# Looking under the lamp post: neither fruitless nor doublesex has evolved to generate divergent male courtship in Drosophila

Jessica Cande, David L. Stern, Tomoko Morita, Benjamin Prud'homme,
Nicolas Gompel

<figure><img src="image_1.png" /></figure>

## To cite this version:

Jessica Cande, David L. Stern, Tomoko Morita, Benjamin Prud'homme, Nicolas Gompel. Looking under the lamp post: neither fruitless nor doublesex has evolved to generate divergent male courtship in Drosophila. Cell Reports, 2014, 8 (2), pp.363-70. 10.1016/j CELREP.2014.06.023. hal-01134524

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<figure><img src="image_2.png" /></figure>
# Looking under the lamp post: neither *fruitless* nor *doublesex* have evolved to generate divergent male courtship in *Drosophila*

Jessica Cande<fnref n="1" />,<fnref n="2" />, David L. Stern<fnref n="2" />, Tomoko Morita<fnref n="2" />, Benjamin Prud'homme<fnref n="1" />,*, and Nicolas Gompel<fnref n="1" />,<fnref n="3" />,†

## SUMMARY

How do evolved genetic changes alter the nervous system to produce different patterns of behavior? We address this question using *Drosophila* male courtship behavior, which is innate, stereotyped and evolves rapidly between species. *D. melanogaster* male courtship requires the male-specific isoforms of two transcription factors, *fruitless* and *doublesex*. These genes underlie genetic switches between female and male behaviors, making them excellent candidate genes for courtship behavior evolution. We tested their role in courtship evolution by transferring the entire locus for each gene from divergent species to *D. melanogaster*. We found that, despite differences in Fru+ and Dsx+ cell number in wild type species, cross-species transgenes rescued *D. melanogaster* courtship behavior, and no species-specific behaviors were conferred. Therefore, *fru* and *dsx* are not a significant source of evolutionary variation in courtship behavior.

## INTRODUCTION

Recent decades have seen enormous progress in our understanding of the molecular causes of evolutionary diversity, especially for the evolution of morphology and physiology (Fletcher et al., 2001; Stern and Orgogozo, 2009). Often, genes that participate directly in the patterning or differentiation of tissues are the targets of evolutionary changes that alter the morphology of these tissues (Ronshaugen et al., 2002; Shapiro et al., 2004; Zhang et al., 2013). There also appears to be a strong bias during evolution toward genetic changes that

co-corresponding authors. gompel@biologie.uni-muenchen.de and benjamin.prudhomme@univ-amu.fr.

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## AUTHOR CONTRIBUTIONS

JC, BP and NG generated the transgenic flies, JC recorded and analyzed courtship video, TM and DS recorded and analyzed song.

<footnote n="1">¹Aix-Marseille Université, CNRS, Institut de Biologie du Développement de Marseille, UMR 7288, 13288 Marseille cedex 9, France</footnote>

<footnote n="2">²Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA</footnote>

<footnote n="3">³Ludwig-Maximilians-Universität München, Fakultät für Biologie, Biozentrum B 02.002, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany</footnote>
alter gene expression levels and patterns, rather than substitutions in protein coding regions (Carroll, 2008; Levine and Tjian, 2003). This is likely because changes in expression patterns can alter specific aspects of morphology without deleterious pleiotropic effects (Stern, 2000). This growing understanding has been built largely through studies of candidate genes (Davis and Patel, 2002; Lynch and Roth, 2011), and studies employing unbiased mapping of the genetic causes of morphological diversity have come to essentially the same conclusions (e.g., (Shapiro et al., 2004; Sucena and Stern, 2000)). Thus, candidate gene approaches can provide valuable insights into the mechanisms of phenotypic evolution.

It is tempting to transfer the candidate gene approach to studies of behavioral evolution. Behavior is generated by the activity of neural circuits, which are anatomical structures patterned by developmental genes and produced by developmental processes. Thus, genes that regulate the development and activity of these circuits would be excellent candidates for behavior evolution. Male courtship behavior in *Drosophila* species is an attractive target for such studies, because not only do *Drosophila* species display extensive variation in courtship behavior (Markow and O'Grady, 2005; Spieth, 1952), but the two genes that act as master regulators of sexual behavior have also been identified in *D. melanogaster*.

Courtship in *D. melanogaster* consists of multiple relatively stereotypical behavioral modules, and includes signaling between males and females in multiple modalities, including visual, auditory, somatosensory, and gustatory inputs (Hall, 1994; Krstic et al., 2009). In *D. melanogaster*, apparently all male sex-specific behaviors are regulated by the activity of two transcription factors, *doublesex* (*dsx*) and *fruitless* (*fru*), which are alternatively spliced in males and females (Baker and Wolfner, 1988; Burtis and Baker, 1989; Demir and Dickson, 2005; Kimura et al., 2008; Manoli et al., 2005; Rideout et al., 2007). *dsx* is required for all external sexually dimorphic features (Hildreth, 1965), as well as some male-specific behaviors such as courtship initiation and song (Kimura et al., 2008; Rideout et al., 2007; Villella and Hall, 1996). Male specific Fruitless (*FruM*) is expressed in ~1200 neurons in the central nervous system, as well as in some peripheral sensory neurons (Lee et al., 2000; Manoli et al., 2005), all of which appear to be involved in courtship behavior or sensing courtship-related cues (e.g. (Datta et al., 2008; Kimura et al., 2008; Stockinger et al., 2005)). Elimination of *FruM* function leads to the complete abrogation of male courtship (Ito et al., 1996; Ryner et al., 1996), while ectopic expression of *FruM* in females is sufficient to induce male courtship behavior (Demir and Dickson, 2005; Manoli et al., 2005). The functional dissection of populations of *FruM* and *DsxM* positive neurons has led to the identification of neurons in the brain that are required to activate male sexual behavior (Kimura et al., 2008) and neurons in the thorax that drive specific subsets of sexual behavior (Clyne and Miesenbock, 2008; Rideout et al., 2007). At a finer level, an individual *FruM* positive neuron and a single *DsxM* positive muscle together are required to produce a single element of courtship song (Shirangi et al., 2013), suggesting that the neuroanatomical substrate regulated by *fru* and *dsx* is highly modular. Studies of morphological evolution have suggested that modularity can contribute to more rapid evolution of new phenotypes (Stern and Orgogozo, 2009; Wagner et al., 2007). Here we test the hypothesis that *Drosophila* courtship has evolved through changes in the *fru* and *dsx* loci.
# RESULTS AND DISCUSSION

## Candidate loci for cross-species transfer of courtship

We surveyed drosophilid species with genome resources (Clark et al., 2007) and then chose three species whose courtship is qualitatively different from *D. melanogaster*. In *D. melanogaster*, males first tap the female and give chase, alternating chasing with bouts of unilateral wing extension (singing), and other behaviors such as abdomen curling and licking (Hall, 1994). While courtship in both *D. yakuba* and *D. persimilis* superficially resembles that of *D. melanogaster*, these species also incorporate unique, species-specific elements (Spieth, 1952). *D. yakuba* males often circle the female slowly while shaking both wings (Fig. 1A, Movie S1). Likewise, *D. persimilis* males punctuate unilateral wing extensions by running in front of the female and extending their proboscis while pumping their abdomen up and down, and will usually also simultaneously hold out their wings, stomp, or wave their T1 legs (Fig. 1A, Movie S1). *D. ananassae* males alternate frequent, short (~1/2 second), bilateral, low-angle wing vibrations with lunges at the female and abdomen curling (Singh and Singh, 2003) (Fig. 1A, Movie S1). Further, all four species differ significantly in their courtship song (Demetriades et al., 1999; Markow and O'Grady, 2005; Waldron, 1964; Yamada et al., 2002).

The expression patterns of FruM and Dsx differ subtly between these fly species in the number of cells in specific clusters in the brain ((Usui-Aoki et al., 2005) and Fig. S1A-F). In addition, both loci contain large introns and upstream non-coding regions (Fig. 1B&C), which suggests that they contain extensive cis-regulatory regions (Nelson et al., 2004). Thus, any test of the role of *fru* and *dsx* in courtship evolution must encompass both protein coding and noncoding potential cis-regulatory sequences. We therefore cloned the entire *fru* locus from *D. ananassae* and *D. persimilis* (Fig. 1B), and the entire *dsx* locus from *D. melanogaster*, *D. yakuba*, *D. ananassae* and *D. persimilis* (Fig. 1C) and made *D. melanogaster* transgenics using BAC recombineering and the P[acman] transgenesis system (Venken et al., 2006). We crossed these transgenes into a *D. melanogaster fru* or *dsx* null background, respectively. This effectively replaced the native *D. melanogaster* locus with a heterologous one from another species or with a *D. melanogaster dsx* transgene as a positive control. We were unable to get transformants for a *D. melanogaster fru* positive control, likely due to the poor integration of constructs of this size (~200 kb, Fig. 1B) (Venken et al., 2009). All *dsx* transgenes rescued the *dsxA/dsxA* intersex phenotype (Hildreth, 1965) with only minor defects in sex comb formation (Fig. S2A). Furthermore, despite significant species-specific differences in sex combs and genital morphology (Fig. S1G), all *dsx* rescue flies resembled *D. melanogaster* wild-type (Fig. S1G & Fig. S2A). Except for the *D. ananassae dsx* transgene, this rescue was sufficient to restore fertility (Fig. S2B). This indicates that *dsx* by itself is not sufficient to specify species differences in sex combs or male genital morphology. The *D. ananassae* and *D. persimilis fruitless* transgenes likewise rescued the non-courting phenotype of hypomorphic *fru* mutant males and the pupal lethality of *fru* null mutants (Table S1, Fig. 2B & C).
## Visual analysis of courtship behavior

When we analyzed courtship, we found that all of the transgenes were largely able to rescue the defects in courtship index (CI) seen in *D. melanogaster* *dsx* or *fru* mutants (Table S1). However, when we looked for qualitative changes in *D. melanogaster* male courtship in the transgenics, we found that none of the qualitative species differences depicted in Fig. 1A were transferred. *D. melanogaster* males carrying heterologous transgenes in combination with *D. melanogaster* *dsx* or *fru* null mutations behaved qualitatively like any other *D. melanogaster* male (examples are shown in Movie S2).

While the transgenes failed to transfer the most obvious species-specific elements of courtship behavior, we wondered if they transferred subtle quantitative differences in courtship steps that are shared across the four species. We therefore quantified not only species-specific aspects of courtship, such as the slow circling seen in *D. yakuba*, but also shared aspects, such as the time spent with wings extended or the angle of the wing relative to the body axis (summarized in Table S1). Many of these behaviors are correlated with each other and we therefore performed principal component analysis to identify the statistically independent elements of these behaviors (Joliffe, 1986).

The first two principal components together explain approximately 60-80% of the variance in each of the three different species comparisons (Fig. 2). Males of all three species, *D. yakuba*, *D. ananassae*, and *D. persimilis*, tend to separate from *D. melanogaster* males most strongly along the first principal component axis (Fig. 2). This separation is less strong for *D. persimilis*, probably because not all *D. persimilis* males performed the proboscis-extension/abdomen pumping combination. Wild-type *D. persimilis* males that did not do this behavior clustered close to *D. melanogaster*, reflecting the overall similarity of their courtship. In all cases, the *dsx* and *fru* transgenics—either alone or in combination as transheterozygotes—as well as the *dsx*<sup>Δ</sup>/*dsx*<sup>Δ</sup> negative control clustered with the *D. melanogaster* wild-type and not with the transgene species of origin. This indicates that the transgenes rescued wild-type *D. melanogaster* behavior and did not appear to transfer any quantitative aspects of male courtship behavior between species.

## Analysis of courtship song

Courtship song provides a potentially more sensitive assay of species differences because multiple details of courtship song are quantifiable and variable between species (Markow and O'Grady, 2005). The four species we studied sing songs that differ qualitatively and quantitatively, which is easily seen in the song traces (Fig. 3A) and heard in the audio files (Movie S3). *D. melanogaster* sings with alternating bouts of sine and pulse song (Schilcher, 1976). *D. yakuba* sings with pulse and clack song with a much longer inter-event interval than *D. melanogaster* song (Demetriades et al., 1999). *D. persimilis* sings polycyclic pulses with an inter-pulse interval approximately twice as long as *D. melanogaster* (Noor and Aquadro, 1998). *D. ananassae* sings short bursts of polycyclic buzzes (Yamada et al., 2002). All transgenic males, with the exception of the *D. persimilis dsx/fru* transheterozygote in a double mutant background, sing like *D. melanogaster*, with alternating bouts of pulse and sine song (Fig. 3B, Movie S3). The *D. persimilis dsx/fru* transheterozygous transgenic flies sing normal pulse song but failed to sing sine song. In *D. melanogaster*, *dsx* and *fru* act in
concert to regulate sine song (Shirangi et al., 2013). It is possible that the *D. persimilis*
*dsx/fru* trangenes are weak hypomorphic alleles that in combination fail to complement
redundant *dsx* and *fru* sine song functions. Regardless, there was no evidence for species-
specific song interconversion. In all cases, even the quantitative features of song, such as the
inter-pulse interval (Fig. 3), resemble *D. melanogaster* song.

**Conclusions**

We tested the hypothesis that elements of species-specific courtship behavior are encoded
by the two known master-control genes for courtship behavior in *D. melanogaster*,
*doublesex* and *fruitless*. We tested the role of both coding and *cis*-regulatory regions by
performing BAC transgenesis of the entire *dsx* and *fru* genes from each of three species into
*D. melanogaster* flies deficient for each of the native loci. Our experiments reveal that all of
these transgenes are sufficient to rescue courtship behavior to near wild-type levels and that
all transgenes rescued normal *D. melanogaster* courtship behavior. We found no evidence
that any qualitative or quantitative details of courtship behavior were transferred between
species.

This result stands in stark contrast to a large and growing body of evidence that the
evolution of candidate genes often contributes to morphological diversity (for example,
(Prud'homme et al., 2006; Tomoyasu et al., 2009)). Unfortunately, there are currently no
other strong candidate genes for courtship behavior available with which to test this
hypothesis further. Instead, other approaches are required to unravel the genetic changes that
underlie behavioral diversity. While the *fru* and *dsx* genes themselves are not substrates for
courtship evolution in *Drosophila* species, the same cannot be said about *Fru*<sup>M</sup> and *Dsx*
positive neurons. It may be possible to leverage the genetic tools developed for these genes
in *D. melanogaster* to examine the function of these cells in other *Drosophila* species using
next-generation genome editing tools such as CRISPR (Gratz et al., 2013). Another
approach is the genetic mapping of strain and species differences (Cande et al., 2012; Gould
et al., 2010; Kitano et al., 2009). However, current methodologies provide only limited
resolution of the genomic regions contributing to behavior differences (Mackay, 2009) and
identification of the causal genes remains a significant challenge.

**EXPERIMENTAL PROCEDURES**

Fly stocks and crosses

The wild-type species stocks used for quantitative analysis were those corresponding to the
sequenced genomes and BAC resources, and are available from the San Diego species stock
center with the following stock numbers: *D. yakuba* #14021-0261.01, *D. ananassae*
#14024-0371.13 and *D. persimilis* #14011-0111.49 and #14011-0111.50. Species-specific
behaviors were confirmed with multiple additional lines (*D. ananassae* #14024-0371.16,
#14024-0371.22 and #14024-0371.34; *D. persimilis* #14011-0111.41; additional *D. yakuba*
lines described in Cande et. al. 2012 (Cande et al., 2012)). The *D. melanogaster* wild-type
used was Canton S. Generation of the custom *dsx* deletion used in the transgene rescue male
flies is described below. *fru*<sup>3</sup> is a *P*-element insertion near the P1 promoter described in
(Goodwin et al., 2000), and the *fru*<sup>DF-Exel6179</sup> deletion is described in (Parks et al., 2004).
The *fru*<sup>AJ96u3</sup> allele (Song et al., 2002) and *dsx*<sup>1</sup> *fru*<sup>P1lexA</sup> (Hildreth, 1965; Mellert et al., 2010) recombinant were the generous gift of Bruce Baker's lab. All transgenes were inserted into the VK16 AttP site on chromosome 2 (Venken et al., 2006), and all *fru* and/or *dsx* alleles were balanced over TM6B. A modified Stinger (Barolo et al., 2000) construct with an AttB site and mini-white marker was knocked into the same VK16 site and used as a negative control. All crosses and species were raised on cornmeal-agar media at 20-22 °C according to standard methods.

## Generation of the *fruitless* and *doublesex* transgenics

Filters spotted with the BAC libraries for *D. yakuba*, *D. ananassae* and *D. persimilis* (DY Ba, DA Ba and DP Ba, respectively, from Arizona Genomics Institute) were probed via standard Southern hybridization with ~500 bp probes labeled through random primer extension by incubating 50ng template DNA in the presence of Klenow and random hexamers to incorporate P<sup>32</sup> dCTP. Labeled filters were analyzed via phosphoimaging on a Fujifilm FLA-5000 phosphoimager. *D. yakuba* *dsx* (coordinates 3R: 6474025-6528771, Flybase release R1.3, cloned from the bac clone at address 27I2), *D. ananassae* *dsx* (scaffold_13340: 14858455-14908776, clone address 21E22), *D. ananassae* *fru* (scaffold_13340: 9713578-9867775, clone address 20H8), *D. persimilis* *dsx* (scaffold 0: 3349345-3409770, clone address 31M5), and *D. persimilis* *fru* (scaffold 0: 8550968-8730455, clone address 36N18) were cloned from the BAC library clones into the P[acman] transgenesis vector using BAC recombineering (Venken et al., 2006). *D. melanogaster* *dsx* (3R: 3749186-3800800) was recombineered from RPCI-98 (CHORI) clone 36E18. Recombineered clones were checked for proper left and right hand integration into P[acman] via PCR, and were also digested with clone-appropriate restriction enzymes and run on a pulse field gel prior to injection. VK16 flies were injected with 100 ng/uL transgene DNA that had been midi-prepped from 100 ml of culture induced 4-6 hours with 0.1% arabinose and purified using a Nucleobond Xtra midi kit (Machery-Nagel reference 740410.50). DNA preps were stored at 4 °C and injected in the next 3 days.

## Generation of the *D. melanogaster* *dsx* deletion

The FDD-0250107 deletion (Parks et al., 2004) was generated by placing the f00683 and d06446 FRT insertions in *trans* in combination with a HS-FLP on X (BSC1929) and heat shocking as described in (Parks et al., 2004). Backcross progeny were screened for the presence of the deletion using two sided PCR with primers: WHXLeft 5' - CCTCGATATACAGACCGATAAAAC-3' WHXRight 5' - TACTATTCCTTTCACTCGCACTTATTG-3' dsxDelF 5' - TGTAGTTGGCCAGGATTAGTGAGC-3' dsxDelR 5' - TGAGTGGTTCGACCTATATCGTC-3' *dsx*<sup>Δ</sup> homozygous flies had an intersex phenotype consistent with previously described *dsx* null mutations (Hildreth, 1965). A *dsx*<sup>Δ</sup> *fru*<sup>Δ</sup> double mutant chromosome was generated by recombining the new *dsx* deletion with *fru*<sup>DF-Exel6179</sup> and screening for recombinants using the *dsx* primers dsxDelF and WHXLeft, and the *fru*<sup>DF-Exel6179</sup> specific primers XP5'minu 5'-TTTACTCCAGTCACAGCTTTG-3' (Parks et al., 2004) and frude 3' 5'-ACACGATCATGTGCAACTGATAAG-3'.
Visual analysis of courtship behavior

Flies were kept at a 20-22°C, 50% humidity, with a constant 12 hour day/night cycle. Male
flies were isolated upon eclosion using a light application of CO2, aged to 4-6 days (except
*D. persimilis*, which were aged 5-7 days) and aspirated into plexiglas courtship chambers
with a hemispherical well 15 mm in diameter and 3 mm deep. Virgin females were handled
similarly, except that they were kept in groups of up to 50 flies. All movies were shot on a
JVC color video camera TK-C1481BEG mounted on a Leica Z6 APO zoom system. Video
capture was performed using Pinnacle Video Capture 1.0.1 software (Elgato systems) set at
640×480 pixels, 25 fps. All movies were shot in the first 3 hours after dawn, and lasted 30
minutes or until copulation, except for the wild-type *D. yakuba* movies which were taken
from (Cande et al., 2012) and are 15 minutes long.

All *D. melanogaster* wild-type and BAC transgene flies were pooled into three lots,
randomized and scored blind. This was not possible with the other wild-type species as they
are readily distinguishable based on external morphology. Video analysis was done using
Annotation 1.0. Courtship index (time spent courting / total time filmed) was calculated for
the full video, while the other parameters were calculated from the first 10 minutes. Wing
extension index (WEI) was calculated as the time spent courting with wing(s) extended /
time spent courting, wing extension frequency as the total number of wing extension events /
time spent courting (likewise with abdomen curling frequency) and wing extension duration
as the average length of time (in seconds) of all wing extension events per fly. Extremely
short wing extensions (< ½ second) were not counted to exclude flicks and scissoring from
the analysis, except for the *D. ananassae* wild-type species, in which nearly all wing
extensions were less than ½ second. Wing angle was measured by drawing one line through
the center of the fly, and another from the tip of the extended or contralateral wing, through
the wing hinge, and connecting to the line through the fly’s center. The angle between these
two lines was measured in Illustrator, and for each fly 10 different wing extensions were
measured at their maxima and averaged. The *D. yakuba* species - specific behavior of wing
shaking while slowly circling was broken into its two components, and each was scored
separately. The *D. persimilis* behavior of running in front of the female, extending the
proboscis, abdomen pumping, holding the wings out and stomping was scored both in its
entirety, and also just the proboscis extension (without licking) element, as this was an
easily recognizable signature of this behavior. All species - specific behaviors were
normalized to the total time spent courting. 16-30 males were scored for each species or
genotype, with the exceptions of the *fru*³/*fru*DF-exel6179 negative control (12 males) and the
*D. persimilis fru*D. per fru; *fru*³/*fru*DF-Exel6179 transgenic (14 males). Males with a CI lower
than 0.1, or that spent less than 100 seconds courting were dropped from additional analysis
after the CI was calculated. This included ~50% of the *dsx*Δ/*dsx*Δ negative control
individuals who were presumably XX rather than XY intersexes. All behavior data analysis
and plotting were done in R (R Development Core Team, 2013). Principal component
analysis was carried out using the prcomp function with centering and scaling, and 95%
confidence ellipses were calculated using dataEllipse. Species specific variables - i.e. slow
circling and wing shaking (*D. yakuba*) and proboscis extensions (excluding licks) and the
proboscis extension/abdomen pumping/stomping combination (*D. persimilis*) - that were not
performed by the species under comparison were not included in a given principal component analysis.

## Analysis of courtship song

Courtship song was recorded and analyzed as described in (Arthur et al., 2013)

## Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

## Acknowledgments

We would like to thank Serge Alonso for his help probing the BAC filters, Justine Magrina for embryo injections, and Michelle Arbeitman and Ilona Grunwald for anti-Dsx and anti-Dlg antibodies, respectively. This work was supported by an HFSP long term fellowship (LT000708/2010-L to JC), FSER, FRM, EURYI, CNRS, and the France-BioImaging/PICsL (ANR-10-INSB-04-01), call "Investissements d'Avenir"), as well as the Howard Hughes Medical Institute. We acknowledge Flybase for information support.

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HIGHLIGHTS

* innate male courtship behavior evolves rapidly in *Drosophila* species

* *fruitless* and *doublesex* are known to control male courtship in *D. melanogaster*

* cross-species transgenics test *fruitless* and *doublesex* for a role in behavior evolution

* *fruitless* and *doublesex* are not involved in courtship behavior evolution
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 1.**</figcaption></figure>

Candidate species courtship behaviors and candidate genes. (A) From left to right: a *D. melanogaster* male licking and abdomen curling with wing extended; a *D. yakuba* male wing shaking and circling (arrow); a *D. ananassae* male singing (arrows indicate wing movements and lunges); *D. persimilis* runs in front of the female (arrow) and extends his proboscis, holds his wings out, stomps and pumps his abdomen up and down (small arrows).
(B) The *D. melanogaster* *fru* locus, showing the *D. ananassae* and *D. persimilis* *fru* transgenes (green bars), the two deletions (*fru*DF-Exel6179 (Parks et al., 2004) and *fru*AJ96u3
(Song et al., 2002), gray bars), and the location of the *fru*³ *P*-element insertion (Goodwin et al., 2000). (C) The *D. melanogaster* *dsx* locus, showing the *dsx* transgenes (the red bar for *D. melanogaster*, the orange bars for cross-species transgenes, and the gray bar for the *dsx* deletion (FDD-0250107) (Parks et al., 2004).
<figure><img src="image_6.png" /><figcaption>B *D. ananassae*</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>C *D. persimilis*</figcaption></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Figure 2.**</figcaption></figure>

Principal component analysis of courtship in wild-type species and heterologous *dsx* and *fru* transgenics. The data are divided by species. In each panel males from the wild-type transgene species of origin – *D. yakuba* (A), *D. ananassae* (B) and *D. persimilis* (C) – are plotted in filled red circles while *D. melanogaster* wild-type are plotted in filled black circles. Species specific transgenes in an endogenous *D. melanogaster* mutant background are plotted as described by the genotype key at the lower right. *fru*<sup>AJ96u3</sup>/*fru*<sup>DF-exel6179</sup> males carry a transheterozygous deletion and are *fru* null. 95% confidence ellipses are indicated.
<figure><img src="image_10.png" /><figcaption>**Figure 3.**</figcaption></figure>

Courtship song in wild-type species and *dsx* and *fru* transgenics. (A) The four wild-type species. (B) *dsx* and *fru* transgenics, the genotypes are the same as in Fig. 2. In both panels a 3 second interval is shown at left and a 100 msec interval of representative song pulses (or pulses and clacks, *D. yakuba*, panel A) is shown at right.