\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,english]{babel}



\usepackage{float}








\begin{document}

\title{Vascularization underlies differences in sexually selected skin
coloration in a wild primate}



\author[1]{Patricia DeLacey}%
\author[1]{Sharmi Sen}%
\author[2]{India A. Schneider-Crease}%
\author[2]{Kenneth Chiou}%
\author[3]{Alemayehu Lemma}%
\author[3]{Ferehiwot Ayele}%
\author[4]{Abebaw Haile}%
\author[5]{Amy Lu}%
\author[1]{Thore J. Bergman}%
\author[1]{Jacinta C. Beehner}%
\author[2]{Noah Snyder-Mackler}%
\affil[1]{University of Michigan}%
\affil[2]{Arizona State University}%
\affil[3]{Addis Ababa University}%
\affil[4]{Ethiopian Wildlife Conservation Authority}%
\affil[5]{Stony Brook University}%


\vspace{-1em}



  
  \date{May 18, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Male reproductive competition can select for condition-dependent,
conspicuous traits that signal some aspect of fighting ability and
facilitate assessment of potential rivals. However, the underlying
mechanisms that link the signal to a male's current condition are
difficult to investigate in wild populations, often requiring invasive
experimental manipulation. Here, we use digital photographs and chest
skin samples to investigate mechanisms of a visual signal used in male
competition in a wild primate, the red chest patch in geladas
(Theropithecus gelada). We analyzed photographs collected during natural
(n=144) and anesthetized conditions (n=38) to understand variability in
male and female chest redness, and we used chest skin biopsies (n=38) to
explore sex differences in gene expression. Male and female geladas
showed similar average redness, but males exhibited a wider
within-individual range in redness under natural conditions. These sex
differences were reflected at the molecular level, with 10.5\% of genes
exhibiting significant sex differences in expression. Subadult males
exhibited intermediate gene expression patters between adult males and
females, pointing to mechanisms underlying the development of the red
chest patch. We found that genes more highly expressed in males were
associated with blood vessel development and maintenance but not with
androgen or estrogen activity. Together, our results suggest male gelada
redness variability is driven by increased blood vessel branching in the
chest skin, providing a potential link between male chest redness and
current condition as increased blood circulation to exposed skin could
lead to heat loss in the cold, high-altitude environment of geladas.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Male reproduction is a zero-sum game that can lead to intense
competition (Weir et al., 2011). Because actual fighting is
energetically costly and exposes both the winner and loser to injury and
infection, rival males can benefit from displaying their strength or
condition using signals that allow for competition without costly
engagement in fighting (Bradbury \& Vehrencamp, 2011; Maynard Smith \&
Parker, 1976). The conspicuous male traits used for rival assessment
(sexually selected signals) allow for conflict resolution at the lowest
cost to both males (Maynard Smith, 1982; Maynard Smith \& Harper, 2003,
1995), and are often condition-dependent, such that they reliably
indicate some aspect of ``quality'' that predicts their ability to win a
physical fight (i.e., body size, body condition, current health status)
(Fisher, 1915; Penn \& Sz\selectlanguage{ngerman}ámadó, 2020; Trivers, 1972; Zahavi, 1977).
Because the expression of the signal is limited by a male's condition,
these signals tend to be honest indicators of ability because only
high-quality males can produce the strongest signals (Weaver et al.,
2017).

While the functional consequences of signals can be observed
noninvasively and are thus relatively well-studied, the mechanisms
underlying these traits are much less well-understood. For example,
although observational data demonstrate that higher ranking male drills
(\emph{Mandrillus leucophaeus} ) exhibit redder lip and groin coloration
than lower ranking males (Marty et al., 2009), identifying the causal
mechanism of this difference would require invasive methods such as
surgical implantation of slow-release hormone devices, social
manipulation, or genetic manipulation, which are often not possible or
ethical to conduct in wild populations (Emlen et al., 2012; Hau et al.,
2000; Karubian et al., 2011). However, capture-and-release programs in
wild populations allow tissue sample collection for genomic and
transcriptomic analyses (Tung et al., 2010), providing a minimally
invasive technique to investigate the molecular correlates of signal
mechanisms without experimental manipulation.

Here we investigated the mechanisms underlying a visual signal that may
mediate male competition in a wild primate: the brilliant red chest
patch in male geladas (\emph{Theropithecus gelada} ), a cercopithecine
primate endemic to the highlands of Ethiopia. Geladas have a
multi-tiered social system in which multiple `reproductive units' (one
dominant adult male, one or more subordinate adult males, up to a dozen
adult females, and related offspring) associate to forage, rest, and
move. The dominant ``leader'' male of units have the majority of the
mating opportunities and display the reddest chests as compared to the
subordinate ``follower'' males (with many fewer reproductive
opportunities) and ``bachelor'' males living in all-male groups (with no
reproductive opportunities) (Bergman et al., 2009; Bergman \& Beehner,
2008). Because geladas live in large, fluid societies where males
frequently congregate and forage with other males that they do not
recognize individually (Bergman, 2010), the chest patch is hypothesized
to be a sexually selected signal that mediates male rival assessment and
allows males to ``size up'' others prior to engaging in conflict
(Benítez, 2016). Bachelor males, which tend to be young adult males
awaiting their chance to overthrow and replace leader males (Pappano \&
Beehner, 2014), are particularly attentive to leader male chest color.
For example, across a one-year study, leaders with redder chests (after
a vigorous ritualized ``vocal display'' accompanied by running, throwing
rocks, climbing trees, or shaking branches) were less likely to be
overthrown by bachelors than their less-red counterparts (Benítez,
2016).

Previous research conducted on the same population has shown that chest
redness in geladas is mediated by increased blood flow with a
concomitant increase in surface temperature. Specifically, redder chests
are associated with (1) increased physical activity (Bergman et al.,
2009; DeLacey et al., 2022) and (2) higher chest skin surface
temperature, whether measured using internal body heat or following the
application of an external heat pack (DeLacey et al., 2022). The
relationship with physical activity could be similar to skin flushing in
humans during exercise, when skin blood flow increases to dissipate heat
generated by muscle contractions (Kenney \& Johnson, 1992).

Sexually selected signals in males are often mediated by testosterone, a
steroid hormone involved in regulation of reproductive function in male
vertebrates (Ketterson \& Nolan, 1999; Plant \& Zeleznik, 2014) that is
associated with reproductive benefits (Enstrom et al., 1997) and
physiological costs (Muehlenbein et al., 2006; Muehlenbein \&
Bribiescas, 2005). High doses of circulating testosterone are known to
dilate vascular networks and increase blood flow, providing an avenue
for testosterone to work alongside a blood flow mechanism to influence
chest redness in geladas (Molinari et al., 2002; Webb et al., 1999).
Testosterone can alter gene transcription by: (1) binding to the
androgen receptor (AR), (2) aromatizing to estradiol and then binding to
estrogen receptor \(\alpha\) (ER\(\alpha\)) or
\(\beta\)(ER\(\beta\)), or (3) converting to
5\(\alpha\)-reductase which binds to AR but cannot convert to
estradiol (Hau \& Wingfield, 2011). Local conversion of testosterone to
estradiol is a particularly strong candidate for chest redness
regulation based on the results of a similar study; in the closely
related male rhesus macaque (\emph{Macaca mulatta} ), increases in
testosterone increased both redness and blood flow in sex skin areas.
Moreover, administration of an aromatase inhibitor (which prevents the
conversion of testosterone to estradiol) decreased skin redness (Rhodes
et al., 1997).

Therefore, to better understand molecular correlates of the chest patch
signal mechanism, we collected chest skin biopsies from male and female
geladas from a wild population in Ethiopia during non-lethal,
immobilizations (hereafter ``capture-and-release'') to explore
differences in gene expression local to the chest patch. Digital
photographs complement this dataset to assess variability in male and
female chest redness both in natural conditions and while under
anesthesia. We tested the hypothesis that male chest redness is a
condition-dependent signal used during rival assessment. We predicted
that, when compared to females, males would have: (1) redder chests and
a larger within-individual range in chest redness under natural
conditions and while under anesthesia, (2) increased expression of genes
associated with vascularization, and (3) increased expression of genes
associated with androgen and estrogen regulation.

\textbf{Materials and Methods}

\emph{Study site and subjects}

Data were collected from wild geladas in the Simien Mountains National
Park, Ethiopia as part of the Simien Mountains Gelada Research Project
(SMGRP). The SMGRP has collected behavioral, demographic, and hormonal
data from a population of wild geladas since 2006 and began conducting
annual capture-and-release campaigns in 2017 under the supervision of
licensed veterinarians and veterinary technicians. A subset of adult
geladas were anesthetized with Telinject blow darts (Telinject USA Inc.)
containing ketamine (\textasciitilde{}7.5 mg/kg) and medetomidine
(\textasciitilde{}0.04 mg/kg). Animals were monitored for temperature,
pulse, and respiration every 10 min during data collection, and sedation
was reversed with atipamezole (0.2 mg/kg). Individuals were monitored
through recovery until they returned to their social unit. All data were
collected with permission from the Ethiopian Wildlife Conservation
Authority, and all research was approved by the Institutional Animal
Care and Use Committee (for the University of Michigan IACUC:
PRO00008871 and Stony Brook University IACUC: 773805 for non-invasive
work; and for the University of Washington IACUC: 4416-01 and Arizona
State University IACUC: 20-1754R for the capture-and-release work) and
followed all laws and guidelines in Ethiopia. This research conformed to
the American Society of Primatologists/International Primatological
Society Code of Best Practices for Field Primatology.

\emph{Photo collection, measurement, and analyses}

To assess patterns in male and female chest redness in geladas, we used
objective color measurement methods for digital photographs taken under
natural conditions (i.e., while conducting daily activities like
resting, grazing, or grooming) and while animals were under anesthesia.
Under natural conditions, chest redness was measured from 144 digital
chest photographs of adult males (\emph{n} =24) and females (\emph{n}
=13) collected between 2008-2010 (range=2-16 photos per individual,
mean=4). We excluded photos taken within 10 minutes of vigorous activity
because chest redness increases with such activity in males (Bergman et
al., 2009; DeLacey et al., 2022). We only included photos taken in March
and April of each year because: (1) this was the only dataset available
for females, and (2) previous analyses have demonstrated that males
exhibit seasonal trends in chest redness that could skew male results if
selected over multiple seasons (Benítez, 2016). While the animals were
under anesthesia, chest redness was measured from photos taken at the
start of anesthetization for both males (\emph{n} =20) and females
(\emph{n} =18). For a subset of individuals (\emph{n} =13 males and
\emph{n} =3 females) we paired the photo taken at the start of
anesthetization with a second chest photo after a heat pack was applied
to one side of the chest. We include a visualization of the within
individual change in redness after temperature treatment in the
supplementary material (\textbf{Fig. S1} ), but we did not run
statistical tests due to the small sample size.

For all photos, we also photographed a color standard, the X-Rite
ColorChecker\selectlanguage{english}\textsuperscript{} Classic chart (hereafter, ``ColorChecker
chart''), to correct for variable light conditions by adjusting the
color in the photograph to the known color levels in the chart squares.
Although the digital camera brand and model was not consistent across
all photographs, f-stop, shutter speed, and white balance settings
remained consistent between chart and chest photos. JPEG format photos
were analyzed in Adobe Photoshop (Adobe Inc. 2022) using color profiles
in the RGB color space created in ColorChecker Camera Calibration
(v2.2.0; X-Rite Inc.) software designed for use with the ColorChecker
chart. We measured redness as the Red to Green Ratio (hereafter,
``Red/Green'') because the value in each RGB channel is only informative
relative to values in the other channels (Bergman \& Beehner, 2008).
Detailed methodology and instructions for photo measurement can be found
elsewhere (DeLacey et al., 2022).

To determine whether males are redder than females during natural
conditions, we constructed a linear mixed-effect model (LMM) with chest
redness as the outcome variable and sex as the predictor variable while
including ID and camera brand as random effects (R packages
\emph{lme4}(Bates et al., 2015) and\emph{lmerTest} (Kunzetsova et al.,
2017)). Next, to assess whether males have a larger range in chest
redness during natural conditions, we ran a linear regression model with
the range in chest redness within an individual (maximum R/G - minimum
R/G for each individual) as the outcome variable and the interaction
between sex and camera brand as the predictor variable. Lastly, to
determine whether males are redder than females under anesthesia at
baseline, we ran a linear regression model with chest redness as the
outcome variable and camera brand and sex as the predictor variables.

\emph{Skin biopsy collection}

We collected chest skin biopsies from 15 adult males, 15 adult females,
6 subadult males, and 2 subadult females during the annual SMGRP
capture-and-release campaigns. Adulthood was determined by the eruption
of the third molar which aligned with our phenotypic metrics of
adulthood for known individuals (McNamara \& Graber, 1975). Biopsies
were collected with a 4mm punch biopsy tool, placed vertically over the
chest patch skin and pressed and rotated in one direction to move the
punch through the skin to the subcutis. The biopsy was then removed,
placed in a 1.5 mL microcentrifuge tube with 0.5 mL RNAlater\selectlanguage{english}, and
frozen in liquid nitrogen within 6 hours. The resulting small wound was
treated with antimicrobial aluminum aerosol bandaging to stop any
bleeding and prevent infection. Upon arrival in the laboratory (still
frozen in a liquid nitrogen vapor shipper), samples were stored at -80\selectlanguage{english}
until RNA extraction.

\emph{RNA extraction, sequencing, and data processing}

DNA and RNA were extracted from chest skin biopsies using TRIzol\selectlanguage{english}
Reagent and the Zymo Quick-DNA/RNA\selectlanguage{english} Microprep Plus Kit (Zymo Research,
Irvine, CA). We quantified RNA integrity (RQN) using a Fragment Analyzer
5200 (Agilent Technology, Inc., Santa Clara, CA). RNA-sequencing
libraries were prepared using 200 ng of total RNA following a recently
developed 3'-based protocol, TM3'seq (Pallares et al., 2020). Libraries
were amplified with 16 PCR cycles. All other procedures followed the
published protocol or manufacturer recommendations. Libraries were
combined in equimolar quantities and sequenced on one lane of an
Illumina NovaSeq S4 flow cell (Illumina Inc.) of 100 bp single-end with
an average of 2.17 million reads per sample mapping to the
transcriptome. Reads were mapped to the \emph{Macaca mulatta}reference
assembly Mmul\_10 (Warren et al., 2020) using kallisto (v0.43.1) (Bray
et al., 2016). We chose the genome of \emph{Macaca mulatta} , a closely
related cercopithecoid, as its richer annotation yielded a higher number
of mapped reads compared to the \emph{Theropithecus gelada} genome
(Chiou et al., 2022).

\emph{Read count normalization}

First, we removed reads mapping to 7 genes encoding ribosomal RNA
subunits and hemoglobin genes to remove the influence of blood
contamination on the skin biopsies. While investigating RNA integrity,
we removed two samples with low RNA quality (retaining samples with RQN
\textgreater{} mean - 2 standard deviations), leaving a final sample
size of 36 geladas (\emph{n} =20 males, \emph{n} =16 females). We then
removed genes with low expression (median TPM \textless{} 10) for either
males or females and 14 Y-chromosome genes (removing sex differences
that are a product of their location on the male-specific Y-chromosome),
which resulted in 10,212 detectably expressed genes for our downstream
analysis. We normalized read counts using the \emph{voom} function in
the R package \emph{limma}(Ritchie et al., 2015).

\emph{Modeling the effect of sex on gene expression}

We used a mixed modeling approach with the R package \emph{EMMREML} to
quantify the effect of sex on gene expression while controlling for
sample collection year, RNA extraction date, RNA concentration, and RNA
quality (Akdemir \& Godfrey, 2015). We used an identity matrix as the
known covariance structure which is required for the \emph{EMMREML}
mixed modeling approach. To focus on gene expression of this putatively
sexually selected trait, we only included adults in this model (\emph{n}
=14 adult males, \emph{n} =14 adult females). We calculated the false
discovery rate (FDR) for each gene using the R package
\emph{qvalue}(Storey et al., 2022). Genes that passed a threshold of a
FDR of 20\% were considered differentially expressed between the sexes:
``male-biased'' if they were more highly expressed in males and
``female-biased'' if they were more highly expressed in females. We then
Z-transformed expression values for this set of male-biased and
female-biased genes across all 36 individuals (\emph{n=} 14 adult males,
\emph{n} =14 adult females, \emph{n} =6 subadult males, \emph{n} =2
subadult females) and averaged the Z-transformed expression levels of
these genes per individual to obtain a composite sex-biased gene
expression score for each individual. Finally, we ran a linear
regression model with expression score as the outcome variable and age
category (adult male, adult female, subadult male, subadult female) as
the predictor variable to investigate whether subadults differed from
adults in sex-biased gene expression.

\emph{Enrichment analyses}

To test our prediction that males would exhibit increased expression of
genes associated with vascularization, we conducted Gene Ontology (GO)
enrichment analyses using the R package \emph{topGO} to identify
biological processes that were enriched in genes differentially
expressed between males and females (Alexa \& Rahnenfuhrer, 2020). We
searched for terms related to angiogenesis, the formation of new blood
vessels, including the phrase ``angio'' and removed those not involved
in angiogenesis (i.e., ``lymphangiogenesis''). We compared the
standardized effect of sex for these angiogenesis genes with the
standardized effect of sex for all other detectably expressed genes with
a Kolmogorov-Smirnov test.

To test our prediction that males would have higher expression of genes
associated with estrogen and androgen regulation, we used the Online
Predicted Human Interaction Database (OPHID) to assess protein-protein
interaction (PPI) networks associated with hormone receptor proteins of
interest: ER\(\alpha\), ER\(\beta\), and AR (Brown \&
Jurisica, 2005). We used ENSEMBL orthology information queried through
the R package\emph{biomaRt} (Durinck et al., 2009) to identify
one-to-one orthologs in the human genome, then filtered and reindexed
our expression matrix to detectably expressed genes in the human genome.
We retained a total of 8,255 human genes for analysis. Next we ran a
query in OPHID to identify the set of human genes involved in PPI
networks with each hormone receptor protein and used \emph{biomaRt} to
convert genes names from UniProt to Ensembl (The UniProt Consortium,
2021). We found the genes associated with PPI networks for
ER\(\alpha\), ER\(\beta\), and AR and compared the
standardized effect of sex for these genes with the standardized effect
of sex for all other detectably expressed genes with human orthologs
with a Kolmogorov-Smirnov test.

\textbf{Results}

\emph{Chest redness in male and female geladas}

Under natural conditions (i.e., not anesthetized), chest redness for
males and females overlapped substantially, with males displaying only
marginally redder chests than females (\(\beta\)=0.11, \emph{P}
=0.06). However, males had a wider range of chest redness within
individuals compared to females (\(\beta\)=0.63, \emph{P}
=0.007, \textbf{Fig. 1} ). While under anesthesia, male geladas did not
have redder chests than females (\(\beta\)=0.04, \emph{P}
=0.40, \textbf{Fig. 2).}

\emph{Sex differences in gene expression}

We then quantified how sex was associated with chest skin gene
expression while including sample collection year in the model (year was
significantly associated with both the first and second principal
component of gene expression; PC1: year \(\beta\)=0.39,
\emph{P} =0.02; PC2: year \(\beta\)=-0.22, \emph{P} =0.003,
\textbf{Table S1, Fig. S2} ). Males and females differed along the first
principal component of gene expression while including year as a
predictor variable (PC1: sex\(\beta\)=-0.39, \emph{P} =0.01,
\textbf{Fig. S3} ; PC2: sex\(\beta\)=-0.13, \emph{P} =0.049).
The results of the principal component analysis did not change when we
removed genes located on sex chromosomes and analyzed only those found
on autosomes (\textbf{Fig. S4} ).

At the level of the individual genes, we found that 10.5\% of the 10,212
detectably expressed genes exhibited significant differential expression
across males and females (\emph{n} genes=1,068, FDR\textless{}20\%), in
a model controlling for the effects of RQN, RNA concentration, RNA
extraction date, and year of sample collection. Of the 1,068
differentially expressed genes, 201 genes were female-biased and 867
genes were male-biased. The average standardized sex-bias gene
expression level for each individual illustrates that subadult males
exhibited an intermediate gene expression pattern that differed from
adult males (\(\beta\)=-0.66, \emph{P} =0.003) and adult
females (\(\beta\)=0.45, \emph{P} =0.04; \textbf{Fig. 3B).} The
sample size for subadult females (\emph{n} =2) was too small to draw
conclusions about sex-bias gene expression trends in this age category.

\emph{Sex-biased genes involved in vascularization}

In line with our prediction that males would exhibit increased
expression of genes associated with vascularization, we found that genes
more highly expressed in males were enriched for biological processes
associated with angiogenesis (K-S Test: D=0.19,\emph{P}
=7.25x10\textsuperscript{-7}; \textbf{Fig. 4} ). Additionally, these
genes in males were also enriched for biological processes associated
with blood pressure regulation and blood vessel maintenance (K-S Test:
D=0.19, \emph{P} =2.59x10\textsuperscript{-5}; \textbf{Fig. S5} ).
Contrary to our predictions, we did not find evidence for increased
expression of genes associated with estrogen or androgen regulation in
males -- including no evidence for indirect effects through
protein-protein networks: there was no enrichment for genes involved in
PPI networks for ER\(\alpha\) (K-S Test: D=0.04,\emph{P} =0.07),
ER\(\beta\) (K-S Test: D=0.05, \emph{P} =0.17), or AR (K-S
Test: D=0.04, \emph{P} =0.46). For subset of individuals for which we
had matched skin biopsies and chest redness at baseline while under
anesthesia (\emph{n} =10 males, \emph{n} =8 females), we did not find a
correlation between the average standardized expression level of genes
involved in PPI networks for ER\(\alpha\), ER\(\beta\),
or AR and chest redness within males or within females (\textbf{Fig.
S6).}

\textbf{Discussion}

We sampled chest skin biopsies from wild geladas to directly measure
putative mechanisms underlying a uniquely evolved sexual signal. We
found that male and female geladas showed a substantial overlap in chest
redness under natural and unmanipulated conditions, but males exhibited
a wider within-individual range in baseline redness under natural
conditions (\textbf{Figs. 1-2)} . Further, subadults displayed an
intermediate gene-expression pattern from adult males and adult females
(\textbf{Fig. 3} ). We also found sex differences in gene expression,
where higher expression in males was associated with angiogenesis, blood
pressure, and blood vessel maintenance, suggesting that blood flow and
vascularization may underlie sex differences in this sexually-selected
signal (\textbf{Fig. 4} ). Contrary to our predictions, genes encoding
proteins that interact with androgen or estrogen were not more highly
expressed in males. Together, these results suggest that males may have
more variable chest redness due to increased blood flow and blood vessel
branching in the chest skin.

Chest photograph measurements revealed an overlap in redness between the
sexes at baseline in both natural and anesthetized conditions, but males
overall exhibited a wider within-individual range in redness. Selection
on an ornamentation trait in one sex can create a correlated response in
the opposite sex within a species (e.g., male and female coloration are
highly correlated in passerines), suggesting that changes in one sex can
be constrained by changes in the other sex (Dale et al., 2015; Poissant
et al., 2010; Potti \& Canal, 2011). In geladas, this overlap in chest
redness between the sexes could simply be the result of a positive
genetic correlation. Alternatively, as male and female color traits
function differently, female chest redness in geladas could have
continued to evolve under a different selective pressure (Dale et al.,
2015; Tobias et al., 2012), and the overlap in redness could be caused
by each sex using the chest patch to communicate different signals. In
males, chest redness varies among males by status (Bergman et al., 2009)
and within males by activity level (DeLacey et al., 2022), suggesting
the chest patch aids in male-male competition. Females have instead
co-opted chest redness to communicate reproductive status through
hormonal and blood signaling as they have the reddest chests late in
gestation when estrogen levels and blood volume are the highest (Hytten,
1985; Roberts et al., 2017). In addition to chest color variation,
gelada females exhibit sexual swellings consisting of cutaneous vesicles
surrounding the chest region where vesicle turgidity varies across the
ovarian cycle, suggesting sexual swellings work in tandem with chest
redness to signal a different aspect of reproductive state (Roberts et
al., 2017).

The sex difference in gelada chest skin gene expression aligns with
findings in humans where a wide variety of tissues exhibit small effects
of sex on gene expression (Lopes-Ramos et al., 2020; Oliva et al.,
2020). However, small expression changes have been shown to have large
phenotypic effects, particularly in the manifestation of disease
(Khramtsova et al., 2019). Within primates, sex-biased gene expression
has also been detected in rank-related genes, immune regulation, and
aging in wild baboons through blood sampling (Anderson et al., 2021; Lea
et al., 2018). The magnitude of sex-biased gene expression has been
shown to increase across development with the greatest differences in
adult tissue (Mank et al., 2010; Perry et al., 2014), and differences
are particularly exaggerated in sexually dimorphic tissues such as
elaborated weaponry (Zinna et al., 2018). Subadult geladas showed an
intermediate pattern of gene expression between that of adult males and
females suggesting that gene expression differences increase at sexual
maturity when sexually selected signals develop for mate acquisition.
Further, an analysis of an avian clade that found the degree of sexual
selection predicts the proportion of male-biased gene expression
(Harrison et al., 2015). This finding is consistent with gelada chest
skin, as we found more genes that were more highly expressed in males
compared to genes that were more highly expressed in females in this
species with a high male reproductive skew.

\selectlanguage{english}\selectlanguage{english}Male geladas expressed genes associated with angiogenesis, blood
pressure regulation, and blood vessel maintenance more highly than
females. The mechanism of increased blood vessel branching in the chest
skin may indicate chest redness is a condition-dependent signal where
the differential costs of signaling based on current body condition
inhibit low-quality males from investing in the signal (Grafen, 1990;
Penn \& Szamado, 2020). We propose energy balance and heat loss as
possible costs associated with producing a red chest. Male geladas may
develop more extensive blood vessel branching in the skin compared to
females through engaging in vocal displays. Post-display chest redness
increases with display rate per hour in gelada males (Benitez, 2016)
which suggests that after frequent activity has built up vascular
networks, an instance of increased blood flow will prompt a larger
increase in chest redness. Among males, leaders spend less time resting,
more time engaging in low-intensity aggression, and produce more calls
per vocal display bout compared to bachelors (Benitez et al., 2016;
Perlman, 2021). The physical effort required to engage in aggression and
vocal displays may contribute to ensuring only high-quality males in
good body condition have red chests (if it is difficult to break the
link between exertion and vascularization). Further, redder chests have
higher surface skin temperatures which indicates the increased blood
flow to this area may also result in heat loss in the cold,
high-altitude environment of the Simien Mountains (DeLacey et al.,
2022). These potential constraints could provide an avenue for chest
redness to communicate current body condition to potential rivals.

Contrary to our predictions, males did not have increased expression of
genes associated with androgen and estrogen regulation in the chest
skin. Although this result could simply indicate that androgen and
estrogen regulation are not important to sex differences in chest
redness, it could also (1) indicate both males and females use the same
androgen and estrogen regulation pathways in the chest skin or (2) be a
product of sequencing skin biopsies in particular as sex-biased genes
have tissue-specific expression profiles (Lopes-Ramos et al., 2020; Yang
et al., 2006). Perhaps we would detect more sex differences in
expression in brain regions involved in the regulation of hormone
secretion rather than the target tissue (Becker et al., 2007).
Additionally, we measured the expression of genes that interact with
estrogen and androgen receptors, but circulating hormones such as
testosterone or changes in androgen receptor density may play a larger
role in regulating redness in primates (Dixson, 1983; Rhodes et al.,
1997; Setchell \& Dixson, 2001). As yet, no relationship has been
identified between testosterone and chest redness in adult male geladas
(DeLacey and Beehner, unpublished data). This may be because this
putative signal is not testosterone dependent, or because we are only
able to measure fecal androgen metabolite levels (capturing an averaged
level of the hormone over the past day) rather than actual circulating
testosterone levels. Further, we may not detect a relationship between
testosterone and chest redness because estrogens directly regulate chest
redness and testosterone only indirectly influences redness through
aromatization to estrogens.

\emph{Limitations}

We only collected skin biopsies from the chest to limit the number of
biopsies collected per individual during the capture-and-release.
However, comparing chest skin gene expression to gene expression in a
non-sexual skin area would allow us to assess (1) whether higher male
expression associated with blood flow and vascularization is unique to
chest skin biopsies and (2) whether both male and female chests are
hotspots for expression of androgen and estrogen related genes. While we
were able to identify interesting sex differences, we were not able to
assess gene expression differences between male status categories
because anesthetizing a leader male to collect a biopsy sample would put
them at risk of losing their status. Lastly, we restricted our male
chest photo dataset to only photos taken in March and April to match our
more limited female chest photo dataset. This facilitated a more direct
comparison between the sexes, but may have reduced our ability to
identify more within-individual variation, particularly in males.

\emph{Conclusion}

Here, we provide mechanistic evidence that gelada chest redness is
linked to increased blood flow near the surface of the skin,
specifically through increased blood vessel branching in the chest skin
in males. Although we did not detect a sex difference in chest skin gene
expression related to androgen and estrogen regulation, future research
into the correlation between fluctuations in fecal testosterone
metabolites and chest redness within males will help determine whether
another aspect of hormonal regulation is involved in chest redness
signaling. Going forward, we hope to better understand whether increased
angiogenesis creates a current-condition signal for males in the cold,
high-altitude environment of the Ethiopian highlands by leveraging
biomarkers of energy balance or heat loss.

\textbf{Acknowledgements}

We are grateful to the Ethiopian Wildlife Conservation Authority for
granting us permission to conduct this research. We also want to thank
the many staff and wardens (Berhanu Gebre, Maru Biadglegn, Abebaw Azanaw
Haile) of the Simien Mountains National Park, our Ethiopian staff
(Esheti Jejaw, Ambaye Fanta, Setey Girmay, Yeshi Dessie, Dereje Bewket,
Atirsaw Aduga, Tariku W/Aregay, Shifarew Asrat), our research assistants
in the field (Clay Wilton, Julie Jarvey, Levi Morris, Tara Regan,
Caitlin Hawley, Peter Clark, Evan Sloan, Megan Gomery, Laura Morrissey,
Emily Downey, Eddy Wild, Liz Babbitt, Maddie Melton, Anna Cryer).

This work was supported by the National Science Foundation (BCS-2041542,
BCS-0715179, BCS-1732231, BCS-1723237, BCS-2010309, BCS-1723228,
IOS-1255974, IOS-1854359), the Leakey Foundation (AWD015438), the Leakey
Foundation Baldwin Award (AWD012312), the National Geographic Society
(NGS-8100--06, NGS-8989--11, NGS-1242, and NGS-50409R-18), the Fulbright
Scholars Program, Nacey Maggioncalda Foundation, Sigma Xi, the
University of Michigan, Arizona State University, University of
Washington, and Stony Brook University.

\textbf{References}

Akdemir, D., \& Godfrey, O. U. (2015). \emph{EMMREML: Fitting Mixed
Models with Known}

\emph{Covariance Structures} . R package version 3.1,

https://cran.r-project.org/web/packages/EMMREML/index.html

Alexa, A., \& Rahnenfuhrer, J. (2020). \emph{topGO: Enrichment Analysis
for Gene Ontology.} R

package version 2.50.0.

Anderson, J. A., Johnston, R. A., Lea, A. J., Campos, F. A., Voyles, T.
N., Akinyi, M. Y.,

Alberts, S. C., Archie, E. A., \& Tung, J. (2021). High social status
males experience

accelerated epigenetic aging in wild baboons. \emph{eLife,} 10.
https://doi.org/10.7554/eLife.66128

Bates, D., Machler, M., Bolker, B., \& Walker, S. (2015). Fitting Linear
Mixed-Effects Models

Using lme4. \emph{Journal of Statistical Software}, 67(1), 1--48.
https://doi.org/10.18637/jss.v067.i01

Becker, J. B., Berkley, K. J., Geary, N., Hampson, E., Herman, J. P., \&
Young, E. (2007). \emph{Sex}

\emph{Differences in the Brain: From Genes to Behavior.} Oxford
University Press.

Benitez, M. E. (2016). \emph{Sizing up strangers: sexual selection and
vocal signals in gelada males}

{[}Doctoral dissertation, University of Michigan{]}. UMich Campus
Repository. https://hdl.handle.net/2027.42/135766

Benitez, M. E., Le Roux, A., Fischer, J., Beehner, J. C., \& Bergman, T.
J. (2016). Acoustic and

temporal variation in gelada (\emph{Theropithecus gelada} ) loud calls
advertise male quality.

\emph{International Journal of Primatology}, 37(4-5), 568--585.
https://doi.org/10.1007/s10764-016-9922-0

Bergman, T. J. (2010). Experimental evidence for limited vocal
recognition in a wild primate:

implications for the social complexity hypothesis. \emph{Proceedings of
the Royal Society B}, 277, 3045--3053.
https://doi:10.1098/rspb.2010.0580

Bergman, T. J., \& Beehner, J. C. (2008). A simple method for measuring
colour in wild animals:

validation and use on chest patch colour in geladas (\emph{Theropithecus
gelada}). \emph{Biological Journal of the Linnean Society}, 94(2),
231--240. https://doi.org/10.1111/j.1095-8312.2008.00981.x

Bergman, T. J., Ho, L., \& Beehner, J. C. (2009). Chest color and social
status in male geladas

(\emph{Theropithecus gelada} ). \emph{International Journal of
Primatology} , 30(6), 791--806.

https://doi.org/10.1007/s10764-009-9374-x

Bradbury, J. W., \& Vehrencamp, S. L. (2011). \emph{Principles of animal
communication: Vol. 2nd}

\emph{Edition} . Sinauer Associates.

Bray, N. L., Pimentel, H., Melsted, P., \& Pachter, L. (2016).
Near-optimal probabilistic RNA-seq

quantification. \emph{Nature Biotechnology} , 34(5), 525--527.
https://doi.org/10.1038/nbt.3519

Brown, K. R., \& Jurisica, I. (2005). Online predicted human interaction
database.

\emph{Bioinformatics} , 21(9), 2076--2082.
https://doi:10.1093/bioinformatics/bti273

Chiou, K. L., Janiak, M. C., Schneider-Crease, I. A., Sen, S., Ayele,
F., Chuma, I. S., Knauf, S.,

Lemma, A., Signore, A. V., D'Ippolito, A. M., Abebe, B., Haile, A. A.,
Kebede, F.,

Fashing, P. J., Nguyen, N., McCann, C., Houck, M. L., Wall, J. D.,
Burrell, A. S., \ldots{} Snyder-Mackler, N. (2022). Genomic signatures
of high-altitude adaptation and chromosomal polymorphism in
geladas.\emph{Nature Ecology \& Evolution}.
https://doi.org/10.1038/s41559-022-01703-4

Dale, J., Dey, C. J., Delhey, K., Kempenaers, B., \& Valcu, M. (2015).
The effects of life history

and sexual selection on male and female plumage
colouration.\emph{Nature} , 527, 367--

370. https://doi.org/10.1038/nature15509

DeLacey, P. M., Perlman, R. F., Sen, S., Schneider-Crease, I., Chiou, K.
L., Lemma, A., Ayele,

F., Higham, J. P., Lu, A., Snyder-Mackler, N., Beehner, J. C., \&
Bergman, T. J. (2022).

Assessing male gelada chest patches: color measurement and physiological
mechanisms.

\emph{Mammalian Biology.}https://doi.org/10.1007/s42991-021-00211-5

Dixson, A. F. (1983). Observations on the evolution and behavioral
significance of ``sexual

skin'' in female primates. In J.S. Rosenblatt, R.A. Hinde, C. Beer, \&
M.-C. Busnel (Eds.), \emph{Advances in the Study of Behavior} (Vol. 13,
pp. 63--106). Academic Press.

Durinck, S., Spellman, P. T., Birney, E., \& Huber, W. (2009). Mapping
identifiers for the

integration of genomic datasets with the R/Bioconductor package biomaRt.

\emph{Nature}

\emph{Protocols}, 4(8), 1184--1191.
https://doi.org/10.1038/nprot.2009.97

Emlen, D. J., Warren, I. A., Johns, A., Dworkin, I., \& Lavine, L. C.
(2012). A mechanism of

extreme growth and reliable signaling in sexually selected ornaments and
weapons. \emph{Science,} 337(6096), 860--864.
https://doi.org/10.1126/science.1224286

Enstrom, D. A., Ketterson, E. D., \& Val Nolan, J. R. (1997).
Testosterone and mate choice in

the dark-eyed junco. \emph{Animal Behaviour}, 54(5), 1135--1146.
https://doi.org/10.1006/anbe.1997.0555

Fisher, R. A. (1915). The evolution of sexual preference. \emph{The
Eugenics Review} , 7(3), 184--192.

Grafen, A. (1990). Biological signals as handicaps. \emph{Journal of
Theoretical Biology} , 144(4), 517--

546. https://doi.org/10.1016/S0022-5193(05)80088-8

Harrison, P. W., Wright, A. E., Zimmer, F., Dean, R., Montgomery, S. H.,
Pointer, M. A., \&

Mank, J. E. (2015). Sexual selection drives evolution and rapid turnover
of male gene

expression. \emph{Proceedings of the National Academy of Sciences of the
United States of America}, 112(14),
4393--4398.\emph{https://doi.org/10.1073/pnas.1501339112}

Hau, M., Wikelski, M., Soma, K. K., \& Wingfield, J. C. (2000).
Testosterone and year-round

territorial aggression in a tropical bird. \emph{General and Comparative
Endocrinology} , 117(1),

20--33. https://doi:10.1006/gcen.1999.7390

Hau, M., \& Wingfield, J. C. (2011). Hormonally-regulated trade-offs:
evolutionary variability

and phenotypic plasticity in testosterone signaling pathways. In

\emph{Mechanisms of Life}

\emph{History Evolution: The Genetics and Physiology of Life History
Traits and Trade-Offs}

(pp. 349--361). Oxford Academic.

Hytten, F. (1985). Blood volume changes in normal
pregnancy.\emph{Clinics in Haematology} , 14(3),

601--612. https://doi.org/10.1016/S0308-2261(21)00496-3

Karubian, J., Lindsay, W. R., Schwabl, H., \& Webster, M. S. (2011).
Bill coloration, a flexible

signal in a tropical passerine bird, is regulated by social environment
and androgens.

\emph{Animal Behaviour} , 81(4), 795--800.
https://doi.org/10.1016/j.anbehav.2011.01.012

Kenney, W. L., \& Johnson, J. M. (1992). Control of skin blood flow
during exercise. \emph{Medicine}

\emph{\& Science in Sports \& Exercise} , 24(3), 303--312.
https://doi.org/10.1249/00005768- 199203000-00005

Ketterson, E. D., \& Nolan, V., Jr. (1999). Adaptation, Exaptation, and
Constraint: A Hormonal

Perspective. \emph{The American Naturalist} , 154(S1), S4--S25.
https://doi:10.1086/303280

Khramtsova, E. A., Davis, L. K., \& Stranger, B. E. (2019). The role of
sex in the genomics of

human complex traits. \emph{Nature Reviews Genetics} , 20(3), 173--190.

https://doi.org/10.1038/s41576-018-0083-1

Kunzetsova, A., Brockhoff, P., \& Christensen, R. (2017). lmerTest
package: Tests in linear

mixed effect models. \emph{Journal of Statistical Software}, 82, 1--26.
https://doi.org/10.18637/jss.v082.i13

Lea, A. J., Akinyi, M. Y., Nyakundi, R., Mareri, P., Nyundo, F.,
Kariuki, T., Alberts, S. C.,

Archie, E. A., \& Tung, J. (2018). Dominance rank-associated gene
expression is

widespread, sex-specific, and a precursor to high social status in wild
male baboons.

\emph{Proceedings of the National Academy of Sciences of the United
States of America} ,

115(52), E12163--E12171.\emph{https://doi.org/10.1073/pnas.1811967115}

Lopes-Ramos, C. M., Chen, C.-Y., Kuijjer, M. L., Paulson, J. N.,
Sonawane, A. R., Fagny, M.,

Platig, J., Glass, K., Quackenbush, J., \& DeMeo, D. L. (2020). Sex
Differences in Gene

Expression and Regulatory Networks across 29 Human Tissues. \emph{Cell
Reports}, 31(12), 107795. https://doi.org/10.1016/j.celrep.2020.107795

Mank, J. E., Nam, K., Brunstrom, B., \& Ellegren, H. (2010). Ontogenetic
complexity of sexual

dimorphism and sex-specific selection. \emph{Molecular Biology and
Evolution,} 27(7), 1570--

1578. https://doi.org/10.1093/molbev/msq042

Marty, J. S., Higham, J. P., Gadsby, E. L., \& Ross, C. (2009).
Dominance, Coloration, and Social

and Sexual Behavior in Male Drills \emph{Mandrillus leucophaeus} .

\emph{International Journal of}

\emph{Primatology,} 30(6), 807.
https://doi.org/10.1007/s10764-009-9382-x

Maynard Smith, J. (1982). \emph{Evolution and the theory of
games.}Cambridge University Press.

https://doi.org/10.1017/CBO9780511806292

Maynard Smith, J., \& Harper, D. (2003). \emph{Animal Signals} . Oxford
University Press.

Maynard Smith, J., \& Harper, D. G. C. (1995). Animal Signals: Models
and Terminology.

\emph{Journal of Theoretical Biology} , 177(3), 305--311.
https://doi.org/10.1006/jtbi.1995.0248

Maynard Smith, J., \& Parker, G. A. (1976). The logic of asymmetric
contests. \emph{Animal Behaviour,}

24(1), 159--175. https://doi.org/10.1016/S0003-3472(76)80110-8

McNamara, J. A., Jr, \& Graber, L. W. (1975). Mandibular growth in the
rhesus monkey (\emph{Macaca}

\emph{mulatta}). \emph{American Journal of Physical Anthropology},
42(1), 15--24. https://doi.org/10.1002/ajpa.1330420104

Molinari, C., Battaglia, A., Grossini, E., Mary, D. A. S. G.,
Vassanelli, C., \& Vacca, G. (2002).

The effect of testosterone on regional blood flow in prepubertal
anaesthetized pigs. \emph{The}

\emph{Journal of Physiology} , 543(Pt 1), 365--372.
https://doi:10.1113/jphysiol.2002.022756

Muehlenbein, M. P., \& Bribiescas, R. G. (2005). Testosterone-mediated
immune functions and

male life histories. \emph{American Journal of Human Biology} 17(5),
527--558. https://doi.org/10.1002/ajhb.20419

Muehlenbein, M. P., Cogswell, F. B., James, M. A., Koterski, J., \&
Ludwig, G. V. (2006).

Testosterone correlates with Venezuelan equine encephalitis virus
infection in macaques.

\emph{Virology Journal} , 3, 19. https://doi.org/10.1186/1743-422X-3-19

Oliva, M., Munoz-Aguirre, M., Kim-Hellmuth, S., Wucher, V., Gewirtz, A.
D. H., Cotter, D. J.,

Parsana, P., Kasela, S., Balliu, B., Vinuela, A., Castel, S. E.,
Mohammadi, P., Aguet, F.,

Zou, Y., Khramtsova, E. A., Skol, A. D., Garrido-Martin, D., Reverter,
F., Brown, A., \ldots{}

Stranger, B. E. (2020). The impact of sex on gene expression across
human tissues.

\emph{Science} , 369(6509). https://doi.org/10.1126/science.aba3066

Pallares, L. F., Picard, S., \& Ayroles, J. F. (2020). TM3'seq: A
Tagmentation-Mediated 3'

Sequencing Approach for Improving Scalability of RNAseq Experiments. G3,
10(1),

143--150. https://doi.org/10.1534/g3.119.400821

Pappano, D. J., \& Beehner, J. C. (2014). Harem-holding males do not
rise to the challenge:

androgens respond to social but not to seasonal challenges in wild
geladas. \emph{Royal Society}

\emph{Open Science} , 1(1), 140081. https://doi.org/10.1098/rsos.140081

Penn, D. J., \& Szamado, S. (2020). The Handicap Principle: how an
erroneous hypothesis

became a scientific principle. \emph{Biological Reviews of the Cambridge
Philosophical}

\emph{Society} , 95, 267--290. https://doi.org/10.1111/brv.12563

Perlman, R. F. (2021). \emph{The energetics of male reproductive
strategies in geladas.} {[}Doctoral

Dissertation, Stony Brook University{]}.

Perry, J. C., Harrison, P. W., \& Mank, J. E. (2014). The ontogeny and
evolution of sex-biased

gene expression in Drosophila melanogaster. \emph{Molecular Biology and
Evolution} , 31(5),

1206--1219. https://doi:10.1093/molbev/msu072

Plant, T. M., \& Zeleznik, A. J. (Eds.). (2014). \emph{Knobil and
Neill's Physiology of Reproduction} .

Academic Press.

Poissant, J., Wilson, A. J., \& Coltman, D. W. (2010). Sex-specific
genetic variance and the

evolution of sexual dimorphism: a systematic review of cross-sex genetic
correlations.

\emph{Evolution: International Journal of Organic Evolution}, 64(1),
97--107. https://doi.org/10.1111/j.1558-5646.2009.00793.x

Potti, J., \& Canal, D. (2011). Heritability and genetic correlation
between the sexes in a songbird

sexual ornament. \emph{Heredity} , 106(6), 945--954.
https://doi.org/10.1038/hdy.2010.142

Rhodes, L., Argersinger, M. E., Gantert, L. T., Friscino, B. H., Hom,
G., Pikounis, B., Hess, D.

L., \& Rhodes, W. L. (1997). Effects of administration of testosterone,

dihydrotestosterone, oestrogen and fadrozole, an aromatase inhibitor, on
sex skin colour

in intact male rhesus macaques. \emph{Journal of Reproduction and
Fertility} , 111(1), 51---57.

https://doi.org/10.1530/jrf.0.1110051

Ritchie, M. E., Phipson, B., Wu, D., Hu, Y., Law, C. W., Shi, W., \&
Smyth, G. K. (2015). limma

powers differential expression analyses for RNA-sequencing and
microarray studies.

\emph{Nucleic Acids Research} , 43(7), e47.
https://doi.org/10.1093/nar/gkv007

Roberts, E. K., Lu, A., Bergman, T. J., \& Beehner, J. C. (2017). Female
reproductive parameters

in wild geladas \emph{(Theropithecus gelada} ). \emph{International
Journal of Primatology,} 38(1), 1--

20. https://doi.org/10.1007/s10764-016-9939-4

Setchell, J. M., \& Dixson, A. F. (2001). Changes in the secondary
sexual adornments of male

mandrills (\emph{Mandrillus sphinx} ) are associated with gain and loss
of alpha status.

\emph{Hormones and Behavior} , 39(3), 177--184.
https://doi.org/10.1006/hbeh.2000.1628

Storey, J. D., Bass, A. J., Dabney, A., \& Robinson, D. (2022).

\emph{qvalue: Q-value estimation for}

\emph{false discovery rate control.} R package version 2.30.0,
http://github.com/jdstorey/qvalue

The UniProt Consortium (2021). UniProt: the universal protein
knowledgebase in 2021. \emph{Nucleic}

\emph{Acids Research,} 49(D1), D480--D489.
https://doi.org/10.1093/nar/gkaa1100

Tobias, J. A., Montgomerie, R., \& Lyon, B. E. (2012). The evolution of
female ornaments and

weaponry: social selection, sexual selection and ecological competition.

\emph{Philosophical}

\emph{Transactions of the Royal Society B: Biological Sciences} ,
367(1600),

2274--2293. https://doi.org/10.1098/rstb.2011.0280

Trivers, R. L. (1972). Parental investment and sexual selection. In B.
G. Campbell (Ed.), \emph{Sexual}

\emph{selection and the descent of man} , \emph{1871-1971} (pp.
136--179). Routledge.

Tung, J., Alberts, S. C., \& Wray, G. A. (2010). Evolutionary genetics
in wild primates:

combining genetic approaches with field studies of natural populations.

\emph{Trends in}

\emph{Genetics}, 26(8), 353--362.
https://doi.org/10.1016/j.tig.2010.05.005

Warren, W. C., Harris, R. A., Haukness, M., Fiddes, I. T., Murali, S.
C., Fernandes, J., Dishuck,

P. C., Storer, J. M., Raveendran, M., Hillier, L. W., Porubsky, D., Mao,
Y., Gordon, D.,

Vollger, M. R., Lewis, A. P., Munson, K. M., DeVogelaere, E., Armstrong,
J., Diekhans, M., \ldots{} Eichler, E. E. (2020). Sequence diversity
analyses of an improved rhesus macaque genome enhance its biomedical
utility. \emph{Science}, 370(6523), eabc6617.
https://doi.org/10.1126/science.abc6617

Weaver, R. J., Koch, R. E., \& Hill, G. E. (2017). What maintains signal
honesty in animal colour

displays used in mate choice? \emph{Philosophical Transactions of the
Royal Society}

\emph{B: Biological Sciences} , 372(1724), 20160343.
https://doi.org/10.1098/rstb.2016.0343

Webb, C. M., McNeill, J. G., Hayward, C. S., de Zeigler, D., \& Collins,
P. (1999). Effects of

testosterone on coronary vasomotor regulation in men with coronary heart
disease.

\emph{Circulation} , 100(16),
1690--1696.\emph{https://doi.org/10.1161/01.CIR.100.16.1690}

Weir, L. K., Grant, J. W. A., \& Hutchings, J. A. (2011). The influence
of operational sex ratio on

the intensity of competition for mates\emph{. The American Naturalist} ,
177(2), 167--176.

https://doi.org/10.1086/657918

Yang, X., Schadt, E. E., Wang, S., Wang, H., Arnold, A. P.,
Ingram-Drake, L., Drake, T. A., \&

Lusis, A. J. (2006). Tissue-specific expression and regulation of
sexually dimorphic

genes in mice. \emph{Genome Research}, 16(8), 995--1004.
https://doi.org/10.1101/gr.5217506

Zahavi, A. (1977). The cost of honesty (further remarks on the handicap
principle). \emph{Journal of}

\emph{Theoretical Biology} , 67(3), 603--605.
https://doi.org/10.1016/0022-5193(77)90061-3

Zinna, R., Emlen, D., Lavine, L. C., Johns, A., Gotoh, H., Niimi, T., \&
Dworkin, I. (2018).

Sexual dimorphism and heightened conditional expression in a sexually
selected weapon

in the Asian rhinoceros beetle. \emph{Molecular Ecology} , 27(24),
5049--5072.

https://doi.org/10.1111/mec.14907

\textbf{Author Contributions}

N.S.-M., T.J.B., J.C.B. and P.M.D. conceived the research and designed
the study. S.S., I.A.S.-C., K.L.C., A. Lemma, F.A., A. Lu, T.J.B.,
J.C.B., and N.S.-M. collected field gelada samples and data, facilitated
by A. A. H. P.M.D. performed photo analyses. P.M.D., K.L.C., and N.S.-M.
generated genomic data. P.M.D. and N.S.-M. performed genomic analyses.
P.M.D., J.C.B., and N.S.-M. wrote the paper. All authors revised and
approved the final manuscript.

\textbf{Conflicts of interest}

On behalf of all the authors, the corresponding author states that there
is no conflict of interest.

\textbf{Data Accessibility Statement}

Data and code are available at
https://github.com/GeladaResearchProject/DeLacey\_et\_al\_chest\_skin\_transcription\_2023

\textbf{Benefit-Sharing Statement}

We developed a research collaboration with scientists from Ethiopia who
have assisted us with gelada capture-and-release campaigns. All
collaborators are included as co-authors. The results of the research
will be shared with the Simien Mountains National Park staff and guides
in the local communities surrounding the park.

\textbf{Figures}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. Adult males had a wider range in chest redness within
individuals compared to females under natural conditions.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. Minimal sex differences in chest redness at baseline
while under anesthesia} . Adult males did not have redder chests than
females while under anesthesia prior to heat application.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3. Subadult males exhibited intermediate expression of
sex-biased genes between adult males and adult females.} Mean,
normalized expression of the 1,068 sex-biased genes for each individual
across all age categories.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4. Genes more highly expressed in males were enriched for
biological processes associated with angiogenesis.} Beeswarm dots
represent individual genes within each subcategory of the angiogenesis
gene ontology category, and the solid vertical lines reflect the average
standardized effect of sex for genes for each subcategory. The solid red
line represents the median standardized effect size of sex for all genes
not in angiogenesis-associated categories.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/fig4/fig4}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

