Org Divers Evol (2010) 10:123–133 DOI 10.1007/s13127-010-0004-4

ORIGINAL ARTICLE

Interglacial refugia and range shifts of the alpine Stenobothrus cotticus (: : )

Dirk Berger & Dragan P. Chobanov & Frieder Mayer

Received: 2 September 2008 /Accepted: 15 April 2009 /Published online: 11 March 2010 # Gesellschaft für Biologische Systematik 2010

Abstract A warming climate leads to shifts in distribution between the two geographically separated populations of ranges to higher latitudes and altitudes. Consequently, cold- S. cotticus studied. We suppose that S. cotticus had a wider adapted alpine species can be trapped in interglacial distribution during colder periods, when its range was Holocene refugia on high mountain summits if they fail to expanded to lower altitudes. This hypothesis is supported expand their ranges to the north. One example is the alpine by the current distribution of the closely related montane S. grasshopper Stenobothrus cotticus. This species was rubicundulus. assumed to be endemic to the southwestern Alps (France, Italy). However, we have found a second refugium in the Keywords Distribution . Glacial expansion . Rila Mountains in southwestern Bulgaria. Analyses of the Interglacial refugia . Stenobothrus cotticus . mitochondrial gene co1 and of phenotypic characters from Stenobothrus rubicundulus morphology and behaviour did not reveal differences

Introduction Electronic supplementary material The online version of this article (doi:10.1007/s13127-010-0004-4) contains supplementary material, which is available to authorized users. Major climatic fluctuations in about 100,000 year cycles are : well documented for the last 700,000 years when cold ice D. Berger F. Mayer ages (glacials) were separated by warm interglacials (Webb Institut für Biologie, Universität Erlangen-Nürnberg, Staudtstraße 5, and Bartlein 1992). Many investigations of plant and 91058 Erlangen, Germany species have shown concordantly that periodical changes in climate during the Quaternary have significantly D. P. Chobanov affected the distribution of species and led to substantial Institute of Zoology, Bulgarian Academy of Sciences, Tsar Osvoboditel blvd. 1, range shifts both in latitude and altitude (e.g. Comes and BG-1000 Sofia, Bulgaria Kadereit 1998; Hewitt 1996, 1999, 2004; Taberlet et al. 1998). The climatic changes among glacial-interglacial Present Address: cycles were not uniform. Warming (interstadials) and D. Berger (*) Senckenberg Naturhistorische Sammlungen Dresden, cooling periods (stadials) lasted from 70 years to several Museum für Tierkunde, millennia; annual mean temperatures could have changed Königsbrücker Landstraße 159, by 10–12°C in fewer than 10 years (Hewitt 1996; Müller et 01109 Dresden, Germany al. 2003). During the glacials the Arctic ice cap progres- e-mail: [email protected] sively expanded southwards. Animal and plant populations Present Address: followed their appropriate environments and moved to the F. Mayer south or to lower altitudes during cold periods. Therefore, Museum für Naturkunde, Leibniz-Institut für Evolutions- und thermophilic species became restricted to small refugia in Biodiversitätsforschung an der Humboldt-Universität zu Berlin, Invalidenstraße 43, the south, while their former ranges were occupied by cold- 10115 Berlin, Germany resistant species. During the interglacials the ice sheets 124 D. Berger et al. retreated northwards and to higher altitudes. Thermophilic help infer the historic biogeography of S. cotticus we species spread out from southern refugia and expanded studied the current distribution of its closest European their ranges northwards or to higher levels in the moun- relative, S. rubicundulus Kruseman & Jeekel, 1967, which tains. In contrast to many others, some cold adapted species is adapted to montane habitats. failed to expand their range to the north and became restricted to small mountain summits (‘sky islands’) that now serve as inter-glacial refugia (DeChaine and Martin Material and methods 2004; Knowles 2000, 2001). of the subfamily Gomphocerinae have Material been studied intensively with the aim of understanding the impact of climatic changes on the distribution of species Bulgarian specimens of S. cotticus were collected at (e.g. Hewitt 1996, 1999). They are widely spread across the Razdela site (42°11′N23°19′E) above Sedemte Rilski Palaearctic and Nearctic and are adapted to a wide range of Ezera tourist hostel and on the surrounding Otovishki Vruh ecological settings. Species diversity is highest in the peak, Topilata range, Vurla peak, Damga peak and Dodov temperate climate zone, and the number of species Vruh peak (42°10′N23°20′E) between 2,300 m and decreases rapidly towards the dry as well as towards the 2,650 m above sea level (m). In order to compare the cold climate zones (Bei-Bienko and Mistshenko 1951). Bulgarian and French populations, a further excursion to Many species expanded their distribution range to the north Col d’Izoard in the French Hautes-Alpes (44°49′N06°44′E, after the last ice age (Cooper et al. 1995; Lunt et al. 1998) site elevation 2,307 m) was made. Furthermore, published and formed hybrid zones at the secondary contacts of distribution records were used to infer the distribution ranges populations that had been separated during glaciation of the closely related species S. cotticus and S. rubicundulus (Bridle and Butlin 2002; Butlin and Hewitt 1985; Hewitt (see the “Electronic Supplementary Material”). Additional 1975; Jiggins and Mallet 2000; Vedenina and von Helversen unpublished records were obtained by revising the collec- 2003). Hybridizing taxa often resemble each other in tions of the National Museum of Natural History, Sofia morphology but can be distinguished easily by their songs. (NMNH); Macedonian Museum of Natural History, Skopje Males generate songs by stridulatory movements of both hind (MMNH); Museum of Blagoevgrad (BHM); L. Stefanov legs rubbing against the forewings. Females respond acous- (Skopje); D. Chobanov (Sofia); D. Berger (Dresden); and O. tically to a singing male if specific song parameters match von Helversen (Erlangen). A distribution map was created their preferences. The songs and, hence, the underlying leg using the utilities of http://www.aquarius.geomar.de/. movement patterns evolve rapidly and can attain high levels The morphological features of ten males and four of complexity (Bull 1979;Elsner1968;vonHelversen1986; females from the Bulgarian population and 11 males and von Helversen and von Helversen 1994; Otte 1972). eight females from the French population were investigat- Therefore, stridulatory leg movement patterns and resulting ed. These included the two and three males, respectively, acoustic signals are highly informative phenotypic traits that of the Bulgarian and French populations for which the assist in the reconstruction of the evolutionary history of song was also recorded, as well as the two genetically populations and closely related species. investigated individuals. In the present study we investigated the biogeography and conservation status of an ecologically highly specialised Morphometric investigations alpine grasshopper, Stenobothrus cotticus Kruseman & Jeekel, 1967. This species was assigned to the subgenus Morphometric measurements were taken from dried grass- Crotalacris Chopard within the genus Stenobothrus Fischer hoppers using an ocular micrometer-equipped (Leitz Wetzlar (Harz 1975) on the basis of wing characters. The species of Germany, Periplan GF 10x) binocular (Zeiss-Jena Citoval Crotalacris show few morphological differences (Harz A10026). The following measurements were taken: Prono- 1975), but their songs are strikingly different (Berger tum length (measured along the median keel), tegmen length 2008;ElsnerandWasser1995a; Ragge and Reynolds (maximum length of the folded right fore wing), and length 1998), thus allow unequivocal identification of the of the right hind femur. Only intact parts of the were different species. Stenobothrus cotticus is currently found measured. only at highest altitudes and was assumed to be endemic to the southwestern Alps (Harz 1975;Voisin2003). Here Song analysis we describe a newly discovered refugium of S. cotticus on the Balkan Peninsula. We use morphological, behavioural Song recordings were made under controlled temperature and genetic traits to investigate the divergence between conditions (∼28°C) in the laboratory of the University the two geographically widely separated populations. To Erlangen-Nürnberg. We limited our acoustic analyses to Interglacial refugia and range shifts of the alpine grasshopper Stenobothrus cotticus 125 courtship songs, because these also contain the elements of Biotechnologie GmbH). The temperature cycle of 94°C for calling songs in S. cotticus and in S. rubicundulus (Berger 30 s, 50°C for 50 s, and 72°C for 90 s was repeated 35 2008; Ragge and Reynolds 1998; own unpublished data). A times. After PCR, two units exonuclease I (E. coli) and one female was placed next to a male to ensure recording of unit Antarctic phosphatase (both from New England courtship songs. Songs were recorded with a 1/2″ condenser BioLabs) in 5 µl H2O were added to the amplification microphone (G.R.A.S. Type 40AF) equipped with a G.R.A. products and incubated at 30°C for 15 min. Both enzymes S. 26AB preamplifier. The signal was amplified by a Brüel were denatured at 80°C for 15 min. & Kjær measuring amplifier (Type 2608). The sound- Cycle sequencing reactions were performed with four producing leg movements were recorded simultaneously internal sequencing primers (ER228: 5′-TAGGAGCAG using an opto-electronic device (von Helversen and Elsner TAAACTTTATTACAAC-3′;ER231:5′-ACTGTAAATA 1977). The signals were A/D-converted by a custom-built TATGATGTGCTCA-3′; ER378: 5′-ATTTTCTTCATA DSP card with sample rates of 125 kHz (leg movements) and GTAATACCTATTAT-3′; ER384: 5′-AAATGTTAGGTTTA 250 kHz (sound). CTCCAATAAATATAA-3′) using the DYEnamic Direct 7- Further song recordings of the Bulgarian population were dZ-dGTP cycle sequencing kit (Amersham) and following carried out at the Institute of Zoology (Sofia) at temperatures the manufacturer’s instructions. Twenty-five cycles at 94°C between 30°C and 35°C, using a built-in PC soundcard for 20 s, 57°C for 30 s, and 72°C for 30 s were performed. (44.1 kHz) and an electret condenser microphone (Knowles The sequences were run on a 4.5% Sequagel XR (National BT-1759-000) equipped with a custom-made preamplifier. Diagnostics) and analysed using a LI-COR DNA sequencer Figures of the song oscillograms were prepared using (model 4200 L). All sequences are available from GenBank Turbolab 4.0 (Bressner Technology, Germany). The fol- (accession numbers FJ555543–FJ555553). lowing song parameters were measured by using custom- A total of 1,000 base pairs (bp) starting at position 215 designed software (W. Schulze) developed in Lab VIEW 7 of the co1 gene were used for pairwise sequence compar- (National Instruments): Song duration (the length of time of isons. Sequence alignment was unequivocal, since no a whole courtship sequence, including all syllables and insertions or deletions were found. Pairwise DNA sequence pauses), syllable duration (sound that is produced by one differences (p-distances) and a neighbour-joining tree were full leg movement cycle, which starts when the leg leaves calculated (Table 3 and Fig. 2). its initial position and lasts until the leg reaches this position again), duration of pauses between syllables, and pulse period (length of a single pulse plus the subsequent Results pause before the following pulse starts) (Fig. 1). Stenobothrus cotticus had been thought to be an endemic Genetic analysis grasshopper of the southwestern Alps (Harz 1975;Voisin 2003). In 2005 a population was discovered in the highest A 1.0 kb fragment of the mitochondrial gene cytochrome parts of the northwestern Rila Mountains in southwestern oxydase subunit 1 (co1) was sequenced in seven Steno- Bulgaria, approximately 1,350 km southeast of the bothrus species, two species of Omocestus Bolivar, and one previously known range of the species. In the Rila species of Chorthippus Fieber (all from Gomphocerinae). Mountains Stenobothrus cotticus was abundant above One individual of each species was sequenced, except that 2,500 m. Other grasshopper species cohabiting with S. for Stenobothrus cotticus one individual each from the cotticus in Bulgaria were Chorthippus parallelus French and Bulgarian populations was included. Tissue (Zetterstedt) up to 2,550 m, variegatus from one hind leg was digested for 4 h at 56°C in TNE (Fischer von Waldheim) above 2,500 m, and sporadically buffer (100 mM NaCl, 100 mM Tris-HCl, 2 mM EDTA, (Linné). Some additional specimens pH 8.0) with 200 µg/ml proteinase K, 2% SDS and 0.04 M of S. cotticus from the eastern Rila Mts. were found in the DTT. DNA was isolated according to a salt-chloroform collection of the National Museum of Natural History in procedure (Müllenbach et al. 1989). Sofia. At the type locality of S. cotticus in the French Hautes- The co1 fragment was amplified with the primers ER224 Alpes (Col d’Izoard, 2,307 m) we observed Gomphocerus (5′-CAGTCTATCGCCTATAAATTCAGC-3′) and ER227 sibiricus, Aeropedellus variegatus, Stauroderus scalaris (5′-AAAAAATGTTAGGTTTACTCCAATAAATA-3′), (Fischer von Waldheim), Myrmeleotettix maculatus which are located in the tRNA-Tyr and the COI gene, (Thunberg), Epipodisma pedemontana (Brunner von respectively. The amplifications were carried out in a Wattenwyl), and Bohemanella frigida (Boheman). volume of 25 µl containing 0.2% BSA, 1 µM of each We did not find obvious morpholological differences primer, 0.25 mM of each dNTP, 0.5 units SAWADY Taq- between the French and Bulgarian populations of S. DNA-Polymerase, reaction buffer and enhancer (peqlab cotticus (Table 1); this includes wing venation and body 126 D. Berger et al.

Fig. 1 Courtship songs of Stenobothrus cotticus from Col d’Izoard in of a French specimen. d Single syllable of a Bulgarian specimen. ML, France (a–c) and from the Rila Mts. in Bulgaria (d). a Overview of a movement pattern of left leg; MR, movement pattern of right leg; OS, whole courtship sequence. b Section of 4 s duration. c Single syllable oscillogram of sound dimensions. In our material, males from the French ic up-and-down movements at a frequency of about 125 Hz population were smaller than those from Bulgaria. However, (Fig. 1). Each upward or downward movement produced a considering the measurements by Kruseman and Jeekel sound pulse. Therefore, each syllable consisted of a pulse (1967) and Harz (1975) for French S. cotticus,nosuch train with a mean pulse period of 3–5 ms. Pulse period did difference in body size is evident. The discrepancy between not change within a syllable nor throughout the whole song. the mean and median values for females from the Rila Mts. After performing several full courtship sequences the male results from a single female that shows conspicuously small sometimes switched to short wing stridulations and noisy dimensions. The other three females fit with published flights. Finally the male tried to jump on the female and measurements. attempted to copulate. We did not find any structural Males of Stenobothrus cotticus from both populations differences in leg movement patterns (see Fig. 1c, d)or performed an uninterrupted courtship display in front of the temporal song pattern (Table 2) between the two geograph- female for up to several minutes. The male started courting ically separated populations. Differences in syllable dura- with a low sound intensity, reached maximum intensity tion between the Bulgarian and French specimens were rapidly and maintained it until the end. The whole song small and in the range of the standard deviation for each consisted of long series of syllables (Fig. 1). Syllable population. It seems that the small variation in the duration duration was 200–300 ms and syllables were separated by of syllables and syllable pauses resulted from variable male pauses of around 600 ms. The duration of both syllables motivation. Slight variation in the pulse period could be and pauses was variable within a courtship sequence. caused by different body temperatures of the recorded Shorter and softer attenuated syllables were generated at animals. the beginning of a courtship sequence and were separated Sequencing 1,000 bp of the mitochondrial gene cyto- by long pauses. Later during courtship syllables became chrome oxidase, subunit 1 (co1), revealed surprisingly little longer, and pauses shorter and more regular. Both hind legs genetic variation among and between the French and were precisely coordinated and performed nearly antidrom- Bulgarian specimens of S. cotticus and other representatives Interglacial refugia and range shifts of the alpine grasshopper Stenobothrus cotticus 127

divergence was found within the subgenus Crotalacris comprising the three species S. cotticus, S. rubicundulus, and S. clavatus Willemse. In addition, these three species and S. fischeri (Eversmann), S. lineatus (Panzer) and S. eurasius Zubovski all showed little interspecific variation in the co1 gene, with a maximum of 1.6% sequence divergence. Only S. stigmaticus (Rambur) showed substan- tial sequence divergence from all other investigated species of Stenobothrus (at least 4.3%). It is noteworthy that in our neighbour-joining tree (Fig. 2) the Bulgarian S. cotticus results as most closely related to S. clavatus. However, the branches in the apical part of the Stenobothrus tree are so short that hardly much relevance can be assigned to this.

Discussion

Animals from the French and Bulgarian populations of S. cotticus closely resemble each other in morphology, songs as well as mitochondrial COI sequences and show distinct morphological and song differences from closely related species of the genus. Stenobothrus cotticus was assigned to the subgenus Crotalacris, which also comprises S. rubi- cundulus (Harz 1975)andS. clavatus (Fig. 2). This Fig. 2 Tree from neighbour-joining analysis of selected European subgenus is characterised by a widened median field of Stenobothrus species based on Kimura-2-parameter distances among the hind wing, a thickened radius apicalis of the hind wing, 1,000 bp sequences of the mitochondrial gene cytochrome oxidase, darkened wings, and by wing stridulation at least during subunit 1 (co1). Chorthippus parallelus used as outgroup. Bootstrap flight (Harz 1975). Morphological evidence suggests a support values≥50% among 1,000 bootstrap replicates given above branches close relationship between S. rubicundulus and S. cotticus, since members of these two species have conspicuously of the genus Stenobothrus (Fig. 2, Table 3). The difference broadened wings, heavily sclerotised veins, and a fused between the single individuals from the two S. cotticus subcosta and radius on the hind wing. Stenobothrus populations was 0.3%. Less than one per cent sequence clavatus is easily distinguished from S. cotticus and S.

Table 1 Variation in three morphological characters in Source Pronotum length [mm] Tegmen length [mm] Hind femur length [mm] Stenobothrus cotticus from the Bulgarian and French (Reference) ♂♀♂♀ ♂ ♀ populations examined in the present and earlier studies Bulgaria: Rila Mountains (present study) Range 3.3–3.7 3.2–4 12.9–14.4 10.2–11.5 9.2–10 9–11.4 Mean±SD 3.4±0.1 3.7±0.3 13.7±0.5 11.1 ± 0.5 9.6±0.2 10.4±0.9 Median 3.4 3.9 13.7 11.4 9.7 10.6 Number of individuals 10 4 10 4 10 4 France: Hautes-Alpes (present study) Range 3–3.5 3.6–4 12.1–13.5 11.1–12.5 8.7–9.4 10–10.8 Mean±SD 3.3±0.2 3.7±0.1 12.9±0.5 11.8±0.5 9.1 ± 0.2 10.4±0.3 Median 3.3 3.7 12.9 11.8 9 10.4 Number of individuals 11 8 5 6 10 8 France: Hautes-Alpes (Kruseman and Jeekel 1967) Range 3.2–3.7 4.0–4.2 13.2–15 11.5–13.3 9.2–10.3 10.8–11.7 France: Hautes-Alpes (Harz 1975) Range 3–3.6 3.8–4.2 12.8–15.3 10.5–13.3 9–10.5 10.5–11.7 128 D. Berger et al.

Table 2 Variation in temporal song parameters of courtship Parameter S. cotticus, Bulgaria: Rila Mts. S. cotticus, France: Hautes-Alpes song in Stenobothrus cotticus from Bulgaria and France Male 1 Male 2 Male 1 Male 2 Male 3

Pulse period [ms] Range 2.1–6.9 1.5–5.5 2.2–8.5 3.6–6.7 2.8–5.7 Mean±SD 3.3±0.4 3.7±0.5 4.0±0.6 4.8±0.4 4.2 ± 0.4 Median 3.3 3.7 4.0 4.7 4.2 (n of pulse periods) 1880 688 779 715 745 Syllable duration [ms] Range 190.5–270.3 203–303.8 150.2–231.9 162.9–274.9 199.3–274.8 Mean±SD 231.3±17.7 272.4±28.5 182.5±14.8 242.3±20.2 247.3±14.9 Median 230.6 278.6 180.6 243 248.5 (n of syllables) 65 9 172 59 49 Pause duration between syllables [ms] Range 522.4–789.5 744.9–1004.6 478.7–727.1 552.6–994.2 555.7–998 Mean±SD 604.2±53.7 818.1±76.5 574.1±33.6 678.8±87 692.8±107.6 Median 598.7 793.3 574.6 653 675.3 (n of pauses) 61 8 171 58 48

rubicundulus, e.g. by the strongly clubbed male antennae that vary between locations and years. Intraspecific differ- that are used to perform additional visual signals during ences in morphological characters, e.g. in wing venation, courtship. Stenobothrus cotticus and S. rubicundulus can be were neither found among populations of S. rubicundulus separated by characters of the wings and external genital nor among S. cotticus populations from the western Alps apertures. In S. rubicundulus the median field reaches the (Harz 1975). In contrast, substantial morphological varia- middle of the elytrum, in S. cotticus it is clearly longer. In tion was found among populations of S. eurasius (Harz contrast to S. cotticus, the fore margin of the alae is apically 1975; Ingrisch and Pavićević 1985; own unpublished data). obliquely truncated in S. rubicundulus.InS. cotticus, when Morphological, behavioural and genetic resemblance be- the stigma on the elytrum is present its position is in the tween the French and Bulgarian populations of S. cotticus distal third of the wing, whereas it is situated in the middle suggests that they have not been separated for long. of the wing in S. rubicundulus. The 10th abdominal tergite Sexually selected traits like courtship songs can evolve in S. rubicundulus bears a pair of acuminate apices laterally rapidly (Mendelson and Shaw 2005; Ritchie and Garcia of the epiproct, in S. cotticus these apices are tiny, not 2005), and species with a bidirectional acoustic communi- acuminate, and located ventrally of the epiproct (see Harz cation system may diverge faster in behavioural traits than 1975: p. 737, pl. 157). In addition, all members of in morphological characters (Heller 2006). This could lead Crotalacris can be identified unmistakably by their to a rapid divergence between allopatric populations. The species-specific songs, which contain characteristic acoustic songs of the closely related species S. cotticus and S. elements (Berger 2008; Elsner and Wasser 1995a, b; Ragge rubicundulus differ in several characters. While the court- and Reynolds 1998; this paper). All these differences ship song of S. cotticus contains only one simple song between S. cotticus and all congeneric species are in strong element (Fig. 1), that of S. rubicundulus comprises several contrast to the great genetic similarity among Stenobothrus complex elements, which are performed in a strict order. At species (Table 2, Fig. 2), but clearly confirm that both the the end of each courtship sequence S. rubicundulus French and Bulgarian populations belong to S. cotticus. produces intense sound by wing stridulation (Berger 2008; Despite the wide geographic separation of the two S. Elsner and Wasser 1995a, b; Ragge and Reynolds 1998). cotticus populations, the analysis of song parameters and While S. cotticus did not show distinct intraspecific several morphological characters, including wing venation, differentiation in the acoustic communication system, the body dimensions, and stridulatory leg movements, did not populations of the closely related and widely distributed reveal obvious differences. The extent of variation in body species S. rubicundulus can be divided in a northwestern size in S. rubicundulus (Harz 1975: p. 776) was found to be (Alps, Slovenia) and a southern (Greece) group according in the same range as in S. cotticus (Table 1). In general, to one courtship-specific song element (a rapid repetition of slight variation of body size in could be explained pulses). Furthermore, Central European populations gener- by environmental differences such as climatic conditions ate this song element by rubbing both legs against the Interglacial refugia and range shifts of the alpine grasshopper Stenobothrus cotticus 129

, forewings, while the males of the Balkan population use only their wings to produce an identical acoustic signal (Elsner and Wasser 1995a, b). 0.003 – S. cotticus France Our preliminary results of small genetic differences in the mitochondrial co1 fragment suggest that substantial interspecific song differences can evolve within a short , evolutionary period. All Stenobothrus species included in our analysis differ strikingly in their courtships songs 0.009– 0.007 S. cotticus Bulgaria (Berger 2008; Ragge and Reynolds 1998). In contrast, with

t the exception of S. stigmaticus the maximum pairwise sequence divergence among all investigated species was no more than 1.6%. This value falls within the range of intraspecific differences of other acridid grasshoppers 0.049– 0.046 0.046 S. rubicun- dulus (Knowles 2000; Lunt et al. 1998). Little variation between species suggests a recent radiation. Resolving phylogenetic relationships among these species might be difficult even if much longer DNA sequences were available, because intra- 0.047 0.016 0.011 0.010 – S. stigma- ticus and interspecific variation could overlap substantially. Nevertheless, our genetic data are consistent with a very recent separation of the French and Bulgarian populations – 0.010 0.046 0.013 0.009 0.008 S. lineatus of S. cotticus. Our results show that Stenobothrus cotticus is not endemic to the southwestern Alps as previously assumed – 0.006 0.010 0.043 0.008 0.003 0.002 S. fischeri (Harz 1975; Voisin 2003). The distribution of this species is highly disjunct, as the Bulgarian locality is separated from the French one by a distance of 1,350 km. Both populations – 0.001 0.007 0.009 0.043 0.007 0.002 0.001 S. eurasius inhabit high mountain peaks usually above 2,000 m. It is unlikely that additional populations of S. cotticus exist in Central and northern Europe, since the distribution of grasshoppers in these regions is well studied (e.g. Baur et – 0.057 0.055 0.055 0.063 0.047 0.060 0.058 0.057 S. clavatus al. 2006; Bellmann and Luquet 1995; Fontana et al. 2002; Harz 1957, 1975;Holst1986;Kočárek et al. 2005; Marshall and Haes 1988; Obenberger 1926; Thorens and Nadig 1997; Voisin 2003). However, with the exception of Greece (Willemse 1984) the fauna of the Balkans is still not – O. haemor- rhoidalis 0.005 0.056 0.055 0.054 0.062 0.046 0.059well investigated. 0.058 0.056 Thus, further isolated populations of S. cotticus could be expected only from high-elevation summits of the Dinaric Mountain chain.

O. ventralis 0.062– 0.063 0.063 0.062 0.062 0.068 0.060 0.065Stenobothrus 0.065 cotticus 0.063 is the only alpine species of its genus in Europe that exhibits low ecological tolerance towards low temperatures. In the Rila Mountains we found it at altitudes between 2,250 m and 2,850 m, where it is 686065 62 46 59 63 47 60 9 43 7 10 43 8 10 46 13 47 16 49 65 58 58 2 3 9 11 46 9 62 54 55 7 6 62 55 55 1 63 56 57 C. paralle- lus – 62 63 5 common on steep ridges with xeric grass communities. This is comparable to the habitats in the southwestern Alps, where S. cotticus was found at altitudes between 1,700 m (Kruseman and Jeekel 1967) or, more commonly, 2,000 m , , France 63(Harz 561975 57; Lemonnier 11999) and 2 2,700 m 8 (Voisin 102003). 46 7 3 In general it inhabits high isolated mountain summits that Bulgaria are often called ‘sky islands’ (e.g. Knowles 2000, 2001). The present highly fragmented distribution of S. cotticus Pairwise DNA sequence differences between species: total numbers of substitutions at bottom left of table diagonal, genetic p-distances at top righ likely represents the remnants of a formerly wider range. Range expansions or contractions with altitudinal shifts Stenobothrus cotticus Stenobothrus cotticus Stenobothrus rubicundulus Stenobothrus fischeri Table 3 Stenobothrus clavatus Genus Species ChorthippusOmocestus parallelus ventralis Omocestus haemorrhoidalis caused by cyclic climatic oscillations are reported for 130 D. Berger et al. several alpine organisms (e.g. DeChaine and Martin 2004, tion of S. rubicundulus (Fig. 3). The latter is a montane 2005; Kropf et al. 2003). In stadial maximum periods in the species with a wide temperature tolerance that today Rila Mts. the mean annual temperatures were more than inhabits dry stony meadows within open forests or in the 10°C lower than today. The timberline was at 1,100– subalpine belt, usually between 1,000 m and 2,000 m (see 1,300 m, while nowadays it is found at about 2,000– “Electronic Supplementary Material”). Its range covers the 2,200 m, corresponding to an altitudinal shift of approxi- mountains of the Balkan Peninsula including the Pindo- mately 1,000 m (Tonkov and Marinova 2005). A similar Dinaric mountain chain, the Peloponnesus (southwards to down-slope shift of the S. cotticus populations in the Rila the Parnon Mts.), the Rhodopean mountain group and Stara Mts. would result in a lower distributional border at about Planina Mts., as far east as the region of Sliven (Fig. 3). 1,200 m. This would have allowed for a wider distribution Further north S. rubicundulus occurs in the southern and a near-continuous distribution area reaching from the Carpathians and the southern Alps (Alpes-Maritimes in Rila Mountains westwards over the Dinaric Mountain chain the West), as high as 2,400–2,560 m. Few isolated localities to the Alps. With a lower limit at 800–900 m (Fig. 3)we are reported from the Apennines in Central Italy and from could trace an uninterrupted mountain bridge from the Rila western Ukraine (“Electronic Supplementary Material”). Mts. via the neighbouring mountains to the north (Verila No syntopic occurrences of S. cotticus and S. rubicundulus Mt., 1,415 m; Vitosha Mt., 2,290 m), small mountains and have been found. highlands of the Kraishte region (central part of western Stenobothrus cotticus is an example of a highly adapted Bulgaria and southeastern Serbia), the Kosovo Pole alpine species currently present in two small refugia. The highland to the Dinaric Mountains. The Dinaric Mountains European mountain ranges and particularly the Balkans are spread northwestwards through the territories of Montene- inhabited by many specialised and endemic forms of gro, southwestern Serbia, Bosnia and Herzegovina, as well different groups of organisms with fragmented distribution as through southern and western Croatia to Slovenia. There patterns (e.g. Nagy et al. 2003). Buresch and Arndt (1926) the Notranjasko highlands provide the transition to the already provided a comprehensive compilation of glacial Alps. However, even if S. cotticus moved to lower altitudes, relict animal species in Bulgaria and Macedonia, including it probably never inhabited the lowlands. In this case, the protists, planarians, copepods, insects (lepidopterans, expansion of S. cotticus must have been constricted to dipterans, coleopterans and hymenopterans), arachnids, mountain ranges during glacial periods, in contrast to most fishes and birds. Disjunct distribution is thought to be a other grasshopper species, which expanded their distribu- result of climatic fluctuations during the Pleistocene tion range through the lowlands during the interglacial (Comes and Kadereit 1998; Hewitt 1996, 1999, 2004; periods (Cooper et al. 1995; Hewitt 1996). Taberlet et al. 1998). The prime examples are boreoalpine This scenario of a much wider distribution of S. cotticus species that show typical distribution patterns, i.e. occur in during colder periods is suggested by the current distribu- England, the Alps, the Carpathians, the Apennines, the

Fig. 3 Current distribution of Stenobothrus cotticus (white triangles) and S. rubicundulus (open circles) in Europe; S. rubicundulus further ranges to western Ukraine (not shown). For localities and corresponding references, see “Electronic Supplementary Material” Interglacial refugia and range shifts of the alpine grasshopper Stenobothrus cotticus 131

Pyrenees and/or the arctic regions of Scandinavia, North (P. keisti, P. styriaca), on high mountains in Montenegro Asia and North America, but are absent from the areas in (P. relicta), in the Carpathians (P. transsylvanica), and in between (Holdhaus 1912). The orders containing the northern Russia (P. poppiusi) (Ander 1949; Bei-Bienko and highest numbers of boreoalpine species are Coleoptera Mistshenko 1951; Ebner 1951; Harz 1975; Koschuh 2008; (Holdhaus and Lindroth 1939) and Lepidoptera (Holdhaus Ramme 1951). We found Gomphocerus and Aeropedellus 1912; Warnecke 1959). Especially the latter have been the co-occurring in the same habitat with S. cotticus in Bulgaria subject of recent studies. Species groups in Erebia and and in the French Alps. Bohemanella frigida was found Glacies as well as Euphydryas cynthia (Lepidoptera) show together with S. cotticus only in the French Alps, but it has disjunct distribution along the European mountain ranges, been reported from high mountain ranges in Bulgaria with the main distribution area often in the Alps. In addition (Drenowski 1936; Ebner 1936). they have been recorded from single high mountains on the Thus, the current distribution of S. cotticus and S. Balkan Peninsula, e.g. in Macedonia, Montenegro and rubicundulus coincides with the southern distribution areas western Bulgaria, the Apennines, Carpathians and the of European boreoalpine insect species, while a northern Pyrenees (Schmitt 2007; Varga and Schmitt 2008). For distribution is lacking. Ander (1949) suggested that the many of the species concerned it is likely that they northern and southern areas of boreoalpine distribution in ‘escaped’ from the mountains and expanded their ranges Europe resulted from different migration events. Whereas during past climatic oscillations. This would explain the the northern populations originate from migrations from isolated occurrence of these species in the Pyrenees and on northeastern populations (northern Russia and Asia) mountains in the northern areas (Warnecke 1959). through cold steppes, the southern populations survived The Erebia rhodopensis species group is of particular glacial stadials at lower altitudes (Ander 1949). interest here as it shows a high degree of correspondence in Alpine ecosystems are of high importance for European distribution with Stenobothrus cotticus: it occurs in the biodiversity and currently function as Holocene refugia. Alpes Maritimes (E. aethiopella) and in the mountain Global warming of the climate in the 19th and 20th ranges of Bulgaria (E. rhodopensis). This distribution is centuries is well documented (Gaillard 2004). During the explained by a pre- or interglacially continuous ancient last century the temperature changes at high elevations were population and postglacial extinction events in the areas in more extreme than for the global averages (Beniston et al. between (Varga and Schmitt 2008). A formerly wider 1997). These changes have severe effects on alpine distribution is likely, because phylogenetic analyses have ecosystems as they lead to an upward altitudinal range shown that in several other still more widely distributed shift of alpine species, and to increased fragmentation of alpine taxa Balkan species and Alpine species cluster in populations, thus putting the latter at higher risk of distant clades (Kropf et al. 2002, 2003; Vargas 2003; Zhang extinction (DeChaine and Martin 2004; Theurillat and et al. 2001). It appears unclear, however, why in the Erebia Guisan 2001). The current temperature level nearly reaches rhodopensis group and in Stenobothrus cotticus only the values of temperature maxima during the Holocene populations in the Alpes Maritimes and in a limited area on (Kinzelbach 2007); therefore, the fragmentation of the S. the Balkans have survived. cotticus populations likely resembles the situation during Further examples of disjunct distributions are given in stadial maxima. In the Rila Mountains only three popula- the literature for various insect groups (e.g. Ander 1949; tions of S. cotticus have been recorded so far, even though Buresch and Arndt 1926; Drenowski 1936; Holdhaus 1912; high-elevation habitats on other mountain summits in the Holdhaus and Lindroth 1939; Pauls et al. 2007; Pittioni region have been visited. The highest elevation there is 1942). This also includes boreoalpine grasshopper species, 2,925 m (Musala summit); thus, range shifts to higher namely Bohemanella frigida, Aeropedellus variegatus, altitudes are restricted. Consequently, such specialised Gomphocerus sibiricus and Podismopsis spp. Besides alpine forms as S. cotticus are currently limited to few occupying wide areas in the north, their distribution is endangered populations. highly fragmented near its respective southern limits. In its southern ranges Bohemanella frigida is known from the Acknowledgements We thank A. Popov (NMNH, Sofia), B. Michaj- European Alps and Bulgaria, Aeropedellus additionally lova (MMNH, Skopje), A. Pulev (BHM, Blagoevgrad), L. Stefanov from the Apennines and Slovenia, and Gomphocerus (Skopje), and O. von Helversen (Erlangen) for providing access to official and personal collections. We are very grateful to F. Willemse furthermore from the Pyrenees and locally from high (Eygelshoven) for supplying inaccessible literature, and to W. Schulze mountains of Serbia, Bosnia, Herzegovina and Montenegro (Erlangen) for technical advice. Furthermore we are indebted to W. (Ander 1949; Bei-Bienko and Mistshenko 1951; Drenowski Schulze (Erlangen), B. Gottsberger (Vienna), J. Ustinova (Karlsruhe), 1936; Ebner 1936, 1937a, b, 1951; Harz 1975; Holdhaus Klaus-Gerhard Heller (Magdeburg), and F. Willemse (Eygelshoven) for comments and fruitful discussions on the manuscript. This study was 1912;Ramme1951).ThefiveEuropeanPodismopsis supported by the Deutsche Forschungsgemeinschaft (DFG) and the species show vicariant distribution in the European Alps Staedtler-Stiftung. 132 D. Berger et al.

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