DOI: 10.5061/dryad.6m905qfws
title: Data from: Divergent male and female mate preferences do not explain incipient speciation between lizard lineages

description: Diversification in sexual signals is often taken as evidence for the
 importance of sexual selection in speciation. However, in order for sexual
 selection to generate reproductive isolation between populations, both
 signals and mate preferences must diverge together. Furthermore,
 assortative mating may result from multiple behavioural mechanisms,
 including female mate preferences, male mate preferences and male-male
 competition; yet their relative contributions are rarely evaluated. Here,
 we explored the role of mate preferences and male competitive ability as
 potential barriers to gene flow between two divergent lineages of the
 tawny dragon lizard, Ctenophorus decresii, which differ in male throat
 coloration. We found stronger behavioural barriers to pairings between
 southern lineage males and northern lineage females than between northern
 males and southern females, indicating incomplete and asymmetric
 behavioural isolating barriers. These results were driven by both male and
 female mate preferences rather than lineage differences in male
 competitive ability. Intrasexual selection is therefore unlikely to drive
 the outcome of secondary contact in C. decresii, despite its widely
 acknowledged importance in lizards. Our results are consistent with the
 emerging view that although both male and female mate preferences can
 diverge alongside sexual signals, speciation is rarely driven by divergent
 sexual selection alone.  
description: <i>Study species and husbandry</i>
 We used 90 adult lizards (&gt;65mm snout-vent
 length; SVL) comprising 21 male and 24 female northern lineage <i>C.
 decresii</i> from Caroona Creek Conservation Park, South Australia
 (-33.4114°S, 139.0945°E), and 21 male and 24 female southern lineage
 <i>C. decresii</i> from private properties around Palmer,
 South Australia (-34.8223°S, 139.1621°E). Lizards were collected in
 September in 2015 and 2016, and subsequently kept in captivity at The
 University of Melbourne, Victoria, Australia, where they were housed
 individually in 55 × 34 × 38cm (length × width × height) opaque plastic
 enclosures containing a layer of sand and a crevice between two ceramic
 tiles for shelter. Housing was maintained at temperatures and lighting
 cycles that mimicked natural seasonal variation, with UV lights (ZooMed T8
 ReptiSun® 10.0 UVB) above each enclosure (30cm), emitting both UVA and UVB
 radiation. A heat lamp was provided to generate a thermal gradient and
 allow the lizards to attain their preferred body temperatures (approx.
 36°C). Lizards were misted with water for hydration and fed live crickets
 dusted with multi-vitamins three times per week. All behavioural trials
 were conducted during the breeding seasons (August–December) in 2016 and
 2017. Research methods used in this study were reviewed and approved by
 the Animal Ethics Committee of The University of Melbourne (1413220.3) and
 the South Australian Wildlife Ethics Committee
 (25/2015).  
 <i>Female-male behavioural
 trials</i> Females are receptive to
 mating approximately 2–3 weeks after emergence from hibernation, and after
 laying their first or second clutch. We conducted mate preference trials
 during these known receptive periods, when females were in good body
 condition (average mass of 16.7g ± 2.9g), though receptivity cannot be
 determined with certainty <i>a priori</i>. Each female was
 paired with both a southern and a northern lineage male, with half of the
 females paired with a southern male first and the other half with a
 northern male first. Females were placed into the first male’s enclosure
 for a period of 24 hours, and then into the second male’s enclosure for
 the subsequent 24 hours. Both encounters were monitored and recorded using
 a Swann DVR8-1525 8 channel 960H digital video recorder with a PRO-615
 camera attached. We conducted a total of 147 trials, with individual
 females paired with one southern and one northern male per reproductive
 cycle, in up to 2 reproductive cycles (average of 3.34 trials, with a
 range of 2–4 trials, per female).  
 Videos were analysed using Behavioural Observation Research
 Interactive Software (BORIS) version 4.1.5 and both female and male
 behaviour was scored. For females, we recorded the number of head-bobs
 (pronounced nodding movement of the head), and combined the number of
 aggressive behaviours (biting and chasing) and times the female fled from
 the male as a measure of “rejection”. For males, we also recorded the
 number of head-bobs (courtship behaviour) as well as the number of
 attempts to copulate, and whether or not copulation was successful. We did
 not analyse the number of successful copulations as copulation was
 observed in only 7 of the 147 trials (although more may have taken place
 under the tile). Lizards were not paired for long enough to ensure mating;
 rather, we were interested in behaviour during initial contact as an
 indicator of mate preference.  
 We tested whether female lineage, male lineage, or
 their interaction predicted: 1) number of copulation attempts, 2) number
 of male head-bobs, 3) number of female head-bobs and 4) number of female
 rejection behaviours using generalised linear mixed models (lme4 package,
 R). Female ID, male ID and pairing number (female’s first or second trial)
 were included as random factors in all models to account for repeated use
 of individuals, and response variables were log transformed to meet model
 assumptions of normality. We performed pairwise comparisons by calculating
 least squares means and confidence intervals using the Satterthwaite’s approximation for
 degrees of freedom (lmerTest package, R).
   <i>Male-male behavioural
 trials</i> A previous
 study investigating aggression levels among morphs of the northern lineage
 found that orange-throated males were significantly more aggressive
 towards territory intruders than yellow, orange-yellow or grey-throated
 males. Therefore, we categorised males into three behavioural groups based
 on lineage and throat colour morph:  southern, northern high aggression
 (orange), or northern low aggression (yellow, orange-yellow, grey). We
 designed trials such that each focal male was matched with three others,
 representing each of the behavioural groups, in random order. Pairs were
 size-matched to minimize the effect of body size on contest outcome, with
 an average difference of 1.59mm ± 1.16mm snout vent length (SVL) between
 competing males.  
 Contest trials were conducted in a neutral 120 × 30 ×
 60cm (length × width × height) enclosure (i.e. not the home enclosure of
 either male). An opaque divider initially separated the enclosure into two
 equally sized holding areas, each containing a layer of sand, ceramic tile
 and heat lamp. Just prior to the trial, males were weighed to obtain a
 measure of body condition as the residuals of a linear model of mass and
 SVL. The designated “focal” and “opponent” males were then placed into the
 separate holding areas and allowed to acclimatise for 48 hours to
 establish residency. At the commencement of the trial, the divider was
 removed and the interaction was recorded from two different angles using
 Panasonic HC-V770M video cameras. Trials were conducted for a maximum of
 25 minutes and monitored to ensure there was no risk of injury to animals
 (as required under the Animal Ethics permit). Consequently, we did not
 record contest outcome (i.e. winner, loser) as some trials were stopped
 before a winner was established. To minimize stress and the potential
 influence of previous contest outcomes, males were not used in a
 subsequent trial for at least 48 hours. We conducted a total of 120 trials
 (involving 42 males), 26 of which were excluded due to no interaction,
 resulting in 94 trials which were used in the statistical
 analysis.  
 We scored focal male behaviour from the video footage
 using BORIS. <i>C. decresii</i> males perform energetic
 displays during territory defence prior to engaging in physical
 aggression. Therefore, we recorded the number of head-bobs, tail flicks
 and push-ups performed by the focal male as a measure of “display
 behaviour”, and combined the duration of chasing and wrestling (involving
 biting) as a measure of “physical aggression”. We also recorded the time
 between the start of the trial and the focal male’s emergence from beneath
 the tile (“latency”), as this is an indicator of individual boldness.
 Display behaviour and physical aggression were divided by the total trial
 duration (minus latency) to account for differences in trial
 lengths.  
 We tested whether behavioural group or body condition
 predicted: 1) focal male latency to emerge, 2) focal male display
 behaviour and 3) focal male physical aggression using generalised linear
 mixed models. We included focal male behavioural group, opponent male
 behavioural group and their interaction, as well as focal male body
 condition and opponent male body condition as predictor variables in the
 models. Additionally, focal male ID and focal male trial number were
 included as random factors in all models to account for repeated use of
 individuals. For models 2 (display behaviour) and 3 (physical aggression),
 the response variables were log transformed to meet model assumptions of
 normality, and we performed <i>post hoc</i> pairwise
 comparisons as detailed above.
description: Includes excel spreadsheets of data collected from female-male
 behavioural trials (female_male_data.xlsx) and male-male behavioural
 trials (male_male_data.xlsx), and an R Markdown file (Analysis.rmd) and
 output document (Analysis.html) with all statistical analyses performed in
 this study.