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Research Article
Revisiting the morphological species groups of West-Palearctic Aphaenogaster ants (Hymenoptera: Formicidae) under a phylogenetic perspective: toward an evolutionary classification
expand article infoEnrico Schifani, Antonio Alicata§, Mattia Menchetti|, Lech Borowiec, Brian L. Fisher#, Celal Karaman¤, Kadri Kiran¤, Wala Oueslati«, Sebastian Salata, Rumsaïs Blatrix»
‡ University of Parma, Parma, Italy
§ University of Catania, Catania, Italy
| Institut de Biologia Evolutiva (CSIC-UPF), Barcelona, Spain
¶ University of Wroclaw, Wroclaw, Poland
# Department of Entomology, California Academy of Sciences, San Francisco, United States of America
¤ Trakya University, Edirne, Turkiye
« University of Tunis El Manar, Tunis, Tunisia
» Université de Montpellier, Montpellier, France
Open Access

Abstract

The West-Palearctic region is a diversity hotspot for the ant genus Aphaenogaster. Species in this region are characterized by high morphological variation, which has led to their subdivisioninto different infrageneric groups. The very first classification in three subgenera, dated 1915, was gradually replaced by eight species-groups. To probe the evolutionary consistency of these species-groups, we sequenced 46 species from all eight species-groups and biogeographic sectors of the region, using one mitochondrial (COI) and six nuclear markers (EPICs), and interpreted the results by integrating qualitative morphology. Our results demonstrate the non-monophyly of all formerly recognized subgenera and species-groups, except for the crocea group. We use the phylogeny and morphological characters to propose a new classification of six monophyletic species-groups (crocea, gibbosa, graeca, pallida, sardoa, subterranea). The pallida, subterranea and sardoa (formerly testaceopilosa) groups attain monophyletic status by reassigning a few taxa. The gibbosa group is to be considered exclusively Western-Mediterranean until further assessments of similar Eastern species. The new graeca group is established by including former members of the splendida and subterranea groups, while the polyphyletic cecconii, obsidiana, and splendida groups are dismissed. Notably, the first is not part of the tropical Deromyrma clade as previously thought, while at least two independent clades which require further investigation are composed of species from both the cecconii and splendida groups, suggesting repeated morphological convergences based on similar ecological adaptations. Finally, A. cardenai is confirmed to be a significantly divergent lineage. In addition, three Aphaenogaster species are moved to different genera: Messor asmaae (Sharaf, 2018) comb. nov., Messor isekram (Bernard, 1977) comb. nov., and Pheidole sarae (Sharaf, 2018) comb. nov. Further studies should address the evolutionary relationships between the clades recovered in this study.

Keywords

apomorphy, biogeography, diversification, Mediterranean fauna, morphological convergence, plesiomorphy, Myrmicinae

1. Introduction

The ant genus Aphaenogaster Mayr, 1853 is part of the tribe Stenammini Ashmead, 1905, along with the genera Goniomma Emery, 1895, Messor Forel, 1890, Novomessor Emery, 1915, Oxyopomyrmex André, 1881, Stenamma Westwood, 1839, and Veromessor Forel, 1917 (Ward et al. 2015). Nowadays, Aphaenogaster includes 210 valid extant species and 17 subspecies, as well as 19 fossil species (Bolton 2022). Their distribution is concentrated in the subtropical regions of the Holarctic realm or in the subequatorial and equatorial areas that constitute the Indomalayan and Australasian realms, while only fewer species occur in the northern Neotropics (Central America and Caribbean), in the Nearctic realm, and in temperate regions of Australasia (Janicki et al. 2016; Guénard et al. 2017). Phylogenomic evidence, however, strongly suggests that, in its current definition, the genus is polyphyletic, and that the subequatorial and equatorial species of Asia, Australia, Madagascar, and Central America should be assigned to a separate genus, provisionally indicated as the “Deromyrma clade” (named after the former subgenus Deromyrma Forel, 1913, currently a junior synonym of Aphaenogaster) (Branstetter et al. 2022). The exclusively Holarctic ‘true’ genus Aphaenogaster is thought to have a Palearctic origin and temperate ancestral habit, and to be the sister genus of the seed-harvesting genus Messor Forel, 1890 (Branstetter et al. 2022). Together, the two form the “Aphaenogaster clade”, and while more likely considered to be a sister group to the ‘true’ Aphaenogaster, Messor may also be phylogenetically embedded within it according to some phylogenetic reconstructions (Gómez et al. 2018; Schär et al. 2020; Branstetter et al. 2022).

The type species of the genus Aphaenogaster, A. sardoa Mayr, 1853, was described from the West-Palearctic region, specifically from the Mediterranean island of Sardinia (Mayr 1853). The Mediterranean region is a widely recognized key biodiversity hotspot for terrestrial organisms (Médail and Quézel 1999) and it hosts the largest portion of the world’s Aphaenogaster diversity, which becomes the vast majority of the species if the Deromyrma clade is not counted. In his catalogue, Borowiec (2014) presented an impressive list of 82 valid taxa, and additional species have been described since then, rising the number at over 100 taxa (Borowiec and Salata 2014; Salata and Borowiec 2016; Gómez et al. 2018; Alicata and Schifani 2019; Bračko et al. 2019; Salata et al. 2021). Interestingly, this diversity is not only a matter of species richness but also remarkable in morphological and ecological terms. The West-Palearctic fauna ranges from long-legged ants running diurnally in open habitats of arid regions (e.g., A. senilis Mayr, 1853), through species with short legs and smaller eyes living in the leaf litter of forest or endogean habitats (e.g., A. pallida, A. subterranea), and troglobiotic and hypogean species with long antennae and slow movements (e.g., A. cardenai, A. cecconii), to specialized granivorous species morphologically convergent with more typical seed-harvesting genera (A. striativentris) (Tinaut and Jiménez Rojas 1991; Caut et al. 2013; Borowiec and Salata 2014; Ortuño et al. 2014; Seifert 2018).

Morphological diversification in particular encouraged the introduction of different infrageneric classifications, often largely based on the West-Palearctic fauna, the first of which was established by Emery (1915). In his work, he presented a subdivision of four subgenera: Aphaenogaster s. str. (comprising species characterized by reduced mesosoma in the queen caste), Attomyrma Emery, 1915 (a large group of species with regular-sized mesosoma for claustral-type queens and heads lacking an elongated neck, type is A. subterranea (Latreille, 1798) described from France), Deromyrma Emery, 1915 (most species having a neck-like elongation of the head, type is A. swammerdami Forel, 1886 described from Madagascar), and Planimyrma Viehmeyer, 1914 (a few Papuan species similar to Deromyrma but whose males possess two spines on the mesonotum and 12 instead of 13 antennal segments, type species is A. loriai (Emery, 1897) described from New Guinea). Emery (1915) divided Deromyrmainto several species-groups, including the cecconii group known from the Mediterranean. According to him, the West-Palearctic fauna was split between three subgenera Aphaenogaster s. str., Attomyrma, and Deromyrma. This framework was gradually abandoned, and eventually Attomyrma, Deromyrma, and Planimyrma were considered junior synonyms of Aphaenogaster (Brown 1973; Smith 1979; Bolton 1982; 1995).

Later Schulz (1994) proposed a subdivision of the very large subgenus Attomyrma (which he still recognized as valid)into six species-groups: gibbosa, obsidiana, pallida, splendida, subterranea, and rothneyi. All of these groups were West-Palearctic and named after Mediterranean species, with the exclusion of the rothneyi group, and this new classification outlived the subgenera. However, several West-Palearctic taxa and most of those from other regions were not mentioned by Schulz (1994), so their status in relation to his classification remained unclear. Two decades later, Boer (2013) followed the same structure as Schulz (1994), mostly focusing on Mediterranean species and entirely abandoning the subgeneric classification of Emery (1915). He replaced the former Aphaenogaster s. str. subgenus by splitting it into a testeceopilosa group, leaving A. sardoa as separate. He also changed the species composition of many of the groups defined by Schulz (1994) without changing their number. In the following years, a number of other studies improved the general understanding of the Mediterranean Aphaenogaster diversity, kept redefining the boundaries of many groups (e.g., Salata and Borowiec 2018), and finally an eighth group was introduced (crocea group, see Alicata and Schifani 2019).

In the last decade, for what concerns the ‘true’ Aphaenogaster, there was the first influx of scattered phylogenetic data produced by the increasing use of molecular phylogenetics, mostly coming from the West-Palearctic or Nearctic faunas (Branstetter et al. 2022; DeMarco and Cognato 2016; Lorite et al. 2017; Centorame et al. 2018; Gómez et al. 2018; Schär et al. 2020). A notable incidental result was the apparent paraphyly of the clades formed by the species of the former subgenera Aphaenogaster s. str. and Attomyrma, but no study was specifically conceived to reconstruct the phylogenetic relationships between the recognized species-groups. A large portion of these groups was not covered by any of these studies, and the consistency of the whole framework was never tested. Moreover, the relationship between the cecconii group and the “Deromyrma clade” was never investigated (Branstetter et al. 2022), despite the fact that Emery (1915) considering the first to be part of the latter.

Our aim was to finally produce a phylogenetic reconstruction covering all the species-groups recognized in the West-Palearctic region, testing for the first time the evolutionary coherence of this framework, including clarifying the groups’ relationship with the tropical “Deromyrma clade”, and trying to determine whether key morphological characteristics traditionally used to characterize these groups were apomorphic or convergent.

2. Materials and methods

2.1. Study area

The West-Palearctic boundaries are here considered to comprise the Mediterranean regions of Africa and Asia. In this definition, we followed the traditional concept of Sclater (1858), and not what was proposed more recently by Holt et al. (2013; also see Wang et al. 2022). Our reasoning behind this choice stems from the fact that the distribution of Aphaenogaster does not comprise the Afrotropical region (Branstetter et al. 2022), but North-Western Africa and the Eastern Mediterranean coast both host a large share of the species thought to be closely related to European taxa (e.g., Borowiec and Salata 2014; Salata and Borowiec 2018; Alicata and Schifani 2019; Salata et al. 2021). This pattern is also visible for other groups, such as vascular plants (Carta et al. 2022).

2.2. Species-groups concept

We define species-groups as mutually exclusive, monophyletic entities comprising multiple closely related species of the same genus.

2.3. Composition of the West-Palearctic species-groups

For the composition of each species-groups, we adopted the most recently published classifications. After each taxon, we report its state-level distribution according to AntMaps (Janicki et al. 2016; Guénard et al. 2017) and recent records, if any, that have not yet been added to its database (Salata et al. 2021; Schifani et al. 2021). Taxa not found in the West-Palearctic region, very few of which were listed by Schulz (1994), are not considered here.

2.3.1. cecconii group

We follow the recent definition by Borowiec and Salata (2014) and Salata and Borowiec (2016), that groups East-Mediterranean species formerly assigned to the subgenus Deromyrma alongside others with similar worker morphology, biogeography, and ecology, resulting in a list of 7 taxa: A. cecconii Emery, 1894 (Greece), A. charesi Salata and Borowiec, 2016 (Greece), A. equestris Borowiec and Salata, 2014 (Türkiye), A. jolantae Borowiec and Salata, 2014 (Greece), A. lykiaensis Borowiec and Salata, 2014 (Türkiye), A. olympica Borowiec and Salata, 2014 (Greece) and A. phillipsi Wheeler and Mann, 1916 (Egypt, Israel and Palestine, Jordan).

Defining combination of morphological characters (after Borowiec and Salata 2014, all characters referred to workers): “Body surface with indistinct microsculpture, shiny across extensive areas; body coloration from yellow to black; head oval with a sharp basal carina or strongly narrowed posteriorly to a neck with a flared collar; antennal scapes long, surpassing the posterior margin of the head by at least 1/3 of their length; basal and mid antennal segments distinctively longer than wide; mesosoma narrow and elongate”

2.3.2. crocea group

According to its very recent definition, the Siculo-Maghrebian crocea group comprises 10 taxa formerly included in the gibbosa, splendida or subterranea groups, grouped together based on male and worker morphology and biogeography (Alicata and Schifani 2019): A. crocea s. str. André, 1881 (Algeria, Morocco), A. crocea croceoides Forel, 1890 (Algeria, Tunisia), A. crocea lenis Santschi, 1911 (Tunisia), A. crocea splendidoides Forel, 1890 (Algeria, Tunisia), A. faureli Cagniant, 1969 (Algeria), A. fiorii Emery, 1915 (Italy, Malta), A. hesperia Santschi, 1911 (Spain: Canary Islands), A. sicula Emery, 1908 (Italy), A. strioloides Forel, 1890 (Algeria, Italy, Tunisia), and A. trinacriae Alicata and Schifani, 2019 (Italy).

Defining combination of morphological characters (based on Alicata and Schifani 2019): worker pigmentation from light brownish to ferruginous or yellow (never blackish); worker appendages and mesosoma not significantly elongate, the latter lacking a deep metanotal groove in profile view; males with an anteriorly gibbous and posteriorly flat mesosoma, head approximately as long as wide.

2.3.3. gibbosa group

This group comprises taxa sharing similar worker and/or male morphology initially thought to be close to the subterranea or the testaceopilosa groups (Emery and Forel 1879; Dalla Torre 1893; Emery 1921). The following 9 taxa, mostly West-Mediterranean, are considered to belong to it (Salata and Borowiec 2018; Gómez et al. 2018; Alicata and Schifani 2019): A. gibbosa s. str. (Latreille, 1798) (France, Italy, Spain, Portugal), A. gibbosa homonyma Emery, 1921 (Algeria, Morocco, Tunisia), A. italica Bondroit, 1918 (Italy, Switzerland), A. mauritanica Dalla Torre, 1893 (Algeria, Morocco, Tunisia), A. muschtaidica Emery, 1908 (Azerbaijan, Georgia, Iran), A. nadigi Santschi, 1923 (Morocco), A. striativentris Forel, 1895 (Spain), A. theryi Santschi, 1923 (Morocco), and A. ulibeli Gómez and Espadaler, 2018 (Spain).

Defining combination of morphological characters (based on Salata and Borowiec 2018; Gómez et al. 2018; Alicata and Schifani 2019): body pigmentation of workers from dark brown to black, their head characterized by longitudinal rugae or reticulation at least on its anterior part of head dorsum, sometimes rugae and reticulation replaced or co-occurring with punctuation, surface between rugae with dense micropunctuaction or smooth and shiny; in workers, funicular segments from 1.5 to 2 times longer than wide and the scapi reach at least 1/5 of its length over the occipital margin of the head; workers’ propodeal spines are always present, short, triangular, inclined at an 45° angle, with a wide base. Males with an anteriorly gibbous and posteriorly flat mesosoma except for A. ulibeli.

2.3.4. obsidiana group

The obsidiana group was defined as a small group of 3 East-Mediterranean species based on worker morphology (Schulz 1994): A. epirotes Emery, 1895 (Albania, Croatia, Bosnia and Herzegovina, Bulgaria, Greece, Israel and Palestine, Italy, Montenegro, Serbia, Slovenia, Türkiye), A. obsidiana (Mayr, 1861) (Azerbaijan, Georgia, Iran, Russia, Türkiye), and A. subcostata Viehmeyer, 1922 (Greece, Türkiye).

Defining combination of morphological characters (from Schulz 1994, all characters referred to workers): “Chubby, squat species with an almost square, rounded head. Antennae just reaching the posterior edge of the head. Median funiculus segments as long as wide. Coloration light red, brown to black, sculpture deep and dense, mostly connected like a net on the head. Hairs thicker than in the previous group, protruding body hairs numerous. Spines of medium length, always prominent. Despite the deep wrinkles, the microsculpture is not developed; smooth and shiny”.

2.3.5. pallida group

A circum-Mediterranean group based on worker morphology, which includes 11 taxa including those presented by Kiran et al. (2008) and Boer (2013), plus their North African subspecies or relatives defined by Cagniant (1996): A. dulcineae Santschi, 1919 (Italy, France, Portugal, Spain), A. finzii Müller, 1921 (Bosnia and Herzegovina, Croatia, Italy, Greece, Macedonia, Montenegro, Serbia), A. foreli Cagniant, 1996 (Algeria, Morocco), A. holtzi (Emery, 1898) (Iran, Türkiye), A. lesbica Forel, 1913 (Greece, Israel and Palestine, Montenegro), A. leveillei s. str. Emery, 1881 (Algeria, Morocco, Tunisia), A. leveillei laurenti (Santschi, 1939) (Morocco), A. pallida (Nylander, 1849) (Italy), A. radchenkoi Kiran and Aktaç, 2008 (Bulgaria, Greece, Türkiye), A. subterraneoides s. str. Emery, 1881 (Azerbaijan, Bosnia and Herzegovina, Bulgaria, Croatia, Cyprus, Greece, Israel and Palestine, Macedonia, Russia, Ukraine), and A. subterraneoides armeniaca Arnol’di, 1968 (Armenia).

Defining combination of morphological characters (from Schulz 1994, all characters referred to workers): “Mostly small narrow forms, with mostly smooth and glazed integument and pale yellow colouring. Epinotum usually with small tooth-like spines, or completely spineless (in A. finzii the spines are longer, but all other characteristics are clearly developed, which is why this species is to be classified here). Hairs long, thinner than other groups. Antennal shaft just exceeding the posterior margin. Median funiculus segments as long as wide”.

2.3.6. splendida group

A mostly East-Mediterranean species-groups recently redefined by Salata et al. (2021) based on worker morphology, and which counts 13 taxa: A. aktaci Kiran and Tezcan, 2008 (Greece, Türkiye), A. dlusskyi Radchenko and Arakelian, 1991 (Armenia), A. festae Emery, 1915 (Bulgaria, Greece, Türkiye), A. hamaensis Salata et al., 2021 (Syria), A. kervillei Forel, 1910 (Israel and Palestine, Lebanon, Syria, Türkiye), A. ovaticeps Emery, 1898 (Albania, Bosnia and Herzegovina, Croatia, Greece, Italy, Montenegro, Serbia, Slovenia), A. peloponnesiaca Salata et al., 2021 (Greece), A. schmitzi Forel, 1910 (Israel and Palestine, Jordan, Syria, Türkiye), A. rugosoferruginea Forel, 1889 (Greece), A. splendida (Roger, 1859) (Algeria, Bosnia and Herzegovina, Cyprus, Egypt, France, Greece, Iran, Israel and Palestine, Italy, Lebanon, Libya, Macedonia, Malta, Montenegro, Portugal, Russia, Spain, Syria, Tunisia, Ukraine), A. syriaca Emery, 1908 (Egypt, Iran, Israel and Palestine, Lebanon, Syria), A. transcaucasica Karavaiev, 1926 (Azerbaijan), and A. vohraliki Salata et al., 2021 (Türkiye).

Defining combination of morphological characters (from Salata et al. 2021): “Workers moderate to large (mesosoma length along Weber’s line: 1.5–3.0), with slim and elongated body; head always longer than wide, slim and oval to elongated (head length on head width ratio: 1.1–1.6); antennae and legs elongate; scape distinctly protruding above the head occipital margin; segments of antennal funicle always longer than wide; body usually yellow to yellowish brown, occasionally brown but never black; head sculpture distinct, with microreticulate background and more or less developed longitudinal to reticulate rugae; surface of mesosoma mostly reticulate and with additional sculpture of longitudinal or/ and reticulate rugae, shiny areas, if present, restricted to pronotal top and sides; gaster shiny, usually smooth or with diffused microreticulation only on the first gastral tergite. Males with gibbous promesonotum, and narrow elongate propodeum, known in detail for only two species (A. festae and A. splendida)”.

2.3.7. subterranea group

A mostly East-Mediterranean group defined by Schulz (1994), Boer (2013), Alicata and Schifani (2019), Galkowski et al. (2019) and Bračko et al. (2019) based on worker morphology, and includes six taxa: A. graeca Schulz 1994 (Greece), A. ichnusa Santschi, 1925 (France, Italy, Spain), A. illyrica Bračko et al., 2019 (Bosnia and Herzegovina, Bulgaria, Croatia, Greece, Macedonia), A. kurdica (Ruzsky, 1905) (Armenia, Azerbaijan, Georgia, Iran, Russia), A. maculifrons Kiran and Aktaç, 2008 (Türkiye), and A. subterranea (Latreille, 1798) (Albania, Austria, Armenia, Azerbaijan, Belgium, Bosnia and Herzegovina, Bulgaria, Czech Republic, France, Georgia, Germany, Greece, Hungary, Iran, Italy, Macedonia, Moldova, Montenegro, Poland, Romania, Russia, Serbia, Slovakia, Slovenia, Spain, Switzerland, Türkiye, Ukraine).

Defining combination of morphological characters (based on Schulz 1994; Bračko et al. 2019): Medium-sized forms, shallow sculpture, large parts of the body shiny. But always at least the front part of the head is dull covered with shallow wrinkles. The posterior edge of the head overruns the antennae. Median funiculus segments are about as long as wide, but not more than 1/2 times as long. Color yellowish-red to dark brown. Head rectangular rounded. Metanotal groove present, deep and narrow. Pronotum and mesonotum form regular convexity, mesonotum not raised above the surface of pronotum, propodeal spines short, not longer than half length of the first segment of antennal funiculus, mesosoma short.

2.3.8. testaceopilosa group

A large Euro-Maghrebian group based on worker and queen morphology (Cagniant 1996; Boer 2013; Cagniant and Galkowski 2013) with 41 taxa: A. afra Santschi, 1933 (Algeria, Morocco), A. atlantis Santschi, 1929 (Morocco), A. balcanica (Emery, 1898) (Albania, Bosnia and Herzegovina, Croatia, Greece, Türkiye), A. balcanicoides Boer, 2013 (Greece), A. baronii Cagniant, 1988 (Morocco), A. campana Emery, 1878 (Italy), A. curiosa Santschi, 1933 (Morocco), A. dejeani Cagniant, 1982 (Morocco), A. depilis s. str. Santschi, 1911 (Algeria, Morocco), A. depilis numida Santschi, 1933 (Tunisia), A. espadaleri Cagniant, 1984 (Morocco), A. fallax Cagniant, 1992 (Algeria, Morocco), A. gemella s. str. (Roger, 1862) (Morocco, Algeria, Portugal, Spain), A. gemella marocana (Forel, 1903) (Morocco), A. iberica Emery, 1908 (Portugal, Spain), A. inermita Bolton, 1995 (Italy, Malta), A. karpathica Boer, 2013 (Greece), A. koniari Cagniant and Galkowski, 2013 (Morocco), A. melitensis Boer, 2013 (Malta), A. miniata Cagniant, 1990 (Morocco), A. picena Baroni Urbani, 1971 (Albania, Croatia, Italy, Slovenia), A. praedo s. str. Emery, 1908 (Algeria, Morocco), A. praedo ellipsoida Santschi, 1933 (Morocco), A. praenoda Santschi, 1933 (Morocco), A. praenoda confinis Santschi, 1933 (Morocco), A. rifensis Cagniant, 1994 (Morocco), A. rupestris Forel, 1909 (Algeria, Morocco), A. semipolita (Nylander, 1856) (Italy), A. senilis Mayr, 1853 (Algeria, France, Italy, Morocco, Portugal, Spain), A. senilis disjuncta Santschi, 1933 (Morocco), A. sicardi Cagniant, 1990 (Morocco), A. simonellii Emery, 1894 (Greece), A. spinosa Emery, 1878 (Italy, France, Switzerland), A. sporadis Santschi, 1933 (Cyprus, Greece, Türkiye), A. testaceopilosa (Lucas, 1849) (Algeria, Morocco, Spain, Tunisia), A. testaceopilosa cabylica Stitz, 1917 (Algeria, Morocco, Tunisia), A. testaceopilosa canescens (Emery, 1895) (Algeria, Tunisia), A. tinauti Cagniant, 1992 (Morocco), A. torossiani Cagniant, 1988 (Morocco), A. weleursseae Cagniant, 1989 (Morocco), and A. wilsoni Cagniant, 1988 (Morocco).

Defining combination of morphological characters (from Boer 2013): “The workers of the A. testaceopilosa-group have a punctate head and mesosoma, while the head is neither elongated, nor collar-shaped. This character combination is absent in the other European species of the genus Aphaenogaster, except for A. sardoa. The punctation is also present in the gyne and male, but only on certain body parts, usually the head. In most species, the anterior portion of the dorsal side of the first gastral tergite is microstriated. […] Originally this subgenus contained all the species here placed in the A. testaceopilosa-group, plus A. sardoa. The reason to exclude this species is that A. sardoa differs in several characters from the other species: 1) the males of A. sardoa have small, short and minutly dentate mandibles, instead of dentate, broad mandibles, 2) the males and gynes of A. sardoa have larger eyes and ocelli, 3) they have broad spherical petioles, and 4) all castes of A. sardoa are yellowish, while those of the A. testaceopilosa-group are blackish or (particularly after preservation) reddish-brown”.

2.3.9. Species not part of any group

We consider A. cardenai Espadaler, 1981 (Spain), A. sardoa s. str. Mayr, 1853 (Algeria, Italy, Morocco, Tunisia), A. sardoa anoemica Santschi, 1910 (Morocco), and A. ujehlyi Szabó, 1910 (Tunisia) not to belong to any group. Although A. cardenai was until recently placed within the splendida group (Espadaler 1981; Boer 2013), Gómez et al. (2018) contested this position and considered it to be only distantly related to the remaining Mediterranean Aphaenogaster based on molecular data (though they did not test its relationship with any species of the splendida group). In morphological terms, A. cardenai is certainly unique in several aspects being characterized by very small eyes (compared to the head size, proportionally smaller than in any other species of the region), very long scapi (only comparable to those of the cecconii group), a significantly developed sculpture interspersed with entirely shiny areas, an elongate mesosoma and very long spines (longer than in any other species of the region). Aphaenogaster sardoa, on the other hand, was long considered to form a single group with the members of the actual testaceopilosa group (i.e. part of the Aphaenogaster s. str. subgenus) until Boer (2013) separated them based on the morphological characters stated above (see under A. testaceopilosa group). Moreover, A. sardoa anoemica and A. ujehlyi are two valid sympatric taxa whose distinctiveness from A. sardoa s. str. is undemonstrated. Most likely due to their dubious status, they were not accommodated in the sardoa group by Boer (2013).

2.3.10. Species of uncertain position

We consider uncertain the position of A. burri (Donisthorpe, 1950) (Türkiye), A. depressa Bolton, 1995 (Türkiye), A. pallescens Walker, 1871 (Egypt), A. saharensis (Bernard, 1953) (Algeria), and A. sangiorgii Emery, 1901 (Greece). Morphology may suggest that the first two could be tentatively associated with the subterranea group, and the latter one to the pallida group, but their statuses are unclear since they are all known from only the holotype specimens, which in the case of A. burri and A. sangiorgii are queens (Emery 1901; Donisthorpe 1950). The identity of A. sangiorgii will be discussed in a dedicated paper (in prep.). Finally, A. pallescens is considered a species incertae sedis or unrecognizable taxon (Emery 1915; Bolton 1995), and the identity of A. saharensis, described from a single male (Bernard 1953), remains unclear.

2.3.11. Species excluded from the West-Palearctic Aphaenogaster list

For reasons we detail in the results section, we do not consider A. asmaae Sharaf, 2018 (Oman), A. isekram Bernard, 1977 (Algeria), or A. sarae Sharaf, 2018 (Oman) as part of the West-Palearctic Aphaenogaster diversity.

2.4. Molecular phylogeny

DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), following the protocol by Cruaud et al. (2019), on whole specimens without damaging the integument, or, in a few cases, on one of the middle legs. One mitochondrial marker (COI, 658 bp, coding for part of the cytochrome c oxidase subunit 1) and six Exon Primed Intron Crossing (“EPIC”) nuclear markers (ant.1, 373 bp; ant.263, 460 bp; ant.346, 391 bp; ant.389, 689 bp; ant.505, 521 bp; ant.1401, 935 bp) were amplified by polymerase chain reaction (PCR) using the primer pairs in Folmer et al. (1994) and Ströher et al. (2013) for COI and EPIC markers respectively. We used the EPIC markers instead of other nuclear markers more commonly used in ant phylogeny (such as those used in Ward et al. 2015) because these more commonly used markers provided little support in a previous phylogeny of the genus Aphaenogaster (DeMarco and Cognato 2016), or showed a lower variability and a lower amplification success than EPIC markers in preliminary tests (see also Centorame et al. 2018, where amplification success of CAD in some European Aphaenogaster species is only 37%). EPIC markers have been successfully used in ant phylogeny in the genera Cataglyphis Foerster, 1850 (Eyer et al. 2017, 2018; Kuhn et al. 2020), Camponotus Mayr, 1861 (Hartke et al. 2019), Plagiolepis Mayr, 1861 (Degueldre et al. 2021) and Tapinoma Foerster, 1850 (Escárraga et al. 2021), and, since they rely on universal primers, they can also be used across a broad taxonomic range. Finally, they may also allow to compare exon and intron fragments although this option was beyond the purpose of this study.

Sanger dideoxy sequencing of PCR amplicons was performed by Eurofins Genomics (Germany) in both directions using the same primers as those used for the initial amplification. Sequences were edited using CodonCode Aligner (CodonCode Corporation, Dedham, MA, USA), and contigs were built from forward and reverse sequences generated for each gene. Conflicting base calls were coded as missing. Sequences were aligned with Muscle (Edgar 2004) using the default settings. Alignments were inspected visually and edited manually using Mesquite v. 3.31 (Maddison and Maddison 2017) when they could be improved. The following substitution models were selected using the Bayesian Information Criteria (BIC) implemented in JModelTest2 v2.1.6 (Darriba et al. 2012) run on the CIPRES Science Gateway (Miller et al. 2010): TIM2 + I + G for COI codon position 1, TIM1 + I + G for COI codon position 2, TIM2 + G for COI codon position 3, HKY + I for ant.1, ant.263 and ant.346, HKY + G for ant.389, ant.505 and ant.1401. The models TIM1 and TIM2 were replaced by the GTR model in MrBayes analyses. Phylogenetic reconstructions were performed using Bayesian inference with MrBayes v. 3.2.7a (Ronquist et al. 2012) on the CIPRES Science Gateway (Miller et al. 2010). Two analyses of four chains were run for 10,000,000 generations, sampling trees every 500 generations and a 25% burn-in for each run. In addition, maximum likelihood phylogenies were constructed with PhyML 3.0 online (http://www.atgc-montpellier.fr/phyml), using automatic model selection by SMS (Lefort et al. 2017) and a standard bootstrap analysis. Phylogenies were produced for all markers concatenated (4027 nucleotides in total), for COI only (658 nucleotides), and for EPIC markers concatenated (3369 nucleotides).

The following numbers of new sequences were produced in this study: 90 for COI (GenBank accession numbers OM896791-OM896880), 82 for ant.1 (OM939213OM939294), 84 for ant.263 (OM939295OM939378), 85 for ant.346 (OM939379OM939463), 67 for ant.389 (OM939464OM939530), 74 for ant.505 (OM939531OM939604), 71 for ant.1401 (OM939605OM939675). Amplification success was 95 % for COI, 88 % for ant.1, 90 % for ant.263, 91 % for ant.346, 72 % for ant.389, 80 % for ant.505 and 76 % for ant.1401. DNA sequence alignments are provided in the Supplementary material S1, while all GenBank accession numbers are provided in the Supplementary material S2.

2.5. Species used for the analysis

A complete list of the specimens sequenced in our investigation, including their geographic origin and collecting data is provided in Supplementary material S2, while this information is reassumed in Figure 1. The following 47 West-Palearctic species, covering all species-groups and biogeographic sectors of the region, were used in the analyses (the % of covered species of each group is expressed in parentheses): cecconii group: A. cecconii, A. charesi, A. olympica (43%); crocea group: A. crocea croceoides, A. fiorii, A. sicula, A. strioloides, A. trinacriae (71%); gibbosa group: A. gibbosa, A. italica, A. mauritanica, A. muschtaidica, A. striativentris, A. ulibeli (75%); obsidiana group: A. epirotes, A. obsidiana (67%); pallida group: A. dulcineae, A. finzii, A. foreli, A. holtzi, A. pallida, A. subterraneoides (86%); splendida group: A. aktaci, A. festae, A. ovaticeps, A. peloponnesiaca, A. rugosoferruginea, A. splendida (50%); subterranea group: A. ichnusa, A. illyrica, A. maculifrons, A. subterranea (80%); testaceopilosa group: A. balcanica, A. campana, A. depilis, A. iberica, A. inermita, A. koniari, A. picena, A. praedo s. l., A. semipolita, A. senilis, A. simonellii, A. spinosa, A. sporadis (39%); species not part of any group: A. cardenai, A. sardoa.

Figure 1. 

Identity and distribution of the samples used for the molecular analyses in this study.

In addition, in order to test a possible relatedness between some West-Palearctic species and members of the tropical Deromyrma clade sensu Branstetter et al. (2022), we also sequenced the following four Malagasy taxa: A. bressleri Csősz and Fisher, 2021, A. gonacantha (Emery, 1899), A. sahafina Csősz and Fisher, 2021, and A. swammerdami Forel, 1886 (Csősz et al. 2021).

Finally, we chose as outgroups the two Stenammini species Stenamma debile (Foerster, 1850) and S. striatulum Emery, 1895 since the genus Stenamma is sister to both the ‘true’ Aphaenogaster and the Deromyrma clade (Branstetter et al. 2022).

Voucher specimens sequenced in this study were marked with unique identifiers which are reported in the Supplementary material S2 alongside their repositories.

2.6. Integrative revision of the species-groups classification

We modify the existing species-groups framework by interpreting the phylogenetic results in light of qualitative morphological characters of the species. Qualitative morphological characters are intended as discrete characters (presence or absence of certain traits) easily observable by trained myrmecologists without the need of detailed numerical recording (Schifani et al. 2022). Modified definitions of existing groups or new species-groups definitions are given only if a strong phylogenetic support backs a clade composed by species bonded by a distinctive set of qualitative morphological characters that should generally allow convincing hypotheses on the possible affiliation of non-sequenced species to that same clade. For the remaining clades, we offer a detailed reporting of the critical issues that must be overcome before a safe group definition can be drafted.

3. Results

3.1. Molecular phylogeny

The sequenced species from the Deromyrma clade (swammerdami group) were recovered as sister to all other investigated species. Similarly, A. cardenai was recovered as sister to all other Mediterraean species, and then, a well-supported clade containing A. striativentris, A. gibbosa, A. ulibeli and A. mauritanica was recovered as sister to all the remaining species. Several highly supported clades were recovered among the remaining species, but the relationships among these clades were poorly supported, hindering any inference on the phylogenetic relationships among them. Most notably, we found as well supported (posterior probability: 0.95–1), a clade containing A. cecconii, A. rugosoferruginea, A. festae and A. splendida, a clade containing A. subterraneoides, A. finzii, A. foreli, A. dulcineae and A. pallida, a clade containing A. illyrica and A. aktaci, a clade containing A. strioloides, A. crocea croceoides, A. sicula, A. fiorii and A. trinacriae, a clade containing A. epirotes, A. holtzi, A. subterranea, A. maculifrons and A. ichnusa, a clade containing A. charesi, A. ovaticeps and A. peloponnesiaca, a clade containing A. striativentris, A. gibbosa, A. ulibeli and A. mauritanica, and a clade containing all the members of the testaceopilosa group plus A. sardoa. The placement of the following species remained unresolved: A. olympica, A. italica, A. muschtaidica and A. obsidiana. This general topology was congruent in both kinds of phylogenetic reconstructions (Bayesian inference and maximum likelihood) (Fig. 2 and Supplementary material S3). The only notable difference between the two reconstruction methods was the support of the clade composed of A. illyrica and A. aktaci, which was strong with Bayesian phylogeny (posterior probability: 1) and only moderate with maximum likelihood (bootstrap value: 0.60).

Figure 2. 

Phylogeny of the West-Palearctic Aphaenogaster ants, including all the species-groups of the region as well as the tropical Deromyrma clade based on a Bayesian analysis of one mitochondrial (COI) and six nuclear (EPICs) markers. Support values represent Bayesian posterior probability values (PP). In the rightmost part of the figure, we present the new species-groups boundaries based on the interpretation of the phylogenetic results that is offered in the Discussion section. Photographs of worker specimens from A. cecconii (CASENT0179868, photo by Erin Prado), A. crocea croceoides (CASENT0907682, syntype, photo by Will Ericson), A. gibbosa (CASENT0914409, photo by Zach Lieberman), A. obsidiana (CASENT0280957, photo by Shannon Hartman), A. dulcineae (CASENT0280959, photo by Michele Esposito), A. splendida (CASENT0280965, photo by S. Hartman), A. subterranea (CASENT0173580, photo by April Nobile), A. testaceopilosa (CASENT0280966, photo by S. Hartman), A. sardoa (CASENT0916080, syntype, photo by Anna Pal), A. cardenai (CASENT0249624, photo by Z. Lieberman), A. swammerdami (CASENT0489647, photo by A. Nobile).

An important difference between phylogenetic reconstructions based on mitochondrial and nuclear markers was that, according to EPIC markers, A. muschtaidica was included in the clade containing A. epirotes, A. holtzi, A. subterranea, A. maculifrons and A. ichnusa, whereas it was outside of this clade following the COI marker. As a whole, the phylogeny based on EPIC markers only was less resolved than that based on COI markers only, but concatenation of both types of markers provided a better result as any of the two marker types taken individually. Every species proved to be monophyletic save the case of A. sporadis, recovered within an unresolved clade with A. balcanica and A. picena.

We obtained the following results concerning the status of each group (Fig. 2):

3.1.1. cecconii group

Status: polyphyletic. The group corresponds to three independent lineages in our tree (one per each species sequenced). In two cases (A. cecconii and A. charesi), the position of these species is strongly supported (posterior probability: 0.95–1) and they are placed as sister to lineages belonging to the non-monophyletic splendida group, while the position of the third (A. olympica) as a sister to A. italica, A. muschtaidica, and A. obsidiana is weakly supported (posterior probability: 0.65).

3.1.2. crocea group

Status: monophyletic. All the sequenced species of the crocea group form a single monophyletic clade with strong support. The two Maghrebian taxa (A. crocea croceoides, A. strioloides) are sister to the species endemic to Italy and Malta (A. fiorii, A. sicula, A. trinacriae).

3.1.3. gibbosa group

Status: polyphyletic. Most of the species are strongly supported in a single clade (posterior probability: 1), however, the position of A. italica and A. muschtaidica is separate in the tree as sister species to A. obsidiana with weak support (posterior probability: 0.65).

3.1.4. obsidiana group

Status: polyphyletic. A. epirotes clusters with high support (posterior probability: 1) as the sister to a group comprising A. holtzi (pallida group) plus all of the species from the subterranea group except A. aktaci. On the other hand, A. obsidiana is positioned separately with a low support (posterior probability: 0.52).

3.1.5. pallida group

Status: polyphyletic. Most species form a well-supported clade (posterior probability: 0.95–1), except A. holtzi which is part of the well-supported clade which is also formed by A. epirotes and most of the subterranea group (posterior probability: 1).

3.1.6. splendida group

Status: polyphyletic. The species of the splendida group form three independent clades in the tree. Aphaenogaster aktaci is strongly supported as the sister species of A. illyrica from the subterranea group (posterior probability: 1). The other species are dividedinto two well-supported clades, each with a species of the cecconii group as its respective sister taxon, one also formed by A. splendida, A. festae, A. rugosoferruginea, and one also formed by A. ovaticeps, A. peloponnesiaca (posterior probability: 0.95–1).

3.1.7. subterranea group

Status: non-monophyletic. All of the species form a well-supported clade except for A. illyrica which is clearly recognized as the sister species of A. aktaci from the splendida group (posterior probability: 1).

3.1.8. testaceopilosa group

Status: paraphyletic. The group is formed by a single clade consisting of two well-supported smaller clades (posterior probability: 1): one which comprises all the European taxa except A. senilis, and the other which is formed by all the North African taxa plus A. senilis. However, A. sardoa is also placed in the latter clade.

3.1.9. Other species

Aphaenogaster cardenai is well-supported in its position as an independent lineage from all the other West-Palearctic Aphaenogaster species (posterior probability: 1). Aphaenogaster sardoa is placed within the testaceopilosa group as mentioned above.

3.1.10. Deromyrma clade

The Malagasy species form a well-supported clade with no close relationship with any of the W-Palearctic Aphaenogaster species-groups (posterior probability: 1).

3.2. New species-groups classification

Based on the available phylogenetic and morphological evidence, we propose to recognize six West-Palearctic species-groups of Aphaenogaster, while commenting the critical issues of the remaining three main clades (Fig. 2). Due to its morphological uniqueness and phylogenetic distance from the rest of the sequenced West-Palearctic species, A. cardenai is kept as a species not belonging to any group. A synoptic list of the West-Palearctic Aphaenogaster fauna, including the new species-groups classifications here proposed is offered in the Supplementary material S4.

3.2.1. crocea group (unchanged)

The definition and composition of this group previously given in the Materials and methods remains unchanged. The group is thought to extend from the Maghreb to Sicily and neighboring regions of Malta and Italy.

3.2.2. gibbosa group (redefined)

The existing morphological definition of the group (see Materials and methods) describes it well but also includes two species (A. italica and A. muschtaidica) whose position is unclear, but which appear to be unrelated to the group. Both have a more Eastern distribution compared to the species which are safely assigned to the group on a phylogenetic basis, as well as A. theryi which occurs sympatrically with A. mauritanica. The affiliation of A. theryi should be established in future studies, while the group should be considered restricted to the W-Mediterranean (Italy almost entirely excluded except for a small North-Western sector where A. gibbosa is thought to occur).

3.2.3. graeca group (newly established)

Our phylogenetic analysis strongly supports a close relatedness between A. aktaci (originally in the obsidiana group and more recently in the splendida group) and A. illyrica (originally in the subterranea group). Aphaenogaster illyrica is very similar to A. graeca morphologically, so that the two were classified in the graeca complex within the subterranea group. Aphaenogaster aktaci shares with them a highly similar morphology, but may also somewhat resemble the darkest species from clades B and C (A. ovaticeps, A. rugosoferruginea). We thus propose to consider the graeca complex as a species-groups considering its independence from the subterranea group and list A. aktaci, A. graeca, and A. illyrica as its members. Further assessments regarding the phylogenetic position of species of the former splendida not sequenced in this study may be relevant, yet none is particularly close to the morphology of the graeca group species which we define according to the following combination of characters (based on workers, see Figure 2): dark brown to reddish, body elongate, appendages long, mesonotum well demarcated, medium-sized spines with a thick base, horizontal or very slightly curved upwards. Mesonotum surmounting the pronotum and separated by a strong promesonotal suture (A. graeca, A. illyrica) or propodeal dorsum lacking transverse surface sculpturing (A. aktaci). The group is considered to inhabit Anatolia and the Balkans.

Figure 3. 

Aphaenogaster workers of species now classifiedinto the new graeca species-groups: A A. aktaci (CASENT0922687, photo by M. Esposito); B A. illyrica (CASENT0872099, holotype, photo by Lech Borowiec); C A. graeca (ANTWEB1041239, paratype, photo by Roland Schultz). Photographs from www.antweb.org.

3.2.4. pallida group (redefined)

The morphological definition of the group should be implemented by highlighting the lack of a strong mesoepinotal furrow which was already used by Alicata and Schifani (2019) to tell apart the crocea group from the subterranea group, and also emphasizing the importance of hairs already reported by Schulz (1994) (Figure 4): this leads to the removal of A. holtzi from the group and restores the monophyly of the pallida group in the tree, while also suggesting to remove A. lesbica which, like A. holtzi, has a strongly reduced head sculpture but in other characters is very similar to the species from the A. subterranea complex. The combination of short ant stout mesosoma, long and abundant hairs (especially on the head, pronotum and gaster), lack of a strong mesoepinotal furrow, shiny integument and brown, often yellowish to greenish pigmentation makes the pallida group one of the most easily recognizable morphologically. This group occurs in the Maghreb, Southern Europe, Anatolia, and Transcaucasia.

Figure 4. 

Lateral profile of Aphaenogaster workers of species from the pallida and subterranea groups. In green, species of the pallida group, with arrows indicating the long and often abundant erect hairs on the dorsal side of the head. In violet, species from the subterranea group with arrows indicating the deep metanotal groove (including A. holtzi and A. lesbica previously attributed to the pallida group, A. epirotes and A. subcostata previously attributed to the obsidiana group). A A. dulcineae (photo by M. Esposito, CASENT0280959); B A. holtzi (CASENT0904178, syntype, photo by W. Ericson); C A. finzii (CASENT0914232, photo by M. Esposito); D A. ichnusa (CASENT0913132, syntype, photo by Z. Lieberman); E A. pallida (CASENT0280960, photo by S. Hartman); F A. maculifrons (CASENT0922688, photo by M. Esposito); G A. subterraneoides (CASENT0281536, photo by Estella Ortega); H A. subterranea (CASENT0172716, photo by A. Nobile); I A. lesbica (CASENT0907690, syntype, photo by W. Ericson); J A. kurdica (CFH000010, photo by Donat Agosti); K A. epirotes (CASENT0281535, photo by E. Ortega); L A. subcostata (FOCOL1221, syntype, photo by Christiana Klingenberg). Photographs from www.antweb.org.

3.2.5. sardoa group (redefined)

The morphological definition of the testaceopilosa group sensu Boer (2013) is modified to restore the original boundaries defined by Emery (1915) for the former Aphaenogaster s. str. genus and establish a new species-groups that contains the former testaceopilosa group members plus A. sardoa. The high similarity of A. sardoa with the species of the former testaceopilosa group was already noted by Boer (2013), so that his set of diagnostic characters for the group, entirely based on workers, would have included A. sardoa and he was forced to complement it with a disclaimer to exclude A. sardoa based on additional characters of males and queens. We therefore refer to the worker-based definition without the complementary part to delimit the sardoa group. However, we also deem important to emphasize that most of the characters chosen by Boer (2013) to separate sardoa based on males and queens from the former testaceopilosa group worked only because he based his study exclusively on the European fauna and neglected the species-rich fauna of North Africa in his definition (Figure 5): it is not true that males of all the other species have larger mandibles and eyes, as the size of mandibles and eyes of A. sardoa is comparable to that of species such as A. curiosa or A. fallax; the notion that queens of A. sardoa have larger eyes compared to the other species does not seem to meet the reality in at least several cases; it is also false that all other species are black and only become reddish after preservation, as some species are genuinely red (e.g. A. praedo, see Cagniant 1969). It is nonetheless true that A. sardoa has an orange-yellowish pigmentation which is lighter than all other species of the genus, and that, as noted by Boer (2013), the petioles of workers and queens are particularly rounded. However, these characters must be autapomorphic.

Figure 5. 

Aphaenogaster species from the sardoa group. Photographs B and D depict A. sardoa specimens from Sicily (queen and male respectively, photos by E. Schifani). Photographs A, C, E are from www.antweb.org: A A. tinauti queen (CASENT0913796, photo by W. Ericson); C A. curiosa male (CASENT0913109, syntype, photo by W. Ericson); E A. praedo worker (CASENT0904158, syntype, photo by Z. Lieberman). Our reassessment suggest that most characters used by Boer (2013) to keep A. sardoa separate from the species of the former testaceopilosa group are not consistent: the eyes of queens and males of A. sardoa are not larger compared to those of other species of the group (A, B), males with relatively small mandibles are not unique to A. sardoa (C, D), and workers of A. sardoa are not the only ones to have a naturally reddish pigmentation (E).

The general aspect and behavior of all species belonging to the sardoa group makes its identification particularly easy even with the naked eye in the field by any experienced myrmecologist. The group is distributed throughout Southern Europe and the Maghreb.

3.2.6. subterranea group (redefined)

The morphological definition of the group should be implemented by removing the ‘shallow sculpture’ as a defining character, which allows to accommodate A. epirotes according to the phylogenetic results. The only species of the former obsidiana group we did not sequence, A. subcostata, shares the same general traits of the subterranea group definition plus a strong sculpture like that of A. epirotes, and is therefore reassigned to the subterranea group on a morphological basis (on the other hand, A. obsidiana lacks a deep mesoepinotal furrow) (Figure 4). Species with a short and stout mesosoma and strong mesoepinotal furrow previously included in the pallida group (namely A. holtzi, A. lesbica) are reassigned to the subterranea group. Species which lack a strong mesoepinotal furrow and/or characterized by an elongate mesosoma are removed (A. graeca, A. illyrica, Figs 2, 3). The group in its new definition is distributed across Southern Europe, Anatolia, and Transcaucasia, with most of its diversity concentrated in the Eastern part of this range.

3.2.7. Clade A (A. cecconii, A. festae, A. rugosoferruginea, A. splendida)

This well-supported clade (boostrap value: 0.95–1) contains the species after which the former cecconii and splendida groups were named, as well as two other species of the splendida group (A. festae and A. rugosoferruginea). Within the former cecconii group, A. cecconii and two non-sequenced species, namely A. lykiaensis and A. phillipsi, are the only ones with a neck-like elongation of the head, which may represent the only criterion to tentatively hypothesize the affiliation of the other members of this former group to the clades recovered in this study (Borowiec and Salata 2014). On the other hand, within the former splendida group, A. festae, A. rugosoferruginea and A. splendida are in broad terms relatively similar one to another and to most of the species hitherto listed alongside them. It is worth noting that a certain morphological contiguity between the two former groups was already revealed by those species of the former splendida group with shiny integument, very long appendages and variably elongate head shape: A. equestris, A. hamaensis, A. kervillei, and A. vohraliki. This clade can be considered entirely Eastern Mediterranean based on the fact that A. splendida seems to be an introduced species in the Western Mediterranean basin (Salata et al. 2021).

3.2.8. Clade B (A. charesi, A. ovaticeps, A. peloponnesiaca)

This well-supported clade (boostrap value: 0.95-1) contains a second group of species from the former splendida group (A. ovaticeps, A. peloponnesiaca) alongside another species from the former cecconii group (A. charesi). The similarities between species of the former cecconii and splendida groups mentioned for clade A should be kept in mind in this second case. In the same way, the difficulty to find morphological criteria allowing to assign the non-sequenced species of both former groups to either clade highlights the need of further molecular and morphological investigationsinto the composition and evolution of clade B. All species are Eastern Mediterranean.

3.2.8. Remaining species

Aphaenogaster italica, A. obsidiana, A. olympica, A. muschtaidica form a clade which has a very weak support (bootstrap value < 0.89 in all nodes), not allowing us to evaluate whether its members are truly closely related. Nonetheless, it is worth noting there is a substantial degree of morphological similarity with the exception of A. olympica (originally in the cecconii group). Aphaenogaster italica and A. muschtaidica (originally in the gibbosa group) and A. obsidiana (originally in the obsidiana group) share the following characters: black pigmentation, lack of deep metaepinotal furrow in profile view, medium to strong sculpture. For what concerns A. obsidiana, it is important to note that its morphology deviates in several aspects from that of A. epirotes and A. subcostata (formerly forming together the obsidiana group and now moved to the subterranea group): i) the background microsculpture of the dorsum of the mesonotum and the sides of the propodeum is distinctively microreticulated instead of dull and shiny (contradicting the group’s definition by Schulz 1994); ii) the antennal scapi are longer (the ratio of their length divided by the arithmetic mean of cephalic length and width is above 0.965, while below 0.930 in A. epirotes and A. subcostata, L. Borowiec unpublished data); the legs are also longer, with the hind femora clearly longer than the first gastral tergite, while approximately as long in A. epirotes and A. subcostata. Interestingly, the presence of A. olympica in the same clade mirrors the presence of A. cecconii in clade A and of A. charesi in clade B, where the cecconii-like morphology is associated with other species which still have an elongate mesosoma and long appendages but to a lesser degree.

3.3. Reassignment of taxa to other genera

Aphaenogaster isekram and A. asmaae are assigned to the genus Messor on a morphological basis. The description of A. isekram as a morphologically particularly aberrant Aphaenogaster species did not takeinto account the existence of extremely similar Messor species such as M. rufotestaceus (Foerster, 1850) (Iran, but AntWeb sample CASENT0264396 from the United Arab Emirates is currently identified with the same name) and M. lamellicornis Arnol’di, 1968 (Kazakhstan) (Bernard 1977). The later described M. boyeri Cagniant, 2006 (Morocco) shares the same worker morphology (Cagniant 2006; Fig. 5), while the other castes are unknown. The morphology of the workers of these species is consistent with Messor and not Aphaenogaster in particular for what concerns the shape of the mandibles strongly curved towards the midline (as emphasized by Boer 2013), but also in the more rectangular head shape and, for what concerns A. isekram, the lack of propodeal spines. Worldwide, the only species of Aphaenogaster with similar traits of mandibles and head shape is A. striativentris which is known to represent an extraordinary case of evolutionary convergence (Tinaut and Jiménez-Rojas 1990; Gómez et al. 2018). On the other hand, the lack of propodeal spines is a very common feature in Messor (for instance, it characterizes 86% of the about 70 West-Palearctic Messor species, see Borowiec 2014) but is very rare in Aphaenogaster (about 2%, as it is only seen in A. inermita and A. pallida in the same region). The description of A. asmaae ignored all the aforementioned Messor species, and the genus attribution by Sharaf et al. (2018) was entirely based on a comparison with A. isekram. Therefore, we consider these taxa as Messor asmaae (Sharaf, 2018) comb. nov., and Messor isekram (Bernard, 1977) comb. nov. based on three elements: i) their morphology is convincingly congruent with traits typical of Messor and extremely rare in Aphaenogaster; ii) species earlier described with the same morphology were all attributed to the genus Messor (although their descriptors ignored it); iii) they live in regions outside the geographical boundaries of all other Aphaenogaster species (i.e., Sahara Desert and Arabian Peninsula) but well within the distribution boundaries of Messor spp.

In addition, A. sarae, according to the description and images presented by Sharaf et al. (2018) presents all key characteristics of a minor worker from the genus Pheidole, including the three-segmented antennal club which is distinctive of Pheidole against Aphaenogaster (the latter having 4 to 5 antennal club segments, see Boer 2013). In addition, just comparing P. sarae to the most well-known West-Palearctic Pheidole species, P. pallidula (Nylander, 1849) (see Seifert 2016), it is easy to recognize the same shape and proportions of the nodes, mesosoma (with a few differences) and head, or approximately the same length and disposition of the erect setae over the body and very similar body sculpture pattern (Fig. 6). As in the previous cases, no argument was made by the descriptor to place the species in Aphaenogaster (Sharaf et al. 2018). Therefore, we consider it as Pheidole sarae (Sharaf, 2018) comb. nov.

Figure 6. 

Names excluded from Aphaenogaster and examples of morphologically similar species belonging to the new assigned genus (photographs from www.antweb.org). A, B Messor asmaae comb. nov., CASENT0922290, photo by M. Esposito; C, D Messor isekram comb nov., CASENT0913609, syntype, photo by W. Ericson; E, F Messor lamellicornis, CASENT0281598, photo by M. Esposito. G Pheidole sarae comb. nov., CASENT0922294, photo by M. Esposito; H Pheidole pallidula, CASENT0249410, photo by Shannon Hartman.

Now that their generic identity has been reassessed, the status of these three taxa should be further investigated to test whether they may be synonyms of other congeneric species.

4. Discussion

While the phylogenetic relationships between many of the clades we recovered have yet to be clarified, our results demonstrate that, following all past schemes, the great morphological diversification of the Aphaenogaster was largely misinterpreted in its evolutionary significance and a new perspective is needed. Almost all of the current and past infrageneric classifications largely defined non-monophyletic groups. This was also the case with the former subdivision in subgenera: Aphaenogaster s. str. would be monophyletic on its own, yet it is nested within Attomyrma, making the latter paraphyletic. The placement of A. cecconii within the former subgenus Deromyrma was also incorrect, as it should belong to Attomyrma, making Deromyrma sensu Emery (1915) polyphyletic. This implies that the morphological delimitation of the true Deromyrma clade sensu Branstetter et al. (2022) is not entirely straight-forward, and that a precise definition may require the examination of further doubtful cases (compare also with Ward and Boudinot 2021). As for the eight species-groups hitherto in use, only the crocea group (Alicata and Schifani 2019) was recovered as monophyletic, while the graeca group is added for the first time. The other pre-existing groups, albeit all non-monophyletic, were characterized by very different situations: some could be redefined with relatively minor changes, while others should be currently abandoned.

On one hand, the monophyly of the pallida, subterranea and testaceopilosa species-groups – the latter renamed sardoa group and now corresponding to the former nominotypical subgenus – was easily achieved by reassigning only a few species on clear morphological bases. The unclear clustering of the specimens identified as A. balcanica, A. picena, and A. sporadis emphasizes the need of a taxonomic revision of the sardoa group, whose boundaries are, however, very clearly defined. On the other hand, the splendida and cecconii group, highly polyphyletic, had to be dismissed. While sequencing all the species from these two groups stands as an important objective for further research, phylogenetic results revealed a very interesting relationship between the two groups: the ‘cecconii-like’ and the ‘splendida-like’ morphologies each evolved independently two or three times, and at least twice ‘cecconii-like’ species were sisters to ‘splendida-like’ species. It is possible that the peculiar ‘cecconii-like’ morphology represents a form of adaptation to the light-avoiding or troglobiotic lifestyle that characterizes the species of this group, since similar traits are exhibited by other troglobiotic Palearctic species, such as the Japanese A. gamagumayaa Naka and Maruyama, 2018 (Borowiec and Salata 2014; Salata and Borowiec 2016; Naka and Maruyama 2018). Notably, the ‘splendida-like’ morphology is also, albeit to a lesser degree, associated with avoidance of sunlight by the slow-moving foraging workers, accomplished by either living in shady and humid environments or foraging at dusk or nocturnally (Salata et al. 2021), and some species formerly assigned to the splendida group show somewhat intermediate characteristics between the two morphotypes. Finally, the possibility that the ‘gibbosa-like’ morphology appears in multiple separate clades remains to be further investigated. More in general, resolving the relationships between the clades will be key to understand how the remarkable morphological diversity of West-Palearctic Aphaenogaster evolved, and which morphologies are ancestral.

When morphology-based infrageneric divisions were defined, in most cases no effort was made to predict which characters were apomorphic or plesiomorphic, or which ones were driven by an adaptive value and which ones were not—tasks that were very difficult or impossible to achieve in many cases without phylogenetic data, or were simply beyond the aims of the systematic works produced at that time. When the crocea group was split from the subterranea group (Alicata and Schifani 2019), subtle differences in the mesosoma of workers were interpreted as characters probably reflecting phylogenetic patterns, while their overall strong similarity was suggested to be the convergent outcome of evolutionary adaptation towards a similar lifestyle. Male morphology and biogeography were key to the formulation of this hypothesis, which ultimately proved to be correct (Alicata and Schifani 2019). Despite the great morphological diversity of Aphaenogaster in the West-Palearctic, our results demonstrate that the detection of morphological characters useful to delimit monophyletic species-groups is rather difficult in many cases, and that complex patterns of evolutionary convergence or retention of ancestral traits may exist. In this environment, an integrative approach of phylogenetics and morphology appears to be necessary. The recognizable presence of a number of morphologically and phylogenetically well-delimited species-groups still represents an advantageous situation compared to other more chaotic Palearctic ant genera (e.g. Temnothorax, Schifani et al. 2022). Any morphology-based hypothesis that certain West-Palearctic groups would include species from outside the region seems very unwise for the time being given the results we obtained (e.g., placing the Nearctic A. ashmeadi (Emery, 1895) in the testaceopilosa group, the Indian A. smythiesii (Forel, 1902) in the subterranea group, and A. pachei (Forel, 1906) or A. sagei (Forel, 1902) in the obsidiana group, according to Schulz (1994), and Boer (2013). Notably, in the phylogeny by Branstetter et al. (2022), East-Palearctic and Nearctic species clustered together.

Integrating phylogenomic data to address the low support of the backbone of our phylogenetic reconstruction, as well as recovering the additional West-Palearctic species which could not be sequenced in this study would help clarify those relationships which could not be resolved here, improving our understanding of the radiation of this genus. Further investigation should also expand to the species from the other, less diverse regions (the East-Palearctic and the Nearctic), which would be important to fully unravel the biogeographic history of the genus (Branstetter et al. 2022). However, the results of the present study offer a first comprehensive evolutionary perspective over the diversification of Aphaenogaster morphologies in the diversity hotspot of the genus, providing a new species-groups framework as a basis for further taxonomic, phylogenetic and evolutionary studies.

5. Authors’ contributions

ES, AA and RB conceived the study and organized the selection of species to be sequenced. RB conducted the molecular analyses. ES and AA curated the morphological parts. ES prepared the first draft of the manuscript. MM and ES curated the graphic parts. All authors participated in the collection and identification of specimens and the preparation of the final draft of the manuscript.

6. Acknowledgements

We are grateful to everyone who helped us collect the specimens used in this study: Volkan Aksoy (Türkiye), Gregor Bračko (Slovenia), Simone Costa (Italy), Antonino Dentici (Italy), Mathias Dezetter (France), Piergiorgio Di Pompeo (Italy), Christophe Galkowski (France), Philippe Geniez (France), Emanuele Genduso (Italy), Vincenzo Gentile (Italy), Kiko Gómez (Spain), Roberto Huertaz (Spain), Konstantinos Kalaentzis (Greece), Claude Lebas (France), Guglielmo Maglio (Italy), David Mifsud (Malta), Francisca Ruano (Spain), Giorgio Sabella (Italy), Andrea Salvarani (Italy), Alberto Sanchez (Spain), Alberto Tinaut (Spain), Roberto Viviano (Italy). Moreover, we wish to thank three anonymous referees as well as the editor Andreas Zwick for their constructive comments which led to a significant improvement of our manuscript.

Funding: Data used in this work were partly produced through the GEMEX technical facilities of the Centre d’Ecologie Fonctionnelle et Evolutive with the support of LabEx CeMEB, an ANR Investissements d’avenir program (ANR-10-LABX-04-01). Support for this research was provided by “la Caixa” Foundation (ID 100010434) to Mattia Menchetti (grant LCF/BQ/ DR20/11790020). The Turkish material used in this project was provided with the support of the projects numbered 109T088 and 111T811 supported by the Scientific and Technological Research Council of Türkiye (TÜBİTAK), and the project numbered 2018-135 of the Trakya University Scientific Research Unit.

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Supplementary materials

Supplementary material 1 

Sequence alignments

Schifani E, Alicata A, Menchetti M, Borowiec L, Fisher BL, Karaman C, Kiran K, Oueslati W, Salata S, Blatrix R (2022)

Data type: .fas.(Alignments are included as FASTA files

Explanation note: The file contains the alignments for all the genetic sequences produced in this study.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (411.64 kb)
Supplementary material 2 

List of investigated samples

Schifani E, Alicata A, Menchetti M, Borowiec L, Fisher BL, Karaman C, Kiran K, Oueslati W, Salata S, Blatrix R (2022)

Data type: .xlsx

Explanation note: The file contains the complete list of samples used in our analyses. For each sample, we provide the following information: voucher identifier, species-level identification, species-groups (this classification does not takeinto account the results of the analyses), label in the phylogenetic trees, specimen code, label for extraction, latitude and longitude (decimal degrees), locality name, name of the collector(s), name of the identifier(s), Genbank accession number for COI and for the 6 EPIC genes sequenced.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (41.23 kb)
Supplementary material 3 

Supplementary phylogenetic trees

Schifani E, Alicata A, Menchetti M, Borowiec L, Fisher BL, Karaman C, Kiran K, Oueslati W, Salata S, Blatrix R (2022)

Data type: .zip

Explanation note: Supplementary file S3: Maximum likelihood phylogeny based on all markers concatenated. — Supplementary file S4: Bayesian phylogeny based on mtCOI. — Supplementary file S5: Maximum likelihood phylogeny based on mtCOI. — Supplementary file S6: Bayesian phylogeny based on all EPIC markers concatenated. — Supplementary file S7: Maximum likelihood phylogeny based on all EPIC markers concatenated.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (46.39 kb)
Supplementary material 4 

Synoptic list of species and new species-groups classification

Schifani E, Alicata A, Menchetti M, Borowiec L, Fisher BL, Karaman C, Kiran K, Oueslati W, Salata S, Blatrix R (2022)

Data type: .svg

Explanation note: The file contains a synoptic list of the West-Palearctic Aphaenogaster and, for each taxon, its classification before and after this study.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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