Accepted on 27 October 2011 Ó 2011 Blackwell Verlag GmbH J Zool Syst Evol Res doi: 10.1111/j.1439-0469.2011.00649.x 1Departamento de Biologı´a Experimental. A´rea de Gene´tica. Universidad de Jae´n. Jae´n. Spain; 2IMEDEA (CSIC-UIB), Miquel Marque´s, 21 Esporlas, Illes Balears, Spain; 3Departamento de Zoologı´a. Facultad de Ciencias. Universidad de Granada. Granada. Spain A new taxonomic status for Iberoformica (Hymenoptera, Formicidae) based on the use of molecular markers Martin Mun˜oz-Lo´pez1,Teresa Palomeque1,Jose´ A. Carrillo1,Joan Pons2,Alberto Tinaut3 and Pedro Lorite1 Abstract The subgeneric subdivision of the genus Formica is still open. In this article, we make a phylogenetic study on several species of the genus Formica and of its closely related genera, Polyergus and Proformica, using sequences of nuclear satellite DNA (stDNA) and the mitochondrial rrnL as molecular markers. Our goal was to shed light on their phylogenetic relationships and particularly on the systematic position of F. subrufa. This species was first included in the subgenus Serviformica, but afterwards a new subgenus (Iberoformica) was established to include only this species. The results show that a stDNA family previously reported in Formica species, with a repetitive unit 129 bp long, is also found in Polyergus rufescens and P. samurai but not in Proformica longiseta. This is the first case of presence of a stDNA family in two different ant genera. In F. subrufa, this stDNA is very divergent relative to those isolated in the remaining Formica species and in the genus Polyergus. The Bayesian analysis of mitochondrial rrnL sequences shows three highly supported groups: F. subrufa, the remaining Formica species studied, and the genus Polyergus, suggesting that parasites (Polyergus species) and hosts (Formica species) are closely related but not sibling species. The combined analysis of nuclear stDNA sequences and mitochondrial rrnL showed their phylogenetic congruence despite their distinct evolutionary dynamics. This analysis did not discriminate between the remaining Formica species that were not grouped according to the subgeneric classification. According to these results, it can no longer be assumed that F. subrufa belongs to the subgenus Serviformica or of the fusca species group. This differentiation was also supported by previous studies based on the morphological characters, molecular and cytogenetic data. Therefore, taking into consideration these arguments and others explained in detail in this article, we propose that the taxon Iberoformica, formerly synonymized subgenus, be raised to a genus status. This genus would be monotypic and only composed, up to the moment, by Iberoformica subrufa (= F. subrufa Roger, 1859). Key words: Satellite DNA – mitochondrial DNA – molecular phylogenies – Iberoformica new status Introduction (F. sanguinea Latreille, 1798), Formica s. str. (F. frontalis The taxonomic classification of the genus Formica Linnaeus, Santschi, 1919) and Iberoformica (formerly F. subrufa) (Lorite 1758 is still disputed. For European Formica species have been et al. 2004). Satellite DNA (stDNA), a highly repetitive DNA distinguished four subgenera (Raptiformica Forel, 1913, characteristic of eukaryotic genomes, is composed of a Coptoformica Muller, 1923, Serviformica Forel, 1913, Formica sequence unit tandemly arranged in long and abundant arrays s. str.) or five subgenera (including Iberoformica Tinaut, 1990) in the constitutive heterochromatin (Palomeque and Lorite (e.g. Donisthorpe 1943; Creighton 1950; Dlussky 1967; Tinaut 2008). Previously, several molecular studies have demonstrated 1990) whereas others considered only species groups (e.g. that monomer sequences indicate phylogenies similar to those Wheeler 1922; Collingwood 1979; Agosti 1994). The problem found with classical markers, and particularly with ribosomal was whether to accept the subgenus as a valid category or use markers, as both exhibit concerted evolution (e.g. Pons and a more cautious classification with the informal species group, Gillespie 2004; Palomeque and Lorite 2008; Martinsen et al. in which the problem of the monophyletic category does not 2009). The consequence of the concerted evolution is an intra- affect the species group organization. Agosti (1994) proposed specific variability lower than the inter-specific divergence. eliminating all subgenera and waiting for future systematic Hence, stDNA may be used as an alternative phylogenetic analyses to determine their validity. One of the most nuclear marker when the sequence of the repetitive unit is controversial issues was the position of F. subrufa Roger conserved across related species (Mesˇ trovic´et al. 2009). In all 1859, which was included in the fusca species group (Wheeler, Formica species studied, this stDNA family was organized as 1913) or in Serviformica subgenus (Santschi, 1919) and finally tandemly repeated 129-bp monomers (Lorite et al. 2004). included in a new monotypic subgenus Iberoformica (Tinaut, Phylogenetic analysis placed all stDNA sequences of Formica 1990). in a highly supported monophyletic group, with the exception A stDNA family was isolated in eight Formica species of the sequences from F. subrufa. Hence, the 129-bp stDNA (Formica 129 bp satellite) and used to infer evolutionary from F. subrufa was considered a subfamily of the 129-bp relationships among four subgenera: Serviformica (F. cunicu- stDNA family from the other Formica species (Lorite et al. laria Latreille, 1798, F. fusca Linnaeus, 1758, F. rufibarbis 2004). Fabricius, 1793 and F. selysi Bondroit, 1918), Raptiformica On the contrary, a molecular phylogenetic study of 20 Formica species using partial mitochondrial cytochrome-b sequences clustered them according to subgeneric classifica- Corresponding author: Pedro Lorite ([email protected]) tions (Goropashnaya 2003). However, other studies using Contributing authors: Martin Mun˜ oz-Lo´pez ([email protected]), Teresa Palomeque ([email protected]), Jose´A. Carrillo another mitochondrial region, NADH dehydrogenase subunit ([email protected]), Joan Pons ([email protected]), 6 and tRNA serine (Goropashnaya et al. 2004a,b), have Alberto Tinaut ([email protected]) not agreed in some cases with those results (Seifert and J Zool Syst Evol Res (2012) 50(1), 30–37 New taxonomic status for Iberoformica 31 Goropashnaya 2004). None of these molecular studies parasitizes F. japonica Motschoulsky, 1866 and F. hayashi included the controversial F. subrufa. Terayama & Hashimoto, 1996 (Liu et al. 2003). Our main goal is to clarify the taxonomic position of F. subrufa by using a classical phylogenetic marker, the 16S mitochondrial DNA (rrnL). This marker has not been used in Material and Methods Formica yet although it has been used successfully in other Material and DNA extraction Hymenoptera (Tanaka et al. 2001; Taylor et al. 2011). Here, Several ant species were used in this study: Formica cunicularia, we also included rrnL sequences of other Formica species and F. fusca and F. subrufa (Jae´n, Spain); F. frontalis and Proformica species from the genera Polyergus Latreille, 1804 and Profor- longiseta Collingwood, 1978 (Granada, Spain); F. rufibarbis and mica Ruzsky, 1902. Molecular studies have shown that the Polyergus rufescens (Tours, France), Formica selysi (Morillon, France), genus most closely related to Formica is Polyergus while F. sanguinea (Tuscany, Italy) and Polyergus samurai (Kyoto, Japan). A population was analysed for each species. A pull of 10–15 adult worker Proformica is closely related to the Polyergus-Formica clade ants were used for genomic DNA extraction according to the (Hasegawa et al. 2002; Brady et al. 2006; Moreau et al. 2006). technique of Heinze et al. (1994). Because the number of available We also investigated whether sequences of the stDNA family individuals from Polyergus samurai was very low, only two workers previously described in Formica are found in those closely were used for DNA extraction in this species. related genera, and, if so, whether they could be used as nuclear phylogenetic markers. The genus Polyergus consists of Satellite-DNA isolation, cloning, sequencing and Southern blot five species (Bolton 2011), all obligatory social parasites of Formica Formica species. Social parasitism is the coexistence of two The sequences of the 129 stDNA family from species used here were obtained elsewhere (Lorite et al. 2004; EMBL accession species of social insects in the same nest, one of the species numbers from AJ238724 to AJ238728, AJ238730 to AJ238732, being parasitically dependent on the other (Tinaut and Ruano AJ308973 to AJ308981, AJ308985, AJ308988 and AJ508813 to 1999; Buschinger 2009). It is widespread in the social bees, AJ508878). wasps and especially in ants (reviewed by DÕEttorre et al. Polyergus rufescencs genomic DNA was restricted with a battery of 2002). Although the mechanism of obligatory social parasitism endonucleases, and fragments were separated by electrophoresis on is not well known, it has been considered a consequence of 2% agarose gels. stDNA fragments produced by the digestion of genomic DNA with Sau3A were eluted from agarose gel and inserted chemical interactions between hosts and parasites (reviewed by into the BamHI site of pUC19 vector. The ligation mix was used to Lenoir et al. 2001; Buschinger 2009). In Hymenopteran social transform E. coli DH5 alpha competent cells that allow the blue-white parasitism, two life-history classes can be differentiated. selection of successful ligation of DNA into the vector. Another