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
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 neubeschriebene 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
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 flies Rhagoletis 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
South of Picos de Europe, Northern
Spain
Bailey et al. 2004; Bridle et al.
2001; Bridle & Butlin 2002;
Bridle et al. 2002a; Bridle et al.
2002b
Schleswig Holstein, Germany
Jacobs 1963
various localities, Switzerland
Baur et al. 2006 and O. v.
Helversen (pers. comm.)
C. jutlandica, Westjutland, Denmark
Nielsen 2003
C. eisentrauti x C. brunneus
“Ticino”-brunneus, Tessin, Switzerland
Ingrisch 1995
C. biguttulus x C. mollis
C. mollis ignifer, Alpine mollis, Le
Broc grasshopper, several populations
in the Alps
Ingrisch 1995; Ragge 1981;
Ragge 1984; Ragge 1987;
Ragge et al. 1990; Ramme 1923
C. rubratibialis, Apenninne biguttulus
Monte Summano, Italy
Ragge 1987; Ragge et al. 1990
Fontana et al. 2002
C. mollis x C. ilkazi
various localities in Turkey
D. Sirin & F. Mayer (pers.
comm.)
C. brunneus x C. bornhalmi
Triest, Italy
Kleukers et al. 2004
C. brunneus x C. jacobsi
C. biguttulus x C. brunneus
Hybridizing species with occasional hybrids in sympatric populations
C. biguttulus x C. brunneus
C. biguttulus x C. miramae
Schönbuch near Tübingen, Germany
Faber 1957
Erlangen, Germany
Kriegbaum (unpubl. recordings)
and O.v. Helversen (pers.
comm.)
various localities, Netherlands
Imst, Tirol, Austria
Middle Volga, Russia
Perdeck 1957
Ragge 1976
Oliger 1974
Interspecific crossings in the laboratory
C. biguttulus x C. brunneus
Elsner & Popov 1978; Flache
1987; Gottsberger & Mayer
2007; Perdeck 1957
C. biguttulus x C. mollis
Helversen & Elsner 1975;
Helversen & Helversen 1975a;
Helversen & Helversen 1975b;
Helversen & Elsner 1977;
Sychev 1979
C. brunneus x C. jacobsi
Saldamando et al. 2005a;
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 distributionfree two-sided all-treatments multiple comparisons based on pairwise ranking, the Dwass-SteelCritchlow-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
b
c
c
100 ms
C. brunneus
d
f
500 ms
pulse
syllable
e
2s
phrase
e
2s
f
500 ms
pulse
syllable
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.
Species/cross
C. biguttulus
SD (N = 35)
CV
big backcross
SD (N = 4)
CV
F1 big x bru
SD (N = 19)
CV
F1 bru x big
SD (N = 18)
CV
F2
SD (N = 6)
CV
bru backcross
SD (N = 7)
CV
C. brunneus
SD (N = 22)
CV
Song
Phrase
Phrase
duration
duration
pause
(s)
(s)
(s)
11.84
3.70
31.2 %
10.37
2.87
27.7 %
8.34
3.45
41.3 %
12.54
3.37
26.9 %
10.81
3.38
31.3 %
14.47
5.66
39.1 %
12.26
4.82
39.3 %
2.07
0.32
18.0 %
0.68
0.05
13.2 %
0.45
0.06
12.8 %
0.56
0.15
27.1 %
0.48
0.19
39.6 %
0.29
0.09
30.1 %
0.18
0.04
21.6 %
2.06
0.57
27.6 %
1.58
0.40
25.3 %
1.74
0.45
25.7 %
1.94
0.39
20.2 %
1.82
0.18
10.1 %
1.68
0.25
14.9 %
1.66
0.54
32.8 %
Nr of
phrases
3.2
0.9
28.1 %
5.3
1.3
24.0 %
4.7
1.7
34.9 %
5.9
1.6
27.3 %
5.5
1.7
29.9 %
8.1
2.6
32.1 %
7.8
2.4
30.8 %
Syllables
Pulses
Syllable
Pulse
Phase-
per
per
period
period
shift
phrase
syllable
(ms)
(ms)
in °
32.5
6.9
21.2 %
5.2
1.5
28.9 %
3.4
1.8
53.8 %
2.0
0.7
32.1 %
3.8
1.6
41.7 %
5.1
1.7
33.7 %
5.3
1.0
18.6 %
6.2
0.5
8.2 %
14.3
3.1
21.7 %
20.2
16.9
84.0 %
32.0
10.5
32.8 %
15.0
10.9
72.7 %
5.3
2.9
54.6 %
3.1
0.1
4.3 %
60.1
7.0
11.6 %
115.3
26.2
22.7 %
193.8
156.7
80.9 %
257.7
106.8
41.4 %
155.1
118.5
76.4 %
50.3
26.5
52.8 %
29.6
4.5
15.1 %
8.6
1.2
14.0 %
9.1
0.9
9.5 %
7.9
0.4
5.6 %
8.0
0.5
5.9 %
9.6
1.0
10.2 %
10.1
1.2
11.8 %
8.8
1.4
16.1 %
85.9
15.2
17.7 %
137.3
14.2
10.3 %
159.6
28.0
17.6 %
165.0
24.4
14.8 %
164.1
37.5
22.9 %
123.6
33.3
26.9 %
131.9
20.6
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
a
C. brunneus x C. biguttulus
2s
100 ms
2s
100 ms
2s
100 ms
2s
100 ms
2s
100 ms
2s
100 ms
b
F2 hybrids
c
c1
C. biguttulus backcross
d
C. brunneus backcross
e
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
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
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
Group comparison
23
1.156
3.066
1.926
1.606
1.297
0.982
1.320
1.800
0.554
0.730
4.598
0.731
4.400
0.890
4.111
0.046
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
*
ns
*
ns
ns
ns
Song duration
5.136
4.865
2.528
4.245
4.324
4.583
1.603
0.720
5.226
5.477
2.707
7.613
7.728
8.367
8.518
8.925
Phrase
duration
**
*
ns
*
*
*
ns
ns
**
**
ns
**
**
**
**
**
2.909
4.075
0.986
0.601
0.911
4.121
0.289
1.613
2.974
4.319
2.552
3.858
5.954
7.639
4.818
8.956
Phrase
number
ns
ns
ns
ns
ns
ns
ns
ns
ns
*
ns
ns
**
**
*
**
5.086
2.945
4.258
2.584
0.397
4.587
4.136
0.766
2.853
5.482
3.555
7.589
5.402
8.376
8.521
8.928
**
ns
*
ns
ns
*
ns
ns
ns
**
ns
**
**
**
**
**
Syllables per
phrase
5.392
4.783
4.214
0.574
4.537
4.596
3.771
0.810
5.315
2.278
4.770
7.656
7.769
8.376
7.142
8.951
**
*
*
ns
*
*
ns
ns
**
ns
*
**
**
**
**
**
Pulses per
syllable
5.392
4.783
4.093
0.803
4.757
4.583
2.357
0.720
5.226
2.660
3.567
7.613
7.728
8.367
6.776
8.925
Syllable
period
**
*
ns
ns
*
*
ns
ns
**
ns
ns
**
**
**
**
**
4.793
5.110
3.612
3.900
2.667
1.375
4.526
4.769
1.900
2.921
0.215
3.307
3.697
2.922
3.445
0.927
*
**
ns
ns
ns
ns
*
*
ns
ns
ns
ns
ns
ns
ns
ns
Pulse period
3.852
3.230
3.010
2.295
1.297
4.583
0.283
0.810
2.771
5.112
0.817
5.152
4.326
8.367
8.389
8.253
ns
ns
ns
ns
ns
*
ns
ns
ns
**
ns
**
*
**
**
**
Phase shift
Table 1.2. Multiple comparisons between all groups in eight parameters with the Dwass-Steel-Critchlow-Fligner test (see Material
and Methods). With seven groups the critical value for P = 0.05 is 4.170 and for P = 0.01 it is 4.882. Abbreviations: big = C.
biguttulus; bru = C. brunneus; big x bru = F1 hybrid C. biguttulus female x C. brunneus male; bru x big = F1 hybrid C. brunneus
female x C. biguttulus male; F2 = F2 hybrid; bru BC = C. brunneus backcross; big BC = C. biguttulus backcross; ns = not
significant; * = significant at the 0.05 P level; ** = significant at the 0.01 P level (not all 21 group comparisons are shown here for
convenience reasons).
Chapter 1: Male songs
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 first and
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
4
phrase duration (s)
song duration (s)
25
20
15
10
5
0
big
bru
x
big
F2
BC
bru
6
4
2
BC
big
bru
x
big
F2
BC
bru
0.4
0.3
0.2
0.1
big
BC
big
big
x
bru
bru
x
big
F2
BC
bru
BC
big
big
x
bru
bru
x
big
F2
BC
bru
bru
big
BC
big
big
x
bru
bru
x
big
F2
BC
bru
bru
big
BC
big
big
x
bru
bru
x
big
F2
BC
bru
bru
30
20
10
14
0.5
0
big
40
0
bru
pulse period (ms)
0.6
big
x
bru
1
50
8
big
2
0
bru
10
0
syllable period (s)
big
x
bru
syllables per phrase
number of phrases
12
BC
big
3
bru
12
10
8
6
4
2
0
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 and C. 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
Number of phrases
Syllables per phrase
Phase shift
Phrase pause
Pulses per syllable
Syllable period
Pulse period
Song duration
0.893*
-0.842*
0.837*
-0.756*
0.435
-0.100
-0.032
-0.059
-0.158
0.208
-0.322
-0.478
0.455
0.074
0.926*
0.918*
-0.391
-0.417
Explained variance (eigenvalue)
Proportion of total variance (%)
3.276
36.41
2.354
26.16
26
Chapter 1: Male songs
3
C. biguttulus
big backcross
F1 (big x bru)
F1 (bru x big)
F2
bru backcross
C. brunneus
PC 2: 26.16 %
2
1
0
-1
-2
-3
-2
-1,5
-1
0
-0,5
0,5
1
1,5
2
PC 1: 36.41 %
Fig. 1.4. The first two principal component axes of nine song parameters of C. 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 of C. 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 of our
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 socalled “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 brunneuslike 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 species C. 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
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Chapter 2: Female preferences
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
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0REFERENCEINDEX
SONGMODEL
0REFERENCEINDEX
SONGMODEL
0HRASEDURATION
C. biguttulus
F1 hybrid big x bru
C. brunneus
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
A
a
600
b
400
a
1200
b
c
800
400
200
0
a
1600
B
a
a
800
Phrase duration (ms)
2000
Response latency (ms)
1000
0
BIG
BIGXBRU
BRUXBIG
BIG
BRU
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
(ms)
U
Z
(2-tailed) P
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 in C. 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.
47
Chapter 3: Natural hybrid population
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).
48
Chapter 3: Natural hybrid population
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 laboratorycrossing 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.
49
Chapter 3: Natural hybrid population
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 computercontrolled 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
50
Chapter 3: Natural hybrid population
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
51
Chapter 3: Natural hybrid population
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).
52
Chapter 3: Natural hybrid population
#JUTLANDICA
S
A
MS
B
&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.
53
Chapter 3: Natural hybrid population
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).
54
38.30 %
11.18
±3.09
5.18-16.87
27.60 %
12.26
±4.82
3.28-27.37
39.3 %
CV
C. jutlandica
SD (N = 20)
range
CV
C. brunneus
SD (N = 22)
range
CV
10.38
F1 hybrids
1.88-19.18
31.21 %
CV
range
5.97-21.70
range
±3.98
±3.70
SD (N = 35)
SD (N = 36)
11.84
55
21.6 %
0.13-0.27
±0.04
0.18
21.36 %
0.44-1.07
±0.16
0.74
23.93 %
0.34-0.95
±0.12
0.51
17.11 %
1.27-2.61
±0.32
2.07
duration (s)
duration (s)
C. biguttulus
Species/cross
Phrase
Song
32.8 %
0.33-2.99
±0.54
1.66
25.68 %
1.13-2.58
±0.40
1.56
23.26 %
1.09-2.65
±0.43
1.83
27.61 %
1.09-3.47
±0.57
2.06
pause (s)
Phrase
30.8 %
5-14
±2.39
7.77
22.74 %
4-9
±1.27
5.60
32.45 %
2-9
±1.71
5.27
28.08 %
2-5
±0.89
3.17
phrases
Nr
of
18.6 %
3.14-7.67
±0.98
5.29
54.62 %
1.50-15.40
±4.21
7.72
56.02 %
1-8
±1.53
2.74
21.23 %
19-48
±6.89
32.45
per phrase
Syllables
per
4.3 %
2.85-3.42
±0.13
3.05
77.24 %
4.60-40.22
±8.97
11.62
58.66 %
4.75-67.60
±15.19
25.90
8.16 %
5.42-7.68
±0.51
6.21
syllable
Pulses
15.1 %
22.55-38.65
±4.47
29.57
35.96 %
53.86-378.70
±84.18
127.62
60.82 %
49.33-586.42
±136.78
224.87
11.56 %
50.36-80.7
±7.04
60.09
period (ms)
Syllable
16.1 %
6.40-11.76
±1.42
8.83
13.24 %
7.07-12.44
±1.23
9.31
5.67 %
7.03-9.28
±0.45
7.96
13.99 %
6.3-11.0
±1.20
(ms)
8.60
period
Pulse
15.6 %
93.58-168.72
±20.59
131.85
21.66 %
75.03-160.68
±22.98
106.10
16.09 %
114.50-210.69
±26.10
162.21
17.70 %
64.2-117.7
±15.20
85.86
of legs(°)
Phase-shift
Table 3.1. Comparison of eight song parameters of C. biguttulus, F1 laboratory hybrids, C. jutlandica and C. brunneus
recorded at 30 ± 2°C. Each value is the mean of all individuals’ mean ± standard deviation (SD), range and the coefficient
of variance (CV). The number of individuals (N) is shown in parentheses.
Chapter 3: Natural hybrid population
Chapter 3: Natural hybrid population
a
3
2,5
2
C. biguttulus
F1
C. jutlandica
C. brunneus
Factor 2: 29.09 %
1,5
1
0,5
0
-0,5
-1
-1,5
-2
-2
-1,5
-1
-0,5
0
0,5
1
1,5
Factor 1: 46.53 %
b
Song parameters
Phrase duration
Number of phrases
Syllables per phrase
Phase shift
Pulses per syllable
Syllable period
Pulse period
PC1
PC2
0,961
-0,823
0,759
-0,710
-0,001
0,047
0,041
0,083
-0,130
-0,608
0,514
0,971
0,956
-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.
56
2
Chapter 3: Natural hybrid population
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 syllablepause 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
57
Chapter 3: Natural hybrid population
#BRUNNEUS
#JUTLANDICA
#BIGUTTULUS
SONGMODEL
&EMALERESPONSEPROBABILITIES
SONGMODEL
SONGMODEL
SONGMODEL
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.
58
Chapter 3: Natural hybrid population
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 to C. 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 of C. 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
#JUTLANDICA ♀
&HYBRID ♀
#BIGUTTULUS ♀
#BRUNNEUS♀
B
A
B
B
2ESPONDINGFEMALES
C
B
B
C
B
B
A
A
B
A
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.
59
Chapter 3: Natural hybrid population
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 between C. 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).
60
Chapter 3: Natural hybrid population
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 of C. 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
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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
1999
Diplomarbeit „Niederschlagsabhängige Rufaktivität einer neotropischen
Froschgemeinschaft“ am Zoologischen Institut der Universität Wien.
1999-2000
Wiss. Angestellte in der Arbeitsgruppe von Prof. Hödl, Institut für
Zoologie der Universität Wien; Forschungsaufenthalte in Arataï,
Französisch Guyana und an der Australian National University,
Canberra, Australien.
2001
Tätigkeit bei der Firma „Brainbows“ Informationsmanagement GmbH
Wien
2001-2002
Wiss. Angestellte in der Arbeitsgruppe von Prof. Hödl, Institut für
Zoologie der Universität Wien; Forschungsaufenthalt an der University
of Sheffield, Großbritannien.
2002-2007
Promotionsstudium am Institut für Zoologie II der Friedrich-Alexander
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 ErlangenNürnberg.
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.
78