Adaptive paternal effects? Experimental evidence that the paternal environment affects offspring performance

doi:10.1890/13-0184.1

Ecology, 94(11), 2013, pp. 2575–2582

! 2013 by the Ecological Society of America

Adaptive paternal effects? Experimental evidence that the paternal

environment affects offspring performance

ANGELA J. CREAN,

1,2,4

JOHN M. DWYER,

1

AND DUSTIN J. MARSHALL

1,3

1

School of Biological Sciences, University of Queensland, Brisbane QLD 4072 Australia

2

Evolution and Ecology Research Centre and School of Biological, Earth and Environmental Sciences,

University of New South Wales, Sydney NSW 2052 Australia

3

School of Biological Sciences, Monash University, Melbourne VIC 3800 Australia

Abstract. The ability of females to adaptively inﬂuence offspring phenotype via maternal

effects is widely ack nowledged, but corresponding nongenetic paternal effects remain

unexplored. Males can adjust sperm phenotype in response to local conditions, but the

transgenerational consequences of this plasticity are unknown. We manipulated paternal

density of a broadcast spawner (Styela plicata, a solitary ascidean) using methods shown

previously to alter sperm phenotype in the ﬁeld, then conducted in vitro fertilizations that

excluded maternal effects and estimated offspring performance under natural conditions.

Offspring sired by males from low-density experimental populations developed faster and had

a higher hatching success than offspring sired by males living in high densities. In the ﬁeld,

offspring survived relatively better when their environment matched their father’s, raising the

possibility that fathers can adaptively inﬂuence the phenotype of their offspring according to

local conditions. As the only difference between offspring is whether they were artiﬁcially

fertilized by sperm from males kept in high- vs. low-density cages, we can unequivocally

attribute any differences in offspring performance to an environmentally induced paternal

effect. Males of many species manipulate the phenotype of their sperm in response to sperm

competition: our results show this plasticity can inﬂuence offspring ﬁtness, potentially in

adaptive ways, raising the possibility that adaptive nongenetic paternal effects may be more

common than previously thought.

Key words: external fertilization; nongenetic inheritance; parental effect; paternal effect; sperm

phenotype; sperm plasticity; Styela plicata.

INTRODUCTION

Populations are linked across generations both

demogra phi ca lly (numer ically) and phenotypically.

While the demographic links among generations are

obvious and well studied (Ca ley et al. 1996), the

population consequences of phenotypic links between

generations are more subtle and have received less

attention (Marshall and Morgan 2011). The best known

sources of nongenetic phenotypic links between gener-

ations are maternal effects, whereby the environment

and/or phenotype of the mother affects the phenotype of

the offspring (Mousseau and Fox 1998). Maternal

effects can take a variety o f f orms ranging from

hormonal ly indu ced mor phol og ica l and behavi or al

changes, to transference of resistance to pollution and

pathogens, to epigenetic modiﬁcation of gene expression

(Uller 2008, Jablonka and Raz 2009). They can buffer

offspring from environmental change, or act as conduits

whereby stress in one generation reduces productivity in

the n ext (Mousseau and Fox 1998). Surprisingly,

variation in offspring phenotype can have a greater

inﬂuence on population structure than variation in

offspring number (Burgess and Marshall 2011a), and

impacts of maternal effects on population dynamics can

persist for up to three subsequent generations (Benton et

al. 2005). Moreover, mothers can adaptively adjust

offspring phenotype to match local conditions, and

hence maternal effects can initiate and direct adaptive

evolution (Galloway and Etterson 2007, Uller 2008).

While the ecological role of maternal effects is

increasingly well recognized, other sources of nongenetic

phenotypic links among generations have largely been

ignored. In particular, the ecological consequences of

nongenetic paternal effects are poorly understood.

Traditionally, paternal effects (with the obvious excep-

tions of paternal care and paternal genetic effects) have

been assumed to be absent or negligible (Mousseau and

Fox 1998); but this assumption has been challenged by a

recent series of studies showing transgenerational

epigenetic effects transferred down the male line

(reviewed in Curley et al. 2011, Jenkins and Carrell

2012), and transmission of compounds in the sperm

cytoplasm and accessory gland products (Avila et al.

2011). In humans, for example, early paternal smoking

has been linked to an increased body mass index in sons,

and paternal grandfather’s food supply has been linked

Manuscript received 30 January 2013; revised 6 May 2013;

accepted 7 May 2013. Corresponding Editor: A. L. Shanks.

4

E-mail: a.crean@unsw.edu.au

2575

to the mortality risk of grandsons (Pembrey et al. 2006).

In mice, paternal obesity reduces sperm function (Bakos

et al. 2011), impairs embryo development and viability

(Mitchell et al. 2011), and diminishes the reproductive

health of offspring for two generations (Fullston et al.

2012). To date, studies of nongenetic paternal effects

have focused on the transgenerational effects of paternal

behavior, nutrition, toxins, and age (Curley et al. 2011).

Thus, with the exception of cases of paternal care, it is

unclear whether nongeneti c paternal effects can be

adaptive, whether they are vehicles for the transfer of

pathological effects, or whether they are simply physi-

ological inevitabilities (Marshall and Uller 2007).

Concurrently, evidence is accumulating that males can

adjust their sperm phenotype in response to their social

environment and perceived risk of sperm competition

(e.g., Cornwallis and Birkhead 2007, Simmons et al.

2007, Crean and Marshall 2008, Immler et al. 2010). The

functional consequences of this plasticity in sperm

phenotype have only been considered in terms of their

effects on fertilization success (Pizzari and Parker 2009),

and thus this potentially important source of paternal

effects remains unexplored. The recent evidence that

sperm can transfer more than just the paternal genome

raises the possibility that plasticity in sperm phenotype

may have transgenerational consequences for offspring

performance, and thus population dynamics (Zeh and

Zeh 2008). In biomedical studies of humans, there has

been increasing speculation regarding the existence of

‘‘adaptive’’ paternal effects (e.g., the ‘‘thrifty telomere

hypothesis’’; Eisenberg 2011), whereby males adjust the

phenotype of their sperm in response to their own

environment in order to increase offspring ﬁtness. While

the potential for such effects is clearly exciting, it has not

been explored empirically.

External fertilizers are an ideal system in which to

empirically examine paternal effects, as eggs are released

before fertilization, precluding the possibility of females

biasing their investment in response to sperm quantity

or quality (Cunningham and Russell 2000, Sheldon

2000). In addition, sessile organisms cannot move after

settlement to cope with environmental heterogeneity,

and therefore developmental and tra nsgener ational

plasticity may be particularly important mechanisms to

ensure offspring success in these species (Galloway and

Etterson 2007, Uller 2008). However, unlike plants, as

both gametes are released prior to fertili zation in

broadcast spawners, paternal effects may be equally as

likely as maternal effects to develop in these systems.

Variation in conspeciﬁc density is an i mportant

ecological driver in marine sessile communities,

directly inﬂuenc ing the availability of food and space,

with strong effects on post-settlement survival and

growth (Allen et al. 2008). We have previously

demonstrated that the broadcast-spawning ascidian

Styela plicata adjusts the phenotype of their sperm

(size, motility, and longevity) in response to changes

in the density of co nspeciﬁcs (Crean and Marshall

2008). Furthermore, signiﬁcant spatial genetic struc-

ture has been detected am ong S. plic ata individuals at

the same loca ti o n as the pres en t st ud y tha t wer e

0–5 m apart (David et al. 2010), suggesting that

dispersal distances are short an d thus the paternal

environment is likely to be a good predictor of the

offspring environment: a requirement for adaptive

transge ne ra ti on a l plastic it y (Gallow ay and Etterson

2007). Here , we manipulate the paternal environment

using the same ﬁeld manipulation previously shown to

induce plasticity in sper m phenotype (Crean and

Marshall 2008). We use sperm collected from males

in these high- and low-density treatments to in vitro

fertilize a common pool of eggs, and track the

performance of offspring. Offspring developmental

performance is mea sured in the laboratory (due to

logistical constraints), and post-metamorphic perfor-

mance is measured in the ﬁeld in both low- and high-

densit y co nditio ns t o te st for adap tive paternal

effects.

M

ATERIALS AND METHODS

Study species and ﬁeld location

We used the solitary ascidian, Styela plicata, as a

mod el system to examine envir onmentally induced

paternal effects, as previous work has shown that they

exhibit plasticity in sperm size, motility, longevity, and

fertilization potential (Crean and Marshal l 2008).

S. plicata is a hermaphroditic broadcast spawner

commonly found in disturbed habitats such as marinas

and harbors throughout eastern Australia (Kott 1972).

We completed all ﬁeld work at the East Coast Marina

(Manly, Brisbane, Australia; 27.4678 E, 153.1838 S), a

sheltered private-access marina consisting of ﬂoating

pontoons. At this site, S. plicata naturally occur in

densities ranging from isolated individuals to large

clumps (e.g., .50 adults on 1 m length of rope; A. J.

Crean, personal observation). Reproductively mature

individuals are present at this site most of the year,

excluding winter months (July to September; A. J.

Crean, personal observation).

Manipulation of the paternal habitat

The paternal envi ronment was manipulated using the

same density manipulation methods that were shown

previou sly to indu ce adapt ive plas tic ity in sper m

phenotype (Crean and M arshall 2008). Brieﬂy, repro-

ductive adults were collected from the ﬁeld site and

randomly allocated to either a high-density (15

individuals) or a low-density (single individual) treat-

ment cage (cage dimensions, 18 3 18 3 18 cm; mesh

size, 1 cm

2

). Treatment cages were suspended from the

pontoons and maintained in the ﬁeld for one month,

and th en a randomly selected h igh-density in dividual

from each cage and all low-density individuals were

transported to the laboratory at the University of

Queensl and. Im portantly, no mortality of tr eatment

ani mals occurred in any of the high- or low-density

ANGELA J. CREAN ET AL.2576 Ecology, Vol. 94, No. 11

cag es, and thus dif ferences in offspring performance

cannot be explained by selection actin g on fathers in

each treatment group. We also collected randomly

selected non-treatment individual s from our ﬁeld site to

be used as mothers in each as say. Data were collected

from three experimental runs beginning in January,

April, and June 2009, respe ctively. Measurements of

time to hatchi ng, hatching success, and larval size were

collected in run one, cell cleavage rate i n run two, and

post-metamorphic survival in runs two and three.

Within each run, multip le trials were conducted (run

one, six trials; run two, ﬁve tr ials; run three, ﬁve trials),

with each trial using a paired, split-clutch design: a

common pool of eggs was split into two groups and

then each group fertilized with sperm from either a

high-density or low-de nsity treatment male. Trials were

initiated consecutively over two days to minimize

temporal differences.

In vitro fertilization

Experimental in vitro fertilization roughly approxi-

mates natural reproduction for external fertilizers, and

can be tightly controlled such that all offspring are

reared under standardized conditions from fertilization

through to deployment in the ﬁeld (Marshall et al.

2008). As we were interested in the nongenetic effects of

the paternal environment on offspring performance, the

sperm from males in density treatments was used in vitro

to fertilize eggs from non-treatment animals in stan-

dardized conditions. Gametes were extracted using

standard strip-spawning techniques (Crean and Mar-

shall 2008). Eggs were harvested from four randomly

selected non-treatment animals, combined together, and

5 mL of this combined egg solution was transferred into

each of two 35 mm diameter petri dishes, one for each

paternal treatment. Eggs from multiple females were

used in each trial to reduce maternal effects and male-

by-female interactions that could confound the inter-

pretation of results. A 5-mL control egg sample was also

put aside and not exposed to sperm to check for any self-

fertilization of eggs (,1% in all trials). We then collected

5 mL of sperm solution from one high-density and one

low-density treatment animal (order of treatments

randomized), and added the sperm to its assigned petri

dish, gently shaking each sample every two minutes to

mix the sperm and eggs. We washed eggs free of sperm

after 15 minutes (so exposure time was constant between

treatments) by gently rinsing each sample through a 100-

lm ﬁlter, and left the eggs to develop in 10 mL of ﬁltered

seawater in a covered petri dish. The concentration of

sperm solution collected from each male was estimated

using a Neubauer improved hemocytometer under 4003

magniﬁcation (three replicate counts per sample). There

was no difference in the average concentration of sperm

collected from males in high- and low-density treatments

(high-density males, 1.719 3 10

8

6 1.861 3 10

7

sperm/

mL [mean 6 SE]; low-density males, 1.608 3 10

8

6 1.627

3 10

7

; paired t ¼ 0.075; df ¼ 15; P ¼ 0.941; Appendix:

Fig. A1).

Cell cleavage rate

To examine the effect of paternal environment on

embryonic cell cleavage rates, we placed each petri dish

with the developing eggs under a dissecting microscope

at 203 magniﬁcation, and used time-lapse photography

to record a digital image every 30 s with PixeLINK

Capture SE software (PixeLINK, Ottawa, California,

USA). As we did not know the exact time of

fertilization, cell development rate was estimated from

the time of the ﬁrst cleavage to the four-cell stage (Rius

et al. 2010). The size of fertilized eggs was measured

from images before the ﬁrst cleavage using Image-Pro

Express (version 5.1; Media Cybernetics, Silver Spring,

Maryland, USA). We estimated total egg area by

digitally tracing around the perimeter of the follicle

cells, and ovicell area by tracing around the intersection

of the follicle cells and ovicell (Crean and Marshall

2008). We measured 15 eggs per paternal treatment per

trial.

Time to hatching and hatching success

To measure development time and calculate hatching

success, fertilized eggs were individually collected with a

micro-pipette and transferred into individual 10 mm

diameter wells with 2 mL of ﬁltered seawater in a 24-well

plate (one plate per replicate ¼ 24 subsamples). These

plates were left in a constant temperature (CT) cabinet

at 228C overnight. Developing embryos were observed

under a microscope (303 magniﬁcation) every 15

minutes (starting 10 hours post-fertilization) to measure

time to hatching. Eggs that had not hatched after 15

hours, and hatched larvae with highly abnormal

morphology, were classed as unsuccessful. Digital

images of successfully hatched larvae were recorded

under a microscope (453 magniﬁcation), and larval area

was measured by tracing around the perimeter of each

larva using Image-Pro Express. Larval area was

calculated from the mean of at least three traces from

different images of the same individual to minimize the

effects of measurement error.

Post-metamorphic survival

To measure the post-metamorphic performance of

offspring sired by fathers that experienced different

environments, we settled larvae sired by low- and high-

density fathers on to 35 mm diameter petri dishes and

then deployed them into the ﬁeld for two weeks.

Although this may seem to be a short time frame to

measure offspring performance, a previous study on

Styela plicata at the same ﬁeld site showed that 99% of

all post-settlement mortality occurred within the ﬁrst

two weeks after settlement (Rius et al. 2009). We also

examined whether the effects of paternal environment

were context dependent by varying the density of settlers

in the ﬁeld. Our analysis therefore included treatments

November 2013 2577ADAPTIVE NONGENETIC PATERNAL EFFECTS

applied at two scales: the paternal density treatment was

applied at the scale of trial, and the offspring density

treatment was applied at the scale of petri dish.

We fertilized eggs with sperm from males in low and

high densities, transferred the embryos from each

treatment into separate 250-mL beakers ﬁlled with

ﬁltered seawater, and left them covered overnight in

the CT cabinet at 228C. Larvae were collected with a

pipette the following morning (approximately 11 hours

after fertilization), and transferred to pre-roughened and

bio-ﬁlmed petri dishes in a drop of water. For low-

density offspring treatments, we transferred ﬁve larvae

to each petri dish (six low-density offspring replicates

per paternal treatment per trial), to ensure that at least

one larva settled on the base of the dish. For offspring

allocated to the high-density treatment, we transferred

between twenty and thirty larvae to each petri dish

(depending on how many larvae successfully hatched),

with four high-density offspring replicates per paternal

treatment per trial. We ﬁrst ensured larvae were free

swimming and not stuck in the water surface layer, and

then placed the covered petri dishes in the CT cabinet

for 24 hours to allow larvae to settle.

In low-density offspring treatments, we marked the

position of a haphazardly selected settler, and removed

all other settlers so that low-density treatments had a

single individual to mirror paternal density manipula-

tions. In high-density offspring treatments, all settlers

were marked and numbered (number of settlers per dish

ranged from 11 to 26; mean ¼ 20 individuals; mean

density ¼ 0.02 individuals/mm

2

). All replicates were then

transported to the marina in an insulated container ﬁlled

with seawater, and deployed in the ﬁeld that afternoon.

Petri dishes were suspended vertically within a rigid

plastic mesh cage (dimensions: 44 3 28 3 18 cm length 3

width 3 height; mesh size 1 cm

2

), as the majority of

S. plicata were observed growing on vertical surfaces at

the ﬁeld site. Replicates were enclosed within cages as

pilot studies showed that ﬁsh are attracted to ﬁeld

equipment and preferentially eat S. plicata settlers (A. J.

Crean, personal observation). These cages were hung

from the pontoons in the marina, approximately 2 m

below the water surface, and left in the ﬁeld for two

weeks. After this time, all settlers were transported back

to the laboratory and examined under a microscope

(303 magniﬁcation) to calculate survival.

Statistical analyses

Hatching success was modeled as a binary response

using mixed-effects logistic regression (logit-link func-

tion and binomial errors), with treatment (high or low

paternal density) included as a ﬁxed effect and trial

included as a random effect. Eggs that did not hatch or

those that hatched with visible lethal deformities were

considered unsuccessful and given a value of zero in this

analysis. Time to hatching was analyzed using mixed-

effects Cox proportional hazards models, again with

paternal density as a ﬁxed effect and trial included as a

random effect. Exploratory Kaplan-Meier plots indicat-

ed that these data met the assumption of proportional-

ity. We ran the Cox models using two ce nsoring

approaches that make different assumptions about the

fate of unhat ched eggs. First, we onl y included

individuals that hatched successfully and no individuals

were censored. This approach ignores the possibility that

some unhatched eggs could have hatched once moni-

toring ceased. Second, we included all individuals that

hatched successfully and those that did not hatch within

the monitoring period. The individuals that did not

hatch were censored. This approach assumes that all

unhatc hed eggs will eve nt ua ll y hatch, d es pit e the

prospect that ma ny of the unhat ch ed e g gs w ere

nonviable. Average estimates of sperm concentration,

cell development time, egg ovicell and total area, and

larval area were calculated for each trial, and paired t

tests used to test for consistent differences between low

and high-density paternal treatments.

Post-metamorphic survival was modeled as a binary

response using mixed-effects logistic regression (logit-

link function and binomial errors), with trial and dish

included as random effects. The number of larvae that

settled in high-density offspring treatments ranged from

11 to 26 individuals per dish, and for this reason we

chose to treat density as a continuous variable rather

than categorical to retain more of the information

contained within the dataset. Hence, ﬁxed effects were

paternal densit y (h igh or low), offsprin g density

(continuous) and their interaction. As survival data

was collected in runs two and three, we checked for a

possible run effect by ﬁtting models that allowed slopes

and intercepts to vary by run (as random effects). All

random-effect terms for run were not signiﬁcant

(Appendix: Table A1) and the ﬁxed-effect estimates

were very similar to those from models that excluded

run. We therefore present data pooled across both runs

for clarity. The signiﬁcance of ﬁxed-effect terms was

assessed using likelihood ratio tests (LRTs) by compar-

ing nested models with and without the variable of

interest. All analyses were completed using the R

statistical program (R Development Core Team 2013),

and the LME4 package (Bates et al. 2013) used to ﬁt all

mixed effects logistic models.

R

ESULTS

Offspring developmental performance

Embryos fertilized by sperm from fathers in low-

density environments were more likely to successfully

complete development and hatch into larvae ( Fig. 1a),

with 69% (95% CI 55.2–79.5) of eggs fertilized by high-

density males successfully hatching into larvae, com-

pared with an 80% (95% CI 71.1–87.6) hatching success

rate of eggs fertilized by low-density males. Paternal

environment also inﬂuenced the time offspring took to

hatch into larvae, with offspring from high-density

males taking longer to complete development than

offspring from low-density males (Fig. 1b). This result

ANGELA J. CREAN ET AL.2578 Ecology, Vol. 94, No. 11

was conﬁrmed by the mixed-effects Cox models,

although the effect of paternal density on hatching time

was more pronounced in the model that excluded all

unhatched eggs (low paternal density coefﬁcient ¼ 0.79,

SE ¼ 0.154, v

2

1

¼ 26.4, P , 0.0001), compared to the

model that censored unhatched eggs (low paternal

density coefﬁcient ¼ 0.50, SE ¼ 0.14, v

2

1

¼ 12.9, P ¼

0.0003). These coefﬁcients can be interpreted as hazard

ratios to assist interpretation of relative treatment

differences. Using the latter model as an example, the

hazard ratio was e

0.5

¼ 1.65. Thus, by the time that 50%

of the eggs hatched in the high-paternal-density treat-

ment, 68% (1 " 0.5

1.65

) had already hatched in the low-

paternal-density treatment.

To determine what drove this difference in time to

hatching, we me asur ed the cleavage rate of eggs

fertilized by males in each environment. Eggs fertilized

by sperm from high-density males took 6% longer on

average to develop from the two-cell to the four-cell

stage than eggs fertilized with the sperm of low-density

males (mean time from two- to four-cell stage: high-

density father ¼ 882 s, low-density father ¼ 830 s; paired

t ¼ 3.104, df ¼ 4, P ¼ 0.036; Appendix: Fig. A1b). Thus,

it appears that differences in time to hatching are driven

by a faster developmental rate of offspring sired by

males in low densities. There was no signiﬁcant

difference between paternal density treatments in the

size of eggs that were fertilized (total egg area, paired t ¼

"0.558, df ¼ 4, P ¼ 0.607; ovicell, paired t ¼"0.623, df ¼

4, P ¼ 0.567; Figs. A1b and c), and therefore differences

in developmental rate between paternal treatments are

unlikely to be driven by differences in egg size.

Offspring post-metamorphic survival

The effect of the paternal environment on the post-

metamorphic survival of offspring was context-depen-

dent, indicated by a signiﬁcant interaction between

paternal and offspring densities (LRT, v

2

1

¼ 5.1, P ¼

0.020; Table 2; Fig. 2a). To investigate this interaction

further, we ﬁtted two sets of post-hoc models; essential-

ly, simple main effects tests for exploring signiﬁcant

interactions (see Quinn and Keough 2002). First, we ran

mixed-effects logistic regressions for each of the paternal

environments to assess the signiﬁcance of slopes with

offspring density. This revealed that offspring density

had a signiﬁcant negative effect on survival of offspring

of low-density males (LRT, v

2

1

¼ 15.2, P , 0.001), but

that offspring from high-density males survived equally

well at all densities (LRT, v

2

1

¼ 1.03, P ¼ 0.310). Second,

we categorized offspring density into thei r original

low- and high-density treatments and analyzed the

relative survival of offspring within each treatment; as

the relative ﬁtness of offspring from each paternal

treatment within each offspring treatment can provide a

more evolutionarily infor mative estimate of ﬁtness

(Kawecki and Ebert 2004, Stanton and Thiede 2005,

Burgess and Marshall 2011b). In low-density offspring

environments, offspring relative survival was higher for

individuals from low-density males, although this

difference was not statistically signiﬁcant (LRT, v

2

1

¼

2.25, P ¼ 0.134; Fig. 2b). In high-density offspring

environments, the relative survival wa s l ower f or

individuals from low-density males (LRT, v

2

1

¼ 10.41,

P ¼ 0.001; Fig. 2b). As there was no difference in larval

size between paternal-density treatments (paired t ¼

1.397, df ¼ 5, P ¼ 0.221), these observed differences in

pos t-me tamorphic survival cannot be explained by

differences in offspring size.

D

ISCUSSION

Conspeciﬁc density in the paternal environment has

transgenerational consequences for offspr ing perfor-

FIG. 1. Effect of manipulating paternal density on offspring

developmental traits. Offspring sired from high-density males

are shown in black; offspring sired by low-density males are

shown in gray. Large points in panel (a) show the probability

(mean 6 SE) of hatching into a larva with a normal phenotype.

The small points linked by lines are the hatching probabilities

for each trial, as estimated by the mixed-effects logistic model.

The box and whisker plots in panel (b) represent raw values of

time from fertilization to hatching. The thick lines are medians,

boxes are 0.25 and 0.75 quantiles, and whiskers indicate

maximum and minimum values accounting for outliers (circles).

November 2013 2579ADAPTIVE NONGENETIC PATERNAL EFFECTS

Adaptive paternal effects? Experimental evidence that the paternal environment affects offspring performance

Summary (3 min read)

INTRODUCTION

MATERIALS AND METHODS

In vitro fertilization

Cell cleavage rate

Post-metamorphic survival

RESULTS

Offspring post-metamorphic survival

DISCUSSION

ACKNOWLEDGMENTS

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