Org Divers Evol (2017) 17:213–228 DOI 10.1007/s13127-016-0313-3 ORIGINAL ARTICLE Evolution and systematics of Green Bush-crickets (Orthoptera: Tettigoniidae: Tettigonia) in the Western Palaearctic: testing concordance between molecular, acoustic, and morphological data Beata Grzywacz1 & Klaus-Gerhard Heller2 & Elżbieta Warchałowska-Śliwa 1 & Tatyana V. Karamysheva 3 & Dragan P. Chobanov4 Received: 14 June 2016 /Accepted: 16 November 2016 /Published online: 8 December 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract The genus Tettigonia includes 26 species distribut- of Tettigonia (currently classified mostly according to mor- ed in the Palaearctic region. Though the Green Bush-crickets phological characteristics), proposing seven new synonymies. are widespread in Europe and common in a variety of habitats throughout the Palaearctic ecozone, the genus is still in need Keywords Tettigonia . mtDNA . rDNA . Phylogeny . of scientific attention due to the presence of a multitude of Bioacoustics poorly explored taxa. In the present study, we sought to clarify the evolutionary relationships of Green Bush-crickets and the composition of taxa occurring in the Western Palaearctic. Introduction Based on populations from 24 disjunct localities, the phylog- eny of the group was estimated using sequences of the cyto- Genus Tettigonia Linnaeus, 1758 presently includes 26 recog- chrome oxidase subunit I (COI) and the internal transcribed nized species (Eades et al. 2016) distributed in the Palaearctic spacers 1 and 2 (ITS1 and ITS2). Morphological and acoustic ecozone and belongs to the long-horned orthopterans or the variation documented for the examined populations and taxa bush-crickets (Ensifera, Tettigonioidea). Tettigonia,popularly was interpreted in the context of phylogenetic relationships known as the Green Bush-crickets, are generally large green inferred from our genetic analyses. The trees generated in orthopterans with moderately slender body and legs and well- the present study supported the existence of three main line- developed wings that inhabit the plant cover searching for ages: BA^—composed of all sampled populations of their food (usually smaller insects or plant tissues). Tettigonia viridissima and the Tettigonia vaucheriana com- Tettigonia is one of the most notable Old World example with plex, BB^—comprising Tettigonia caudata, Tettigonia two centers of diversity: one in the Mediterranean–Pontic re- uvarovi, and the Tettigonia armeniaca complex, and BC^— gion (see, e.g., Ramme 1951;Pinedo1985;Chobanovetal. consisting of Tettigonia cantans. The present study provides 2014) and another in the Japanese archipelago (see Ichikawa the first phylogenetic foundation for reviewing the systematics et al. 2006; Kim et al. 2016). Both regions are characterized by a similar number of endemic taxa and insufficient knowledge regarding the taxonomy and systematics of Green Bush- crickets (Ichikawa et al. 2006; Chobanov et al. 2014; own * Beata Grzywacz unpublished data). [email protected] Despite the fact that several species of Green Bush-crickets are quite well known and have been the subject of detailed 1 Institute of Systematics and Evolution of Animals, Polish Academy neuro-ethological studies (e.g., Zhantiev and Korsunovskaya of Sciences, Sławkowska 17, 31-016 Krakow, Poland 1978; Schul 1998), others remain poorly known from single 2 Grillenstieg 18, 39120 Magdeburg, Germany specimens, and even nowadays, the discovery of new species 3 Institute of Cytology and Genetics of the Siberian Branch of the continues (Ogawa 2003;Ichikawaetal.2006;Chobanovetal. Russian Academy of Sciences, Novosibirsk, Russia 2014; Storozhenko et al. 2015). Data on the systematics of this 4 Institute of Biodiversity and Ecosystem Research, Bulgarian genus involve piecemeal morpho-acoustic studies conducted Academy of Sciences, 1 Tsar Osvoboditel Boul, 1000 Sofia, Bulgaria for geographically restricted areas or focused on 214 Grzywacz B. et al. morphological groups of species (e.g., Heller 1988;Rhee In bush-crickets, the songs of closely related species, 2013;Chobanovetal.2014). Our recent morphological and especially those that speciated in allopatry, usually have a acoustic studies on Tettigonia, concentrated on the Western lineage-specific amplitude–temporal pattern that enables Palaearctic, revealed a number of conflicts within the pub- recognition for systematic purposes and for drawing con- lished data when trying to identify certain populations and clusions about paths of speciation (e.g., Heller 1990, 2006; develop hypotheses about the systematics of the group (own Chobanov and Heller 2010). In Tettigonia, differences be- unpublished data). The latter further supports the need to use tween species have been observed in the time and frequen- new markers to test the systematic position of some taxa and cy domains, while particular song-recognizing mecha- unravel the evolutionary history of this genus. nisms may depend on the geographic and ecological pref- The evolution of acoustic communication systems in erences of the species (Schul 1994; Schul et al. 1998). orthopterans has led to high levels of acoustic specializa- Hence, we use the male calling song as an additional clue tion. As acoustic signals are important for intraspecific and for evolutionary assumptions as well as for testing the var- sex recognition as well as for interspecific isolation iation of song types according to genetic or morphological (Paterson 1985; Hochkirch and Lemke 2011), they are of units. Thus, the present study indirectly vindicates the sig- great significance for studying the processes underlying nificance of acoustic recognition systems and song special- evolutionary radiation. Acoustic diversity within bush- ization patterns in this genus. cricket genera varies from very low with a more or less uniform pattern of the male calling song (cf. Heller 2006; Çıplak et al. 2009)toveryhighwithagreatvarietyofsong types, especially in sympatric taxa, even within groups of Material and methods closely related species (cf. Heller 1988, 1990, 2006; Chobanov and Heller 2010,etc.). Taxon sampling and morphological identification In Tettigonia, song differences between species (especially well expressed in sympatric taxa) may express in different The species used in this study and their sampling locali- syllable arrangement and repetition rate, echeme length, and ties are presented in Table 1 and Fig. 1. This dataset con- duty cycle. Some differences are also found in the carrier tains 66 Tettigonia specimens from 33 disjunct localities/ frequency of the song. In some species (e.g., Tettigonia populations and five outgroup taxa representing two cantans), females are not very sensitive to the conspecific tettigoniid subfamilies (Tettigoniinae: Amphiestris Fieber, structure of the song and thus may respond to heterospecific 1853 (Tettigoniini), Onconotus Fischer von Waldheim, males (Schul et al. 1998), while in other species (i.e., 1839, Paratlanticus Ramme, 1939, Platycleis Fieber, Tettigonia caudata), females rely on the minimum duty cycle 1853; Saginae: Saga pedo (Pallas, 1771)). Due to the of the echemes, thus neglecting the fine song structure (Schul properties of the chosen DNA fragments (high amount 1998). In Tettigonia viridissima, song recognition based on of interspecific variation providing good phylogenetic sig- temporal clues has been shown to be more complicated. nal at a generic level but some risk of false results at a Here, females evaluate the pause within disyllabic echemes higher systematic level due to convergencies and phenom- and respond only to the species-specific echeme structure ena like long branch attraction), we choose taxa that, ac- (Schul 1998). cording to published data, are closely related and/or have In the present study, we aim to evaluate phylogenetic rela- close ancestral position to Tettigonia (in the case of tionships within Tettigonia. We based our study on a genetic Saginae) (e.g., Gorochov 1995;Songetal.2015). dataset that was used as a basis for mapping acoustic and Used samples of Tettigonia have preliminary been identi- morphological characters in an attempt to track the evolution- fied using original descriptions and published reviews (e.g., ary paths of the acoustic communication in this genus. For Bolívar 1914; Chopard 1943; Ramme 1951;Harz1969; these purposes, sequences of the mitochondrial cytochrome Massa 1998;Chobanovetal.2014 and references therein). c oxidase subunit I (COI) gene and the nuclear internal tran- All specimens listed in Table 1 were morphologically related scribed spacers 1 and 2 (ITS1 and ITS2) were used that have to existing taxa based on available literature and museum previously been widely employed in phylogenetic studies of specimens. Apart from own material, the following specimens grasshoppers (e.g., Cooper et al. 1995; Chapco and from public collections that refer to the studied taxa were Litzenberger 2002) and bush-crickets (Ullrich et al. 2010; studied: Allegrucci et al. 2011; Boztepe et al. 2013; Çiplak et al. Tettigonia acutipennis Ebner, 1946—male, holotype, 2015). The DNA sequences selected for the present study BKleinasien 1914 | Marasch, Tölg. | coll. R. Ebner^ have different modes of evolution and inheritance history, (Naturhistorisches Museum Wien (NHMW)); male, Hakkari and thus, they may reveal different aspects of the speciation (the Natural History Museum London (NHM)); two males, history of the examined lineages. BTurkey: |