lovegrove 2014 escroto placenta mamiferos

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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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).



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.




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.



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)


Intercept 35.39 1.13 31.44 <0.001 1.000 77.93

Slope 0.15 0.34 0.44 0.664


Intercept 36.35 0.92 39.07 <0.001 0.634 76.64

Slope 0.02 0.29 0.06 0.950


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)


Intercept 34.37 1.43 24.0 <0.001 1.000 44.33

Slope 0.12 0.36 0.32 0.756


Intercept 34.27 1.39 24.70 <0.001 0.873 46.21

Slope 0.13 0.35 0.38 0.712


Intercept 33.66 1.29 26.16 <0.001 48.03

Slope 0.20 0.36 0.56 0.592




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.



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




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



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.




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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