Aphaenogaster ichnusa Santschi, 1925, bona species, and Redescription of Aphaenogaster subterranea (Latreille, 1798) (Hymenoptera, Formicidae)

Casevitz-Abstract Morphological and molecular investigation conducted in France and the Tyrrhenian islands reveal that Aphaenogaster subterranea is composed of two distinct species. We propose to raise A. subterranea var. ichnusa Santschi, 1925, described from Sardinia, to the species status, A. ichnusa Santschi, 1925 stat. nov . This species differs from A. subterranea by having shorter propodeal spines and a less sculptured cuticle. Phylogenetic reconstruction using the mitochondrial marker COI shows that the two species form well separated clades, with a genetic distance (K2p) of 9.8 %. In the studied area, the two species are parapatric, A. ichnusa occurring in Sardinia, Corsica and the Mediterranean area of continental France, and A. subterranea occurring north of this area. We propose a redescription of both species and designation of a neotype for A. subterranea .


Introduction
. Those characters, albeit stable within the Mediterranean population, were most often considered irrelevant in systematics. Indeed, lighter individuals occur naturally in all populations and the sculpture of the cuticle varies to some extent within most species of the genus Aphaenogaster Mayr, 1853. The differences between Mediterranean and non-Mediterranean specimens could correspond to mere intraspecific variability, or could result from particular environmental conditions under Mediterranean climate (ants tend to have a lighter color when developing at higher temperature).
Aphaenogaster subterranea has a broad distribution, from Western Europe to Asia Minor and Caucasus, throughout which it has a rather homogeneous morphology. Thus, the ancient authors described few varieties or sub-species, although they were quite prolific with this respect in other genera. Interestingly, Santschi (1925) described the taxon ichnusa as a mere variety of A. subterranea. The type specimen is from Sorgono, Sardinia. According to Santschi, the variety ichnusa differ from the typical subterranea by its yellow-brownish color, by the seventh article of the funiculus more elongated and by the propodeal spines weaker. In his revision of the palearctic Aphaenogaster, Finzi (1930) did not find any difference in the size of the funiculus segments between the specimens of ichnusa and of subterranea he had access to. He only noticed, again, the small and triangular propodeal spines. Baroni Urbani (1971), in his catalog of the ants of Italy, only reported the existence of the variety ichnusa, considered as endemic to Sardinia.
The type of A. subterranea var. ichnusa, of which high quality pictures can be viewed online thanks to the AntWeb project (available from http://www.antweb.org. Accessed 4 July 2018. Specimen CASENT0913132), shows the same combination of morphological characters as specimens from Corsica and the Mediterranean coast of France. We present a morphological and molecular analysis of specimens of A. subterranea collected in Sardinia, Corsica and continental France, showing that the taxon ichnusa should be considered as a valid species, distinct from A. subterranea sensu stricto. We propose hereafter the redescription of A. ichnusa stat. nov. and of A. subterranea, and the designation of a neotype for A. subterranea.

Morphometrics
The following characters were measured (all measures are in mm): HL: maximum length of head, measured along a median line from vertex to anterior margin of clypeus. HW: maximum width of head, including the eyes. SL: maximum length of scape. The condylar bulb is excluded from the measurement.. ED: maximum diameter of the eye. ML: maximum length of mesosoma (alitrunk). MW: maximum width of mesosoma, measured on pronotum. PSL: length of propodeal spine, measured from the center of the propodeal spiracle to the tip of the spine. PW: maximum width of petiole. PL: maximum length of petiole. PPW: maximum width of post-petiole. PPL: maximum length of post-petiole.

Mitochondrial DNA analysis
New DNA sequences for part of the gene coding for the cytochrome c oxidase subunit I (COI, 658 bp) were obtained either within the framework of the International Barcode of Life project (iBOL) or at the Center for Evolutionary and Functional Ecology (CEFE) in Montpellier, France. In the latter case, each specimen was used entirely for DNA extraction, whereas in the former case, only one leg was used and vouchers were stored at the CEFE. After DNA extraction, the COI fragments were amplified through polymerase chain reaction (PCR) using the primer pairs LepF1: 5'-ATTCAACCAATCATAAAGATAT-3' and LepR1: 5'-TAAACTTCTGGATGTCCAAAAA-3' (Hebert et al., 2004). Sanger dideoxy sequencing of the unpurified PCR amplicons was performed in both direction 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 using the ambiguity code. Sequences were aligned with Muscle (Edgar, 2004). Alignments were inspected visually and edited manually using Mesquite v. 3.31 (Maddison and Maddison, 2017) when they could be improved. Genetic distances were estimated in Mesquite using the Kimura 2-parameter model (K2p).
A partition scheme was defined with PartitionFinder (Lanfear et al., 2016) for each phylogenetic analysis, using the Akaike Information Criteria for nucleotide substitution model selection. Prior data blocks were defined by codon position. Phylogenetic reconstructions were performed using Bayesian inference with MrBayes v. 3.2.6 (Ronquist et al., 2012). Two analyses of four chains were run for 1 000 000 generations, sampling trees every 500 generations with 25 % burn-in samples discarded for each run. The phylogenetic tree was rooted using Messor structor (Latreille) as outgroup.

Redescription of Aphaenogaster subterranea (Latreille, 1798) and designation of a neotype
The type specimen of A. subterranea designated by Latreille could not be found in the collection of the National Museum of Natural History (MNHN) in Paris, France, and we consider it is lost. In order to fix a neotype and paraneotypes, we collected worker specimens from one nest at Brive, terra typica of the species. The neotype is deposited at MNHN. Nine workers from the same nest, designated as paraneotypes, are deposited at MNHN, from which a new neotype could easily be designated in case of loss or destruction of the neotype. Ten workers of this same nest have also been deposited at the Senckenberg Museum für Naturkunde of Görlitz, Germany.
Material examined for morphometrics:  Fig 1) Head brown, of the same color as the rest of the body, longer that wide, widening from mandibles to occiput. Eyes rather small, c. 1/6 of head length, located lateraly at midlength between occiput and clypeus. Scapes long, overreaching the occipital margin distinctly, very slightly arched, their diameter increasing from base to apex. Funicular segments c. 1.5 times longer than wide, antennal club weakly pronounced, of four segments. Clypeus almost smooth, with a few weak longitudinal carinae, anteriorly emarginate. Frontal carinae short, frontal lobes narrow. Head weakly sculptured, with a few striae extending from frontal carinae and around the eyes. Space above antennal fossae weakly reticulate. The sculpture of the cuticule vanish towards the occiput, which is entirely smooth.
Mandibles strong, rounded, with a few longitudinal striae, masticatory margin armed with c. 4 relatively strong apical teeth followed by c. 4 basal denticles more or less distinct. The whole head is covered by long, semi-erect, setae. Many semi-erect to semi-decumbent setae on the scape.  Mesosoma evenly brown, light brown or dark brown, with long erect setae, more numerous on pronotum and mesonotum. Promesonotum very rounded, separated from propodeum by a guttershaped groove. Propodeal spines clearly distinct, pointed shaped, oriented at c. 45°, of a length almost equal to the distance between their tips. Cuticle weakly sculptured: sides of pronotum with weak longitudinal striae, space between wrinkles smooth or slightly irregular, summit of pronotum almost smooth. Mesonotum a bit more sculptured, with sometimes a few transversal striae at the summit. Mesopleuron et propodeum much more sculptured, with marked longitudinal ridges separated by a dense reticulation mainly visible on the lower part of the mesopleuron, summit of propodeum with transversal ridges. Space between propodeal spines almost smooth with a few fine transversal striae.
Petiole and post-petiole brown. Petiole with a long anterior peduncle, summit of node rounded, anterior and posterior faces convex. Post-petiole rounded. Cuticule smooth on nodes, slightly sculptured on the peduncle of the petiole and on the lower lateral parts of petiole and post-petiole. Nodes of petiole and post-petiole with a few long semi-erect setae directed backwards.
Gaster smooth and shiny, of the same color as mesosoma or sometimes slightly darker in the lighter individuals. All gaster segments with long erect or semi-erect setae, and with sparse pubescence made of thin and appressed hairs.
Appendices long, light brown, with many decumbent or semi-decumbent setae, and a few semi-erect setae on the interior border of femurs and tibias.
We did not examine the type specimen directly but we viewed the high quality pictures of it accessible online thanks to the AntWeb project (available from http://www.antweb. org. Accessed 4 July 2018. Specimen CASENT0913132) Material examined for morphometrics:    (Table 1, Fig 1) Whole body brown yellow, some nests contain darker individuals. Head characters as for A. subterranea, but sculpture of cuticle finer, carinae on clypeus almost indistinguishable, and smooth space on the occiput larger. Continental specimens may be more sculptured as those from Corsica and Sardinia. Cuticular pattern on mesosoma as for A. subterranea, but weaker, summit of pronotum and mesonotum and space between propodeal spines entirely smooth. Propodeal spines short, triangular, sometimes reduced to mere carinated angles in small individuals. The setae have the same distribution as for A. subterranea. Petiole, gaster and appendices as for A. subterranea, setae on tibias finer and more decumbent.

Mitochondrial DNA analysis
For this study, 34 new COI sequences were generated (22 by iBOL and 12 by the CEFE), with GenBank accession numbers MH138330 to MH138350, MH138398 and MH630453 to MH630464. Sequences are also publicly available on the dataset "DS-ASUB -Aphaenogaster subterranea and Aphaenogaster ichnusa" of the BOLD -Barcode of Life Data Systems (Doi: dx.doi.org/10.5883/DS-ASUB). The dataset included 658 characters. The two species form two well supported clades (Fig 3) with a K2p genetic distance of 9.8 %. The average intraspecific genetic distance is 0.06 % for A. subterranea and 0.7 % for A. ichnusa. For A. ichnusa, the genetic distance between mainland France and the Tyrrhenian islands (Corsica and Sardinia) is 1.2 %. Only one specimen, from Caussols, shows a disagreement between morphology and COI (Fig 3). This specimen was collected on a meridional plateau at >1000 m asl, which hosts cold tolerant insect communities although it is close to the Mediterranean cost. Introgression between the two species is likely to explain this disagreement. Schulz (1994) defined the A. subterranea species group which was considered to contain a dozen species. Our study, along with others in progress (see Salata and Borowiec, 2018), reveal that this group is more diversified that previously thought.

Conclusion
Although our study focuses on France, which represents only a small part of the distribution range of the A. subterranea group, the two species here show roughly parapatric distributions that seem driven by climate. Aphaenogaster ichnusa seems to be restricted to areas under the influence of the Mediterranean climate whereas A. subterranea is found in more temperate environments. Although the Tyrrhenian islands are located in the Mediterranean region, both A. subterranea and A. ichnusa are reported from Sicily (Schifani and Alicata, 2018), and only A. ichnusa in Corsica and Sardinia. In Sicily, A. subterranea is found above 1200 m asl and A. ichnusa at lower altitudes (Schifani and Alicata, 2018), confirming opposite climate preferences between the two species. Our sampling includes a few sites on the transition zone between Mediterranean and temperate climates where the two species are detected a few km apart. Nevertheless, the two genetic clades are clearly distinct, suggesting efficient reproductive isolation although the two species may occur in sympatry in certain regions.