Temperature Effects on the Temporal Properties of Calling Songs in ...

C 2006) Journal of Insect Behavior, Vol. 20, No. 1, January 2007 ( DOI: 10.1007/s10905-006-9061-0

Temperature Effects on the Temporal Properties of Calling Songs in the Crickets Gryllus fultoni and G. vernalis: Implications for Reproductive Isolation in Sympatric Populations Yikweon Jang1,2,3 and H. Carl Gerhardt1 Revised October 17, 2006; accepted October 24, 2006 Published online: December 16, 2006

Two closely related wood-cricket species, Gryllus fultoni (Orthoptera: Gryllidae) and G. vernalis, produce similar calling songs, consisting of 3-pulse chirps. Analysis of field and laboratory recordings of calling songs showed that, after correction to a common temperature, there was a divergence in chirp and pulse rates between far allopatric populations of G. fultoni and populations sympatric with G. vernalis. To determine whether the divergence in calling songs potentially provides reproductive isolation between G. fultoni and G. vernalis throughout the temperature range over which these insects sing, we recorded calling songs of lab-reared G. fultoni and G. vernalis populations between 18 and 28◦ C. Mean chirp rate significantly differed between sympatric and far allopatric G. fultoni populations as well as between sympatric G. fultoni and G. vernalis populations. Although there was a significant difference in mean pulse rate between sympatric G. fultoni and G. vernalis populations, pulse rate did not differ between sympatric and far allopatric G. fultoni populations in the laboratory study. Considering the magnitudes of differences in calling song characters discriminated by females of G. fultoni and the mean differences and the variability in calling song characters between the two species, the joint difference in chirp and pulse rates may

1 Division

of Biological Sciences, University of Missouri, Columbia, MO 65211, USA. of Life Science, Ewha Womans University, Seoul, 120–750, Republic of Korea. 3 To whom correspondence should be addressed; e-mail: [email protected]. 2 Department

33 C 2006 Springer Science+Business Media, LLC 0892-7553/07/0100-0033/1

34

Jang and Gerhardt

be adequate for species discrimination over most of the range at which these crickets breed. KEY WORDS: calling song; Gryllus fultoni; Gryllus vernalis; reproductive character displacement; reproductive isolation.

INTRODUCTION Temperature has a profound effect on acoustic communication in insects and frogs (Walker, 1962; Gerhardt and Huber, 2002). Except for some katydids and cicadas, in which singing itself can generate heat in the stridulatory muscles and raise body temperature (e.g., Josephson, 1984; Sanborn, 2001), many call characteristics of ectotherms depend on ambient (or water) temperature. Temperature can fluctuate widely during a season or even a single night, and these variations may present a problem for sympatric species in the context of mate recognition. The structures of calling songs are often similar among closely related sympatric species, and differences in the same specific calling song characters may be important in species recognition. As temperature changes, the rates of change in such characters often may also differ between species ( = non-parallel slopes of temperature regressions; see Walker, 1962). One consequence is that differences in calling song characters between two sympatric species may be sufficient for species recognition at some temperatures, but too narrow for reliable differentiation to occur at other temperatures. Overlap or insufficient differences in the values of key identifying call properties may, in turn, result in heterospecific pairing. The consideration of temperature effects on calling songs is particularly critical for assessing reproductive character displacement (RCD), a geographic pattern in which differences in reproductively isolating traits of two taxa are greater in sympatry than in allopatry (Brown and Wilson, 1956; Grant, 1972; Howard, 1993; Gerhardt and Huber, 2002). This pattern of sympatric divergence arises from selection against the adverse consequences of hybridization between sympatric species (Dobzhansky, 1940; Howard, 1993). This process is termed reinforcement (sensu Blair, 1955) and may contribute to speciation (Servedio and Noor, 2003). Thus one criterion for reinforcement is that the species differences in the trait promote species isolation in areas of sympatry. Furthermore, the pattern of RCD as well as reproductive isolation might be expected to be maintained under various environmental conditions that are typically encountered in the field. Thus, for example, a complete assessment of RCD includes an analysis of sympatric divergence in species-identifying call characteristics over the entire range of breeding temperature.

Temperature Effects on the Temporal Properties

35

Fig. 1. Oscillograms of the G. fultoni (a) and G. vernalis (b) calling songs. Calling songs of both G. fultoni and G. vernalis consist of repeating chirps, each usually consisting of three pulses. See the main text for definitions of calling song characters.

In the eastern United States, two wood-cricket species, Gryllus fultoni (Alexander) and G. vernalis Blatchley (Orthoptera: Gryllidae), occur together in an area between eastern Kansas and northwest of the Appalachian Mountains (Jang and Gerhardt, 2006a). The two species have similar calling song structure, consisting of three-pulse chirps (see Fig. 1). Alexander (1957) first noted qualitative differences in calling songs between sympatric and allopatric populations of G. fultoni. A formal analysis of geographic variation in calling songs of G. fultoni, corroborated by studies of the songs of laboratory-reared crickets, showed that values of chirp and pulse rates were highest in sympatric populations, lowest in far allopatric populations, and intermediate in the near allopatric populations that were located close to sympatry (Jang and Gerhardt, 2006a). As a result, divergence in chirp and pulse rates between the two species was greater in areas of sympatry than in areas of allopatry. The pattern of female mate discrimination in G. fultoni also parallels the divergent pattern of chirp and pulse rates between sympatric and far allopatric populations (Jang and Gerhardt, 2006b). At a common temperature, this female selectivity would

36

Jang and Gerhardt

be expected to significantly reduce heterospecific mating attempts in sympatry, consistent with predictions of RCD. There was no statistical difference in calling song characters between sympatric and allopatric G. vernalis populations (Jang et al., 2007). In this study, we recorded calling songs of both G. fultoni and G. vernalis at temperatures ranging from 18 to 28◦ C to assess temperature effects on calling songs. Because calling songs recorded in the field may be influenced by unknown environmental differences between sympatric and allopatric populations during development or adult period, we used crickets that were raised in the laboratory under the standard conditions. The temperature range that we recorded for this study largely coincides with the temperature range over which both G. fultoni and G. vernalis produce calling songs in the field. We discuss the temperature effects on calling songs of both G. fultoni and G. vernalis from a viewpoint of RCD and reproductive isolation between the two species in sympatry.

METHODS Study Populations Adults of both species were caught in the field in May 2002, and their progeny were reared from eggs to adults in the lab. Four populations of G. fultoni used for this study were as follows: one far allopatric population from Jackson, Georgia (JK, 35; approximately 250 km to the nearest sympatry); one near allopatric population from Rising Fawn, Georgia (RF, 23; approximately 34 km to the nearest sympatry); and two sympatric populations from Dawson Springs, Kentucky (DS, 27) and Goreville, Illinois (GR, 19). The letters and numerals in parentheses indicate the abbreviation for the locality and the number of field-caught females used for egg collection, respectively. In addition, crickets from one sympatric population of G. vernalis from Dawson Springs, Kentucky (DS, 35), were studied. Crickets were reared in plastic bins (33 × 50 × 29 cm) with holes on the side for ventilation. Both juvenile and adult crickets were provided with cricket chow (Fluker Farms, Baton Rouge, Louisiana, USA), lettuce, and shelter ad libitum. All crickets were maintained at 23 ± 1◦ C and with a 14 h:10 h, light:dark photoperiod during development. Virgin male crickets were drawn randomly from each population for recording and were housed in individual containers (12 × 12 × 9 cm). Age of crickets was the number of days between the final molting and recording. Males aged between 7 and 40 days were recorded during the scotophase.


37

Calling Song Recording and Analyses Because ambient temperature is a good predictor of cricket body temperature and because calling does not appreciably raise body temperature above ambient temperature (Toms et al., 1993; Martin et al., 2000), we measured air temperature near crickets. Cricket calling songs were recorded in a temperature-controlled anechoic chamber (3 × 3 × 2 m). Each male was housed in a container (12 × 12 × 9 cm) with a screened lid. The temperature of the room was measured using a thermocouple probe (Model 450-AKT, Omega Engineering, Inc., Stamford, Connecticut, USA) that was placed in the chamber about 10 cm from calling males. Adequate numbers of calling songs were recorded within three temperature ranges, which encompassed a large proportion of the natural calling temperatures (see Jang and Gerhardt, 2006a): 18–20◦ C; 22–23◦ C; and 26–28◦ C. Crickets were allowed to acclimate to a particular calling temperature for at least 30 min, and each male was recorded within at least two different temperature ranges. A Sennheiser microphone (ME 66 shotgun head + K6 powering module; frequency response: 50–20,000 Hz ± 2.5 dB) was placed 58 cm directly above the container. Output from the microphone was fed into a UA5 audio interface (Edirol Corp., Bellingham, Washington, USA) operating at a sampling rate of 44.1 kHz. The audio interface was connected to a PC with which singing crickets were recorded for 1 min. Cricket signals were analyzed using Raven 1.1 (Cornell Laboratory of Ornithology, Ithaca, New York, USA) installed on a PC. The calling songs of G. fultoni and G. vernalis consist of repeating chirps, each usually comprising three pulses (Fig. 1). We measured the beginning and end of all pulses in at least 12 consecutive chirps for statistical analyses. The beginning and end of a pulse were defined as the first and last digitized samples, respectively, whose absolute values exceeded three times the absolute digitized value of the peak background noise level. Pulse duration (PD) is defined as the time between the start and the end of a pulse. Pulse period is the time between the start of one pulse and the start of the subsequent pulse. Pulse rate (PR) is the inverse of the pulse period. Chirp period is the time between the start of one chirp and the start of the subsequent chirp. Chirp rate (CR) is the inverse of the chirp period. Carrier frequency (CF) refers to the frequency with the most acoustic energy. Values of CF were obtained from spectrogram analyses with 124-Hz filter bandwidth, 512-sample Fast Fourier Transformation size, and the Hanning window function. Calling song characters analyzed in this study were CR, PR, PD, and CF. The values of a calling song character obtained from all chirps in a recording were averaged to represent the value of the calling song character for the recording.

38

Jang and Gerhardt

Statistical Analyses We organized the calling song recordings into two data sets: (1) a data set consisting of recordings of G. fultoni from one near allopatric (RF), and one far allopatric (JK), and two sympatric populations (DS and GR); and (2) a data set consisting of recordings of G. fultoni from two sympatric populations (DS and GR) and of G. vernalis from one of the same sympatric populations (DS). Analysis of the first data set addressed the question of whether the three groups of G. fultoni (sympatric, near allopatric, and far allopatric) differed in calling song characters throughout the tested temperature range. Analysis of the second data set addressed the question of whether G. fultoni and G. vernalis in sympatry differed in calling song characters throughout the tested temperature range. All statistical procedures in this study were conducted with SPSS (ver. 11, SPSS Inc., Chicago, Illinois, USA). To test whether sympatric and allopatric G. fultoni populations differed in their calling song characters, we employed repeated measures analysis of covariance (ANCOVA) with linear mixed models (Mixed procedure, see SPSS, 2001). The ANCOVA models included two covariates (temperature and age) and two independent variables (zone and locality). The actual temperature was used for all analyses, unless otherwise indicated. Zone indicated whether a population was sympatric or allopatric. Locality, which referred to the population of the individual, was nested within zone. ANCOVA models were not converged when age and temperature were random variables, thus we set them to fixed-effect variables. The effects of repeated measurements were estimated using the unstructured variance-covariance structure for all calling song characters. Because age (F ≤ 3.623, P ≥ 0.072) and all interactions involving age (F ≤ 4.182, P ≥ 0.055) were not significant for any calling song character, the repeated measures ANCOVA were performed without age in subsequent statistical analyses. We used the same statistical procedures to test whether sympatric populations of G. fultoni and G. vernalis differed in calling song characters throughout the tested temperature range. We used the repeated measures ANCOVA on the sympatric data set. The variable “zone” was replaced with “species” in this data set, which indicated whether an individual belonged to G. fultoni or G. vernalis. We also used least-squares linear regression analyses to visualize the effects of temperature on male calling song characters for each population. Calling song characters were distributed normally (P ≥ 0.063, Kolmogorov-Smirnov test with Lilliefors significance correction) in all populations.


39

RESULTS Temperature Variation in the Field In the field, males of sympatric populations of G. fultoni were recorded at temperatures between 16.24 and 27.33◦ C (Fig. 2; n = 92; see Jang and Gerhardt, 2006a, for more information), and males of allopatric populations were recorded at temperatures between 14.47 and 26.89◦ C (n = 99). Males of sympatric populations of G. vernalis were recorded at temperatures between 17.52 and 27.22◦ C (n = 90). Thus, the temperatures at which males of both G. fultoni and G. vernalis produced calling songs overlapped in a wide range. Notice that most males were recorded at temperatures between 18 and 28◦ C, the temperature range that we used for recording in this study (Fig. 2).

G. fultoni in sympatry G. fultoni in allopatry G. vernalis

25

Frequency

20 15 10 5 0 14

18 22 Temperature (°C)

26

Fig. 2. Temperatures at which calling songs of G. fultoni and G. vernalis were recorded in the field. The temperature conditions for recordings were not planned; rather, this graph shows the temperature ranges within which males of G. fultoni and G. vernalis produce calling songs. Calling songs of G. fultoni were recorded from seven allopatric and six sympatric localities including JK, RF, DS, and GR (Jang and Gerhardt, 2006a). Field recordings were made between dusk and midnight on rainless nights. During each recording night, ambient temperature was continuously monitored every 30 s using a HOBO data logger (model H08-004-02, Onset Computer Corp., Pocasset, Massachusetts, USA) that was placed 5 cm above the ground near calling males. See Jang and Gerhardt (2006a) for more information about materials and methods of field recording.

40

Jang and Gerhardt

Table I.

Results of Repeated Measures ANCOVA on Calling Songs of Sympatric and Allopatric G. fultoni Populations (n = 112) Component

Num. df

Denom. df

F

P

(1) Chirp rate Zone Temperature Locality (Zone) Zone × Temperature

2 1 1 2

31.763 30.075 43.976 30.488

1.691 263.445 3.831 3.759

0.201