DOI: 10.5061/dryad.41ns1rnmn
title: Behavioral estimates of mating success corroborate genetic evidence for pre-copulatory selection

description: In promiscuous species, fitness estimates obtained from genetic parentage
 may often reflect both pre- and post-copulatory components of sexual
 selection. Directly observing copulations can help isolate the role of
 pre-copulatory selection, but such behavioral data are difficult to obtain
 in the wild and may also overlook post-copulatory factors that alter the
 relationship between mating success and reproductive success. To overcome
 these limitations, we combined genetic parentage analysis with behavioral
 estimates of size-specific mating in a wild population of brown anole
 lizards (Anolis sagrei). Males of this species are twice as large as
 females and multiple mating among females is common, suggesting the scope
 for both pre- and post-copulatory processes to shape sexual selection on
 male body size. Our genetic estimates of reproductive success revealed
 strong positive directional selection for male size, which was also
 strongly associated with the number of mates inferred from parentage. In
 contrast, a male’s size was not associated with the fecundity of his mates
 or his competitive fertilization success. By simultaneously tracking
 copulations in the wild via the transfer of colored powder to females by
 males from different size quartiles, we independently confirmed that large
 males were more likely than small males to mate. We conclude that body
 size is primarily under pre-copulatory sexual selection in brown anoles,
 and that post-copulatory processes do not substantially alter this
 pre-copulatory selection. Our study also illustrates the utility of
 combining both behavioral and genetic methods to estimate mating success
 to disentangle pre- and post-copulatory processes in promiscuous species.
description: We studied an island population of brown anole lizards
 (<em>Anolis</em> <em>sagrei</em>) in the Guano
 Tolomato Matanzas Natural Estuarine Research Reserve in northern Florida
 (29°37′53′′ N, 81°12′ 46′′ W) using procedures approved by the University
 of Virginia Animal Care and Use Committee (protocol 3896). To assay the
 reproductive success of males, we sampled all adults and juveniles of the
 population at four different times during the breeding season (March, May,
 July, and October) in 2019. This population has been the focus of a
 long-term mark-recapture study since 2015, such that most adults in the
 2019 breeding season were first captured, marked, and genotyped as
 juveniles in 2017 or 2018. We measured the snout-vent length (SVL, nearest
 1 mm) and body mass (nearest 0.01g) of all individuals prior to releasing
 them at their exact site of capture the following day. We extracted DNA
 from adults captured in the population and used SNPPIT 2.0 (Anderson 2012)
 to assign genetic parentage. At two points in the middle of the breeding
 season (May and July), we also tracked copulations in the wild by dusting
 the venters of adult males with fluorescent powder as we released them and
 tracked the transfer to females to evaluate if and the extent to which
 body size mediates higher reproductive success through mating
 success.
description: # Behavioral estimates of mating success corroborate genetic evidence for
 pre-copulatory sexual selection in the wild \[Access this dataset on
 Dryad](10.5061/dryad.41ns1rnmn) The findings in this paper are drawn from
 a mark-recapture and genotyping study carried out on a focal island
 population of brown anole lizards along the intra-coastal waterway in
 Guano Tolomato Matanzas Natural Estuarine Research Reserve in Florida.
 Although animals in this population have been tracked between 2015 and
 2019 part from a long-term ongoing study, this dataset primarily focuses
 on the adult male population present on the island in 2019, their
 reproductive fitness, and pre-and post-copulatory components of fitness.
 We asked if selection due to total reproductive success on a measure of
 body size (here, body mass) is primarily due to higher mating success
 (pre-copulatory selection) or is primarily due to competitive
 fertilization success (post-copulatory success). We also tested alternate
 hypotheses for stronger selection on larger male body size by assessing
 patterns of assortative mating and if selection on male body size was also
 driven by the average fecundity of females. Based on the methods, the
 datasets provided illustrate individual identity of animals (e.g., ID,
 Sex, Age, Cohort), capture information (Year, Month of capture, New or
 recaptured), genetic fitness of individuals
 (total_no_of_offspring(Reproductive success), total_no_of_mates (Mating
 success), For males only: Average per mate fecundity, competitive
 fertilization success, unadjusted proportion paternity; For females only:
 Average female fecundity), data collected from powdering study to measure
 behavioral mating success (Males: size class and color of powder applied;
 Females only: color of powder detected, Size class classification, Mating
 status, Incidence of multiple mating, secondary contact). There are two
 separate datasets provided. The first Males_2019.csv file details adult
 males who were captured from March to July 2019. From this dataset: a)
 Selection gradients reported in Fig. 1 were measured from adult males
 captured in March only, where fitness measures were standardized for this
 subset of individuals. b) Males captured in May and July were part of the
 fluorescent powdering study, the results and analyses from which are
 illustrated in Fig. 2. c) The annual fitness data for adults has also been
 specified in the same dataset to illustrate Fig. 4 and 5 respectively. d)
 Finally the dataset titled Females_2019.csv illustrates the identity,
 morphology, behavioral mating success, and genetic fitness of females
 captured in May and July. Data from this file was used to illustrate Fig.
 2 and Fig. 3. ## Description of the data and file structure Variables used
 in Dataset and what they refer to: The following variables are present in
 the dataset. Although there are 3 separate datasets, they have all been
 collected from the same population described in the paper **Bhave et al.
 (2023)**. How data under each variable was collected is explained in the
 respective Methods section for each analyses described above. ### **A)
 Males\_2019.csv** This dataset was utilized for estimating linear and
 non-linear selection gradients. All individuals in this dataset were
 included to estimate selection on body mass due to reproductive success
 and mating success. However, individuals with no offspring were excluded
 from estimating selection due to average per-mate fecundity(off_year = 0).
 Individuals that had no offspring or had all offspring with a single
 female who also had no other mates were excluded(mna_PCS = 1) from
 estimates of competitive fertilization success(m_PCS). Relative fitness
 for selection analyses was estimated by dividing individual fitness with
 the average fitness of all animals that were successfully genotyped and
 were present in the dataset in March (off_year = 0 or &gt;0). NAs in any
 column would indicate that the values could not be ascertained or
 determined for that given column. **ID**: Continuous variable, Unique
 identifying ID number given to a captured individual. Each individual is
 distinguished based on the order in which toes are clipped. **Sex**: (Sex
 at birth) Categorical variable, M = male, F = female **CapDate**: Date of
 capturing animal on field at the start of the breeding season in March
 2019. Written in yyyy-mm-dd format or mm-dd-yyyy **Month**: Mentions month
 in which animals were captured. Note the months indicate when the sampling
 trip began (although some sampling trips may end in the following month).
 This column can help filter males for the specific subset used for the
 analyses. **Recap**: Categorical N/R. N = New capture ~ Captured for the
 first time in the mark-recapture study, R = Recaptured ~ Animal has been
 captured and tracked between 2015 and 2018. **DaR**: Status at release
 (Dead/Alive). Records if the animal died of unnatural causes due to
 mishaps at capture or was known to be alive at the time of release back on
 the site of capture. **Cohort**: Assignment based on the year in which the
 individual was known to have been born (e.g., 2017 would imply the
 individual was born in 2017). NA values indicate Cohort could not be
 ascertained and subsequently Age could not be determined. **Age**:
 Determined based on the annual difference between the Cohort in which the
 animal was born and the year in which it was captured (Age = 0 is a
 juvenile animal, 1 = animal entering the first breeding season (~ 1 year
 old), 2 = animal entering the second breeding season (~2 year old). NA
 values indicate unknown cohort and thus, unknown age. **SVL**: Known as
 snout-vent-length. Measured in millimeters using a ruler. Estimates the
 animal's length from the tip of its snout to its venter on the date
 of capture. Used as a common measure of body size. If an animal was not
 measured at the time of capture, it is indicated by an NA. **Mass**:
 Measured body weight of animals (in grams) on the date of capture. If an
 animal was not measured at the time of capture, it is indicated by an NA.
 **off_year**: Estimated number of offspring sired in the year 2019 based
 on genetic parentage. A p-value corrected with FDR at &lt;0.05 was
 considered as a successful parentage assignment. Used as a measure of
 annual reproductive fitness. NA values indicate that the individual was
 not included in parentage analysis and thus could not be assigned
 offspring. Zero values, on the other hand, indicate individuals that were
 included in parentage analysis but were not assigned offspring suggesting
 they did not successfully mate or sire offspring. **mat_year**: Estimated
 unique number of females with which males sired offspring in 2019 based on
 genetic parentage assignments. Used as a measure of annual mating success.
 NA values indicate that the individual was not included in parentage
 analysis, could not be assigned offspring , and thus could not be assigned
 any mates. Zero values, on the other hand, indicate individuals that were
 included in parentage analysis but had no assigned offspring indicating
 they did not successfully mate. While in theory, it could indicate that
 they mated but females did not lay eggs sired by the given male, we
 considered these components inestimable. **t_FEC**: Total number of
 offspring produced by the unique mates of the focal male. It is an
 estimate of the total fecundity of all partners of a male. This measure
 was not included in the selection analyses. NA values indicate that males
 either were not successfully genotyped or that males were genotyped and
 included in parentage analyses but were not assigned any offspring
 (off_year = NA or 0). **m_FEC**: Average per mate fecundity. Calculated by
 dividing t_FEC by mat_year to estimate the average number of offspring
 produced across all females that a male sired offspring with. NA values
 indicate that males either were not successfully genotyped or that males
 were genotyped and included in parentage analyses but were not assigned
 any offspring (off_year = NA or 0). **mna_PCS**: Unadjusted siring
 success. Calculated as the average proportion of offspring sired by a male
 amongst all the offspring produced by female mates. Calculated as the
 ratio between off_year and t_FEC. Selection on male body mass due to this
 measure is demonstrated in Fig. S3. NA values indicate that males either
 were not successfully genotyped or that they were not assigned included in
 parentage analyses but did not have any offspring assigned (off_year = NA
 or 0). **m_PCS**: Competitive Fertilization success. Calculated by scaling
 the average proportion paternity of individuals across all the females who
 had more than 2 mating partners (following Devigili et. al. 2015). To
 calculate this metric, one would need the total number of mates that each
 female partner of a male had, which cannot be displayed in this dataset
 for reasons of brevity. We estimated selection on body mass due to this
 measure of competitive fertilization success. NA values indicate, males
 either were not successfully genotyped, males sired no offspring or sired
 all offspring with the same female and had no known competing males thus
 rendering competitive fertilization success inestimable. **Size_class**:
 Allotted size quartile that males were distributed in based on their body
 mass in the given month. **Color**: Specifies the color of fluorescent
 powder applied to males based on the size quartile they belonged to in the
 given month (orange, pink, yellow, green) **Notes:** Specifies anything of
 note in the experiment. Blank values mean that there are no comments
 against those individuals. Specifically indicates a male that was
 accidentally included in the powdering experiment but was ascertained as
 an individual belonging to Cohort 2019 (sexually immature juvenile male).
 ### **B) Females\_2019.csv** **ID**: Continuous variable, Unique
 identifying ID number given to a captured individual. Each individual is
 distinguished based on the order in which toes were clipped. Few cases
 have missing IDs since these animals were captured and measured directly
 on the field and were eventually excluded from the dataset before
 analyses. NA values indicate animal could not be linked to an ID, although
 they were toe-clipped previously. These NA values tended to occur in
 conjunction with captures in May which was the only time we captured,
 measured, identified females directly on the field, and released them back
 to their site of capture on the same day. We considered these as data
 points in the study primarily to assess behavioral estimates of mating
 success. **Sex**: (Sex at birth) Categorical variable, M = male, F =
 female. Since this subset consists of only Females, M is not present.
 **CapDate**: Date of capturing animal on field at the start of the
 breeding season in March 2019. Written in yyyy-mm-dd format or mm-dd-yyyy
 **Month**: Mentions month in which animals were captured. Note the months
 indicate when the sampling trip began (although some sampling trips may
 end in the following month). This column can filter females for the
 specific subset used for the analyses (May or July) **Recap**: Categorical
 N/R. N = New capture ~ Captured for the first time in the mark-recapture
 study, R = Recaptured ~ Animal has been captured and tracked between 2015
 and 2018. **DaR**: Status at release (Dead/Alive). Records if the animal
 died of unnatural causes due to mishaps at capture or was known to be
 alive at the time of release back on the site of capture. **Cohort**:
 Assignment based on the year in which the individual was known to have
 been born (e.g., 2017 would imply the individual was born/captured as a
 juvenile in 2017).NA values indicate that cohort of individuals could not
 be identified if it was newly captured as an adult or a previously clipped
 animal could not be accurately identified **Age**: Determined based on the
 annual difference between the Cohort in which the animal was born and the
 year in which it was captured (Age = 0 is a juvenile animal, 1 = animal
 entering the first breeding season (~ 1 year old), 2 = animal entering the
 second breeding season (~2 year old) and, so on). NA values indicate the
 Cohort could not be ascertained confidently. **SVL**: Known as
 snout-vent-length. Measured in millimeters using a ruler. Estimates the
 animal's length from the tip of its snout to its venter on the date
 of capture. Used as a common measure of body size. **Mass**: Measured body
 weight of animals (in grams) on the date of capture. NA values indicate
 that males either were not successfully genotyped or that males were
 genotyped and included in parentage analyses but were not assigned any
 offspring. **off_year**: Estimated number of offspring produced in the
 year 2019 based on genetic parentage. A p-value corrected with FDR at
 &lt;0.05 was considered as a successful parentage assignment. Used as a
 measure of annual reproductive fitness as well as a measure of total
 fecundity of the female. NA values indicate that the individual was not
 included in parentage analysis and thus could not be assigned offspring.
 Zero values, on the other hand, indicate individuals that were included in
 parentage analysis but were not assigned offspring suggesting they did not
 successfully mate or produce viable offspring. **mat_year**: Estimated
 unique number of males with which females produced offspring in 2019 based
 on genetic parentage assignments. Used as a measure of annual mating
 success. NA values indicate that the individual was not included in
 parentage analysis and thus could not be assigned offspring. Zero values,
 on the other hand, indicate individuals that were included in parentage
 analysis but were not assigned offspring suggesting they did not
 successfully mate. **fec_year**: Average per mate fecundity. Calculated by
 dividing off_year by mat_year to estimate the average number of offspring
 produced across all males that a female produced offspring. NA values
 indicate that the individual was not included in parentage analysis and
 thus could not be assigned offspring. We also assumed that individuals who
 did not have any offspring assigned, did so because they did not mate
 successfully even though it is likely this may be due to other factors
 like not producing viable eggs etc. As a result, we considered fec_year
 inestimable for these individuals too. **Color_Present**: Categorical
 (Yes/No). Specifies if females had color present in and around their
 venter under UV light suggesting a potential transfer of fluorescent
 powder from males during mating. Blank values indicate that these
 individuals were not part of the powdering experiment.
 **Could_segregate**: Categorical (Yes/No). Specifies if the observer could
 reliably identify the color on the venter. In cases, where we could not
 (Could \_segregate = No), those copulations were omitted from further
 analysis. **Non_copulation contact**: Categorical (Yes/No). Indicates if
 the color was on a part of the body that is usually not in direct contact
 during mating and is not considered as a copulation event.
 **Multiple_mating**: Categorical (Yes/No). Indicates if two or more colors
 were detected on the venter, suggesting instances of mating with males
 belonging to more than one size quartile. Even if females could have mated
 with more than one male from the same size quartile, we did not consider
 that as an instance of multiple mating. If multiple mating = Yes, we chose
 to report the copulation as a new entry in the dataset, even if all other
 morphological and fitness data for the ID remained the same. **Color**:
 Indicates the identified color of fluorescent powder present on the
 female. Categorical variable with one of the following entries - none,
 orange, pink, green or yellow. **Mating**: Indicates if an individual
 copulated or not, as determined by transfer of fluorescent powder.
 Categorical (1/0 where 1 = Copulated, 0 = Did not copulate).
 **Size_class_mating**: Indicates the size category of males with which the
 females were detected to have mated (In May, yellow = S1, green = S2, pink
 = S3, orange = S4; whereas in July, orange = S1, pink = S2, green = S3,
 yellow = S4). S0 indicated that a mating was not detected (Color = none).
 ## Sharing/Access information Links to other publicly accessible locations
 of the data:- NA Data was derived from the following sources:- Data was
 collected as part of this study. ## Code/Software We used RStudio to write
 scripts to wrangle and analyze the data. Following is the complete list of
 packages we used in analyzing and plotting the data. The Rcode used to
 analyze the dataset to reproduce figures and analyses in the paper is
 available upon request. Requests may be sent to the Corresponding Author:
 Rachana Bhave at the following email address -
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