Download Contribution of X chromosomal and autosomal genes to species

CONTRIBUTION OF X
CHROMOSOMAL AND AUTOSOMAL
GENES TO SPECIES DIFFERENCES IN
MALE COURTSHIP SONGS OF THE
DROSOPHILA VIRILIS GROUP SPECIES
SELIINA
PÄÄLLYSAHO
Department of Biology,
University of Oulu
OULU 2001
SELIINA PÄÄLLYSAHO
CONTRIBUTION OF X
CHROMOSOMAL AND AUTOSOMAL
GENES TO SPECIES DIFFERENCES IN
MALE COURTSHIP SONGS OF THE
DROSOPHILA VIRILIS GROUP SPECIES
Academic Dissertation to be presented with the assent of
the Faculty of Science, University of Oulu, for public
discussion in Kuusamonsali (Auditorium YB210),
Linnanmaa, on December 21st, 2001, at 12 noon.
O U L U N Y L I O P I S TO, O U L U 2 0 0 1
Copyright © 2001
University of Oulu, 2001
Manuscript received 26 November 2001
Manuscript accepted 28 November 2001
Communicated by
Doctor Michael G. Ritchie
Docent Tapio Heino
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(URL: http://herkules.oulu.fi/isbn9514265831/)
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OULU UNIVERSITY PRESS
OULU 2001
Päällysaho, Seliina, Contribution of X chromosomal and autosomal genes to species
differences in male courtship songs of the Drosophila virilis group species
Department of Biology, University of Oulu, P.O.Box 3000, FIN-90014 University of Oulu, Finland
2001
Oulu, Finland
(Manuscript received 26 November 2001)
Abstract
In sympatric Drosophila species, songs produced by male wing vibration during courtship are an
effective mechanism preventing interspecific matings and maintaining sexual isolation between
different species. These songs can vary greatly even between closely related species. The aim of this
study was to localise X chromosomal and autosomal genes affecting species differences in male
courtship song and to study their interaction in the D. virilis group species. Various genes were
probed by in situ hybridisation on the X chromosomes of six species of the group, which enabled us
to use localised RFLP markers in QTL studies, as well as to compare gene arrangements of different
species.
Genetic analyses of differences between the songs of D. virilis and D. littoralis showed that
species-specific song traits are affected both by X chromosomal and autosomal genes. The X
chromosomal gene(s) having a major impact on pause and pulse length in male song were found to
be located at the proximal region of the chromosome. Precise localisation of the song genes was,
however, not possible due to multiple chromosome rearrangements restricting recombination
between RFLP markers located on this area. The same problem was faced when studying hybrids
between D. flavomontana and D. montana with less diverged X chromosomal gene arrangements.
Interaction between the X chromosomal and autosomal song genes in determining male song
traits was studied in four species belonging to the virilis and montana phylads of D. virilis group.
The long pauses in courtship song were found to be mainly caused by X chromosomal song genes
(or maternal / cytological factors), while pulse length was determined by X chromosomal genes
interacting with autosomal genes. This confirms the important role of X chromosomal gene(s) in
song evolution in the montana phylad species. The direction of dominance in hybrid songs suggests
that the songs of the montana phylad species have been affected by directional selection favouring
shorter pulses and longer pauses between sound pulses during their evolution.
The levels and patterns of DNA polymorphism in an X-linked fused (fu) gene was studied in
different D. montana populations. These studies revealed that D. montana populations are
significantly but not completely isolated, and that a selective sweep at fu (or at a gene linked to fu)
may be the reason for the reduced levels and patterns of variability of this gene in Finnish D.
montana populations. The methods used in this study will be utilized to study variation in 'song
genes' in the future.
Keywords: courtship song, DNA sequence variation, Drosophila virilis, in situ hybridisation, inversion, RFLP markers
Acknowledgements
The present work was carried out at the Department of Biology, Division of Genetics,
University of Oulu. I warmly thank Docent Anneli Hoikkala, my supervisor, for all her
support and guidance during my studies. She always had time to discuss my various
questions with overwhelming patience. Warm thanks are also due to Docent Jouni Aspi,
who was my second supervisor, for helping me especially in statistical problems. I am
very grateful to Professor Pekka Pamilo and Professor Emeritus Seppo Lakovaara for
providing excellent working facilities at the department. Many warm thanks to my
collaborators at the department and to the former and present members of our research
group. Especially I would like to thank Dr. Jaana O. Liimatainen, for always advising me
in my minor and major problems, and M.Sc. Susanna Huttunen, Ms. Mari SaarikettuKänsälä and Dr. Leena Suvanto, for many inspiring discussions, and in particular, for
their friendship. I also want to express my thanks to the staff who have helped in
cultivating the flies.
I wish to thank Docent Tapio Heino and Doctor Michael G. Ritchie for their valuable
comments on my thesis. Michael G. Ritchie also skilfully revised the English language,
all remaining mistakes are mine. I would particularly like to acknowledge Dr. Michael B.
Evgen’ev for his invaluable advice and guidance, and Dr. Jorge Vieira, my co-author, for
pleasant collaboration. I want to thank my parents, Tuulikki and Sampo Päällysaho, for
always being there for me and especially for good genes. I also thank my brother Kustaa
Päällysaho and all my friends and relatives for their support and encouragement. Finally,
I am deeply grateful to my fiancé Juha Yli-Hemminki who tirelessly shared everything
with me.
This work was financially supported by Graduate School of Evolutionary Ecology,
Betty Väänänen Foundation and the University of Oulu.
Oulu, November 2001
Seliina Päällysaho
Abbreviations
AFLP
bp
CN
FRE
Gln
His
IPI
kb
PAUSE
PCR
PL
PN
PTL
QTL
RAPD
RFLP
TE
amplified fragment length polymorphism
base pair
the number of cycles in a sound pulse
the carrier frequency of the song
glutamine
histidine
the interpulse interval
kilo base pair
the distance from the end of a sound pulse to the beginning of the next
one
polymerase chain reaction
the length of a sound pulse
the number of sound pulses in a pulse train
the length of the sound pulse train
quantitative trait loci
random amplified polymorphic DNA
restriction fragment length polymorphism
transposable element
List of original papers
The thesis is based on the following publications, which are referred to in the text by their
Roman numerals.
I
Hoikkala A, Päällysaho S, Aspi J & Lumme J (2000) Localization of genes affecting
species differences in male courtship song between Drosophila virilis and D.
littoralis. Genet Res 75: 37-45.
II
Päällysaho S, Huttunen S & Hoikkala A (2001) Identification of X chromosomal
restriction fragment length polymorphism markers and their use in a gene localization
study in Drosophila virilis and D. littoralis. Genome 44: 242-248.
III
Päällysaho S (2001) In situ hybridisation analysis of the X-linked genes in the species
of the virilis group of Drosophila. Genetica, in press.
IV
Päällysaho S, Aspi J, Liimatainen JO & Hoikkala A (2001) The role of X
chromosomal song genes in evolution of species-specific courtship songs in D. virilis
group species. (submitted).
V
Päällysaho S, Vieira J & Hoikkala A (2001) Sequence variation at the fused locus
shows divergent selection in Drosophila montana populations. (submitted).
Contents
Abstract
Acknowledgements
Abbreviations
List of original papers
1 Introduction..................................................................................................................... 9
1.1 Male courtship songs ............................................................................................. 10
1.2 The genetic basis of song divergence ......................................................................11
1.3 Chromosomal evolution in Drosophila virilis group species ..................................11
1.4 Gene localisation.................................................................................................... 13
1.5 Purpose of this study .............................................................................................. 14
2 Materials and methods .................................................................................................. 15
2.1 Flies........................................................................................................................ 15
2.2 Song recording and analysis................................................................................... 15
2.3 Molecular methods................................................................................................. 16
2.4 In situ hybridisation ............................................................................................... 16
2.5 Sequence analysis .................................................................................................. 16
2.6 QTL mapping methods........................................................................................... 17
3 Results and discussion .................................................................................................. 18
3.1 The localisation of genes affecting species differences in male song traits............ 18
3.1.1 Genetic basis of the song traits...................................................................... 18
3.1.2 Gene localisation with visible markers.......................................................... 19
3.1.3 Gene localisation with molecular markers .................................................... 19
3.2 Comparison of the X chromosomal gene arrangement in the D. virilis group
species .......................................................................................................................... 20
3.3 Evolution of species-specific songs in the montana phylad ................................... 22
3.3.1 Interaction between X chromosomal and autosomal genes........................... 22
3.3.2 Dominance and the direction of song evolution ............................................ 23
3.4 Sequence variation in fused gene in D. montana populations ................................ 23
4 Conclusions and plans for further research ................................................................... 26
References........................................................................................................................ 28
1 Introduction
The genetic basis of species differences in traits maintaining sexual isolation is one of the
central questions in speciation theories (reviewed e.g. by Orr 2001, Turelli et al. 2001).
Despite its importance, surprisingly few attempts have been made to find genes causing
the interspecific divergence in sexual traits. These kinds of data are crucial to find out
whether speciation proceeds by the accumulation of multiple genetic factors with
relatively small effect (Lande 1981) or whether it involves novel genetic processes like
the fixation of genes with a large effect (Coyne et al. 1994), chromosomal
rearrangements (reviewed by Rieseberg 2001), mobilisation of transposable elements
(e.g. Zelentsova et al. 1999) and/or genomic resetting (reviewed by Rose & Doolittle
1983). It is also unclear if the same loci are responsible to both intraspecific variation and
interspecific differences (Nuzhdin & Reiwitch 2000). In cases where reproductive
isolation between two species is controlled by a small number of loci with large effects,
speciation may evolve rapidly (e.g. Bradshaw et al. 1995, Doi et al. 2001). There are,
however, also examples of cases where a large number of loci are required for population
divergence (e.g. True et al. 1997, Ting et al. 2001).
One approach to study ‘speciation genes’ is to produce hybrids between closely related
species. Research on interspecific hybrids is often complicated. The main problems with
interspecific crosses are caused by postmating isolation, such as defects in hybrid
viability and fertility (e.g. Noor et al. 2001), and by restricted recombination due to
species differences in gene arrangement (Evgen’ev 1971). Hence, material for these kinds
of studies is not easy to find.
The Drosophila virilis species group (Diptera, Drosophilidae) offers a good
opportunity to study the genetic basis of species differences. The group contains twelve
species or subspecies and is currently subdivided into four phylads (Spicer 1991, 1992).
There are excellent reviews on the genetics, biology and evolution of species comprising
this group (e.g. Patterson & Stone 1952, Throckmorton 1982). The phylogeny among the
species is well established, and the approximate time of divergence between virilis and
montana phylads has been estimated as 9.0 ± 0.7 Mya (Nurminsky et al. 1996). Most
species of the group can be crossed with each other to obtain partially fertile F1 progeny,
and in some cases also viable F2 progeny and backcrosses (see Patterson & Stone 1952,
Throckmorton 1982), which is a prerequisite for the studies on interspecific hybrids.
10
There are also chromosome maps (e.g. Gubenko & Evgen’ev 1984), visible and
molecular markers (Alexander 1976, Schlötterer & Harr 2000), and a library of P1 clones
(Lozovskaya et al. 1993) available for a key species of the group, D. virilis.
1.1 Male courtship songs
In sympatric Drosophila species reproductive isolation involves both prezygotic (sexual
and habitat isolation) and postzygotic (sterility or inviability of hybrids) isolation
mechanisms, the former ones being significantly stronger than the latter ones (Coyne &
Orr 1997, Noor 1997). Songs produced by male wing vibration during courtship are an
effective mechanism preventing interspecific matings and maintaining sexual isolation
between species in wild (Liimatainen & Hoikkala 1998). These songs are evolutionary
labile and sometimes their divergence between taxa precedes the evolution of sexual
isolation and hybrid sterility (Gleason & Ritchie 1998). Thus the genes affecting male
song may be called ‘speciation genes’.
Courtship songs can vary largely between closely related species (Hoikkala et al.
1982). In the species of the D. virilis group, the male courtship songs play an important
role both in species recognition (Liimatainen & Hoikkala 1998) and in sexual selection
within the species in the wild (Aspi & Hoikkala 1995). The songs of the montana phylad
species (D. kanekoi, D. ezoana, D. littoralis, D. flavomontana, D. lacicola and D.
montana) can easily be distinguished from each other by the length of the sound pulses
and interpulse intervals. In some of these species the males produce only one sound pulse
during each wing extension and vibration, which results in species-specific songs with
long pauses (in D. littoralis about 300 ms) between successive sound pulses. The males
belonging to the virilis phylad (D. a. americana, D. a. texana, D. novamexicana, D.
lummei, D. borealis and D. virilis) keep their wing extended while producing a train of
sound pulses, where the pulses follow each other without any pause. The songs of these
species are structurally very similar to each other. The species of the montana phylad are
effectively sexually isolated from each other, while the isolation between the species of
the virilis phylad is in many cases poor (e.g. Patterson & Stone 1952, Throckmorton
1982). Hence, the more the courtship songs of the D. virilis group species differ from
each other, the more effectively these species are sexually isolated (Hoikkala et al. 1982).
Song divergence can be enhanced by selection. In directional selection one direction is
more favourable and the alleles acting in that direction will be expected to show
dominance over their alternative alleles at all loci (Lawrence 1984). This leads to
unidirectional dominance, like in the songs of virilis phylad, where song evolution has
been suggested to go towards longer and denser pulse trains (Hoikkala & Lumme 1987).
Selection can also be stabilizing, in which case dominance is expected to be
ambidirectional and incomplete, if present (Lawrence 1984).
11
1.2 The genetic basis of song divergence
Although male courtship songs are strongly implicated in sexual isolation, the genetic
basis of courtship song differences between Drosophila species is not well understood.
Traditionally courtship song differences have been studied with the aid of visible
phenotypic markers (e.g. Hoikkala & Lumme 1984, Hoikkala & Lumme 1987, Pugh &
Ritchie 1996), which are not necessarily neutral with respect to behavioural characters.
Recently developed neutral molecular markers e.g. restriction fragment length
polymorphism markers (RFLPs), amplified fragment length polymorphism markers
(AFLPs), microsatellites, random amplified polymorphic DNA markers (RAPDs) and P1
clones (reviewed e.g. by Rafalski & Tingey 1993, Vieira et al. 1997a, Mueller &
Wolfenbarger 1999) have made it possible to analyse species differences in courtship
song more thoroughly, giving an opportunity to trace the genetic changes associated with
speciation.
Many genes affecting the male behaviour in D. melanogaster are known to be located
on the X chromosome (Yamamoto et al. 1997). Hoikkala and Lumme (1987) have shown
that also in several species of the D. virilis group (D. littoralis, D. flavomontana, D.
lacicola) species-specificity of the courtship songs is largely caused by X chromosomal
gene(s). These authors have suggested that an X chromosomal major change has occurred
during the separation of the two major D. virilis group phylads (virilis and montana
phylad; Spicer 1991, 1992) allowing variation in inter pulse interval and further in pulse
length and cycle number in the species of the last-mentioned phylad.
The founders of neo-Darwinism have proposed that most adaptive changes in
populations are polygenic. However, Quantitative Trait Loci (QTL) studies provide
examples of major genes affecting a variety of traits (e.g. Mitchell-Olds 1995, Liu et al.
1996, Zeng et al. 2000). According to Ritchie and Phillips (1998), the likelihood of major
genes (and hence the possibility of rapid speciation) varies with the mode of signalling,
such as pheromonal systems showing many more examples of major gene effects than
acoustic systems. Differences in acoustic signals may be based on the cumulative effects
of very mild changes in several genes, e.g. Dmca1A, slowpoke and maleless loci (Peixoto
& Hall 1998), but there are also examples of major genes, such as period (Kyriacou &
Hall 1980).
1.3 Chromosomal evolution in Drosophila virilis group species
The D. virilis species group exhibits six major karyotypes (Throckmorton 1982). The D.
virilis karyotype, which is considered a primitive form in this group, consists of five pairs
of rod chromosomes and a pair of dots (Patterson & Stone 1952). In contrast to the
karyotypic variation observed in most of the 12 species in this species group, wild
populations of D. virilis exhibit no chromosomal polymorphism. In the D. virilis group,
the majority of chromosomal rearrangements between species are fusions and inversions
(e.g. Hsu 1952, Patterson & Stone 1952, Throckmorton 1982). As a consequence, the
content of chromosome is generally conserved in different species even though the
chromosomal elements are reorganised during speciation (Loukas & Kafatos 1986).
12
Although the evolution of many species-specific autosomal inversions can be traced,
there are several discrepancies in the estimated number and location of inversions on the
X chromosome. This is mainly due to the large number of X chromosomal inversions,
which may be up to four times more frequent than is observed in chromosome 3, for
example (Vieira et al. 1997b).
Classically genome evolution has been studied by direct observation of polytene
chromosomes of different species or interspecific hybrids. In hybrids, only homeologous
chromosomal sections of the parent species conjugate, which enables definition of all
major chromosomal rearrangements (e.g. Hsu 1952, Krimbas & Loukas 1984, Papaceit &
Prevosti 1989). This kind of approach is limited to closely related species, which can be
crossed with each other. In situ hybridisation (Pardue & Gall 1969) provides a powerful
and reliable method that allows unambiguous localisation of labelled DNA probe on the
larval polytene chromosomes. The technique enables direct comparisons of genome
organisation in different Drosophila species and may help to solve problems in the
analysis of chromosomal evolution in less closely related species (e.g. Whiting et al.
1989, Kress 1993, Ranz et al. 1997).
Gene rearrangements are routinely used as a tool to construct phylogenetic trees for
different Drosophila species groups. The generally accepted dogma relies on the
assumption that inversions are monophyletic in origin, i.e. each rearrangement appears as
a unique mutational event, and therefore identical inversions found in different strains or
species are consequences of a shared ancestor in which the rearrangement arose. There is,
however, evidence that inversions may not always be monophyletic. In natural
populations mobile elements have been found to cause endemic rearrangements (Lyttle &
Haymer 1992). Caccone et al. (1998) have suggested that cytologically similar inversions
may originate in parallel in different species due to the presence of genes vulnerable to
chromosome breaks. In D. virilis group species, inversion breakpoints have been found to
occur often in specific loci, which represent ‘hot spots’ for the insertion of transposable
elements (TEs) (Zelentsova et al. 1999). The congruity of rearrangement breakpoints and
chromosomal sites of transposable elements leads to an interesting possibility, that the
mobile elements may have played an important role in genomic reorganisation and
evolution of the virilis species group (Zelentsova et al. 1999, Evgen’ev et al. 2000).
Intraspecific chromosomal variation (particularly inversions) within and between
populations complicates comparisons of gene arrangements. A number of inversions are
present in most D. melanogaster populations at inconsistent frequencies (Lemeunier et al.
1986), some of them varying in season and latitude (e.g. Stalker 1980). The most variable
species of the D. virilis group, D. montana, consists of three subspecies or forms
(standard, giant, and Alaskan-Canadian montana), and has at least 30 heterozygous
inversions (see Moorhead 1954, Stone et al. 1960). The populations of the standard form
are virtually free of inversion polymorphisms on the X chromosome (Hsu 1952), but
many strains of giant montana and Alaskan-Canadian montana exhibit a number of X
chromosomal polymorphisms (Stone et al. 1960). These forms differ from the standard
type by at least four X-linked heterozygous inversions (Stone et al. 1960). Another
species showing large amounts of intraspecific chromosomal variation is D. a.
americana. Hsu (1952) tested 61 strains of this species and found eight autosomal and
two X-linked heterozygous inversions. Uncertainties in taxonomical ranks in the early
literature complicate the interpretation of cytological differences between the species and
13
chromosomal polymorphism within the species. In 1952 Hsu designated two forms of
montana complex as yampa (this form was initially found by Warters 1944) and superior
strains, which were later reevaluated and described under the names D. flavomontana and
D. borealis, respectively (Patterson 1952).
1.4 Gene localisation
Almost the entire euchromatic genome of D. melanogaster has been sequenced and the
species is predicted to have about 13 600 genes (Adams et al. 2000). This achievement is
a landmark in gene hunting and a herald of a new era of exploration and analysis.
However, effort is still needed to map the genes as well as to ascertain the function of the
genes with an unknown phenotype. Since genome rearrangements are frequent in the
genus Drosophila, localisation of genes and constructing genetic maps of the
chromosomes have to be done for each species under study separately.
One goal at gene localisation studies is to find major and minor genes affecting the
trait, to study their interaction and, if lucky, to clone major genes affecting the studied
trait. Quantitative trait loci (QTL) analysis is a combination of linkage mapping and
traditional quantitative genetic analysis, and it provides great potential to resolve the
number and location of genes influencing a continuous trait. QTL mapping involves
construction of genomic maps and allows identification of any relationship between the
studied traits and polymorphic markers (which should be localised prior to using them in
QTL). A significant association between the molecular marker and the trait may be an
indication of a QTL locus at or near the marker locus. Association between the marker
and the studied trait can be estimated by using e.g. single-marker analysis, interval
mapping or composite interval mapping (e.g. Lynch & Walsh 1998). In single-marker
analysis, which is the simplest method, the distribution of trait values is examined
separately for each marker locus. This method indicates a simple linkage of a QTL locus
and a marker locus. More advanced methods (e.g. interval or composite interval mapping)
increase the power of linkage detection and give more precise estimates of the QTL
effects and position.
Knowledge on the chromosomal location of the genes is also needed for studies on
DNA sequence variation in different parts of the genome. The level of DNA variation is
largely dependent on the occurrence of recombination at the studied site (Begun &
Aquadro 1992). The amount of recombination is known to vary depending on the
chromosome region; e.g. in D. melanogaster the telomeric ends and the proximal parts of
all chromosomal arms are known to be regions of low recombination (Ashburner 1989).
In D. virilis the telomeric region of the X chromosome shows suppression of
recombination (as in D. melanogaster), but there is no evidence for suppression of
recombination (in contrast with D. melanogaster) at the proximal end of the X
chromosome (Vieira & Charlesworth 1999). In addition, recombination is reduced inside
small heterozygous inversions and close to inversion breakpoints (Andolfatto et al. 2001).
The genes located on the regions of low recombination have been found to exhibit low
variability, making them inconvenient for studies of DNA polymorphisms (studies
reviewed by Charlesworth & Charlesworth 1998).
14
1.5 Purpose of this study
The main aim of this study was to identify and localise putative genes affecting male
courtship song. I wanted to find out whether the differences in male songs are based on
the effects of several genes with a minor effect or a few genes with a large effect, and
whether these genes can be localised on a certain chromosome(s) or chromosome
segment(s). In the first paper (I), we studied the relative contribution of the X
chromosome and autosomes to differences in male song traits between two species, D.
virilis and D. littoralis. We also studied whether different traits of the male song are
affected by the same genes or gene clusters, and whether the song genes can be localised
by using classical crossing methods. In paper II, we continued the gene localisation
project by identifying and localising by in situ hybridisation six X chromosomal RFLP
markers for D. virilis and D. littoralis. We also investigated conjugation of the
homeologous X chromosomes in the salivary glands of interspecific hybrid female larvae
to find out whether recombination would be possible in chromosome regions under
investigation. Linkage between RFLP markers and the gene(s) affecting species
differences in male song between D. virilis and D. littoralis was studied in the F2
progeny.
In paper III, the gene arrangement of four other species belonging to the D. virilis
group (D. a. americana, D. flavomontana, D. lacicola and D. montana) was probed by in
situ hybridisation. X chromosomal conjugation of the species pair having the most similar
gene order (D. flavomontana and D. montana) was studied in interspecific hybrids, and
the occurrence of recombination in hybrid females was examined with the aid of three X
chromosomal RFLP markers. In paper IV, we studied how the X chromosomal song
genes of D. virilis and D. flavomontana interact with the autosomal song genes of
different species, and whether the song traits show dominance in interspecific F1 males.
Information on the location of an X-linked fused (fu) gene (obtained in paper III) was
utilized in paper V to study the levels and patterns of DNA variability in this gene in
different D. montana populations. In the future we plan to use the same kind of methods
to study variation of putative song genes in the same populations.
2 Materials and methods
The materials and methods are given here only briefly. More detailed descriptions of
molecular methods, such as primer sequences and PCR conditions, are found in the
original papers (II-V).
2.1 Flies
The fly strains used in papers I, II, III and IV were isofemale strains or marker stocks,
which had been maintained in the laboratory for several years. Wild-caught D. montana
males for paper V were collected during the summers 1999 and 2000 from Utah, USA
and from two locations in Finland (Oulu and Oulanka). Wild-type D. montana strains
used in paper V are maintained at the Department of Biology, University of Oulu,
Finland. Some of these strains have been obtained from the Drosophila Species Stock
Center, Tucson, USA. Inter/intraspecific hybrids were detained in papers I, II, III and IV.
In these crosses the female is always marked prior the male.
The strains were maintained in uncrowded culture bottles on Lakovaara’s malt
medium in continuous light and 19o C. Males and females were sexed under light CO2
anaesthesia and collected in separate vials no later than 2 days after their emergence and
used in song recordings and/or crosses when sexually mature. In papers II, III and V the
culture bottles were transferred into 4o C and the larvae were given some extra yeast two
days before dissecting their salivary gland chromosomes.
2.2 Song recording and analysis
In papers I and IV the male courtship songs were recorded when the male was courting a
female(s). The recording chamber was made of a Petri dish (diameter 5.0 cm, height 0.7
cm) covered with a nylon net. The floor of the chamber was covered with a moistened
filter paper. The male songs were recorded using a Sony TC-FX33 cassette recorder and a
JVC-condenser microphone. The recordings were made at 19 ± 1oC.
16
The male courtship songs were analysed with the SIGNAL Sound Analysis System (
Engineering Design). The traits analysed were the length of the pulse train (PTL), the
number of pulses in a pulse train (PN), the number of cycles in a pulse (CN), the length
of the pulse (PL), the interpulse interval, i.e. the time from the beginning of a pulse to the
beginning of the next pulse (IPI), the distance from the end of the pulse to the beginning
of the next one (PAUSE), and the carrier frequency of the song (FRE), determined from
Fourier spectra.
2.3 Molecular methods
Genomic DNA (papers II, III and V) was isolated from adult male flies using QIAamp
DNA Mini Kit (QIAGEN) according to the instructions of the supplier. Amplified PCR
products were purified from agarose gel (Glenn & Glenn 1994) and sequenced
automatically with an ABI PRISM 377 (Applied Biosystems). For the RFLP method
(papers II and III), restriction endonucleases were chosen based on the DNA sequences so
that the enzyme would either cut the PCR product or leave it complete, depending on the
origin of the allele. Species-specific digestion patterns were determined from agarose gel.
2.4 In situ hybridisation
In situ hybridisation has been performed in papers II and III according to Heino (1994),
except for some minor modifications. Briefly, polytene chromosome preparations from
the salivary glands of female third instar larvae were dissected in Drosophila Ringer
solution and squashed in 45% acetic acid on a slide. The cover slip was flipped off in
liquid N2 and the slides were dehydrated in 96% cold ethanol. The probes were labelled
with digoxigenin-11-UTP by the random priming method. Hybridisation was carried out
overnight (at 37o C) in 40% formamide, 4xSSC and 100 ng sonicated herring sperm.
After hybridisation the slides were washed and incubated with digoxigenin antibody
conjugate. Finally the slides were stained with Giemsa and mounted in Permount.
Photographs with in situ hybridisation signals were obtained by Zeiss Axiolab phase
contrast microscope, using Achroplan 100x/1,25 oil Ph3 or Achroplan 40x/0,65 Ph2
objectives.
2.5 Sequence analysis
DNA sequences (papers II, III and V) were edited and aligned using SequencherTM
version 4.0 (Gene Codes Corporation) and Dnasis (Hitachi Software Engineering Co). In
paper V the estimates for the nucleotide variation were calculated using DnaSP software
version 3.50 (Rozas & Rozas 1999) and Proseq version 2.43 (http://helios.bto.ed.ac.uk/
evolgen/filatov/proseq.html).
17
2.6 QTL mapping methods
The genes affecting differences in male courtship song traits were traced with QTL
mapping in papers I and II. Transmittance of genetic markers together with a specific
value of the male song trait was studied by a single marker analysis. Here the null
hypothesis is that a given marker is unlinked to the putative QTL, i.e. the recombination
fraction between the marker and the gene(s) affecting the studied trait is 0.5. The
hypothesis can be examined by testing for a nonzero slope to the regression line of trait
value on marker indicator (Doerge et al. 1997).
3 Results and discussion
3.1 The localisation of genes affecting species differences in male song
traits
3.1.1 Genetic basis of the song traits
Song variation among Drosophila species is typically controlled by multiple genes (e.g.
Pugh & Ritchie 1996, Noor & Aquadro 1998, Williams et al. 2001). Differences between
the songs of D. virilis and D. littoralis males, affected by major X chromosomal gene(s),
are an exception (Hoikkala & Lumme 1987). Ritchie and Phillips (1998) have suggested
that major genes are more likely to be found for qualitative than quantitative trait
differences (e.g. the presence and absence of song types in D. pseudoobscura and D.
persimilis is possibly controlled by major genes whereas differences in interpulse interval
are not; Ewing 1969). This could reflect the possibility that a single gene may block the
expression of a polygenically controlled behavioural trait rather than that this gene would
control a complex behaviour (Ritchie & Phillips 1998).
In paper I we studied the genetic basis of species differences between D. virilis and D.
littoralis in various song traits: pulse train length (PTL), pulse train number (PN), cycle
number (CN), carrier frequency (FRE), pause length (PAUSE) and pulse length (PL).
Together the two last traits make up interpulse interval (IPI), which is a commonly used
trait in song analysis (e.g. Ewing 1969). The songs of reciprocal F1 hybrid males between
the two species differed from each other both by pause length and pulse length suggesting
that these traits are affected by X chromosomal or maternal factors. The same song traits
differed between backcross hybrids (virilis x littoralis) x virilis and (virilis x littoralis) x
littoralis, which indicates that in addition to X chromosomal genes, also autosomal genes
have a major impact on pause length and pulse length. The songs of the males from the
(virilis x littoralis) x littoralis backcross covered the whole range of variation in pause
and pulse length found in virilis x littoralis F1 hybrids and in D. littoralis, but only a few
of these males had pause length close to that of D. littoralis (Figure 2 in paper I). This
19
suggests that the autosomal genes of D. littoralis affecting the latter trait must be
homozygous to have a full effect on male song. We did not find any evidence of the
effects of maternal factors on male song characteristics.
3.1.2 Gene localisation with visible markers
Visually scored phenotypic markers have traditionally played an important role in gene
localisation studies (e.g. Tan 1946, Coyne et al. 1994, Doi et al. 2001, Ting et al. 2001).
D. virilis has about 200 visible mutant genes which can be used as markers (Alexander
1976). In paper I we used D. virilis and D. littoralis strains with different phenotypic
markers to localise X chromosomal and autosomal genes affecting the male song.
In backcross (virilis x littoralis) x virilis, only the X chromosomal marker gene white
(w) was linked with genes affecting male song (Table 2 in paper I). It explained about 20
% of the variation in pulse length in the songs of these backcross hybrids. In D. virilis,
the w locus is located at the proximal end of the X chromosome (site 13C; Lozovskaya et
al. 1993). The fact that yellow (y) marker (site 1D; Lozovskaya et al. 1993) was not
linked with male song genes, shows that the song genes are not randomly distributed
along the X chromosome, and that they are closer to white than yellow locus. Among the
autosomal markers, the cardinal (cd) marker (close to site 41E on the fourth
chromosome; Gubenko & Evgen’ev 1984) explained about 30 % of the variation in pause
length (Table 2 in paper I).
In the backcross (virilis x littoralis) x littoralis all X chromosomal marker genes
showed significant linkage with song gene(s) (Table 2 in paper I). The linkage was
strongest for the markers notched (nd) and apricot (ap), which explained 30 – 40 % of
variation in pause length, pulse length and cycle number. The exact cytological location
of these markers in D. littoralis is not known, but they are located near the proximal end
of the X chromosome. Among the autosomal markers only cinnabar (cn) showed linkage
with song genes, explaining more than 20 % of the variation in pause length (Table 2 in
paper I). In D. littoralis, cn is probably located close to the chromocenter of the fused
chromosomes 3-4 (paper I).
The data suggest that the traits, which differ most between the songs of D. virilis and
D. littoralis, are affected by several major and minor genes located on the X chromosome
and autosomes. Single marker analysis also suggested that the major X chromosomal
gene(s) is located inside a large inversion at the proximal end of the X chromosome of D.
littoralis. Single marker analysis has low statistical power and it also gives less exact
estimates of QTL effects and gene location than interval or composite interval mapping
(Liu 1998). Lack of crossing over (due to the species differences in gene arrangement),
however, prevented us from using more precise statistical methods.
3.1.3 Gene localisation with molecular markers
As discovered in paper I, visible marker genes have several disadvantages which hamper
their use in gene localisation studies. Phenotypic markers are often limited in number and
20
their exact cytological location is not always known. Furthermore, the expression of these
mutant markers may not be unambiguous and they can have pleiotropic effects on fly
behaviour. To avoid these problems, we continued to localise the major X chromosomal
gene(s) implicated in paper II by identifying neutral RFLP markers for six X
chromosomal loci (Dmca1A, fused, nonA, para, period and white) for D. virilis and D.
littoralis. Three of these markers (period, Dmca1A and nonA) are in loci where the genes
have previously been shown to affect male courtship song in D. melanogaster (Kyriacou
& Hall 1980, Rendahl et al. 1992, Peixoto et al. 1997). Accordingly, period, Dmca1A and
nonA can be considered as ‘candidate genes’ affecting male song traits.
The RFLP markers were first localised by in situ hybridisation on larval polytene
chromosomes of D. virilis and D. littoralis, which revealed differences in gene
arrangements of the two species at the proximal end of the X chromosome. The RFLP
markers Dmca1A, nonA and para, and the phenotypic marker ap (see paper I) are located
in D. littoralis in an inverted order compared to D. virilis (Figure 3 in paper II). This
indicates the presence of at least one inversion, but probably even more extensive
chromosome rearrangements. The finding was confirmed by observing interspecific F1
hybrid female larvae. In hybrids, the chromosome structure and gene arrangement were
similar at the distal end of the paternal and maternal X chromosome, which allowed
conjugation and recombination around the per marker locus. On the contrary, conjugation
was totally prevented at the proximal end of the X chromosome, which also inhibited
recombination between RFLP markers located on this area.
In paper II, QTL analysis was carried out with molecular markers for about half of the
males (80/151) of the (virilis x littoralis) x littoralis backcross studied in paper I. The
RFLP markers located at the proximal part of the X chromosome (Dmca1A, fused, nonA,
para and white) showed high linkage with gene(s) affecting male song traits (Table 4 in
paper II). These five markers were always inherited together with the phenotypic marker
nd. Although we found segregation between phenotypic markers nd and ap in paper I (3
males out of 151), the data in paper II suggest no recombination between homeologous
chromosomes in hybrid females in this area. This discrepancy is most likely due to a
failure in classifying ap phenotypes correctly, i.e. the song gene(s) does not have to be
inside the area inverted in D. littoralis (see paper I), but it may also be located on the area
between the inversion and the centromere. Hence, among the song genes of D.
melanogaster, nonA and Dmca1A (but not per) are possible candidates for causing
species differences in male song in the D. virilis group species.
3.2 Comparison of the X chromosomal gene arrangement in the D.
virilis group species
We found in papers I and II that a gene or a gene cluster located at the proximal region of
the X chromosome has a major effect on the differences between male courtship songs of
D. virilis and D. littoralis. The gene arrangements of these species appeared to be
different at the proposed site of song gene(s), which prevented more precise localisation
of the gene(s). In the genus Drosophila, genome rearrangements are frequent and thus the
location of specific genes can vary a lot even between closely related species. In paper
21
III, I compared the location of the five RFLP markers in six species belonging to D.
virilis group to find species pairs with more potential for gene localisation studies.
The RFLP markers (see paper II) were localised by in situ hybridisation on the
polytene chromosomes of four additional species of the D. virilis group: D. a. americana,
D. flavomontana, D. lacicola and D. montana. In the montana phylad, rearrangements of
the X chromosome appear to have occurred multiple times (D. montana has at least 11
fixed X-linked inversions when compared to the D. virilis chromosome; Vieira et al.
1997b), and in these species it is difficult to use the cytological map of D. virilis in gene
localisation studies. Hsu (1952) has determined species-specific inversion breakpoints for
most species belonging to the virilis phylad. The D. a. americana strain used in paper III,
however, had additional rearrangements, and therefore only relative locations of the
markers are also given for this species (Figure 3 in paper III).
In QTL analysis the conjugation of homeologous chromosomes and the occurrence of
recombination are prerequisites for successful gene localisation. Evgen’ev (1971, 1976)
has shown for hybrids between D. virilis and D. a. texana that the sections of
chromosomes characterised by a high frequency of conjugation in salivary gland
chromosomes exhibit a high frequency of crossing over in hybrid females. In paper III, I
studied these aspects in D. flavomontana and D. montana, the species pair having the
most similar gene order among studied species. Interspecific F1 hybrids between these
species showed conjugation in most parts of the homeologous X chromosomes. The
synapsis was, however, disturbed by two overlapping inversions near the proximal end of
the X chromosome and some additional minor aberrations at the distal end of this
chromosome (Figure 4 in paper III). Because the F1 hybrids of D. flavomontana and D.
montana exhibited normal pairing in a chromosome section between the RFLP marker
nonA and the inversion, there was a chance for recombination to occur between nonA and
the other markers. However, all markers were always inherited in a species specific
configuration in F2 hybrids, which indicated no crossing over in F1 hybrid females.
Recombination was evidently reduced not only within the heterozygous inversion, but
also at the region flanking the inversion breakpoints.
Intraspecific variation in chromosomes complicates studies of interspecific differences
in gene arrangement, but it may also offer new possibilities to find homologous gene
arrangements in different species. To begin studies of X chromosomal inversion
polymorphism in D. littoralis populations, I examined the salivary gland chromosomes of
F1 female hybrids from crosses between six different D. littoralis strains. These strains
were found to diverge from each other by one large inversion (see Figure 3 in paper II) at
the proximal third of the X chromosome, three Finnish strains having a similar gene
arrangement and the rest of the strains (Swedish, Swiss and Russian) having an
alternative arrangement (unpublished results). Unfortunately, also the alternative gene
arrangement differed too much from the gene arrangement of D. virilis, and in paper II it
would not had been possible to improve gene localisation studies by choosing another D.
littoralis strain (with alternative gene arrangement).
22
3.3 Evolution of species-specific songs in the montana phylad
3.3.1 Interaction between X chromosomal and autosomal genes
We studied the interaction between X chromosomal and autosomal song genes in four
species belonging to virilis and montana phylads of the D. virilis group in paper IV.
Hoikkala and Lumme (1987) have suggested earlier that during the separation of these
two phylads, an X chromosomal major change has allowed variation in the interpulse
interval. This song trait consists of two components, pause and pulse length, and it tends
to be highly variable among Drosophila species, but much less variable within species
(e.g. Cowling & Burnet 1981, Ritchie & Gleason 1995). In paper IV we studied songs of
hybrid males having the X chromosome and one set of autosomes from D. virilis or D.
flavomontana, and the second set of autosomes from D. virilis, D. flavomontana, D.
montana or D. littoralis.
Both maternal and paternal factors contributed to pause length, although the autosomal
and Y chromosomal effects were much smaller than the X chromosomal effects (Figure
3a in paper IV). The long pauses in courtship songs were found mainly to be caused by X
chromosomal song genes (or maternal / cytological factors), when D. flavomontana
(member of montana phylad) was used as a maternal species (Figures 4a & 4b in paper
IV). Pulse length did not show consistent maternal or paternal effects in any cross (Figure
3b in paper IV). However, significant differences between the crosses, where D.
flavomontana was used as a maternal species, suggest that X chromosomal genes interact
with autosomal genes in determining the length of this song trait (Figures 5a & 5b in
paper IV). Thus X chromosomal gene(s) do indeed play an important role in song
evolution in montana phylad species. In contrast, when crossing D. virilis (a member of
virilis phylad) with D. montana phylad species, X chromosomal (or maternal) factors of
D. virilis did not interact with the autosomal components of the paternal species. This
was seen as a lack of variation in pulse length. In the virilis phylad the differences
between species in each sound trait have previously been found to be determined mainly
by autosomal genes (Hoikkala & Lumme 1987).
In paper IV the mean pause length of F1 and F2 hybrids from the cross D.
flavomontana x D. montana was found to be intermediate between the values of the
parental species (Figure 6 in paper IV). The fact that the F2 males had significantly longer
sound pulses than the F1 males suggests that in addition to X chromosomal genes,
recessive autosomal genes of D. flavomontana also affect the pulse length. The F2 data
consisted of only one male with the D. montana X chromosome. The song of this male
resembled the song of D. montana, which again implies a strong role of the X
chromosome in determining species-specific song characters. Even though the
homeologous chromosomes conjugated in interspecific hybrids of D. flavomontana and
D. montana, recombination was reduced (paper III), which prevented localisation of song
genes in D. flavomontana and D. montana hybrids.
23
3.3.2 Dominance and the direction of song evolution
Hoikkala and Lumme (1987) have found both additive and dominance variance in pulse
train length, pulse number, pulse length, cycle number, cycle length and interpulse
interval in species belonging to the virilis phylad (D. a. americana, D. a. texana, D.
novamexicana, D. lummei and D. virilis). The direction of dominance was for longer
pulse trains, higher pulse and cycle number, and shorter interpulse interval and cycle
length, which suggests that the song evolution in these species has gone towards longer
and denser pulse trains (Hoikkala & Lumme 1987). In contrast to the virilis phylad, the
male courtship songs in the montana phylad have evolved to become species-specific.
In our data (paper IV) the direction of dominance suggests that the songs of the
montana phylad species have been affected by directional selection favouring shorter
pulses and longer pauses between successive sound pulses. In all crosses (except in the
cross between D. flavomontana and D. littoralis) autosomal genes affecting pause length
showed dominance towards longer pauses irrespective of the maternal species. Pulse
length showed dominance towards shorter pulses, when the paternal species was from the
montana phylad, but not when the paternal species was D. virilis.
It has been proposed that sexual selection exercised by the females could be a driving
force in song evolution (Suvanto et al. 2000). D. montana females have been found to
prefer males with short and dense (high frequency) sound pulses (Aspi & Hoikkala 1995,
Ritchie et al. 1998) and the song of this species shows directional dominance in the same
direction (Suvanto et al. 2000). Although dense pulse trains are very common among
Drosophila species, some species such as D. littoralis have songs where both pauses and
pulses are exceptionally long. Evolution of this kind of song might be due to the same X
chromosomal genes which cause long pauses perhaps also affecting the length of sound
pulses (paper I). Also, D. robusta males (Hoikkala, unpublished results) and the males of
melanica group species (Ewing 1970) produce songs with long pauses between sound
pulses. These species are closely related to the D. virilis group (see Throckmorton 1975).
3.4 Sequence variation in fused gene in D. montana populations
In paper III, I localised five genes on the X chromosome of several D. virilis group
species, which provides a good impetus to study DNA polymorphism in these genes. As
genes located in regions of low recombination are usually less variable than genes located
in regions of normal recombination (Begun & Aquadro 1992), it is important to know the
location of the studied genes, and whether this location may be affected by gene
rearrangements within the species. The gene chosen for study object in paper V was fused
(fu), which is a segment-polarity gene encoding a serine-threonine kinase (Preat et al.
1990). We investigated the levels and patterns of DNA polymorphism in a short fragment
of this gene in D. montana, a member of D. virilis group. In this species, the fu gene is
located in Moorhead’s map (Moorhead 1954) at cytological position S6 (paper III). The
same region in D. virilis is known to experience normal recombination (Vieira &
Charlesworth 2000). D. montana is distributed from the Atlantic coast of North America
across Canada and northern Asia to Scandinavia (Throckmorton 1982) and the
24
populations exhibit variation in morphological characters, chromosome structure and
behaviour (see for instance Aspi et al. 1993, Moorhead 1954, Lakovaara & Hackman
1973, Suvanto et al. 1999), which makes D. montana a good model for studies of
population structure, selection and speciation.
Vieira and Hoikkala (2001) have previously analysed most of the coding region of fu
(2.4 kb) and a short fragment of the suppressor of sable (su(s)) gene (469 bp) for two D.
montana populations. These authors suggested that a selective sweep at fu or at a gene
linked to fu may have affected the levels and patterns of variability at this gene in a
Finnish D. montana population. This suggestion was based on the low level of silent site
variability in fu in the Finnish Oulanka population. Vieira and Hoikkala (2001) found the
sequence of one male from the Oulanka population to define 12 new polymorphic sites
relative to the sequences of the remaining 24 Oulanka males. Nine of the polymorphic
sites, confined to a small 145 bp region, were present in the Utah population (USA),
which raised a question of a possible gene conversion event between Oulanka and Utah
populations. In paper V we have analysed the variable region of the fused gene for 45
additional wild-caught males from Finland (Oulu and Oulanka) and for 18 males
representing laboratory strains originating from different parts of the world. The new data
were combined with the earlier data of Vieira and Hoikkala (2001).
Vieira et al. (2001) have found several fixed differences in D. americana associated
with the alternative chromosome arrangements at the fused1 locus. Although D. montana
populations exhibit a large amount of chromosomal polymorphism (Stone et al. 1960),
only one of our study strains in paper V appeared to have an inversion on the X
chromosome. The fu sequence of this strain showed no difference to the sequences of the
strains with the standard gene arrangement. This is not surprising as fu was found to be
located in the middle of the inverted segment (Figure 1 in paper V), and recombination is
usually greatly suppressed only near inversion breakpoints (Andolfatto et al. 2001). The
strain with an inversion was, however, excluded from data analysis in paper V.
The haplotype structure of Finnish, Japanese, Alaskan-Canadian and American
standard and giant D. montana populations is given in Figure 2 in paper V. As this Figure
shows, we found in our new data set a second male with a diverged fu sequence in
Finland (this time from Oulu). Finnish populations showed the lowest and the American
and Japanese populations the highest variability (see Table 3 in paper V). Recombination
level (C = (8Nec)/3, where Ne is the effective population size for X chromosomal loci and
c is the recombination frequency per nucleotide site in females; Hudson 1987, Vieira et
al. 2001) could be estimated reliably for only the American Standard (C = 0.054) and for
the Finnish population (C = 0.056 with the two divergent sequences removed, otherwise
C = 0). As the power of test statistics for detecting selection in regions with a normal
level of recombination is usually low if one does not assume recombination (Wall 1999),
recombination was included in neutrality tests when feasible.
There are several kinds of methods developed to test the neutrality of sequence
variation, and the choice of the test should depend on which alternative hypothesis the
investigator regards most reasonable (Wall 1999). Large sample sizes in the present study
(compared to those of Vieira and Hoikkala 2001) gave us a chance to find out whether the
low polymorphism found in Finnish D. montana population was combined with deviation
from neutrality. Feasible tests were also made for the largest American sample, American
standard, where we wanted to find out whether the deviation from neutrality previously
25
detected using Wall’s (1999) B and Q tests (Vieira & Hoikkala 2001) also implied
selection, or whether it was due to the violation of other assumptions of the test.
Significance of all test statistics was determined by coalescent simulations (Wall 1999).
We first made Tajima’s D test (Tajima 1989), which is based on the relationship of the
number of segregating sites and the average number of nucleotide differences estimated
from pairwise comparisons. This test gave non-significant values (-1.3217 for the whole
population and 0.8875 when the sequences of the two deviating males were removed).
Non-significant values of Tajima’s D are consistent with less recent sweeps and weaker
selection, as well as with the neutral model (Simonsen et al. 1995), and so the test does
not totally reject deviation from neutrality. We then made Wall’s B and Q test statistics
(Wall 1999), which are based on the proportion of pairs of adjacent segregating sites that
are congruent (i.e. have consistent genealogies; Wall 1999). These tests, sensitive
especially to detect balancing selection (Filatov & Charlesworth 1999), detected
deviations from neutrality. Our data shows that the Finnish D. montana lacks derived
mutations in fu gene present in American D. montana as well as in D. flavomontana, D.
borealis and D. lacicola. It is suggested that deviation from neutrality in Finnish D.
montana could be due to a selective sweep, where the target of selection was linked with
an ancestral haplotype.
Our data in paper V show that the studied D. montana populations have diverged, but
that there is still some gene flow between populations. The fu gene had three nonsingleton replacement polymorphisms in the region analysed in paper V. The Gln/His
polymorphism (G/C on site 286) was shared by almost all populations (Figure 2 in paper
V) at different frequencies. Here the Gln haplotype (G on site 286) can be regarded as the
ancestral one as it is found in all species of the D. virilis group studied (see paper V).
Furthermore, comparison of the sequences of Finnish Gln and His haplotypes with those
of the other species of the D. virilis group (Table 4 in paper V) shows that eight out of 11
fixed differences between the His and Gln haplotype sequences in Finnish population are
derived in His haplotypes. The frequency and distribution of the derived His haplotype (C
on site 286) in different populations suggest that, although differentiated, the D. montana
populations studied are not completely isolated. The two Finnish His haplotype males
have probably obtained their ‘American-like’ sequence (between sites 241 and 378) by an
ancient gene conversion event. Vieira and Hoikkala (2001) have sequenced nearly the
whole fu gene from one of the two males, and the data show no deviation from other
Finnish males in any other region of this gene.
Based on differences in the structure of the flies’ genitalia, Lakovaara & Hackman
(1973) have been able to distinguish Finnish D. montana (described as D. ovivororum)
from American and Japanese D. montana. Our study gives further evidence of the
divergence between the Scandinavian and North American populations. Although
individuals of laboratory strains originating from different populations mate freely
(Suvanto et al. 2000), the finding that D. montana laboratory strains from different
continents differ significantly in male song traits (A Hoikkala, S Huttunen, S Päällysaho,
M Evgen’ev and J Aspi; unpublished results) and that strains also differ in hydrocarbons
(Suvanto et al. 2000), give further support to the hypothesis of significant divergence
among D. montana populations.
4 Conclusions and plans for further research
The aim of the present work was to study the genetic background of male courtship song
traits in D. virilis group species and to develop novel methods for such studies. As a first
step we used visible marker genes to localise the X chromosomal and autosomal genes
affecting differences in male song traits between D. virilis and D. littoralis (I). This study
revealed that it is not possible to localise or even detect all loci affecting song traits with a
few markers. By analysing the songs of interspecific backcross hybrid males we were,
however, able to show that both X chromosomal and autosomal genes have a major
impact on pause and pulse length, even though none of the autosomal markers used in
this study showed linkage with the latter trait.
In paper II we identified RFLP markers which appeared to be superior to traditional
phenotypic markers in QTL studies. RFLP markers can be localised by in situ
hybridisation on polytene chromosomes, they are easy to deal with and they show
unambiguous species-specific digestion patterns. Precise localisation of the song gene(s)
was, however, prevented due to the lack of recombination between homeologous X
chromosomes of D. virilis and D. littoralis. Lack of recombination also prevented gene
localisation attempts in another species pair (D. flavomontana and D. montana) having
less diverged X chromosomes (III, IV). We were, however, able to study the interaction
between the X chromosomal and autosomal song genes (IV) and to confirm the important
role of the X chromosomal genes in song evolution of the montana phylad species. Our
data suggest that the songs of montana phylad species have been affected by directional
selection favouring shorter pulses and longer pauses during their evolution.
In D. melanogaster several mutant genes (studies reviewed by Yamamoto et al. 1997)
have been found to affect male courtship song. We show in paper V how the effects of
selection can be studied in putative song genes at the DNA level. Our first study has been
carried out on the fused gene, but we are starting similar studies on candidate song genes
like the sodium channel gene paralytic, which has been found to increase the length of
the interpulse interval in D. melanogaster flies carrying a mutant allele (Peixoto & Hall
1998). Whether the genes inducing variation within the species could also play a role in
inducing species differences in male song is not known.
A growing amount of evidence suggests that speciation may involve novel genetic
processes such as mobilization of transposable elements (TEs). In the future, I plan to
27
take a part in studies concerning the role of these elements in promoting chromosomal
polymorphism and pre/post-zygotic isolation leading to speciation. In some species of the
D. virilis group the distribution of TE’s, such as Penelope and Ulysses (Evgen’ev et al.
2000), show associations with inversion breakpoints. My aim is to study the contribution
of transposable elements to the evolution of behavioural traits, like male song, through
the induction of chromosome rearrangements. In the light of the present study it seems
unlikely that the differences in male song traits could be due to the cumulative effects of
mild changes in several genes. It rather seems that the species differences in male song
are affected by more drastic genetic changes involving one or a few major and several
minor genes located on the X chromosome and autosomes.
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