Bulletin of Insectology 65 (2): 301-310, 2012 ISSN 1721-8861 New data on Reticulitermes urbis and Reticulitermes lucifugus in Italy: are they both native species? Silvia GHESINI, Mario MARINI Dipartimento di Scienze Biologiche, Geologiche e Ambientali, Università di Bologna, Italy Abstract In Italy, three species of subterranean termites in the genus Reticulitermes exist: the native R. lucifugus (Rossi), the North- American R. flavipes (Kollar), and R. urbis Bagneres, Uva et Clement. R. urbis is a recently described species, that is suspected to have been introduced from the Balkans to France and Italy, where until the present study it had been found only in a few urban areas. The distribution of R. urbis in Italy is still poorly understood. We collected 22 Reticulitermes samples in 17 Italian localities in order to define better the distributions of R. urbis and R. lucifugus and to determine whether R. urbis can be found also in natu- ral environments. We identified termite species by sequencing a portion of the mitochondrial gene encoding for the cytochrome oxidase subunit II (COII), that we also used for the construction of haplotype networks and for phylogenetic analysis. We found R. urbis in three urban areas in the north, and in seven natural areas in the south. So, in Italy, R. urbis is more diffused than previ- ously thought. The same pattern of distribution, restricted to urban areas in the north and including also natural environments in the south, is seen in the native R. lucifugus, and may be explained by the climatic requirements of these species. The lower genetic variability of R. urbis observed in Italy and France, compared with the Balkans, may be explained without implying an introduc- tion by man. Moreover, many insect species exist with a range similar to that of R. urbis. For these reasons, we think that R. urbis could be native in Italy. Key words: introduced species, native species, Italy, subterranean termites, mitochondrial DNA. Introduction kanensis Clement, the Israeli species Reticulitermes cly- peatus Lash and some other taxa, not yet described, dis- Three species of the genus Reticulitermes (Isoptera Rhi- tributed in eastern Greece, Turkey, Crete, and Cyprus notermitidae) are known to occur in Italy. Reticuliter- (Velonà et al., 2010; Ghesini and Marini, 2012). mes lucifugus (Rossi) is found in many localities across While the status of R. lucifugus and R. flavipes as, re- most of the Italian territory. The recently described spe- spectively, a native and an introduced species in Italy, is cies Reticulitermes urbis Bagneres, Uva et Clement commonly accepted, the status of R. urbis still presents (Bagnères et al., 2003) has been found in some locali- some elements of doubt, and arguments supporting both ties in the Adriatic side of the Po Plain and in Apulia its indigenous origin and its introduction by man have (Marini and Mantovani, 2002; Luchetti et al., 2004). been suggested (Uva et al., 2004; Luchetti et al., 2007; The North-American species Reticulitermes flavipes Leniaud et al., 2010). (Kollar), whose presence in Europe is long time known When the existence of R. urbis as a distinct species (Kollar, 1837; Jucci, 1924; Feytaud, 1925; Austin et al., was demonstrated (Clément et al., 2001), only a few 2005; Dronnet et al., 2005), has recently been found in Italian and French populations, confined to urban areas, two localities in Lombardy (Ghesini et al., 2010; 2011). were known. Because Italian localities with R. urbis Three subspecies of R. lucifugus exist: R. lucifugus presence are situated on the Adriatic side of the penin- lucifugus in the Italian peninsula, R. lucifugus corsicus sula, it was thought possible that the core of R. urbis in Sardinia and on the coasts of Tuscany, and a third range could be in the Balkan Peninsula. This hypothesis subspecies not yet described in Sicily and Trieste (Marini was supported by the finding that the defensive secre- and Mantovani, 2002; Luchetti et al., 2004). Outside Italy, tions of R. urbis have affinities with those of the Greek R. lucifugus lucifugus is found in southern France and in species R. balkanensis (Quintana et al., 2003). Termite Bastia (Corse), while R. lucifugus corsicus is found in surveys carried out from Istria down to Peloponnese ac- southern France and Corse (Lefebvre et al., 2008). tually allowed to identify R. urbis populations in many Outside Italy, R. urbis is distributed in western Greece localities, in natural environments (Luchetti et al., 2005; and Peloponnese, in Croatia and in southern France 2007). Therefore, R. urbis was thought to be native to (Luchetti et al., 2007; Leniaud et al., 2010). the Balkan Peninsula, and introduced by man in Italy In the phylogeny of European Reticulitermes species, and France (Luchetti et al., 2007). Analyses on mito- two main clades are identified, the first one including chondrial DNA sequences and microsatellites have con- west-Mediterranean species and the second one includes firmed that the genetic diversity of R. urbis is higher in east-Mediterranean species (Velonà et al., 2010). R. the Balkan Peninsula than in Italy and France (Leniaud lucifugus belongs to the west-Mediterranean clade, to- et al., 2010). These results, together with the observa- gether with the Franco-Iberian species Reticulitermes tion that, in Italy and France, the distribution of R. urbis grassei Clement and Reticulitermes banyulensis Clem- mitochondrial DNA haplotypes is not concordant with ent. R. urbis belongs to the east-Mediterranean clade, geographic regions, have been interpreted as supportive together with the Greek species Reticulitermes bal- of the hypothesis that R. urbis was introduced by man (Leniaud et al., 2010). The introduction of termites by ral environments in the same regions and in the other man, through the displacement of infested wood, regions along the Adriatic and Ionian coasts. We con- wooden objects or soil is in fact a possible occurrence, sidered natural environments as those areas that are not as documented by many cases (Vargo and Husseneder, urbanized, and where old scrubs or woods exist, because 2009). areas with primary vegetation are scarce in Italian During the 20th century R. lucifugus has been reported plains. In northern regions (Veneto, Friuli-Venezia in more than 200 localities in continental Italy (Sbrenna Giulia), no termites were found in natural environments, and Micciarelli Sbrenna, 2008), but the identification of and all the samples were collected in urban areas. In these samples cannot be trusted, because, before R. ur- southern regions (Apulia, Basilicata, Calabria), termites bis was recognized as a distinct species, all Reticuliter- were found and collected in natural environments. mes findings in Italy were automatically identified as R. Seventeen localities were sampled, and 22 samples lucifugus. Recent analyses of samples collected in some were collected (figure 1). In most localities, a single ter- of these localities have shown that the species involved mite colony was found. In five localities, two termite is actually R. urbis (Marini and Mantovani, 2002; colonies, separated by a distance of at least 500 m, were Luchetti et al., 2004). Up to now, the presence of R. ur- sampled. The two samples from the same locality will bis in Italy is ascertained in eight urban areas, both in be indicated as 1 and 2. the North-East and in the South-East, in particular in Veneto, Emilia-Romagna, and Apulia (Marini and Man- Genetic analysis tovani, 2002; Luchetti et al., 2004). It is possible that A preliminary analysis of the samples collected revealed the presence of R. urbis in Italy is still underestimated. that they all belonged to the genus Reticulitermes. The In this study, we examine Reticulitermes populations identification of Italian Reticulitermes species based on from the Adriatic and Ionian sides of Italy, with the pur- morphological characters is not sufficiently reliable, pose of clarifying R. urbis and R. lucifugus distributions, therefore, in order to determine our samples, we se- verifying whether R. urbis is present in natural environ- quenced a portion of the mitochondrial gene encoding ments, and comparing the two species from an ecologi- for the cytochrome oxidase subunit II (COII). This por- cal and genetic (mitochondrial DNA) point of view. tion is a good marker for the identification of species and subspecies in the genus Reticulitermes and has been used in many studies on European termites (Marini and Materials and methods Mantovani, 2002; Luchetti et al., 2007, Leniaud et al., 2010). Sampling Total DNA was extracted from termite heads with a Termite collection surveys were conducted during CTAB protocol (Doyle and Doyle, 1987). Two indi- 2009 and 2010. Surveys were initially carried out in viduals for each sample were analyzed. For each indi- northern regions, in some localities where termite activ- vidual, a 684-bp portion of the mitochondrial gene ity was reported by homeowners, then extended to natu- COII, encoding for 228 amino acids, was amplified. Figure 1. Sampling localities in northern and southern Italy. 302 Amplification was obtained with the primers TL2-J- obtained with MrBayes 3.1.2 (Huelsenbeck et al., 2001; 3034 = mtD-13 (5’-AAT ATG GCA GAT TAG TGC Ronquist and Huelsenbeck, 2003). Convergence was A-3’) and TK-N-3785 = mtD-20 (5’- GTT TAA GAG reached after two million generations (average standard ACC AGT ACT TG-3’). deviation of split frequencies <0.005). Trees were sam- PCR was performed in a 50 µl mixture using GoTaq® pled every 100 generations, and the first 5000 trees were Flexi DNA Polymerase kit (Promega, USA), following discarded as burn-in, after graphic visualization. Trees the manufacturer’s protocol. Reaction conditions were were edited with the software FigTree 1.3.1, available set as follows: initial denaturation at 94 °C for 5 minutes; online at http://tree.bio.ed.ac.uk/software/figtree/.