Multiple paternity in clutches of common lizard Lacerta vivipara: data

Molecular Ecology (2004)

, 719–723 doi: 10.1046/j.1365-294X.2004.02102.x

Blackwell Publishing, Ltd.

SHORT COMMUNICATION

Multiple paternity in clutches of common lizard

Lacerta

vivipara

: data from microsatellite markers

D. LALOI,

M. RICHARD,

J. LECOMTE,

†

M. MASSOT

and J. CLOBERT

Laboratoire d’Ecologie, UMR 7625, Université Pierre et Marie Curie, 7 quai Saint Bernard, case 237, 75252 Paris cedex 05, France;

†

Laboratoire Ecologie, Systématique et Evolution, UMR 8079, bât. 362, Université Paris Sud, 91405 Orsay cedex, France

Abstract

The common lizard (

Lacerta vivipara

) is a small live-bearing lacertid that reproduces once

a year. In order to document the poorly known mating system of this species, we present

here an assessment of multiple paternity using microsatellite markers. Paternities were

established within 122 clutches belonging to two wild populations from contrasted areas

and to four seminatural enclosed populations. The proportion of multiply sired clutches

was found to be very high (between 50.0% and 68.2%) and similar among populations,

which suggests that the mating system of this species may be insensitive to environmental

and population conditions.

Keywords

Lacerta vivipara

, Lizards, mating system, microsatellites, multiple paternity

Received 23 September 2003; revision received 20 November 2003; accepted 20 November 2003

Introduction

Multiple mating in female animals constitutes one of the

challenging questions about mating systems. In this broad

field of investigation, studies in vertebrates have been domin-

ated for a long time by works on birds and mammals (e.g.

Birkhead & Møller 1992; Smuts & Smuts 1993; Reynolds

1996; Jennions & Petrie 2000) while reptiles, and especially

lizards, have been understudied until recently. In an extens-

ive review, Olsson & Madsen (1998) found evidence of

multiple copulations in 12 out of 20 lizard species studied

for their mating systems. Nevertheless, most of these data

are based on behavioural observations or on counting of

mating scars resulting from the male’s mouthgrip on the

female’s abdomen during copulation. Such data generally

do not indicate if multiple copulations come from the same

male or from different partners, nor if they lead to effective

multiple paternity. Indeed, recent lizard studies using

molecular markers have revealed that effective paternity

can contrast with the estimation given by matings observa-

tions (Olsson

et al

. 1994, 1996b; Gullberg

et al

. 1997; LeBas

2001). Some of these studies also provided original data

concerning mating systems, for instance on mate choice and

sperm competition (Olsson & Madsen 1998).

Lacerta vivipara

is a small lacertid (adult snout-vent

length = 50–70 mm, females on average larger than males)

living in peatbogs and moist heathlands, widely distributed

throughout Eurasia. This live-bearing lizard reproduces

once a year and has a short mating period in April–May.

With regard to mating system in natural populations,

Bauwens & Verheyen (1985) have detected multiple (two

to three) mating scars in females and they have suggested

that different scars may be from different partners as they

could occur within one to three days. Nevertheless, we

assume that mating scars cannot be used to assess accur-

ately the numbers of partners in this species, at least

because a male can sometimes inflict multiple scars during

a single copulation (personal observation). Behavioural

observations on captive individuals by Heulin (1988) have

proved that females can copulate with more than one male.

However, as the experimental situation (three males and

three females in a small enclosure) may enhance the oppor-

tunity for copulation, this author suggested that polyandry

might be weaker in natural populations. Such a divergence

was observed in the closely related species

Lacerta agilis

where females may mate with more than 15 males in few

days in captivity while they rarely mate more than five to six

times in the wild (Olsson

et al

. 1996a; Olsson & Madsen 1998).

Finally, even if multiple matings occur in

L. vivipara

, there is

no evidence that they lead to effective multiple paternity

within clutches. Here, we present an assessment of multiple

Correspondence: D. Laloi. Fax: (+ 33) (0)1 44 27 35 16; E-mail:

[email protected]

720

D. LALOI

ET AL.

Molecular Ecology

, 13, 719–723

paternity using microsatellite DNA loci. In order to investigate

variations of the level of multiple paternity, we checked

families from three populations with different environmental

conditions: one natural population from a high altitude area,

one natural population from a low altitude area, and four

experimental populations maintained in seminatural enclos-

ures. This experimental population was located at low

altitude but it was originally constituted with lizards com-

ing from the same area as the first studied wild population.

Materials and Methods

Collection of samples in natural populations

We collected samples in two wild populations in 2000. The

first population is located in mountains of southern France

(Mont Lozère, Lozère, 44

′

N, 3

′

E) at an altitude of

1420 m. It has been part of an ongoing demographic and

behavioural study for the past 15 years (Massot & Clobert

2000). The second population is located in northern France

(Forêt d’Orient, Aube, 48

′

N, 4

′

E) at an altitude of

about 110–180 m.

To obtain clutches, pregnant females were captured

in early July and kept in the laboratory until parturition.

This period corresponds to the second month of gestation,

parturition occurring generally in July or early August.

Females were housed in individual terraria with damp soil

and a shelter. To facilitate thermoregulation, we provided

an incandescent lamp as a heat source for 6 h per day. Each

female was also supplied with water and

Pyralis

larvae.

After birth, tissue samples were obtained by cutting 2–

3 mm tail tip of each female and their offspring, a non-

destructive technique since lizards practice natural tail

autotomy to escape predators. In clutches with incomplete

hatching success, we also took tissue samples from dead

born embryos. Then, all females and their viable hatchlings

were released at the place where the female was captured

within five days after birth, i.e. before juvenile dispersal

(Clobert

et al

. 1994). The mother’s capture point is likely

to be close to the offspring natal site as females are highly

sedentary during gestation (Bauwens & Thoen 1981).

Semi-natural enclosed population

We analysed multiple paternity in seminatural enclosed

populations which has been part of a long-term experi-

mental study of demography and dispersal (Boudjemadi

et al

. 1999a). These populations were originally constituted

with lizards collected during July 1995 from Mont Lozère

and brought to the Ecological Research Center of Foljuif

near Paris, France (42

′

N, 2

′

E, altitude 200 m). They

were randomly distributed into four seminatural enclosures

(10

10 m) covered with nets to exclude avian predators,

each enclosure receiving six postgravid females and their

litter, four adults males, and five yearlings of each sex (age

and sex structure similar to natural populations). During

spring 1997 and 1998, gravid females were captured and

kept in the laboratory until they gave birth. The rearing

conditions were similar to those applied for the study of

wild populations. Regular monitoring allowed us to obtain

tissue samples from all males (i.e. putative fathers) in

addition to those of the females and their offspring.

Microsatellite amplification and analysis

Genomic DNA was extracted from ethanol-preserved

samples using Perfect gDNA Blood Mini Isolation kit for

animal blood (Eppendorf). We applied the manufacturer’s

protocol except for the use of a small piece of tissue instead of

blood. To obtain genotypic profiles, we used six highly poly-

morphic microsatellite DNA loci characterized in

Lacerta

vivipara

: Lv-3–19, Lv-4–72, Lv-4-alpha, Lv-2–145, Lv-4-X

and Lv-4–115 (Boudjemadi

et al

. 1999b). Primer sets were

redesigned for multiplexing purposes. Microsatellite poly-

morphism was analysed using the fluorescent dye detec-

tion method. One primer of each locus was labelled, with

VIC (green) for Lv-3–19, Lv-4-alpha and Lv-2–145, NED

(black) for Lv-4–72 and Lv-4–115, and 6-FAM (blue) for

Lv-4-X. Polymerase chain reaction (PCR) was carried out

in 10

L of a mixture containing 15–50 ng of DNA, 50–

200 n

of each primer, 300

of dNTPs, 1

L of Qbiogene

10X incubation buffer (50 m

KCl, 10 m

TrisHCl, 1.5 m

MgCl

, 0.1% TritonX-100, pH 9.0) and 0.25 U of

Taq

DNA

polymerase (Qbiogene). A PCR multiplex (for Lv-3–19,

Lv-4–72, Lv-4-X and Lv-4–115) and simplex (for Lv-4-alpha

and Lv-2–145) was performed in a GeneAmp PCR System

9700 thermocycler, using the following profile: an initial

denaturing step of 12 min at 94

C; followed by 30 cycles

of 15 s at 94

C; 15 s at 53

C (56

C for loci Lv-2–145 and

Lv-4-alpha) and 30 s at 72

C, a final extension step at 72

for 10 min. Electrophoreses were performed with an ABI

310 automated sequencer. Allelic size was determined

using

genescan

software version 2.1 by reference to the

genescan

ROX 400HD size standard and by comparison

to previously scored samples.

Assessment of paternity and analyses

In natural populations, mothers and their offspring were

genotyped for five microsatellite loci (all but Lv-2–145).

Because actual genotypes of putative fathers remained

unknown, the extent of multiple paternity was inferred

from the genotypes of juveniles after subtraction of mater-

nal alleles. Multiple paternity was first detected by the

presence of three or more paternal alleles per locus. Then,

the minimum number of fathers to explain genetic composi-

tion of each clutch was estimated applying a conservative

reconstruction of the possible paternal genotypes. In the

MULTIPLE PATERNITY IN THE COMMON LIZARD

721

Molecular Ecology

, 13, 719–723

enclosed populations, all individuals including potential

fathers were genotyped for the six microsatellite loci. Patern-

ity assignments were then carried out by a likelihood

approach using the

cervus

software version 2.0 (Marshall

et al

. 1998). To prevent false identification of father due to

genotyping or reading errors, particularly in natural popu-

lations where offspring’s genotypes could not be compared

to putative fathers, we ignored additional fathers when the

detection relied on a single locus in a single hatchling.

Conversely, such a precaution may lead to some under-

estimation of the number of fathers. This, however, only

occurred in one clutch from the Mont Lozère population.

Results and Discussion

Forty-five females and their 256 offspring from the Mont

Lozère, as well as 15 females and their 79 offspring from

the Forêt d’Orient, were genotyped for five loci. Consider-

ing all loci, both populations do not deviate significantly

from Hardy–Weinberg equilibrium. Among the 45 clutches

from the Mont Lozère, one clutch with only two hatchlings

was excluded from the estimation of multiple paternity as

it was not possible to detect more than two paternal alleles

in such a situation. Multiple paternity was detected in

30 (68.2%) of the 44 remaining clutches, 28 clutches (63.6%)

being sired by at least two males and two clutches (4.6%) by

at least three males (Table 1). Among the 15 clutches from

the Forêt d’Orient, one clutch with only two hatchlings

was excluded from the analysis of paternity. Multiple patern-

ity was detected in seven clutches (50.0%), six clutches

(42.9%) being sired by at least two males and one clutch

(7.1%) by at least three males (Table 1). In both popula-

tions, clutch size does not differ between singly sired and

multiply sired clutches (

= 0.39,

> 0.10 for the Mont Lozère

population;

= 0.13,

> 0.10 for the Forêt d’Orient popula-

tion; see Table 2 for details on clutches’ characteristics).

From the semi-natural enclosed populations, a total of

670 individuals were genotyped for six loci, including 26

clutches in 1997, 36 clutches in 1998 and all adults. The data

from the four enclosures were pooled since the respective

populations did not differ according to the number of

mates (

= 3.88,

> 0.10). However, data from 1997

and 1998 are presented separately as the population

size increased significantly between these two years,

which might affect the pattern of matings. Considering all

loci, the population does not deviate significantly from

Hardy–Weinberg equilibrium. The exclusionary power of

paternity assignments vary between 0.937 and 0.999

according to enclosure and year. Multiple paternity was

detected in 15 out of 26 clutches in 1997 (57.7%) and in 24

out of 36 clutches in 1998 (66.7%), with, respectively, six

clutches in 1997 (23.1%) and 10 clutches in 1998 (27.8%)

sired by more than two males (Table 1). Extreme cases of

multiple paternity were two clutches sired by four males

(one clutch of six hatchlings in 1997 and one clutch of 13

hatchlings in 1998) while up to five males were found in

one clutch of 11 hatchlings obtained in 1998. Clutch size

does not differ between singly sired and multiply sired

clutches (

= 0.37,

> 0.10 in 1997;

= 0.06,

> 0.10 in 1998;

see Table 2 for details on clutches’ characteristics).

At first sight, the pattern of multiple paternity seems

to differ between surveyed populations with respect to

the number of fathers: clutches sired by more than two

Table 1 Extent of multiple paternity in clutches of common lizard.

Number of fathers were obtained after inference of paternal haplo-

types from hatchlings genotypes in wild populations and after

paternity assignment in the seminatural enclosed populations

Number (%) of clutches with

father

fathers

3–5

fathers

Population from Mont Lozère 14 (31.8) 28 (63.6)2(4.6)*

Population from Forêt d’Orient 7 (50.0)6 (42.9)1 (7.1)*

Enclosed population — 1997 11 (42.3)9 (34.6)6 (23.1)

Enclosed population — 1998 12 (33.3) 14 (38.9) 10 (27.8)

*In natural populations, the proportion of litters with 3–5 fathers

is probably underestimated as a result of the conservative method

applied for detection of paternity.

Clutch size*

% multiply

sired clutches**N Mean ± SE min max

Population from Mont Lozère 45 5.7 ± 1.6 2 9 68.2

Population from Forêt d’Orient 15 5.4 ± 2.0 2 10 50.0

Enclosed population — 1997 26 6.2 ± 2.6 2 13 57.7

Enclosed population — 1998 36 4.9 ± 2.7 1 12 66.6

*Including dead born embryos. **Clutches with only two juveniles were excluded from

the estimation of multiple paternity.

Table 2 Summary of clutches’ characteristics

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D. LALOI

ET AL.

Molecular Ecology

, 13, 719–723

males appear quite rare in natural populations while

they constitute about a quarter of the clutches in the

semi-natural populations. Nevertheless, since the males

(i.e. the putative fathers) remained unknown in natural

populations, the conservative method applied for detec-

tion of paternity may have led to some underestimation

of the actual number of fathers. Indirect evidence for this

underestimation can be given applying the inference of

paternity from juveniles’ genotypes, when males are

unknown, to our data set from enclosed populations. This

leads to an identical estimation of the number of multiply

sired clutches, but to fewer fathers in these clutches

than the number obtained by paternity assignment to

known males (only 4.3% of 1997 clutches and 3.6% of 1998

clutches appeared to be sired by more than two males

when paternity was inferred from hatchlings’ genotypes,

while 23.1% and 27.8% were, respectively, found by patern-

ity assignment). Given that this methodological bias

can explain differences in paternity estimation between

natural and enclosed populations, we suspect that the

pattern of paternity does not differ markedly between

the different populations studied. Indeed, when we pooled

the clutches in two classes, singly sired vs. multiply sired

clutches, the level of multiple paternity did not differ

significantly between populations (

= 2.05,

> 0.10).

This constancy of multiple paternity level among various

sites suggests that the mating system of

Lacerta vivipara

may be insensitive to environmental and population

conditions.

In lacertids, multiple paternity often relate to the co-

existence of conflicting male mating strategies. Most cases

refer to territorial species where territory-holding males

sired the greater part of the hatchlings while floaters may

achieve some matings, leading to multiply sired clutches.

For example, 23–62% of clutches are multiply sired in

Scelophorus virgatus

(Abell 1997), 25% in

Ctenophorus ornatus

(LeBas 2001), 65–82% in

Eulamprus heatwolei

(Morrison

et al

2002). Cases of multiple paternity in nonterritorial lizards

are more scarce. In

Lacerta agilis

, females have been found

to accept courtship by several males (Olsson

et al

. 1994,

1996a) and four out of five clutches (80%) were sired by

more than one male (Gullberg

et al

. 1997). Males of this

species guard their present female between several hours

to several days following copulation, which is an un-

common behaviour in lizards but can be expected in such

a competitive situation. In

Lacerta vivipara

, males do not

form territory (Avery 1976), they do not exhibit any type of

mate guarding and, until now, there has been no evidence

of various male mating strategies. Further studies should

thus address other possible reasons that could lead to an

almost constant pattern of single vs. multiple paternity in

this species. This might include studies on the benefits of

multiple mating, especially for females, as well as on the

possible existence of more than one male strategy.

Acknowledgements

We thank Sandrine Meylan and Jean-François Le Galliard for

encouraging discussions, and three anonymous referees for help-

ful comments on a previous version of the manuscript.

Supplementary material

The following material is avaialbale from

http://www.blackwellpublishing.com/products/journals/

suppmat/MEC/MEC2102/MEC2102sm.htm

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David Laloi works on behavioural ecology with a particular inter-

est in the plasticity of behaviour. In parallel with the development

of genetic markers for ecological purposes, Murielle Richard works

on reproductive behaviour in lizards and birds. Jane Lecomte’s

research subjects include the dynamics of fragmented animal

populations and ecological risk assessment related to trans-

genic plants cultivation. Manuel Massot studies the evolution of

dispersal, phenotypic plasticity, senescence, and consequences of

climate’s changes, mainly based on a long-term survey of common

lizard’s populations. Jean Clobert is investigating population

dynamics, mating systems, evolution of life history traits and

dispersal.