Bird-song functions, such as mate attraction, species recognition, and territory defense, are closely linked to individual fitness. Thus, habitat characteristics that affect song transmission and degradation should exert a strong influence on the evolution of song attributes. Whereas different acoustic environments may influence the evolution of song characteristics, factors such as body size and evolutionary history of taxa are expected to constrain the amount of environmental variation in song properties. In the present study, we controlled for phylogeny and examined the effects of body mass and habitat structure on variation in song structure of 30 taxa of Phylloscopus and Hippolais warblers, which are a closely related group of birds that occupy a wide variety of habitats and show high variation in vocalizations. Habitat structure was strongly correlated with temporal characteristics of songs but not with most of the frequency-related attributes that we measured. Only the highest frequencies of songs varied with habitat structure. As predicted, species occupying closed habitats avoided the use of rapidly modulated signals and had song structures that minimized reverberation. Body mass covaried significantly with most of the song attributes. Smaller species used higher frequencies and had more notes in their songs compared with larger species. Received 9 May 1996, accepted 26 July 1996.

Division of Biological Sciences and Montana Cooperative Wildlife Research Unit, University of Montana, Missoula, Montana 59812, USA THE MAJOR FUNCTIONS OF BIRD SONG, such as mate attraction, species recognition, and the es- tablishment and defense of territories, are close- ly linked to individual fitness (Catchpole and Slater 1995). Thus, songs that maximize effec- tive communication of signals to intended re- ceivers should increase the fitness of the sing- ing individual. Concurrently, songs and acous- tic communication in general are subject to spherical-spreading and frequency-dependent attenuations, reverberations, and irregular am- plitude fluctuations caused by habitat structure (Wiley and Richards 1982, Wiley 1991). Thus, the acoustic environment may exert a strong influence on the evolution of song attributes. Previous studies of habitat associations of song properties led to predictions that lower fre- quencies should be favored more in closed hab- itats than in open habitats (Chappuis 1971, Mor- ton 1975, Cosens and Falls 1984, Waas 1988), although, theoretically, lower frequencies should always be favored because they travel the farthest regardless of habitat (Wiley and Richards 1982, Wiley 1991). High frequencies have a greater tendency to be scattered by fo- liage than do low frequencies, which are af- fected only by objects larger than foliage (Wiley  E-mail: abadyaev@selway.umt.edu 40 1991). Thus, high frequencies may be more con- strained by habitat (Wiley 1991). Reverberation is thought to influence the length of notes (also referred to as "elements") and the amount of time between notes (Tubaro and Segura 1995). Consequently, birds that live in habitats with numerous scattering surfaces (e.g. leaves and branches) may: (1) avoid using rapidly modu- lated signals, (2) use shorter notes, and (3) put more space between notes to avoid extensive reverberation (Nottebohm 1975, Wasserman 1979, Richards and Wiley 1980, Gish and Mor- ton 1981, Handford 1981, Anderson and Conner 1985, Sorjonen 1986, Handford and Lougheed 1991, Wiley 1991). Song frequencies also may be constrained by body size because the mass of a vibrating struc- ture affects the frequency that is produced most efficiently (Morton 1975, Bowman 1979, Wall- schager 1980, Shy 1983, Ryan and Brenowitz 1985). Hence, inter-habitat variation in songs could be confounded by unequal distribution of different-sized birds among habitat types (Ryan and Brenowitz 1985). In addition, evo- lutionary history and relatedness of species can confound variation in song structures exerted by habitat parameters (Ryan and Brenowitz 1985), especially if different taxa are unevenly distributed among habitat types (Wiley 1991). In this paper we use a phylogenetic approach Phylloscopus collybita abietinus -r- SYLLABLE '" RAN v z o u. I I I_F INT NTLEN Fig. 1. Sonogram of Phylloscopus collybita abietinus showing variables measured. HF = highest frequency; INT = internote interval; RAN = frequency range; LF = lowest frequency; NTLEN = note length. A typical syllable consisting of two notes also is identified. to examine habitat associations of 30 taxa of Phylloscopus and Hippolais warblers. These gen- era are especially well-suited for this study. Most of the Phylloscopus and Hippolais warblers are poorly differentiated in plumage characteristics and morphology but show extensive variation in vocalizations, even on a subspecies level (e.g. Dementiev and Gladkov 1954, Cramp 1992; see Appendix). These warblers are widely distrib- uted and occupy a broad variety of habitats (De- mentiev and Gladkov 1954, Cramp 1992). In addition, the phylogenetic relationships of these birds, especially Phylloscopus warblers, have been studied extensively (Ticehurst 1938, Martens 1980, Haffer 1991, Cramp 1992, Richman and Price 1992, Helbig et al. 1995 ), providing an opportunity to control for species and subspe- cies relatedness. We statistically controlled for phylogeny and examined the effects of body mass and habitat structure on variation in song structure of Phyl- loscopus and Hippolais warblers. Specifically, we tested the following predictions: (1) frequency characteristics of song should correlate with body mass, and high frequencies should vary with habitat type; (2) temporal characteristics of songs should be affected by habitat structure. METHODS Songs used in the study were obtained from the song identification guide by Veprintsev (1987). Re- cordings on these disks contain complete songs and calls of several individual birds of each species. We used Canary 1.0 (Cornell Laboratory of Ornithology) software to measure the following variables (see Fig. 1): (1) highest frequency (HF; kHz), (2) lowest fre- quency (LF), (3) range of frequency distribution (RAN), (4) dominant frequency (PEAK; i.e. the most preva- lent frequency within a song), (5) number of notes (NT), (6) number of syllables (SYLL), (7) length of notes (NTLEN), (8) song length (SLEN), and (9) in- ternote interval (INT). Means of songs of at least three to four individuals per species or subspecies were averaged to obtain values given in the Appendix. We gathered published data on body size and hab- itat type for all 30 taxa (Dementiev and Gladkov 1954, Veprintsev 1987, Cramp 1992). We ranked nesting habitat from open to closed in the following se- quence: (1) open fields, steppes, and deserts; (2) bush- es and subalpine bushes; (3) intermediate between bushes and forest habitats, gardens; (4) coniferous forests; and (5) deciduous forests. Published data on habitat distribution were used in conjunction with habitat types listed on the source of the recordings (Veprintsev 1987). The phylogeny of Phylloscopus and Hippolais species used in this study was constructed by summarizing available systematics data (Ticehurst 1938, Martens 1980, Haffer 1991, Cramp 1992, Richman and Price 1992, Helbig et al. 1995). Our phylogenetic hypothesis (see Fig. 2) was largely constructed based on molec- ular data for Phylloscopus warblers (Richman and Price 1992, Helbig et al. 1995). However, the resulting phy- logenetic tree is consistent with findings in most oth- er classification studies as well. Branch lengths were set as equal because they were available only for a few species in this study (Richman and Price 1992, Helbig et al. 1995). Plots of standardized contrasts against the variances of the untransformed contrasts showed no significant correlation, justifying the use of equal branch lengths (Purvis and Rambaut 1995). To control for species relatedness, we calculated independent contrasts between nodes for the traits of interest (Felsenstein 1985) using the CAIC software package (Purvis and Rambaut 1995). We then exam- ined the relationships between the variables by cal- ß e. bre-os  elle  eates   P .   P .beta   H,oa ta Fig. 2. Phylogenetic hypothesis for the Phylloscopus and Hiolais warblers. culating linear regressions on these contrasts (Gar- land et al. 1992, Grafen 1992). All regressions were forced through the origin (Garland et al. 1992). Anal- yses of independent contrasts of habitat types were conducted following Martin and Badyaev (1996). The numbers of syllables and notes were square-root transformed; all other song parameters and body mass were log-transformed before the analyses. RESULTS Song characteristics were highly intercorre- lated (Table !). The taxa we studied used widely ranging types of syllables in songs, thereby complicating the direct comparison of original song variables among species. Therefore, we used principal components (PC) analysis to de- scribe variation in song characteristics (Table !). Principal components were constructed such that PC I (32% of variation in the model) ac- counted for most of the variation in frequency- related variables, and PC II (22.5% of variation) accounted for most of the variation in temporal characteristics of songs (Table !). Phylloscopus and Hippolais warblers of different size were equally represented across habitat types; body mass did not vary with habitat openness (Pear- son r = -0.!, P = 0.6). Among frequency-related song parameters, only the highest frequency varied significantly with habitat openness (Table 2). Frequency pa- rameters were strongly affected by body mass; TABLE 1. Pearson correlation coefficients of song properties and eigenvectors for principal components analyses of song variables (corrected for phylogeny using linear contrasts; Purvis and Rambaut 1995). See Methods for acronyms of song variables. SLEN HF LF SYLL NT RAN PEAK INT NTLEN HF 0.34* LF -0.10 0.01 SYLL 0.43* 0.50** -0.31 NT 0.59** 0.01 -0.10 0.43** RAN 0.34* 0.76*** -0.58*** 0.50** -0.03 PEAK -0.00 0.20 0.50** -0.14 -0.06 -0.12 INT 0.00 0.17 0.02 -0.24 -0.49** 0.27 NTLEN 0.25 0.28 -0.02 0.26 -0.35** 0.34* HT a -0.33* 0.16 0.07 -0.20 -0.51'* 0.15 MASS b -0.07 -0.33* 0.05 -0.17 0.20 -0.36** PC I c 0.42 0.47 0.50 PC II c -0.60 0.10 -0.06 0.24 0.17 0.25 0.14 -0.28 -0.37** -0.46** 0.53 0.40 *,P < 0.1; **, P < 0.05; ***, P < 0.001. ß Habitat type.  Body mass (g). ' Only loadings - [0.40[ are shown. smaller species used the highest frequencies and had the widest frequency range compared with larger species (Table 2, Fig. 3). Body mass also was strongly correlated with temporal charac- teristics of song; smaller species had shorter in- tervals between notes and used shorter notes compared with larger species (Table 2). In con- trast to the frequency characteristics of songs, temporal parameters were strongly affected by habitat structure. Species in closed habitats used fewer notes and longer notes, and had longer intervals between notes compared with their open-habitat relatives (Table 2, Fig. 4). TABLE 2. Standardized coefficients from multiple re- gression of song properties on habitat and body mass (corrected for phylogeny using linear con- trasts; Purvis and Rambaut 1995). Sources of variance Habitat Body Variable F openness mass Frequency properties Highest frequency 3.78** 0.41'* -0.39* Lowest frequency 0.04 0.03 0.03 Dominant frequency 2.31' 0.37 -0.27* Frequency range 2.44** 0.14 -0.39** PC I 3.67** 0.32 -0.39** Temporal properties No. of syllables 1.09 -0.19 -0.16 No. of notes 1.69 -0.35 0.29 Internote interval 2.69* 0.22 -0.42** Song length 0.82 -0.24 -0.04 Note length 3.56** 0.14 -0.48** PC II 6.22** 0.45** -0.52** *, P < 0.1; **, P < 0.05. DISCUSSION The results support our predictions that tem- poral characteristics of song vary strongly with -0.08 Fig. 3. -0.06 -0.04 -0.02 0.00 0.02 0.04 0.06 BODY MASS 2 GREATER 0.00 0.25 0.50 0.75 1.00 1.25 1.50 HABITAT :: MORE CLOSED Partial-regression residual plots of inde- pendent linear contrasts illustrating relationship be- tween frequency-related characteristics of songs (i.e. PC I), body mass (upper), and habitat openness (low- 3 2 0 [ -3 -0.06 -004 -0.02 0.i 0.02 0.04 (c) 0.06  BODY MASS  GREATER o.5 o.;5 HABITAT  MORE CLOSED Fig. 4. Partial-regression residual plots of inde- pendent linear contrasts illustrating relationship be- tween temporal characteristics of songs (i.e. PC II), body mass (upper), and habitat openness (lower). habitat openness, whereas frequency attributes largely are unaffected by habitat structure. Only the highest frequencies varied significantly with habitat structure (Table 2). Closed habitats are thought to cause greater attenuation in songs, which in turn would affect the upper band of acceptable frequencies (Wiley 1991). Thus, only the highest frequencies are expected to vary with habitat. The highest frequencies might also be affected by different levels of background noise among habitat types (Ryan and Brenowitz 1985). Temporal attributes of song should vary with habitat because reverberation rises with increasing habitat closeness (e.g. Wiley and Richards 1982). As we have shown, birds in closed habitats avoid the use of rapidly mod- ulated signals, use shorter notes, and have more space between notes (potentially to minimize reverberation). These findings are consistent with other studies (Wiley 1991, Tubaro and Se- gura 1995). Body mass was a significant constraint on most song attributes (Table 2, Figs. 3 and 4). In par- ticular, smaller species use higher frequencies and shorter notes than do larger species (Table 2). This is consistent with the finding that body size is positively correlated with the mass of the song-producing structures in birds (Morton 1975, Ryan and Brenowitz 1985). It is thought that in vocalizations intended for long-range communication, body mass should strongly limit the lower range frequency because more energy is required to produce and transmit low-fre- quency sounds (Ryan and Brenowitz 1985). Al- though song structure predictably varied with habitat type, singing behaviors such as use of high perches and song-flights also could max- imize song transmission (Jilka and Leisler 1974, Wilczynski et al. 1989). However, courtship song-flights, such as the one of P. sibilatrix, are very rare among these warbler species. We did not examine the relationship between singing height and song structure. These species used a wide variety of habitats, and taxa with greater number of species are needed to simultaneously control for effects of habitat, singing height, and body mass. Although habitat structure and body mass significantly affected song properties in Phyl- loscopus and Hippolais warblers, a large amount of variance in song characteristics (46.9%) re- mained unexplained. These warblers, especial- ly Phylloscopus, vary widely in such biological parameters as mating system, territory size, and population density (reviewed in Cramp 1992). These factors may determine functions of songs, i.e. whether songs are intended for long- or short-range communication, and thus can in- fluence song structure and composition (e.g. Salomon and Hemim 1992). Species recognition frequently is invoked to explain high variation in characteristics of vo- calizations, especially in Phylloscopus warblers (e.g. Salomon and Hemim 1992). The degree of sympatry varies within Phylloscopus (Cramp 1992); thus, the variation we observed, especial- ly among subspecies, may be influenced by the presence of closely related species (Salomon 1989, Salomon and Hemim 1992). 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Species SLEN HF LF SYLL NT RAN PEAK INT NTLEN Phylloscopus proregulus 2.192 7.738 2.735 3.7 19.3 5.005 4.35 57.01 94.39 P. i. inornatus 1.433 8.460 3.925 4.5 4.0 4.545 5.39 58.50 129.50 P. i. humei 2.192 8.200 3.400 3.0 7.0 4.800 5.30 53.00 89.00 P. fuscatus 1.160 6.003 2.773 1.3 6.7 3.230 5.16 91.10 118.57 P. griseolus 1.163 5.437 2.607 3.0 9.0 2.830 3.83 32.83 73.27 P. collybita abietinus 2.865 6.810 3.190 3.0 11.0 3.630 5.13 162.00 106.50 P. c. brevirostris 2.898 7.140 2.800 3.0 11.0 4.330 4.52 168.50 105.00 P. c. tristis 5.648 6.775 2.710 3.5 31.0 4.070 4.52 86.52 101.90 P. c. menzbieri 2.556 7.310 3.110 3.0 8.0 4.200 4.26 238.80 105.80 P. c. fulvescens 2.136 7.410 2.760 2.0 7.0 4.650 4.17 267.70 73.00 P. c. lorenzii 2.587 6.900 3.090 2.0 8.0 3.810 5.30 289.00 80.50 P. c. sindianus 3.873 6.230 2.920 3.0 15.0 3.310 4.35 109.00 205.00 P. neglectus 1.113 6.450 2.400 10.0 11.0 4.050 4.35 34.80 69.30 P. t. trochilus 2.513 7.265 2.200 7.0 17.3 5.063 3.65 63.30 134.95 P. t. yakutensis 2.742 6.950 1.880 8.0 18.0 5.070 1.88 68.75 102.50 P. sibilatrix 2.823 7.620 3.070 1.0 29.0 4.550 4.87 98.30 36.30 P. coronatus 1.722 5.987 2.823 2.0 8.7 3.163 4.78 94.57 132.07 P. occipitalis 4.536 7.510 2.980 6.0 22.0 4.530 3.91 74.30 127.70 P. tenellipes 1.727 6.755 5.400 1.0 24.5 1.350 5.70 12.50 44.65 P. borealoides 2.584 7.887 4.290 1.3 9.3 3.590 4.67 47.87 256.27 P. b. borealis 2.316 6.360 2.040 2.0 18.0 4.320 4.61 41.00 32.80 P. b. xanthodryas 2.025 5.750 2.470 3.0 20.0 3.290 4.43 39.30 54.70 P. trochiloides nitidus 3.091 7.600 2.980 4.0 13.0 4.620 4.96 72.30 112.30 P. t. viridanus 1.507 6.570 2.880 3.0 14.0 3.690 3.82 112.50 83.00 P. t. plumbeitarsus 1.846 8.050 2.500 4.0 15.0 5.550 5.30 47.30 110.50 P. schwarzi 1.459 6.440 1.850 3.0 24.5 4.585 3.48 17.00 50.80 Hippolais icterina 5.951 7.520 1.540 5.0 23.0 5.990 3.48 63.50 145.00 H. pallida 2.881 7.280 1.700 6.0 18.0 5.580 -- -- -- H. languida 6.132 4.640 2.190 2.0 39.0 2.450 3.82 63.50 61.00 H. c. caligata 2.765 4.840 1.780 1.5 19.0 3.060 3.04 80.00 102.30 H. rama 5.816 6.520 1.670 15.0 47.0 4.850 3.82 54.50 109.00