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Fall 12-16-2020
Evidence of Incipient Song Divergence in a Hawaiian Population of Warbling White-eyes
Jesse D. Robinson CUNY Hunter College
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This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] Running head: EVIDENCE OF INCIPIENT SONG DIVERGENCE
Evidence of Incipient Song Divergence in a Hawaiian Population of Warbling White-eyes
by
Jesse Robinson
Submitted in partial fulfillment of the requirements for the degree of Master of Arts in Animal Behavior and Conservation, Hunter College City University of New York
2020
December 16, 2020 Dr. Caroline Dingle Date Signature of first reader
December 16, 2020 Dr. Ofer Tchernichovski Date Signature of second reader
EVIDENCE OF INCIPIENT SONG DIVERGENCE 2
ABSTRACT
Natural or human-mediated founder events can lead to changes in avian communication signals, potentially impacting the trajectory of evolution. Warbling white-eye (Zosterops japonicus) was introduced from Japan to the Hawaiian Islands between 1929 – ca. 1937. I recorded primary songs in Hawai‘i (O‘ahu, Big Island) and collected archival recordings, then conducted a comparative analysis between introduced and native song types which revealed significant differences in the O‘ahu and Big Island populations. To test for behavioral responses that corresponded to these differences, I presented conspecific playback stimuli (intra-island, inter- island, native) to individuals in Hawai‘i (O‘ahu, Big Island). Big Island individuals increased responses to songs from their own island across a range of indices. O‘ahu individuals responded at equal rates, indicating response asymmetry between these populations.
Keywords: Song divergence, isolation, asymmetry, warbling white-eye, Hawaiian Islands
EVIDENCE OF INCIPIENT SONG DIVERGENCE 3
TABLE OF CONTENTS
Abstract...... 2
Acknowledgements...... 4
List of figures...... 5
List of tables...... 6
Introduction...... 7
Methods...... 11
Results...... 25
1. Acoustic analysis...... 25
2. Behavioral analysis...... 31
Discussion...... 37
Conclusion...... 43
Supplementary tables...... 44
Appendix...... 48
References...... 49
EVIDENCE OF INCIPIENT SONG DIVERGENCE 4
ACKNOWLEDGMENTS
I acknowledge that field research for this study was conducted on lands inhabited by the indigenous Kanaka ‘Ōiwi peoples and yielded to the United States of America by Queen
Lili‘uokalani under duress and protest.
This research was supported in part by a Thesis Research grant from the Animal Behavior and
Conservation Graduate Program, Hunter College. I’m deeply indebted to: Dr. Caroline Dingle and the Dingle Lab (University of Hong Kong) for endless support, insight, mentorship, and many time zone-challenged Zoom calls; Dr. Ofer Tchernichovski (Hunter College) for invaluable help with design and analysis; Dr. Diana Reiss (Hunter College) for commitment to seeing this project through; Matthew Medler (Macaulay Library at The Cornell Lab of
Ornithology) for assistance with song archives; Jay McGowen (eBird) for equipment consultation; Dr. Patrick Hart (University of Hawai‘i) for field correspondence; Raedelle Van
Fossen (Lyon Arboretum) and Adam Williams (Hawai‘i DLNR) for logistical support; Lyndsay
Hage (ABC), Amanda Rodriguez (ABC), and Amanda Puitiza (ABC) for student support; the people of Hawai‘i for their “aloha spirit”; and to Valerie Snow, who supported this project with heroic patience and endless encouragement.
Mahalo nui loa!
EVIDENCE OF INCIPIENT SONG DIVERGENCE 5
LIST OF FIGURES
Figure 1. Warbling white-eye (Zosterops japonicus)...... 12
Figure 2. The Hawaiian Islands...... 14
Figure 3. Spectrogram of four white-eye songs...... 15
Figure 4. O‘ahu: experimental sites...... 16
Figure 5. Big Island: experimental sites...... 17
Figure 6. Spectrogram with robust selection annotations...... 19
Figure 7. Pictogram of experimental setup...... 21
Figure 8. Scatterplots of significant MANOVA results...... 27
Figure 9. Spectrogram of song exemplars from three populations...... 28
Figure 10. Scatterplot of PC1/PC2 regression output...... 30
Figure 11. Scatterplot of LDA canonical variates...... 31
Figure 12. Histogram of Chi-square test of Independence response rate results...... 32
Figure 13. Histogram of Kruskal-Wallis behavioral response results...... 34
Figure 14. Histogram of Kruskal-Wallis extended vigilance results...... 35
Figure 15. Histogram of Kruskal-Wallis latency-to-response results...... 36
EVIDENCE OF INCIPIENT SONG DIVERGENCE 6
LIST OF TABLES
Table 1. Acoustic parameters...... 19
Table 2. Behavioral ethogram...... 22
Table 3. Summary results: MANOVA...... 26
Table 4. Summary results: PCA residuals carried forward for univariate ANOVAs...... 29
Table 5: Summary Results: Behavioral/latency-to-response variables...... 37
Supplementary Table S1. Descriptive statistics: Acoustic variables...... 44
Supplementary Table S2. PCA loading variables...... 44
Supplementary Table S3. LDA structure matrix...... 45
Supplementary Table S4. Summary results: Chi-square test of Independence...... 45
Supplementary Table S5. Descriptive statistics: Behavioral response variables...... 46
Supplementary Table S6. Descriptive statistics: Latency-to-response variables...... 47
Appendix: Archival recordings...... 48
EVIDENCE OF INCIPIENT SONG DIVERGENCE 7
Evidence of Incipient Song Divergence in a Hawaiian population of Warbling White-eyes
INTRODUCTION
Island ecosystems have long been of interest to students of evolutionary biology (Darwin,
1859; Wallace, 1869; Mayr, 1963; MacArthur & Wilson, 1967; Grant, 1999). In contrast to the mainland, where broad population distribution allows for wider gene flow, the isolation of island biota often simplifies phenotypic differentiation and speciation through the causal processes of founder events, ecological selection, or genetic drift (Templeton, 1980; Baker et al., 2006; Losos
& Ricklefs, 2009; reviewed in Provine, 2004). The adaptive radiation common to island species has been studied in morphological and molecular patterns of evolution (Clegg et al., 2002a;
2002b; Grant, 2002; Moyle et al., 2009). Less studied are behavioral phenotypes, such as those involved in pre-mating and mating signals, which play a major role in mate choice and may mediate evolutionary processes such as reproductive isolation and speciation (Slabbekoorn &
Smith, 2002; Uy et al., 2018).
Oscine passerines are among the most rapidly evolving and speciose vertebrate lineages
(Wyles et al., 1983), potentially due to the role of song learning in accelerating rates of allopatric speciation (Lachlan & Servedio, 2004). For many oscine passerines, including songbirds, hummingbirds, and parrots, species-typical song is a phenotypically plastic trait, essential for conspecific recognition and reproductive success (Searcy, 1922; Rothstein et al., 1986;
Catchpole & Slater, 1995; Ballentine et al., 2004). Normal song acquisition relies heavily on mimetic learning from conspecific social models during critical windows of development
(Thorpe, 1958; Marler & Tamura, 1964; Baptista & Petrinovich, 1984; Nelson et al., 2001; reviewed in Alpin, 2019). Any social or cultural transmission of learned traits is subject to a EVIDENCE OF INCIPIENT SONG DIVERGENCE 8
number of copy “errors”, which can quickly generate novel phenotypes and promote population divergence (Mundinger, 1980; Pfennig et al., 2010; reviewed in Alpin, 2019). Accelerated changes in learning (Lachlan & Servedio, 2004) which lead to a sufficient structural divergence may constitute a barrier to reproduction (Slabbekoorn & Smith, 2002; reviewed in Price, 2008).
As noted by Cushing (1941), “…it is conceivable that an initial sexual isolation may arise entirely as the result of environmental influences without the occurrence of genetic change” (see also Immelmann, 1975; Grant & Grant, 1997). The effects of learning and plasticity on the rapid evolution of phenotypic traits, and ultimately speciation, are often context-dependent, with empirical examples of phenotypic changes both reducing gene flow (Haavie et al., 2004) and also expanding population connectivity (Grant & Grant, 1997).
Island introductions or expansions further alter the trajectory of these learning processes
(reviewed in Grant, 2001), where song variations likely arise through the loss of repertoire elements when immature birds and/or a small subset of the adult population colonize a geographically isolated area (Thielcke, 1973). This process, called a ‘cultural founder effect’
(Baker & Jenkins, 1987), is akin to a genetic founder event, defined as, “the loss of genetic variation that occurs when a new population is established by a small number of individuals from a larger population” (Mayr, 1942; 1954; reviewed in Mayr, 2000). The impact of founder events on vocal communication commonly include reductions in song and note types (e.g. fox sparrow,
Passerella iliaca: Naugler & Smith, 1991; singing honeyeater, Meliphaga virescens: Baker,
1996; Chinese hwamei, Garrulax canorus: Tu & Severinghaus, 2004) as well as simplified note types, fewer note combinations, and increased note stereotypy (e.g. Christmas Island warbler,
Acrocephalus aequinoctialis: Milder & Schreiber, 1989; blue tit, Parus caeruleus palmensis:
Schottler, 1995). The loss of note or syllable repertoire has been observed most prominently in EVIDENCE OF INCIPIENT SONG DIVERGENCE 9
mainland-island comparisons. For example, in an introduced chaffinch (Fringilla coelebs) population on Chatham Island, the pool of note types was significantly reduced compared to the mainland population (Baker & Jenkins, 1987). In comparisons of remote island and mainland populations, the introduced island population of Japanese bush warblers (Cettia diphone) exhibited a rapid decrease in frequency range, lower number of inflections, and simplified note types (Hamao & Ueda, 2000; Hamao, 2013; 2015). By contrast, the sub-oscine rainbow lorikeet
(Trichoglossus haematodus), whose song is largely innate rather than learned, showed no evidence of a cultural founder effect following an extreme population bottleneck after introduction of fewer than 10 individuals to Perth, Western Australia (Baker, 2014).
Once established on a new island, multiple local ecological and sexual selection pressures also act upon song production in island ecosystems which could lead songs to diverge from their parental types (Kroodsma & Miller, 1996). For example, in the gray-breasted wood-wren
(Henicorhina leucophrys), habitat-dependent song divergence is a likely driver of reproductive divergence via assortative mating (Dingle & Slabbekoorn, 2008).
Another impact of introduction to islands is the rapid adaptive radiation of species, known most famously from the extensive studies of Darwin's finches on the Galapagos Islands
(Grant, 1999). An equally interesting example of rapid phenotypic diversification is the genus
Zosterops (Gill, 1973; Moyle et al., 2009). Called “the great speciator” (Diamond et al., 1976) and a “textbook example of rapid speciation” (Lim et al., 2019), Zosterops is a highly speciose genera distributed widely throughout the Eastern Hemisphere; especially known for having colonized numerous islands throughout East and Southeast Asia, the Indian Ocean, and Tropical
Pacific (Gill & Donsker, 2019). Many of the approximately 100 species or subspecies are single island endemics (Mees, 1961; Pratt et al., 1987). Because of this, Zosterops is an ideal taxon to EVIDENCE OF INCIPIENT SONG DIVERGENCE 10
examine evolutionarily divergent patterns and the diversification of acoustic signaling following island introduction.
The Hawaiian Islands (16,636 km2) – a sprawling archipelago comprised of over 100 atolls, reefs, islets, and eight major islands – are among the most remote on Earth, located approximately 2,400 km from the nearest landmass (Macdonald et al., 1983). [Hereafter,
“Hawai‘i” and “Hawaiian Islands” refers to the island group, whereas the “Big Island”, as it’s known colloquially, refers to the Island of Hawai‘i.] Despite this extreme isolation, Hawai‘i has been at the epicenter of exotic species release, with more introductions than any location in the world, relative to size (Loope & Mueller-Dombois, 1989). Over 170 avian species have been released, either intentionally or by escape, and at least 54 species have naturalized (Pyle, 2002).
Among these is the warbling white-eye (Zosterops japonicus), intentionally introduced from
Japan to O‘ahu, Hawai‘i in 1929 to control insect pests (Caum, 1933). Success led to multiple subsequent introductions on Kaua‘i, parts of Maui Nui (made up of Maui, Moloka‘i, Lāna‘i, and
Kaho‘olawe islands), and the Big Island (Berger, 1981). While introduction dates were not well documented, peak importation occurred between 1929 – ca. 1937 (Foster, 2009).
In the present study, I examined the potential divergence of song features from their native forms in the introduced white-eye populations of Hawai‘i. I recorded songs and collected archival recordings to conduct a comparative spectrographic analysis of three isolated populations (O‘ahu, Big Island, Japan), illustrating the surprising variety of ways that isolation has influenced song divergence in this species. To explore behavioral changes that may correspond to this apparent divergence, I presented playback stimuli from conspecific populations (inter-island, intra-island, native) to individuals on two Hawaiian Islands (O‘ahu,
Big Island) at the onset of their breeding season and recorded behavioral responsiveness across a EVIDENCE OF INCIPIENT SONG DIVERGENCE 11
suite of indices. I hypothesized that individuals would indicate the recognition of song types particular to their own population by increased behavioral responsiveness, such as reduced latency, increased duration of interest, vocalizations, agonistic postures, and extended vigilance.
Despite widespread abundance across the Eastern Hemisphere, there are limited studies of vocal and behavioral communication in genus Zosterops (e.g. Slater, 1991; 1993; Robertson, 1996;
Potvin et al., 2011; Baker, 2012; Potvin & Parris, 2012; Potvin, 2013), with scant empirical analysis of Zosterops japonicus vocalizations to date (e.g. Hamao et al., 2013). Therefore, this study offers insights into song features unique to this species and advances our basic knowledge of white-eye behavior in Hawai‘i.
METHODS
Research aims
In this study, I recorded novel songs in Hawai‘i and collected archival recordings to conduct the first comparative analysis between introduced Hawaiian white-eyes and their native population, Japan. I then tested whether song divergence corresponds with behavioral change through a series of playback experiments. I created playback stimuli of white-eye songs from intra-island, inter-island, and native (Japan) populations; presented each at random to individuals at the onset of breeding season (January – February); and observed responses across a suite of behavioral measurements using 10 sec interval sampling methods.
Study species
Introduced to the Hawaiian Islands between 1929 – ca. 1937, Zosterops japonicus
[hereafter, “white-eye”] was established on O‘ahu by the early 1930’s (Caum, 1933) and was soon ubiquitous on all major islands (Berger, 1972). The success of white-eyes is owed to a EVIDENCE OF INCIPIENT SONG DIVERGENCE 12
flexibility of habitat and diet (Scott et al., 1986; Pratt et al., 2009). They thrive in a wide range of habitats, from low-elevation urban areas to montane rain forests up to 3100 m
(Mountainspring & Scott, 1985) and are omnivorous, feeding on arthropods and over thirty varieties of native and exotic nectar and fruit (Guest, 1973). Breeding season in Hawai‘i begins in late January and ends with the formation of large winter flocks in August (Guest, 1973).
Adult white-eyes range 10.0 – 11.5 cm; 7.5 – 13.9 g. Sexes are alike: olive-green with a lemon-yellow throat and upper breast; pale grey underparts and belly; grey legs; slate-grey, pointed bill; blackish spot behind bill and black loral line continuing under a prominent white eye-ring (Van Riper & van Balen, 2020). The scientific nomenclature Zosterops reflects this latter feature, derived from the Ancient Greek [ζωστήρ ωπος] for “eye-girdle” (Jobling, 2018)
[Figure 1].
Figure 1. Adult warbling white-eye (Zosterops japonicus), Hawai‘i, HI. Photo by Peter Roberts, 2017. Retrieved from Macaulay Library [archive ML63638051].
Inter-island migration has not been observed in Hawai‘i (Van Riper & van Balen, 2020), except for rare pelagic events when individuals or small flocks have been sighted miles from land, presumably blown to sea (Ely, 1971; Amerson & Shelton, 1976; Howard et al., 2013). EVIDENCE OF INCIPIENT SONG DIVERGENCE 13
Rather, white-eyes appear sedentary (Guest, 1973), potentially an example of the “behavioral flightlessness” phenomenon common to island avifauna, where despite being able to do so, populations exhibit reduced propensity to migrate across water barriers (Diamond, 1972).
Therefore, it is presumed that populations on O‘ahu and the Big Island (215 km apart) are currently isolated from each other and the result of multiple direct introductions from Japan, followed by a natural range expansion across each island once established [Figure 2].
Male Zosterops have an expansive repertoire of discrete note types (Cynx, 1990) which are rearranged in series to form unique combinations that appear to be continuous and represent enormous diversity (e.g. silvereye, Zosterops lateralis: Slater, 1993; Baker, 2012; Potvin &
Parris, 2012; Potvin, 2013). A typical white-eye song consists of a 2-7 sec (M = 3.35; SD ±
1.80) sequence containing between five and 40 notes (M = 17.36; SD ± 9.33), though occasionally longer (J. Robinson, pers. obs.). The order and number of notes is variable between songs, but individual phrases are occasionally repeated as introductory signatures and as multi- syllabic memes embedded within a song [Figure 3]. Male white-eyes sing throughout the day, with the highest frequency during an early dawn chorus where songs are produced in repetitive bouts lasting approximately 30-45 minutes (Guest, 1973). EVIDENCE OF INCIPIENT SONG DIVERGENCE 14
Figure 2. The eight major Hawaiian Islands. Red arrows indicate islands where experiments and song recordings took place: O‘ahu and Big Island (Island of Hawai‘i) (215 km apart).
EVIDENCE OF INCIPIENT SONG DIVERGENCE 15
1 2 3 4 5 6 7 8 1 3 9 10 11 12 13 14 15 16
17 18 19 20 21 16 16 22 23 8 ? 8 1 3 9
1 2 3 4 5 6 24 18 25 25 26
19 14 11 14 ? 27 28 7 23 19 16 29
Figure 3. Four songs of a Big Island white-eye song bout. Numbers below notes designate note types identified by the author. Color indicates multi-syllabic repeated sequence. [archive ML129119].
EVIDENCE OF INCIPIENT SONG DIVERGENCE 16
Study sites
Two replicate playback experiments were conducted: on O‘ahu, Hawai‘i (1,545 km2) from 28 January – 5 February 2020 and on the Big Island, Hawai‘i (Island of Hawai‘i:
10,430 km2) from 6 February – 13 February 2020. Geospatial coordinates for 10 experimental locations were selected from the eBird Basic Dataset, a citizen-sourced archive of worldwide bird observations (2019a; 2019b) [Figures 4 and 5]. Each location was a low- to mid-elevation, publicly accessible birding area where multiple white-eye observations had been made during the same period in previous years by active eBird users. State Park locations were accessed with permission from the Hawai‘i Department of Land and Natural Resources, Division of State
Parks. Between 0645 – 1100 and 1600 – 1830, trails, roads, or open grounds were walked until subjects were opportunistically discovered by sight or vocalization. To reduce duplicate sampling, each site within a location was only visited once
Figure 4. O‘ahu study sites: (1) Lyon Arboretum, (2) Wa‘ahila Ridge Trail, (3) Kea‘iwa Heiau State Recreation Area, (4) Queen Kapi‘olani Garden. (5) University of Hawai‘i (UH) Manoa campus. EVIDENCE OF INCIPIENT SONG DIVERGENCE 17
Figure 5. Big Island study sites: (1) Hāpuna Beach State Recreation Area, (2) Kekaha Kai State Park, (3) Maka‘eo Walking Path, (4) Kealakekua Bay Historical Park, (5) Manuka State Wayside.
Song sampling
Novel songs of male white-eyes were recorded (24-bits/48 kHz) in Hawai‘i using a shotgun microphone (Azden SGM-250) connected to a portable digital recorder (Tascam DR-
70D) with an 18 dB in-line phantom powered preamp (TritonAudio FetHead). A modular windshield (Rode BLIMP) was used to minimize low frequency interference. 18 individuals were opportunistically recorded during the dawn chorus on two islands (Big Island, N = 10;
O‘ahu, N = 8). Songs were natural (i.e. not a response to playback stimuli). EVIDENCE OF INCIPIENT SONG DIVERGENCE 18
To increase sample sizes, in situ recordings were supplemented with archival recordings from the Macaulay Library at The Cornell Lab of Ornithology (macaulaylibrary.org); eBird
(ebird.org); and xeno-canto citizen science project (xeno-canto.org) [see Appendix]. Archived songs were selected for quality (four/five stars or A/B rating) and date of recording (less than 20 years old).
Acoustic Analysis
All song recordings were transformed into spectrograms and salient acoustic features were annotated in the time-frequency domain in Raven Pro 1.6.1 (Center for Conservation
Bioacoustics, 2019) within a 256 Hann window and 50% time grid overlap, spacing of 86.1 Hz, and a discrete Fourier transform size of 512 samples. A spectral analysis was undertaken at the population level (mean of songs per bird) to account for intra-individual variation and test for patterns of divergence between populations. Nine acoustic features were selected: 95% frequency (95% of the summed energy in a note); peak frequency; bandwidth (90%); number of
PFC inflections (the number of changes in the sign of the derivative [slope] of frequency, measured with the robust peak frequency contour [PFC] function); aggregate entropy (a measure of total disorder in a note); song duration; note length; notes per song; and ‘delivery rate’
(number of notes per second of song) (following Dingle & Slabbekoorn, 2018) [Table 1, Figure
6]. Differences in aggregate entropy may reflect variation in recording conditions; however, where considerable structural differences exist (e.g. between ‘simple’ and ‘complex’ note types) aggregate entropy measurements are capable of discriminating between notes (Fournet et al.,
2018).
The precision of measurements in Raven Pro can be influenced by several factors, including observer bias. Therefore, the robust signal measurement tools within Raven Pro EVIDENCE OF INCIPIENT SONG DIVERGENCE 19
interface, which are less sensitive to (i.e. robust against) variations by the user, were utilized to produce a more precise range of signal components within selection fields (Charif et al., 2010).
Table 1. Acoustic parameters.
95% Frequency (Hz) 95% of summed energy in the spectrogram selection Bandwidth (90%) (Hz) The difference between 5% frequency (above which 95% of the energy is contained) and 95% frequency (below which 95% of the energy is contained) Peak frequency (Hz) Frequency with the highest spectral peak, or average power PFC inflection points (n) Number of changes in the sign of the derivative (slope) of the frequency on the sound spectrogram; measured using the robust peak frequency contour (PFC) function in Raven Pro Aggregate entropy (bits) Total disorder in a note, measured as the proportion of energy in each frequency bin times its log (base 2), summed over all frequency bins within a selection Delivery rate (n) Number of notes produced per second of song Song duration (s) Duration of song sequence production from first to last note Note length (s) Length of discrete notes Notes per song (n) Cumulative number of notes per song
Figure 6. Spectrogram annotations of six notes in a warbling white-eye song. Numbers represent discrete notes. Aqua boxes indicate user selections within Raven Pro 1.6 interface. Light green line indicates 95% frequency. Dark green line indicates peak frequency. Bandwidth (90%) is indicated as the difference between purple and blue lines. PFC inflections are indicated as changes in the yellow line. EVIDENCE OF INCIPIENT SONG DIVERGENCE 20
Playback sequences
Playback sequences were created from songs in the archives of The Macaulay Library and xeno-canto [see Appendix]. To account for intra-individual variability and reduce pseudoreplication (Hurlbert, 1984; Kroodsma, 1989; McGregor, 2000; Kroodsma et al., 2001), songs were selected from 24 unique individuals (Big Island, N = 8; O‘ahu, N = 8; Japan, N = 8).
At the time of the experimental design, there were no ‘good’ quality (four/five stars or A/B rating) O‘ahu songs in these archives, therefore eight wild O‘ahu individuals were recorded in situ prior to the experiment. Song sequences were prepared for presentation in OcenAudio
(2019, version 3.7.6), a 64-bit audio editing software. None of the recordings used in the playbacks were from test subjects or their neighbors.
Playback experiment
Conspecific playback stimuli were divided into three treatment groups: intra-island, inter- island, and native (Japanese). A research randomizer (www.randomizer.org) was used to generate random stimulus presentation assignments throughout the experiments. The red-billed leiothrix (Leiothrix lutea) is an introduced passerine, whose low elevation habitat overlaps with white-eyes in Hawai‘i (Male et al., 2020). To test if white-eyes responded to any stimulus provided, song exemplars of this heterospecific species were randomly presented using the above methods. These songs were from the Macaulay Library archives [see Appendix].
Playbacks were presented opportunistically when an individual was visually located.
Trials were conducted from 0645–1100 and 1600–1830, when activity is highest (Guest, 1973).
Each bird received a single trial and only one trial was conducted per site (even if no response was elicited). To minimize the chance of an individual overhearing a previous stimulus, all trials EVIDENCE OF INCIPIENT SONG DIVERGENCE 21
took place >100 m apart (following Gray, 1988). In total, 198 individual birds were presented conspecific playback trials (Big Island, N = 99; O‘ahu, N = 99), with equal sample sizes between treatment groups, and 36 individuals were presented heterospecific playback trials (Big Island, N
= 13; O ‘ahu, N = 23).
Each trial consisted of placing a Bluetooth speaker (JBL CLIP-3) approximately 2 m above ground within a putative territory; broadcasting a playback at natural amplitude (80 dB at
1 m from the source; McGregor et al., 1992) for 120 sec; and observing the subject for an additional 60 sec in silence after the playback. The observer stood 10 m from source [Figure 7].
Figure 7. Pictogram of the experimental setup.
Quantifying response to playbacks
Playback trials were visually observed for behavioral responses throughout the 180 sec trial period using a 10 sec instantaneous point sampling method (Martin & Bateson, 2007). A suite of nine parameters represented reliable responses to playbacks (following Kikkawa &
Kakizawa, 1981; Martin & Martin, 2001; Nelson & Soha, 2004; Searcy et al., 2006; Jankowski et al., 2010; Pegan et al., 2015; Freeman et al., 2016). These included: duration of interest, EVIDENCE OF INCIPIENT SONG DIVERGENCE 22
closest approach distance, duration of vocalizations, agonistic postures (wing flutter, supplant, binocular gaze), extended vigilance (time spent < 10 m from speaker after playback ended), latency to first response, latency to closest approach, latency to first vocalization, and latency to first agonistic response [Table 2].
Nominal categories estimated the focal subject’s distance from the speaker: “5” (>10 m),
“4” (5-10 m), “3” (2-5 m), “2” (1-2 m), and “1” (<1 m) (adapted from Dingle & Slabbekoorn,
2018). A closer approach was inferred to indicate a stronger response. Category “5” was used when no bird was observed or if the focal individual was >10 m from the speaker. These data were recorded into Insight: Behavioral Timer (version 1.3.2) (Radloff, 2019), a behavioral observation touchscreen application.
Table 2. Ethogram of behavioral responses.
LATENCIES Time from onset of stimulus period First approach (s) < 10 m from speaker Closest approach (s) Closest distance to speaker Vocal response (s) First vocal response Agonistic response (s) First wing flutter, binocular gaze, supplant (or combination) Wing flutter: lowered wings, vibrating wing tips Binocular gaze: horizontal posture, head forward, open bill Supplant: direct aerial ‘attack’, contact with speaker BEHAVIORAL RESPONSES Approach distance (m) Closest approach distance at any point during trial Interest duration (s) Cumulative time spent <10 m during trial Vocal duration (s) Time of vocal response during trial Agonistic duration (s) Time of agonistic response during trial Extended vigilance (s) Time spent <10 m from speaker after stimulus period
Statistics
For the acoustic data, three analyses were utilized: (1) one-way multivariate analysis of variance (MANOVA), (2) principal component analysis (PCA), and (3) linear discriminant function analysis (LDA). The MANOVA aimed to test for the linear composite vector of EVIDENCE OF INCIPIENT SONG DIVERGENCE 23
acoustic variable means between independent groups. There were no univariate outliers in the data, as assessed by inspection of boxplots and Mahalanobis distance (p > 0.001). Statistically significant results were carried forward for further univariate one-way ANOVA analyses using a
Bonferroni adjusted alpha. As sample sizes were unequal, Tukey’s post hoc tests were used where relevant to conduct multiple pairwise comparisons. To minimize the risk of a Type II error, I conducted a PCA which grouped the acoustic variables in a smaller number of components. Correlation matrices were first judged for sampling adequacy with the Kaiser-
Meyer-Olkin (KMO) test and their condition was examined with Bartlett's sphericity test.
Reflective of the small sample size, the KMO value = 0.63, indicating a ‘mediocre” suiting for factor analysis (Kaiser, 1974). However, this value was above the acceptable 0.50 value and correlation matrixes were well-conditioned by Bartlett's test (χ2(36) = 392.56, p < 0.001), therefore I proceeded. Varimax rotation was used to simplify the expression. PCA resulted in three principal components that explained 84.16% of the total variance in the data. A univariate analysis of variance (ANOVA) was carried out on principal components with eigenvalues > 1.
Tukey’s Honest Significant Difference (HSD) post hoc tests were used for ANOVAs which returned significant results. I used an LDA to determine whether songs could be accurately identified to population based on these song characteristics. Canonical discriminant functions were judged using Wilks' lambda (Λ). Post-hoc assignment probabilities were obtained by jack- knifed cross validation. As the acoustic parameters were on different scales, all were re-scaled for inclusion in PCA and LDA, using a standardizing function such that their means = 0 and their standard deviations = 1.
Chi-square tests of Independence were conducted to test if individuals responded to conspecific and heterospecific stimuli with equal likelihood (Yes/No) on both islands (N = 234). EVIDENCE OF INCIPIENT SONG DIVERGENCE 24
All expected cell frequencies were greater than five. Adjusted values were then transformed into
Chi-square values and compared using a Bonferroni correction.
Behavioral response scores were not normally distributed as assessed by visual inspection of boxplots and Shapiro-Wilke’s tests (all dependent variables, p < 0.05). Therefore, non- parametric Kruskal-Wallis H-tests were used to determine if there were differences in behavioral responses between multiple playback stimuli treatment groups on each island. To control for
“false discovery rate” (FDR), all p-values were assigned ranks and the Benjamini-Hochberg
(1995) adjusted critical value (0.05) was calculated. Where relevant, subsequent pairwise comparisons were performed using Dunn's (1964) procedure. Significant values were then adjusted by a Bonferroni correction for multiple comparisons. Experiments were analyzed independently [hereafter: “Big Island Experiment” and “O‘ahu Experiment”].
All statistical analyses were conducted using IBM SPSS Statistics for Windows version
26.0 (IBM Corp., Armonk, NY, USA).
Funding & ethics statement
This research was supported by an Animal Behavior & Conservation (ABC) program student research grant from the Hunter College Foundation (#020520). The warbling white-eye is not an endangered or protected species in the Hawaiian Islands and no individuals were physically manipulated during this research.
RESULTS
Acoustic analysis
Acoustic analysis was based on 18 recordings made by the author and 24 archival recordings (xeno-canto, N = 11; Macaulay Library, N = 7; eBird, N = 6) for a total of 349 songs EVIDENCE OF INCIPIENT SONG DIVERGENCE 25
from 42 male individuals: 181 songs from 16 individuals on the Big Island (mean of 11.3 songs per individual); 102 songs from 12 individuals on O‘ahu (mean of 8.5 songs per individual); and
66 songs from 14 individuals in Japan (mean of 4.7 songs per individual). Song duration ranged from 1.9 to 14.8 sec (M = 3.35; SD ± 1.80) with inter-song intervals ranging from 1.6 to 18.7 sec
(M = 3.71; SD ± 3.15). To account for intra-individual variation, means were computed from an average of songs per individual on each island. Consequently, the population samples for subsequent statistical tests were: Big Island, N = 16; O‘ahu, N = 12; Japan, N = 14 [descriptive statistics in Supplementary Table S1].
Song variables differed significantly between island populations (MANOVA: F2, 41 =
3.477, p < 0.001, Wilks’ Λ = 0.248, partial η2 = 0.502). Follow-up univariate ANOVAs with a
Bonferroni adjusted alpha showed that five of nine acoustic variables were significantly different: 95% frequency, bandwidth, note length, number of inflection points, and aggregate entropy. Tukey’s post hoc comparisons showed that O‘ahu populations had significant acoustic differences from both the Big Island and Japan: 95% frequency (p = 0.02, p = 0.044), bandwidth
(p = 0.009; p = 0.008), aggregate entropy (p = 0.007; p = 0.008) (for O‘ahu/Big Island;
O‘ahu/Japan, respectively). Note length in O‘ahu songs was shorter than length in songs from
Japan (p = 0.002) but did not differ from songs from the Big Island. Number of inflection points
(changes in slope of the frequency) were significantly reduced on the Big Island when compared to Japan (p = 0.012) [Table 3, Figure 8].
EVIDENCE OF INCIPIENT SONG DIVERGENCE 26
Table 3. Summary results: MANOVA and Tukey’s post hoc comparisons testing for differences in song variables between Big Island, O‘ahu, and Japan (N = 42, the number of individuals compared). Wilks’ Λ= 2 0.248, F = 3.477, p < 0.001, ηp = 0.502. Bold indicates significance.
FACTOR Statistics post hoc tests 2 F2, 41 p ηp O/B O/J B/J FREQUENCY (95%) 4.586 0.016 0.19 0.02 0.044 0.962 PEAK FREQUENCY 0.936 0.401 0.046 – – – BANDWIDTH 6.466 0.004 0.249 0.009 0.008 0.99 NOTE LENGTH 6.466 0.003 0.264 0.139 0.002 0.129 SONG DURATION 1.099 0.343 0.053 – – – NOTES PER SONG 0.503 0.609 0.025 – – – DELIVERY RATE 2.228 0.121 0.103 – – – INFLECTION POINTS 4.676 0.015 0.193 0.578 0.168 0.012 AGGREGATE ENTROPY 6.649 0.003 0.254 0.007 0.008 0.997
EVIDENCE OF INCIPIENT SONG DIVERGENCE 27
Figure 8. Scatterplots of significant variables, MANOVA. (A) 95% frequency, (B) bandwidth, (C) PFC inflection points, (D) aggregate entropy. X-axis represents individuals. Blue circles represent Big Island (N = 16). Gray circles represent Japan (N = 14). Orange circles represent O‘ahu (N = 12).
EVIDENCE OF INCIPIENT SONG DIVERGENCE 28
A.
B.
C.
Figure 9. Song exemplars (A) Big Island [archive ML129122], (B) O‘ahu [recorded by JR], (C) Japan [archive XC507025]. EVIDENCE OF INCIPIENT SONG DIVERGENCE 29
PCA was conducted to group song variables in a smaller number of components. Three principal components were obtained with eigenvalues > 1 which explained 84.16% of the total variance: PC1 (51.26%), PC2 (18.73%), and PC3 (14.16%). The first rotated component (PC1) was most heavily loaded by bandwidth (0.923), aggregate entropy (0.914), 95% frequency
(0.876), note length (0.876), PFC inflection (0.676), and delivery rate (-0.594). The second rotated component (PC2) was most heavily loaded by song duration (0.946), notes per song
(0.662), and delivery rate (-0.519). The third component (PC3) was most heavily loaded by peak frequency (0.864) [loading variables in Supplementary Table S2]. PC1 differed significantly
between island populations (ANOVA: F2, 41 = 4.763, p = 0.014). Tukey’s HSD post hoc tests showed that O‘ahu songs differed most significantly from Japan songs (p = 0.015), and that there were no significant differences between O‘ahu/Big Island, or Big Island/Japan. PC2 and PC3
did not differ between populations (PC2, ANOVA: F2, 41 = 0.855, p = 0.433; PC3, ANOVA: F2, 41
= 2.136, p = 0.132) [Table 4, Figure 10].
Table 4. Summary of PCA residuals carried forward for univariate ANOVAs with Tukey’s HSD post hoc tests where relevant. Bold indicates significance.
COMPONENT Statistics post hoc tests 2 F2, 41 P ηp O/B O/J B/J PC1 (51.26%) 4.763 0.014 0.196 0.691 0.015 0.068 PC2 (18.73%) 0.855 0.433 0.042 – – – PC3 (14.16%) 2.136 0.132 0.099 – – –
EVIDENCE OF INCIPIENT SONG DIVERGENCE 30
Figure 10. Scatterplot of PC1/PC2 regression values between populations (indicative of bandwidth, aggregate entropy, 95% frequency, note length, PFC inflection, and delivery rate). PC1 explains 51.26% of total variance. PC2 explains 18.73%.
LDA was used to determine whether songs could be accurately identified to population based on song characteristics. Variable correlations within the discriminant function were significant for: 95% frequency (p = 0.016); bandwidth (p = 0.004); note length (p = 0.003), PFC inflection (p = 0.015), and aggregate entropy (p = 0.003) [structure matrix in Supplementary
Table S3]. Canonical discriminant Function 1 had an eigenvalue = 1.636, which explained
75.5% of total variance (Wilks' Λ = 0.248, p < 0.001). Function 2 had an eigenvalue = 0.532 which explained the remaining 24.5% of variance (Wilks' Λ = 0.653, p = 0.06). The classification function correctly assigned song features to their respective populations at a cross- validated probability of 64.3% (Big Island = 81.3%, O‘ahu = 66.7%, Japan = 42.9%) [Figure
11].
EVIDENCE OF INCIPIENT SONG DIVERGENCE 31
Figure 11. Scatterplot of multivariate spatial distributions of white-eye songs between Big Island, O‘ahu, and Japan (N = 42). Canonical variates are from a linear discriminant analysis (LDA) of nine acoustic measures. Classification function resulted in 64.3% of cross-validated group values correctly assigned: Big Island (81.3%); O‘ahu (66.7%); Japan (42.9%).
Behavioral Analysis
There were significant and strong associations (Cohen, 1988) between the playback treatments and response types (Yes/No) on each island. The heterospecific control group responses were distinct from conspecific treatment responses in both experiments (p < 0.001)
[Supplementary Table S4, Figure 12]. Therefore, it was determined that white-eyes were responding to conspecific, not heterospecific, stimuli. The heterospecific control group was removed from further analysis.
EVIDENCE OF INCIPIENT SONG DIVERGENCE 32
*** *** *** ***
Figure 12. Chi-square tests of Independence (N = 234). Response (Y/N) to playback treatment groups. (A). Big Island Experiment: χ2(3) = 47.22, p < 0.001, Cramer’s V = 0.644, (B). O‘ahu Experiment: χ2(3) = 39.66, p < 0.001, Cramer’s V = 0.570. [***] indicates post hoc significance (p < 0.001).
The remaining 198 trials (Big Island, n = 99; O‘ahu, n = 99) had equal sample sizes between treatment groups [descriptive statistics in Supplementary Tables S5 and S6]. In the Big
Island Experiment, individuals significantly increased responses to song types from their own island and decreased responses to song types from O‘ahu across all metrics: approach (p = 0.03), interest (p < 0.001), vocal (p < 0.001), and agonistic postures (p < 0.001) [Table 5, Figure 13].
There were also significant differences between intra-island song types and song types from
Japan: agonistic postures (p = 0.04). To test for extended vigilance, individuals were observed for an additional 60 sec immediately following the end of a playback stimulus. In the Big Island
Experiment, vigilance to intra-island song types was significantly greater than inter-island
(O‘ahu) song type vigilance (p = 0.002). This indicates that Big Island birds quickly lost interest in O‘ahu songs after the playback stimulus ended but retained a strong interest in their own EVIDENCE OF INCIPIENT SONG DIVERGENCE 33
island songs [Figure 14]. Latency scores in the Big Island Experiment also indicated that responses occurred nearer to the onset of a playback treatment when an intra-island song was presented than when an inter-island (O‘ahu) or native (Japan) song was presented. The most significant differences in latency were between the Big Island/O‘ahu: first approach (p < 0.001), first agonistic posture (p < 0.001); and Japan/O‘ahu: first approach (p < 0.001), first vocalization
(p = 0.003) [Table 5, Figure 15].
In the O‘ahu Experiment, birds responded asymmetrically to playback treatments [Table
5]. Across most behavioral metrics there were similar response rates between playback treatments, indicating that individuals on O‘ahu did not discriminate between divergent song types. Individuals did exhibit increased responsiveness in one metric type, agonistic posture (p =
0.003). When presented songs from their own island, birds showed significant increases in agonism, particularly when compared with native (Japan) songs (p = 0.003) [Figure 13]. O‘ahu birds also showed reduced latency to first agonistic posture (p = 0.02), with the most significant differences between O‘ahu/Big Island (p = 0.02) [Figure 15]. Extended vigilance was observed in the O‘ahu population; however, after correcting for multiple comparisons, the trend in between intra-, inter-island, and native stimuli was rendered nonsignificant.
EVIDENCE OF INCIPIENT SONG DIVERGENCE 34
A. B. a,c x a,c
b x x x x a,b b x a
C. D. a,c a x a,c x x x x,y b b,c
b y
Figure 13. Behavioral responsiveness: (A) approach distance, (B) duration of interest, (C) duration of vocalization, (D) duration of agonistic posture. X-axis represents playback treatment types. Lower case letters above bars indicate groups (a,b,c) (x,y) that are not significantly different, error bars are standard errors, confidence interval = 95%.
EVIDENCE OF INCIPIENT SONG DIVERGENCE 35
a x a,b
x b x
Figure 14. Extended vigilance. Lower case letters above bars indicate groups (a,b,c) (x,y) that are not significantly different, error bars are standard errors, confidence interval = 95%.
EVIDENCE OF INCIPIENT SONG DIVERGENCE 36
A. B. a x a a,b x x x b x x
a,b b
C. b a,b y x,y a x
Figure 15. Latencies (measured in seconds from initiation of playback stimulus) to (A) closest approach, (B) first approach, (C) first agonistic posture. X-axis represents playback treatment types. Lower case letters above bars indicate groups (a,b,c) (x,y) that are not significantly different, error bars are standard errors, confidence interval = 95%.
EVIDENCE OF INCIPIENT SONG DIVERGENCE 37
Table 5. Results summary: Behavioral responsiveness to playback stimuli: Big Island and O‘ahu Experiments. Kruskal-Wallis H-tests with Benjamini-Hochberg false discovery rate (FDR) and adjusted Bonferroni post hoc pairwise corrections. Bold indicates significance.
BIG ISLAND EXPERIMENT
BEHAVIORAL RESPONSE Statistic post hoc tests H(2) p p(FDR) O/B O/J B/J APPROACH DISTANCE 7.444 0.024 0.027 0.032 1.0 0.105 INTEREST 23.96 0.001 0.009 0.001 0.008 0.202 VOCALIZATION 14.149 0.001 0.005 0.001 0.034 0.755 AGONISTIC POSTURE 19.336 0.001 0.003 0.001 0.157 0.043 VIGILANCE 11.701 0.003 0.005 0.002 0.663 0.094 LATENCY APPROACH 22.549 0.001 0.002 0.001 0.001 1.0 LATENCY CLOSEST 11.416 0.003 0.004 0.725 0.003 0.093 LATENCY VOCAL 6.221 0.045 0.045 0.056 0.173 1.0 LATENCY AGONISTIC 19.052 0.001 0.002 0.001 0.08 0.094 OAHU EXPERIMENT
Statistics post hoc tests H(2) p p(FDR) O/B O/J B/J APPROACH DISTANCE 2.484 0.298 0.447 – – – INTEREST 5.31 0.07 0.157 – – – VOCALIZATION 0.49 0.783 0.881 – – – AGONISTIC POSTURE 11.703 0.003 0.027 1.0 0.003 0.06 VIGILANCE 6.09 0.048 0.144 0.175 0.061 1.00 LATENCY APPROACH 0.09 0.956 0.956 – – – LATENCY CLOSEST 1.844 0.398 0.512 – – – LATENCY VOCAL 5.306 0.07 0.126 – – – LATENCY AGONISTIC 7.555 0.023 0.104 0.838 0.019 0.298 Note: O = O‘ahu; B = Big Island; J = Japan. (N = 199)
DISCUSSION
This study presents spectrographic and experimental evidence of rapid incipient divergence between-song features of three isolated populations of warbling white-eyes (O‘ahu,
Big Island, Japan). Since introduction, the O‘ahu population has undergone a significant shift in frequency, bandwidth, note length, and aggregate entropy. The Big Island population has undergone a significant reduction in the number of inflection points (changes in slope of the frequency) within notes. These results point to reduced song complexity and simpler note EVIDENCE OF INCIPIENT SONG DIVERGENCE 38
structures following multiple founder events and subsequent colonization in Hawai‘i approximately 90 years ago. Playback results showed an asymmetric response pattern between islands. The Big Island population responded strongly to song types from its own island and weakly to songs from O‘ahu and Japan. The O‘ahu population responded equally to song types from all three populations across most indices, only showing a strong response in agonistic posture to song types from its own island.
Song divergence
Variation in complexity between introduced and native populations may provide insight into the role that song plays in mediating behaviors, such as mate choice and individual recognition (Slabbekoorn & Smith, 2002). The overall rates of vocal evolution are higher among taxa with learned rather than innate songs (Mason et al., 2016), and song complexity can be a strong predictor of male quality (Spencer et al., 2003) and genetic diversity (Ballentine et al.,
2004). The cultural founder effect predicts that song complexity will be reduced in island systems, as the population bottleneck of the founding generation will reduce the proportion of total vocal diversity (Thielcke, 1973). During this process, isolated populations may exhibit a consolidation of syllabic features, potentially leading to the extinction of ancestral traits or the emergence of novel phenotypic traits in different populations (Lynch & Baker, 1994; Pfennig et al., 2010). For example, the territorial song in an island population of singing honeyeaters
(Meliphaga virescens) was lost following island introduction, such that individuals did not respond to playback stimuli of mainland conspecifics just 25 km away (Baker, 1994). In an island population of western gerygones (Gerygone fusca), 37% of the population produced a unique song that was not present in the mainland population (Baker et al., 2003). Further, if populations experience variant ecological, acoustic, or social selection pressures, there is EVIDENCE OF INCIPIENT SONG DIVERGENCE 39
potential for particular song features to be favored in one population but not the other (Hunter &
Krebs, 1979; Potvin & Parris, 2012). For example, the New Zealand population of chaffinches
(Fringilla coelebs) was introduced from England about 100 years ago, and their songs now have fewer trills but conclude with a more complex structure, likely a result of the transmission properties of the dense New Zealand forests (Jenkins & Baker, 1984). Meanwhile, white-eyes in the northern islands of the Ryukyu Archipelago favor songs with fewer notes and shorter trills when compared to southern islands, despite being geographically similar (Hamao et al., 2013).
In my examination of song spectrograms, I found high variability in notes and note combinations among white-eye individuals, potentially indicating an open-ended system of vocal innovation. I was able to detect limited but discrete, multi-syllabic sequences within songs, which were repeated, frequently as an introductory sequence [Figure 3], suggesting individual repertoire elements may exist but would require larger samples to determine. Zosterops songs and calls are known to be variable at small scales (Habel et al., 2015), and Baker (2012) found a similar, potentially open-ended system in the closely-related silvereye (Z. lateralis), suggesting that direct comparison between individuals creates a challenge for vocal researchers of these species.
Within-group comparisons of Big Island, O‘ahu, and Japan populations suggest that divergence appears to be significant primarily among individuals on O‘ahu. Much of the acoustic variance within the O‘ahu population appears to be located in one sub-population, recorded at the UH Manoa site in Honolulu, but without longer-term and larger sampling, it is not possible to say if these differences are the result of a small number of outlier individuals or a larger song culture fragmentation within this population. One hypothesis is that multiple founder events produced several independent population bottlenecks from divergent ancestries in the EVIDENCE OF INCIPIENT SONG DIVERGENCE 40
O‘ahu population, which allowed novel phenotypic traits to emerge separately within the population following colonization, while the Big Island may have undergone either a single colonization event from Maui (as suggested by Bryan, 1959) or a reduced number of founder events, compared to O‘ahu. Poor documentation during the importation period makes this difficult to determine.
Asymmetrical response to playback
Individuals in the Big Island Experiment increased interactions with playback stimuli from their own island, exhibiting behaviors that indicated they discriminated between the songs presented. Songs representing their own island population elicited significantly heightened levels of responsiveness, whereas songs representing the inter-island population (O‘ahu) and native population (Japan) elicited reduced levels of responsiveness. Robertson (1996) showed that silvereyes (Z. lateralis) could accurately recognize their mates’ contact call when presented with three call type playbacks: mate, familiar, and stranger. The increased behavioral responsiveness across all metrics on the Big Island suggests a similar recognition of song features particular to their own population. The asymmetric response to playback types in the
O‘ahu Experiment suggests a lower degree of differentiation between songs in this population.
Although asymmetric responses across populations of the same species or subspecies have received considerable attention in a wide variety of taxa, including insects (e.g. Polynesian field crickets, Teleogryllus oceanicus: Tinghitella & Zuk, 2009), anurans (e.g. green-eyes treefrogs, Litoria genimaculata: Hoskin et al., 2005), birds (e.g. black-throated blue warblers,
Dendroica caerulescens: Colbeck et al., 2010; gray-breasted wood-wrens, Henicorhina leucophrys: Dingle et al., 2010), and marine mammals (e.g. orcas, Orcinus orca: Riesch et al.,
2016), relatively few studies have considered divergent sexual signals as the drivers of response EVIDENCE OF INCIPIENT SONG DIVERGENCE 41
variation. Kaneshiro (1976; 1980) hypothesized that at the initial stages of island colonization, when population size is small, extremely choosy females would be unlikely to mate, and strong selection pressures would be imposed on less discriminating females. This model has been criticized for its generality (Arnold et al., 1996) and failed to gain strong empirical or theoretical support; however, in this case, three assumptions of the model are met: (1) the ‘derived’ lineage was established through a founder event by an ‘ancestral’ taxon, (2) no gene flow exists between populations, and (3) the ancestral lineage has not been subject to severe bottlenecks since the founder event (Giddings & Templeton, 1983). The shift of male phenotypic trait complexity and the (potential) relaxation of female preference following introduction and rapid colonization on
O‘ahu offers a clue into the response asymmetry between Hawaiian islands, adding further evidence to the growing body of literature on asymmetric variation and the role that founder events play in the evolution of sexual traits.
Recognition errors associated with divergent signals should arise more frequently when dispersal is non-existent between populations (Wright & Dorin, 2001). Given the isolation of these populations for approximately 90 years, and the pronounced acoustic differences, particularly between O‘ahu and the other populations, it is perhaps not surprising that individuals on Big Island responded less strongly to O‘ahu songs. What is more surprising is that individuals on O‘ahu responded equally to all three song types. Slater (1991) presented playback stimuli to silvereyes in a neighbor-stranger model (50 m distance between subjects); subjects showed no behavioral response differences between treatments, similar to O‘ahu birds in this playback study.
Reduced differential responsiveness between populations, despite strong differences in song, has also been reported in Gambel's white-crowned sparrows (Zonotrichia leucophyrs EVIDENCE OF INCIPIENT SONG DIVERGENCE 42
gambelii) (Nelson, 1998). Nelson suggested that as a consequence of differences in the timing of learned song, individuals in one population differentiated between the latter portion of the song, where much of the asymmetry between populations exists, while another population attended primarily to the beginning of songs (Nelson, 1988; 1998). Black-throated blue warbler
(Dendroica caerulescens) song asymmetry follows the reverse pattern: introductory notes show the greatest differences, while terminal trills exhibit more subtle differences between populations
(Colbeck et al., 2010). It is possible that white-eye individuals follow one of these models; for example, in the Big Island population, individuals could attend to introductory notes, while the
O‘ahu population may attend to another part of the song. This will require further investigation, although the detection of repeated multi-syllabic introductory sequences is promising [Figure 3].
Another possibility is that O‘ahu individuals did recognize songs particular to their own island, as evidenced by the decreased latency to agonistic response and increased duration of agonistic postures during playback trials. In this case, the other behavioral metrics either had little explanatory power, or another confound, such as the timing of the experiment (28 January –
5 February), precluded a strong interest in behavioral responsiveness.
Whatever the mechanism, the increased discrimination of song types in the Big Island population provides evidence that vocal interactions among males can have important behavioral consequences for patterns of mate choice, with gradual trait divergence potentially affecting gene flow (Irwin et al., 2001). It remains to be tested whether female preferences are reflected in male responses to playback, but a similar asymmetric female response pattern could theoretically lead to further sexual isolation and offer strong evidence of the importance of sexual signaling in the process of speciation (Ritchie, 2007; Slabbekoorn & Smith, 2008).
EVIDENCE OF INCIPIENT SONG DIVERGENCE 43
CONCLUSION
In summary, this study presents spectrographic and experimental evidence of rapid incipient song divergence in an introduced species, the warbling white-eye (Zosterops japonicus). I concluded: (1) acoustic structures of the introduced populations differ significantly from native populations, (2) these differences are most significant in the O‘ahu population, potentially the result of early and repeated founder events, (3) primary song recognition and discrimination of intra-island conspecifics clearly exists in the Big Island population, while an asymmetric response pattern exists in the O‘ahu population, (4) identifiable acoustic features
(frequency, bandwidth, note length, number of inflections, and aggregate entropy) can be used to successfully predict song features specific to populations, and (5) a sub-population of O‘ahu white-eyes appears to have diverged substantially from both intra- and inter-island song systems, potentially indicating a large-scale acoustic fragmentation among this population, although larger sampling is required to determine.
The remote biogeography of Hawai‘i offers a “natural” opportunity investigate the cultural evolution of birdsong, and the isolation of neighboring island populations is ideal for comparative frameworks. That the genus Zosterops is known as a “textbook example” of rapid phenotypic diversification (Diamond et al., 1976; Moyle et al., 2009; Lim et al., 2019) makes the underexamined Hawaiian white-eye population a fruitful research model. Future researchers might extend these analyses to additional Hawaiian Islands where white-eyes are abundant or where larger samples can be collected throughout the breeding cycle. A genetic analysis of the
Hawaiian population could help address many unanswered questions of ancestral provenance and potentially contribute to the ongoing taxonomic revision of this rapidly diversifying genus.
EVIDENCE OF INCIPIENT SONG DIVERGENCE 44
SUPPLEMENTARY TABLES
Table S1. Descriptive statistics: Mean ± SD for acoustic variables (N = 42).
Individuals ACOUSTIC VARIABLES Big Island O‘ahu Japan (n = 16) (n = 12) (n = 14) 95% FREQUENCY 5926.81 ± 178.28 5360.27 ± 909.80 5857.48 ± 190.98 PEAK FREQUENCY 7181.45 ± 414.65 6935.25 ± 551.39 7175.73 ± 753.08 BANDWIDTH 1811.32 ± 138.74 1545.04 ± 415.60 1874.54 ± 200.44 NOTE LENGTH 0.096 ± 0.009 0.083 ± 0.022 0.122 ± 0.025 SONG DURATION 3.41 ± 0.89 3.67 ± 1.63 4.05 ± 1.37 NOTES PER SONG 17.06 ± 3.74 19.13 ± 8.45 18.62 ± 6.96 DELIVERY RATE 5.14 ± 0.99 5.40 ± 1.52 4.57 ± 0.4 PFC INFLECTION PTS 7.28 ± 1.76 8.84 ± 4.75 11.59 ± 1.48 AGG. ENTROPY 4.02 ± 0.08 3.78 ± 0.3 4.02 ± 0.16 Note: Number in parentheses indicates individuals from each population.
Table S2. Summary of loading variables in the PCA with eigenvalues >1 and the proportion of variance of these PCs explained (N = 42).
ACOUSTIC VARIABLES PC1 PC2 PC3 BANDWIDTH 0.923 0.050 0.148 AGG. ENTROPY 0.914 0.084 0.041 95% FREQUENCY 0.876 0.058 0.376 NOTE LENGTH 0.876 0.135 -0.384 PFC INFLECTIONS 0.676 0.193 -0.427 DELIVERY RATE -0.594 -0.519 0.004 SONG DURATION -0.280 0.946 0.137 NOTES PER SONG -0.652 0.662 0.12 PEAK FREQUENCY 0.277 -0.122 0.864 PROPORTION OF TOTAL VARIANCE 51.26% 18.73% 14.16% Note: Total variance explained = 84.16%
EVIDENCE OF INCIPIENT SONG DIVERGENCE 45
Table S3. LDA structure matrix of pooled within-groups correlations between discriminating variables and standardized canonical discriminant functions (N = 42).
ACOUSTIC VARIABLES FUNCTION 1 FUNCTION 2 AGG. ENTROPY 0.456 0.017 BANDWIDTH 0.45 0.032 95% FREQUENCY 0.378 -0.056 PEAK FREQUENCY 0.169 -0.053 PFC INFLECTIONS 0.048 0.666 NOTE LENGTH 0.396 0.438 DELIVERY RATE -0.175 -0.347 SONG DURATION 0.0 0.326 NOTES PER SONG -0.003 0.165
Table S4. Summary results: Chi-square tests of independence between playback treatment type and response (No/Yes), Big Island and O‘ahu Experiments (N = 238). Bold indicates significance.
BIG ISLAND EXPERIMENT O‘AHU EXPERIMENT TREATMENT Response Response No Yes No Yes BIG ISLAND 1 32 5 28 (-2.8) (2.8) (-1.4) (1.4) [3.0] [97.0] [15.2] [84.8] {0.9} {28.1} {4.1} {23.0} O‘AHU 8 25 3 30 (0.9) (-0.9) (-2.3) (2.3) [24.2] [75.8] [9.1] [90.9] {7.0} {21.9} {2.5} {24.6} JAPAN 1 32 4 29 (-2.8) (2.8) (-1.8) (1.8) [3.0] [97.0] [12.1] [87.9] {0.9} {28.1} {3.3} {23.8} CONTROL (L. lutea) 12 3 17 6 (6.4) (-6.4) (6.3) (-6.3) [80.0] [20.0] [73.9] [26.1] {10.5} {2.6} {13.9} {4.9} Note: Numbers represent observed response frequencies. Adjusted crosstabulation results appear parenthesis. Percentages within individual playback treatments appear in brackets. Cumulative percentages of total responses per island appear in braces.
EVIDENCE OF INCIPIENT SONG DIVERGENCE 46
Table S5. Descriptive statistics: Behavioral responsiveness to playback stimuli, Big Island and O‘ahu Experiments (N = 199).
BIG ISLAND EXPERIMENT
BEHAVIORAL RESPONSE Playback Treatment Big Island O‘ahu Japan APPROACH DISTANCE 1.33 ± 0.78 4.73 ± 4.35 3.12 ± 2.85 (1.0) (5.0) (2.0) INTEREST 124.24 ± 38.33 64.24 ± 50.06 105.45 ± 37.76 (130.0) (70.0) (110.0) VOCALIZATION 52.12 ± 41.82 19.39 ± 22.35 40.30 ± 37.54 (50.0) (10.0) (30.0) AGONISTIC POSTURE 22.73 ± 27.07 1.82 ± 8.82 8.18 ± 14.02 (20.0) (–) (–) VIGILANCE 34.55 ± 22.79 14.85 ± 22.10 21.82 ± 22.98 (40.0) (–) (10.0) O‘AHU EXPERIMENT
Playback Treatment Big Island O‘ahu Japan APPROACH DISTANCE 2.12 ± 2.84 2.70 ± 3.31 3.18 ± 3.55 (1.0) (1.0) (1.0) INTEREST 83.33 ± 51.58 104.55 ± 55.46 78.48 ± 48.93 (90.0) (110.0) (70.0) VOCAL RESPONSE 40.30 ± 41.19 36.06 ± 36.91 33.03 ± 34.41 (30.0) (30.0) (30.0) AGONISTIC POSTURE 13.33 ± 16.89 17.58 ± 20.47 3.64 ± 7.42 (–) (10.0) (–) VIGILANCE 16.67 ± 20.41 26.97 ± 24.81 14.55 ± 21.52 (–) (20.0) (–) Note: Numbers represent mean values ± SD; median values in parenthesis; (–) indicates median of zero.
EVIDENCE OF INCIPIENT SONG DIVERGENCE 47
Table S6. Descriptive statistics: Latency of responses to playback treatments: Big Island Experiment and O‘ahu Experiment (N = 199).
BIG ISLAND EXPERIMENT
Latency (s) TREATMENT First Closest First First Approach1 Approach2 Vocalization3 Agonistic4 BIG ISLAND 31.21 ± 19.65 72.12 ± 28.15 63.33 ± 49.73 114.55 ± 64.04 (30.0) (70.0) (50.0) (100.0) O‘AHU 82.42 ± 61.19 95.45 ± 55.29 106.67 ± 68.81 174.55 ± 21.81 (70.0) (90.0) (90.0) (180.0) JAPAN 33.03 ± 30.36 56.97 ± 33.68 72.12 ± 57.27 143.94 ± 57.22 (20.0) (50.0) (50.0) (180.0) O‘AHU EXPERIMENT
Latency (s) First Closest First First Approach1 Approach2 Vocalization3 Agonistic4 BIG ISLAND 56.06 ± 57.28 83.64 ± 53.67 64.55 ± 60.73 122.42 ± 63.35 (30.0) (70.0) (40.0) (180.0) O‘AHU 50.61 ± 50.43 7211.70.73 ± 83.64 ± 65.47 108.18 ± 66.17 (30.0) 45.43 (60.0) (110.0) (60.0) JAPAN 51.82 ± 51.87 67.58 ± 48.67 71.16 ± 62.53 149.09 ± 56.92 (30.0) (50.0) (70.0) (160.0) Note: Numbers represent mean values ± SD; median values in parenthesis. Latency (s) between onset of playback stimulus and (1) when the focal individual moved <10 m from speaker [APPROACH]; (2) moved to the closest distance from speaker at any point during trial [CLOSEST]; (3) exhibited first vocalization [VOCAL]; (4) exhibited first agonistic posture [AGONISTIC].
EVIDENCE OF INCIPIENT SONG DIVERGENCE 48
APPENDIX
Table A1. Archival recordings used in playback experiments and acoustic analysis.
Used in Used in Archive Recordist Location Experimental Acoustic Playback Analysis XC175490 Lane, D. Hakalau, Hawai‘i Y Y XC252716 Mark, T. Pu‘u ‘Ō‘ō, Hawai‘i Y Y XC385605 Elman, U. Volcano NP, Hawai‘i Y Y XC172706 Muzika, Y. Tokyo, Japan Y Y XC172824 Muzika, Y. Tokyo, Japan Y Y XC193175 Torimi, A. Kyoto, Japan Y Y XC285853 Boesman, P. Miyakejima, Japan Y Y XC285872 Boesman, P. Miyakejima, Japan Y Y XC285946 Boesman, P. Miyazaki, Japan Y Y XC285947 Boesman, P. Miyazaki, Japan Y Y XC507025 Jacobs, M. Okinawa, Japan Y Y ML102005 Ord, M. Unknown, Hawai‘i Y Y ML129119 Budney, G.F. Hakalau, Hawai‘i Y Y ML129120 Budney, G.F. Hakalau, Hawai‘i Y Y ML129122 Budney, G.F. Hakalau, Hawai‘i Y Y ML281334 Boesman, P. Nagano, Japan N Y ML281569 Boesman, P. Miyazaki, Japan N Y ML281722 Boesman, P. Okinawa, Japan N Y EB101778451 O’Brien, J. Tantalus, Hawai‘i N Y EB148990341 Hung, T.W Tokyo, Japan N Y EB150753591 Harlan, H. Waiwai, Hawai‘i N Y EB203924361 del Hoyo, J. Pu‘u‘Ualakaa, Hawai‘i N Y EB92476681 Lincoln, S. Kaimuki, Hawai‘i N Y EB182552831 Floyd, T. ‘Aiea, Hawai‘i N Y ML6091* Burr, T. Kiawewai, Hawai‘i Y (control) N ML32513* Robbins, C. Unknown, Hawai‘i Y (control) N ML14175* Ward, W. Honolulu, Hawai‘i Y (control) N ML5128* Pratt, D. Polipoli, Hawai‘i Y (control) N ML6018* Schallenberger, R. Koolau FR, Hawai‘i Y (control) N ML6074* Burr, T. Upper Hana, Hawai‘i Y (control) N Note: XC = xeno-canto; ML = Macaulay Library; EB = eBird. [*] indicates (Leiothrix lutea).
EVIDENCE OF INCIPIENT SONG DIVERGENCE 49
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