Interspecific Hybridization Between the Grasshoppers Chorthippus Biguttulus and C

Home , Chorthippus, Chorthippus biguttulus, Chorthippus brunneus, Chorthippus mollis, Gomphocerinae, Species concept

Interspecific hybridization between the grasshoppers Chorthippus biguttulus and C. brunneus (Acrididae; Gomphocerinae)

Den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades

im Jahre 2007

vorgelegt von

Brigitte Gottsberger aus Botucatu, Brasilien Als Dissertation genehmigt von den

Naturwissenschaftlichen Fakultäten der Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 11.01.2008

Vorsitzender der Promotionskommission: Prof. Dr. E. Bänsch

Erstberichterstatter: PD Dr. F. Mayer

Zweitberichterstatter: Prof. Dr. B. Ronacher Contents

CONTENTS

Zusammenfassung...... 5 Introduction...... 7 Chapter 1...... 13 Behavioral sterility of hybrid males in acoustically communicating grasshoppers (Acrididae, Gomphocerinae) 1 Abstract...... 13 2 Introduction...... 14 3 Materials and methods...... 16 3. 1. Animals and crossing experiments...... 16 3. 2. Song recordings...... 16 3. 3. Terminology of song description and measured parameters...... 17 4 Results...... 18 4. 1. Songs of parental species...... 18 4. 2. Songs of F1 hybrids...... 21 4. 3. Songs of F2 hybrids...... 24 4. 4. Songs of backcrosses...... 26 4. 5. Principal component analysis of song parameters...... 27 5 Discussion...... 28 5. 1. Behavioral sterility of male hybrids...... 28 5. 2. Neuronal control of stridulation...... 29 Chapter 2...... 32 Dominant expression of song preferences in F1 hybrid females contribute to sexual isolation between two sympatric grasshopper species

1 Abstract...... 32 2 Introduction...... 33 3 Material and Methods...... 35 3.1. Study animals...... 35 3.2. Interspecific crossing experiments...... 35 3.3. Female preference tests...... 36

Contents

3.4. Female signals and response latencies...... 37 4 Results...... 38 4.1. Preference of parental species...... 38 4.2. Song preferences of F1 hybrid females...... 41 4.3. Female signals and response latencies...... 41 5 Discussion...... 43 5.1. Expression of female preferences...... 43 5.2. Signals of females...... 45 5.3. Strength of hybridization barriers...... 45 Chapter 3...... 47 Evolution of songs and female preferences in a natural hybrid population between the grasshopper species Chorthippus biguttulus and C. brunneus 1 Abstract...... 47 2 Introduction...... 48 3 Material and Methods...... 49 3.1. Study animals...... 49 3.2. Male songs: recordings and analysis...... 50 3.3. Female preference...... 50 3.3.1. Playback experiments of females with artificial sounds...... 50 3.3.2. Song models for female preference tests...... 51 3.3.3. Playback experiments of females with male calling songs...... 51 4 Results...... 54 4.1. Male calling songs...... 54 4.2. Female preference...... 57 4.2.1. Playback experiments with model songs...... 57 4.2.1. Playback experiments with natural male songs...... 59 5 Discussion...... 60 5.1. Hybrid origin of C. jutlandica...... 60 5.2. Origin of the hybrid population...... 61 References...... 63 Erklärung...... 76 Lebenslauf...... 77 Danksagung...... 78

Zusammenfassung

Die zwei europäischen Feldheuschreckenarten Chorthippus biguttulus und C. brunneus kommen in ihrem Verbreitungsgebiet, das sich über ganz Mitteleuropa erstreckt, weitestgehend sympatrisch und häufig syntop vor. Beide Arten unterscheiden sich kaum in ihrer äußeren Morphologie, in ihrer Ökologie und in neutralen genetischen Markern. Im Gegensatz dazu produzieren die Männchen beider Arten artspezifische Gesänge und die Weibchen besitzen artspezifische Gesangspräferenzen, was eine effektive prägame Hybridisationsbarriere zur Folge hat. Dennoch werden in der Natur immer wieder Hybride zwischen den beiden Arten gefunden. Dies wirft die Frage auf, weshalb sich die beiden Arten trotz gelegentlicher Hybridisierung nicht vermischen und stattdessen vielfach syntop vorkommen.

Um dieser Frage nachzugehen, untersuchte ich Verhaltensmerkmale beider Geschlechter von C. biguttulus, C. brunneus, und der interspezifischen Hybriden. Die akustischen Isolationsmechanismen wurden experimentell außer Kraft gesetzt, so dass Kreuzverpaarungen zwischen den Arten durchführbar waren. Die Gesänge wurden mit einer Positionsapparatur aufgenommen, die das akustische Signal und die Beinbewegungen der beiden Hinterbeine, die bei Feldheuschrecken für die Gesangsproduktion verantwortlich sind, simultan aufzeichnete. Da paarungsbereite Weibchen auf artspezifische Gesänge antworten, ist es möglich, die Präferenzen der Weibchen für bestimmte Gesangsparameter experimentell zu untersuchen. Hierfür verwendete ich einen computergesteuerten Versuchsaufbau, der den Tieren akustische Signale vorspielte und die Antworten der Weibchen mit Hilfe eines Mikrophons registrierte.

Im ersten Kapitel der Arbeit analysierte ich die Spontangesänge der Männchen von C. biguttulus, C. brunneus und deren Hybriden. Die Gesänge der Hybridmännchen (F1, F2 Generation und Rückkreuzungen) waren hinsichtlich der Anzahl an Versen pro Gesang und der Versdauern intermediär ausgeprägt. Im Gegensatz dazu war die artspezifische Silbenstruktur der Gesänge der Elternarten weitestgehend verloren gegangen. Gelegentlich in den Hybridgesängen auftretende Silben waren sowohl in ihrer Dauer als auch in ihrer Struktur sehr unregelmäßig. Die Gesänge der Hybridmännchen sind wegen der fehlenden Silbenstruktur für C. biguttulus Weibchen unattraktiv. Da sie über zu lange Verse verfügen, lehnen auch C. brunneus Weibchen Gesänge von Hybriden ab.

Zusammenfassung

Im zweiten Kapitel ging ich der Frage nach, inwieweit Hybridweibchen und Weibchen der beiden Elternarten gegen die Hybridgesänge selektieren. Dazu wurden den Weibchen künstlich generierte Gesänge vorgespielt, die in Versdauern und Silbenmuster variiert wurden. Für C. biguttulus Weibchen war das Silbenmuster innerhalb eines Verses ein entscheidendes Kriterium, während C. brunneus Weibchen allein aufgrund der Versdauer selektierten. Ein überraschendes Ergebnis war, dass die Hybridweibchen ein ähnliches Lautschema wie C. biguttulus Weibchen hatten. Auch für Hybridweibchen musste das Silbenmuster einem biguttulus-Silbenmuster entsprechen. Die Präferenz für Silbenmuster wird offenbar dominant vererbt. Bei der Verwendung von biguttulus-Silbenmustern akzeptierten die Hybridweibchen eine große Bandbreite von recht kurzen bis zu sehr langen Versdauern, was auf eine intermediäre Vererbung dieses Merkmals deutet. Die Ergebnisse zeigen, dass Hybridweibchen die Hybridmännchen nicht als Paarungspartner wählen, sondern eindeutig C. biguttulus Männchen bevorzugen. Die Gesänge von Hybridmännchen werden demnach von keinem Weibchen akzeptiert, weshalb Hybridmännchen verhaltenssteril sind. Dies stellt eine postzygotische Isolationsbarriere dar, die auch bei einer gelegentlichen Hybridisierung zwischen C. biguttulus und C. brunneus in der Natur erhalten bleibt.

Im dritten Kapitel der vorliegenden Arbeit wurde C. jutlandica, eine erst kürzlich neu- beschriebene Art aus der C. biguttulus Artengruppe aus Jütland in Dänemark untersucht. Der Vergleich der Gesänge von im Labor erzeugten F1 Hybriden (C. biguttulus x C. brunneus) und C. jutlandica-Männchen zeigte, dass es sich bei der Population von C. jutlandica um Hybride zwischen C. biguttulus und C. brunneus handelt. Die Gesänge von C. jutlandica Männchen und F1 Hybriden waren sowohl in den Vers- als auch in den Silbendauern sehr ähnlich. Allerdings tendierten C. jutlandica Männchen dazu regelmäßigere Silben zu bilden als F1-Hybride. Auch in den Gesangspräferenzen von C. jutlandica und der Hybridweibchen zeigten sich Übereinstimmungen, vor allem in der Bevorzugung von strukturierten Gesängen. Chorthippus jutlandica Weibchen antworteten auf Gesänge von C. biguttulus, C. jutlandica und F1 Hybridmännchen, jedoch nie auf Gesänge von C. brunneus. Diese Ergebnisse legen nahe, dass eine isolierte C. biguttulus Population in West-Jütland lokal mit C. brunneus hybridisierte. Da C. jutlandica Weibchen C. brunneus Männchen ablehnen, können C. jutlandica und C. brunneus sympatrisch vorkommen. Sollte C. biguttulus sich nach West-Jütland ausbreiten, so ist zu erwarten, dass es zumindest aufgrund der Gesänge und Gesangspräferenzen keine effektiven Hybridisationsbarrieren zwischen C. biguttulus und C. jutlandica gibt und es somit zur Vermischung beider Formen kommen wird.

Introduction

Hybridization has long been considered as a rare phenomenon. Because hybrids often suffer from sterility, hybridization has been regarded as an evolutionary dead end (Mayr 1963). In plants speciation by hybridization has been mainly documented by the mechanism of polyploidization. In the case of animals, some scientists have neglected hybridization as playing a significant role in evolution. In 2001 Mayr noted that the formation of a new species by non-polyploid hybridization in plants is very rare, and that in animals nothing corresponding has been found (Mayr 2001). But he admitted that introgressive hybridization between species is not seldom, at least in some groups, particularly when their habitat is disturbed by men. In recent studies examples of natural hybridization in plants and animals are increasing, including hybridizing taxa that remain distinct despite gene exchange (Barton and Hewitt 1989; Mallet et al. 2007). Thus there is currently a change of view in science concerning the impact of hybridization as an important evolutionary mechanism that could lead to speciation.

The most accepted speciation concept is the biological species concept. It was Dobzhansky (1937) who formulated it first and Mayr modified it (Mayr 1942) where he defined that species are “groups of actually or potentially interbreeding populations, which are reproductively isolated from other such groups”. Consequently, the evolution of biological barriers to gene flow between members of two different populations leads to reproductive isolation and thus to speciation.

Classically two types of reproductive isolating barriers have been distinguished according to time within the life cycle of organisms (Dobzhansky 1937). Isolation can occur before fertilization (prezygotic barriers) or after fertilization (postzygotic barriers). Furthermore prezygotic isolation can occur either before mating (premating barriers) or after mating (postmating barriers). One type of premating prezygotic isolation occurs when potential mates from populations do not meet, either because they are separated in time (temporal isolation) by e.g. breeding at different times, or gene flow between sympatric species is impeded because species prefer different habitats (habitat isolation). In this case not geographic separation is meant, but for example cases of host-specific insects whose mating and oviposition are restricted to a single plant. The tephritid fliesRhagoletis pomonella inhabits apples and hawthorn, whereas its close relative R. mendax mates and oviposits only on blueberry (Feder 1998). Another type

Introduction

of premating prezygotic isolation occurs when individuals of two populations meet, but they do not mate (behavioural or sexual isolation). This can occur when courtship behaviours differ between individuals of two populations like differing songs or calls in birds, anurans and many insects; different pheromones in moths or light displays at different rates in fireflies (Espmark et al. 2000; Gerhardt and Huber 2002; Greenfield 2002). Postmating prezygotic isolation happens when mating takes place but either gametes are not transferred to the female (mechanical isolation) or when males gametes are actually transferred but eggs are not fertilized (gametic isolation). The gametic isolation can be either noncompetitive, when intrinsic problems with the transfer, storage or fertilization of heterospecific gametes occurs in single fertilizations (Palumbi 1998), or competitive, when the fertilizations problems arise when the heterospecific gametes compete with the conspecific gametes (Gregory and Howard 1993; Howard et al. 1998).

After mating and a successful zygote formation postzygotic isolating mechanisms can operate. They are classified into three types. The first type is hybrid inviability, which means that the hybrid has reduced viability and does not survive long enough to reproduce. Second, hybrid sterility occurs when hybrids are not fertile. The most famous example is the mule, the offspring of a female horse and a male donkey. The third type of postzygotic isolation occurs when at least F1 hybrids are viable and fertile, but the offspring of hybrids are inviable or sterile (hybrid breakdown). In recent years a more detailed view came up, and the postzygotic isolating barriers have been specified. In Coyne and Orrs book on speciation (2004) they further divide the postzygotic barriers into extrinsic and intrinsic mechanisms. Intrinsic barriers are the ones mentioned above. Extrinsic postzygotic barriers depend on the environment, either biotic or abiotic (Coyne and Orr 2004). This can be ecological inviability, where hybrids develop normally but suffer from lower viability because they cannot find an appropriate ecological niche, and behavioural sterility, respectively. Behavioural sterility means that hybrids have normal gametogenesis but are less fertile than parental species because they cannot obtain mates due to their aberrant behaviour. The hybrids may have intermediate phenotypes that make them unattractive to the choosing sex (Coyne and Orr 2004). This sexual selection disadvantage of hybrid offspring is currently getting in focus in studies on hybridizing sympatric species (Stratton and Uetz 1986; Seehausen et al. 1999; Jiggins and Mallet 2000; Naisbit et al. 2001; Turelli et al. 2001; Henry et al. 2002). One prominent example in animals are ducks. It is known that in Anseriformes 42% of all studied species hybridize in nature (Grant and Grant 1992). There is no intrinsic sterility and inviability in many duck hybrids, and it is therefore likely that introgression is limited by postzygotic behavioural isolation based on ecological intermediacy of hybrids or behavioural sterility caused by their intermediate morphology or sexual preferences (Johnsgard 1960; Brodsky and Weatherhead 1984; Mank et al. 2004). Coyne and Orr (2004) even state that such barriers may be as strong as behavioural isolation between

Introduction

the pure species. In acridid grasshoppers it was shown that some sympatric species hybridize and several hybrid populations were recorded in the past (see Table 1). Crosses between many species in laboratory hybridization experiments confirmed that there are no obvious intrinsic postzygotic barriers (Helversen and Helversen 1975a; Helversen and Helversen 1975b; Perdeck 1957; Saldamando et al. 2005b). Hybridization in gomphocerine grasshoppers, at least in some groups, seems to be a quite common phenomenon (see Table 1). Beside many records of hybridization in the Chorthippus biguttulus species group, there are also those on the C. parallelus group (Butlin and Hewitt 1985; Bella et al. 1992; Butlin et al. 1992) and the C. albomarginatus group (Vedenina and Helversen 2003; Vedenina et al. 2007). The mating behaviour of gomphocerine grasshoppers through primarily acoustic communication provides unique conditions to study the mechanisms and effectiveness of hybridization barriers and thus predestines grasshoppers for the investigation of speciation processes.

Acoustic communication in orthopteran insects acts as localization, species recognition and sexual selection mechanisms. Sound production in insects is attained mostly by mechanical action of appendages, although other modes of sound production exist like substrate vibration or by tymbal devices (Greenfield 2002). The predominant mode of orthopteran sound production is stridulation. Gomphocerine grasshoppers produce the sound by rubbing both hind legs against the tegminal veins of the forewings. On the inner side of the hind legs a stridulatory file with pegs is located and the tegminal veins on the wings have raised edges (Elsner and Popov 1978; Gerhardt and Huber 2002; Bailey 2003). By the use of an opto-electronic device it is possible to record the sound and the underlying leg movements synchronously (Helversen and Elsner 1977). In some grasshopper species the two legs move with a typical phase difference that causes the pauses in the temporal pattern of one side to be obscured by the sound produced by the other side. The behavioural significance of this masking has been studied in detail in the grasshopper C. biguttulus (Kriegbaum and Helversen 1992; Helversen and Helversen 1997; Balakrishnan et al. 2001). The songs of males are species-specific. There is an enormous diversity of song patterns and the amplitude modulation can be quite complex (Helversen and Helversen 1994; Ragge and Reynolds 1998). Closely related species, like e.g. the three main species in the C. biguttulus group C. biguttulus, C. brunneus and C. mollis, which are morphologically and genetically very similar have astonishing different songs, which are easily distinguishable. They are used as the main taxonomically criterion to identify species (Faber 1957; Elsner 1974; Helversen and Helversen 1981; Ragge et al. 1990).

Introduction

Table 1. List of hybrid populations, single records and interspecific crossings in the laboratory of different species of the Chorthippus biguttulus species group and references.

Hybridizing species forming local populations

Bailey et al. 2004; Bridle et al. South of Picos de Europe, Northern 2001; Bridle & Butlin 2002; C. brunneus x C. jacobsi Spain Bridle et al. 2002a; Bridle et al. 2002b Schleswig Holstein, Germany Jacobs 1963 Baur et al. 2006 and O. v. C. biguttulus x C. brunneus various localities, Switzerland Helversen (pers. comm.) C. jutlandica, Westjutland, Denmark Nielsen 2003

C. eisentrauti x C. brunneus “Ticino”-brunneus, Tessin, Switzerland Ingrisch 1995

C. mollis ignifer, Alpine mollis, Le Ingrisch 1995; Ragge 1981; Broc grasshopper, several populations Ragge 1984; Ragge 1987; C. biguttulus x C. mollis in the Alps Ragge et al. 1990; Ramme 1923 C. rubratibialis, Apenninne biguttulus Ragge 1987; Ragge et al. 1990 Monte Summano, Italy Fontana et al. 2002 D. Sirin & F. Mayer (pers. C. mollis x C. ilkazi various localities in Turkey comm.) C. brunneus x C. bornhalmi Triest, Italy Kleukers et al. 2004 Hybridizing species with occasional hybrids in sympatric populations

Schönbuch near Tübingen, Germany Faber 1957 Kriegbaum (unpubl. recordings) Erlangen, Germany and O.v. Helversen (pers. C. biguttulus x C. brunneus comm.) various localities, Netherlands Perdeck 1957 Imst, Tirol, Austria Ragge 1976 C. biguttulus x C. miramae Middle Volga, Russia Oliger 1974 Interspeciﬁc crossings in the laboratory

Elsner & Popov 1978; Flache C. biguttulus x C. brunneus 1987; Gottsberger & Mayer 2007; Perdeck 1957

Helversen & Elsner 1975; Helversen & Helversen 1975a; C. biguttulus x C. mollis Helversen & Helversen 1975b; Helversen & Elsner 1977; Sychev 1979 Saldamando et al. 2005a; C. brunneus x C. jacobsi Saldamando et al. 2005b C. biguttulus x C. rubratibialis Schmidt 1987

10 Introduction

The grasshopper songs act as the main premating reproductive isolation barrier between species (Perdeck 1957). Members of the subfamily Gomphocerinae have a bidirectional acoustic communication system. Males produce the songs, and receptive conspecific females answer by replying with songs themselves. The probability of replying is a good predictor of the female’s subsequent acceptance of a male for mating (Perdeck 1957; Kriegbaum 1989; Helversen and Helversen 1994; Klappert and Reinhold 2003). Therefore, the females can easily be tested in playback experiments. The advantages of preference studies with insects are that songs are not learned and the selectivity of the female responses are unaffected by experience. The tested stimuli can be varied artificially in many parameters and can exceed the parameters of natural male songs (Ritchie et al. 1998; Schul 1998; Ritchie 2000; Helversen et al. 2004). Therefore preferences of individual females can be assessed precisely (Wagner 1998).

The focus of this thesis is on the sexual signals that contribute to the maintenance of reproductive isolation of the two grasshopper species Chorthippus biguttulus and C. brunneus. These two species of acridids occur sympatrically over a wide range of Europe and mostly syntopically in the same habitats (Ragge and Reynolds 1998; Ragge et al. 1990). The occasional occurrence of hybrids between the two species (Faber 1957; Perdeck 1957; Ragge 1976; Ingrisch 1995; H. Kriegbaum unpubl. data, O. v. Helversen, pers. comm.), shows that the premating hybridization barrier between this species pair is not complete yet. No obvious intrinsic postzygotic incompatibilities are known since hybrids are viable and fertile (Perdeck 1957). Additionally, mate choice is based almost exclusively on differences in the acoustic signals of males and can be tested by playback experiments (Perdeck 1957; Helversen and Helversen 1994; Butlin et al. 1985; Helversen et al. 2004). Therefore, this species pair is ideal for studying female preferences as they are closely related, morphological and genetically very similar and it is possible to hybridize them in the laboratory.

The sexual signals that I have studied are male song characters and the preferences of females to these signals. I studied these characters in the pure species, in interspecific laboratory hybrids and in a natural hybrid population between these species. Results are presented in three chapters.

More specifically, in the first chapter I tested whether there are certain kinds of intrinsic postzygotic effects. It was possible to produce hybrids and the hybrids showed normal behaviours and were fertile. I studied the phenotype of hybrids and compared them with the parental species. First, I quantified differences between males of the two Chorthippus species, and compared them with the songs of hybrids. I asked whether the hybrid songs could function as a postzygotic extrinsic reproductive barrier in form of behavioural sterility by analysing

11 Introduction

different song parameters and comparing them to the preferences of the females of the parental species. In the second chapter I specified the female preferences for the male signals of the parental species and those of the hybrid females by performing playback experiments. The main aim was to find out whom the hybrid females would accept as mating partners. I measured how preferences were inherited and specified which song characters were important for females. Hence I investigated the effectiveness of the premating barrier between the two species, and what influence the preferences of females could have on gene flow between the pure species and hybrids, respectively.

The third chapter pursues the case of a natural occurring hybrid population between C. biguttulus and C. brunneus in Denmark. I asked what the differences between laboratory hybrids and a natural occurring hybrid population are, and furthermore how such a hybrid population could stay stable despite the possibility to backcross to one parental species.

12 Chapter 1: Male songs

Chapter 1

Behavioral sterility of hybrid males in acoustically communicating grasshoppers (Acrididae, Gomphocerinae) *

1 Abstract

The effectiveness of hybridization barriers determines whether two species remain reproductively isolated when their populations come into contact. We investigated acoustic mating signals and associated leg movements responsible for song creation of hybrids between the grasshopper species Chorthippus biguttulus and C. brunneus to study whether and how songs of male hybrids contribute to reproductive isolation between these sympatrically occurring species. Songs of F1, F2 and backcross hybrids were intermediate between those of both parental species in terms phrase number and duration. In contrast, species-specific syllable structure within phrases was largely lost in hybrids and was produced, if at all, in an irregular and imperfect manner. These divergences in inheritance of different song parameters are likely the result of incompatibility of neuronal networks that control stridulatory leg movements in hybrids. It is highly probable that songs of hybrid males are unattractive to females of either parental species because they are intermediate in terms of phrase duration and lack a clear syllable structure. Males of various hybrid types (F1, F2 and backcrosses) are behaviorally sterile because their songs fail to attract mates.

* published with F. Mayer as co-author in J. Comp. Phys. A (2007) 193: 703-714

13 Chapter 1: Male songs

2 Introduction

One fundamental question in speciation research concerns the evolution of reproductive isolation between populations and maintenance of hybridization barriers. Interspecific reproductive barriers can be classified in prezygotic and postzygotic isolation mechanisms according to the time when they occur during the life cycle (Dobzhansky 1937; Coyne and Orr 2004). Intrinsic postzygotic incompatibilities such as hybrid inviability or sterility have been seen as the classic driving force behind speciation and maintenance of species (reviewed in Coyne and Orr 2004). Hybrids might also be subject to extrinsic postzygotic isolation by being behaviorally sterile. This means that hybrids may be “unfit” because some of their mating traits or preferences are intermediate compared to parental species and therefore they may be unable to find mating partners (Stratton and Uetz 1986; Seehausen et al. 1999; Jiggins and Mallet 2000; Naisbit et al. 2001; Turelli et al. 2001; Henry and Wells 2002).

In order to understand the role behavioral sterility of hybrids plays in evolution and maintenance of reproductive isolation, it is important to learn how sexually selected male signals contribute to behavioral isolation. Acoustically communicating grasshoppers are ideal model organisms for exploration of these questions, because intersexual acoustic communication traits are known to play a decisive role in species recognition, mate localization and sexual selection (Faber 1957; Jacobs 1963; Otte 1974; Kriegbaum 1989; Kriegbaum and Helversen 1992; Helversen and Helversen 1994). In addition, interspecific hybrids can be generated in the laboratory in order to study the inheritance of behavioral traits, their neuronal control and their role as a postzygotic behavioral hybridization barrier. Grasshoppers of the subfamily Gomphocerinae produce elaborate songs by rubbing both hind legs against a tegminal vein of the forewing (Elsner 1974; Helversen and Helversen 1975b; Helversen and Helversen 1994). The complex leg movement patterns, their coordination and hence the emitted songs differ between closely related species (Harz 1975; Ragge and Reynolds 1998).

In this study we investigated two closely related species, Chorthippus biguttulus (Linnaeus, 1758) and C. brunneus (Thunberg, 1815), which occur sympatrically across most of Europe and often even syntopically. These grasshopper species hybridize in nature occasionally (Faber 1957; Jacobs 1963; Ragge 1976; Ingrisch 1995), but despite potential hybridization events, both species remain reproductively isolated and do not form a hybrid zone, although local hybrid populations exist. Hybrids do not suffer from obvious developmental or fertility deficiencies that would indicate strong intrinsic postzygotic barriers (Perdeck 1957; our own observations). It is unknown how Chorthippus biguttulus and C. brunneus remain effectively

14 Chapter 1: Male songs

reproductively isolated, and thus distinct biological species, while other grasshopper taxa with divergent calling songs readily hybridize in the context of secondary contact (Reynolds 1980; Butlin and Hewitt 1985; Stumpner and Helversen 1994; Ingrisch 1995; Bridle and Butlin 2002; Vedenina and Helversen 2003; Kleukers et al. 2004; Saldamando et al. 2005a; Saldamando et al. 2005b; Bridle et al. 2006; Vedenina et al. 2007).

Hybridization studies in grasshoppers have revealed strikingly different results with respect to hybrid songs. Substantial variability in hybrid song parameters and even novel song elements were found in hybrids between Chorthippus biguttulus and C. mollis (Helversen and Helversen 1975a) and in hybrids between C. albomarginatus and C. oschei (Vedenina and Helversen 2003; Vedenina et al. 2007). No such increase in song complexity was found in crosses between C. jacobsi and C. brunneus (Saldamando et al. 2005a). The songs of both reciprocal F1 hybrids differed markedly in some interspecific crosses (Helversen and Helversen 1975a) but not in others (Saldamando et al. 2005a). In the study of the C. albomarginatus x C. oschei hybrids some song parameters were expressed intermediately and some were highly biased to one parental species (Vedenina et al. 2007). In the crosses between Chorthippus biguttulus and C. mollis at least some hybrid males combined song elements of both parental species within a song (Helversen and Helversen 1975a).

To investigate whether behavioral sterility in the form of aberrations in song generation may be responsible for the observed reproductive isolation of the commonly sympatric species Chorthippus biguttulus and C. brunneus, we generated hybrids in the laboratory and studied characters of male calling songs that are likely crucial to pre- and postmating hybridization barriers between the two species. We investigated male calling songs and associated leg movements responsible for song creation of both species and several types of interspecific hybrids. Since female preferences of both parental species are largely known (Helversen and Helversen 1983; Helversen 1984; Charalambous et al. 1994; Helversen and Helversen 1997; Klappert and Reinhold 2003; Helversen et al. 2004), we discuss to what degree hybrids may suffer from behavioral sterility and whether neuronal incompatibilities in hybrids result in behavioral phenotypes that are neither attractive to females of C. biguttulus nor to those of C. brunneus.

15 Chapter 1: Male songs

3 Materials and methods

3. 1. Animals and crossing experiments

For convenience, the first mentioned species in a cross is always the female. Animals were collected as nymphs from various parts in southern Germany (Erlangen and Seewiesen, Bavaria) and Austria (Kühtai, Tyrol) and were raised in the laboratory. Animals were kept in plastic breeding cages (44 x 44 x 44 cm) and were fed with orchard grass (Dactylis glomerata) and annual bluegrass (Poa annua) ad libitum. Light and additional heat was provided for 12 hours each day with a 40 W bulb inside the cage. Cages were monitored daily. At the day of imaginal molt males and females were separated and housed in different cages. Adult grasshoppers were marked individually by a color code on their pronotum and wings with paint markers (Edding 780). To obtain interspecific crosses between the two species we removed the elytrae of the males to mute them and thus to exclude the heterospecific song stimuli. It was already shown by Perdeck (1957) that the removal of the wings had no influence in copulation attempts of males. We followed the crossing method described in Helversen and Helversen (1975a), wherein both sexes were stimulated by conspecific songs while females were placed together with mute heterospecific males. Even with this strong intervention it was difficult to obtain interspecific mating between C. biguttulus and C. brunneus. Six crosses between C. biguttulus and C. brunneus were obtained in 2002 and F1 hybrids were raised in the consecutive year. Three crosses between C. brunneus and C. biguttulus were obtained in 2003 and the offspring were raised in 2004. Only the latter F1 hybrids were crossed to obtain an F2 generation while only C. biguttulus x C. brunneus F1 hybrids were used to produce backcrosses. Egg pods were stored in moist sand in Petri dishes at 6°C over winter, and were raised in the successive year in late spring or early summer. Nymphs were kept separately according to crossings and families and were fed with Poa annua grass. After the imaginal molt, hybrids were bred and marked like the parental species described above.

3. 2. Song recordings

Parental songs of 35 C. biguttulus and 22 C. brunneus males were recorded. We recorded songs of 19 males of the C. biguttulus x C. brunneus cross, 18 males of the C. brunneus x C. biguttulus cross, six males of F2 hybrids, four males of the F1 x C. biguttulus cross, six males from C. brunneus x F1 crossings and one male of the F1 x C. brunneus cross. Data of the last

16 Chapter 1: Male songs

mentioned two crossings were pooled as no obvious differences were found.

Songs of male grasshoppers were recorded in the lab using a 1/2” condenser microphone (G.R.A.S. Type 40AF; frequency response 3 Hz - 25 kHz ±3 dB) equipped with a G.R.A.S. 26AB preamplifier. The songs were amplified by a Brüel & Kjær measuring amplifier (Type 2608). Simultaneously we recorded the movements of both hind legs with an opto-electronic device (Helversen and Elsner 1977). Song and leg signals were digitized using a custom developed three channel AD-converter with 16-bit resolution and 250 kHz (song) / 125 kHz (leg movements) sampling rate. The recordings were analyzed with Turbolab 4.0 (Stemmer software) and custom-designed software developed in LabVIEW 7 (National Instruments). Recording temperature was held constant at 31 ± 2°C. All recordings were of spontaneous calling songs and were obtained from untethered intact animals with two stridulatory hind legs.

3. 3. Terminology of song description and measured parameters

The terminology used to describe songs of grasshoppers is not standardized and sometimes confusing (Elsner 1974; Robinson and Hall 2002). For this reason, we here include a glossary of terms. We follow the nomenclature of Helversen and Helversen (1994), which uses the terms song, phrase, syllables and pulses (shown in Fig. 1.1). We measured the parameters in the oscillograms of digitized sound, unless the movement itself was the matter of measurement (e.g. phase shift of legs).

Pulse: Each partial or uninterrupted upward or downward leg movement produces a pulse. We measured pulse period from beginning of one pulse to beginning of the following pulse in recordings of intact animals. Pulse duration and pulse pause was not measured as the phase shifts between legs usually cause a masking of the pauses in recordings of intact animals. Syllable: Pulses are grouped to syllables. One syllable consists of one full cycle of upward and downward movements of legs (chirp in Elsner 1974; echeme in Ragge and Reynolds 1998). The sound is produced between the moment when the femur leaves the position of rest and the moment when it returns to the resting position again. We measured syllable periods and number of pulses per syllable. Phrase: A series of syllables forms a phrase; (sequence of first order in Elsner 1974; echeme-sequence in Ragge and Reynolds 1998). We measured phrase durations, pauses between phrases and number of syllables per phrases. As first phrases often differ in duration compared to the following ones, we analyzed them separately.

17 Chapter 1: Male songs

Song: A series of phrases separated by pauses. We counted number of phrases comprising a song and measured total song duration from the beginning of first phrase until the end of the last phrase. Phase shift between legs: To make phase shift between legs comparable between species we measured the time interval difference between highest point of second and first leg (D1) and the time interval difference from the upper reversal point of first leg and the next upper point of this same leg (D2) and calculated (360 °/D2) x D1 to assess the phase shift in degrees (°).

In total, we measured nine song parameters for each parental species, for the F1 and F2 hybrids and the backcross progeny. Mean values of measured individuals, one standard deviation and coefficients of variance (CV) for each group (among- individual variation) are shown in Table 1.1. Wilcoxon signed rank tests for related samples were applied to evaluate differences of durations of first phrases to subsequent phrases. To compare parameters between groups, we first applied a Kruskal-Wallis global test. As a post-hoc test, we chose a distribution- free two-sided all-treatments multiple comparisons based on pairwise ranking, the Dwass-Steel- Critchlow-Fligner (DSCF) test (Hollander and Wolfe 1999). The results of the DSCF test are shown in Table 1.2. Burkard Pfeiffer (pers. comm.) programmed the test for R Version 2.4.0 GUI (© R Foundation for Statistical Computing, 2006; http://www.r-project.org). To find out which variables were correlated and which could explain our dataset best, we applied a principal component analysis (PCA) to the nine measured parameters (Bortz 1999). Before calculation, the PCA data were log transformed to meet assumptions of normality. The PCA was performed using SPSS 11.0.4 (© SPSS Inc. 1989-2005).

4 Results

4. 1. Songs of parental species

The songs of Chorthippus biguttulus consisted usually of three (minimum two to maximum five) phrases (Fig. 1.1a). The first phrase was always the longest and lasted on average for 3.06 ± 0.62 s. The mean duration of subsequent phrases was 2.07 ± 0.32 s (Table 1.1). Duration of first phrases differed significantly from the following phrases (Wilcoxon test Z = -5.159; P < 0.001). Chorthippus biguttulus calling songs showed a characteristic syllable structure. The first pulse in each syllable had the highest amplitude generated by an accentuated down stroke of both hind legs (Fig. 1.1c). Each phrase consisted of 19 to 48 syllables (32.5 ± 6.9). Syllables

18 Chapter 1: Male songs

C. biguttulus a

phrase b 2 s b

c c 500 ms

syllable pulse 100 ms d C. brunneus

phrase e 2 s e

f 500 ms f

syllable pulse 100 ms Fig. 1.1. Male songs of Chorthippus biguttulus and C. brunneus. The two upper traces show the hind leg movement patterns. The oscillograms of the emitted sound are shown underneath: a total song, b one phrase and c detail of a phrase showing syllable and pulses of C. biguttulus; d total song, e two phrases and f detail of a phrase showing syllable and pulses of C. brunneus. The black arrow indicates the two-stepped downstroke of leg movement of C. brunneus.

19 Chapter 1: Male songs

usually comprised six pulses (rarely four or more than six pulses) and the mean syllable period was 60.1 ± 7.0 ms. Leg movement pattern of the two hind legs differed slightly from each other. One leg preceded the other by a phase shift of 85.9 ± 15.2 ° (Table 1.1). This slight asynchronous movement of the hind legs masks pauses between pulses. A stridulatory leg can not produce sound when it is in its upper or lower position and hence only intact animals can avoid pauses between pulses.

Table 1.1. Measurements of nine song parameters of C. biguttulus, C. brunneus and their hybrids recorded at 30 ± 2°C. Each value is the mean of all individuals’ mean, one standard deviation (SD) and the coefficient of variance CV( ). Number of individuals (N) is shown in parentheses; big = C. biguttulus, bru = C. brunneus.

Song Phrase Phrase Syllables Pulses Syllable Pulse Phase- Nr of Species/cross duration duration pause per per period period shift phrases (s) (s) (s) phrase syllable (ms) (ms) in ° C. biguttulus 11.84 2.07 2.06 3.2 32.5 6.2 60.1 8.6 85.9 SD (N = 35) 3.70 0.32 0.57 0.9 6.9 0.5 7.0 1.2 15.2 CV 31.2 % 18.0 % 27.6 % 28.1 % 21.2 % 8.2 % 11.6 % 14.0 % 17.7 % big backcross 10.37 0.68 1.58 5.3 5.2 14.3 115.3 9.1 137.3 SD (N = 4) 2.87 0.05 0.40 1.3 1.5 3.1 26.2 0.9 14.2 CV 27.7 % 13.2 % 25.3 % 24.0 % 28.9 % 21.7 % 22.7 % 9.5 % 10.3 % F1 big x bru 8.34 0.45 1.74 4.7 3.4 20.2 193.8 7.9 159.6 SD (N = 19) 3.45 0.06 0.45 1.7 1.8 16.9 156.7 0.4 28.0 CV 41.3 % 12.8 % 25.7 % 34.9 % 53.8 % 84.0 % 80.9 % 5.6 % 17.6 % F1 bru x big 12.54 0.56 1.94 5.9 2.0 32.0 257.7 8.0 165.0 SD (N = 18) 3.37 0.15 0.39 1.6 0.7 10.5 106.8 0.5 24.4 CV 26.9 % 27.1 % 20.2 % 27.3 % 32.1 % 32.8 % 41.4 % 5.9 % 14.8 % F2 10.81 0.48 1.82 5.5 3.8 15.0 155.1 9.6 164.1 SD (N = 6) 3.38 0.19 0.18 1.7 1.6 10.9 118.5 1.0 37.5 CV 31.3 % 39.6 % 10.1 % 29.9 % 41.7 % 72.7 % 76.4 % 10.2 % 22.9 % bru backcross 14.47 0.29 1.68 8.1 5.1 5.3 50.3 10.1 123.6 SD (N = 7) 5.66 0.09 0.25 2.6 1.7 2.9 26.5 1.2 33.3 CV 39.1 % 30.1 % 14.9 % 32.1 % 33.7 % 54.6 % 52.8 % 11.8 % 26.9 % C. brunneus 12.26 0.18 1.66 7.8 5.3 3.1 29.6 8.8 131.9 SD (N = 22) 4.82 0.04 0.54 2.4 1.0 0.1 4.5 1.4 20.6 CV 39.3 % 21.6 % 32.8 % 30.8 % 18.6 % 4.3 % 15.1 % 16.1 % 15.6 %

Chorthippus brunneus songs consisted of 5 to 14 (mean 7.8 ± 2.4) phrases (Figs. 1.1d and 3). The first phrase in a calling song lasted for 0.16 ± 0.04 s, while subsequent phrases were slightly longer (0.18 ± 0.04 s; Wilcoxon test Z= -2.873 P = 0.004). The mean syllable period of C. brunneus was 29.6 ± 4.5 ms (Table 1.1). Syllables of C. brunneus are generated by different leg movement patterns compared to C. biguttulus. Each leg performs a three-step movement, comprising one straight upstroke and a two-step downstroke (marked by an arrow in Fig. 1.1f). A syllable in C. brunneus consists of a first pulse of high amplitude and two subsequent pulses

20 Chapter 1: Male songs

of lower amplitude (Fig. 1.1f). For example the first pulse is generated by the first down-step of one leg (middle trace in Fig. 1.1f), and the second down-step of the contra-lateral leg (upper trace in Fig. 1.1f). Pauses within a syllable result from synchronized turning points of the two hind legs or a stop during the downstroke (Fig. 1.1f). Therefore a clear pulse-pause structure was visible in C. brunneus resulting in a typical syllable structure (Fig. 1.1f). The mean phase shift between the peak positions of the hind legs was 131.9 ± 20.6 ° (Table 1.1).

Songs of the parental species C. biguttulus and C. brunneus differed quite significantly in phrase duration, number of phrases per song, number of syllables per phrase, number of pulses per syllable, syllable period and phase shift between legs (Table 1.2). Song duration and pulse period were not significantly different P( > 0.05; Table 1.2). The duration of pauses between phrases did not differ significantly neither between the two pure species nor between all seven groups of study animals (C. biguttulus, C. brunneus, F1 hybrids C. biguttulus x C. brunneus and reciprocal, F2 hybrids and backcrosses (Kruskal-Wallis test; df = 6; χ2 = 11.68; P = 0.069), therefore, this parameter was excluded from the multiple comparisons.

4. 2. Songs of F1 hybrids

The songs of F1 hybrid males were intermediate between the songs of both parental species with respect to phrase duration and number (Table 1.1; Fig. 1.3). Most comparisons of song parameters between parental species and both reciprocal F1 hybrids yielded highly significant differences (Table 1.2). This was not the case for pulse period and song duration. In addition, C. brunneus and C. brunneus x C. biguttulus F1 hybrids did not differ in number of phrases per song (Table 1.2).

The characteristic syllable structures of both parental species, as described above disappeared almost completely in the songs of both reciprocal F1 hybrids. The leg movement pattern of F1 hybrids consisted mainly of simple up and down oscillations of homogenous amplitude with only few exceptions (Fig. 1.2). The phase shift between both hind legs of F1 hybrids was 159.6 ° and 165.0 °, respectively (Table 1.1) and thus almost an antidromic leg movement pattern (Fig. 1.2). If at all, only the first phrase contained some syllables and was occasionally interrupted by one or two short pauses (Fig. 1.2a). The subsequent phrases often began with a high up and downstroke of both hind legs which resulted in loud pulses. This high amplitude movement, that is production of a syllable, and a stepped downstroke occurred occasionally amidst the phrases in C. biguttulus x C. brunneus F1 hybrids (Fig. 1.2a) but almost never in the reciprocal C. brunneus x C. biguttulus F1 hybrids (Fig. 1.2b). This resulted

21 Chapter 1: Male songs

C. biguttulus x C. brunneus

2 s 100 ms C. brunneus x C. biguttulus b

2 s 100 ms F2 hybrids c

2 s 100 ms c1

C. biguttulus backcross 2 s 100 ms d

2 s 100 ms C. brunneus backcross

2 s 100 ms Fig. 1.2. Male songs of a C. biguttulus x C. brunneus F1 hybrid, b C. brunneus x C. biguttulus F1 hybrid, c and c1 two individuals of F2 hybrids (C. bru/C. big x C. bru/C. big), d C. biguttulus backcross (C. big/C. bru x C. big) and e C. brunneus backcross (C. bru x C. big/C. bru). The two upper traces show the hind leg movement patterns. The oscillograms of emitted sound are shown underneath. At the right side always the third phrase of songs a – e are shown.

22 Chapter 1: Male songs

C. ** ** ** * ** ns ** ns ns ns * ns ns ns ns ns 5.112 8.253 8.389 8.367 4.326 5.152 0.817 2.771 0.810 0.283 4.583 1.297 2.295 3.010 3.230 3.852 Phase shift C. brunneus C. ns ns ns ns ns ns ns ns * * ns ns ns ns ** * 5.110 0.927 3.445 2.922 3.697 3.307 0.215 2.921 1.900 4.769 4.526 1.375 2.667 3.900 3.612 4.793 backcross; ns = not Pulse period ** ** ** ** ** ns ns ** ns ns * * ns ns * ** period 8.925 6.776 8.367 7.728 7.613 3.567 2.660 5.226 0.720 2.357 4.583 4.757 0.803 4.093 4.783 5.392 Syllable C. biguttulus male; bru x big = F1 hybrid F1 = big x bru male; ** ** ** ** ** * ns ** ns ns * * ns * * ** = 0.01 it is 4.882. Abbreviations: big = P 8.951 7.142 8.376 7.769 7.656 4.770 2.278 5.315 0.810 3.771 4.596 4.537 0.574 4.214 4.783 5.392 syllable Pulses per Pulses per C. brunneus C. ** ** ** ** ** ns ** ns ns ns * ns ns * ns ** level (not all 21 group comparisons are shown here for here shown are comparisons group 21 all (not level P backcross; big BC = 8.928 8.521 8.376 5.402 7.589 3.555 5.482 2.853 0.766 4.136 4.587 0.397 2.584 4.258 2.945 5.086 phrase female x female Syllables per Syllables per = 0.05 is 4.170 and for ** * ** ** ns ns * ns ns ns ns ns ns ns ns ns C. brunneus P P Phrase 0.911 8.956 4.818 7.639 5.954 3.858 2.552 4.319 2.974 1.613 0.289 4.121 0.601 0.986 4.075 2.909 C. biguttulus C. number ** ** ** ** ** ns ** ** ns ns * * * ns * ** 8.925 8.518 8.367 7.728 7.613 2.707 5.477 5.226 0.720 1.603 4.583 4.324 4.245 2.528 4.865 5.136 Phrase level; ** = significant at the 0.01 the at significant = ** level; duration P ns ns ns * ns * ns ns ns ns ns ns ns ns ns ns ; big x bru = F1 hybrid F1 = bru x big ; 4.111 0.046 0.890 4.400 0.731 4.598 0.730 0.554 1.800 1.320 0.982 1.297 1.606 1.926 3.066 1.156 male; F2 = F2 hybrid; bru BC = Song duration C. brunneus C. bru big x bru bru x big big x bru bru x big bru x big F2 F2 F2 F2 big BC bru BC big x bru bru x big big x bru bru x big Multiple comparisons between all groups in eight parameters with the Dwass-Steel-Critchlow-Fligner test (see Material (see test Dwass-Steel-Critchlow-Fligner the with parameters eight in groups all between comparisons Multiple C. biguttulus ; bru = bru ; Group comparison Group big big big bru bru big x bru big bru big x bru bru x big big bru big BC big BC bru BC bru BC Table 1.2. Table and Methods). With seven groups the critical value for biguttulus female x 0.05 the at significant = * significant; convenience reasons).

23 Chapter 1: Male songs

in the lowest syllable number per phrase value and longest syllable period in C. brunneus x C. biguttulus F1 hybrids compared to all other groups investigated (Table 1.1; Fig. 1.3). The pulse per syllable variability among F1 hybrid individuals was even up to ten times higher than in C. biguttulus and C. brunneus (Table 1.1; compare CVs: parental species 8.2 % and 4.3 %, F1 hybrids 84.0 % and 32.8 %, respectively). Consequently, also the variability of F1 hybrids in the number of syllables per phrase was two to three times higher than in the parents (Table 1.1). In all other parameters measured, the among-individual variability in F1 hybrids had a magnitude similar to the parental species (Table 1.1).

Like C. biguttulus (but unlike C. brunneus) all F1 hybrids produced moderately longer first phrases than the subsequent phrases. In both reciprocal crosses, durations of firstand subsequent phrases were significantly different C.( biguttulus x C. brunneus first phrases: 0.58 ± 0.21 s; N = 19; second to last phrases see Table 1.1; Wilcoxon test Z = -3.260; P = 0.001 and C. brunneus x C. biguttulus first phrases: 0.65 ± 0.23 s; N = 18; Z = -2.243; P = 0.025).

The C. biguttulus x C. brunneus and reciprocal C. brunneus x C. biguttulus F1 hybrids differed significantly only in song duration and number of pulses per syllable (Table 1.2). All other parameters were not significantly different (Table 1.2). However, although number of phrases per song did not differ significantly between reciprocal hybrids (Table 1.2),C. brunneus x C. biguttulus F1 hybrids had a tendency to sing more phrases per song (4 - 9) than C. biguttulus x C. brunneus F1 hybrids (2 - 7; Fig. 1.3).

4. 3. Songs of F2 hybrids

The songs of F2 hybrids (C. bru/C. big x C. bru/C. big) usually resembled those of F1 hybrids and not those of the parental species (Fig. 1.2c). Except for pulse period (Table 1.2), no significant differences between F1 and F2 hybrids were found among any of the study parameters. F2 hybrids showed high song variability (Fig. 1.2c 1) and thus the comparison of F2 hybrids to parent species revealed a very heterogeneous picture. Differences between C. biguttulus and F2 hybrids were found in phrase duration, number of phrases per song, syllables per phrase and phase shift (Table 1.2). Between C. brunneus and F2 hybrids significant differences were found in phrase duration, pulses per syllable and syllable period. Leg movement patterns were more variable in F2 than in F1 hybrids. Some F2 individuals showed a uniform pattern comparable to many F1 hybrids (Fig. 1.2c), whereas others included some two-step elements in their movements like that of C. brunneus (Fig. 1.2c 1). In addition, phrase duration showed a higher coefficient of variance in F2 than in F1 hybrids (Table 1.1). Interestingly, C. biguttulus x C. brunneus F1 hybrids (but not F2 hybrids) were most variable in six of the eight song parameters measured.

24 Chapter 1: Male songs

25 4

20 3 15 2 10 1

song duration (s) 5 phrase duration (s)

0 0 big BC big bru F2 BC bru big BC big bru F2 BC bru x x x x big bru big bru big bru 12 50 bru big

10 40 8 30 6 20 4 10 number of phrases 2 syllables per phrase 0 0 big BC big bru F2 BC bru big BC big bru F2 BC bru x x x x big bru big bru big bru big bru 0.6 14 0.5 12 10 0.4 8 0.3 6 0.2 4 pulse period (ms) syllable period (s) 0.1 2 0 0 big BC big bru F2 BC bru big BC big bru F2 BC bru x x x x big bru big bru big bru big bru

Fig. 1.3. Boxplots of six song parameters of C. biguttulus (big), C. brunneus (bru), C. biguttulus x C. brunneus (big x bru) and C. brunneus x C. biguttulus (bru x big) F1 hybrids, F2 hybrids (F2), C. biguttulus backcross (BC big; C. big/C. bru x C. big) and C. brunneus backcross (BC bru; C. big/C. bru x C. bru and C. bru x C. big/C. bru). Parameters are song duration, phrase duration, number of phrases per song, syllables per phrase, syllable period and pulse period. The dots in the boxes show the mean and the whiskers on the bottom extend from the 10th percentile and top 90th percentile.

25 Chapter 1: Male songs

4. 4. Songs of backcrosses

The C. brunneus backcross hybrids (C. big/C. bru x C. bru and C. bru x C. big/C. bru) showed several similarities to their backcross parent (Table 1.1; Fig. 1.2e). They produced many brunneus-like two-step elements in their leg movements (Fig. 1.2e). Three song parameters differed significantly between the songs of C. brunneus backcross hybrids and C. brunneus, namely, phrase duration, pulses per syllable and syllable period (Table 1.2), whereas five parameters differed significantly between F1 hybrids andC. brunneus backcrosses (Table 1.2).

In contrast, C. biguttulus backcross hybrids (C. big/C. bru x C. big) resembled in most song parameters F1 and F2 hybrids and not C. biguttulus (Fig. 1.2d). The same was true for the leg movement patterns. Only three parameters were significantly different between all F1 hybrids to C. biguttulus backcrosses, namely phrase duration, syllables per phrase and pulses per syllable (all just P = 0.05; Table 1.2). A significant difference was found between the songs of C. biguttulus backcross hybrids and C. biguttulus in phrase duration, syllables per phrases, pulses per syllable, syllable period and phase shift (Table 1.2). Variability among individuals in backcross progeny was mostly lower than variability among individuals in F1 and F2 hybrids (Table 1.1).

Table 1.3. Factors loading from the two first principal component from a PCA with nine song parameters of C. biguttulus, C. brunneus and their F1 and F2 hybrids and backcrosses (total N = 111). Rotation method is varimax with Kaiser normalization. Before analyses data were log 10 transformed to meet assumptions of a normal distribution. Asterisks (*) indicate loadings were the correlation coefficient R > 0.700.

Song parameters PC 1 PC 2 Phrase duration 0.893* 0.208 Number of phrases -0.842* -0.322 Syllables per phrase 0.837* -0.478 Phase shift -0.756* 0.455 Phrase pause 0.435 0.074 Pulses per syllable -0.100 0.926* Syllable period -0.032 0.918* Pulse period -0.059 -0.391 Song duration -0.158 -0.417

Explained variance (eigenvalue) 3.276 2.354 Proportion of total variance (%) 36.41 26.16

26 Chapter 1: Male songs

C. biguttulus big backcross F1 (big x bru) 2 F1 (bru x big) F2 bru backcross C. brunneus 1

PC 2: 26.16 % -1

-2

-3 -2 -1,5 -1 -0,5 0 0,5 1 1,5 2 PC 1: 36.41 % Fig. 1.4. The first two principal component axes of nine song parameters ofC. biguttulus, C. brunneus, F1, F2 hybrids and backcross progeny. The song parameters with the highest loadings on PC1 were phrase duration, phase shift, number of phrases and syllables per phrase. The song parameters with the highest loadings on PC2 were pulses per syllable and syllable period (compare Table 1.3).

4. 5. Principal component analysis of song parameters

A multivariate analysis was performed to quantify the variations in songs among both parental species and four classes of hybrids. In Table 1.3 we show the first two principal components (PC) axes for nine song parameters. The first factor (PC1) of the principal component analysis was composed of the parameters phrase duration, phase shift, number of phrases and syllables per phrase. These parameters contributed 36.41 % to PC1. The second factor (PC2) was composed of the parameters pulses per syllable and syllable period. These parameters contributed 26.16 % to PC2 (Table 1.3). Therefore, PC1 comprised the gross temporal parameters concerning phrases and number of phrases, with the exception of the phase shift, which is relevant for the fine structure of songs. The second factor (PC2) included fine temporal structures of songs.

27 Chapter 1: Male songs

F1 and F2 hybrids were nearly intermediate between parental species at PC1, showing a bias towards C. brunneus (Fig. 1.4). Low variation was found at PC2 for parental species, whereas hybrids showed high variability here and not intermediacy. Nevertheless, the backcrosses to C. biguttulus fell into the cluster of the F1 and F2 hybrids, whereas the backcrosses to C. brunneus shifted noticeably towards its parental species (Fig. 1.4).

5 Discussion

5. 1. Behavioral sterility of male hybrids

The calling songs of all hybrid males (F1, F2, and both reciprocal backcross hybrids) were intermediate between the calling songs of males of the two parental species Chorthippus biguttulus and C. brunneus in terms of phrase number and duration, although there was a shift in duration towards C. brunneus (Table 1.1; Fig. 1.3). The average phrase duration of hybrid calling songs was about 500 ms, that is twice to three times as long as in C. brunneus. There was no overlap in phrase duration between range between hybrids and C. brunneus, except for some individual C. brunneus backcross hybrids. Female choice experiments have shown that C. brunneus females are highly sensitive to phrase duration (Weih 1951; Ellegast 1984; Helversen and Helversen 1994). They respond only to songs with phrases that last between 50 and 300 ms and prefer songs with phrases between 110 and 130 ms. Therefore, phrase duration alone seems to be sufficient for females of C. brunneus to discriminate against hybrid males and to prevent acceptance of hybrid males by C. brunneus females. Song discrimination against hybrids is even more probable if females combine phrase duration with other song parameters such as phrase number or pulse period (Butlin et al. 1985).

Phrase durations of hybrid and C. biguttulus male calling songs also did not overlap but this parameter alone is unlikely to prevent backcrosses between hybrid males and females of C. biguttulus. Female choice experiments in C. biguttulus showed that females respond to phrase durations that are typically found in hybrid males (Helversen 1972; Helversen and Helversen 1994), although conspecific males do not produce such short phrases. Therefore, phrase duration alone would not prevent mating between hybrid males and females of C. biguttulus.

28 Chapter 1: Male songs

Female choice experiments have also shown that females of C. biguttulus primarily recognize conspecific songs according to a species-specific syllable structure. Females respond only to male songs that show a stereotypic and characteristic syllable structure throughout all phrases (Helversen 1972; Helversen and Helversen 1997; Balakrishnan et al. 2001). This sensitivity of females to regular syllables and the lack of such syllables in all male hybrid songs leads to the prediction that hybrid songs are highly unattractive to females of C. biguttulus. Many hybrid songs consisted of phrases that showed no fine temporal structure resembling the characteristic syllable structures in parental species. Only occasionally did hybrid males produce songs having syllables that were highly variable in duration (Figs. 1.2 and 1.3). Therefore, the lack of regular syllables likely represents an effective mating barrier between C. biguttulus females and hybrid males of various hybridization degrees. This makes backcrosses of hybrid males with C. biguttulus females unlikely.

These findings strongly suggest that backcrosses of hybrid males to females ofC. brunneus are primarily prevented by phrase duration while the missing syllable structure prevents backcrosses to females of C. biguttulus. This unattractiveness of hybrid males (including F1, F2 hybrids and first-generation-backcrosses) to females of both parental species likely plays an important role in reproductive isolation between C. biguttulus and C. brunneus, which occur in sympatry across most of their species’ distribution ranges (Ragge et al. 1990). The behavioral disadvantage of hybrid males is an essential but not a sufficient requirement to prevent gene flow and to maintain reproductive isolation between the two species. The viability ofour hybrids provided no indication of postzygotic handicaps, because hybrid males and females did not suffer from obvious developmental deficiencies or reduced fecundity (Perdeck 1957 and this study). Therefore, in order to understand the rare occurrence of natural hybrids and thus the effectiveness of the hybridization barrier between C. biguttulus and C. brunneus, it will be necessary to study the preferences of hybrid females.

5. 2. Neuronal control of stridulation

Hybrids between C. biguttulus and C. brunneus mostly displayed simple patterns of leg movements consisting of many straight up- and down oscillations and a phase shift of nearly 180° (antidromic leg movements). The leg movement pattern is similar to the leg movement pattern of C. mollis males during the production of vibratory syllables (the so- called “Schwirrlaut”-elements) of the calling song (Elsner 1974; Elsner and Popov 1978). The only obvious difference is a much smaller phase shift in C. mollis than in the C. biguttulus x

29 Chapter 1: Male songs

C. brunneus hybrids. The straight up and down oscillations may represent a hypothetical archetype of a grasshopper song consisting of a temporally structured array of pulses. This simple pattern of leg movements may have originally evolved from wing movements (Elsner and Popov 1978), because leg movements as well as wing movements are to a great extent controlled by the same “bifunctional muscles”. It was also shown in C. biguttulus that the frequency of stridulatory leg movements is similar to the flight frequency (Elsner and Popov 1978).

Stridulatory leg movement pattern is generated by thoracic neuronal networks (Elsner 1975; Elsner and Popov 1978; Hedwig 1992; Hedwig and Heinrich 1997; Heinrich and Elsner 1997). Consequently, the strikingly different movement patterns of C. biguttulus and C. brunneus must be ascribed to differences in the thoracic neuronal stridulatory network. The loss of structural elements within a phrase giving rise to a simple leg movement pattern as seen in hybrids suggests that the two derived neuronal networks of C. biguttulus and C. brunneus may be incompatible in hybrids. Hybrids only sporadically produced leg movement elements characteristic of C. biguttulus and C. brunneus and these were never as clear as in the pure species. Hemisection of the metathoracic ganglion complex (Ronacher 1989, 1991) and intracellular recordings and staining of interneurons within the metathoracic ganglion complex (Hedwig 1992; Ocker and Hedwig 1996) revealed that the stridulation of grasshoppers is controlled by hemisegmental pattern generator subunits. The split of the metathoracic ganglion in C. biguttulus led to a disturbance of the rhythm of pauses leading to a much higher variation of syllable durations with much longer syllables than in intact individuals (Ronacher 1989). These results show that a precise neuronal coordination in the metathoracic ganglion is required to produce the complex leg movements as observed in males of C. biguttulus and C. brunneus. Therefore an incomplete or disturbed development of neuronal network connections between the hemisegmental pattern generator subunits is a likely reason for rare and irregular syllables in songs of hybrids.

In contrast to syllable structure, phrase duration was intermediate in hybrids. The on- and offset of stridulation, and therefore the duration of phrases, are controlled by the central neurons in the brain and descending commando neurons, which switch on and off the metathoracic pattern generators (Elsner and Huber 1969; Hedwig and Heinrich 1997; Heinrich et al. 2001). The balanced neuronal control of intermediate phrase durations in hybrids suggests that no neuronal incompatibilities exist in these probably homologous central nervous elements of C. biguttulus and C. brunneus.

30 Chapter 1: Male songs

A similar study of song analysis of Chorthippus. biguttulus and C. mollis hybrids (Helversen and Helversen 1975a) yielded a completely different result than that found here between C. biguttulus and C. brunneus. Songs of many F1 hybrid males were generated by even more complex leg movement patterns than observed in both parental species. Some parameters had an intermediate form and others were more or less superimposed (Helversen and Helversen 1975a). Songs of C. mollis males consist of an array of many “Schwirrlaute” that are separated by pauses. As mentioned above, the “Schwirrlaut” is generated by homogenous straight up and down oscillations. Many male hybrids between C. biguttulus and C. mollis produced “Schwirrlaute” that showed the syllable structure of C. biguttulus. Therefore, structural song elements of both species were combined by the hybrids without conflict. So, in this case, the effect was additive and resulted in songs of high complexity by these hybrids that exceeded those of both parental species. The observation that during one song one leg performed the C. biguttulus pattern whereas the other leg performed the C. mollis pattern simultaneously lead to the hypothesis that two parallel and, to some extent, independent pattern generating neuronal circuits were developed (Helversen and Helversen 1975a; Helversen and Elsner 1977). Their outputs converge in a common final pathway, probably at the motoneuron level, and lead to the superimposed pattern of the hybrid song.

These two examples of interspecific hybrids illustrate how differently neuronal circuits control signal structure. In one case, C. biguttulus x C. brunneus, derived neuronal networks are lost in hybrids, resulting in a simple leg movement pattern. In another case, C. biguttulus x C. mollis, partly distinct networks are combined and complex patterns are generated by superimposition. Apart from providing important insights into the effectiveness of hybridization barriers and, hence, reproductive isolation, these studies show that hybridization experiments may lead to a better understanding of the neural control of stridulation

31 Chapter 2: Female preferences

Chapter 2

Dominant expression of song preferences in F1 hybrid females contribute to sexual isolation between two sympatric grasshopper species

1 Abstract

In this chapter I question why two sympatric grasshopper species, Chorthippus biguttulus and C. brunneus (Gomphocerinae) remain distinct species, despite occasional hybridisation between them. Therefore I performed hybridisation experiments and accessed female preferences for song parameters of the pure species and the interspecific F1 hybrids. Females of gomphocerine grasshopper respond to species-specific male calling songs with reply songs. I conducted playback experiments with virgin females using model songs. Two parameters, namely phrase duration and syllable pattern were varied ranging from biguttulus to brunneus- like songs.

For C. biguttulus females the syllable pattern of songs is the crucial character, whereas for C. brunneus females the syllable pattern is of minor importance but phrase duration is the most important character. In F1 hybrid females I found an interesting twofold inheritance. The F1 females accepted a range of phrase durations from brunneus phrase durations up to the longest phrases tested. The preference for phrase duration may be inherited intermediate. But in preferences for syllable pattern F1 hybrid females clearly behaved like C. biguttulus females. Hence I assume that the preference for syllable patterns is inherited dominantly. Consequently, as calling songs of hybrid males between C. biguttulus and C. brunneus lack a correct syllable structure, females will not choose these males as mating partners. If hybrids between the two species occur in nature females will rather cross back to biguttulus, than to hybrids or brunneus males. Thus the dominant expression of the syllable pattern strengthens the isolating barrier between the two sympatric species and helps to maintain species boundaries despite introgression.

32 Chapter 2: Female preferences

2 Introduction

There is growing evidence for interspecific hybridization between sympatric species especially within radiating lineages (Grant et al. 2005; Mallet 2005; Mallet 2007). This raises the question how species boundaries are maintained despite hybridization. Crucial factors are frequency and fitness of hybrids that both influence the extent of gene flow between hybridizing taxa. The fitness of hybrids can be reduced by (i) genetic incompatibilities that cause sterility or inviability, (ii) hybrid inviability due to environmental effects or (iii) sexual selection against hybrids. The latter case, which has also been named behavioural sterility of hybrids, has recently gained in interest. Although evidence accumulates that speciation by sexual selection may be particularly fast in several groups of animals the discrimination of females against hybrids was studied only in a small number of species (Shaw 2000; Kirkpatrick and Ravigné 2002; Haesler and Seehausen 2005).

Two generally distinct types of behavioural sterility are known. First, intrinsic behavioural hybrid sterility occurs, when hybrids have behavioural anomalies like deficiencies that prohibit courting or mating (Coyne 1989; Wu and Hollocher 1998; Coyne and Orr 2004). The other is extrinsic behavioural hybrid sterility, which is mainly caused by the fact that hybrids have intermediate behaviour, which is not attractive to the choosing sex (Servedio and Noor 2003). For example, Stratton and Uetz (1986) showed that both sexes of F1 hybrids between two wolf spider species were completely sterile through their intermediate behaviour. Another study showed that hybrids mate readily with each other, but not with parentals (Naisbit et al. 2001). Most studies showing behavioural sterility of hybrids also show that ecological factors (environmental cues or predation) contribute to the extent hybrids suffer from disadvantages compared to parental species (Vamosi and Schluter 1999; Naisbit et al. 2001; Höbel and Gerhardt 2003; Nosil et al. 2007).

To assess female preferences of interspecific hybrids between sympatric well separated species provides the possibility to study if and to what extent males are behaviourally sterile, to learn how preferences are inherited, and to find out which behavioural traits are important in maintaining species integrity (Beukeboom and van den Assem 2002; Rodríguez et al. 2006).

Acoustic experiments with insects communicating by sound allow a detailed analysis and multiple testing of female preferences (Ritchie et al. 1998; Schul 1998; Ritchie 2000; Helversen et al. 2004). Preferences of females for acoustic signal characteristics not represented in natural populations can be studied performing playback experiments with synthetic stimuli.

33 Chapter 2: Female preferences

This allows to assess, which components of a signal are of crucial importance for females to choose (Wagner 1998).

Grasshoppers of the subfamily Gomphocerinae have a bidirectional acoustic communication system. Males produce calling songs, and receptive conspecific females answer by producing response songs. The probability of replying is a good predictor of the female’s subsequent acceptance of a male for mating (Perdeck 1957; Helversen and Helversen 1994; Klappert and Reinhold 2003). I studied two gomphocerine grasshopper species Chorthippus biguttulus and C. brunneus. They occur sympatrically over a wide range of Europe and mostly syntopically in the same habitats (Ragge et al. 1990; Ragge and Reynolds 1998). Occasionally hybrids between the two species occur (Faber 1957; Perdeck 1957; Ragge 1976; Ingrisch 1995; H. Kriegbaum unpublished data, O. v. Helversen, pers. comm.), showing that the premating hybridization barrier is not complete. No obvious intrinsic postzygotic incompatibilities are known since hybrids are viable and fertile (Perdeck 1957). Additionally, mate choice is based almost exclusively on differences in the acoustic signals of males and can be tested by playback experiments (Perdeck 1957; Butlin et al. 1985; Helversen and Helversen 1994; Helversen et al. 2004). Therefore, this species pair is ideal for studying female preferences as they are closely related, morphological and genetically very similar and it is possible to hybridize them in the laboratory.

I wanted to find out why despite occasional hybridization the two species remain distinct. Thus I first assessed female preference for the two parental speciesC. biguttulus and C. brunneus. I investigated the preference functions by changing the phrase duration and syllable to pause ratio in small steps and tested females with computer controlled artificial stimuli. This approach allowed to estimate the degree of reproductive isolation between the species and moreover to find out, which parameters of male songs are crucial to females. Secondly, I examined the preferences of hybrid females. Finally, I measured the phrase duration and latency of female response songs and discuss how signals and preferences of females are inherited.

34 Chapter 2: Female preferences

3 Material and Methods

3.1. Study animals

I collected third and fourth instar larvae of C. biguttulus and C. brunneus in southern Germany (Erlangen and Seewiesen, Bavaria) and Austria (Kühtai, Tyrol). Animals were kept in plastic breeding cages (44 x 44 x 44 cm) and were fed with orchard grass (Dactylis glomerata) and annual bluegrass (Poa annua) ad libitum. Light and heat was provided for 12 hours each day with a 40 W bulb inside the cage. Cages were monitored daily. After imaginal moult both sexes were separated and housed in different cages. Animals were marked individually on their pronotum and/or wings with paint markers (Edding 780). Male songs were recorded in the laboratory at 30 ± 2 °C with a 1/2” G.R.A.S. microphone (Type 40 AC). Signals were amplified using a Brüel & Kjær amplifier (Type 2608) and digitized with a custom built PCI-DSP D/A converter (16bit/250 kHz).

3.2. Interspecific crossing experiments

Crossing experiments were done in small gauze cages (8 x 7 x 6 cm) under a 60 W bulb to obtain a temperature between 35 °C and 40 °C. I only used motivated singing males and virgin females, which were responding to conspecific male songs in the breeding cages prior to experiments. First, I placed one female with one heterospecific male in a gauze cage and waited a minimum of 3 h up to several days. No copulation was observed in a total of 59 experiments with one C. biguttulus female and one C. brunneus male and in total 36 experiments in the reciprocal situation. In a second series of experiments I muted males by cutting off the fore-and back wings. Males were still able to perform leg movements but no sound was emitted. The abscission of the wings did not influence the courtship behaviour of males. Before and during crossing experiments females were stimulated by conspecific calling songs from a recorder or from singing males, which were placed around the females’ gauze cage. Six copulations were obtained in a total of 186 experiments with a C. biguttulus female and a mute C. brunneus male. Three reciprocal crosses occurred in a total of 143 experiments with a C. brunneus female and a mute C. biguttulus male. After copulation females were kept isolated and egg pods were

35 Chapter 2: Female preferences

collected until the female’s death. Egg pods were embedded in moist sand in Petri dishes for about two months at room temperature and thereafter at 6 °C for at least six months for diapause. Hatching was initiated by incubating the egg pods at room temperature. In hybrids the first mentioned species is always the mother, and the second mentioned species is the father species.

3.3. Female preference tests

All female preference tests were performed using virgin females at an age of at least six days after final moult. Females start to react to the species-specific male calling five to six days after imaginal moult (Kriegbaum and Helversen 1992). The behavioural tests were performed in a sound-attenuated thermostatic chamber with a constant temperature of 30 ± 1 °C. A female was kept 15 cm from the loudspeaker in small gauze cage and was provided with some blades of grass and moist sand as egg-laying substrate. A computer-controlled set-up played-back synthetic sound stimuli and registered female reply songs automatically. Thus, there was no disturbance and no observer bias during the tests. Analog white noise was amplitude-modulated by an IBM compatible computer and played-back with custom-built power amplifiers and emitted by tweeter loudspeaker with a flat response from 2 to 40 kHz (Dynaudio D21/2, Skanderborg Denmark). Signal amplitude was calibrated to a constant intensity level of 70 ± 2 dB SPL (peak) with a Brüel & Kjær sound level meter (Type 2231 and a Brüel & Kjær ½ inch condenser microphone 4133) at the position of the animal. A condenser microphone (Type MCE-101; 50 to 12000 Hz) registered the female response inside the chamber (Helversen 1979; Helversen and Helversen 1983).

Experiments were performed as described in (Helversen and Helversen 1983). I used song models that consisted of square modulated white noise. Two song parameters were altered to simulate a transition from C. biguttulus to C. brunneus male calling songs. I tested five different combinations of syllable and pause duration: a typical C. biguttulus song pattern with 80 ms long syllables separated by 12 ms pauses (song model 1), a C. brunneus pattern that consisted of 10 ms syllables with 4 ms pauses (song model 4), two intermediate syllable/pause durations of 40/8 ms (song model 3) and 20/6 ms (song model 4), respectively and one pattern without any pauses (song model 5) which served as a control. The second altered parameter was phrase duration. The five song models were played back with eleven different phrase durations (90 ms, 130 ms, 180 ms, 250 ms, 350 ms, 500 ms, 700 ms, 1000 ms, 1400 ms, 2000 ms and 2800 ms). Thus a total of 55 different song models

36 Chapter 2: Female preferences

were presented to females. Each sound stimulus consisted of three identical phrases. Pauses between phrases were set to 6 s, when females did not reply, and 2 s when females answered with a response song. The order of the stimuli was pseudo-randomized. Between playbacks of two stimuli a pause of one minute was included. If no response song was registered within one session (the playback of all 55 sound stimuli), presentation was stopped for 30 minutes. When females responded, the program immediately continued with a next session. Each female was tested on average with 21 sessions (N = 69; SD = 6.2).

Female preference was measured for each song stimuli as the proportion of tests to which the female produced a response song. Unmotivated or highly motivated and hence possibly unselective females were excluded from further analysis if the maximum response probability to any song stimuli was below 15 % or if they responded to a control song stimuli (song model 5 tests) in more than 15 % of all sessions. An exception was made for C. brunneus females. Even highly selective females responded to short song stimuli of the control group (lacking pauses within phrases). Therefore, only females with a response rate higher than 15 % to control song models longer than 500 ms duration were excluded. To control for differences in motivation levels among days, females and species, I normalized each response profile by setting the maximum response rate of a female to 100%. Preference indices were calculated by dividing the response probabilities by 100. I analyzed in total 27 C. biguttulus, 13 C. brunneus, 17 C. biguttulus x C. brunneus and 12 C. brunneus x C. biguttulus females.

3.4. Female signals and response latencies

The latency (i.e. the time between the end of the song stimulus and the beginning of the response song) and duration of the female’s response songs were registered automatically. The number of measured phrases differed between individuals (from 60 up to 5000 per female). Thus I calculated a weighted mean per female. Phrase durations and response latencies were measured in 25 C. biguttulus, 13 C. brunneus, 16 C. biguttulus x C. brunneus and 12 C. brunneus x C. biguttulus females (Fig. 2.4). Statistical tests (Mann- Whitney U test, Kruskal- Wallis test and ANOVA) were performed using SPSS 11.0.4 (© SPSS Inc. 1989-2005).

37 Chapter 2: Female preferences

4 Results

4.1. Preference of parental species

Females of Chorthippus biguttulus and C. brunneus showed high response probabilities to song stimuli that resemble conspecific song parameters (Fig. 2.1). Median response probabilities of C. biguttulus females to songs with a syllable to pause ratio of 80 to 12 ms were above 70% if phrases had a duration of at least one second and remained high until the longest phrase duration tested, lasting 2800 ms. This range covers the phrase duration of natural male calling songs (Fig. 2.1). In contrast to C. biguttulus, males of C. brunneus produce short phrases with a median duration of 212 ms (Fig. 2.1) and phrase consists of pulses and pauses of approximately 10 ms and 4 ms (Gottsberger and Mayer 2007). Females of C. brunneus tested with songs with a pulse to pause ratio of 10 to 4 ms responded frequently to phrase durations between 90 and 350 ms and preferred phrases of 130 ms (Fig. 2.1b). Surprisingly, this peak in phrase duration is below the 95 % confidence interval of natural male songs (Fig. 2.1; median = 212 ms; lower 95% CI 212 ms). The two species differed significantly in their response probability for all phrase durations tested, if females were tested with conspecific syllable to pause ratios (Table 2.1).

I also tested females with intermediate song stimuli by varying phrase duration as well as the syllable to pause ratio within a phrase (Fig. 2.2). Females of both species were not only sensitive to the phrase duration but also to syllable patterns within phrases. Response probabilities of C. biguttulus females were highest for a 80 to 12 ms ratio of syllables and pause (song models 1), decreased at a 40 to 8 ms ratio (song models 2), and there was almost no reaction at all to the shorter syllable to pause ratios (Fig. 2.2). Song models with a brunneus-like syllable pattern (song models 4) evoked no response of C. biguttulus females. While females of C. biguttulus were sensitive to the syllable to pause ratio, females of C. brunneus were particularly sensitive to the phrase duration of a signal. In all four groups of song models females responded to songs stimuli with phrase durations between 90 ms and 350 ms. Nevertheless, females of C. brunneus were also sensitive to the syllable to pause ratio, as response probabilities decreased continuously from a brunneus-like to a biguttulus-like syllable to pause ratio (Fig. 2.2). In summary, response probabilities of both species were highest for song models, which resembled conspecific male songs. Heterospecific and intermediate song models provoked minor or no responses at all.

38 Chapter 2: Female preferences A 2800 2800 2800 2600 2600 2600 2400 2400 2400 2200 2200 2200 SONGMODEL SONGMODEL 2000 2000 2000 1800 1800 1800 1600 1600 1600 1400 0HRASEDURATION 1400 1400 0HRASEDURATION

1200 1200 1200 1000 1000 1000 800 800 800 600 600 600

400 400 400 200 200 200 0 0 0

0 0

80 60 40 20 80 60 40 20 2ESPONSEPROBABILITY 2ESPONSEPROBABILITY 100 100 A B B

Fig. 2.1. Boxplots of response probabilities of a C. biguttulus females (N = 27) to song model 1 and b C. brunneus females (N = 13) to song model 4, respectively. The illustrations of the respective song models are shown at phrase duration of 300 ms. Boxplots a1 and b1 represent phrase duration of calling songs of C. biguttulus males (a1; mean = 2.17 s; SD = 0.435) and C. brunneus males (b1; mean = 0.22 s; SD = 0.05).

39 Chapter 2: Female preferences

SONGMODEL 0REFERENCEINDEX

0REFERENCEINDEX SONGMODEL

SONGMODEL

0REFERENCEINDEX

SONGMODEL

0REFERENCEINDEX

0HRASEDURATION C. biguttulus C. brunneus F1 hybrid big x bru F1 hybrid bru x big Fig. 2.2. Female preference of C. biguttulus (N = 27), C. brunneus (N = 13), C. biguttulus x C. brunneus (N = 17) and C. brunneus x C. biguttulus (N = 12) F1 hybrid females to song models 1 to 4. Song models varied in syllable to pause durations (model 1: 80/12 ms; model 2: 40/8 ms; model 3: 20/6 ms; model 4: 10/4 ms). Mean preference + SE are shown. The illustrations of the respective song models are shown at phrase duration of 300 ms.

40 Chapter 2: Female preferences

4.2. Song preferences of F1 hybrid females

F1 hybrid females of both reciprocal crosses showed high response probabilities to the same song stimuli, which were also favoured by C. biguttulus females (Fig. 2.2). Response probabilities increased towards longer phrase durations starting at about 350 ms. Hybrid females preferred a syllable to pause ratio of 80 to 12 ms, which is characteristic for songs of C. biguttulus males (song model 1). The shortening of syllables and pauses (song model 2) reduced response probabilities of F1 hybrid females, but their response rate to model 2 was on average slightly higher than the response rate of females of C. biguttulus. Response probabilities to song model 3 (syllable to pause ratio of 20 to 6 ms) were low, reaching only a maximum of 35 %. Stimuli of brunneus-like songs (song model 4) evoked low to zero response rates in F1 hybrid females (Fig. 2.2).

4.3. Female signals and response latencies

Phrase duration of female response songs differed significantly between C. biguttulus,

C. brunneus and both reciprocal F1 hybrids (ANOVA F3, 62 = 54.9, P < 0.001; Fig. 2.3a). Females of C. biguttulus had the longest, C. brunneus the shortest and F1 hybrids intermediate phrase durations (Fig. 2.3a). Post hoc Tukey tests showed no significant difference of means between C. biguttulus x C. brunneus and C. brunneus x C. biguttulus F1 hybrids (P = 0.99), but F1 hybrids differed significantly from both parental species. I found no significant differences in response latencies (the duration between the end of a song stimulus and the beginning of a response song) between both parental species and the two reciprocal F1 hybrids (ANOVA

F3, 62 = 1.4, P = 0.24; Fig. 2.3b). Thus the duration of female response songs of F1 hybrids were intermediate between those of both parental species whereas the latency in response, i.e. the readiness to answer did not differ among the parental species and hybrids.

41 Chapter 2: Female preferences

1000 A 2000 B a a a

800 1600 a a

600 1200 b b

400 Response latency (ms) 800 c Phrase duration (ms)

200 400

0 0 BIG BIGXBRU BRUXBIG BRU BIG BIGXBRU BRUXBIG BRU Fig. 2.3. Female response song characteristics. Comparisons of a phrase duration (mean ± SE) and b latencies (mean ± SE) between C. biguttulus, F1 reciprocal hybrids and C. brunneus females. Significant differences at P < 0.05 in Tukey multiple comparison tests are indicated by different letters; big = C. biguttulus (N = 25); bru = C. brunneus (N = 13); big x bru = C. biguttulus x C. brunneus F1 hybrids (N = 16); bru x big = C. brunneus x C. biguttulus F1 hybrids (N = 12).

Table 2.1. Results of Mann- Whitney U- tests comparing female preference of C. biguttulus for song model 1 (N = 27) and of C. brunneus females for song model 4 (N = 13) in the 11 tested phrase durations.

Phrase duration U Z (2-tailed) P (ms) 90 17.0 -4.950 < 0.001 130 21.0 -4.960 < 0.001 180 43 -4.191 < 0.001 250 106 -2.088 0.037 350 108.5 -1.977 0.048 500 78.5 -2.864 0.004 700 8.5 -4.889 < 0.001 1000 8.0 -4.890 < 0.001 1400 0 -5.109 < 0.001 2000 0 -5.094 < 0.001 2800 0 -5.193 < 0.001

42 Chapter 2: Female preferences

5 Discussion

5.1. Expression of female preferences

The investigation of two parameters of song preference in F1 hybrid females from crosses between the two grasshopper species C. biguttulus and C. brunneus revealed two modes of expressions: dominant and intermediate.

Preferences of F1 hybrid females of both reciprocal crosses between Chorthippus biguttulus and C. brunneus for syllable structures within a phrase were not intermediate between those of both parental species. Instead, they resembled the female preferences of one parental species (C. biguttulus), but not of the other (C. brunneus). Chorthippus biguttulus as well as F1 hybrid females had highest response rates for the biguttulus-like syllable pattern. Response rates of hybrids decreased to almost zero towards songs with short phrases, which is characteristic for songs of C. brunneus males. These results are in line with studies on C. biguttulus. Females respond best to syllables longer than 30 ms in durations and not longer than 100 ms (Helversen and Helversen 1983; Helversen and Helversen 1997). If syllables (and phrases) get shorter or if short pauses are included response probabilities drop dramatically (Helversen and Helversen 1997; Balakrishnan et al. 2001; Helversen et al. 2004).

Despite a number of studies on song preferences of hybrid females with different parental taxa (Bentley and Hoy 1972; Helversen and Helversen 1975b; Butlin and Hewitt 1985; Reinhold 1998; Ritchie and Phillips 1998) a dominant expression of a song preference parameter was never found in hybrid females within the Orthoptera. I am aware of only one study, which found dominance effects similar to this results. Crossing experiments between Drosophila ananassae and D. pallidosa showed that F1 hybrid females showed the same mating pattern as D. ananassae females (Doi et al. 2001). The authors concluded that the female discrimination mechanisms of D. ananassae are dominant.

43 Chapter 2: Female preferences

This study shows that a complete haploid set of the C. biguttulus genome is sufficient to develop a functional neuronal filter that allows F1 hybrid females to select male songs with regular biguttulus-like syllables. This indicates that a neuronal filter for syllable detection as in C. biguttulus may be absent in C. brunneus, which would not be surprising since songs of C. brunneus lack syllables. The expression of a neuronal filter in one but not the other parental species probably contributed to the development of an almost perfect C. biguttulus-like neuronal filter for syllables in F1 hybrid females.

F1 hybrid females did not loose the sensitivity for phrase duration. In contrast to preferences for syllable durations, preferences for phrase durations were intermediate in hybrid. The neuronal filter mechanisms evaluating phrase duration may be homologous inC. biguttulus and C. brunneus females. These neuronal filters seemed to be combined in hybrids without incompatibilities. Hybrid females accepted long phrases as females of C. biguttulus do, but they responded already to shorter phrases by which they shifted towards C. brunneus phrases. Intermediate inheritance of certain parameters was found in various studies in orthopterans. Female preferences for intermediate phrase durations were also shown for the closely related pair of grasshoppers C. brunneus und C. jacobsi (Bridle et al. 2006), and preferences of two races of the bushcricket Ephippiger ephippiger for numbers of syllables were intermediate in hybrid females (Ritchie 1992).

The lack of obvious differences in song preference parameters between both reciprocal F1 hybrids makes a major X-chromosomal and/or maternal effect unlikely. Contrasting, a number of studies including some on Orthoptera suggest that X- chromosomal genes can have a significant effect on sexually selected traits (Reinhold 1998; Ritchie and Phillips 1998). When Chorthippus biguttulus was crossed with C. mollis, strong maternal effects in preferences of hybrid females were found. Chorthippus biguttulus x C. mollis hybrids showed no intermediate preferences, but either preference for songs of one parental species, in most cases like the mother species or combined preference patterns of both parents (Helversen and Helversen 1975b). The F1 hybrid females between Teleogryllus oceanicus and T. commodus preferred song of the appropriate reciprocal hybrid type suggesting a sex-linked inheritance (Bentley and Hoy 1972). In the C. parallelus group the inheritance patterns there seemed to be sex-linked or maternal effects (Butlin and Hewitt 1988). In crosses between Poecilimon races (Reinhold 1994) found X-chromosomal or maternal effects.

44 Chapter 2: Female preferences

5.2. Signals of females

The response songs of hybrid females were intermediate in phrase duration between parentals. Thus, concerning phrase duration, songs of females were inherited like male signals (compare Gottsberger and Mayer 2007). In contrast the preferences for the songs of males were not inherited intermediate but I found a more complex inheritance pattern. The hybrid female’s own song patterns are not coupled to the preference for a certain pattern. This fact stands again the hypothesis of genetic and functional coupling between signals of females (and males) and the preferences of females for these signals. Some authors have assumed genetic coupling in Orthoptera (e.g. Bentley and Hoy 1972; Hoy et al. 1977), but other inheritance studies also did not find “genetic coupling” of signals and preferences (Helversen and Helversen 1975b; Bridle and Jiggins 2000; Bridle et al. 2006). In the species of the C. biguttulus group the synchronization of signals and the receiver system seems to have evolved by a coevolutionary process and are apparently not functionally coupled (Helversen and Helversen 1975b).

No differences between both parental species and F1 hybrids were found in respect of latency of female response. The latency to answer indicates the ability to answer and to react properly and timely to a stimulus but also depends on motivation of females (Helversen et al. 2004). The hybrid females show therefore the same vigour and motivation as pure species females like it has been observed in other hybridization studies on caeliferan grasshoppers (Helversen and Helversen 1975b; Saldamando et al. 2005b). Therefore hybrid females seem to have no postzygotic deficiencies concerning the detection of signals and reaction to these.

5.3. Strength of hybridization barriers

The similarities between the preferences of both reciprocal F1 hybrid females and C. biguttulus females indicate that hybrid females will readily and most exclusively backcross to males of C. biguttulus. Songs of F1 hybrid males are even unattractive to F1 hybrid females because they contain, if at all, only occasionally a syllable structure. In addition, syllables were never produced in a regular manner by hybrid males, and they were rather long compared to the syllables of C. biguttulus (Gottsberger and Mayer 2007). The songs of hybrid males are also unattractive for females of C. brunneus and C. biguttulus.

45 Chapter 2: Female preferences

The uniparental backcross of F1 hybrid females to C. biguttulus males strengthens the reproductive isolation between the two parental species C. biguttulus and C. brunneus. Both species rarely hybridize in nature (Perdeck 1957; Ragge 1976) and F1 hybrids do not show signs of reduced viability (Gottsberger and Mayer 2007). F1 hybrid females will backcross with C. biguttulus males as long as males of C. biguttulus are common. In contrast, mating probabilities of hybrid males will be very low since their song parameters do neither match the preferences of hybrid females nor the preferences of females of both parental species. Such restriction of F1 hybrid reproduction to one sex (females) that mates to one parental species (C. biguttulus) prevents proceeding hybridization and limits unidirectional gene flow from C. brunneus to C. biguttulus. Therefore, dominant expression of song preferences in F1 hybrid females likely represents an effective evolutionary mechanism that allows occasional hybridization without merging of the two species, although both species largely overlap in their distribution range and occur syntopically at many locations (Ragge and Reynolds 1998; Ragge et al. 1990).

46 Chapter 3: Natural hybrid population

Chapter 3

Evolution of songs and female preferences in a natural hybrid population between the grasshopper species Chorthippus biguttulus and C. brunneus

1 Abstract

Interspecific hybridisation could play a major role in the evolution of novel signals and in the diversification in animals. I investigated whether a gomphocerine grasshopper, C. jutlandica, occurring in West-Jutland, Denmark, could be of hybrid origin between the species C. biguttulus and C. brunneus. It is known that the premating isolation mechanisms between the two species are strong and they occur in sympatry throughout most of their distribution range despite occasional hybridization. I compared songs of males and female preferences of C. jutlandica with those of hybrids between C. biguttulus and C. brunneus reared in the laboratory. F1 hybrids and C. jutlandica were extremely variable in male song parameters as well as in female preferences. Male calling songs of F1 hybrids and C. jutlandica were very similar, but the latter tended to produce more regular syllables. Preferences of C. jutlandica and laboratory hybrid females resembled each other, but C. jutlandica females were less critical concerning syllable pattern. Females of C. jutlandica accepted C. biguttulus, C. jutlandica and F1 hybrid songs, but not songs of C. brunneus. The results confirm a hybrid origin of C. jutlandica. The C. jutlandica population has more in common with C. biguttulus and is clearly separated from C. brunneus. This population maintains its distinctiveness because C. biguttulus does not occur in West-Jutland.

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2 Introduction

The role of hybridization in evolution and speciation is far from being understood and is subject of intense actual discussions (Rieseberg and Wendel 1993; Arnold et al. 1999; Barton 2001). Different outcomes result from interspecific hybridization. One possibility is that introgression and gene flow leads to a hybrid swarm (Seehausen 2004; Llopart et al. 2005a) or formation of a hybrid zone (Barton and Hewitt 1981; Barton and Hewitt 1989; Harrison 1993; Jiggins et al. 1996; Jiggins and Mallet 2000; Hewitt 2001). In the latter case, hybridization between spatially overlapping species can lead to hybrid individuals, which occur quite abundant at certain localities. Another possible result of interspecific hybridization is allopatric introgression, when genes of one taxon introgress into an allopatric population of a second taxon (Anderson and Stebbins 1954; Mallet 2005). Hybridization can also lead to the formation of a new species, and thus in speciation. The increasing number of cases of hybrid speciation in animals suggests that such a mechanism is probably not as rare as previously thought (Rieseberg and Wendel 1993; Karrenberg et al. 2007; Mallet 2007). Further aspects of hybridization are that the number of hybridizing species seems to be higher in rapidly radiating groups (Seehausen et al. 1999; Shaw 2002; Bell and Travis 2005; Llopart et al. 2005b; Schwarz et al. 2005; Mavárez et al. 2006), phenotypes of hybrids can be extreme compared to any parental species (“transgressive segregation”, Rieseberg et al. 1999) and hybridization is often accomplished with new adaptations which allow hybrids to colonize novel habitats, to which parental species were not adapted to (Dowling and Secor 1997; Arnold and Emms 1998).

Hybridization, as a mechanism creating diversity and speciation rather then leading to dilution of species, was a long neglected phenomenon, especially in animals (Mallet 2001; Mallet 2007) but now examples of interspecific hybridization in animals are increasing (Grant et al. 2005; Gompert et al. 2006; Mallet et al. 2007). In this chapter I investigate the question whether hybridization can lead to novel mating signal, which could lead to an effective premating reproductive isolation barrier between parental species and hybrids. Therefore I analyzed the reproductive behavior of a grasshopper population, which is of putative hybrid origin. This taxon was recently described as a new species (Chorthippus jutlandica, Nielsen 2003) and belongs to the species rich C. biguttulus group. The distribution of C. jutlandica is restricted to West-Jutland in Denmark. Species of the C. biguttulus group are widely distributed in Central Europe, morphologically and genetically very similar, and it is known that syntopically distributed species occasionally hybridize in nature (Faber 1957; Perdeck 1957; Ragge 1976).

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The main factor causing reproductive hybridization barriers are the species-specific calling songs of the males, and the precise concerted female preferences for the conspecific songs. No obvious intrinsic postzygotic barrier have evolved, as hybridization studies in the laboratory indicate (Perdeck 1957; Gottsberger and Mayer 2007).

In this study I test the hypothesis if C. jutlandica originated by interspecific hybridization. A method for detecting hybrid origin is to demonstrate that hybridization generated functional diversity (Seehausen 2004). It has to be shown that traits in hybrids have been acquired from two different parental species. One way to test this is to cross individuals of the putative parental species and to investigate the inherited traits. Therefore I compared data obtained from the C. jutlandica population in Denmark with data of laboratory hybrids. I performed a behavioral study comparing male songs and female preferences of hybrids between C. biguttulus x C. brunneus with C. jutlandica. Finally, I discuss how a stable hybrid population might have evolved, despite the possibility of backcrossing to one parental species.

3 Material and Methods

3.1. Study animals

Chorthippus jutlandica animals were collected as nymphs or adults during July 2004 and August 2005 at Vejers Strand (N 55°36.917’ O 08°07.164’) and Børsmose Strand (N 55°40.311’ O 08°08.764’) in West-Jutland, Denmark. Males and females were brought to Erlangen and kept separately in plastic breeding cages (44 x 44 x 44 cm) and were fed on fresh grass (Dactylis glomerata). Light and additional heat was provided for 12 hours each day with a 40 W bulb inside the cage. Adult grasshoppers were individually marked with paint markers (Edding 780).

Chorthippus biguttulus was collected in Erlangen and surroundings (Bavaria, Germany) and C. brunneus originated from localities in Erlangen and Denmark. F1 hybrids were laboratory- crossing animals between C. biguttulus females and C. brunneus males and the reciprocal cross (Gottsberger and Mayer 2007). I crossed F1 hybrid individuals (from C. biguttulus female x C. brunneus male crosses) to obtain an F2 generation.

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3.2. Male songs: recordings and analysis

I recorded songs of male grasshoppers in the lab with a ½ inch condenser microphone (G.R.A.S. Type 40AF) equipped with a G.R.A.S. 26AB preamplifier. The songs were amplified by a Brüel & Kjær measuring amplifier (Type 2608). Simultaneously I recorded the movements of both hind legs with an opto-electronic device (Helversen and Elsner 1977). Song and leg signals were digitized using a custom developed three channel AD-converter with 16-bit resolution and 250 kHz (song) / 125 kHz (leg movements) sampling rate. The recordings were analyzed with Turbolab 4.0 (Stemmer software) and custom-designed software (by W. Schulze) developed in LabVIEW 7 (NATIONAL INSTRUMENTS). During recordings temperature was held constant at 31 ± 2°C. Only intact spontaneous calling males with two stridulatory hind legs were recorded.

I measured eight song parameters (song terminology according to Gottsberger and Mayer 2007 and Helversen and Helversen 1994): total song duration, duration of phrases (without first phrases in songs), number of phrases per song, syllable period, number of syllables per phrase, pulse period, number of pulses per syllable and phase shift between legs. Song measurements of both reciprocal F1 hybrid males were pooled (see Chapter 1). I performed a principal component analysis (PCA) of seven song parameters (Fig. 3. 2). Total song duration was excluded from PCA as no difference was found between C. biguttulus, C. brunneus, the F1 lab hybrids and C. jutlandica (Kruskal-Wallis χ2 = 3.203, df = 3, P = 0.361).

3.3. Female preference

3.3.1. Playback experiments of females with artificial sounds

The playback experiments were performed with virgin C. brunneus, C. biguttulus and C. jutlandica females. Animals were placed in a small gauze cage in a sound-attenuated thermostatic chamber with a constant temperature of 30 ± 1°C. Females were provided with some blades of grass and sand as egg-laying substrate during experiments. A computer- controlled set-up played-back sound and recorded female reply songs automatically. Analog white noise was amplitude-modulated by an IBM compatible computer and played-back with custom-built power amplifiers and emitted by tweeter loudspeaker with a flat response from 2 to 40 kHz (Dynaudio D21/2, Skanderborg Denmark). Signal amplitude was calibrated to a constant intensity level of 70 ± 2 dB SPL (peak) with a Brüel & Kjær sound level meter (Type

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2231 and a Brüel & Kjær ½ inch condenser microphone 4133) at the position of the animal. A condenser microphone (Type MCE-101; 50 to 12000 Hz) registered the female response inside the chamber (Helversen and Helversen 1983).

3.3.2. Song models for female preference tests

Experiments were performed as described in Helversen and Helversen (1983). I used song models that consisted of square modulated white noise. Two song parameters were altered to simulate a transition from C. biguttulus to C. brunneus male calling songs. I tested five different combinations of syllable and pause duration: a typical C. biguttulus song pattern with 80 ms long syllables separated by 12 ms pauses (song model 1), a C. brunneus pattern that consisted of 10 ms syllables with 4 ms pauses (song model 4), two intermediate syllable/pause durations of 40/8 ms (song model 3) and 20/6 ms (song model 4), respectively and one pattern without any pauses (song model 5).

The second altered parameter was phrase duration. The five song models were played back with eleven different phrase durations (90 ms, 130 ms, 180 ms, 250 ms, 350 ms, 500 ms, 700 ms, 1000 ms, 1400 ms, 2000 ms and 2800 ms). Thus a total of 55 different song models were presented to females. Each sound stimulus consisted of three identical phrases. Pauses between phrases were set to 6 s, when females did not reply, and 2 s when females answered with a response song. The order of the stimuli was pseudo-randomized. Between playbacks of two stimuli a pause of one minute was included. If no response song was registered within one session (the playback of all 55 sound stimuli), presentation was stopped for 30 minutes. When females responded, the program immediately continued with a next session. Each female was tested on average at 21 sessions with all 55 sound stimuli (SD = 5.14; N = 80). I analyzed in total 13 C. brunneus, 27 C. biguttulus and 40 C. jutlandica females.

3.3.3. Playback experiments of females with male calling songs

To access the strength of the reproductive isolation barrier, I tested 20 C. biguttulus, 24 C. brunneus, 33 C. jutlandica and 9 F2-hybrid females with different male songs. Females were tested with individual male calling songs of 10 C. biguttulus, 10 C. brunneus, 10 C. jutlandica and 5 F1 C. biguttulus x C. brunneus and 5 reciprocal F1 hybrids. Songs were recorded as described above, high-pass filtered (3 kHz) and resampled to 96 kHz with the program Spark ME (Version OSX 2.10). To normalize amplitude for all sounds, I selected a 100 ms interval at approximately the maximum amplitude of the sound from each recording, measured the RMS value (in µpa), calculated an amplifying factor and amplified all recordings

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to the same amplitude level. This was performed with the program Amadeus II (3.8.3.). Songs were stored on a Macintosh G4 Computer (Apple Computer Inc.) as AIFF-files. Playback experiments were carried out with the software iTunes 5 (Apple Computer Inc.). Male songs were D/A converted by an external 24Bit/96kHz USB audio interface (M-Audio; Quattro USB) and amplified by means of two custom built stereo amplifiers. Songs were played back via four loudspeakers (Technics 10TH400C), each positioned 30 cm from females. In the range of females the loudest part of each calling was calibrated to amplitudes of 70 ± 3 dB SPL (Brüel & Kjær ½ inch condenser microphone 4133; Brüel & Kjær sound level meter 2231; RMS measure).

Each female was illuminated by a 25 watt filament bulb. During experiments temperature within the gauze cages was kept constant at 30 ± 2°C. Motivated females were placed in small gauze cages (7 x 7 x 7 cm). Four females were tested simultaneously. Test chambers were separated by 60 x 60 cm sized wooden plates coated with 5 cm foam. Females were always tested three times with all 40 male songs. The order of songs was pseudo-randomized in each test. Songs were played at intervals of 2 min to minimize the influence of previous male song on female responses. Between tests of the 40 songs a pause of 15 min was included. I counted female responses to each male song until 30 s after end of signal. Females occasionally sing spontaneously without stimulation. Thus answers of females after 30 s were not counted as such.

I took the percentage of females answering to a song category (C. biguttulus, C. brunneus, F1 hybrids and C. jutlandica) as a measure for female preference (see Fig. 3.4). Differences in preferences between females to the four male songs types were tested with the distribution free two-sided all treatment Dwass-Steel-Critchlow-Fligner (DSCF) post hoc test (Hollander and Wolfe 1999). The critical values for four groups comparison are 3.633 for P = 0.05; 4.403 for P = 0.01 and 5.309 for P = 0.001. Statistical test were performed with SPSS 11.0.4 (© SPSS Inc. 1989-2005) and R, Version 2.4.0 GUI (© R Foundation for Statistical Computing, 2006; http://www.r-project.org).

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#JUTLANDICA

S A

&HYBRIDBIGXBRU

C S

D MS

Fig. 3.1. Comparison of sound and corresponding leg movements of C. jutlandica males with laboratory F1 hybrids males (C. biguttulus female x C. brunneus male). The two upper traces show movements of left and right hind legs and bottom trace show the corresponding oscillogram of emitted sound. a song of C. jutlandica from Denmark, West-Jutland, Vejers Strand (N 55° 36.917’ E 08° 07.164’; 13.VIII.2004; leg. B. Gottsberger), recorded at 34°C. b detail of third phrase with seven syllables. c Song of F1 hybrid reared in the lab, recorded at 31° C. d detail of third phrase with three syllables.

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4 Results

4.1. Male calling songs

Males of C. jutlandica produced calling songs composed of four to nine phrases and each phrase consisted of a series of syllables (Fig. 3.1). Phrases had durations of 0.74 ± 0.16 s and were separated by pauses of 1.56 ± 0.40 s (± SD; Table 3.1). Syllables were generated by a variable number of up-and down-oscillations of the hind legs, leading to a variable number of pulses per syllable (Fig. 3.1b; Table 3.1). Syllables started with a loud pulse that was generated by a fast down-stroke of both hind legs. A short resting phase of hind legs at a high position terminated a syllable and resulted in syllable pause.

The calling songs of C. jutlandica males resembled those of F1 hybrids between C. biguttulus and C. brunneus in phrase structure and leg movement patterns (Fig. 3.1; Table 3.1). Song duration (U = 314; P = 0.349) and number of phrases per song (U = 330.5; P = 0.498) were not significantly different between C. jutlandica and F1 hybrids. However, differences in the fine structure of songs existed (Fig. 3.1d). Within a phrase F1 hybrid males moved continuously both hind legs. This resulted in the lack of C. jutlandica-characteristic resting phases of one hind leg at a high position and thus a clear syllable structure in the songs of F1 hybrids was lost. Nevertheless, the movements of both hind legs showed syllable structures also in F1 hybrids although in a less regular repetition. Significant differences between C. jutlandica and F1 hybrids were found for the following song parameters (Table 3.1): phrase duration (U = 88; P < 0.01), phrase pause (U = 227; P = 0.017), syllables per phrase (U = 97.5; P < 0.01), pulses per syllable (U = 133.5; P < 0.01), pulse period (U = 88; P < 0.01) and phase shift of legs (U = 46; P < 0.01).

Despite these differences in song parameters between C. jutlandica and F1 hybrids, they were much smaller than the differences between the songs of F1 hybrids and both parental species (C. biguttulus and C. brunneus) respectively (Table 3.1). In terms of number of phrases and phrase duration, the songs of F1 hybrids and C. jutlandica were intermediate between those of C. biguttulus and C. brunneus. In contrast, syllables were much longer in C. jutlandica and F1 hybrids than in C. biguttulus and C. brunneus. Whereas C. biguttulus and C. brunneus showed low variance between individuals in syllables per phrase, pulses per syllable and syllable periods, F1 hybrids and C. jutlandica showed high levels of inter-individual variance (Table 3.1).

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C. brunneus Phase-shift of legs(°) 85.86 ±15.20 64.2-117.7 17.70 % 162.21 ±26.10 114.50-210.69 16.09 % 106.10 ±22.98 75.03-160.68 21.66 % 131.85 ±20.59 93.58-168.72 15.6 % and Pulse p e r i o(ms) d 8.60 ±1.20 6.3-11.0 13.99 % 7.96 ±0.45 7.03-9.28 5.67 % 9.31 ±1.23 7.07-12.44 13.24 % 8.83 ±1.42 6.40-11.76 16.1 % ), range and the coefficient the and range ), SD C. jutlandica Syllable period (ms) 60.09 ±7.04 50.36-80.7 % 11.56 224.87 ±136.78 49.33-586.42 60.82 % 127.62 ±84.18 53.86-378.70 35.96 % 29.57 ±4.47 22.55-38.65 15.1 % Pulses per syllable 6.21 ±0.51 5.42-7.68 8.16 % 25.90 ±15.19 4.75-67.60 58.66 % 11.62 ±8.97 4.60-40.22 77.24 % 3.05 ±0.13 2.85-3.42 4.3 % , F1 laboratory hybrids, Syllables phrase per 32.45 ±6.89 19-48 21.23 % 2.74 ±1.53 1-8 56.02 % 7.72 ±4.21 1.50-15.40 54.62 % 5.29 ±0.98 3.14-7.67 18.6 % C. biguttulus Nr Nr phrases of 3.17 ±0.89 2-5 28.08 % 5.27 ±1.71 2-9 32.45 % 5.60 ±1.27 4-9 22.74 % 7.77 ±2.39 5-14 30.8 % ) is shown in parentheses. N Phrase pause (s) 2.06 ±0.57 1.09-3.47 27.61 % 1.83 ±0.43 1.09-2.65 23.26 % 1.56 ±0.40 1.13-2.58 25.68 % 1.66 ±0.54 0.33-2.99 32.8 % Phrase duration (s) 2.07 ±0.32 1.27-2.61 % 17.11 0.51 ±0.12 0.34-0.95 23.93 % 0.74 ±0.16 0.44-1.07 21.36 % 0.18 ±0.04 0.13-0.27 21.6 % ). The number of individuals ( ). Song duration (s) 11.84 ±3.70 5.97-21.70 31.21 % 10.38 ±3.98 1.88-19.18 38.30 % 11.18 ±3.09 5.18-16.87 27.60 % 12.26 ±4.82 3.28-27.37 39.3 % CV Comparison of eight song parameters of Species/cross C. biguttulus SD (N = 35) range CV F1 hybrids SD (N = 36) range CV C. jutlandica SD (N = 20) range CV C. brunneus SD (N = 22) range CV Table Table 3.1. ( deviation standard ± mean individuals’ all of mean the is value Each 2°C. ± 30 at recorded of variance (

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3 a

2,5

2 C. biguttulus F1 C. jutlandica 1,5 C. brunneus

0,5

Factor 2: 29.09 % 0

-0,5

-1

-1,5

-2 -2 -1,5 -1 -0,5 0 0,5 1 1,5 2 Factor 1: 46.53 %

b Song parameters PC1 PC2

Phrase duration 0,961 0,083 Number of phrases -0,823 -0,130 Syllables per phrase 0,759 -0,608 Phase shift -0,710 0,514 Pulses per syllable -0,001 0,971 Syllable period 0,047 0,956 Pulse period 0,041 -0,305

Fig. 3.2. a Distribution of song parameters in C. biguttulus, C. brunneus, laboratory F1 hybrids and C. jutlandica along the two principal component axes PC1 and PC2. Percentages indicate the proportion of song variation explained by each PC. b Factors loading from the first two principal component of a PCA with seven song parameters. Total number of individuals was 114.

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I performed a principal component analysis (PCA) including all song parameters to quantify the song differences between the three taxa, C. biguttulus, C. brunneus and C. jutlandica as well as the F1 hybrids (Fig. 3.2). The first axis of the PCA accounted 46.53% to variation of song parameters (Fig. 3.2a). Especially phrase duration, number of phrases per song, syllables per phrase and phase shift contributed to this axis (Fig. 3.2b). Along PC1 C. biguttulus and C. brunneus were most divergent. F1 hybrids and C. jutlandica largely overlapped and were intermediate between these two species. F1 hybrids showed some overlap with C. brunneus values, whereas C. jutlandica values were slightly shifted towards C. biguttulus (Fig. 3.2a). The second axis (PC2) was composed of number of pulses per syllable and syllable period and accounted 29.09% to variation of song parameters (Fig. 3.2b). Along PC2 C. biguttulus and C. brunneus were separated and did not overlap. Both species showed little variation along PC2 if compared with the high variation in C. jutlandica and F1 hybrids, which was caused primarily by the highly variable number of pulses per syllable. Along PC2 C. jutlandica overlapped more noticeably with C. biguttulus than F1 hybrids (Fig. 3.2a).

4.2. Female preference

4.2.1. Playback experiments with model songs

Response probabilities of C. jutlandica females were highly variable in comparison with those of C. brunneus and C. biguttulus (Fig. 3.3). Females of C. brunneus had a narrow preference function answering only to short phrases with a maximum duration of 400 ms. They did almost not react to changes in syllable-pause duration, since they responded to all five song models tested (Fig. 3.3). In contrast, females of C. biguttulus were very sensitive to syllable- pause durations that closely resembled those of C. biguttulus songs since they only responded to the first two song models, whereas the other three song models evoked almost no answers and were therefore unattractive for C. biguttulus females (Fig. 3.3). Mean female response probabilities of C. biguttulus females surpassed a 50 % threshold if phrase durations exceeded 800 ms. Therefore preferences of C. biguttulus and C. brunneus were well separated by the phrase duration.

Chorthippus jutlandica females were highly variable in respect of their song preferences. With the exception of the C. brunneus song model (model 4), all song models were responded to with response probabilities up to 100% (Fig. 3.3). Only C. jutlandica females responded to model 5, which consisted of continuous white noise without any pauses. Among 40 tested C. jutlandica females, 14 had high response probabilities to this song model (Fig. 3.3). Other females did not respond to this song model at all. Eleven out of 40 C. jutlandica females

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#BRUNNEUS #JUTLANDICA #BIGUTTULUS

SONGMODEL

SONGMODEL &EMALERESPONSEPROBABILITIES

SONGMODEL

0HRASEDURATIONMS Fig. 3.3. Response probabilities of C. brunneus (N = 13), C. jutlandica (N = 40) and C. biguttulus (N = 27) females with five song models of artificial stimuli to assess female preference functions. Each model is characterized by a specific syllable to pause duration (model 1: syll-pause = 80-12 ms; model 2: syll-pause = 40-08 ms; model 3: syll-pause = 20-06 ms and model 4: syll-pause = 10-04 ms. In model 5 no pauses between syllables are included, thus phrases consisted of continuous white noise. Each model type was presented in 11 phrase durations, from 90 ms until 2800 ms, shown on the x-axes. On y-axes female response probabilities in percent are shown. Each gray curve is the preference function of one individual female. Black curves represent the mean of tested females.

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responded just to song model one and two and thus behaved like females of C. biguttulus (Fig. 3.3). The remaining 15 C. jutlandica females responded with high probabilities to the three first song models, but not to song models 4 and 5.

4.2.1. Playback experiments with natural male songs

Chorthippus brunneus females responded solely to natural songs of the conspecific males (Fig. 3.4). The remaining songs of the other three taxa were responded only sporadically. Females of C. biguttulus mostly preferred conspecific songs, but in mean 55% of females also answered to songs of C. jutlandica males. Nevertheless, number of responding females of C. biguttulus to songs of C. biguttulus and C. jutlandica differed significantly (DSCF test,W = 4.0287, P = 0.05). F2 hybrid females responded frequently to C. jutlandica songs (Fig. 3.4), but response rates did not differ significantly from response rates toC. biguttulus songs (W = 3.632, 0.1 > P > 0.05). Response rate to C. biguttulus and C. jutlandica male songs was quite variable between F2 females and ranged from below 20% until 75%. Chorthippus jutlandica females preferred firstly songs of conspecific males, and secondly songs ofC. biguttulus males. Although response rates to songs of C. jutlandica and C. biguttulus were not significantly different

(W = 3.329, 0.1 > P > 0.05) there was a trend to respond more frequently to C. jutlandica songs than to C. biguttulus songs. Only females of C. jutlandica responded also to some of the F1

#BRUNNEUS♀ #BIGUTTULUS ♀ &HYBRID♀ #JUTLANDICA♀ B A

B B C B

C 2ESPONDINGFEMALES

A B A B A B A A

MALESONGS Fig. 3.4. Female preferences tested with natural songs of C. brunneus, C. biguttulus, F1 hybrid and C. jutlandica males. Same letters stand for no significant difference; different letters stand for significant difference between percentage of responding females with P ≤ 0.05, tested with DSCF post-hoc tests.

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hybrid male songs, although at a much lower rate as to C. jutlandica (W = 5.363, P < 0.01) and to C. biguttulus (W = 5.250, P < 0.01). Chorthippus jutlandica females were the least selective group, as they answered to three of the four tested male songs groups.

5 Discussion

5.1. Hybrid origin of C. jutlandica

Several lines of evidence support the hypothesis that C. jutlandica originated by interspecific hybridization betweenC. biguttulus and C. brunneus. First, many song parameters of C. jutlandica were rather similar to those of F1 hybrids between C. biguttulus and C. brunneus but not to those of pure C. biguttulus and C. brunneus (Figs. 3.1 and 3.2). Phrase durations and number of phrases in songs of C. jutlandica and F1 hybrids were intermediate between those of C. biguttulus and C. brunneus. Some males of C. jutlandica showed simple leg movement patterns and generated few or nearly no syllables. Both characters are characteristic for F1 hybrids (Fig.3. 1; Gottsberger and Mayer 2007).

Second, there was a high variability concerning some song parameters in F1 hybrids and C. jutlandica, but not in the parental species C. biguttulus and C. brunneus.

Third, also females of C. jutlandica showed higher levels of variability than females of C. biguttulus and C. brunneus. Using song preference tests with artificial sound stimuli I found three groups of C. jutlandica females. The first group of females responded mostly to the first two song models, which were characterized by long syllables and with a syllable-pause duration ratio of about 5:1, which is known as an optimal syllable-pause duration ratio for C. biguttulus females (Helversen 1972; Helversen and Helversen 1997). Thus these females showed a narrow preference spectrum, which may result in selection of males performing clear syllable structures in their songs. The second group of females responded to the first three but not to the fourth and fifth song model and thus had a broader preference spectrum. The third group of females can be characterized as the group with the least selective song preferences. Along with the first three song models, these females responded with high response probabilities also to the last but not the fourth song model. Females of C. biguttulus preferred long phrases of song models 1 and 2. Females of C. brunneus were not sensitive to the syllable-pause structure of songs, but preferred short phrase durations between 100 and 300 ms (Fig. 3.3).

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This variability of song preferences in C. jutlandica was confirmed by playback experiments using natural male songs. Whereas C. brunneus and C. biguttulus females usually only responded to songs of their conspecific males, females ofC. jutlandica responded to songs of C. jutlandica, C. biguttulus and albeit in lower numbers also to songs of F1 hybrids (Fig. 3.4). The F2 hybrid females had preferences similar to C. jutlandica females, but no F2 female responded to songs of F1 hybrid males.

5.2. Origin of the hybrid population

The large variation in song parameters and female preferences in C. jutlandica could be the result of a recent hybrid origin. It is likely that interspecific hybridizations between C. biguttulus and C. brunneus lead at least initially to a small number of hybrids since one of the parental species (i.e. C. brunneus) still coexists with C. jutlandica. Low population density can relax sexual selection, since individuals cannot be as choosy when encounters with mates are rare (Dowling and Secor 1997). Low population densities facilitate backcrosses in general and backcrosses of hybrid females to males of C. biguttulus in particular since F1 hybrid females show preferences for songs of C. biguttulus males. The songs of C. jutlandica did not match exactly with songs of F1 and F2 hybrids between C. biguttulus and C. brunneus. Instead, C. jutlandica songs seem to resemble mostly backcrosses to C. biguttulus because of the more frequent occurrence of syllables and longer phrases in C. jutlandica songs than in songs of F1 or F2 hybrids (Gottsberger and Mayer 2007). These similarities between songs of C. jutlandica and C. biguttulus could also be the result of sexual selection since F1 hybrid females show higher response rates to songs of C. biguttulus males than to songs of F1 males (Boake 2002; Higgins and Waugaman 2004; Olvido and Wagner 2004; see also Chapter 2).

There are two main factors why a hybrid population between the parental species C. biguttulus and C. brunneus became established in western Jutland, while occasional hybridization between both species (Faber 1957; Ragge 1976; Ingrisch 1995; Baur et al. 2006) did not result in fusion of them throughout the most part of these species’ distribution ranges.

The first factor is the expression of male songs and female preferences in hybrids. Chorthippus jutlandica currently occurs sympatrically and syntopically with C. brunneus. Although C. jutlandica is of hybrid origin, clear differences in song and female preference remained between C. jutlandica and C. brunneus. The most important song parameter for C. brunneus females are characteristic short phrase durations (Ellegast 1984; Butlin and Hewitt 1986). Males of C. jutlandica do not sing such short phrases. Interestingly, females of

61 Chapter 3: Natural hybrid population

C. jutlandica did not react to songs of C. brunneus males although C. jutlandica females are generally unselective in their song preferences. The discrimination of C. jutlandica females against songs of C. brunneus represents an important hybridization barrier between both taxa and thus allows their coexistence in western Jutland (Nielsen 2003). Interestingly, F1 and F2 hybrids of C. biguttulus x C. brunneus also were selective against C. brunneus male songs (Fig. 3.4). Thus the reproductive barrier against C. brunneus can be strong in hybrids from the first generation on and it still is in the natural hybrid population in Jutland.

The second factor for the hybrid population of West-Jutland is that probably a small number of C. biguttulus immigrated to western Jutland, where C. brunneus is common but C. biguttulus is not present (Nielsen 2003). In Jutland C. biguttulus is only known from Middle to East-Jutland (Nielsen 2003). Due to the initially low population size of C. biguttulus they mated with the probably much more common C. brunneus. The resulting hybrids expressed hybridizations barriers towards C. brunneus since the male songs of this species have too short phrase durations for hybrid females of the first generation. The novel signalling parameters led to a effective hybridization barrier between C. jutlandica and C. brunneus and thus both taxa coexist syntopically in West-Jutland.

Additionally, ecological factors might play an important role in the maintenance of the hybrid populations (Arnold et al. 1999). West-Jutland shows a typical coastal dune habitat. Dunes are very dynamic habitats, with rapid changing conditions. Novel habitats could be formed quite easily, which could lead to a local (parapatric) isolation of the first hybrids in this habitat. And it is possible that hybrids could have a higher fitness in the new habitat. This example represents a case of allopatric introgression, which formed a stable hybrid population (Anderson and Stebbins 1954; Mallet 2005). As a result of the hybridization novel signalling parameters evolved rapidly. There is already a tendency in the evolution of reproductive isolation, as C. jutlandica males have more structured songs than F1 hybrids. Given time and if the sensitive hybrid population will not be disturbed, it is possible that a new species could evolve through the hybridization event in Jutland. Hybridization in Chorthippus grasshoppers should be seen as a process with evolutionary significant consequences and hybridization can generate functional diversity (Arnold 1997; Arnold and Emms 1998; Seehausen 2004).

In case of a secondary contact of C. biguttulus with C. jutlandica, this hybrid population would most probable be expunged. Thus it is not correct to treat C. jutlandica as a new species (see Nielsen 2003) according to the biological species concept, because the hybridization barrier to C. biguttulus is not present yet.

62 References

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75 Erklärung

Erklärung

Hiermit erkläre ich, Brigitte Gottsberger, dass ich diese Arbeit selbstständig verfasst habe und keine anderen als die von mir angegebenen Literatur Quellen und Hilfsmittel benutzt habe.

Wien, Oktober 2007

76 Lebenslauf

Lebenslauf

Brigitte Gottsberger geboren am 8.12.1971 in Botucatu, Brasilien Österreichische Nationalität

1978-1981 Grundschule Collégio La Salle in Botucatu, Brasilien

1981-1983 Privatschule Collégio Dom Bosco in São Luís, Brasilien

1983-1984 Ludwig- Uhland Schule, Grundschule mit Förderstufe, Gießen, BRD

1984-1991 Gymnasium an der Liebig Schule, Gießen, BRD mit Abiturabschluss

1991-1999 Studium der Biologie an der Universität Wien, Österreich

Diplomarbeit „Niederschlagsabhängige Rufaktivität einer neotropischen 1999 Froschgemeinschaft“ am Zoologischen Institut der Universität Wien.

Wiss. Angestellte in der Arbeitsgruppe von Prof. Hödl, Institut für Zoologie der Universität Wien; Forschungsaufenthalte in Arataï, 1999-2000 Französisch Guyana und an der Australian National University, Canberra, Australien.

Tätigkeit bei der Firma „Brainbows“ Informationsmanagement GmbH 2001 Wien Wiss. Angestellte in der Arbeitsgruppe von Prof. Hödl, Institut für 2001-2002 Zoologie der Universität Wien; Forschungsaufenthalt an der University of Sheffield, Großbritannien. Promotionsstudium am Institut für Zoologie II der Friedrich-Alexander 2002-2007 Universität, Erlangen-Nürnberg

77 Danksagung

Danksagung

Viele Leute haben mir bei der Erstellung dieser Arbeit geholfen und ich möchte dafür meinen herzlichen Dank aussprechen. An erster Stelle möchte ich mich bei meinem Doktorvater Priv. Doz. Dr. Frieder Mayer für seine umfassende Betreuung und seine Unterstützung bedanken. Er hat mir mit vielen Ideen und den intensiven Diskussionen meiner Ergebnisse sehr geholfen. Ich bedanke mich bei Herrn Prof. Bernd Ronacher, der sich bereit erklärt hat das Zweitgutachten meiner Arbeit zu übernehmen. Bei der Durchführung der Versuche zum Lautschema der Weibchen, bei der Benutzung der Positionsapparatur und bei zahlreichen kleineren und größeren Problemen waren mir vor allem Wolfram Schulze und Maria Bauer eine wertvolle Hilfe. Vielen Dank! Dirk Berger danke ich für die Hilfe beim Fangen der Heuschrecken und für die zahlreichen Gespräche und wichtigen Kommentare zu meiner Arbeit. Burkard Pfeiffer danke ich für die Hilfe bei den statistischen Auswertungen. Für das Korrekturlesen und für wichtige Anregungen während meiner Arbeit möchte ich mich bei Dirk Berger, Frieder Mayer, Otto von Helversen, Matthias Hennig, Bernd Ronacher, Wolfram Schulze, Jana Ustinova und Varja Vedenina recht herzlich bedanken. Die Versuche und Auswertungen des Kapitels über C. jutlandica wurden gemeinsam mit Ullrike Schöbel und Wolfram Schulze durchgeführt. Gefördert wurde diese Arbeit durch die Deutsche Forschungsgemeinschaft und durch das Hochschul- und Wissenschaftsprogramm der Friedrich-Alexander Universität Erlangen- Nürnberg. Viele Menschen am Institut für Zoologie II haben dazu beigetragen, dass ich eine schöne Zeit in Erlangen verbrachte und viel Spaß hatte. Danke an Olli Behr, Angela Bruns, Dagmar Dachlauer, Ute Fehn, Tina Kapitza, Mirjam Knörnschild, Nicolai Kondratieff, Ulrich Marckmann, Martina Nagy, Monika Otter, Andrea Ross, Volker Runkel, Ralph Simon, Deniz Şirin und Saskia Wöhl. Ich danke meiner Familie, die stets an mich geglaubt hat. Und am meisten danke ich Thomas Platzer für seine Geduld und seine Liebe.