation concern. Colletes brevigena, Andrena sardoa, A. griseobalteata, Sphecodes crassanus, Panurgus corsicus, Hylaeus deceptorius, Hoplitis bihamata and H. fasciculata had previously been assessed as being at moderate risk of extinction, while Lasioglossum prasinum was classified as Near Threatened in European and Italian red lists. The study is a locality-based inventory rather than a population-trend analysis, and it did not test the causes of rarity or the effectiveness of any management measure. Its principal contributions are the first peninsula-level checklist, six additions to the documented Sardinian fauna, new Sardinian COI sequences deposited in international databases and an expanded record of plant–bee associations. The authors conclude that further field sampling, specialist morphological analysis, molecular studies and formal species delimitation are needed to resolve the taxonomic boundaries highlighted by the survey and to determine whether the newly recorded species have restricted Sardinian ranges or had been overlooked elsewhere on the island.

Subject of Research: Integrative inventory of wild-bee diversity, DNA barcodes and plant associations in northeastern Sardinia

Article Title: Integrating taxonomic, genetic and ecological data to explore the species richness of wild bees ( Hymenoptera , Apoidea , Anthophila ) of the Culuccia Peninsula (NE Sardinia, Italy)

Article References: Annessi, M., Riccieri, A., Marconi, M., Rossi, S., & Di Giulio, A. (2025). Integrating taxonomic, genetic and ecological data to explore the species richness of wild bees (Hymenoptera, Apoidea, Anthophila) of the Culuccia Peninsula (NE Sardinia, Italy). Journal of Hymenoptera Research, 98, 117-145. https://doi.org/10.3897/jhr.98.138933

Image Credits: AI Generated

DOI: 10.3897/jhr.98.138933

Keywords: wild bees, Sardinia, Culuccia Peninsula, species richness, DNA barcoding, COI sequences, pollination, integrative taxonomy, flower visitation, Mediterranean ecology, Integrating, taxonomic