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Herpetological Review, 2008, 39(1), 46–50.
© 2008 by Society for the Study of Amphibians and Reptiles
Genetic Evidence for Single Season Polygyny in
the Northern Leopard Frog (Rana pipiens)
GREGORY A. WILSON1
TARA L. FULTON1
Alberta Conservation Association, Edmonton, Alberta, T6H 4P2, Canada
DANNA M. SCHOCK
Detroit Zoological Society, Royal Oak, Michigan 48067, USA
CYNTHIA A. PASZKOWSKI1
DAVID W. COLTMAN1
1Department of Biological Sciences, University of Alberta
Edmonton, Alberta, T6G 2E9, Canada
A species’ mating system is an important life history character-
istic that impacts many aspects of its ecology and evolution. Ani-
mal mating systems are differentiated by the number of mates each
gender has in a breeding season. Many temperate pond-breeding
anurans display what is likely a polygynandrous mating system,
which may vary with the operational sex ratio of a population
(Halliday and Tejedo 1995; Lode et al. 2004). There is genetic
evidence for multiple paternity in some anuran amphibians, where
eggs from a single mass are fertilized by more than one male
(D’Orgeix and Turner 1995; Laurila and Seppa 1998; Lode and
Lesbarreres 2004; Roberts et al. 1999; Sztatecsny et al. 2006).
However, the ability of males to fertilize eggs from more than one
female during a breeding season has not been demonstrated using
genetic techniques. In the course of a larger study, we had the
opportunity to look for this phenomenon in a population of North-
ern Leopard Frogs (Rana pipiens).
What is known about the mating system of R. pipiens suggests
that males may be capable of breeding with multiple females in a
single season. Rana pipiens is an explosive breeder, and the breed-
ing season can last from a few days to a few weeks (Gilbert et al.
1994 and references therein). If female synchrony is high, there is
a limited time when males can fertilize eggs, which could result in
a functionally monogamous mating system where only full-sibs
and unrelated individuals will be present among the progeny pro-
duced in a given year. However, as is true for other anuran spe-
cies, males sometimes remain at breeding sites after being ob-
served in amplexus (C. A. Paszkowski, unpubl. data), which sug-
gests they may be attempting to find additional mating partners.
Males can increase their fitness by producing offspring with more
than one female in a breeding season (Arnold and Duvall 1994;
Clutton-Brock and Vincent 1991). If males are successful in fer-
tilizing eggs from more than one female, then half-sibs will also
Herpetological Review 39(1), 2008 47
be found within a cohort. In laboratory studies, male R. pipiens
have been observed breeding with more than one female during a
reproductive bout, although a decline in fertilization rates may
have resulted (Paula Jackman, Environment Canada, pers. comm.).
Rana pipiens is considered a Species of Special Concern by the
Committee on the Status of Endangered Wildlife in Canada
(COSEWIC 2000) as its numbers have been declining, especially
in the western part of its range (Rorabaugh 2005). During a study
on the conservation genetics of 14 populations of R. pipiens from
across central and western Canada, we found that the distribution
of genotypes from 31 tadpoles sampled from Belza Pond, Grass-
lands National Park, Saskatchewan, deviated significantly from
linkage equilibrium at 10 of 28 pairwise locus comparisons (see
Results). This was unusual, as no locus pairs deviated from link-
age equilibrium when all populations were considered as a whole
(data not shown). In this larger scale study, “populations” were
defined as either individuals from a single pond or genetically
similar nearby ponds. Deviation from linkage equilibrium can occur
if related individuals are present in a sample. Rana pipiens lays up
to 7000 eggs per breeding season (Corn and Livo 1989), so it is
possible that multiple larvae sampled from a pond may be from
the same egg mass. Because the Belza population was the only
one from which tadpoles were sampled (as opposed to terrestrial
juveniles and adults) we predicted that the distinct pattern observed
at Belza was a result of sampling some combination of full-sibs
(tadpoles likely originating from the same egg mass sharing both
parents), maternal half-sibs (tadpoles likely originating from the
same egg mass sharing a mother but not a father), and paternal
half-sibs (tadpoles likely originating from different egg masses
sharing a father but not a mother). To test this we estimated the
genetic relationships among the tadpoles from Belza to determine
if the sampled individuals shared a familial relationship. If pater-
nal half-sibs are present in the population, this would be the first
genetic confirmation that multiple fertilizations with different fe-
males in a single breeding season are possible by male R. pipiens,
and increases our knowledge of the mating system in this species.
Materials and Methods.—Grasslands National Park,
Saskatchewan, is located within the mixed-grass prairie ecosys-
tem of North America. Belza Pond is ca. 20 × 20 m, located at
49°09'128"N, 107°31'546"W within Grasslands National Park. It
is 500 m from the Frenchman River, but areas between ponds are
quite dry and movement of frogs among ponds is likely infrequent
and highly dependent upon rare weather patterns that cause high
rainfalls. As Belza Pond is spring-fed and in a valley, it generally
holds water throughout the year, despite having a maximum depth
of 150 cm. The average precipitation at the Val Marie,
Saskatchewan, weather station (approximately 10 km from Belza
Pond) over a 12-month period from July to the following June
from 1990 to 2004 was calculated as 410.19 mm (s.d. 104.47 mm,
National Climate Data and Information Archive, Environment
Canada 2007; years with missing data were not included in the
analysis). Annual precipitation for 2003–2004 was 397.1 mm.
Tailclips were collected from 31 tadpoles of R. pipiens at Belza
Pond on a single date (19 July 2004). Tadpoles were collected
opportunistically, but explicit effort was made to sample animals
from throughout the pond. Little is known about the Belza popu-
lation of R. pipiens, but at the time of sampling no adults and
approximately 100 young of year were observed. Tadpoles were
at a minimum of Gosner Stage 42 (where forelimbs erupt, Gosner
1960), and therefore capable of having moved throughout the pond
DNA was extracted using a DNeasy Tissue Kit (Qiagen). For
microsatellite analysis, we used loci Rpi100, Rpi101, Rpi103,
Rpi104, Rpi107, Rpi108 (Hoffman et al. 2003), Rp193, and Rp415
(Hoffman and Blouin 2004a). Each PCR amplification contained
120 µM dNTPs, 0.27 µM each primer (one of which was dye-
labeled), 1.5 U Taq polymerase, 1X PCR buffer (10 mM Tris buffer,
pH 8.8, 0.1% Triton X-100, 0.16 mg/mL bovine serum albumin,
50 mM KCl) and ~40 ng genomic DNA in a final volume of 15µL.
Rpi101 and Rpi193 were amplified with 2.0 mM MgCl2, while all
other PCR reactions contained 2.5 mM MgCl2. PCR cycling con-
ditions were as follows: 1 min at 94°C; 3 cycles of 30 s at 94°C,
20 s at 44°C, 5 s at 72°C; 30 cycles of 15 s at 94°C, 20 s at 45°C,
1 s at 72°C; 30 min extension at 72°C. PCRs were performed on
an Eppendorf Mastercycler and electrophoresed on a 3100-Avant
Genetic Analyzer (ABI).
All loci were examined for deviations from Hardy-Weinberg
equilibrium and linkage disequilibrium using GENEPOP 3.4
(Raymond and Rousset 1995). Queller and Goodnight’s (1989)
relatedness was calculated with KINSHIP 1.3.1 (Goodnight and
Queller 1999). Genotypes for 1000 pairs of unrelated individuals
were also simulated with this program by choosing alleles at each
locus based on the allele frequencies in the Belza population
sample. The relatedness values from simulated unrelated individu-
als can be compared to those observed in the Belza population to
determine if the Belza individuals had higher relatedness values
(and were thus more closely related) than would be expected by
chance. Individuals were assigned to full-sib families nested within
half-sib groups using COLONY 12 (Wang 2004). This program is
known to be effective at identifying full- and half-sib groups in a
number of species (e.g., Carlsson et al. 2007; A. J. Wilson et al.
2005). The COLONY program utilizes maximum likelihood and
allows for errors through allelic dropout and mistyping. Both types
of error rates were set to 0, 0.01 and 0.02, and runs were per-
FIG. 1. Frequency histograms of Queller and Goodnight’s (1989) relat-
edness values for the 31 Belza tadpoles (black) and 1000 simulated unre-
lated individuals (white). Relatedness values are higher in the Belza popu-
lation than in individuals that are simulated to be unrelated, suggesting
the presence of related individuals in our sample.
48 Herpetological Review 39(1), 2008
formed at each of these settings with 10 initial random seeds.
All individuals but one were sequenced for an 812 base pair
(bp) fragment of mitochondrial DNA, corresponding to bases 27-
838 of the NADH dehydrogenase subunit 1 (ND1) gene. The re-
gion was amplified using newly designed primers, RpND1F (5'
GGT TCA AAT CCC CTT ACT A 3') and RpND1R (5' AGT TGG
TCA TAG CGG AAT CGT G 3'), in a 25 µl reaction containing
1X PCR buffer, 2.5 mM MgCl2, 160 µM dNTPs, 0.4 µM each
primer, 1 U Taq polymerase, and ~50 ng genomic DNA. Cycling
conditions were: 5 min at 95°C; 35 cycles of 1 min at 94°C, 1 min
at 54°C, 90 s at 72°C; 5 min extension at 72°C. PCR products
were purified using the QIAquick PCR purification kit (Qiagen)
and directly sequenced using the forward primer and the BigDye
v.3.1.1 (Applied Biosystems) chemistry following the
manufacturer’s protocol. Individuals with variable sites near the
priming region were also sequenced using a reverse primer
RpND1R.int (5' TTG AGG ATA CCG AGG CAG AGC 3') for
sequence confirmation. Unincorporated dye terminators were re-
moved using the DyeEx 96 kit (Qiagen) and the fragments were
resolved using an Applied Biosystems 3730 capillary sequencer.
Sequences were analyzed, basecalled, and aligned with ABI Prism
SeqScape software v.2.1 (Applied Biosystems).
Results.—The number of alleles in the Belza Pond population
per microsatellite locus ranged from 2 to 10, with a mean of 5.25.
The Belza population was significantly out of Hardy-Weinberg
equilibrium when the exact Hardy-Weinberg test was used
(Haldane 1954) and all loci were considered (χ2
16 = 31.16, P <
0.05). We also observed significant (P < 0.05) linkage disequilib-
ria for 10 of 28 locus comparisons. Two of these tests remained
significant following Bonferroni correction for multiple tests (P <
0.0018). Neither linkage nor Hardy-Weinberg disequilibrium was
observed in the 13 other populations of R. pipiens we examined
from across western and central Canada (data not shown). The
distribution of pairwise relatedness values for the Belza popula-
tion and unrelated individuals simulated from this population’s
allele frequencies are shown in Fig. 1. On average, unrelated indi-
viduals have a relatedness value of 0. This increases to 0.25 for
individuals related at the half-sib level, and 0.5 for full-sibs.
Twenty-five of the 465 observed pairwise comparisons within the
Belza population (5.4%) had relatedness values at the full-sib level
or higher. Four of these comparisons (0.9%) were higher than any
of the 1000 simulated r-values for unrelated individuals, and 28
(6.0%) were outside of the upper bound of the 95% confidence
interval of the distribution of simulated unrelated individuals (-
0.425, 0.473), suggesting a high degree of relatedness in these
samples. The shapes of these distributions were significantly dif-
ferent from one another (G-test for heterogeneity based on counts
grouped by 0.1; d.f. = 14; P < 0.0005).
Relatives were common in our sample, as only two individuals
from the Belza population did not assign to a nested half-sib group
generated by COLONY (full-sib groups 8 and 9 in Fig. 2). The
most common arrangement of full-sib groups within half-sib groups
(after examining all error rates and initial seeds) and the mito-
chondrial haplotypes possessed by these individuals is shown in
Fig. 2. Other arrangements were similar, and saw either full-sib
group 4 merged with full-sib group 3 or full-sib group 2 merged
with full-sib group 1. Half-sib group B was the largest, containing
4 full-sib groups and 22 individuals (71% of the sampled popula-
tion). Full-sib group membership ranged from 1 (groups 1, 2, 8,
and 9) to 10 (32% of the sampled population, group 5). Two dif-
ferent mtDNA haplotypes were observed in the Belza population,
differing by three synonymous mutations. These mutations all
occurred in the first 580 bp of our sequenced region which over-
lapped the region sequenced by Hoffman and Blouin (2004b), and
thus their haplotype numbering system was used. One of the nested
half-sib groups (group B) possessed both of the haplotypes ob-
served at Belza. Individuals in full-sib groups 3 and 4 shared hap-
lotype 1 (GenBank accession AF548568) and all other individu-
als shared haplotype 7 (GenBank AF548575). The individual for
which mtDNA sequence was unavailable was assigned to full-sib
Discussion.—Sampling tadpoles from the same age cohort al-
lowed us to examine parentage in a population of R. pipiens using
genetic techniques. Our results show that sib-groups may be com-
monly encountered among tadpoles. When a sample of individu-
als from a population consists of related individuals, the genetic
material they share is overrepresented. For example, if a pair of
full-sibs sharing 50% of their genetic material is sampled, then
the shared material will occur in the sample not once, but twice.
As populations in both Hardy-Weinberg and linkage equilibrium
are assumed to consist of unrelated individuals, the presence of
genetic material shared between relatives violates this assumption
resulting in the rejection of these hypotheses. Under the assump-
tion that the entire population is randomly mating, larvae analyzed
from Belza did not represent a random sampling of the genetic
diversity in this population, as suggested by the rejection of the
null hypotheses of Hardy-Weinberg and linkage equilibria. This is
likely due to the presence of related individuals within our sample,
which is also supported by the fact that pairwise relatedness val-
ues in the Belza population are elevated over what would be ex-
pected in an unrelated sample of individuals (Fig. 1).
FIG. 2. Partitioned full-sib groups (1–9) nested within half-sib groups
(A–E) and the sample size for each full-sib group based on microsatellite
data. The largest square represents the sample from Belza Pond,
Saskatchewan. Gray full-sib squares possess mitochondrial haplotype 1
and white squares possess haplotype 7.
Herpetological Review 39(1), 2008 49
The presence of related tadpoles in our Belza sample allowed
us to test if male R. pipiens are capable of reproducing success-
fully with more than one female per breeding season. Sibship analy-
sis identified high membership in full- and half-sib groups. These
patterns could be explained by multiple paternity (a female breed-
ing with multiple males) which is known to occur in anurans
(D’Orgeix and Turner 1995; Laurila and Seppa 1998; Lode and
Lesbarreres 2004; Roberts et al. 1999; Sztatecsny et al. 2006), or
multiple fertilizations by males, which has not been documented.
Within half-sib group B (Fig. 2), two full-sib groups possess hap-
lotype 1 while the other two full-sib groups possess haplotype 7.
The only way this pattern can occur is if a single male bred with at
least two females that possessed different mitochondrial
haplotypes. This does not mean, however, that all other half-sib
groups we observed were paternal half-sibs. If multiple paternity
occurs, all members of the half-sib group must share the same
mitochondrial haplotype, as it is maternally inherited. However,
paternal half-sibs will also possess identical mitochondrial
haplotypes if their mothers share the same haplotype. Therefore,
if nested full-sib groups within a half-sib group share the same
mitochondrial haplotype, it is impossible to determine which par-
ent they have in common. This lack of resolution typified half-sib
group A (Fig. 2), where both nested full-sib groups possessed hap-
Males entering amplexus and successfully breeding with more
than one female in succession is the best explanation for our ob-
servation of different haplotypes occurring within a half-sib group.
However, this is not the only possible explanation for the phe-
nomenon observed. Spermatozoa can survive for a short period of
time in aquatic environments, and may be able to diffuse into
nearby egg masses if amplexus is synchronous and pairs occur in
close proximity (Laurila and Seppa 1998). However, spermato-
zoa diffusion has been deemed unlikely in other anurans (Lode et
al. 2005; Sztatecsny et al. 2006). Consequently, we do not con-
sider diffused spermatozoa a likely explanation.
This first genetic evidence that wild male anurans are able to
reproduce successfully with more than one female offers new in-
sight into anuran breeding systems. Some male R. pipiens are able
to sire offspring from multiple egg masses in a single breeding
season. This suggests that remaining at a breeding site after fertil-
izing one female’s eggs is a good reproductive strategy for males
and will increase their fitness, as they are sometimes able to mate
with an additional female. This finding also has repercussions for
the sampling of any anuran population for genetic study if sam-
pling unrelated individuals is the goal. As the majority of tadpoles
captured were related at least at the half-sib level, adults should be
targeted to ensure a representative sample of the genetic diversity
within a population. It should be noted, however, that the large
number of half-sibs in our sample was likely exacerbated by the
small size of this population and its isolation from other popula-
tions, which would both result in a higher proportion of related
individuals than would be found in a large population with gene
flow between regions.
Our results also apply to the selection of source individuals to
be used in translocation efforts to restore or augment anuran popu-
lations if maximizing genetic diversity is a management goal (e.g.,
Goossens et al. 2002; Moritz 1999; G. A. Wilson et al. 2005). The
use of subadults or adults as founding stock is more likely to re-
sult in a representative sampling of the genetic diversity present
in the source population. However, since survivorship of translo-
cated adults can be low (Dodd and Seigel 1991), obtaining
subsamples from many egg masses may maximize genetic diver-
sity in translocated animals while minimizing the effect on the
Acknowledgments.—Thanks to R. Sissons of Parks Canada for sample
collection. We thank J. Austin and M. Mahoney for their helpful reviews.
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© 2008 by Society for the Study of Amphibians and Reptiles
A Fluorescent Vertebrate:
the Iberian Worm-lizard
Blanus cinereus (Amphisbaenidae)
DAVID P. MAITLAND
ADAM G. HART*
Department of Natural and Social Sciences
University of Gloucestershire, Cheltenham, GL50 4AZ, UK
*Corresponding author; e-mail: email@example.com
Luminescence is the emission of radiation in excess of thermal
radiation (Leverenz 1968). In biological systems there are two main
types of luminescence: chemiluminescence (including biolumi-
nescence) which results from biochemical reactions, and photolu-
minescence (including fluorescence) which results from low-en-
ergy photo-excitation (visible light, UV) (Leverenz 1968). Lumi-
nescence is found sporadically among different groups of organ-
isms (Herring et al. 1990).
Most of the known bioluminescent organisms are marine, and
few are bioluminescent over their entire body surface (Farrant
1997). Bioluminescent terrestrial vertebrates are unknown, but
photoluminescencent fluorescence (where UV light is absorbed
and re-emitted at longer wavelengths without a detectable after-
glow i.e., phosphorescence; Leverenz 1968) is well documented
for some vertebrate structures such as parts of bird feathers (Arnold
et al. 2002). Among other terrestrial organisms whole-body fluo-
rescence is documented in scorpions (Fasel et al. 1997; Frostet al.
2001), and has not been shown previously in vertebrates (although
anecdotal reference is made to this phenomenon in young West-
ern Banded Geckos [Coleonyx variegatus] Stahnke 1972; and the
blind snake Leptotyphlops humilis, Hulse 1971).
We show here for the first time that the Iberian Worm-lizard
Blanus cinereus (Vandelli) 1797 (Reptilia, Lacertilia,
Amphisbaenidae) (Gans 2005) fluoresces blue/green light over its
entire body when irradiated by UV radiation.
Materials and Methods.—During our undergraduate terrestrial
biology field course a live specimen of the amphisbaenian Blanus
cinereus was collected (and released after examination) in the
Algarve, Portugal, along the sea cliffs of Albuifera at the west end
of Praia de Oura Beach during April 2006. Digital photographs
were taken using a Canon 1Ds mark II camera (at 16.7 megapixels)
in Adobe RGB, 12 bit RAW capture mode at ISO 100 and 400.
Macro lenses of 65mm and 100mm focal length and bulb expo-
sure settings were used for fluorescent light capture. White light
images were taken using electronic flash. Images were converted
and analyzed using Adobe CS2 and RGB pixel values were con-
verted to wavelengths using “efg’s Computer Lab”
“WaveLengthToRGB” program in the “SpectraLibrary.PAS” avail-
Blanus cinereus was found to fluoresce using an excitation wave-
length of 368 nm supplied by a portable Ushio ultraviolet lamp,
Blacklight Blue (BLB), fitted with a low-pressure mercury-arc
lamp code F4T5BLB that emits a narrow band of UV radiation
with a sharp spectral peak at 368 nm. Specifications and emission
spectra for the lamp are available at:
Five representative fluorescent regions around the head and body
of the worm lizard were sampled for RGB color pixel values (8
bit) using the eyedropper tool in “Info Palette” of Adobe Photoshop
CS2. Ten readings were sampled at each of the five regions and
the mean (± SD) was obtained from these. Similar measurements
were obtained from the central non-fluorescing scale regions, and
background (soil) for comparison.
Results.—Blanus cinereus fluoresces with a blue-green light be-
tween 437 and 550 nm (mean 465 ± 28 nm; ± SD; N = 5) when
excited by UV at 368 nm (Fig. 1). Fluorescence between, and
around, scale edges occurs over the entire body but is brightest
around the head and tail tip (Fig. 1). A fluorescent scale (brille)
covers the eyes (Fig. 1). The polished surfaces of some scales might
reflect UV, but we were unable to quantify this (Fig. 1b). No phos-
phorescence was observed.
Discussion.—The fluorophor (Leverenz 1968) responsible for
fluorescence in this amphisbaenian is unknown. However, intact
scorpion cuticle fluoresces in the range 440–560 nm (Fasel et al.
1997) as a result of at least two fluorophors; beta-carboline and 7-