DOI: 10.5061/dryad.t76hdr8ds
title: How relaxed preferences facilitate the evolution of novel animal signals

description: The evolution of novel animal signals is critical to the generation of
 biodiversity. Here, we explore how new sexual signals become established.
 This process is challenging to explain because if receiver preferences are
 coupled with existing signals, then most receivers should discriminate
 against new signals. We investigated an underappreciated hypothesis:
 relaxed receiver preferences facilitate novel signal evolution by allowing
 new signals to establish a foothold. Further, we probed the mechanistic
 underpinnings of relaxed preferences by combining field-based and common
 garden approaches, allowing us to investigate evolution and plasticity as
 mechanisms. We capitalized on the Pacific field cricket, Teleogryllus
 oceanicus, a species that has recently evolved multiple novel acoustic
 signals (e.g., purring and rattling) in response to an eavesdropping
 parasitoid fly only found in the crickets’ introduced range in Hawaii. To
 test the hypothesis that selection associated with high search costs in
 introduced populations leads to relaxed mating preferences and determine
 whether such relaxation is plastic, we conducted sound preference
 (phonotaxis) trials with females from the cricket’s native range
 (Australia and French Polynesia, where the fly is absent) and its
 introduced range (Hawaii, where the fly is present). We presented females
 with novel songs plus the typical, ancestral song. Differences in
 phonotactic behavior between the lab and field settings would indicate
 plasticity in preferences. Using Generalized Linear Mixed Models (GLMM)
 with whether the female cricket was phonotactic to a song (y/n) as a
 response variable, we found that Australian and French Polynesian females
 were quite plastic; they discriminated strongly against most songs in the
 field, but were much more phonotactic to rattling and the typical song in
 the lab. However, Hawaiian females exhibited little plasticity and were
 consistently highly responsive to the rattling and typical songs in the
 lab and field. This pattern points to a loss of ancestral plasticity in
 female preferences sometime after colonizing Hawaii, resulting in
 heightened responsiveness to all songs, allowing novel signals to
 establish. We then asked which specific preference function traits
 (tolerance, strength, and/or responsiveness) differed among regions to
 better understand what is ‘relaxed’ about preferences in Hawaii by
 generating individual and region-level preference functions for females
 from each region with respect to the purring, rattling, and ancestral
 stimuli. When examining whether females were phonotactic or not and how
 quickly they contacted stimuli, responsiveness, tolerance, and preference
 strength differed among regions. Finally, we developed a
 computational model to estimate the distances at which female T. oceancius
 from Australia and French Polynesia should be able to hear and use the
 purring, rattling, and typical calling songs. A model of mean
 peak frequencies and amplitudes among each song type revealed dramatic
 differences in effective hearing distances across song types. Our results
 provide insight into how novel signals gain a foothold when they initially
 invade, after which coupled preferences may eventually evolve, perhaps
 leading to reproductive isolation. Alternatively, relaxed preferences may
 remain longer term, facilitating the maintenance of signal diversity
 within populations.
description: <em>Field-based Phonotaxis Tests</em>
 We collected reproductively mature adult female <em>T.
 oceanicus</em> at six field sites: two replicate sites in
 Queensland, Australia (Cairns and Daintree) in February of 2023, two in
 French Polynesia (Tahiti and Mo’orea) in December of 2023, and two in
 Hawaii (Hilo and Wailua) in December of 2022 (Figure S1, Table S1) using
 methods that are not biased with respect to song, sex, or life stage.
 Briefly, rather than locating individuals by sound, we swept by foot in
 the fields where the crickets are found, collecting animals visually such
 that all life stages, sexes, and morphs were encountered (following
 Tinghitella <em>et al.</em> 2018; Tinghitella <em>et
 al.</em> 2021). Upon collection, we took the animals to local field
 stations where we housed females in 15L plastic storage containers with
 rabbit food <em>ad libitum</em>, egg cartons, and cotton with
 water under natural day-night cycles (at a density of ~30-40 females per
 container). We acoustically isolated females from calling males for 24
 hours before conducting phonotaxis trials. Our first
 question was whether female crickets from Australian, French Polynesian,
 and Hawaiian populations differ in their phonotactic responses to the
 typical song and newly evolved songs in Hawaii. We addressed this in the
 field using standardized phonotaxis trials in which we tested each
 female’s response to the following stimuli: a loop of five purring calling
 songs (played in immediate succession), a loop of four rattling calling
 songs, a loop of four typical calling songs, and a silent negative
 control. All loops were previously used in Tinghitella <em>et
 al.</em>, 2018, 2021, and Gallagher <em>et al.</em>
 2022. We randomized the order in which the stimuli were played. We
 conducted all trials between 2-8 hours after sunset (the active period
 when females search for males) at 24-26°C and under red light (following
 Tinghitella <em>et al</em>, 2021). In each trial, we placed a
 single female cricket under a plastic cup at one end of an arena (50 cm
 wide × 195 cm long × 25 cm tall) 1m away from an AOMAIS Sport II Bluetooth
 speaker that was positioned in the arena’s center. After the female
 adjusted to the arena, we played the first stimulus track, lifting the cup
 and allowing her to walk about the arena. The typical <em>T.
 oceanicus</em> loop was broadcast at 70 dBA, the rattling loop at 60
 dBA, and the purring loop at 53 dBA (at one meter away), reflecting
 biologically realistic amplitudes (Tinghitella et al. 2018; Gallagher et
 al 2022, 2024). We confirmed amplitudes using a class 1 PCE-430 sound
 level meter set to “A” weighted measurements with fast integration time.
 We played each stimulus for a maximum of one minute or until the female
 contacted the speaker. If the female demonstrated positive phonotactic
 behavior, but did not contact the speaker in the first minute, we gave her
 a second minute of playback to allow potential contact. We considered a
 female to be positively phonotactic when she moved in the direction of the
 speaker in a classic zigzag pattern without following the wall or circling
 the arena. In addition to noting whether the female was positively
 phonotactic, we recorded the maximum distance traveled and the latency to
 contact the speaker for each stimulus (following Tinghitella et al. 2021).
 One observer relayed behavioral observations to a recorder while wearing
 earplugs so they were blind to the stimulus. Given the low levels of
 phonotaxis exhibited by Australian females to our stimuli, we created a
 supernormal stimulus (Supplemental Methods) to confirm that females were
 indeed sexually mature and phonotactic (Figure S2).
 <em>Lab-based Phonotaxis Tests</em>
 To investigate the impacts of environment and experience on
 female preferences, we repeated our phonotaxis trials in the lab after
 rearing animals to at least the F2 generation (to avoid transgenerational
 effects). We reared animals in a common garden, but separated by
 population, in 15 L plastic containers in a clock-shifted, temperature and
 humidity-controlled room set to a 12:12 light:dark cycle and 25 °C. Each
 15 L container housed a mixed sex group with access to ad libitum food
 (Fluker’s Cricket Chow for juveniles and Kaytee rabbit chow for adults),
 water from moistened cotton, and an egg carton. Prior to trials, we
 haphazardly chose females from each of the six lab reared populations at
 the antepenultimate instar, the instar prior to which they develop
 auditory organs (Kämper, 1992), and isolated them in a sound-insulated
 incubator inside of 1.89 L containers; thus, females were reared in
 silence (eliminating song experience as a source of variation in
 preference behavior). We ensured their virginity at the time of trials and
 checked isolated females twice a week for eclosion to adulthood. We
 conducted phonotaxis trials on adult females during their scotoperiod when
 they were 7-21 days post-eclosion. All equipment and the phonotaxis
 protocol were identical to the field trials except that we conducted
 lab-based trials in a temperature-controlled room set to 25 °C. All sample
 sizes can be found in Table S1. <em>Statistical
 Analyses</em> To address the hypothesis that
 relaxed preferences facilitate the success of novel sexual signals and
 gain insight into the underlying mechanisms, we first asked how
 phonotactic behavior depended on region (Australia, French Polynesia,
 Hawaii), song type (purring, rattling, typical, silence), and whether
 trials were conducted in the field or lab. Using the
 <em>lme4</em> package (Bates 2015) in R Studio version
 2024.12.1, we ran a Generalized Linear Mixed Model with a binomial
 distribution in which phonotaxis (yes/no) was the response variable and
 region, song type, and lab vs field plus all two-way interactions were
 included as main effects. Individual ID was a random effect to account for
 repeated testing of females with the four stimuli. In this model, a
 significant interaction between region and song type would indicate that
 there are differences in female preference among regions. As the lab vs.
 field effect indicates whether preferences are plastic, finding an
 interaction between region and lab vs. field would indicate that regions
 differ in the extent to which their preferences are plastic, as would
 occur if plasticity had evolved. This could occur in several ways. For
 instance, plasticity might be selected for in Hawaii if it facilitates
 mating where search costs are high, or ancestral plasticity might be lost
 if it is costly to maintain, leading Hawaiian females to be consistently
 highly responsive. And, a significant song type by lab vs field effect
 would support the hypothesis that plasticity is
 stimulus-dependent. Next, we constructed preference
 functions using our field phonotaxis data to examine which preference
 traits (see below) differed among regions in order to gain insight into
 the manner in which preferences are relaxed in Hawaii. Preference
 functions describe signal attractiveness as a function of variation in
 signalling traits (Neelon et al. 2019) and can take on many shapes (e.g.,
 open-ended or closed) described by quantifying preference traits
 (Rodríguez et al., 2006, 2013; Fowler-Finn & Rodríguez 2012a,
 2012b; Kilmer et al. 2017; Neelon et al., 2019). We investigated three
 preference function traits: tolerance (acceptance of trait values that
 deviate from the peak preference), responsiveness (mean response level
 across all signal trait values), and strength (how much attractiveness
 declines as signal values deviate from the peak). An individual with a
 relaxed preference might, for instance, have high responsiveness, be more
 tolerant, and/or have low preference strength relative to an individual
 with a preference strongly coupled to a particular signal (Figure 1). We
 used PFunc (Kilmer et al. 2017) to visualize the shape of individual and
 region-level preference functions for the three song types (typical,
 rattling, and purring songs). Note that because novel songs are
 categorically different from the typical song, differing in multiple
 signal traits (e.g., dominant frequency, bandwidth), we visualized
 preference functions across song types. Preference function traits
 (tolerance, responsiveness, and preference strength) were extracted from
 individual-level functions fit to two measures of female responses:
 phonotaxis (yes/no) and latency to contact. General linear models compared
 these values across regions. Having found that Hawaiian
 females are more responsive to novel songs than those from elsewhere
 across the crickets’ range, particularly in field studies, we next
 explored one mechanism, enhanced locomotor behavior, that may underlie
 that pattern. More locomotory females may be more likely to wander close
 enough to the quieter purring or rattling males to hear their songs and
 then pursue them as mates. We asked whether females from different regions
 differed in the distance they travelled during the silent negative control
 trials. These data were non-normally distributed, so we ran Kruskal-Wallis
 tests on distance traveled during silence by region in both the field and
 lab and conducted pairwise post-hoc comparisons with Mann-Whitney-Wilcoxon
 tests. Additionally, given that rattling males are only
 present in Hilo (not Wailua), and purring males are only present in Wailua
 (not Hilo), we investigated potential local adaptation, using a GLMM
 comparing phonotaxis to these two songs (rattling and purring) between
 these two populations with female ID as a random effect. If local
 adaptation has occurred, we expect females to be more responsive to the
 novel song found in their respective populations. If coevolution with
 silent flatwing males is critical, Kauai females (where the population was
 more than 95% silent for at least 60 generations; Bailey <em>et
 al.</em> 2024) should be the most relaxed of all.
 <em>Model of Effective Hearing
 Distance</em> Finally, because the pattern of
 female responses to purring and rattling songs across the crickets’ range
 differed, with phonotaxis to purring being rare overall (see results), we
 developed a computational model to determine how far away female
 <em>T. oceanicus</em> are likely to be able to hear and use
 purring, rattling, and typical songs in the context of long-distance mate
 location. Using previously published neural audiogram data in <em>T.
 oceanicus</em> (Atkins & Pollack, 1986; Fullard <em>et
 al</em>., 2010) and the sound intensity levels and peak frequencies
 of different types of cricket calling songs, we built a model estimating
 effective hearing distances for each song type. We based the model on peak
 frequencies of the purring, rattling, and typical songs reported in
 (Gallagher et al. 2022) and sound intensity levels from (Wikle
 <em>et al.</em> 2025), modeling responses to the average
 dominant frequency +/- 1 standard deviation and the average amplitude +/-
 1 standard deviation. The model accounts for non-frequency-dependent
 damping of sound with distance and frequency-dependent attenuation due to
 atmospheric absorption. Detailed modeling methods can be found in (Wikle
 et al, 2025), where we previously modeled the effective hearing distances
 to these songs for the parasitoid fly. There is substantial
 inter-individual variation in spectral characteristics (dominant frequency
 and bandwidth) and amplitude of novel songs (Gallagher <em>et
 al.</em> 2023). All of these likely influence effective hearing
 distances, and our model examines two important axes of variation
 (amplitude and dominant frequency) that characterize differences among
 song types, but we are unfortunately not able to account for variation in
 bandwidth in this model. Several previously published
 neural audiograms based on ON1 (omega neuron 1- responsible for
 directional processing of cricket songs) recordings from <em>T.
 oceanicus</em> have been collected from lab-reared animals that
 originated from Australia (Atkins & Pollack, 1986; Kostarakos et
 al., 2009; Fullard et al., 2010) and Mo’orea (Fullard et al, 2010). Since
 purring and rattling songs contain frequencies that extend well above 10
 kHz (Tinghitella et al, 2021; Gallagher et al. 2022), we digitized neural
 audiograms from (Atkins & Pollack, 1986) and (Fullard et al,
 2010), both of which describe auditory tuning of ON1 up to 40 kHz (whereas
 (Kostarakos et al. 2009) only examines neural responses from 3-6kHz). We
 used Meazure 2.0 (written by Baron Roberts, C Thing Software) to capture
 data points across three audiograms. As the neural audiograms from these
 two studies were conducted for different frequency sampling points, we
 imported the captured data into MATLAB, used linear interpolation between
 frequency sampling points, and averaged across the three interpolated
 audiograms to produce an average <em>T. oceanicus</em>
 audiogram (Figure S3). We determined thresholds for song peak frequencies
 from the interpolated average neural audiogram. Following the approach
 described in (Wikle <em>et al.</em> 2025), we calculated
 attenuation curves over distance starting at peak amplitude for nine
 different frequency and amplitude combinations for each song type and
 determined the distance at which a particular song's sound pressure
 level reached the threshold identified from the average neural audiogram
 required to elicit a neural response. We calculated these estimated
 hearing distances for each song type.
description: # How relaxed preferences facilitate the evolution of novel animal signals
 Dataset DOI:
 [10.5061/dryad.t76hdr8ds](https://doi.org/10.5061/dryad.t76hdr8ds) ##
 Description of the data and file structure The phonotaxis and behavior
 data for both the lab and field are located in the file titled
 "Welsh_behavior_data.csv." The data used to make the heatmap are
 located in the file titled "Welsh_heatmap.csv," and the hearing
 model data for Figure 5 are located in
 "Welsh_hearing_model.csv." The data used to make Figure S2 in
 the supplement are in "Welsh_supernormal.csv." ### Files and
 variables ## Welsh_behavior_data.csv **tripID:** season and year in which
 the field trip to collect data took place. W22.23 indicates winter
 2022-2023, F24 indicates Fall 2024, etc. Lab trials have an "na"
 since the data were not associated with a field trip **region:** one of
 three broad locations with cricket populations- Australia, French
 Polynesia, and Hawaii. Each region had two replicate populations
 **population:** one of six individual locations with crickets across the
 three regions- Wailua and Hilo in Hawaii, Tahiti and Moorea in French
 Polynesia, and Cairns and Daintree in Australia **Lab_field:** indicates
 whether the trials took place in the lab vs the field **femaleID:** unique
 ID for each female tested, used as a random effect in models **date:**
 date on which trial took place in MM/DD/YYYY format **start_time:** time
 at which trial started **observer:** initials of the data collector who
 was observing the female cricket and making the behavioral data calls
 **song:** the stimulus that was played to the female crickets- silence,
 purr, rattle, and typical **phonotaxis:** whether or not the cricket was
 phonotactic, indicated with a "y" for "yes" and a
 "n" for "no" **time_contact:** time it took the
 cricket to contact the speaker (in seconds). "na" if the cricket
 did not contact the speaker **distance:** maximum longitudinal distance
 the cricket traveled during the first minute of each trial (in cm)
 **mating_status:** whether the female was a virgin when tested.
 "virgin" for all lab trials and "na" for all field
 trials since mating status is unknown for field-caught individuals
 **dev_exp:** sound environment that female crickets experienced during
 development. "na" for field-caught crickets since developmental
 environment was unknown. "silence" if lab crickets were raised
 in silence and "cricketroom" if lab crickets were raised with
 all songs in the main cricket room ## Welsh_hearing_model.csv **song:**
 stimulus modelled **effective_hearing_distance:** distance at which a
 female cricket can hear the stimulus (m) ## Welsh_supernormal.csv
 **tripID:** season and year in which the field trip to collect data took
 place. W22.23 indicates winter 2022-2023, F24 indicates Fall 2024, etc.
 Lab trials have an "na" since the data were not associated with
 a field trip **population:** one of six individual locations with crickets
 across the three regions- Wailua and Hilo in Hawaii, Tahiti and Moorea in
 French Polynesia, and Cairns and Daintree in Australia **femaleID:**
 unique ID for each female tested, used as a random effect in models
 **date:** date on which trial took place in MM/DD/YYYY format
 **start_time:** time at which trial started **observer:** initials of the
 data collector who was observing the female cricket and making the
 behavioral data calls **song:** the stimulus that was played to the female
 crickets- typical and supernormal_stimulus **phonotaxis:** whether or not
 the cricket was phonotactic, indicated with a "y" for
 "yes" and a "n" for "no" **time_contact:**
 time it took the cricket to contact the speaker (in seconds).
 "na" if the cricket did not contact the speaker **distance:**
 maximum longitudinal distance the cricket traveled during the first minute
 of each trial (in cm) ## Code/software **Welsh_et_al_2025.Rmd** is an R
 Markdown file that is fully annotated for all analyses and figures
 included in the main manuscript. All necessary packages are included in
 the script. **Welsh_et_al_2025_supplement.Rmd** is an R Markdown file that
 is fully annotated for all analyses and figures included in the
 supplement. All necessary packages are included in the script.