DOI: 10.5061/dryad.mcvdnck82
title: Experience and trust: The benefits of mate familiarity are realized through sex-specific specialization of parental roles in cassin’s auklet

description: Maintaining a pair bond year after year (perennial monogamy) often
 enhances reproductive success, but what familiar pairs are doing
 differently to improve success is unknown. We tested the hypothesis that
 endocrine changes mediate improvements in parental attendance in known-age
 Cassin’s auklets Ptychoramphus aleuticus, which we found limited
 evidence for. Instead, we found sex-specific parental roles in familiar
 pairs. Males modulated their nest attendance depending on the attendance
 of their mate, but the direction depended on mate familiarity.
  We suggest his flexibility may be mediated by prolactin. In a
 historical dataset, females with a familiar mate laid larger eggs that
 hatched into more robust chicks, but larger eggs correlated with lower
 female body condition. In study birds, attendance by males and females in
 good condition predicted chick weight, but attendance by females in poor
 condition did not, suggesting female-specific energetic constraint. Our
 findings suggest that males and females contribute differently to their
 joint reproductive fortunes, and that improvements in their respective
 roles may result in the benefits of mate familiarity. Since improved
 reproductive success is presumed to be a main benefit of maintaining a
 long-term pair bond, these results suggest a new avenue of research in the
 evolution of monogamy.
description: <em>Fieldwork and sample collection: </em>We
 conducted our study on Southeast Farallon Island (37.6989° N, 123.0034° W;
 Farallon Islands National Wildlife Refuge, California, USA) in 2019 and
 2021. These two years represented a positive El Niño Southern Oscillation
 (ENSO) event where on average only 8% of followed pairs fledged a chick
 (2019), and a negative ENSO event (2021) where 65% of pairs fledged a
 chick [1]. Many individuals of the Southeast Farallon Island Cassin’s
 auklet population are of known age and have known reproductive histories
 [2], allowing us to disentangle the confounding effects of age and mate
 familiarity. Nest boxes were checked every 15 days until an incubating
 bird was found. If the individual was of known age, it was measured and
 evaluated for inclusion in the study. All morphometrics are therefore from
 birds in their first two weeks of incubation. We weighed known-age
 incubating adults between 13:00 and 15:00 with a Pesola spring scale (+/-
 1g), measured wing cord with a wing rule (+/- 1mm) and measured bill depth
 with a digital calliper (+/- 0.1mm). The following day, we measured the
 mate. We checked nests every 5 days to determine egg fate, and after
 hatching, chicks were weighed every 5 days until fledging to measure the
 maximum weight attained. We acknowledge that sex is a
 nonbinary phenotype and that treating it as such can obscure important and
 relevant biological information, and inadvertently support social agendas
 that cause real harm [3]. For brevity and clarity, we refer to the
 egg-laying member of the pair as “female” and the non-egg laying member of
 the pair as “male”. Males have deeper bill depths compared to females, but
 there is significant overlap [4]. To sex birds accurately, we used
 bill-depth based sex estimations from multiple previous years for all
 individuals that had been measured before with a different mate (63 of 76
 individuals in 2019 and 66 of 80 individuals in 2021), assuming a pair was
 one male and one female. Sex-specific body condition was estimated using
 the residual of the model mass~bill depth+wing cord. This model was built
 using morphometrics for this colony from 1973 to 2021 (N =
 7964). Pairs were selected for the study if there was
 at least one known-age mate. We attempted even representation of
 individuals in their first or second breeding attempt (i.e., 2-3 years
 old; “young”; in this population many individuals breed for the first time
 at 2 years) or had more years of previous breeding (i.e., 5+ years old;
 “old”.) If the pair contained an individual of unknown age, the pair was
 classified based on the known-age mate, but because ages of mates are only
 weakly correlated (coefficient = 0.39, SE = 0.051, p &lt; 0.001,
 R<sup>2</sup> = 0.14, N = 353), we used the categorical
 classification of pairs as “old” or “young” for pair selection purposes
 only. We used individual age for 97 known-age birds and band year-based
 age estimation for 51 non-known-age mates of known-age birds in our
 models. Although there were 9 individuals mistakenly classified because
 they were not thought to be known-age and were later discovered to have
 been banded as chicks, there was no difference in the mean age between
 experience groups (student’s t test, p = 0.3). When we compared newly
 formed pairs (“inexperienced”) to pairs that had been observed breeding
 together at least once before (“experienced”) in our analyses, we used a
 categorical variable to describe mate familiarity (i.e., “experienced” or
 “inexperienced”; “pair experience”). When exploring changes over time, we
 used the number of years the pair had been observed breeding together
 (“bond duration”). In this population, known-age individuals are usually
 recaptured yearly from age 2 or 3 until senescence, so it is unusual to
 have gaps in our knowledge of their reproductive history. We followed 79
 auklet pairs over two years, 19 inexperienced pairs and 20 experienced
 pairs in 2019 and 19 experienced and 21 inexperienced pairs in 2021. A
 total of 145 individuals were used in this study, 23 of which contributed
 to the study in both years. To measure nest attendance,
 nest boxes of were fitted with radio frequency identification (RFID)
 readers and both individuals were fitted with a passive integrated
 transponder (PIT) tag on the tarsus, a method that is reliable for
 determining nest attendance and causes little disturbance [5,6]. Our RFID
 readers were programmed to continuously record from 2 circular antennas
 deployed at the nest entrance to record tagged birds entering or exiting
 the nest box. Tags and readers were deployed 1-2 days after birds were
 weighed and measured. We measured nest attendance rate as the proportion
 of nights an individual was present at the nest. During incubation,
 presence at night means the bird was either finishing an incubation bout
 or relieving its mate. During chick rearing, presence at the nest at night
 was assumed to be prey delivery. Every effort was made to equalize the
 number of nights of recordings across nests, but it was not always
 possible as readers occasionally ran out of battery or malfunctioned. We
 accounted for this variation in our statistical analysis by weighting
 models by the number of nights of RFID recording.
 Physiological sampling was conducted 10 days after the egg was
 first observed during incubation (May 13-16 in 2019 and April 5-17 in
 2021) between 10:00 and 19:00 PST and when the chick was 10 days old (June
 17-24 in 2019 and April 28-May 19 in 2021) between 22:00 and 0:00, as
 Cassin’s auklets are only present on the island at night during chick
 rearing. A one-time measurement will not fully capture the range of
 hormones across a breeding period, which can be inconsistent within an
 individual [7]. However, repeated sampling would cause levels of
 disturbance that nesting auklets may not tolerate and we were unwilling to
 risk triggering nest abandonment, given that this population has
 significantly declined since monitoring began in the 1970’s [2]. We
 therefore limited our disturbance of the colony, at the cost of a lower
 likelihood of finding correlations between behaviour and hormone levels.
 We measured PRL in plasma and CORT in faeces, since faecal CORT is less
 susceptible to momentary stressors from investigator disturbance [8]. An
 analysis of faecal CORT data collected in this population in 2021 show no
 correlation between CORT and time of collection (generalized linear mixed
 model with faecal sample emulsification as a random effect, p = 0.61). To
 our knowledge, there is no existing assay for measuring PRL in avian
 faeces, but it takes much longer than CORT to respond to handling stress
 [9]. Other studies have found little evidence of circadian rhythm in
 plasma PRL[10]. We collected a faecal sample by holding the bird over a
 Pyrex container for no more than 1 minute, then collected a 150µL blood
 sample from the brachial vein into heparinized capillary tubes to measure
 PRL. Plasma and faecal pellets were frozen at -10°C in the field. After
 the field season, plasma samples were frozen at -80°C and faecal samples
 at -15°C. <em>Laboratory methods</em>: We
 dried faecal samples in a fume hood, then weighed and homogenized them
 before rehydrating with 2mL dH<sub>2</sub>O. To validate our
 radioimmunoassay protocol, we made a pool from three samples, added 1 and
 0.25 ng CORT and performed serial dilution to confirm parallelism to the
 standard curve. We measured 1.05 ng and 0.15 ng CORT in these pooled
 samples. Steroids were extracted in duplicate following Krause et al. [11]
 across two assays. Mean percent recovery was 61.5% (SD = 20.6%).
 Intra-assay CV was 3.66% and inter-assay CV was 7.16%. 26 out of 194
 faecal samples were emulsified by dichloromethane during steroid
 extraction, which decreased the resulting CORT measurement by 6.8 ng/mg
 (SE = 1.2, t-value = 8.70, p &lt; 0.001). There was no evidence that
 emulsified samples were nonrandomly distributed among any variables of
 interest (Supplementary table 1). We determined plasma
 concentrations of PRL via a heterologous radioimmunoassay as detailed in
 Cherel et al. [12] in 20µL duplicates across two assays. The detection
 limit was 0.99 ng/mL. The intra- and inter-assay CVs were 11.20 and
 12.56%, respectively. Pooled plasma samples of Cassin’s auklet produced a
 dose–response curve that paralleled chicken prolactin standard curves,
 validating our PRL radioimmunoassay for this species.
 <em>Historical Data: </em>We used historical data of
 known-age Cassin’s auklet egg length and diameter from Southeast Farallon
 Island (the same population as our study birds) from 1992-2017 (N = 783).
 Egg length and diameter of the widest point were measured using digital
 callipers (+/- 0.1mm) at the same time as adults were measured in early
 incubation. Egg measurements of the study birds were not available because
 objective 3 was added post-hoc, in response to interesting patterns we
 observed in our results for the first two objectives. Accordingly, egg
 measurements were not in our original study design and regular egg
 measurement was discontinued from the monitoring protocol in 2018. We
 calculated egg volume as 0.5202LD<sup>2</sup>-0.4065 [13],
 where L = length and D = diameter. These data were paired with information
 on pair experience, female age, wing cord (a proxy of body size), body
 condition, hatching success, and the maximum weight of the chick. Adult
 and chick morphometrics were collected using the same protocol as for our
 study birds, and body condition was calculated using the same model. We
 omitted one female with an anomalously low residual body condition of -58,
 which was likely due to measurement error (population mean = -1, SD =
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description: # Experience and trust: The benefits of mate familiarity are realized
 through sex-specific specialization of parental roles in cassin’s auklet
 [https://doi.org/10.5061/dryad.mcvdnck82](https://doi.org/10.5061/dryad.mcvdnck82) The data presented here are endocrine, behavior, and fitness metrics from Cassin's auklets collected in 2019 and 2021 on Southeast Farallon Island, California. Endocrine measurements are levels of fecal corticosterone and plasma prolactin. Behavioral measurements were the proportion of nights an individual was present at the nest during both incubation and chick rearing. Fitness metrics include hatching and fledging success, and the maximum weight the chick attained. There is also some basic information about each bird in the dataset, such as its age if known, sex, and body condition. It also includes information on egg volumes from individuals where egg morphometrics were taken between 1992 and 2017, and information about the females that laid those eggs. ## Description of the data and file structure Behavior_Code.RMD is a markdown file of the code used for statistical analysis and generating figures. The spreadsheet behavior_data.xlsx includes all data necessary to run the markdown file. The first sheet (“readme”) includes a description of all columns in the data sheets. Note that all empty cells indicate data that were unavailable, generally because the bird was not recaptured for sampling. ## Code/Software To run the markdown file, install the following R packages: readxl, dplyr, ggplot2, emmeans, lme4, tidyverse, car, lmerTest, bbmle, ggpubr, cAIC4, sjPlot, DescTools, openxlsx, interactions, and jtools. Download the provided metadata and replace the filepaths in the markdown file to where it is stored. You should then be able to run the code, starting from the top and working down. There is code at the end that you will not be able to run, as it requires raw data rather than summaries. This code is included for transparency. The code is commented to explain what each section is doing.