Lack of Evolutionary Divergence in Courtship Songs ...

Journal of Insect Behavior, Vol. 13, No. 2, 2000

Short Communication

Lack of Evolutionary Divergence in Courtship Songs of Drosophila pseudoobscura Subspecies Mohamed A. F. Noor,1,3 Marcus A. Williams,1 Diana Alvarez,2 and Manuel Ruiz-Garcıá2 Accepted November 11, 1999; revised December 2, 1999 KEY WORDS: courtship song; wingbeat; sexual isolation; Drosophila. It’s just the same old song but with a different meaning since you’ve been gone. —Holland, Dozier, and Holland as interpreted by the Four Tops (1965)

INTRODUCTION Elements of courtship song have been implicated as means by which females select conspecific mates in several Drosophila species (e.g., Ewing and Bennet-Clark, 1968; Cowling and Burnet, 1981; Kyriacou and Hall, 1982; Hoikkala and Lumme, 1987; Crossley and Bennet-Clark, 1993; Tomaru et al., 1995). Courtship songs differ greatly among closely related Drosophila species (e.g., Chang and Miller, 1978; Cowling and Burnet, 1981; Geng et al., 1989; Hoikkala et al., 1994; Ritchie and Gleason, 1995), suggesting that their rate of divergence is rapid. If courtship song is generally important in causing sexual isolation between species, such rapid divergence may help to ‘‘drive’’ the process of speciation. In a recent survey, Gleason and Ritchie (1998) compared the rates of divergence in courtship song, sexual isolation, and postmating isolation (hybrid sterility) among species of the Drosophila willistoni group. They found that courtship song diverged the most rapidly, followed by sexual isolation, and finally by hybrid sterility. This finding may suggest that court1

Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803. 2 Department of Biology, Pontificia Universidad Javeriana, Carrera 7 No. 43-82, Bogota, Colombia. 3 Correspondence should be sent to Mohamed A. F. Noor, e-mail: [email protected] 255 0892-7553/00/0300-0255$18.00/0  2000 Plenum Publishing Corporation

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Noor, Williams, Alvarez, and Ruiz-Garcıá

ship song has driven speciation in this group, though other interpretations are possible. To evaluate the generality of Gleason and Ritchie’s (1998) observation on the relative speed of courtship song divergence, we have compared the courtship song of two Drosophila pseudoobscura subspecies: D. p. pseudoobscura and D. p. bogotana. Drosophila p. pseudoobscura is found along much of western North America, while D. p. bogotana is geographically isolated in the vicinity of Bogota, Colombia. The estimated divergence time between these subspecies based on genetic data is 75,000 to 150,000 years (Schaeffer and Miller, 1991; Jenkins et al., 1996; Alvarez et al., 1999a), and these taxa have been used extensively in studies of speciation (e.g., Prakash, 1972; Orr, 1989; Noor, 1995a; Wang et al., 1997). We have also detected virtually no genetic differentiation among populations within these subspecies (Alvarez et al., 1999a; Noor et al., 2000). The courtship songs of D. pseudoobscura and its sibling species, D. persimilis and D. miranda, have already been shown to differ in their interpulse interval (IPI) and intrapulse frequency (Waldron, 1964; Noor, 1998). However, these species possess strong sexual isolation and postmating isolation, so comparisons of relative rates of divergence are not useful. In contrast, hybrid males between D. p. pseudoobscura and D. p. bogotana are sterile when the mother is D. p. bogotana but fertile in the reciprocal hybridization (Prakash, 1972). Sexual isolation between these subspecies is low but statistically significant (Noor, 1995a). As moderate postmating and sexual isolation exist between these subspecies, substantial courtship song differences should be detectable between them if song generally diverges more quickly.

METHODS Stocks All flies were maintained on standard dextrose–yeast–agar medium at 20⬚C in a 12 : 12 light–dark cycle. The D. pseudoobscura Flagstaff stock was a combination of three isofemale lines collected in Flagstaff, Arizona, in 1993, and has been used extensively in previous behavioral studies (e.g., Noor, 1995b; Noor, 1996; Noor, 1997b; Noor and Aquadro, 1998). The D. pseudoobscura Goldendale strain was collected from Goldendale, Washington, in 1996 (Noor and Aquadro, 1998). The D. p. bogotana isofemale lines from Susa and Sutatausa were collected in 1997 from the corresponding locations in Colombia.

Courtship Songs in Drosophila pseudoobscura

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Pairings All males and females used in this study were separated by sex and housed for 8 days at 20⬚C after eclosion prior to assay. On the last day, males were separated into individual food vials to reduce crowding-mediated courtship inhibition (Noor, 1997a). Recordings were done in two sets: males from Flagstaff, Susa 6 and Sutatausa 5 were assayed in February, 1999, and males from Goldendale, Susa 2 and Sutatausa 3 were assayed in June, 1999. Hence, one line of D. p. pseudoobscura and two lines of D. p. bogotana were assayed simultaneously to control for environmental effects on courtship song elements, which are known to exist in this species (Noor and Aquadro, 1998). Further, all strains from each set were assayed on the same mornings to control for day-to-day environmental effects, and individual recordings were alternated among the three strains to control for withinday environmental effects. Song Analyses We measured courtship song parameters by confining a single male with a single female inside an upgraded INSECTAVOX (Gorczyca and Hall, 1987) at room temperature (21 ⫾ 1⬚C) beginning at 0900 hr. Generally, about 15 recordings were done each morning, and no recordings were made on or after 1100 hr. The INSECTAVOX was upgraded such that light was transmitted through a clear plastic cylinder into the chamber, hence reducing the chamber heating that often occurred in older models. The INSECTAVOX was connected through an amplifier to a Macintosh Powerbook running Canary sound analysis software. At least twenty individuals from each strain were recorded. We measured IPI directly from the waveform tracings of Canary, and calculated intrapulse frequencies as the inverse of time between the first two consecutive peaks in a sound pulse. Mean values from each male were then used in the analyses. We used analyses of variance (ANOVAs) to test for heterogeneity among strains within a set (month) in courtship song parameters. These statistical analyses were performed using StatView software or by software written by the authors (nested ANOVA). The nested analyses of variance had low power since only two strains from one subspecies and one strain from the other were compared, but they are presented to show partitioning of variation. RESULTS The courtship songs of Drosophila pseudoobscura bogotana were very similar to those of D. p. pseudoobscura in both their mean interpulse

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intervals and mean intrapulse frequencies (Tables I and II). Table I presents the maximum and minimum mean per male mean values for each of these parameters. The range of individual mean values overlapped greatly between the subspecies within each of the two sets. Further, the mean values and ranges for the courtship song elements of the D. p. pseudoobscura strains were consistent with previously reported mean values for these song characters in numerous other strains of this subspecies (Waldron, 1964; Noor and Aquadro, 1998). Hence, courtship song cannot be used to reliably distinguish D. pseudoobscura subspecies (Noor, 1998). Though large and highly significant differences were observed between the two sets (months) of recordings (p ⬍ 0.0001), these differences likely result from environmental differences present in the laboratory at the times of the recordings. In the first set, the two bogotana strains each had significantly higher average IPIs than the Flagstaff pseudoobscura strain (p ⬍0.0001), but there was no significant heterogeneity among the strains in intrapulse frequency. In the second set, there was no significant heterogeneity among the strains in IPI, but the Susa line of D. p. bogotana had a significantly lower intrapulse frequency than either D. pseudoobscura from Goldendale (p ⬍ 0.0001), or D. p. bogotana line 3 from Sutatausa (p ⫽ 0.0110). contributing to a nearly significant variation among strains overall (Table II). However, in this set, the Goldendale D. pseudoobscura line had a higher mean IPI than either of the two D. p. bogotana strains. Table I. Comparison of Courtship Song Elements in Subspecies of Drosophila pseudoobscura Subspecies

Mean ⫾ SE

Strain

Minimum

Maximum

Interpulse interval Set 1 bogotana bogotana pseudoobscura Set 2 bogotana bogotana pseudoobscura

Susa 6 Sutatausa 5 Flagstaff

42.6 ⫾ 0.6 ms 42.7 ⫾ 0.9 ms 39.0 ⫾ 0.6 ms

35.4 ms 36.5 ms 35.7 ms

46.0 ms 51.5 ms 44.7 ms

Susa 2 Sutatausa 3 Goldendale

36.5 ⫾ 0.3 ms 37.4 ⫾ 0.4 ms 37.9 ⫾ 0.5 ms

34.6 ms 34.8 ms 33.4 ms

39.3 ms 40.0 ms 43.5 ms

Intrapulse frequency Set 1 bogotana bogotana pseudoobscura Set 2 bogotana bogotana pseudoobscura

Susa 6 Sutatausa 5 Flagstaff

255 ⫾ 14 Hz 238 ⫾ 5 Hz 232 ⫾ 4 Hz

214 Hz 214 Hz 201 Hz

508 Hz 307 Hz 288 Hz

Susa 2 Sutatausa 3 Goldendale

228 ⫾ 5 Hz 253 ⫾ 8 Hz 261 ⫾ 4 Hz

183 Hz 207 Hz 233 Hz

275 Hz 314 Hz 325 Hz


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Table II. Nested Analysis of Variance Results for Interpulse Interval and Intrapulse Frequency, Also Partitioning the Observed Variation df

SS

MS

F

p

% Var

Interpulse interval Set 1 Subspecies Strains Error Set 2 Subspecies Strains Error

1 1 58

180.6 6.1 597.3

180.6 6.1 10.3

2968 5.91

⬍0.001 ns

40.6 0a 62.3

1 1 57

12.0 8.9 195.7

12.0 8.9 3.4

1.35 2.59

ns ns

3.0 7.1 89.8

Intrapulse frequency Set 1 Subspecies Strains Error Set 2 Subspecies Strains Error

1 1 58

2931 3255 97,554

2931 3255 1682

0.90 1.94

ns ns

0a 4.4 96.2

1 1 57

5573 6462 41,896

5573 6462 735

0.86 8.79

ns ⬍0.005

0a 29.0 74.4

a

The nested ANOVA yielded a negative number for the amount of variation attributable to these characters.

Partitioning the variation using nested analyses of variance showed that most of the variation was generally observed among males within strains (Table II). As mentioned above, some variation was observed among strains within the D. p. bogotana subspecies for intrapulse frequency, though there was a difference between the subspecies surveyed in the first set for IPI (see above). We discuss the significance of these findings below.

DISCUSSION Although significant differences in some courtship song elements were observed between some strains of Drosophila pseudoobscura pseudoobscura and D. p. bogotana, there were no trends suggesting consistent differences between these subspecies in any courtship song elements. This finding seems to contradict the assertion of Hoenigsberg (1995) about evolutionary forces driving divergence in sexual behaviors in D. p. bogotana. Previous investigations have noted some sexual isolation and hybrid sterility between these subspecies (Prakash, 1972; Noor, 1995a). Similarly, chromosomal rearrangement frequencies and some meristic morphological characteristics differ between North American and Colombian D. pseudoobscura subspecies (Ruiz-Garcıá and Alvarez, 1997; Alvarez et al., 1999b). However, de-

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spite the evolution of these many differences, the courtship songs of these subspecies have not diverged from each other substantially. In contrast, although there is no sexual isolation among strains within these subspecies (e.g., Anderson and Ehrman, 1969), we have observed significant differences among strains in courtship song elements (Noor and Aquadro, 1998, and this study), as also noted by Ritchie and Gleason (1995) in D. willistoni. The evolutionary significance of such variation within the D. pseudoobscura subspecies yet no divergence between them is unclear, but it might suggest that song traits evolve in a selectively neutral manner in this species so long as they are within a range of values. A previous study suggested that courtship song IPI in particular may not contribute to species discrimination between Drosophila pseudoobscura and D. persimilis (Noor and Aquadro, 1998). Despite a large difference between these species in IPI, hybrid male mating success to females of either species appeared uncorrelated with differences in song IPI. Our current result may suggest that divergence in IPI between these species occurred after reproductive isolation between them was already strong. The lack of divergence in courtship song between Drosophila pseudoobscura subspecies contrasts an observation in the Drosophila willistoni group (Gleason and Ritchie, 1998), where courtship song elements (and interpulse interval in particular) diverged between taxa more quickly than sexual isolation or postmating isolation evolves. Other authors have also suggested that courtship song, and IPI in particular, may be important in species recognition in a variety of Drosophila species (e.g., Cowling and Burnet, 1981). Our negative result is important because it demonstrates that courtship song does not universally diverge between taxa faster than the evolution of known forms of reproductive isolation, and thus courtship songs may not be universally important in the speciation process in Drosophila. ACKNOWLEDGMENTS We thank K. McCutcheon for generating preliminary data. H. McGuire, M. Ritchie, and an anonymous reviewer provided helpful comments on the manuscript. This work was funded by National Institutes of Health Grant GM58060-01, subcontracted through J. Hey at Rutgers University to MAFN and MAW. REFERENCES Alvarez, D., Noor, M. A. F., and Ruiz-Garcıá, M. (1999a). Comparative genetic structure among Colombian and North American Drosophila pseudoobscura populations using five microsatellite loci. Unpublished ms.


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