DOI: 10.5061/dryad.k6djh9wgd
title: Data for: Kin recognition for incest avoidance in Damaraland mole-rats, Fukomys damarensis

description: Across taxa, breeding among close relatives is usually avoided because it
 incurs fitness costs to offspring. Incest is often averted through the
 dispersal of either sex from the natal area to breed. In some philopatric
 species, association among relatives extends into adulthood, and an
 ability to discriminate kin may be required for individuals to reduce
 inbreeding risk. Here, we aim to determine the mechanism of kin
 recognition for incest avoidance in the Damaraland mole-rat Fukomys
 damarensis, a cooperative breeder characterized by extreme reproductive
 skew. Pairs of opposite-sex adults were formed in the laboratory and,
 within pairs, genetic relatedness and degree of familiarity were
 manipulated through cross-fostering experiments. We found that unfamiliar
 pairs were more likely to engage in sexual behaviours and bred more
 successfully than familiar pairs, regardless of their genetic
 similarity.  Females paired with unfamiliar males were also more
 likely to exhibit reproductive activation, characterized by increased
 levels of oestradiol and progesterone. This study shows that in Damaraland
 mole-rats, inbreeding avoidance can be achieved through a discrimination
 mechanism that relies on association during rearing, and that ovulation is
 induced by mating. This study advances our understanding of incest
 avoidance in species with constrained dispersal.
description: The experiment was initially carried out on 32 pairs of
 opposite-sex individuals that were either: (i) familiar kin
 (<em>n</em> = 8); (ii) familiar non-kin
 (<em>n</em> = 8); (iii) unfamiliar kin (<em>n</em>
 = 8); or (iv) unfamiliar non-kin (<em>n</em> = 8), in a full
 factorial treatment design.  Behavioural observations
 were carried out to quantify copulation, defined as one individual
 mounting another and attempting intromission with pelvic thrusts, and sex
 foreplay, defined as the rapid succession of bites, sparring, sniffs,
 passes, and drumming. A full ethogram of these and other observed mole-rat
 behaviours are presented in Supplementary Table S1A. Behavioural
 observations consisted of focal and scan sampling. Focals were carried out
 on the female. One two-hour focal session was carried out immediately
 after pairing (day 0, approximately 1000 SAST), and another one-hour focal
 session the following day (day 1, approximately 0800 SAST). Focal
 behaviours were sampled as ‘states’, recorded with a start and an end
 time, or ‘events’, recorded at observation without a duration
 (Supplementary Table S1B). Weekly 12-hour scan sessions were carried out
 for eight weeks, starting 2-8 days after pairing (approximately 0700
 SAST). Four pairs were observed concurrently during each session
 (<em>n</em> = 8 individuals). Scan sessions comprised a
 combination of instantaneous and continuous sampling (Supplementary Table
 S1C). Behavioural states were recorded every four minutes, generating 180
 instantaneous samples per individual. In between instantaneous sampling,
 events and states of short duration were recorded continuously. For both
 focals and scans, observations were recorded using Observer 11XT pocket
 version 3.2. Urine samples were collected to quantify
 oestradiol (E2) and progesterone (P4) and determine the effect of
 treatment on reproductive activation. Samples were collected 2-4 days
 before pairing to establish baseline E2 and P4 levels. Samples were
 subsequently collected on day 1, then every 3 days until day 90, and every
 7 days between day 90 and day 270. E2 and P4 levels were quantified using
 high-performance liquid chromatography-tandem mass-spectrometry
 (Supplementary Material S2). All pairs were closely
 monitored for nine months following pairing. To investigate reproductive
 success, we tested the effect of treatment on the: (i) probability of
 successful gestation following reproductive activation; (ii) total number
 of pups produced; and (iii) total number of litters produced.
description: # Data for: Kin recognition for incest avoidance in Damaraland mole-rats,
 Fukomys damarensis
 [https://doi.org/10.5061/dryad.k6djh9wgd](https://doi.org/10.5061/dryad.k6djh9wgd) ## Description of the data Data sets and code are provided for 1. Behavioural Analysis and 2. Hormone Analysis. The experiment was carried out on 31 pairs of opposite-sex individuals that were either: (i) familiar kin (*n* = 8); (ii) familiar non-kin (*n* = 8); (iii) unfamiliar kin (*n* = 7); or (iv) unfamiliar non-kin (*n* = 8), in a full factorial treatment design. Behavioural observations were carried out to quantify copulation, defined as one individual mounting another and attempting intromission with pelvic thrusts, and sex foreplay, defined as the rapid succession of bites, sparring, sniffs, passes, and drumming. Behavioural observations consisted of focal and scan sampling. Focals were carried out on the female. One two-hour focal session was carried out immediately after pairing (day 0, approximately 1000 SAST), and another one-hour focal session the following day (day 1, approximately 0800 SAST). Focal behaviours were sampled as ‘states’, recorded with a start and an end time, or ‘events’, recorded at observation without a duration (Supplementary Table S1B). Weekly 12-hour scan sessions were carried out for eight weeks, starting 2-8 days after pairing (approximately 0700 SAST). Four pairs were observed concurrently during each session (*n* = 8 individuals). Scan sessions comprised a combination of instantaneous and continuous sampling. Behavioural states were recorded every four minutes, generating 180 instantaneous samples per individual. In between instantaneous sampling, events and states of short duration were recorded continuously. For both focals and scans, observations were recorded using Observer 11XT pocket version 3.2. Urine samples were collected to quantify oestradiol (E2) and progesterone (P4) and determine the effect of treatment on reproductive activation. Samples were collected 2-4 days before pairing to establish baseline E2 and P4 levels. Samples were subsequently collected on day 1, then every 3 days until day 90, and every 7 days between day 90 and day 270. E2 and P4 levels were quantified using high performance liquid chromatography-tandem mass-spectrometry. All pairs were closely monitored for nine months following pairing. To investigate reproductive success, we tested the effect of treatment on the: (i) probability of successful gestation following reproductive activation; (ii) total number of pups produced; and (iii) total number of litters produced. Observations of sexual behaviour were compared across treatments using tweedie GLMMs with rates of copulation and sex foreplay specified as response variables. Rates were analysed as counts/hour for focal data and counts/12 hours for scan data. Pair and session ID were included as random effects, to account for multiple observations of pairs. We compared hormone levels within 60 days of pairing across treatments using tweedie GLMMs, with E2 and P4 specified as response variables and female ID as a random effect. For P4, we specified the model to estimate a dispersion parameter for each treatment to avoid issues of  heteroscedasticity in the residuals. To investigate reproductive success, we compared the timing of reproductive activation between treatments. Two females from the unfamiliar non-kin treatment took over twice as long to activate their reproductive axis than the next longest female in this treatment, so to consider the possibility that these may be outliers, models were sequentially ran with none, one and both of these potential outliers removed. We used generalized poisson (all data) and gamma (outliers removed) GLMMs with log link and specified the number of days between pairing and reproductive activation as the response variable (one model for each activation criterion). We also compared the likelihood of successful gestation between treatments, specified in a binomial GLMM as whether females produced their first litter within one hundred days of activation. Finally, the number of pups and the number of litters produced was compared using poisson GLMMs with Pair ID specified as random effect. ## File structure **Focal_durations.csv** This data was used to calculate mean focal duration. **Variables** FocalFileID: Unique Identifier for focal session FocalDate: Date of focal StartTime: Start time of focal StopTime: End time of focal ObservationDuration: Focal duration in seconds Focal: 0 = focal on day 0 (pairing day), 1 = Focal on day 1 (1 day after pairing) **Focal_obs.csv** This data was used for analysing behavioural data from focal sessions. **Variables** AnimalID: Identity of experimental subject       ExperimentalGroup: Identity of experimental pair         ColonyRef: Numeric identifier of experimental pair Treatment: type of pairs; FR = Familiar Related; FUR = Familiar Unrelated; UFR = Unfamiliar Related; UFUR = Unfamiliar Unrelated   Kinship: Relationship between individuals within experimental pair    FocalFileID: Unique Identifier for focal session               ObservationDuration: Focal duration in seconds           SexforCount: counts of sex foreplay behaviours observed during focal               SelfgroomCount: counts of self-grooming observed during focal SparrCount: counts of sparring observed during focal CopulationCount: counts of copulation observed during focal               BiteCount: counts of biting observed during focal         SniffCount: counts of sniffing observed during focal    OvaggCount: counts of overt aggression observed during focal **Scan_obs.csv** This data was used for analysing behavioural data from scan sessions. **Variables** AnimalID: Identity of experimental subject       Sex: M=Male; F=Female ExperimentalGroup: Identity of experimental pair         ColonyRef: Numeric identifier of experimental pair Treatment: type of pairs; FR = Familiar Related; FUR = Familiar Unrelated; UFR = Unfamiliar Related; UFUR = Unfamiliar Unrelated   Kinship: Relationship between individuals within experimental pair    StartDate: Date of scan               TotalDuration: Total scan duration in seconds ContinuousDuration: Total scan duration minus time taken to record instantaneous sampling SfCount: counts of sex foreplay observed during scan SfDuration: duration of sex foreplay states observed during scan in seconds  CopCount: counts of copulation observed during scan              BiteCount: counts of biting observed during scan          PullTailCount: counts of tail pulling observed during scan         SparrCount: counts of sparring observed during scan ChaseCount: counts of chasing observed during scan                AggCount: counts of aggressive interactions observed during scan      GroomCount: counts of allogrooming observed during scan   SniffCount: counts of sniffing observed during scan     SubmissCount: counts of submissive behaviours observed during scan           PumpCount: counts of body pumps observed during scan                       HudCount: counts of huddling observed during scan  SelfGCount: counts of self-grooming observed during scan     SocCount: counts of social interactions observed during scan               PassCount: counts of tunnel passes observed during scan \** ** **RA_MS.csv: reproductive activation info** **Variables** AnimalID: Identity of experimental subject ExperimentalGroup: Identity of experimental pair Treatment2: Determine whether the male and the female from the experimental pair are familiar or unfamiliar with each other at time of pairing  Criteria: Criteria used to determine the time point of the experimental subject reproductive activation (described in supplementary material) Activation date: Date of reproductive activation determined under "Criteria" Treatment: type of pairs; FR = Familiar Related; FUR = Familiar Unrelated; UFR = Unfamiliar Related; UFUR = Unfamiliar Unrelated ParturitionDate: Date of female subject first parturition ActivationParturition_DayDiff: Difference in number of days between reproductive activation and and first female subject first parturition of female subjects. **Abortion_P4_MS.csv** This file only provide the information required to highlight the probable termination of unsuccessful gestation or of a luteal phase on the figures of female subjects' estradiol and progesterone profile provided in supplementary material (blue vertical lines in Fig. S2D). The time point corresponding to the termination of unsuccessful gestation or of a luteal phase was determined based on the sudden and profound decrease in urinary progesterone. **Variables** AnimalID: Identity of experimental subject. P4_End_Day: Day progesterone decrease likely to mark the end of the luteal phase or the unsuccessful gestation. P4_End_Date: Date progesterone decrease likely to mark the end of the luteal phase or the unsuccessful gestation. **SuccessfullMating_MS** This file only provide the information required to highlight the time window during which the mating that led to a successful conception - gestation - parturition gestation. This information was gathered through microsattelite analyses showing that 3 litters had been sired by a male immigrants that were experimentally introduced in foreign groups for a maximum of 2 hours, and thus for which the date of successful copulation is known, for the purpose of a another study. Successfull mating occurred 91, 93 and 95 days pre-parturition and the range of 91-95 is shown as dotted black vertical lines in Fig. S2D. **Variables** AnimalID: Identity of experimental subject ExperimentalGroup: Identity of experimental pair Mating: Count of days post-pairing of the successful mating **IB_Hormones_MS.csv** **Variables** AnimalID: Identity of experimental subject ExperimentalGroup: Identity of experimental pair Treatment: Type of pairs; FR = Familiar Related; FUR = Familiar Unrelated; UFR = Unfamiliar Related; UFUR = Unfamiliar Unrelated Treatment2: Determine whether the male and the female from the experimental pair are familiar or unfamiliar with each other at time of pairing ConceptionCategory: Category of first successful conception (that lead to a parturition) created for plotting purposes; Conception30 = within or around 30 days of pairing not preceded by luteal phase or abortion; Conception60 = around 60 days post-pairing and not preceded by luteal phase or abortion; Abortion = successful conception preceded by luteal phase and/or abortion; ConceptionLate = successful conception much later than 60 days and not preceded by luteal phase and/or abortion PlotCat: Variable created for plotting purpose; all levels described in ConceptionCategory above except FamHigh = female from familiar pairs with high levels of E2; FamMedium = female from familiar pairs with intermediate levels of E2; FamLow = female from familiar pairs with low levels of E2 SampleID: ID of urine sample  UrineDate: Date of urine sample            DatePairing_DayDiff: number of days between urine sampling date and pairing date DateTime: specifies whether SampleID was collected before pairing (Baseline) or after (PostPairing) SamplingPeriod_Months: variable created for plotting purposes. 2M = SampleID collected within 2 month of pairing; 9M = SampleID collected towards the end of the experiment between 200 and 270 days post-pairing. NPACID: ID of urine sample extract for hormone analyses                        Batch: ID of batch on which NPACID was put on mass spectrometer  UrineVolume: Volume of urine used for NPACID                             NextParturition_ParturitionDayDiff: Count of days between UrineDate and date of next parturition Hormone: Hormone ID QuantificationCategory: ND = non-detectable, D = Detectable, Q = Quantifiable; Conc: urinary concentration in ng/ml **ExperimentInfo_MS.csv** **Variables** AnimalID: AnimalID: Identity of experimental subject Sex: M=Male; F=Female. Note that the analyses in the MS are performed on female exclusively ExperimentalGroup: Identity of experimental pair Treatment: Type of pairs; FR = Familiar Related; FUR = Familiar Unrelated; UFR = Unfamiliar Related; UFUR = Unfamiliar Unrelated Treatment2: Determine whether the male and the female from the experimental pair are familiar or unfamiliar with each other at time of pairing       PairingDate: Date of experiment start (removal of male and female from their groups to create pairs)                ParturitionDate: Date of first parturition post-pairing                   EstimatedConceptionDate_87: Estimated date of first successful sonception, assuming a gestation length of 87 days.     ConceptionPairing_DayDiff_87: Number of days between pairing and first successful conception, assuming a gestation length of 87 days (female are certainly pregnant at this point) ParturitionPairing_DayDiff: Number of days between pairing and first parturition         ConceptionCategory: - ConceptionCategory: Category of first successful conception (that lead to a parturition) created for plotting purposes; Conception30 = within or around 30 days of pairing not preceded by luteal phase or abortion; Conception60 = around 60 days post-pairing and not preceded by luteal phase or abortion; Abortion = successful conception preceded by luteal phase and/or abortion; ConceptionLate = successful conception much later than 60 days and not preceded by luteal phase and/or abortion # Code/Software Code is provided for Behavioural Analysis and Hormone analysis for use in R. All data analyses were performed in R version 4.2.1, using Generalized Linear Mixed Models (GLMMs) specified in the *glmmTMB* package. **R Code Hormone Data** **R Code Behavioural Data** Annotated R codes to generate the content of the manuscript