There are only two Aphaenogaster species from the subterranea group in the western Mediterranean: A. ichnusa Santschi, 1925, from south-western Europe, and A. subterranea (Latreille, 1798), also occurring in central and eastern Europe. Historically, the two species have been widely misunderstood: A. ichnusa was long considered a Sardinian endemic subspecies of A. subterranea, while its continental populations were misidentified as A. subterranea s. str. Recently, A. ichnusa was elevated to species rank and its worker caste was redescribed with that of A. subterranea, allowing for their correct identification. Yet their distribution was documented in detail only for France and Sardinia. Furthermore, no morphological characters were described to distinguish the males and queens of the two species. By investigating private and museum collections, 276 new records of A. ichnusa are provided here and 154 of A. subterranea from the western Mediterranean. Additionally, qualitative and quantitative morphological characters were combined to identify their males and queens. We present the new southernmost, easternmost, and westernmost distribution limits for A. ichnusa. Based on our results, this species is widely distributed in Italy and Catalonia (Spain), also occurring on several Mediterranean islands, avoiding areas with continental climate and high altitudes. Sicily is the only island to host the less thermophilous A. subterranea, which otherwise extends westward to Galicia (Spain). Sympatric occurrence is not rare along the contact zone. Additional natural history observations are reported regarding foraging habits, associated myrmecophiles, habitat preferences, and colony structure in the two species.

Ants, Aphaenogaster ichnusa, biogeography, Mediterranean, Myrmicinae, Stenammini

The genus Aphaenogaster Mayr, 1853 belongs to the myrmicine tribe Stenammini Ashmead, 1905 (Ward et al. 2015) and currently counts 206 valid species and 16 valid subspecies (Bolton 2023). The true genus Aphaenogaster is almost exclusively Holarctic, while the tropical species belong to the “Deromyrma” clade that awaits formal separation (Branstetter et al. 2022). Most Aphaenogaster ant species inhabit the West Palearctic, where six species groups have been identified (Schifani et al. 2022a).

The subterranea group currently includes eight species [A. epirotes (Emery, 1895), A. holtzi (Emery, 1898), A. ichnusa Santschi, 1925, A. kurdica Ruzsky, 1905, A. lesbica Forel, 1913, A. maculifrons Kiran & Aktaç, 2008, A. subcostata Viehmeyer, 1922, A. subterranea (Latreille, 1798)], mostly residing in the eastern Mediterranean and Caucasus region, where several putative undescribed species also exist (Borowiec and Salata 2017; Schifani et al. 2022a). Ten taxa formerly included in this group that are distributed in the Maghreb region and around Sicily, were recently assigned to the distinct crocea group according to morphological and genetic evidence (Alicata and Schifani 2019; Schifani et al. 2022a). The only three North African records of A. subterranea are based on outdated taxonomy (Forel 1890; Bernard 1967; Cagniant 1970; Henine-Maouche et al. 2022), and most likely represent misidentifications of species of the crocea group (Alicata and Schifani 2019; Schifani et al. 2022a). For instance, a worker labeled A. subterranea in the Forel’s collection of Geneve Natural History Museum (AntWeb identifier CASENT0907686) is in fact A. strioloides (see Schifani et al. 2021a).

Only two species belonging to the subterranea group are recognized in West Europe: A. subterranea (Latreille, 1798) (terra typica: mainland France) and A. ichnusa Santschi, 1925 (terra typica: Sardinia). The first was described using the spelling “subteranea” (Latreille 1798), which was later corrected by Latreille himself (Latreille 1802).

Aphaenogaster subterranea has a wide distribution, from Iberia to Anatolia, and north to central Europe, while the range of A. ichnusa is restricted to West Europe and does not occur in the Balkans (Borowiec and Salata pers. comm., 2022; MM unpublished data). Until recent years A. ichnusa was considered as a Sardinian endemic and a subspecies of A. subterranea. Their separation as two distinct species was only recently demonstrated based on worker morphology and mitochondrial DNA (Galkowski et al. 2019). The recent recognition implies that reliable data over the distribution of the two species is scarce in the sympatric range, where they were historically confused. Apart from Sardinia, Galkowski et al. (2019) reported the presence of A. ichnusa in Corsica and southern mainland France. More recently, only a few scattered records were published from mainland Italy, Sicily, and mainland Spain (Schifani and Alicata 2018; García et al. 2020; Schär et al. 2020; Zara et al. 2021; Bazzato et al. 2022; Scupola et al. 2022). However, a detailed picture of the distribution of A. ichnusa and A. subterranea in these regions is lacking.

The investigation of specimens from private and museum collections allowed us to gather reliable distribution data of A. ichnusa and A. subterranea from the west Mediterranean basin, and to fill the current gaps of knowledge. Among them, we investigated several complete nest samples allowing us to morphologically characterize the sexual castes of the two species and provide tools for their identification.

In this study, we focused our investigation on the West European region, where A. ichnusa is present and co-occurs with A. subterranea. We re-identified worker specimens belonging to the subterranea species group found in the authors’ personal collections and the Natural History Museum of Milan. Specimen identification was performed using the morphological characters illustrated by Galkowski et al. (2019): A. ichnusa differs from A. subterranea in the shorter and thicker propodeal spines, which are triangular, the feebler surface sculpturing of the pronotal sides, and paler pigmentation (Fig. 1).

Figure 1. 

Workers of Aphaenogaster ichnusa (on the left, sample from Monte Petroso, Sicily) and of A. subterranea (on the right, sample from Parma, Italian peninsula). Cropped images highlight the differences in surface sculpturing of the pronotal sides and in the shape of the propodeal spines.

Queens and males associated with identified workers were examined to detect further distinctive morphological features. In total, we studied 16 queens from nine colonies and 17 males from seven colonies of A. ichnusa, as well as 16 queens from eight colonies and 19 males from nine colonies of A. subterranea. Samples were chosen from across the study region and included specimens from their terrae typicae (see Suppl. material 1). Due to the unknown, but possibly significant, dispersal capabilities, queens and males unassociated with workers as proof of successful colony establishment were not considered informative for the distribution range of the two species. Morphometric data for the descriptions was produced by taking pictures with a Carl Zeiss Stemi 2000-C stereomicroscope equipped with a CMEX PRO-5 DC.5000p digital camera and measurements taken with ImageFocus 4 software. Three quantitative morphological characters were defined for this study based on those presented by Wagner et al. (2017) and Seifert (2019) (Fig. 2):

Figure 2. 

Diagnostic quantitative morphological characters recorded on males (A) and queens (B) of Aphaenogaster ichnusa and A. subterranea.

PrW Males only. In dorsal view, maximum width of the dorsal plate of the propodeum at the level of the propodeal spiracles.

SPWI Males only. In dorsal view, maximum width of the dorsal plate of the propodeum at the level of the propodeal spiracles.

MCMssctm Queens only. In dorsal view, quantification of stickman-like or reticulate microsculpture units on the mesoscutum: the number of connected lines building units and being separated by line intersections and by flections angled > 30° is counted. Very short lines are also considered full counts. Arithmetic means of three units per specimen are taken.

In addition to means to distinguish males and queens of A. ichnusa from those of A. subterranea, we provide identification keys that tell them apart from the other Aphaenogaster species occurring in the study area (i.e., A. cardenai, A. italica, A. ovaticeps, and species from the crocea, gibbosa, pallida, splendida, and sardoa groups; see Schifani et al. 2022a).

All characters are given in μm as mean ± sd (min, max). A complete list of the investigated material and of the morphological data is provided in the Suppl. material 1.

The morphological distinction of the subterranea group species in the region is relatively easy (Schifani et al. 2022a), and male-based separation of A. ichnusa from A. subterranea is normally straight-forward as demonstrated by our data. While distinction of queens and workers can be more difficult at first sight, queens are safely separable at adequate magnification and workers are normally easy for a trained eye. Colony samples of A. ichnusa with longer spines and colony samples of A. subterranea with shorter ones may occur, always requiring a certain attention to other characters such as spine shape, pronotal sculpture and pigmentation. The different ecological requirements of the two species are often helpful but the two may occur sympatrically. While Galkowski et al. (2019) reported a possible introgression event, we did not encounter colony samples with intermediate morphological characters based on subjective evaluation.

While A. ichnusa inhabits areas with a Mediterranean climate, A. subterranea in the same region occurs only at higher elevations or in specific microclimates. However, A. subterranea is frequent at the sea level in continental climatic zones where A. ichnusa is absent (e.g., most of mainland France, the Po Plain). We show that A. ichnusa is frequent across a vast region of the western Mediterranean (Wang et al. 2023, including Italy’s Tyrrhenian and Ionian coasts, and part of the Adriatic one. Moreover, it is the sole species found on all investigated islands with the only exception of Sicily, where A. subterranea is found at higher elevations in the Etna and the Sicilian Apennines alongside other continental species (Schifani et al. 2021b, 2022b).

Notably, both A. ichnusa and A. subterranea seem to have a more limited distribution in Iberia, where they seem to be restricted to northern Spain. The reliability of few old records from southern Spain appears doubtful in the absence of more recent findings (e.g., Santschi 1919). No historic records exist from Portugal (Arcos et al. 2022), yet our data suggest that targeted investigations may discover A. subterranea near the northern border with Spain. The wide distribution of A. ichnusa is apparently reflected in the fact that in some old keys, descriptions and drawings attributed to A. subterranea were found to depict A. ichnusa instead (e.g., Emery 1916 key to the Italian fauna with a drawing of an A. ichnusa male). Aphaenogaster subterranea may have been temporarily introduced to Madagascar (Csősz et al. 2021).

Colonies of A. ichnusa and A. subterranea are often found nesting under rocks, and both species may be especially abundant in shady forest habitats with sufficient humidity and leaf litter (Castracani et al. 2010, referring at least part of the records to A. ichnusa based on voucher checked; Seifert 2018, assuming Central-European records to represent A. subterranea; Zara et al. 2021; Bazzato et al. 2022). Albeit rarely, at least A. ichnusa may adapt significantly to urban environments: in summer days, we observed workers foraging in a shady courtyard entirely made of concrete within a historic palace of Florence.

However, despite their ecological success and wide distribution, still relatively little is known ca. the habits of these species besides anecdotal reports. They are probably mostly predators and scavengers, but at least A. ichnusa was also observed by the authors to tend root aphids, suggesting that trophobiosis may also play an important role in the diet of both. In addition, we observed isopods (Platyarthrus sp.), oribatid mites, silverfish, leaf beetle larvae (Chrysomelidae: Clytrinae), and planthoppers nymphs (Cixiidae) in A. ichnusa nests, and isopods (Platyarthridae in A. subterranea nests, in which also Cixiidae were reported by Lörinczi (2012). In shady habitats, we observed minimal daytime foraging by both species. However, on other occasions, workers of A. ichnusa mostly started to forage outside the nest at dusk. Perhaps the foraging activity is often conducted within the leaf litter or in endogean microspaces, but none of these species is truly subterranean (Ortuño et al. 2014).

Some of us also observed cooperative food transport in A. subterranea at least. Furthermore, A. subterranea workers are known to use tools: they drop small debris into liquid food and transport food-soaked tools back into their nest (Lőrinczi et al. 2018; Módra et al. 2022). This behavior is deemed to be a complex strategy developed to compensate for the inability to ingest and carry into the body large quantities of liquid food (Lőrinczi et al. 2018; Módra et al. 2022). Notably, we found multiple times numerous dealate queens (up to five) in some colonies of A. ichnusa and A. subterranea, suggesting at least occasional polygyny in both species.

An integrative approach is needed to clarify whether hybridization or introgression between the two species have a significant role as observed in other ants (Seifert 2018). Nuptial flights of A. subterranea and A. ichnusa seemingly overlap in late summer/early autumn. However, the relatively large size of the contact zone compared to the distribution range of A. ichnusa, and the consistent morphological differentiation from A. subterranea, suggest some adaptation to counter hybridization. At the same time, the data we are providing could facilitate future studies focused on the ecology and niche partitioning of these species.

We are grateful to Rumsaïs Blatrix for sharing with us the raw species distribution data used by Galkowski et al. (2019), as well as to all the collectors who contributed to gather specimens relevant to this paper (see the list in the Suppl. material 1). We also thank the referees Alexander Wild and Sebastian Salata as well as the editor Brendon Boudinot for their comments on an earlier version of this manuscript.

Support for this research was provided by “la Caixa” Foundation (ID 100010434) to Mattia Menchetti (grant LCF/BQ/ DR20/11790020).