Global Amphibian Declines, Loss of Genetic Diversity and Fitness: A Review

Abstract

It is well established that a decrease in genetic variation can lead to reduced fitness and lack of adaptability to a changing environment. Amphibians are declining on a global scale, and we present a four-point argument as to why this taxonomic group seems especially prone to such genetic processes. We elaborate on the extent of recent fragmentation of amphibian gene pools and we propose the term dissociated populations to describe the residual population structure. To put their well-documented loss of genetic diversity into context, we provide an overview of 34 studies (covering 17 amphibian species) that address a link between genetic variation and >20 different fitness traits in amphibians. Although not all results are unequivocal, clear genetic-fitness-correlations (GFCs) are documented in the majority of the published investigations. In light of the threats faced by amphibians, it is of particular concern that the negative effects of various pollutants, pathogens and increased UV-B radiation are magnified in individuals with little genetic variability. Indeed, ongoing loss of genetic variation might be an important underlying factor in global amphibian declines.

Full text

Diversity 2010, 2, 47-71; doi:10.3390/d2010047
diversity
ISSN 1424-2818
www.mdpi.com/journal/diversity
Review
Global Amphibian Declines, Loss of Genetic Diversity and
Fitness: A Review
Morten E. Allentoft
1,
* and John O’Brien
2
1
School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch,
New Zealand
2
Brannoxtown, Trim, Co. Meath, Ireland; E-Mail: johnob@ucd.ie
* Author to whom correspondence should be addressed; E-Mail: mea39@student.canterbury.ac.nz;
Tel.: +61-422502486.
Received: 4 November 2009 / Accepted: 26 December 2009 / Published: 5 January 2010
Abstract: It is well established that a decrease in genetic variation can lead to reduced
fitness and lack of adaptability to a changing environment. Amphibians are declining on a
global scale, and we present a four-point argument as to why this taxonomic group seems
especially prone to such genetic processes. We elaborate on the extent of recent
fragmentation of amphibian gene pools and we propose the term dissociated populations to
describe the residual population structure. To put their well-documented loss of genetic
diversity into context, we provide an overview of 34 studies (covering 17 amphibian
species) that address a link between genetic variation and >20 different fitness traits in
amphibians. Although not all results are unequivocal, clear genetic-fitness-correlations
(GFCs) are documented in the majority of the published investigations. In light of the
threats faced by amphibians, it is of particular concern that the negative effects of various
pollutants, pathogens and increased UV-B radiation are magnified in individuals with little
genetic variability. Indeed, ongoing loss of genetic variation might be an important
underlying factor in global amphibian declines.
Keywords: amphibian conservation; fitness; genetic diversity; genetic drift; inbreeding
OPEN ACCESS

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1. Introduction
Biodiversity is in the midst of another period of mass extinction, probably comparable to those of
the palaeontological past [1,2]. However, the fundamental difference with regard to the current crisis is
that it has not been brought about by stochastic or catastrophic events, but is instead directly
attributable to anthropogenic effects (e.g., [3,4]). With more than 40% of the World’s amphibian
species in decline [5] and an estimated extinction rate over 200 times that of their natural background
rate [6], amphibians are possibly of greater conservation concern than any other vertebrate group
(although see [7]).
The proposed factors behind these declines are almost as many as they are complex. Amphibians
are generally believed to be sensitive to environmental perturbations, partly because of their central
place in the food chain (being both prey and predator), because they often utilise both terrestrial and
aquatic habitats, and can have very different feeding ecologies at different stages of their life cycles.
Recent research has documented and suggested an array of causes associated with this crisis. In
addition to the ongoing and very obvious destruction and fragmentation of habitat [8-11], these include
effects of climate change [12,13], increased UV-radiation due to ozone depletion (e.g., [14,15]),
predation or competition by invasive species [16,17], pollution (e.g., [18,19]), road-kill [20,21],
over-harvesting for human consumption [22] and diseases [23,24]. Of particular concern is the rapid
and global spread of chytridiomycosis (e.g., [25,26]), caused by a pathogenic fungus, which has been
described as posing the greatest threat to biodiversity of any known disease [1]. Further, many of these
factors can work synergistically in complex interactions [27,28]. For example, it has been argued that
climate change has caused an increase in virulence of the chytrid fungus [29]. Similarly, increased UV-
penetrance [30] and agro-chemicals [31] have both been shown to cause immuno-suppression in
amphibians, thereby further increasing their vulnerability to circulating pathogens.
In this review we deal with another factor which, perhaps, has been somewhat overlooked in
discussions of this topic, i.e., genetic effects as a potential key factor in amphibian global declines and,
in particular, reduced fitness due to eroded genetic diversity. We summarise the available literature to
investigate the extent to which lower genetic variation renders amphibian populations prone to reduced
fitness. It seems very timely to provide an overview of our current knowledge on this subject, because
identifying the key factors contributing to the declines is more important than ever for these highly
endangered animals and, although the issue of low genetic variation has been addressed in many
isolated studies of amphibians, no attempt has been made to compile this information in a cohesive and
comprehensive review. It is also timely because, while maintaining a high level of genetic diversity is
ever so often hailed as an important objective in many conservation contexts, its critical importance in
wild populations remains controversial and more clarification is needed to understand the underlying
processes and their relative importance.
The concept that populations with low levels of genetic variability possess higher extinction risk is
not new [32,33]. Populations with low genetic variation have a higher probability of becoming
genetically inbred (see Textbox), with the potential consequence of lowered fitness. Furthermore, the
inherent genetic variability of many populations is considered ‘adaptive’ to changing environmental
conditions, thereby acting as a buffer against stochastic and catastrophic events (e.g., [34,35]). For that
reason, genetic diversity is perceived as a vital pillar of biodiversity, deserving of protection under

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international conventions and national legislature, such as the Convention on Biological Diversity
(CBD 1992), the Habitats Directive of the European Union (Council Directive 92/43/EEC 1992), and
the National Strategy for the Conservation of Australia’s Biological Diversity (DEST 1996), amongst
others.
1.1. Why Are Amphibians Particularly Vulnerable
Our objective here is not to present a treatise on the theory behind genetic problems in small
populations (but see Textbox for an overview) as detailed discussions of this topic have been presented
elsewhere for a vast array of fauna. Here we focus specifically on amphibians, but before engaging in a
review of the literature examining genetic diversity-fitness correlations (GFCs), we need to clarify why
the discussion is particularly relevant to this taxonomic group. Below, we present a four-point
argument as to why we believe amphibians are prone to severe loss of genetic diversity in recent times.
They include a combination of obvious anthropogenic impacts and natural life-history traits that
operate synergistically to increase the impact of genetic effects: (1) amphibian breeding strategy; (2)
recent, vast and rapid declines in population sizes; (3) habitat fragmentation; (4) a typically low
dispersal capability compared to most other vertebrates.
1.1.1. Small effective population sizes and whole clutch mortality
Loss of genetic diversity by genetic drift and inbreeding is directly linked to the effective
population size (see Textbox). In large stable populations with random mating and many breeders each
year, genetic drift and inbreeding are minimal and genetic diversity is maintained across generations.
Amphibians often have very low effective population sizes [36,37], with just a small proportion of the
reproductively-capable individuals contributing to the gene pool each mating season [38-40]. Also,
breeding success and mortality rates can be highly variable between years [41-43]. While overlapping
generations, or “genetic compensation” through a decrease in polygonous matings [44], may provide a
buffer in this context and minimise unfortunate genetic effects, a few consecutive “bad” years, can
cause a highly fluctuating population size with strong genetic drift as a consequence [45]. Equivalent
to a very low number of effective breeders, genetic drift can reach extreme levels if survival among the
offspring is genetically non-random. For example, if only one or a few “lucky” egg clutches survive
predation or a desiccation event, an entire cohort can, in theory, be comprised solely of siblings. This
issue of selective mortality causing fast genetic drift has been discussed mainly in relation to directly-
developing amphibians, where extreme levels of genetic differentiation have been observed among
populations even in continuous habitat (e.g., [46,47]). Dubois [48] takes this theory one step further,
and suggests that dramatic genetic drift events by whole clutch mortality could perhaps explain the
tremendous and fast radiation of amphibian fauna on Sri Lanka.

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1.1.2. Population declines
This point is arguably the most straightforward and needs only brief mention here. Over 40% of
known amphibian species are considered to be in decline [5,49] and therefore many local amphibian
populations must be experiencing a depletion of genetic diversity due to increased genetic drift and
inbreeding (see Textbox). Destruction of habitat, introduction of competitors or predators, road-kill,
pollution and other environmental hazards compound the effects of already small effective population
sizes and thus, reinforce the negative genetic effects attributable to small populations. A direct link
between genetic variability and population size in wild populations was documented in a large,
influential meta-study by Frankham [50], and this association has also been shown specifically for
amphibians [51,52]. In terms of maintaining genetic diversity, small effective population sizes and
population declines do not necessarily impose a problem if a genetic influx is maintained to counteract
the effects of drift in the local gene pools. However, as we discuss below, gene flow is effectively
prevented in many localities today.
1.1.3. Habitat fragmentation
This point is central to our discussion and, therefore, we discuss it here in some detail. Many
amphibian species in natural environments can be considered to form meta-populations [43,53] due to
their requirements for non-continuous habitat types (e.g., ponds). The gene flow and colonisation
dynamics that characterise meta-population systems ensures that equilibrium between founding and
extinction of localised populations is maintained (e.g., [54]). It is widely acknowledged that
anthropogenic fragmentation of natural habitats constitutes one of the greatest threats to terrestrial
biodiversity [55-58], but also from a strictly genetic viewpoint difficulties can arise when large
contiguous populations become fragmented, or former meta-populations transform into isolated islands
of sundered populations. As a result, the isolated sub-populations become the units on which genetic
drift, inbreeding and selection act [35,59-61] and without the ameliorating influence of gene flow, their
concerted effects impose a more rapid erosion of genetic diversity, exacerbating fitness reductions and
extinction risks. A large number of studies have documented the negative impacts of recent habitat
fragmentation on amphibians (e.g., [8,9,62,63]) but, in relation to the present review, the most
illustrative ones are those comparing genetic variation and/or gene flow in fragmented versus
non-fragmented landscapes in populations of the same species. For example, Arens et al. [64]
compared genetic diversity in moor frog (Rana arvalis) populations in two areas fragmented by roads
and agriculture, but differing in time since the establishment and intensity of the barriers. Higher
genetic differentiation and lower genetic diversity was documented among sub-populations in the most
intensively cultivated area. Andersen et al. [51] provided evidence of how fragmentation has
contributed to bottlenecking and subsequent inbreeding in the European tree frog Hyla arborea.
Hitchings and Beebee [65] showed lower genetic diversity and twice the differentiation among urban
common frog (Rana temporaria) populations compared to populations from rural habitat, despite study
sites in the urban setting being in greater proximity. Lesbarrères et al. [66] documented profound
genetic structuring and significantly lower genetic variation in sub-populations of agile frog (Rana
dalmatina) sampled on either side of a highway, compared to populations sampled far from trafficked

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roads. Large roads have also been identified as effective barriers for common frog (Rana temporaria)
dispersal [67] and, similarly, Vos et al. [68] showed that roads and railways worked as barriers to gene
flow and represented a higher explanatory value to the genetic differentiation between populations of
moor frog (Rana arvalis) than geographic distance between populations. A study by Spear and
Storfer [69] emphasized that fragmentation can take many shapes, e.g., tailed frogs (Ascaphus truei)
prefer low levels of solar radiation, explaining why absence of forest (whether due to natural changes
or anthropogenic activity) was identified as a key restrictor to gene flow. Likewise, recent logging
accounted for higher genetic differentiation and lower genetic diversity in populations of giant
salamander (Dicamptodon tenebrosus) [70]. Although associations of recent habitat fragmentation and
genetic variation in amphibian gene pools have been most intensively examined in Europe and North
America, the trend is by no means unique to the northern hemisphere (e.g., [71-74]).
Low genetic diversity in amphibian populations is of course not always attributable to human
activities. For example, a clear valley-mountain effect has been demonstrated in Columbia spotted
frogs (Rana luteiventris), with a negative correlation of genetic diversity and elevation [75]. The agile
frog (Rana latastei) has demonstrated a strong east-west gradient in genetic diversity, suggesting a loss
of genetic variability through numerous consecutive founder effects as the species expanded westward
from a glacial refugium in Eastern Europe [76]. Similarly, a study of the Natterjack toad (Bufo
calamita) across Europe revealed a remarkable decrease in genetic diversity with increasing northern
latitude that could not be easily attributed to anthropogenic effects [77]. In an elegant study,
Ficetola et al. [78] were able to show that the genetic signal in Rana latastei was jointly shaped by
postglacial colonization patterns and recent fragmentation, but that fitness, in this case hatching
success, was only affected by the latter. It can be a difficult task to discriminate between low genetic
diversity as a result of recent fragmentation or as a remnant of the phylogeographic history (e.g., [62]),
but from a conservation perspective, an attempt to make the distinction is important. The risk of
inbreeding depression is greatly enhanced in the former; whereas populations with a long history of
low diversity may have had time to purge deleterious alleles by natural selection (see Textbox).
We propose the term dissociated populations to describe the residual populations that arise where
former meta-population structures have experienced major disruption to gene flow because of human-
mediated loss of connectivity in the landscape. As discussed above, this has serious negative
implications for the preservation of genetic diversity in many local amphibian gene pools.
1.1.4. Low dispersal rates
Amphibians are often described as highly site-philopatric and demonstrate poor dispersal
capabilities, even in natural environments [79-83]. This is commonly explained by a dependence on
moist habitats and slow terrestrial movement capabilities [84,85]. Although these limitations are
clearly not true for all amphibians and should not be accepted by default [86,87], it is not unreasonable
to use this generalisation when comparing amphibian migration rates [88] or levels of inter-population
genetic differentiation (e.g., [47,89]), with that of other vertebrates. Consequently, many amphibian
sub-populations might be prone to high, background genetic drift because of limited connectivity,
causing a state of natural fragmentation of the gene pool. Further, in modern landscapes, genetic influx
is often severely reduced, making it even more difficult for dissociated populations to counter-balance

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the effects of genetic drift on local gene pools. Low recruitment of dispersing individuals is believed to
play a major role in amphibian declines in fragmented landscapes [9,90,91] and the genetic effects of
restricted gene flow are, as discussed above, well-documented. From a long-term genetic viewpoint,
species of low vagility seem more vulnerable to anthropogenic obstructions since even moderate
fragmentation can completely hamper gene flow. However, it has been argued that highly dispersing
amphibian species actually suffer more from fragmentation in the short term [9]. This seems somewhat
counter-intuitive, but dispersing amphibians are exposed to higher mortality risk in modern landscapes,
for example as road-kill [92]. Likewise, Funk et al. [93] suggested that the introduction of barriers in
the landscape has more severe and immediate effects on species with high dispersal rates, since this
trait plays an important role in their overall population survival, compared to species adapted to a less
dynamic situation.
While we have no intention of presenting a single stereotyped image of amphibians, we still argue
that a combination of the four points mentioned above apply to the vast majority of amphibian
populations. Consequently, it seems reasonable to suggest that the current loss of genetic diversity in
amphibians is likely to be greater than in many other taxa (especially vertebrates). However, large
scale meta-studies, beyond the scope of this article, are needed to examine that hypothesis.
2. Genetic Diversity and Fitness in Amphibians
In the previous section we have outlined why we believe amphibians are experiencing a rapid and
ongoing loss of genetic diversity in many parts of the world. Here, by reviewing the available
literature, we assess the impact of this “molecular erosion” on fitness in amphibians. We discuss the
findings of 34 published studies with direct relevance to this theme (see Appendix 1). The considerable
number of publications addressing what might appear to be a narrow, taxon-specific topic serves to
confirm our contention of a general concern shared by many researchers in the field, and also the
relevance of compiling and interpreting the available information in this review.
2.1. Genetic-Fitness Correlations
Firstly, how and why do genetic-fitness correlations (GFCs) arise? Finite populations lose genetic
variability due to genetic drift, and a distinct negative correlation is expected between loss of genetic
variation and individual/population fitness (see Textbox). In support of this, reduced fitness
(i.e., inbreeding depression) has been reported from practically all species where inbreeding has been
documented [94,95]. Though many publications show that GFCs exist and can sometimes be
established with surprisingly limited genetic information compared to optimal theoretical predictions,
the underlying mechanism is still debated. Three hypotheses have been proposed for a positive
association between genetic variability and fitness (reviewed in [96]): (1) a ‘direct effect’ whereby
GFCs arise through selection acting directly on the loci under study, with higher fitness in
heterozygotes (overdominance). This is pertinent to the study of, for example, the MHC genes
involved in immuno-competence; (2) a ‘local effect’ where selection acts on genes linked to the
molecular markers being studied and GFCs arise because of linkage disequilibrium (associative
overdominance) and (3) A ‘general effect’ where genetic variation at the molecular markers under

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study reflects the level of genome-wide heterozygosity, and variance in fitness is caused by variance in
inbreeding. Obviously, the ‘direct effect’ hypothesis can only be applicable to molecular markers
subjected to selective pressure, such as allozymes. Microsatellites, on the other hand, are neutral
genetic markers and the most widely used in present day population genetics. Though GFCs have been
established countless times with these markers, the degree to which microsatellites represent genome-
wide heterozygosity has been questioned [97], and the relative importance of local and general effects
remains controversial (e.g., [98]). Allozyme data, often used in earlier GFC studies, typically have low
variability and hence little power to detect true differences in genome-wide heterozygosities between
individuals. While highly variable microsatellites may be preferable due to their selective neutrality,
they lack the potential ‘direct effect’ offered by allozymes. Moreover, Lesbarrères et al. [99] showed
how detectability of GFCs was highly environmentally-dependent in Rana temporaria populations.
Thus, GFC studies should preferably be carried out in a context-driven setting. This point is perhaps
corroborated by quick review of Appendix 1 where, in those two instances where tests were made
concurrently in wild and laboratory set-ups, only the wild populations demonstrated significant GFCs.
Despite these caveats, meta-analyses by Reed & Frankham [100], Coltman & Slate [101] and
Chapman et al. [102] generally found weak but yet positive and significant correlations
between proxies of genetic variability and fitness from a variety of datasets representing many
taxonomic groups.
2.2. Review of GFCs in Amphibians
The 34 publications that we consider here represent 17 species of amphibians: 16 anurans and one
caudate (Appendix 1). Published works dealing with GFCs in caecilians could not be found; although
Gower and Wilkinson [103] reviewed the conservation status of this enigmatic amphibian group. The
bias towards the anuran Order may reflect species richness within the group, but also the relative ease
with which large numbers of anuran eggs and larvae can be collected and reared in a controlled
environment. We have not attempted to carry out quantitative meta-analyses on these datasets, as we
feel the experimental set-ups are so heterogeneous as to render any potential correlations meaningless
and because comprehensive investigations of GFCs already exist [100-102]. This review simply serves
to provide an overview to examine the extent and context in which these genetic effects have been
identified in amphibians. Nineteen of the 34 publications we consider directly investigated a
correlation between a measure of genetic diversity and fitness (Appendix 1). In the remaining 15
studies, the authors based “GFC-like” interpretations on more indirect evidence from, for example, the
experimental setup (e.g., [104]), the known demographic history of the sampled populations [90,105],
or by correlating fitness with other relevant genetic measures such as the inbreeding coefficient [51] or
degree of genetic isolation [65], rather than genetic variability per se. For every fitness trait
investigated in each study, we have listed a simple “yes” or “no”, respectively confirming or rejecting
a GFC or a GFC-like association (Appendix 1).
Perhaps displaying the omnipresence of the problem and/or a relative ease of testing these
associations in amphibians, GFCs have clearly been documented in many amphibian species and for
many different fitness traits. Likewise, strong correlations have been described using different types of
genetic markers, for both wild populations and laboratory set-ups, and under normal as well as

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stressful conditions (Appendix 1). In fact, for 15 of the 19 publications directly investigating GFCs, a
positive correlation was demonstrated in one or more of the fitness traits under scrutiny suggesting that
the individuals were affected in at least one life stage. In nine of these studies, GFCs were documented
in all investigated fitness traits. In contrast, only four of these 19 studies rejected an association in all
measured fitness traits (Appendix 1). So, there is a considerable body of evidence in favour of GFCs in
amphibians, albeit offset by a number of studies where such links were not uncovered (as discussed
further below). In addition, convincing GFC-like associations have been shown in several studies
(Appendix 1). However, a qualitative assessment of the documented GFCs in amphibians seems more
appropriate here than actual numbers, which are likely skewed by a publication bias [102]. Also, it is
important to note that an individual needs only to be affected in one trait to be affected overall.
2.2.1. Measures of fitness
In the first publication to assess a link between fitness and genetic diversity in amphibians,
Nace et al. [104] showed in 1970 how post-metamorphic survival considerably declined in highly
inbred lineages of Rana pipiens in the laboratory. The objective of that particular study was not related
to understanding the ecology of amphibians, but rather, the observation of lowered fitness was simply
used to confirm the successful development of gynogenetic individuals, which are useful in
experimental genetic research. Samollow and Soule [106] and Pierce and Mitton [107] represent the
first attempts to directly correlate genetic diversity (measured as heterozygosity in allozyme loci) with
fitness traits in amphibians, and here the results are indeed discussed in an ecological context. Both of
these studies demonstrated how survival and larval growth in wild populations are clearly linked to
levels of genetic diversity and they successfully managed to assess the importance of genetic diversity
for survival during different life history phases. For example, while the periods of winter and
metamorphosis are both highly stressful for amphibians, causing increased mortality, only survival
through the former phase appears to be correlated with heterozygosity [106]. The authors hypothesise
that the genetic basis for surviving climatic events that vary in time, space and severity must be very
different from that underlying survival under a developmental constant such as metamorphosis.
Of the >20 different fitness-related traits listed in Appendix 1, survival (under a variety of settings
and life stages) is arguably the most prominent fitness parameter and certainly the most frequently
assessed. This is, conceivably, because survival is the fitness attribute most exposed to selective
pressure, and therefore most likely to reveal a correlation where one exists. The most illustrative
example of survival being directly linked to genetic diversity is provided by Schmeller et al. [108],
who compared the age of individual adult Rana perezi with allozyme variability and showed a
significant positive correlation. Their conclusion was that the more genetic variation a frog possesses,
the longer it lives. Hitchings and Beebee [109] showed a significant relationship between larval
survival and genetic diversity in Bufo bufo populations and, in accordance with our discussion above
of habitat loss and genetic effects, isolated urban populations appeared much less viable than their
rural counterparts. A very similar pattern was apparent in Rana temporaria populations in the study by
Johansson et al. [110], where small isolated populations displayed less genetic variability and higher
larval mortality than populations in continuous habitat. Thus, again, direct links emerge between
recent, human-mediated changes of the landscape and genetic diversity and fitness. In addition, that

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same study also demonstrated a significant loss of variation in quantitative genetic traits in the
fragmented landscape, suggesting that these amphibian populations are not only experiencing reduced
fitness but also a serious loss of adaptive potential. The authors concluded: “...our results indicate that
agriculturally-induced habitat fragmentation may increase the role of random genetic drift to such an
extent that both genetic variability in neutral marker genes and mean values of fitness-related traits are
reduced...” Likewise, Halverson et al. [111] documented a staggering seven-fold increase in survival
from the least to most heterozygous individuals within a single Rana sylvatica population, measured in
the wild.
Growth, and especially larval growth, has been assessed in many of the publications listed in
Appendix 1. Large larvae often have lower predation risk, are competitively superior to smaller larvae,
and metamorphose earlier and at larger body size, which is closely linked to post-metamorphic
fitness (summarized in [112]). However, this fitness parameter appears less intimately linked with
genetic diversity measures than survival. Eight studies document a GFC or GFC-like association using
larvae growth, whereas another five find no such association. This is perhaps not surprising, since
impaired growth is a less detrimental factor than survival in terms of overall fitness, and hence the
selection pressures operating on this trait must be less pronounced. Regardless, the results of several
studies confirm a direct relationship between growth and genetic diversity (e.g., [99,107,110,113]).
Interestingly, Rowe et al. [52] detected a significant GFC in growth rates of Bufo calamita larvae and
their variability at eight microsatellites, but no correlation with larval survival, demonstrating
that the relative strength of these signals is highly variable and can only truly be assessed on a
case-by-case basis.
The propensity for physical abnormality in larvae also represents a fitness trait often assessed in
amphibians (Appendix 1). In an allozyme survey of British Bufo bufo populations, Hitchings and
Beebee [109] documented a correlation between low genetic diversity and physical abnormalities in
small isolated populations found in urban environments. Similar effects have been observed in Rana
sylvatica [15] and Rana temporaria [65], although the failure to document any correlation between
malformations and individual heterozygosity within a population of Ambystoma tigrinum [114]
confirms that other factors are also responsible for amphibian deformity. Indeed, physical
abnormalities may arise due to a variety of factors, and investigating GFCs in this context without
considering other biotic and abiotic factors, could prove misleading.
Mitton et al. [115], measured oxygen consumption as a proxy for fitness in Ambystoma tigrinum
and correlated it with genetic variation in allozymes. Interestingly, active O
2
consumption (the aerobic
capacity) of an individual was positively correlated with its genetic variation; whereas standard O
2
uptake (cost of maintenance) showed a negative correlation. Both results suggest a very direct fitness
advantage in heterozygotes. The study of Rowe and Beebee [116] on Bufo calamita is notable in that
the experimental set-up facilitated investigation of competitive potential in two populations that
differed markedly in levels of genetic variation and inbreeding. Individuals from the large genetically
diverse population performed significantly better in competition, even when reared in a shared pond
representing the local environment of the small, inbred population. In this context, any adaptations
conferring local advantage on the small inbred population appear to be largely over-ruled by much
greater general fitness among individuals from the genetically diverse population. Clutch size [117]
and hatching success [78] have both shown a significant GFC. The latter study is especially

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interesting in that it effectively manages to isolate the effects of long term genetic loss due to long term
dispersal patterns from more recent habitat fragmentation, with fitness only negatively affected
by the recent genetic loss, again providing a compelling argument for the direct link between
human-mediated landscape changes and reduced amphibian viability because of genetic effects.
2.2.2. Limitations
Not all of the studies listed in Appendix 1 could detect a genetic effect on fitness. Several
explanations for this are available, but the most obvious one is simply that no GFC exists. If only
limited variance exists in the level of inbreeding among individuals, for example because the entire
population is either highly inbred or highly outbred, then no correlation is expected [98,118]. This
could perhaps explain why no GFCs were detected in studies of large healthy populations of A.
tigrinum [114], B. calamita and R. temporaria [119] in contrast to studies that compared individuals
from relatively inbred and outbred populations (e.g., 51,116]). On the other hand, the latter approach
has been criticised because environmental heterogeneity over the geographic range sampled can
potentially create spurious relationships [120]. Other reasons for an apparent lack of GFCs could be
small sample sizes or limitations in the genetic markers applied. Chapman et al. [102] emphasize that
these correlations are normally weak and that many highly polymorphic microsatellites are needed to
effectively detect them. The limitations in less variable markers such as allozymes would be even more
pronounced, unless loci under direct effects (displaying overdominance) are used. Also, the
investigated fitness trait(s) might not be under great selection pressure in the given experimental setup.
In general, life history fitness traits are believed to be more strongly affected by inbreeding compared
to morphological and physiological traits [101]. This prediction is perhaps reflected in Appendix 1
where survival—a life history trait—appears more intimately linked with genetic variability than
growth, which is a morphological trait. Finally, purging of the mutational load (see Textbox) in
previous generations might have obscured an otherwise expected relationship between heterozygosity
and fitness. Despite these limitations, the fact that associations between genetic variability and fitness
are documented so vigorously here confirms the applicability of these approaches and, more
importantly, provides a convincing argument for the contribution of genetic factors to fitness
components and the overall viability of amphibian populations. Although representing a highly
interesting topic, it is slightly less important, from a conservation perspective, whether these
associations arise through direct, local or general effects.
2.2.3. GFCs and synergisms
We now turn our focus towards a few very pertinent studies that are of crucial importance to
understanding the magnitude of some of the threats amphibians presently face (summarized in our
Introduction). It is well known that low levels of genetic diversity can be accompanied by higher
susceptibility to emerging pathogens [121-124]. Thus, it is particularly worrying that one of the
greatest direct threats to amphibians appears to be from pathogens. Different strains of Ranavirus, for
example, are responsible for amphibian mass mortality in Europe and the USA [125]. Pearman and
Garner [126] tested susceptibility to this virus in individuals from populations with low versus high

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genetic diversity in Rana latastei. As suspected, limited mortality was recorded in individuals from the
genetically variable population, whereas there were few survivors in the genetically depauperate
population. As mentioned in the Introduction, the global occurrence of the pathogenic chytrid fungus is
of major concern in amphibian conservation, but despite an urgent relevance, we could not find any
study that examines a direct link between susceptibility to chytridiomycosis and genetic variability in
the host. However, Parris [127] investigated the response to chytrid infection in artificial hybrids
compared to pure strains of two closely-related ranids and documented a reduction in several fitness
traits in all strains, but greater effects were seen among hybrids. This study suggests that the fungus
attacks a variety of genotypes indiscriminately, but also that severe outbreeding can have a negative
effect on individual resistance to the disease. The latter result is perhaps not surprising, since other
negative outbreeding effects, in terms of larval malformations and size, have been documented even
within the same ranid species in crosses between two populations separated by just 130 km [89].
Bridges and Semlitsch [128] showed that variation in the ability of Rana sphenocephala (Lithobates
sphenocephalus) larvae to tolerate an insecticide was closely linked with additive (heritable) genetic
variance, indicating that resistance to such pollutants could perhaps evolve, but only if a high level of
genetic diversity was maintained. Pierce et al. [129] uncovered significant differences between
survival of larvae from different egg clutches when exposed to low pH, and hypothesised an additive
genetic basis for this effect. Several other studies have documented a clear genetic basis for local
adaptations in fitness-related characters in amphibians investigated under a range of set-ups [130-133].
Such results emphasize the crucial evolutionary importance of maintaining high levels of genetic
diversity, especially where amphibians are challenged with the introduction of novel chemical
compounds and physical alterations of the environment, which can require a rapid adaptive response to
prevent extinction.
Increased exposure to UV-B light due to recent ozone depletion is believed to be one of many
important factors in amphibian declines (e.g., [134-136]). Weyrauch and Grubb [15] examined the
synergistic effect of reduced genetic diversity and UV-B exposure and showed that populations of
Rana sylvatica with low genetic diversity had higher larval mortality rates than populations with
higher diversity when both were exposed to direct sunlight. Because of the well-documented and on-
going genetic erosion of amphibian gene pools, these synergistic effects between genetic diversity and
known threats are a major concern and emphasise the difficulties in isolating cause-and-effect
relationships if simplified tools and single parameters are employed.
These latter studies provide a tantalizing glimpse as to how GFCs may underlie global amphibian
declines. While a variety of causes have been suggested and, indeed, demonstrated in recent years, the
effects of these threats are propagated by genetic effects and their concomitant contribution to
individual and/or population fitness. We have only considered briefly the role of additive genetic
variation and heritabilities in this review, although they undoubtedly contribute to the ability of
amphibians to respond to novel threats given their link to evolutionary potential.
3. Conclusions
A growing body of research shows that many amphibian populations are experiencing a depletion
of genetic variation and we offer a four-point argument to explain this progressive deterioration in

Diversity 2010, 2
58
genetic variability. We introduce the term dissociated populations to describe the effect of
contemporary loss of habitat connectivity, causing fragmented gene pools and allowing genetic drift to
operate on small isolated units. The link between modern anthropogenic landscape modifications and
loss of genetic diversity is direct, unambiguous and well-documented.
Theory predicts that loss of genetic variation will lead to reduced fitness because of inbreeding
effects and loss of adaptability and, indeed, in the vast majority of the 34 publications assessing this
link in amphibians, clear correlations have been presented. We note that GFCs have been demonstrated
across many different fitness traits in amphibians, using a variety of genetic markers and are evident
both in laboratory settings, as well as wild populations. Therefore, it is hardly controversial to suggest
that the ongoing reduction of genetic diversity is significantly reducing fitness in many wild amphibian
populations. Controversy may arise, however, in trying to establish the relative importance of genetic
factors compared to more direct threats, such as habitat destruction or spread of chytridiomycosis.
General discussions on the role of genetic effects in extinction have been provided elsewhere, with
examples both in favour of (e.g., [137,138]) and against (e.g., [139]) the process.
This review is not aimed at resolving that particular dispute, but we do wish to emphasize that an
assessment of isolated effects of low genetic diversity (e.g., inbreeding depression) can rarely provide
a complete picture of the potential magnitude of the problems facing threatened species. Several of the
papers included in Appendix 1, show that GFCs are often more pronounced and negative genetic
effects more severe under stressful conditions. Hence, concluding that populations without overt signs
of inbreeding depression are genetically viable, might prove short-sighted. Perhaps even more
importantly, this review also highlights that the effects of several currently know threats to amphibians
are magnified when low genetic variability prevails. Therefore, assessment of the impacts of these
threats would likely gain accuracy and credibility by also discussing their genetic element. Although
large-scale meta-studies are imperative to investigate this further, we propose that genetic depletion
could be a major underlying factor in global amphibian declines, increasing susceptibility to many of
the direct threats amphibians are currently subjected to.
Additionally, given that the greatest (amphibian) species richness and extinction threat lies in
tropical regions, efforts should be made to balance the present significant temperate species bias in
studies of genetic diversity. It is extremely important to increase our knowledge on the genetic
consequences accompanying the ongoing habitat destruction which takes place in many tropical
areas today.
Finally, we hope that this review will serve as yet another strong argument for conservation
biologists as to why population connectivity is of crucial importance in maintaining population fitness
and, just as importantly, in maintaining adaptive potential in a world experiencing massive
environmental and climatic changes.
4. Textbox
4.1. Genetic Drift and Inbreeding—Big ‘Players’ in Small Population Genetics
Each new generation of any sexually-reproducing species represents a sample of the parental gene
pool. Since only one of the two (in diploids) alleles from each parent is passed on to the next

Diversity 2010, 2
59
generation and not all individuals may successfully mate, or may differ in the number of progeny
produced, the allele frequencies in any given locus are prone to stochastic changes across generations.
In time, some alleles will either become fixed or lost to a population unless new mutations or incoming
gene flow counteract these processes. This random loss of heterozygosity is termed genetic drift and
the magnitude is inversely proportional to the effective population size (N
e
). In finite populations,
genetic drift will eliminate heterozygosity at a rate of 1/(2N
e
) per generation, if no selectional
constraints interfere. Thus, small populations lose genetic variation faster than larger ones.
Consider the following equations:
T
fix
= [-4N
e
(1-p)ln(1-p)]/p
and:
T
loss
= [-4N
e
p×ln(p)]/1-p
where T
fix
and T
loss
are the number of generations expected to pass before allele fixation [140] and
allele loss [141] occur, respectively. Clearly, allelic losses and fixations will occur much more rapidly
in small populations. Genetic drift serves to decrease genetic variation within populations but,
concurrently, increases genetic differentiation among populations because stochastic processes fix
different alleles in different gene pools (e.g., [142]). Isolated populations will drift apart genetically
over time, at a rate determined by the sizes of the populations.
Inbreeding is classically considered to arise through consanguinous mating of related individuals,
causing allele frequencies to deviate from Hardy-Weinberg proportions. However, in most wild
populations, inbreeding effects associated with random mating (i.e., genetic drift) are often more
relevant. In small populations, there is a high probability that two related individuals will mate by
chance and produce inbred progeny. Further, in a small population with low genetic diversity, the
probability is relatively high that an individual will inherit two copies of the same allele for any given
locus, thereby becoming autozygous for that locus. If that allele happens to be a recessive deleterious
one, signs of inbreeding depression will emerge, even if the parents were essentially unrelated. The
level of autozygosity in an individual describes the proportion of alleles being identical by descent
(e.g., [143]), and the concomitant negative effects are indistinguishable from “true” inbreeding events
between related individuals. Given that genetic drift and inbreeding in small populations are
inextricably linked, we consider them together here, and their co-dependence and effects can be
visualised through the mathematic relationship:
H
t
/H
0
= [1 - (1/2N
e
)]
t
= 1 - F
t
[140]
where H
t
and H
0
are measures of heterozygosity at times t and 0 respectively, N
e
is the effective
population size and F is the inbreeding coefficient. We can recognize the element 1/2N
e
from above as
the loss of heterozygosity due to genetic drift. Genetic drift and inbreeding events affect the gene pool
by increasing homozygosity and, as a consequence, reveal the phenotypic effects of deleterious
recessive alleles normally present in low frequencies. Inbreeding depression is manifested by a
reduction in ‘fitness’ attributes such as survival, growth, reproductive success, etc. While the impact of
increasing homozygosity of recessive lethal alleles on ‘fitness’ is obvious, it is often the effect of
sub-lethal alleles that contributes most to inbreeding depression and extinction risk [144]. Recessive
lethal alleles are exposed to strong selection pressure and thus are rapidly eliminated (purged) from the
gene pool [145]. In contrast, sub-lethal alleles can persist, both as homozygotes and heterozygotes, and

Diversity 2010, 2
60
can even increase in frequency in small populations where strong genetic drift might override the
purging effects of selection. In any case, the efficiency of purging has been questioned recently [146]
even if such alleles could be purged, new sub-lethal alleles will arise by chance through mutation and
as a result, a mutation-selection balance persists. In addition, balancing selection, either associated
with heterogeneous environmental conditions [147] or through overdominance, plays a significant
role in the maintenance of genetic diversity at ‘fitness’ loci. For example, there are instances where
heterozygous individuals at some loci are ‘fitter’ (overdominance) than homozygotes, even in the
absence of recessive deleterious alleles. This effect is very pronounced in the immuno-competence
MHC gene complex [148,149]. Therefore, when genetic drift reduces the proportion of heterozygotes
in the gene pool, the population as a whole is rendered less ‘fit’. Aside from these potential direct
negative effects of reduced genetic variability, a limiting responsiveness to novel environmental
threats (climate change, pesticides, competition with invasive species, disease, predation etc.) might
develop as well. Because of reduced adaptive potential, genetic effects act to diminish the reproductive
capabilities of isolated populations, and thus their persistence. Overall, the effects of reduced genetic
diversity are of grave concern to small populations. Not only do such effects limit the evolutionary
potential of populations, but in many cases they are also directly and clearly correlated with reduced
fitness [100], potentially perpetuating a downward spiral towards local extinction. It is tempting to
propose the relatively simple solution of establishing links between isolated breeding assemblages to
counteract the negative effects of genetic drift. For example, a management strategy involving artificial
mixing of lineages was successfully utilised to address inbreeding in captive populations of the Puerto
Rican crested toad Peltophyrne lemur [150]. However, caution must be exercised before considering
such artificial gene flow [151]. Outbreeding depression, causing a negative GFC, may arise due to
the disruption of co-adapted gene complexes, either between genes or between genes and the local
environment. Reduced fitness in outbred individuals has been reported from a diversity of plants and
animals, including amphibians [89].
Acknowledgements
We wish to thank three anonymous reviewers for their encouraging and constructive comments on
an early draft of this manuscript.

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© 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
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Appendix 1. An overview of 34 studies that examine a link between genetics and fitness in amphibians.
Taxon Reference Molecular markers Fitness trait GFC Comments
Ambystoma tigrinum Mitton et al. [115]^ 8 allozymes oxygen consumption yes
larvae growth yes
Ambystoma tigrinum Pierce & Mitton [107]^ 7 allozymes early larvae growth (in wild/lab) yes*/yes
late larvae growth (in wild) no*
Ambystoma tigrinum Williams et al. [114]^ 6 microsatellites and mtDNA malformations in adults no*
malformations in larvae no*
Bufo boreas Samollow & Soule [106]^ 9 allozymes survival during metamorphosis no*
survival during winter yes*
Bufo bufo Hitchings & Beebee [109]^ 27 allozymes larvae malformation yes
larvae survival yes
Bufo calamita Rowe & Beebee [113] inbred vs outbred population larvae survival yes* Higher survival in outbred population
larvae growth yes*
Bufo calamita Rowe & Beebee [116] inbred vs outbred population larvae competitive potential yes* Outbred larvae superior in competition
Bufo calamita Rowe et al. [52]^ 8 microsatellites larvae survival no
larvae growth yes
Bufo calamita & Rana temporaria Rowe & Beebee [119]^ 5 and 7 microsatellites larvae growth no
larvae development rate no
Hyla arborea Andersen et al . [51] 12 microsatellites larvae survival yes Lower fitness in inbred populations
Hyla arborea Edenhamn et al. [152] 18 allozymes (only one variable) hatching success no High fitness despite very low genetic variation
larvae survival no
Hyla cinerea McAlpine [117]^ 8 allozymes (in parents) clutch size yes
hatching success yes
parent body size no*
parent mating success no*
Hyla cinerea McAlpine & Smith [153]^ 8 allozymes mating success no* Heterozygosity in one locus did affect fitness though
adult survival no*
Pseudacris clarkii Whitehurst & Pierce [112]^ 9 allozymes larvae growth no
larvae development rate yes
Rana blairi & Rana sphenocephala Parris [127] hybridization experiment larvae suscept. to chytrid fungus yes F1 hybrids more suscept. than parental genotypes
Rana blairi & Rana sphenocephala Parris et al. [154] hybridization experiment larvae suscept. to predation no F1 hybrids and parents performed equally well
Rana blairi & Rana sphenocephala Parris et al. [155-158] hybridization experiments various larvae/juvenile fitness traits diff. results Various hybridization experiments
Rana latastei Ficetola et al . [78]^ 6 microsatellites hatching success yes

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Taxon Reference Molecular marke rs Fitne s s trait GFC Comme nts
Rana latastei P e a rman & Garner [126]^ 6 mic rosatellite s Ranavirus and larvae survival yes
Rana lessonae Sjogren [90] not assessed hatching success no* High fitness despite sampling isolated edge populations
Rana lessonae & Rana esculenta Planade et al . [159] hybridization experiment tadpole parasite load no Hybrids and parental strains had same parasite load
Rana perezi Schmeller et al. [108]^ 13 allozymes age (a dult survival) yes*
Rana pipiens Nace et al. [104] gynogenetic experiment postmetamorphic survival yes Inbred, (uniparental) strains had reduced fitness
Rana ridibunda Zeisset & Beebee [105] 14 RAPD's and 5 microsatellites larvae survial no High fitness despite known founder effect
larvae growth no
Rana sylvatica Halverson et al. [111]^ 10 microsatellites larva e survival (in wild/lab) yes*/no
larvae growth (in wild/lab) no*/no
larvae development rate (in wild/lab) no*/no
Rana sylvatica Weyrauch & Grubb [15]^ 25 RAPD 's hatching success yes
larvae survival yes
larvae malformation yes
larvae suscept. to UV-B yes
Rana sylvatica Wright & Guttman [160]^ 7 allozymes larvae growth no*
Rana temporaria Hitchings & Beebee [65] 16 allozymes larvae survial yes Reduced fitness in fragmented urban populations
larvae malformation yes
Rana temporaria Johansson et al . [110]^ 7 microsatellites larvae survival ye s
larvae growth yes
Rana temporaria Lesbarreres et al. [99]^ 8 mic rosa tellites larvae survival ye s
larvae growth yes
larvae development rate yes
Rana temporaria Lesbarreres et al. [161]^ 8 micros a tellites larvae growth yes*
larvae development rate no*
Rana temporaria Sagvik et al. [89] outbreeding experiment larvae growth yes Outbreeding reduces fitness: a negative GFC
larvae malformation yes
hatching success no
^ = The study provides a direct measure of genetic diversity and correlates it against fitness (a true GFC study)
* = Fitness was measured in wild populations
Clarifying comments are provided when the results refer to a GFC-like study rather than a direct correlation between genetic diversity and fitness