Geographical Variation in the Song of Chrysoperla ...

Geographical Variation in the Song of Chrysoperla plorabunda (Neuroptera: Chrysopidae) in North America CHARLES S. HENRY AND MARTA MARTINEZ WELLS Department of Ecology and Evolutionary Biology, The University of Connecticut, Storrs, Connecticut 06269

Ann. Entomo!. Soc. Am. 83(3): 317-325 (1990) ABSTRACT Green Iacewings of the widespread North American Chrysoperla plorabunda (Fitch) are morphologically and ecologically homogeneous but polymorphic with respect to their substrate-borne courtship songs. At least three distinct song morphs can be recognized. One of these, here called PI, best corresponds to C. plorabunda as originally defined. Geographical variation in six major features of this PI song is described, from individuals native to Connecticut, Idaho, Washington, and Oregon. Differences between eastern (Connecticut) and western (Idaho, Washington, Oregon) populations are slight, exhibiting statistical significance only for frequency (pitch) measures. Additional small differences between the sexes are probably artifacts of the experimental protocol. The results are compared with preliminary data on the P2 and P3 song morphs, which are found sympatrically with PI in western North America. We conclude that the PI song morph is a valid taxonomic entity, synonymous with C. plorabunda as currently defined, and that the other song morphs probably represent additional, cryptic species. KEY WORDS

GREEN LACEWINGS of

Insecta, Chrysoperla plorabunda, courtship songs, sibling species

the genus Chrysoperla Steinmann produce unique, low-intensity songs that are propagated through leaves, grass, or thin twigs between courting individuals (Henry 1979). Both sexes sing, establishing prolonged duets that have been shown to prevent the hybridization of closely related species in the field (Henry 1985a,b). The songs can thus be viewed as important, if not the principal, premating barriers to reproduction among some species within the genus. Consequently, the origin and genetic basis of these songs, and their variation within and among taxa, speak to more general issues concerning mechanisms of animal speciation (Henry & Johnson 1989). Experiments designed to assess the responses of individuals to songs (Henry 1985b; unpublished data) indicate that the success or failure of courtship is a function of how well the songs of two insects match in their measurable traits. Seemingly minor but consistent song differences, perhaps based on single allelic substitutions, might produce nearly complete reproductive isolation between two populations, resulting in speciation. In Chrysoperla, mechanisms involving song divergence seem to have generated swarms of nearly identical, reproductively isolated but sympatric song morphs in western North America and central Europe (Henry 1983a,b). Results of female choice tests (unpublished data) support the hypothesis that the North American song morphs are real species. However, for this to be true, songs must be invariant within a song morph and show sharp discontinuities between different morphs. Because several morphs are geographi-

cally widespread, it is important that the variation of the songs within each morph across its range be documented. Chrysoperla plorabunda (Fitch) is a common green lacewing that occurs in North America from coast to coast and from Alaska and the provinces of Canada to Mexico (Agnew et al. 1981). Synonymized with palearctic C. carnea (Stephens) by Tjeder (1960), it is acoustically unlike any known Old World populations of the latter species and exhibits considerable postmating incompatibility with Swiss C. carnea in the laboratory (Henry 1983a). It should therefore be considered a separate species. Within North America, C. plorabunda includes at least three song morphs, herein referred to as PI, P2, and P3 (Henry 1985a). In the strict sense, C. plorabunda is the PI morph because only PI occurs in New York state where the type and paratypes were collected (Fitch 1856). The song of Connecticut and New York specimens of this morph has been fully described (Henry 1983c). However, confirmed individuals of PI have been collected from widely separated locales, including Virginia, Illinois, Montana, Idaho, Washington, and Oregon (Henry 1985a; C.S.H., unpublished data). In our paper, the songs of PI individuals from several of these different geographical populations are described and compared with one another. Variance within and between populations is analyzed after compensating for the effects of temperature on the song features. We present preliminary comparisons of the songs of PI with those of the P2 and P3 morphs of C. plorabunda and evaluate the rele-

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middle, and terminal part of each volley; and frequency drop, calculated by subtracting the terminal from the initial frequency of each volley. The shortest repeated unit (SRU) was the single Materials and Methods volley. In total, the songs of 23 individuals from Living specimens of C. plorabunda were taken Idaho, four from Washington, seven from Oregon, from four principal locales in North America. Those and 10 from Connecticut were analyzed in detail. hereinafter referred to as Connecticut were col- At least 10 SRU's of the PI song of each insect lected in overgrown fields near the campus of the were examined. University of Connecticut, Storrs, in July-SeptemSome analyses made before 1986 were perber 1983, elevation 200 m. Idaho insects were col- formed on the screen of an oscilloscope, using a lected by Dr. James B. (Ding) Johnson in Moscow, ruler and counting the cycles per unit time. Later during September 1988 from a residential area, evaluations were enhanced by computer methods: elevation 800 m. Washington specimens were ob- recorded songs were digitized, transferred to an tained from a neighborhood in Walla Walla in IBM personal computer, and examined using exSeptember 1986, elevation 400 m. Oregon insects isting commercial programs that perform detailed were collected near the summit of Mary's Peak, 20 waveform and spectral (frequency) analyses (see km west of Corvallis, elevation 500 m, from the Henry [1989] for details). Data were reduced furbranches of scattered hemlock and spruce growing ther within an electronic spreadsheet and transin a field in September 1987. ferred to the programs Asystant Plus (1986), CSS Because all known song morphs of C. plorabun- (1988), Sigma-Plot (1987), and SAS (SAS Institute da are morphologically indistinguishable, field-col- 1982) for statistical and graphical interpretation. lected insects were segregated by sex and main- A two-way analysis of variance (ANOVA) was run tained in our laboratory at the University of to compare the mean values of song features beConnecticut until the song of each individual could tween the two sexes and among the four geographbe recorded and analyzed. Breeding populations ical populations. Scheffe's test (Scheffe 1953) showed from each geographical area were then established, which populations differed from each other. using only confirmed PI individuals. These laboLacewing songs are strongly influenced by temratory colonies of adults were maintained on long- perature. Because data were taken at various temday photoperiods of 17:7 (L:D) and fed a Wheast- peratures, a method for adjusting all measurements based diet described in earlier papers (Henry to a single temperature was needed. We did this 1980a). Eggs and new hatchlings were removed by recording many songs of eastern and western from the colony, placed individually in 18-ml (7 insects at several temperatures ranging from 19.5 dram) vials, and provided with ether-killed Dro- to 30°C. Data for a given individual at a certain sophila spp. Until their songs could be assessed, temperature were averaged, and all such individnew adults were segregated by sex to protect the ual means were then used to calculate an "eastern" breeding population from accidental contamina- and a "western" temperature regression equation tion by larvae of other morphs or species. for each of the six song features (Henry 1983c). The vibrational songs of single or paired C. plo- Those equations were used to normalize the aprabunda were recorded from the plastic wrap cov- propriate subsets of raw data to 27°C so that staner of a small experimental arena using a piezoe- dardized means and confidence intervals could be lectric transducer as described previously (Henry calculated and compared. In addition, the slopes 1979, 1980b; Henry & Johnson 1989). The signals and intercepts of the regression equations describwere electronically amplified and recorded on cas- ing eastern lacewings were compared with those sette tapes. Individuals or couples were induced to of western ones using a forward step wise regression sing by playing back previously recorded or arti- analysis to highlight statistically significant differficially synthesized PI songs through a 12-cm cone ences. This special analysis of covariance compared loudspeaker located 5 cm above the arena (Henry each dependent variable with temperature and with two dummy variables, one coding for the effects 1989). of geographical location and the other for location Analysis of recorded songs involved frequency x temperature. and time (duration and interval) measurements of various song components. In PI C. plorabunda, the Other song morphs of C. plorabunda were obsong consists of a series of basically similar volleys tained from sites in western North America. The of abdominal vibration, repeated at regular inter- P2 morph occurred sympatrically with PI at the vals (Henry 1983a). The rate of abdominal vibra- Mary's Peak, Oreg. locale. It also was collected tion determines the pitch or frequency of the vol- farther south in California in mid-September 1987 ley, which changes from the beginning to the end in the conifers of Sequoia National Park (2,400 m) of the volley. Thus, six measures were sufficient to and at the Forest Home Campground in the San characterize the song: duration of each volley in Bernardino Mountains (1,650 m). Specimens of P3 milliseconds; interval between volleys, measured were collected with PI or P2 or both at the Moscow, between the starts of two consecutive volleys; fre- Idaho, site, at Mary's Peak, in Sequoia National quency, in Hertz or cycles per second, of the initial, Park, and in the San Bernardino Mountains. Advance of these findings to chrysopid systematics and speciation.

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= EASTERN: Y=4.30x-16.74, R 2 =0.93. N=37

O = WESTERN: Y=4.02x-24.07. R 2 =0.79, N=34

N

IE (J

100-

z

O

Ld

o LU

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60-

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19 20 21 22 23 24 25 26 27 28 29 30 31 TEMPERATURE, °C Fig. 1. Effect of temperature on the initial frequency (pitch) of the song volleys of eastern (Connecticut) versus western (Idaho, Washington, Oregon) populations of the PI morph of C. plorabunda. Each symbol represents the mean of all songs delivered at a given temperature by an individual.

ditionally, they were taken in September 1987 from evergreens in the mountains east of Medford, Oreg. (1,400 m) and from mixed conifers and scrub oak in Strawberry Canyon, Berkeley, Calif. (50 m) and Isabella Lake, Bodfish, Calif. (1,000 m). A special population of P3 individuals was discovered by Livy Williams and Ding Johnson in January 1983 living in the low deciduous and evergreen shrubs of the xeric, unforested Kofa Mountains of Arizona (1,200 m). All populations were maintained, reared, and analyzed, and compared using the methods described for PI. Voucher specimens have been deposited in the personal collection of C.S.H. or in the collection of the Connecticut State Museum of Natural History, University of Connecticut, Storrs. Results The calculated values of the temperature regression equations for six features of the songs of eastern and western C. plorabunda are shown in Table 1. The effect of temperature on one song feature, initial frequency (pitch), is shown graphically in Fig. 1 for eastern and western populations. Regression slopes were positive for frequency measures and negative for duration and interval; that is, the pitch of the song increased with increasing temperature, whereas the length and spacing of song volleys decreased at higher temperatures. The equations for each song feature were generally sim-

ilar between eastern and western populations, although several slope and intercept differences were statistically significant (Table 1, asterisks). Volley interval regressions were notable for showing negligible change in either slope or intercept across the North American continent. Table 2 shows the average values in each population of the six song features, adjusted to 27°C with the regression equations. Some of these data are shown graphically in Fig. 2 and 3. An ANOVA detected few consistent differences among the means of the four populations or between the means of the two sexes (Table 3). Only the pitch (frequency) of abdominal vibration exhibited significant geographical variation. Specifically, initial and midvolley frequencies were higher in Connecticut lacewings than in insects from the other locales. Although these differences were not large, they were more pronounced for the initial frequencies, which averaged nearly 100 Hz in Connecticut lacewings but 90 Hz or less in the three western populations. The analysis also revealed a minor but statistically significant difference in the volley spacing (interval) of males versus females when all populations were combined: volley interval averaged 1,095 ms in males compared with 996 ms in females. In all other respects, the songs of males and females were indistinguishable. Analysis of the songs of P2 and P3 morphs of C. plorabunda has not been completed and will be treated in future papers. Preliminary results (Fig.


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Table 1. Regression against temperature for the principal song features of eastern versus western populations of the C. plorabunda PI song morph Song feature

Population"

Regression equation*1

R2c

Volley duration

Eastern Western Eastern Western Eastern Western Eastern Western Eastern Western Eastern Western

y = -39.27* + 1,631 y = -32.35* + 1,416 y = -78.76x + 3,186 y = -77.08* + 3,125 y = 4.30* - 16.74 y = 4.02* - 24.07 y = 2.77* + 0.74 y = 2.12* + 6.22 y = 0.95* + 18.39 y=1.31* + 7.20 y = 3.33* - 34.75 y = 2.72* - 31.63

0.83 0.56 0.86 0.77 0.93 0.79 0.78 0.84 0.65 0.60 0.91 0.56

Volley interval Initial frequency Middle frequency Ending frequency Frequency drop

F ratiod Slopes

Intercepts

1.49

4.23**

0.03

0.39

0.48

132.4***

110.8* 2.79* 91.77*

0.38 7.43*** 0.08

" Eastern, Connecticut; Western, Idaho, Washington, Oregon. Values represent averaged data from 10 eastern and 23 western individuals. b y = a* + b: a, slope of regression; b, y intercept; *, observed value; y, calculated value. c 2 R , coefficient of determination. d Calculated from a multiple regression model. All comparisons, df = 67. Significant differences: *, P = 0.05; **, P = 0.01; ***, P = 0.001.

4 and 5) indicated that volleys of P2 are longer and farther apart that those of PI. P3 songs were composed of even longer volleys (>3 s long and 5 or more s apart), and several volleys rather than a single volley constituted the SRU. Frequency characteristics of the volleys of all three forms were quite similar.

Discussion Crucial to the validity of a study such as this is the proper temperature compensation of all measurements. Because the temperature regression equations differed significantly between western and eastern populations of C. plorabunda (Table

1250 r T

T

1000 OR CO

CO

(N=7)

750

WA (N=4)

ID 500

(N=23)

250

DURATION

INTERVAL

Fig. 2. Mean volley duration and volley interval compared by an ANOVA across four geographical populations of the PI morph of C. plorabunda (see Table 3). The principal value at each site is the mean of the separate means of all individuals (n) of a population. Error bars represent 99% CIs. OR, western Oregon; WA, Walla Walla, Wash.; ID, Moscow, Idaho; CT, Storrs, Conn.

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***

OR (N=7)

WA (N=4)

ID (N=23)

INITIAL PORTION

END PORTION

MIDDLE PORTION

Fig. 3. Mean initial, middle, and ending volley frequencies compared by ANOVA across four geographical populations of the PI song morph of P. plorabunda (see Table 3). The principal value at each site is the average of the separate means of all individuals (n) of a population. Error bars represent 99% CIs; asterisks indicate significant differences at p < 0.001. OR, western Oregon; WA, Walla Walla, Wash.; ID, Moscow, Idaho; CT, Storrs, Conn.

of Chrysoperla. These data strongly suggest that PI is a distinct taxonomic unit and that the name C. plorabunda should be reserved for that song morph alone. Our study did reveal some measurable, statistically significant pitch differences between the songs of western and Connecticut PI populations: the initial and middle pitches of each volley are higher in eastern individuals than in western ones. However, such small pitch differences could be detected only by sensitive spectral analysis. It is therefore doubtful that the insects themselves can discriminate among songs based on pitch differences, and preliminary tests (unpublished) show that females are just as likely to answer a recording of an allopatric PI song as one made by a sympatric individual. The reason for this may be related to the transmission characteristics of vibrational songs in lightweight substrates as discussed in Michelsen et al. (1982). Excitation of any thin, sticklike substrate

1), we chose to correct the song features of the two populations separately. Oregon, Idaho, and Washington lacewings were treated together as the "western" unit because no consistent differences could be detected among them (Table 2). Tight constraint of the slopes of volley duration, volley interval, and initial frequency suggests a common mechanism of temperature control operating in eastern and western populations for each of these three song features. The PI song morph of C. plorabunda exhibits extraordinary phenotypic stability in its song over a vast geographical range. In particular, average duration and interval of the song volleys are strikingly invariant characteristics. Consequently, individuals from different geographical locales cannot be told apart reliably by song features alone. Provided that it can be induced to sing, a PI individual from any part of North America is very easy to distinguish from any other morph or species

Table 2. Average song characteristics of different geographical populations of C. plorabunda normalized to 27°C Abdominal vibration frequency, i strokes/s ± SD

PI population Oregon Washington Idaho Connecticut

Initial 7 4 23 10

84.49 ± 89.14 ± 83.70 ± 99.21 ±

3.58 1.21 6.44 3.12***

Middle 62.35 ± 65.50 ± 63.42 ± 75.01 ±

1.81 0.56 2.85 3.37***

End 42.09 ± 41.88 ± 42.94 ± 43.93 ±

4.05 3.21 2.66 2.14

Drop

Volley duration, ms

Interval between volleys, ms

42.42 ± 5.12 47.13 ± 3.39 40.72 ± 7.21 55.35 ± 2.78*

529.34 146.62 578.09 ± 47.92 540.40 ± 90.53 572.96 ± 40.60

1,027.19 ± 1,076.59 ± 1,042.75 ± 1,062.94 ±

" Asterisks mark populations that differ significantly from all others in a particular feature: *, P = 0.05; ***, P = 0.001.

72.98 104.04 130.93 72.20

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Table 3. Analysis of variance of the six major song features of C. plorabunda, by four geographical localities and by sex. The analysis uses the mean values measured in 4 4 individuals By locality Song features Duration Interval Initial frequency Middle frequency End frequency Frequency drop

F-Value

P-Value

0.907 0.367 21.62*** 47.28*** 0.870 15.81***

0.4494 0.7795 0.0000 0.0000 0.4678 0.0000

By sex F-Value P-Value 0.068 3.786** 0.610 0.223 0.008 0.428

0.7847 0.0565 0.4457 0.6443 0.8887 0.5242

Asterisks mark features that show significant variation among the measured populations: **, P = 0.01; ***, P = 0.001.

by a vibrational signal produces regularly spaced nodes of relatively intense resonance along the length of the stick. Between those nodes, vibration is reduced or absent. In addition, the spacing between the nodes is a function of pitch (frequency), so the nodes will appear at different locations for different frequencies. A frequency-modulated signal such as that produced by PI C. plorabunda will appear at nearly all points along the length of the stick, because each of its multiple frequencies will excite a different set of nodes along the substrate. At any given site on the twig, an insect will "hear" a subset of the original signal, which will consist of a series of volleys characterized by a single tone or frequency rather than a swept (frequency-modulated) tone. Thus, in a typical natural P1 Morph

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environment and on a given substrate, only the interval between the volleys will be perceived in exactly the same way by all individuals. Consequently, frequency, and to a lesser extent volley duration, are features that can vary considerably without consistently affecting the perception of the songs by the lacewings themselves. We expect, and find, volley interval to exhibit the least amount of geographical variation of the six measured traits. There also are some sexual differences in the songs. On average, the spacing between volleys (volley interval) in the songs of males is slightly greater than that measured in the songs of females. However, this disparity is an artifact of the methods used for data collection. It has been shown (Henry 1980b) that volley interval in C. plorabunda gradually decreases during the course of a long song (sequence of consecutive volleys). Whereas the first few volleys of an individuals song may be only 600-800 ms apart, later ones of the same series will exhibit intervals of 1,200-1,400 ms or more. Therefore, individuals that typically produce only 5-10 volleys at a time when they sing will display shorter average spacing between their volleys than those that produce 20-30 volleys in each song. Females of C. plorabunda do not sing as readily or energetically as males, and they stop singing just a few seconds after starting unless dueting with males. Consequently, most of our data for females are derived from short song sequences and consist of closely spaced "start-up" volleys, whereas male data are extracted from longer songs. This difference is

i

P2 Morph Z) Q_

'' P3 Morph

0.00

2.50

7.50 5.00 TIME, in SECONDS

10.00

12.50

Fig. 4. Digitized oscillographs of the vibrational songs of the PI, P2, and P3 morphs of the C. plorabunda sibling species complex. Each trace represents a 12-s sample of data.

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C. plorabunda

P1 Morph

LU

P2 Morph

Q_