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Cool sperm: why some placental mammals have a scrotum
B. G. LOVEGROVE
School of Life Sciences, University of KwaZulu-Natal, Scottsville, South Africa
Keywords:
body temperature;
mammals;
scrotum;
spermatogenesis;
testes.
Abstract
Throughout the Cenozoic, the fitness benefits of the scrotum in placental
mammals presumably outweighed the fitness costs through damage, yet a
definitive hypothesis for its evolution remains elusive. Here, I present an
hypothesis (Endothermic Pulses Hypothesis) which argues that the evolu-
tion of the scrotum was driven by Cenozoic pulses in endothermy, that is,
increases in normothermic body temperature, which occurred in Boreothe-
ria (rodents, primates, lagomorphs, carnivores, bats, lipotyphylans and
ungulates) in response to factors such as cursoriality and climate adaptation.
The model argues that stabilizing selection maintained an optimum temper-
ature for spermatogenesis and sperm storage throughout the Cenozoic at the
lower plesiomorphic levels of body temperature that prevailed in ancestral
mammals for at least 163 million years. Evolutionary stasis may have been
driven by reduced rates of germ-cell mutations at lower body temperatures.
Following the extinction of the dinosaurs at the Cretaceous–Palaeogeneboundary 65.5 mya, immediate pulses in endothermy occurred associated
with the dramatic radiation of the modern placental mammal orders. The
fitness advantages of an optimum temperature of spermatogenesis out-
weighed the potential costs of testes externalization and paved the way for
the evolution of the scrotum. The scrotum evolved within several hundred
thousand years of the K-Pg extinction, probably associated initially with the
evolution of cursoriality, and arguably facilitated mid- and late Cenozoic
metabolic adaptations to factors such as climate, flight in bats and sociality
in primates.
Introduction
The evolution of the mammalian scrotum is one of the
great enigmas of evolutionary biology (Wislocki, 1933;
Ruibal, 1957; Freeman, 1990; Gallup et al., 2009; Kleis-
ner et al., 2010). Dangled between the legs and home
to the male’s genetic arsenal, the testes are highly vul-
nerable to physical and thermal damage. Surprisingly,
though, a definitive explanation for the evolution of
the scrotum remains elusive.
The scrotum is a sac-like thermoregulatory structure
that houses the testes at a temperature lower than the
core body temperature (Tb; Moore, 1926; Wislocki,
1933; Ruibal, 1957; Setchell, 1998). The cremasteric
muscles in the scrotum contract and relax to draw the
testes closer to or allow them to dangle further away
from the body in order to maintain the testes at an
‘optimal’ temperature for spermatogenesis (sensu
Moore, 1926; Tsperm) and sperm storage of 34–35 °C(Setchell, 1998; Gallup et al., 2009; Mawyer et al.,
2012). In humans, the scrotal temperature is main-
tained about 2.7 °C lower than the Tb (Momen et al.,
2010).
Bedford (1978a) argued that the ‘prime mover in
evolution of scrotum’ was the cauda epididymis and
that it is the regulated temperature of sperm storage,
maturation and capacitation (see Bedford, 2004 for
review) that poses the fitness benefit rather than sper-
matogenesis within the testes. He maintains that ‘. . .tes-ticular descent is seen as a merely mechanistic event
which enables the cauda epididymis to project from the
body, but has no significance for the biological function
of the testis as such. . .’ (Bedford, 1978a). By this
reasoning, it would seem that the pendulant testis acts
Correspondence: B. G. Lovegrove, School of Life Sciences, University of
KwaZulu-Natal, Private Bag X01, Scottsville 3209, South Africa.
Tel.: +27-33-2605113; fax: +27-33-2605105;
e-mail: [email protected]
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801JOURNAL OF EVOLUT IONARY B IO LOGY ª 20 1 4 EUROPEAN SOC I E TY FOR EVOLUT IONARY B IO LOGY
doi: 10.1111/jeb.12373
mainly as an anchor for the epididymis within the
scrotum. Compared with the other major group of en-
dotherms, birds, in mammals, sperm storage at a regu-
lated temperature lower than the core in the
epididymis compensates for a lower sperm production
rate, more complex maturation and a slower rate of
sperm transport (Bedford, 2004).
Notwithstanding the relative fitness benefits of a
cooled epididymis vs. the cooled testis, four nonmutual-
ly exclusive hypotheses currently dominate explana-
tions for the evolution of the scrotum: the Cool
Spermatogenesis Hypothesis (Moore, 1926), the Gallop-
ing Hypothesis (Frey, 1991), the Mutation Hypothesis
(Short, 1997) and the Activation Hypothesis (Gallup
et al., 2009). For nearly a century, the Cool Spermato-
genesis Hypothesis has argued that Tsperm and the
maintenance of sperm viability during storage in the
epididymis is about 34–35 °C (Moore, 1926; Appell
et al., 1977). Scrotal temperatures that approach those
of the core Tb compromise fertility (Moore, 1926; Bed-
ford, 1978b, 2004; Setchell, 1998), particularly if evapo-
rative cooling of the scrotum is impaired (Momen et al.,
2010). The Galloping Hypothesis (Frey, 1991) proposes
a trade-off between testes vulnerability (fitness cost)
and the avoidance of strong intra-abdominal pressure
fluctuations during galloping which impair spermato-
genesis in abdominal testes (fitness benefit). The Muta-
tion Hypothesis maintains that the testis is a ‘hot spot’
for germ-cell mutations and that the lower tempera-
tures of the scrotum reduce the rates of mutation on
the Y chromosome through mutagenic metabolites
(Short, 1997). The Activation Hypothesis posits that the
storage of sperm at a lowered temperature ensures that
they undergo ‘thermal shock’ during ejaculation into
the higher temperatures of the female, which increases
their motility and hence the probability of a successful
insemination (Gallup et al., 2009).
Four testis locations have been mapped onto the
mammalian phylogeny: testicondy (the embryonic,
abdominal, nondescended state), descended ascrotal,
scrotal and the marsupial condition (Werdelin & Nil-
sonne, 1999; Kleisner et al., 2010). These studies
arrived at opposing conclusions concerning the ances-
tral location of the testes, primarily because of the dif-
ferent mammalian phylogenies that were available at
the time. Whereas Werdelin and Nilsonne (1999)
argued that the scrotum was the ancestral (plesiomor-
phic) condition which was subsequently lost in more
recently evolved lineages, Kleisner et al. (2010) con-
cluded that testicondy was plesiomorphic and that the
scrotum is a derived, apomorphic structure, certainly in
placental mammals. These latter authors also argued
that (i) the scrotum evolved twice independently, in
the marsupials and the placental mammals, (ii) the
scrotum was lost early during the evolution of the Lip-
otyphla (moles, hedgehogs, solenodons, shrews, moon-
rats, gymnures), and (iii) there is an ‘equivocal’ state
basal to Laurasiatheria (bats, pangolins, lipotyphlans,
carnivores and ungulates) and Euarchontoglires
(rodents, treeshrews colugos, lagomorphs and primates;
Kleisner et al., 2010).
Attempts to test hypotheses that scrotal mammals
may have higher Tbs than ascrotal mammals (e.g.
Moore, 1926) did so without the benefit of contempo-
rary phylogenetic approaches (Wislocki, 1933; Johnson
& Omland, 2004; O’Leary et al., 2013). For example, it
was not possible, as it is now, to apply likelihood-based
methods to fit alternative models of trait evolution (e.g.
Brownian motion, Ornstein Uhlenbeck), identify
changes in the rate of evolution at various positions on
a phylogeny, or reconstruct continuous or binomial
ancestor character states (O’Meara et al., 2006; Revell &
Reynolds, 2012; Revell et al., 2012; Revell, 2013).
The maximum variation in Tb among extant mam-
mals exceeds 10 °C, which emphasizes the contrasting
quantitative levels of mammalian endothermy (Clarke
et al., 2010; Lovegrove, 2012a). Using an ancestral char-
acter reconstruction that employed maximum parsi-
mony, the Tb of the ancestral therian was estimated to
be about 34.2 °C (Lovegrove, 2012b), which falls well
below the 20th percentile of extant mammal Tbs (Love-
grove, 2012a) but comfortably within the range of
Tsperm (Setchell, 1998). In extant placental mammals,
Tbs < 35 °C have been argued to be basoendothermic,
possibly reflecting the outcome of either stabilizing
selection around the plesiomorphic state or a reduction
(reversal) from higher mesoendothermic (35 °C ≤ Tb≤ 37.9 °C) or supraendothermic (Tb > 37.9 °C) states at
some period during the Cenozoic (Lovegrove, 2012a).
As a direct measure, as well as a proxy for metabolism,
the Tb variation between extant mammals has been
related variously to factors such as climate and latitude
(Lovegrove, 2003, 2005), cursoriality (Lovegrove, 2004,
2012a,b) and muscle power (Clarke & P€ortner, 2010).If the central argument of the Cool Spermatogenesis
Model that the primary function of the scrotum is to
maintain spermatogenesis and sperm storage at temper-
atures lower than the core Tb is valid, then any factor
associated with selection for enhanced endothermy
during the Cenozoic must implicitly and theoretically
be tied to the evolution of externalized testes. More-
over, as the pinnacle of endothermy (supraendother-
my) was attained in the Cenozoic within digitigrade
and unguligrade mammals (Lovegrove, 2012b), the
evolution of cursoriality may be the primary, but not
exclusive, evolutionary force involved in the selection
for the placental scrotum.
In this study, I restricted analyses to the placental
mammals because of the recent progress in resolving
the placental mammal phylogeny (O’Leary et al., 2013).
I tested the predictions that the evolution of the scro-
tum in placental mammals during the Cenozoic was
associated with (i) the evolution of higher Tbs, that is,
the evolution of the meso- and supraendothermic pulses
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JOURNAL OF EVOLUT IONARY B IOLOGY ª 2014 EUROPEAN SOC I E TY FOR EVOLUT IONARY B IO LOGY
802 B. G. LOVEGROVE
(sensu Lovegrove, 2012b), (ii) the increases in body size
(Lovegrove & Mowoe, 2013; Slater, 2013) and (iii) the
evolution of cursoriality (Lovegrove, 2012a; Lovegrove
& Mowoe, 2013). The Tb and body mass of ancestral
states were reconstructed from mean values for mam-
mal families using stochastic character mapping sampled
from Bayesian posterior probability distributions (Boll-
back, 2006; Revell, 2012, 2013). I also investigated
exceptional changes in the rate of evolution of body size
and Tb (Revell et al., 2012). Phylogenetically informed
generalized least squares analyses (GLS) and analysis of
covariance (ANCOVA; Pinheiro et al., 2013) were
employed to test for the influences of the scrotum and
cursoriality on body temperature.
Materials and methods
All statistical analyses were conducted using various
packages in R version 3.0.2 (2013-09-25; R Core Devel-
opment Team, 2012). Data for the mean Tbs of 83
mammal families were obtained from Lovegrove
(2012b) (see Data S1). The topology of the phylogeny
was obtained from Lovegrove (2012b) and the diver-
gence dates from O’Leary et al. (2013). Data for aquatic
families (seals, hippos, whales and dolphins) were omit-
ted from the analysis on the basis of the different ther-
mal conductivities of water and air and the resultant
alternative location of the testes (Kleisner et al., 2010).
Ancestral reconstructions
Testis location data were obtained from Lunn (1948),
Freeman (1990) and Kleisner et al. (2010) and were
simplified to three states: (i) embryonic, undescended,
testicond (ascrotal), (ii) descended, pendant, scrotal
(scrotal) and (iii) descended, ascrotal, inguinal or a
reversal from a descended scrotal state to a descended
inguinal state (inguinal). The testis location of ancestral
mammal states was reconstructed using stochastic char-
acter mapping sampled from Markov chain Monte Car-
lo (MCMC) Bayesian posterior probability distributions
(Bollback, 2006; Revell, 2012, 2013) using the package
PHYTOOLS (Revell, 2012). Stochastic character maps
were generated using MAKE.SIMMAP and the posterior
probability densities using DENSITYMAP. A plot of the
posterior density map was generated using PLOTSIM-
MAP, and the posterior probabilities of nodes of interest
were obtained using DESCRIBE.SIMMAP (Revell,
2012). For this binomial reconstruction, the scrotal and
inguinal data were lumped (nontesticond), but subse-
quently identified graphically on the character map.
Also using the PHYTOOLS package, the ancestral
character state of the body masses and Tbs of mammal
families were reconstructed using CONTMAP. The map-
ping relies upon states estimated at internal nodes using
maximum likelihood with FASTANC and was plotted
with PLOTSIMMAP and CONTMAP.
Rates of evolution
The largest increases and decreases in the rate of evolu-
tion of body mass and Tb were identified using EVOL.-
RATE.MCMC, MINSPLIT and POSTERIOR.EVOLRATE
in the PHYTOOLS package (Revell et al., 2012).
100 000 generations were simulated and sampled every
200 generations. The first 25% of these samples were
discarded as burn-in (Revell et al., 2012). Branches with
posterior probabilities > 0.01 were identified (blue dots)
and plotted on a map of the reconstructed body mass
and Tb values. These dots indicate the probabilities of
the highest increases and decreases in the rate of evolu-
tion identified by the differences between the initial (r21) and final (r22) rates of evolution along specific
branches. Thus, the branches merely identify the major
patterns in evolutionary rate changes without ranking
the magnitude of evolutionary change or the exact
position along the branch where the rate change
occurred. Default starting values in EVOL.RATE.MCMC
were used in the simulations.
Body mass, scrotal position and cursoriality
Using the package NMLE (Pinheiro et al., 2013), alter-
native mixed-effects models (Brownian motion, Pagel’s
model and the Ornstein–Uhlenbeck model) were fitted
to mean Tb values for mammalian families as a function
of log10 body mass with testis location and a binomial
cursoriality index added as fixed factors. The homoge-
neity of model variances was verified using a Shapiro
test for normality (SHAPIRO.TEST). The lowest AIC
value of each model was used to identify the best-fit
model. The cursorial indices were obtained from Love-
grove (2004) and Carrano (1999). In short, with a few
exceptions (some hystricognath rodents families), plan-
tigrade mammals were coded as noncursorial and digiti-
grade and unguligrade mammals as cursorial.
Macroscelidea (elephant shrews) were coded as curso-
rial (see Lovegrove & Mowoe, 2014).
Results
Reconstruction of the testis location
Stochastic character mapping of the testis location on
the phylogeny of extant mammal families predicted an
86% probability that the ancestral placental mammal
was ascrotal (Figs 1 and 2, model 1). The probability
that the Afrotherian and Xenarthran (South American
armadillos, sloths and anteaters) ancestors were ascrotal
was also 86% (Figs 1 and 2, model 1). Thus, because
the Afrotheria and Xenarthra are testicond, the highest
probability is that the embryonic location of abdominal
testes dorsal to the kidneys was the plesiomorphic,
eutherian condition. The probability that the ancestor
of Boreotheria (Laurasiatheria plus Euarchontoglires)
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Evolution of the placental scrotum 803
was ascrotal was only 3.4% (Figs 1 and 2, model 1),
which suggests that the scrotum evolved very rapidly
after the K-Pg boundary.
Within Boreotheria, the scrotum does not occur in
Lipotyphla, Rhinocerotidae, Tapiridae, Pholidota, in one
family of Chiroptera (Pteropodidae), or within certain
32.3 39.6Tb (°C)
32.5 million years 32.5 million years
Scrotal Ascrotal
Fig. 1 Left phylogeny: maximum-likelihood reconstruction of the ancestral body temperatures of the eutherian mammal families. Blue dots
indicate branches with the highest posterior probabilities (> 0.01) of evolutionary rate changes (increases and decreases). The dots are
centred on each branch and do not indicate the precise time along the branch where the change took place. Right phylogeny: The stochastic
posterior probability density map of the ancestral scrotal state in eutherian families. Branches in blue indicate the proportional probability of
an ascrotal state, either as the testicond (plesiomorphic) state, the inguinal state or a reversal from a scrotal condition, whereas red branches
indicate the probability of a fully scrotal condition. As the illustration of the probabilities of greatest evolutionary interest are masked by the
rapid evolutionary changes which occurred within the first several hundred thousand years of the K-Pg boundary (sensu O’Leary et al.,
2013), the ordinal-level data have been extracted and graphically presented in Fig. 2 using arbitrary branch lengths (model 1).
ª 2 0 14 THE AUTHOR . J . E VOL . B I OL . 2 7 ( 2 0 1 4 ) 8 0 1 – 81 4
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804 B. G. LOVEGROVE
rodent groups (Hystricognatha, Bathyergidae, Spalaci-
dae, Rhizomyinae; Fig. 1), and in aquatic families
within the Pinnipedia and Cetacea (not shown in
Fig. 1). The ascrotal state in these families can be attrib-
uted to either (i) secondary internalization and retreat
of the testis to a subcutaneous, inguinal position or (ii)
an intermediate testicular location, that is, descended
but inguinal.
The mean body size of the ancestral eutherian was
estimated at 895 g (lower 95% CI = 359 g, upper 95%
CI = 2233 g, Fig. 3). The reconstruction of the ancestral
eutherian Tb predicted a mean (� 95% CI) of
36.22 � 0.56 °C (Fig. 1). Within the extant families,
60.7% display a mean Tb which was higher than this
predicted ancestral value (Fig. 1). Visually, the three
notable pulses towards supraendothermy occurred with
the Lagomorpha, Artiodactyla and Carnivora (Fig. 1;
however, see Evolutionary Rates analysis below). Less
dramatic pulses (increases) occurred in the Soricidae
(shrews), some rodent families (Myocastoridae, Cavii-
dae, Dasyproctidae, Echimyidae, Arvicolidae and Cricet-
inae), and some haplorhine primates (Cercopithecidae,
Aotidae and Cebidae).
Evolutionary rates
The largest number of exceptional changes in the rate
of evolution of body size occurred within the Rodentia
and the Primates (Fig. 3). The most exceptional
increase within the rodents occurred along the branch
subtending the subterranean families Rhizomyinae
(344 g) and Spalacinae (173 g), which are at least dou-
ble the mass of their closest relatives (Fig. 3). The most
exceptional decrease in body size occurred within the
Deomyinae, which is comprised mostly of spiny mice
(Acomys) and brush-furred mice (Lophuromys). The sec-
ond most exceptional increase occurred within the
Procyonidae (2.9 kg) which, typified by the racoons,
are on average more than double the size of their clos-
est relatives the weasels (Mustelidae; 1.1 kg; Fig. 3).
However, apart from the clustering of rate changes
within Rodentia, there were three exceptional basal
increases in body mass that are not easy to visualize in
Fig. 1: (i) the branch subtending all nonxenarthran
mammals, (ii) the branch subtending Boreotheria and
(iii) the branch subtending Euarchontoglires.
The largest proportion of changes in the rate of evo-
lution of Tb occurred again within Rodentia, and to a
lesser extent in the Primates (Fig. 1). The notable rate
increases occurred within the Neotropical Caviidae and
the Arvicolidae and Cricetidae (Fig. 1). Within Lip-
otyphla, the Soricidae (shrews) showed an exceptional
increase in the rate of change of Tb. Several rate
changes in the early Cenozoic are highly noteworthy,
but again not easy to visualize; the rate of Tb increase
in all nonxenarthran eutherians, Euarchontoglires and
Carnivora (Fig. 1). The highest decreases in the rate of
evolution of Tb occurred within several xenarthran
families, the Afrosoricida (Tenrecidae, Macroscelidea
and Chrysochloridae) and Rhinocerotidae (Fig. 1).
Body temperature, testis location and body mass
The empirical mean (� SD) Tb for the three testis loca-
tions was Tb = 34.32 � 1.59 °C for testicond mammals,
Tb = 36.42 � 1.30 °C for inguinal mammals and Tb =37.00 � 1.34 °C for scrotal mammals (Fig. 4a). The
best-fitting phylogenetically independent ANCOVA models
that tested for differences in Tb after controlling for
log10 body mass were the Brownian motion model and
Pagel’s model, which showed no significant differences
in Tb between the three testis locations (Table 1).
However, both of these models showed significant
ScandentiaPrimatesLagomorphaRodentiaLipotyphlaPholidotaCarnivoraChiropteraPerrisodactylaArtiodactylaAfrotheriaXenarthra
RR E
uarch
Lau
r.
Bo
reoth
eria
Model 1
ScandentiaPrimatesLagomorphaRodentiaLipotyphlaPholidotaCarnivoraChiropteraPerrisodactylaArtiodactylaAfrotheriaXenarthra
R
S
S
Model 2
Eu
archL
aur.
Bo
reoth
eriaScandentiaPrimatesLagomorphaRodentiaLipotyphlaPholidotaCarnivoraChiropteraPerrisodactylaArtiodactylaAfrotheriaXenarthra
Model 3S
SS
Eu
archL
aur.
Bo
reoth
eriaTesticondScrotalInguinalReversal from scrotal to inguinal
Fig. 2 Graphical depictions of the three possible evolutionary
outcomes of the evolution of the scrotum in the eutherian orders.
The branch lengths in all three models are arbitrary (Pagel, 1992).
Model 1: The scrotum evolved once only within Eutheria in the
ancestor of Boreotheria. The posterior probabilities of the ascrotal
(blue) and scrotal (red) states obtained from the stochastic
probability density map shown in Fig. 1 are shown as pie
diagrams. The symbols ‘R’ within orange circles indicate reversals
from a scrotal to an ascrotal state (secondary internalization),
whereas the letter ‘S’ within yellow circles indicates independent
evolutionary appearances of the scrotum. Model 2: The scrotum
evolved twice, independently in the nonlipotyphlan Laurasiatheria
and Euarchontoglires. Model 3: The scrotum evolved three times,
independently in the Carnivora, Euarchontoglires and the clade
including the Chiroptera, Artiodactyla and Perissodactyla.
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Evolution of the placental scrotum 805
heteroscedasticity. Pagel’s lambda value was 0.917 con-
firming strong phylogenetic signal in the data. Hetero-
scedasticity was owed to a strongly left-skewed
distribution of Tb which could not be overcome
through regular log10 transformation of the variables.
Figure 4a illustrates that the skewed distribution is
caused by the low Tbs of the testicond mammals. On
average, the Tb of testicond mammals was 2.68 °Clower than that of the scrotal mammals, whereas the Tb
of the inguinal mammals was a mere 0.58 °C lower
than that of the scrotal mammals. Consequently, the
inguinal mammals were lumped with the scrotal mam-
mals for further analyses.
The Brownian motion model and Pagel’s model again
gave the best fit to the phylogenetic ANCOVA that investi-
gated the difference in Tb between testicond and non-
testicond mammals after controlling for body mass
effects (Table 1). Both models had homogeneous resid-
32.5 million years
Log10 body mass (g)0.80 6.51
Fig. 3 Maximum-likelihood
reconstruction of the ancestral body
masses of the eutherian mammal
families. Blue dots indicate branch
length showing the highest posterior
probabilities (> 0.01) of evolutionary
rate changes, both increases and
decreases. The dots are centred on each
branch and do not indicate the precise
time along the branch where the
change took place.
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806 B. G. LOVEGROVE
uals and showed significant influences of testis location
on Tb (Table 1). These models also showed significant
effects of body mass on Tb (Table S3).
The influence of body mass was investigated further
using GLS models of Tb as a function of log10 body
mass. However, the residuals of the model for the com-
plete data set deviated significantly from a normal dis-
tribution (e.g. Brownian motion model, Shapiro test;
W = 0.94, P ≤ 0.001) despite log-transformation of both
variables. The data were consequently analysed sepa-
rately for each testis location (Table 2, Fig. 4a). The
residuals of these separate models were normally dis-
tributed. For the scrotal mammals, the Brownian
motion model and Pagel’s model provided the best fit
to the data and showed a significant allometry of Tb as
a function of body mass (Table 2, Fig. 4a). The Brown-
ian motion model and Pagel’s model also provided the
best fit to the data for the inguinal and testicond mam-
mals but, in contrast to scrotal mammals, there was no
significant allometry of Tb and log10 body mass
(Table 2, Fig. 4a).
The influence of cursoriality
The mean (� SD) Tb of cursorial mammal families
(Tb = 37.73 � 0.99 °C) was 1.74 °C higher than that of
the noncursorial families (Tb = 35.99 � 1.56 °C;Fig. 4b) and 1.51 °C higher than that of the
reconstructed estimate of the placental ancestor. Pagel’s
model provided the best fit to a phylogenetically inde-
pendent ANCOVA testing for differences in Tb between
cursorial and noncursorial mammals after controlling
for body mass effects (Table 1). This model showed a
significant difference between the Tb of cursorial and
noncursorial mammals, but the residuals of the model
were heteroscedastic (Table 1). However, ANCOVA mod-
els testing for the differences in Tb between scrotal cur-
sorial and scrotal noncursorial mammal families had
homoscedastic residuals (Table 1). The Brownian
motion model was the best-fitting model and showed a
significant difference in Tb (Table 1). Thus, the mean
(� SD) Tb of scrotal cursorial mammal families
(Tb = 37.99 � 0.83 °C) was significantly 1.54 °C higher
than that of the noncursorial scrotal families
(Tb = 36.45 � 1.26 °C).
Discussion
The various approaches employed here to test hypothe-
ses for the evolution of the scrotum provided mixed
results. As discussed later, the ancestral character
reconstructions and evolutionary rate determinations
were influenced by the presence of ghost lineages in
the phylogeny of extant mammal families. Ghost lin-
eages are inferred to exist in reconstructions, but lack
Table 1 Summary of phylogenetic analyses of covariance (ANCOVA)
testing for the differences in body temperature between various
variables after controlling for body mass effects. Complete statistics
for each model are provided in the Supporting information.
Evolutionary model
Model residual
variability Res. d.f. P AIC
Testis location (testicond, inguinal, scrotal)
Brownian motion Heteroscedastic 80 0.369 290.9
Pagel (k = 0.917) Heteroscedastic 80 0.314 291.5
Ornstein Uhlenbeck Homoscedastic 80 0.007 317.5
Testis location (testicond, nontesticond)
Brownian motion Homoscedastic 80 0.002 280.5
Pagel (k = 0.829) Homoscedastic 80 < 0.001 279.3
Ornstein Uhlenbeck Homoscedastic 80 < 0.001 289.6
Locomotion (cursorial, noncursorial)
Brownian motion Heteroscedastic 80 0.316 290.6
Pagel (k = 0.730) Heteroscedastic 80 0.002 285.3
Ornstein Uhlenbeck Heteroscedastic 80 < 0.001 305.2
Locomotion of scrotal mammals (cursorial, noncursorial)
Brownian motion Homoscedastic 48 0.045 153.3
Pagel (k = 1.214) Homoscedastic 48 0.051 154.8
Ornstein Uhlenbeck Homoscedastic 48 0.089 164.2
Table 2 Statistics from PGLS models of the relationship between
body temperature and log10 body mass of testicond, inguinal and
scrotal mammals.
Value SE t P lambda AIC
Scrotal mammal families only (n = 51)
Brownian motion (Shapiro–Wilk test: W = 0.970, P = 0.225)
Intercept 35.47 0.55 64.20 <0.001 1.000 156.5
Slope 0.53 0.17 3.06 0.004
Pagel (Shapiro–Wilk test: W = 0.965, P = 0.141)
Intercept 35.62 0.61 58.47 <0.001 1.249 157.9
Slope 0.48 0.19 2.52 0.015
Ornstein Uhlenbeck (Shapiro–Wilk test: W = 0.975, P = 0.354)
Intercept 35.29 0.39 91.39 <0.001 165.5
Slope 0.64 0.13 4.89 <0.001
Inguinal mammal families only (n = 21)
Brownian motion (Shapiro–Wilk test: W = 0.932, P = 0.151)
Intercept 35.39 1.13 31.44 <0.001 1.000 77.93
Slope 0.15 0.34 0.44 0.664
Pagel (Shapiro–Wilk test: W = 0.928, P = 0.125)
Intercept 36.35 0.92 39.07 <0.001 0.634 76.64
Slope 0.02 0.29 0.06 0.950
Ornstein Uhlenbeck (Shapiro–Wilk test: W = 0.928, P = 0.127)
Intercept 35.82 0.84 43.51 <0.001 79.14
Slope 0.02 0.27 0.08 0.934
Testicond mammal families only (n = 11)
Brownian motion (Shapiro–Wilk test: W = 0.947, P = 0.602)
Intercept 34.37 1.43 24.0 <0.001 1.000 44.33
Slope 0.12 0.36 0.32 0.756
Pagel (Shapiro–Wilk test: W = 0.947, P = 0.602)
Intercept 34.27 1.39 24.70 <0.001 0.873 46.21
Slope 0.13 0.35 0.38 0.712
Ornstein Uhlenbeck (Shapiro–Wilk test: W = 0.944, P = 0.570)
Intercept 33.66 1.29 26.16 <0.001 48.03
Slope 0.20 0.36 0.56 0.592
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Evolution of the placental scrotum 807
fossil records to support their existence (Sidor & Hop-
son, 1998). The reconstruction of the location of the
ancestral eutherian scrotum did, however, confirm
Kleisner et al.’s (2010) argument that the scrotum is a
derived trait. However, despite this conformation, the
evolutionary history of the lipotyphlan testis descendus
remains equivocal, yet is crucial to the understanding
of the evolution of the scrotum. There are three possi-
ble evolutionary models.
Model 1: The scrotum evolved once only within Eu-
theria in the ancestor of Boreotheria, but became sec-
ondarily internalized in Lipotyphla and Pholidota after
full scrotal descendus in the boreotherian ancestor
(model 1, Fig. 2).
Model 2: The scrotum evolved twice independently
within Eutheria, once in the ancestral nonlipotyphlan
Laurasiatheria and once within Euarchontoglires
(model 2, Fig. 2).
The testis location in Lipotyphla was not a conse-
quence of secondary internalization but, rather, the
intermediate descendus stage between testicondy and
full scrotal descendus (descended inguinal location).
The ascrotal status of Pholidota represents a true sec-
ondary internalization.
Model 3: The intermediate, descended, inguinal loca-
tion was inherited by Pholidota from Lipotyphla. The
scrotum would then need to have evolved indepen-
dently three times within Eutheria; once within Eu-
archontoglires, once within Carnivora and once within
the ancestor of the Chiroptera, Artiodactyla and Peris-
sodactyla (model 3, Fig. 2).
Although the maximum-likelihood reconstruction of
the ancestral testicular location supports model 1, the
alternative models should be dismissed with consider-
able caution if they are based purely upon a character
reconstruction that omitted extinct taxa from the phy-
logeny. Reconstructions that rely on data for extant
species only are weakened by ghost lineages generated
by missing soft-tissue data. The scrotum and the testes
do not fossilize, and hence, there are no data available
on the testis location of extinct taxa, despite sound
skeletal and stratigraphic information for extinct taxa
within each clade. Thus, in the case of the lipotyphlan
reconstruction, ghost lineages may generate putatively
false evolutionary reversals, such as that shown in
model 1, rather than provide possible confirmation of
the retention of a plesiomorphic state, such as that
shown in model 2. This is a common problem associ-
ated with the reconstruction of physiological traits, for
example, as noted in previous attempts at Tb recon-
struction (Lovegrove, 2012b). I discuss broader implica-
tions of this topic later.
It is intuitive when choosing evolutionary models
that predict the derivation of new traits to choose those
that predict the least number of evolutionary events.
Model 3 suggests that the scrotum may have evolved
three times independently in placental mammals. How
realistic are these models?
The scrotum evolved in marsupial lineages indepen-
dently of Eutheria (Kleisner et al., 2010), so it has
evolved with certainty at least twice, or a maximum of
four times (accepting model 3), in mammals. Model 3
is probably the least realistic because the ascrotal state
is found in all armoured mammals, so it is reasonable
to expect secondary testis internalization in pangolins
(Pholidota). Armoured mammals that roll up into a
protective ball such as pangolins do are either testicond
(armadillos, tenrecs) or inguinal (hedgehogs). Never-
theless, the evaluation of the validity of models 1 and 2
will require additional approaches to those attempted
here. For example, in Lipotyphla, it may be possible to
0 1 2 3 4 5 6
Bod
y te
mpe
ratu
re (°
C)
32
33
34
35
36
37
38
39
40
TesticondScrotalInguinal
Log10 body mass (g)0 1 2 3 4 5 6
Bod
y te
mpe
ratu
re (°
C)
32
33
34
35
36
37
38
39
40
Non-cursorsCursors
11
21
25
58
(a)
(b)
51
Fig. 4 Body temperature as a function of log10 body mass of (a)
testicond, inguinal and scrotal mammals and (b) cursorial and
noncursorial mammals. The regression line (red line) in (a) was
calculated from the Brownian motion model (lowest AIC value)
and is fitted to the data for scrotal mammal families only (see
Supporting information for regression statistics). The box plots
show the median (horizontal line), 25th percentile (bottom of
box), 75th percentile (top of box), 5th percentile (bottom
whisker), 95th percentile (top whisker) and the outliers (symbols)
for each category in each graph. The numbers in white squares
indicate the sample sizes.
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808 B. G. LOVEGROVE
examine the evolutionary history of testis migration
using an anatomical examination of structures such as
the inguinal ligament (Lunn, 1948).
Body temperature and body mass reconstruction
Similar interpretation constraints apply to the recon-
struction of Tb, although there are certainly meaningful
patterns that are noteworthy and important. From a
graphical inspection of the Tb reconstruction in Fig. 1, I
intuitively would have expected that the highest rates
of evolutionary change would have been identified on
the branches leading to the Artiodactyla, Lagomorpha
and Carnivora (the orange/red lineages in Fig. 1). These
are the clades that display mid-Cenozoic supraendo-
thermic pulses that I identified previously (Lovegrove,
2012b). Yet, only the carnivoran expectation was borne
out. Again, ghost lineages are highly likely to be
involved, and the Lagomorpha provide a good illustra-
tion of the problem.
Lagomorpha is the sister order to the Rodentia and
diverged 64.6 mya from the latter very soon after the
K-Pg extinction event (O’Leary et al., 2013). The two
extant lagomorph families, Leporidae (hares and rab-
bits) and Ochotonidae (pikas) split 31.7 mya (O’Leary
et al., 2013). The mean Tbs of the Leporidae and Ocho-
tonidae are Tb = 38.9 °C and Tb = 39.6 °C, respectively(Supporting information), highly supraendothermic,
and around 2.5 °C higher than that of their common
ancestor with Rodentia. Thus, we must recognize the
supraendothermy of the Lagomorpha to represent a
profound physiological apomorphy in mammals, and
certainly one which should provide valuable informa-
tion on the evolution of endothermy. The problem,
though, is the long period of ghost evolutionary history
along the branch leading from the split with the Rod-
entia to the origin of the extant families (32.9 million
years), and the long branches leading from the origin
of the two families to the present.
The topology of the clade subtending the extant
lagomorph families could, of course, be resolved to
finer detail by analysing Tb at the generic or species
level, thus eliminating ghost lineages. However, the
same approach cannot be applied to the branches basal
to the appearance of the extant lagomorph families –there are no Tb data for the extinct lagomorph ances-
tors. Thus, in terms of character reconstruction at the
level of family, ghost lineages can mask exceptional
evolutionary rate changes because trait changes occur
over very long periods of uninterrupted ghost history.
It is not surprising, therefore, that the majority of
exceptional Tb rate changes identified in this study
occurred on branches with seemingly innocuous Tbchanges within the Rodentia, especially within the mu-
roid lineages. These lineages underwent spectacular
and rapid Miocene adaptive radiation and their current
phylogenetic reconstruction lacks ghost lineages
(Michaux & Catzeflis, 2000; Michaux et al., 2001; Hu-
chon et al., 2002; Fabre et al., 2012). Exceptional eleva-
tion in the ancestral carnivoran Tb was detected
because, relative to the lagomorph and artiodactyl lin-
eages, the carnivore lineage incorporated fewer ghost
lineages (Fig. 1).
My estimated body mass of the ancestral placental
mammal was several-fold higher than that estimated in
an extensive genomic and phenomic phylogenetic
reconstruction of the placental mammals (O’Leary et al.,
2013). Again, though, I would argue that my value is
an overestimate and is a consequence of ghost lineages.
Of course, this problem can be resolved easily on its
own, because body masses can be estimated for extinct
species, but this was not the objective of this study.
The comparative method
As useful and promising as character reconstruction
and evolutionary rate determinations through maxi-
mum-likelihood procedures are, they are limited at
present in their capacity to provide realistic probabilities
whenever missing data generate ghost lineages deep
within phylogenetic hierarchies. However, phylogeneti-
cally informed model selection processes (Johnson &
Omland, 2004) using the comparative method hold bet-
ter promise. Pagel’s model or a Brownian motion model
consistently gave the best fit to the various models
applied to the data.
Heteroscedasticity made it statistically unreliable to
quantify an allometric relationship between Tb and
body mass of the combined data set. However, it is
highly noteworthy that a significant allometry exists in
scrotal mammals, but not in inguinal or testicond mam-
mals. These data confirm previous observations of cor-
related evolution between Tb and body mass in large,
scrotal placental mammals (Lovegrove, 2012a,b; Love-
grove & Mowoe, 2013). Additional salient observations
were that the mean Tb of nontesticond mammal fami-
lies was significantly higher than that of testicond fami-
lies and, within scrotal mammals, there was a
significant influence of cursoriality. Thus, the highest
Tbs have been attained in the largest, cursorial, scrotal
mammals. There are no ascrotal supraendothermic fam-
ilies.
Irrespective of the individual validity of the three
evolutionary models presented here, it seems certain
that the first appearance of the scrotum in placental
mammals occurred 64.8 mya with the evolution of
Boreotheria (see also Kleisner et al., 2010). Moreover,
the scrotum evolved in tandem with elevated Tbs and
increased body sizes surprisingly quickly and early in
the Cenozoic. Despite the limitations of ghost lineages,
Palaeocene pulses in body mass and Tb were confirmed
here in the ancestors of the major eutherian lineages
(Laurasiatheria and Euarchontoglires). Thus, perhaps
the important question that we need to ask at this
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Evolution of the placental scrotum 809
point is not why the scrotum evolved in such spectacu-
lar fashion but, rather, why there was such strong
selection for elevated Tbs and increased body size so
early in the Cenozoic? Holistic answers to this question
would require delving into the myriad of hypotheses
on the evolution of endothermy, which is beyond the
scope of this study. So, notwithstanding the value of
these endothermy models, I present, instead, a brief
synopsis of the major patterns that were thought to be
involved in the evolution of endothermy, as a prece-
dent to the presentation of a new model on the evolu-
tion of the scrotum.
The antiquity of cool sperm
It has been suggested (Lovegrove, 2012b) that endo-
thermy may have evolved in association with the major
increases in relative brain size that occurred in ancestral
mammals 200 mya with the evolution of nocturnalism
(Rowe et al., 2011). Fur is a critical necessity for endo-
thermy and evolved in mammaliaforms (the immediate
precursors to true mammals) with some certainty by
165 mya (Zhou et al., 2013). The ancestor of mono-
tremes and marsupials was, de facto, also furred, and as
these two lineages diverged ~228 mya (Zheng et al.,
2013), the origin of endothermy can, using this line of
argument, be pegged to at least this date. Thus, in all
mammals except some scrotal marsupials (see Lunn,
1948), the highest Tsperm in Jurassic and Cretaceous tes-
ticond mammals would have been 34–36 °C for at least
163 million years. Tsperm has seemingly remained stable
over the past 228 million years despite the evolution of
elevated Tbs in meso- and supraendothermic mammals
during the Cenozoic (Lovegrove, 2012b). I would sug-
gest that stabilizing selection has maintained Tsperm at
the lower plesiomorphic Tbs of the ancestral ascrotal
mammals throughout the Cenozoic. At present, perhaps
the best explanation for the driving force of this resis-
tance to evolutionary change is the fitness benefit of
the reduction in the rate of germ-cell mutations (Short,
1997).
The Endothermic Pulses Model
The model that I present here for the evolution of the
scrotum in placental mammals argues that selection for
increased Tb and the elevation of the level of endo-
thermy, what I have termed the ‘Cenozoic supraendo-
thermic pulses’, was the driving force for testis
externalization. Many factors have been implicated in
the elevation of a regulated metabolic rate in mammals
during the Cenozoic (see Lovegrove, 2012a for review),
but several are predominant; those involved with
increased muscle power (Clarke & P€ortner, 2010),
increased locomotory efficiency and speed (Garland &
Janis, 1993; Janis & Wilhelm, 1993; Lovegrove, 2004,
2012b; Lovegrove & Mowoe, 2013) and climate adapta-
tion (Lovegrove, 2003; Withers et al., 2006). More
ancient Mesozoic selection associated with factors such
as aerobic capacity (Bennett & Ruben, 1979), parental
care (Farmer, 2000, 2003) and assimilation efficiency
(Koteja, 2000, 2004) preceded the Cenozoic forces and
was debatably involved in the initial stages of the evo-
lution of endothermy in mammals (see Lovegrove,
2012a for review). The question, though, is whether
the Cenozoic selection forces acted in concert, or
whether one force was primary to the others?
As selection for climate adaptation was likely to have
commenced only with the onset of global cooling fol-
lowing the Early Eocene Climate Optimum (ca.
50 mya; see Zachos et al., 2001), selection for locomo-
tory efficiency, speed and muscle power probably pre-
ceded that for climate adaptation and other factors.
Indeed, although fossil morphological trends suggest
that cursoriality in typical large-bodied cursors, such as
ungulates and carnivores, commenced in the mid-
Cenozoic in response to global cooling (Janis & Wil-
helm, 1993), in the euarchontoglirid lineage, ‘micro-
cursoriality’ (sensu Rathbun, 1979) evolved as early as
the Palaeocene in the Macroscelidea (elephant shrews;
Lovegrove & Mowoe, 2014) prior to the major Ceno-
zoic increases in body size (Lovegrove & Mowoe,
2013). It also evolved in Palaeocene Lagomorpha
(Asher et al., 2005). The stem fossils of these orders
show elongations of hind limb bones typical of fast-
moving cursors (Asher et al., 2005; Zack et al., 2005).
For example, the hind limb of the Palaeocene lago-
morph Gomphos is almost identical in bone proportions
to modern rabbits and hares (Asher et al., 2005). I have
suggested that micro-cursoriality in stem macroscelids
and lagomorphs seemed to have evolved in a forested
environment (Lovegrove & Mowoe, 2014), unlike later
in the Cenozoic when global cooling, more open land-
scapes and grasslands drove the evolution of cursoriality
as well as increased body size in ungulates (Janis, 1993;
Edwards et al., 2010; Figueirido et al., 2012; Lovegrove
& Mowoe, 2013).
Within Laurasiatheria, geometric bone comparisons
between the ungual phalanx and proximal radial head
outlines of extinct and extant taxa suggest that cursori-
ality also evolved in Palaeocene species such as Pachyae-
na gracilis (Mesonychia), Hyracotherium (Perissodactyla)
and Phenacodus (Condylarthra; Macleod & Rose, 1993).
Postcranially, Pachyaena is most similar to tapirs, suids
and capybaras, which are all excellent swimmers,
strengthening the argument that cursoriality in eutheri-
ans may have been achieved via multiple pathways
(Oleary & Rose, 1995). Phenacodus is placed in the fam-
ily Phenacodontidae (Thewissen & Babcock, 1992). The
earliest phenacodontid was Tetraclaenodon puercensis
from the Torrejonian North American Stage (63.3–60.2 mya). It had bunodont molars and was estimated
to have weighed about 12 kg. Phenacodus species from
slightly later North American Stages bore lophodont
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810 B. G. LOVEGROVE
molars and ranged between 15 and 87 kg (Thewissen,
1990). Thus, in addition to the rapid appearance of dig-
itigrade limbs and cursoriality, Palaeocene laurasiatheri-
ans also showed a dramatic increase in body size
compared with the 6–245 g ancestral placental mammal
(O’Leary et al., 2013), and exhibited increasingly com-
plex molars allowing for more efficient food processing.
I have argued elsewhere that the driving force for the
evolution of increased body sizes in ungulates was
probably associated with the optimum body size for the
efficient fermentation of cellulose (Lovegrove, 2012a;
Lovegrove & Mowoe, 2013).
The incredible speed with which the major mamma-
lian orders radiated within several hundred thousand
years of the K-Pg boundary (O’Leary et al., 2013)
makes it quite realistic to accept that the cursorial char-
acteristics of the ancestral euarchontoglirids and laurasi-
atherians may have been inherited from the ancestral
boreotherian (model 1, Fig. 2). However, irrespective of
the presumed multiple pathways involved in the later
diversification of cursorial styles within Laurasiatheria
and Euarchontoglires, the evolution of cursoriality was
arguably coupled with selection for greater muscle
power which is strongly correlated with elevated body
temperatures (Clarke & P€ortner, 2010). The mainte-
nance of a homoeothermic, elevated Tb requires a con-
comitant sustained elevation in metabolic rate because
Tb and metabolic rate are correlated in a slow-fast met-
abolic continuum (Lovegrove, 2003). These energetic
dependencies occur in extant cursors; basal metabolic
rate is positively correlated with maximum running
speed (Lovegrove, 2004), and Tb is positively correlated
with the metatarsal/femur ratio (a proxy for running
speed; Lovegrove, 2012b). Thus, in terms of the Cool
Spermatogenesis Model, the scrotum would have
evolved as a thermoregulatory mechanism to external-
ize the testes from the core Tb and permit spermatogen-
esis and sperm storage to occur at the cooler,
plesiomorphic temperatures.
Once the scrotum evolved, I would argue that it
paved the way for metabolic adaptation to factors other
than those associated with cursoriality. For example,
small mammals, which have high surface-area-to-vol-
ume ratios and are hence susceptible to high heat
fluxes, display strong latitudinal influences on Tb and
metabolic rate (Lovegrove, 2003). High latitude small
mammals exposed to the lowest ambient temperatures
display the highest levels of endothermy, presumably
to facilitate thermogenesis (Lovegrove, 2003). The
exceptional rate increase in Tb of the Nearctic soricid
shrews (identified in this study) compared with their
Asian and African crocidurid counterparts is a good
example that has been discussed elsewhere in more
detail (Lovegrove, 2012a). Another good example is the
Tb rate increase identified here in the arvicolid rodents,
high latitude species, compared with other rodent
families.
In addition to climate adaptation, in certain lineages,
such as Primates, the scrotum may also have facilitated
the evolution of complex social systems and prolonged
life histories which are correlated with increased brain
sizes that bear metabolic costs reflected in higher basal
metabolic rates and Tb (Aiello & Wheeler, 1995; Isler &
van Schaik, 2006; Barrickman et al., 2008). The scro-
tum may also have facilitated the evolution of flight in
the Chiroptera. Although the Tbs of bats measured
under basal conditions are low relative to other scrotal
mammals, during flight there is a substantial increase
in Tb and testes descent into the scrotum occurs (Car-
penter, 1986).
The inguinal state
The high Tb of certain inguinal mammals, such as sori-
cid shrews seem to challenge the Endothermic Pulses
Model as well as the Cool Spermatogenesis Model,
upon which it is based. How do very small mammals
produce and store viable sperm with a Tb > 37 °C? One
likely explanation is that the subcutaneous position of
the epididymis and testis allows for a higher degree of
temperature regulation (Bedford, 2004) than is cur-
rently appreciated, on both a daily and seasonal basis.
For example, a typical core-to-skin temperature gradi-
ent in small mammals is around 2 °C (Lovegrove et al.,
1991), so a hypothetical inguinal mammal with des-
cended testes with a core Tb = 38 °C would have a skin
temperature of about 36 °C, which is the putative
upper limit of Tsperm. Thus core-to-skin gradients may
be effective at maintaining the testes at Tsperm in small
inguinal mammals because of their comparatively larger
surface-area-to-volume ratios relative to larger mam-
mals. Added support for a cooler subcutaneous location
is that lipotyphlans such as hedgehogs are able to sea-
sonally retract the testes into the abdominal position
during the nonbreeding season (Lunn, 1948). More-
over, even in those inguinal species not known to show
seasonal, testicular retraction, a scrotal-like outpocket-
ing of the abdominal wall in the unguinal region
becomes more pronounced and protuberant during the
breeding season (Weir, 1974; Freeman, 1990).
Cursorial cavid rodents are interesting in this regard
because they display the high Tbs associated with curso-
riality (Lovegrove, 2012b), yet the ancestral hystricog-
nath underwent a secondary testicular internalization
and all extant Neotropical hystricognath rodents retain
the reversal (Fig. 1). A possible explanation is that the
majority of cursorial cavids are semi-aquatic and that
the inguinal subcutaneous location is sufficient to
ensure that spermatogenesis and sperm storage occur
within the upper limits of Tsperm.
Future research needs to measure and compare tes-
ticular and epididymal temperatures with core body
temperatures. At present, there are too few data to per-
form this comparison.
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Evolution of the placental scrotum 811
Testicond oddities
The testicond elephant-shrews (Macroscelidea) are chal-
lenging to the Endothermic Pulses Model as well as the
Galloping Model because they are the most cursorial of
all small mammals < 1 kg (Carrano, 1999; Lovegrove &
Mowoe, 2014) and have a mean Tb = 37.3 °C (Support-
ing information). Other testicond Afrotherians (ele-
phants, hyraxes, dugongs, aardvarks, golden moles and
tenrecs) do not conflict as severely with existing models
because they maintain Tbs equal to or < 36 °C (Love-
grove, 2012b), and they are not considered to be curso-
rial (Carrano, 1999). Hyraxes, for example, have viable
sperm despite their testicondy (Bedford & Millar, 1978).
Short (1997) argued that an ‘elevated’ Tb in elephants,
and hence the assumed increase in germ-line mutations
per generation, may offset the fitness cost of their extre-
mely long generation time relative to all other mammals.
This argument, though, cannot apply to elephant-
shrews, whose generation times are similar to those of
other small mammals. It is possible that the extensive
employment of daily torpor by elephant-shrews, which
involves a profound daily reduction in Tb during the cir-
cadian rest and active phases (Lovegrove et al., 1999,
2001; Mzilikazi & Lovegrove, 2004, 2005), may offset or
reduce the ‘damage’ that putatively occurs to sperm at
the higher Tbs when the animals are active. However,
further research needs to focus on how spermatogenesis
and sperm storage in the nonbasoendothermic, testicond
mammals seems to occur successfully at comparatively
high Tbs.
Validity of activation model
As a closing observation, if we accept the major trends
in the evolution of endothermy that have been out-
lined above, then the Activation Hypothesis (Gallup
et al., 2009) is incompatible with the Endothermic
Pulses Model. Sperm transference between sexes for at
least 163 million years in ancestral testicond mammals
would not have involved a ‘thermal shock’, so it is
unlikely that this requirement should be a Cenozoic
phenomenon which evolved in scrotal, placental mam-
mals only.
Conclusions
This study employed various phylogenetic maximum-
likelihood procedures (ancestral reconstruction, evolu-
tionary rate changes, GLS and phylogenetic ANCOVA) to
test the principal hypothesis that scrotal mammals have
higher body temperatures than ascrotal mammals. The
results of the comparative methods gave the most reli-
able tests of the hypotheses and confirmed that (i) non-
testicond mammals have higher Tbs than testicond
mammals, (ii) scrotal, cursorial mammals have higher
Tbs than scrotal, noncursorial mammals, and (iii) there
was a significant allometry between Tb and body size in
scrotal mammals but not ascrotal mammals. Stochastic
character mapping of the testis location (testicond, ingui-
nal, scrotal) confirmed that the scrotum first appeared in
placental mammals in the ancestor of Boreotheria. How-
ever, three evolutionary models are presented showing
that the scrotum could have evolved as many as three
times independently within Boreotheria. The ubiquity of
whether the inguinal testis location in Lipotyphla repre-
sents a secondary internalization is critical to the evalua-
tion of these models. A new model on the evolution of
the scrotum is presented which argues that the evolution
of the scrotum was driven by the endothermic pulses
which occurred in placental mammals during their
explosive radiation following the extinction of the nona-
vian dinosaurs at the K-Pg boundary. It is proposed that
the initial selective force for increased Tb was associated
with the evolution of cursoriality in the Palaeocene. The
evolution of the scrotum facilitated the subsequent evo-
lution of elevated metabolic levels in response to other
selective forces during the Cenozoic, such as those
involved with climate adaptation.
Acknowledgments
This research was financed by NRF competitive and
incentive grants, and UKZN Research Office incentive
awards. I thank Danielle Levesque and Shaun Welman
for comments on the draft manuscript. Liam Revell
patiently assisted me with the implementation of PHY-
TOOLS.
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Supporting information
Additional Supporting Information may be found in the
online version of this article:
Data S1 Data selection criteria.
Table S1 Body temperature and body mass data coded
as testicond, inguinal, or scrotal, and cursorial or non-
cursorial.
Table S2 Statistics of analysis of covariance (ANCOVA)
testing for the difference in body temperature as a func-
tion of testis location [testicond (n = 11), inguinal
(n = 21), and scrotal (n = 51)].
Table S3 Statistics of analysis of covariance (ANCOVA)
testing for the difference in body temperature as a func-
tion of testis location (testicond, n = 11, and nontestic-
ond, n = 72 mammal families).
Table S4 Statistics of phylogenetically independent
analyses of covariance (ANCOVA) testing for the differ-
ence in body temperature between cursorial and non-
cursorial mammal families.
Table S5 Statistics of phylogenetically independent
analyses of covariance (ANCOVA) testing for the differ-
ence in body temperature between cursorial and non-
cursorial scrotal mammal families.
Data deposited at Dryad: doi:10.5061/dryad.s5k0t
Received 16 September 2013; revised 11 March 2014; accepted 12
March 2014
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