3.5 Patterns of genomic turnover and genomic vulnerability across
environmental space
We used a GF approach to determine associations between SNPs and
environmental variables and map environmentally-associated genomic
turnover across the total study area. A total of 1396 SNPs (45% of all
SNPs) had R2 values > 0 (0.01-0.704,
average 0.33). When testing model performance, the number of SNPs with
R2 values > 0 for all of the randomized
datasets fell below the number observed for the real data (Fig. S2) and
the mean R2 value generated for the real dataset fell
within the upper 95% quartile of values generated for the randomized
datasets (Fig. S3), both indicating that the GF model shows a stronger
association between environmental and genomic variation for our dataset
relative to the set of randomized datasets. Precipitation of the coldest
quarter, precipitation of the warmest quarter, and latitude were the
most important environmental predictors of genomic turnover (Fig. 3b).
Projected associations between allele frequencies and precipitation of
the coldest quarter, precipitation of the warmest quarter, and latitude
revealed areas of pronounced genomic turnover across the Cameroonian
highlands (pink to orange), forest-savanna ecotone of south-central
Cameroon (orange to green) and across the equator (green to blue) (Fig.
3c). There was also a moderate gradient from the coast to the interior
of Gabon (dark blue to light blue).
Predictions of environmentally associated genomic turnover under future
climate change projections showed the same general pattern of
genotype-environment associations across the landscape relative to
current predictions (Fig. S4). However, there were notable differences
in patterns of genomic turnover between present and future climate
change scenarios that were relatively consistent across both RCPs.
Subtracting the current predictions from the future predictions under
climate change projections (RCP 2.6 & RCP 8.5) for the year 2080
revealed two distinct hotspots of genomic vulnerability (Fig. 3d & Fig.
S5): one centered around the lower Sanaga basin and a second in the far
southeastern region of Cameroon.
4. DISCUSSION
We adopted a comprehensive statistical approach to disentangling the
effects of environment, geographic distance, and landscape barriers on
phenotypic and genomic variation in the African puddle frog P.
auritus . Overall, we find that environmental variation plays an
important role in shaping patterns of morphological and genomic
differentiation. This is in addition to, but independent of, geographic
distance. In particular, seasonal patterns of temperature and
precipitation appear to be key in driving patterns of diversification in
this tropical region, in keeping with a recent meta-analysis conducted
of environmentally-mediated selection across the tropics (Siepielski et
al., 2017). We also find that environmentally heterogeneous landscapes
are important generators of patterns of high phenotypic and genomic
variation suggesting that they may play an important role in promoting
and maintaining biodiversity.
Our first hypothesis posited that temperature and precipitation are the
most important drivers of morphological differentiation. We found that
temperature evenness and precipitation seasonality were significant
predictors of all measures of phenotypic variation. Both temperature and
precipitation are known to be important factors influencing amphibian
development, growth, and population dynamics and are expected to be key
determinants of survival under climate change (Ficetola & Maiorano,
2016; Pounds et al., 1999). Our linear regression results show that body
size increases with temperature evenness and decreases with
precipitation seasonality. Thus, larger bodies are expected to be found
in more uniform habitats with less variation in temperature and
precipitation. This is somewhat consistent with the converse water
availability hypothesis such that in areas with less intense wet and dry
periods there is likely more water available year-round, allowing for
investment in growth.
Body size, relative leg length, and head shape are predicted to vary
along the transition zone between forest and savanna in central Cameroon
where patterns of elevated morphological divergence have also been
reported in a sunbird (Smith et al., 2011). The Cameroonian highlands
and coastal regions of the Gulf of Guinea also appear to be associated
with variation in body size and head shape, possibly due to strong
ecological gradients associated with elevation and precipitation in
these regions. Differences in skull shape morphology have been linked to
the type, size, and speed of prey consumed in frogs and other amphibians
(Emerson, 1985; Kaczmarski et al., 2017; Van Buskirk & Schmidt, 2000;
Vega-Trejo et al., 2014) indicating that head shape might be at least
partly adaptive. Head morphology has also been shown to exhibit
considerable developmental plasticity in response to changes in
temperature and could have important consequences for post-larval
survival (Tejedo et al., 2010). There are also strong gradients
predicted in both body size and relative leg length differentiation from
the coast to the interior of Gabon, likely influenced by the degree of
variation found in the population from Gamba. These frogs may be an
example of cryptic speciation considering they have smaller body sizes
but larger relative leg lengths compared to the rest of the samples and
also given their high levels of genomic differentiation from other
sites. There is also evidence that P. auritus exhibits complex
patterns of spatial niche partitioning (Zimkus et al., 2010) and
extraordinary patterns of diversification (Gvoždík et al., 2020).
Although we cannot disentangle the effects of genetic adaptation and
phenotypic plasticity, it is important to note that tropical ectotherms
are considered to be particularly sensitive to changes in temperature
and/or precipitation so that even subtle shifts in these variables could
have profound impacts on fitness (Deutsch et al., 2008; Ficetola &
Maiorano, 2016).
Our second hypothesis stated that IBE will influence genomic
differentiation more than IBB or IBP. Contrary to many phylogeographic
studies that have been carried out previously in central Africa, we did
not find evidence for an effect of landscape barriers or Pleistocene
refugia on population genomic differentiation. These findings are in
stark contrast to many previous studies that have placed emphasis on the
role of Pleistocene refugia and/or rivers (Anthony et al., 2007;
Bohoussou et al., 2015; Eriksson et al., 2004; Nicolas et al., 2011)
with the exception of Bell et al. (2017) where rivers were not important
in reed frog diversification. However, our findings provide strong
support for the role of environment, specifically seasonal variation in
patterns of precipitation, as the most important environmental factor.
Geographic distance is also consistently identified as a strong
predictor of genomic differentiation. The role of IBD was supported by
findings from our Mantel tests, the significance of geographic distance
in GDM, and the significance of latitude, but not longitude, in
predicting genomic turnover in GF analyses.
Patterns of environmentally-associated genomic differentiation reported
here are consistent with previous investigations of gene-environmental
associations in this region. For example, precipitation has been shown
to be an important predictor of patterns of genetic variation in central
African lizards (Freedman et al., 2010), chimpanzees (Mitchell et al.,
2015), birds (Smith et al., 2011), and forest antelope (Ntie et al.,
2017). In the present study, precipitation of the coldest quarter is
highest in the Cameroon highlands, and decreases progressively
throughout central Cameroon and Gabon (Fig. S6a), mirroring shifts in
genomic turnover observed in P. auritus. Conversely,
precipitation of the warmest quarter is highest in most of Gabon,
especially along the coast and decreases towards Cameroon (Fig. S6b).
Both of these patterns demonstrate shifts in genomic differentiation
throughout the highlands, across the equator, and subtly from coastal to
inland Gabon. Gradients in rainfall not only shape the distribution of
forest cover but also present potentially strong selection pressures on
the phenology of P. auritus since the timing and duration of
amphibian reproductive events are very sensitive to rainfall levels
(Corn, 2005; Ficetola & Maiorano, 2016).
GDM also identified precipitation seasonality as a significant predictor
of genomic turnover. This environmental variable is linked to seasonal
patterns in rainfall availability that are inverted across the Equator
separating Cameroon and Gabon. Rainforests either side of the equator
have their own distinct seasonal patterns of rainfall (Heuertz et al.,
2014) such that the dry season in central Cameroon coincides with the
rainy season in northern Gabon and vice versa. This seasonal inversion
could be responsible for the shift in genomic variation observed inP. auritus across the equator. It has been hypothesized that
these contrasting patterns of seasonal rainfall could lead to
reproductive isolation and speciation across this region (Heuertz et
al., 2014). Future work should look more closely at the seasonal
inversion hypothesis and how heterogeneous annual patterns of rainfall
influence genomic differentiation in other rainforest species.
Our third hypothesis was that areas of greatest
environmentally-associated genomic turnover are associated with strong
environmental gradients across the landscape. Areas of elevated genomic
turnover in P. auritus appear to correspond to known ecological
gradients. Genomic turnover is predicted to be high throughout the
forest-savanna ecotone region south of the montane region in Cameroon
where rainforest habitat in the south gradually transitions to savanna
in the north. These findings are consistent with patterns of high
intraspecific genomic diversity across this ecotonal region in the
rainforest bird Andropadus virens (Zhen et al., 2017) and
soft-furred mouse Praomys misonnei (Morgan et al., 2020). There
is also high genomic turnover in P. auritus across the Cameroon
highlands, reflecting both elevation and distance from the coast. The
Cameroon highlands are a known biodiversity hotspot, especially for
amphibian richness and endemism (Amiet, 2008; Herrmann et al., 2005;
Pauwels & Rodel, 2007; Zimkus & Gvoždík, 2013) so that elevated
genomic turnover in this region is to be expected. Mountain ranges and
elevational gradients are often recognized as important drivers of
genetic heterogeneity and, as is the case here, are important for the
conservation of evolutionary potential. Overall, our results show a
strong role for environment in shaping genomic differentiation such that
areas of elevated genomic turnover span regions of strong ecological
transition, providing further support for the role of environmental
gradients and ecotones in shaping adaptive diversification.
Our fourth hypothesis stated that patterns of environmentally-associated
genomic variation reflect those observed for phenotypic variation.
Patterns of environmentally-associated morphological and genomic
variation are relatively similar in Cameroon. RF and GF projections
suggest that the forest-savanna ecotone in Cameroon is predicted to
result in elevated morphological and genomic variation. These
projections are based on the aggregated effects of the significant
environmental variables which are primarily related to seasonality.
Western Cameroon is characterized by more densely forested areas with
pronounced precipitation seasonality, which then transitions south to
habitats that include both forest and savanna and experience especially
high seasonal variability in temperature and precipitation (Sesink Clee
et al., 2015; Smith et al., 2011). Our findings are consistent with
previous examples indicating that seasonality in moisture levels and
precipitation are key explanatory variables for both morphological and
genomic variation within this region (Smith et al., 2011), and thus
provides further support for the role of environmental variation in
driving diversification. In Gabon, we find relatively uniform patterns
of genomic variation relative to patterns of morphological variation.
While seasonal variation is less pronounced relative to Cameroon, Gabon
harbors a variety of heterogeneous habitats, such as narrow, coastal
alluvial plains, extensive wetlands, patches of savanna, and low
elevation mountain zones (Lee et al., 2006), many of which may present
unique selection pressures contributing to phenotypic variation.
Finally, we posited that genomic vulnerability is predicted to be
highest in areas expected to undergo the greatest environmental change.
We identified several areas of high genomic vulnerability where
populations may be more susceptible to climate change under future
projections. In the present study, the Sanaga River delta area in
southwest Cameroon and an area in the southeast of the country, north of
Lobéké and Nki National Parks, are predicted to be regions of greatest
genomic vulnerability. While most of the Sanaga River is unprotected,
the Douala Edea Wildlife Reserve falls within this area and constitutes
an important target for continued protection. Genomic vulnerability may
be an important metric to incorporate into conservation prioritization
as it may also indicate areas where populations are already susceptible
to present-day environmental pressures. For example, Bay et al. (2018)
have recently shown that yellow warbler (Setophaga petechia )
populations with the highest genomic vulnerability were also
experiencing the largest population declines. Therefore, areas of high
genomic turnover and vulnerability may be important targets for future
conservation efforts since the former serves as centers of high adaptive
potential whereas the latter signal susceptibility to environmental
change.
Although we adopted a genome-wide approach in the present study, our SNP
dataset is only likely to capture a fraction of the total number of loci
in the genome that constitute targets for selection and/or regions of
the genome that may be linked loci under selection. Further research
should focus on linking genotypic variation to phenotypic traits under
selection to more fully understand the evolutionary significance of
divergence across ecological gradients as well as examine the relative
importance of genetic versus environmental factors in contributing to
the observed morphological variation.
Understanding the ecological and historical processes involved in
diversification is important not only for increasing our knowledge of
evolutionary mechanisms, but also for making evolutionarily informed
conservation decisions to protect biodiversity and prioritize new area
for preservation in the light of rapid climate change. By taking a
robust statistical approach to disentangling competing drivers of
differentiation, we show that environmental factors rather than
historical barriers to gene flow are largely responsible for patterns of
morphological differentiation and genomic turnover in our study species.
These findings, therefore, highlight the importance of preserving
heterogeneous environments, such as environmental gradients, in
maintaining species adaptive evolutionary potential and underline the
importance of considering evolutionary processes in the design of future
protected areas.
ACKNOWLEDGEMENTS
We thank the Agence Nationale des Parcs Nationaux, ANPN (permit
#AE130012), Centre National de la Recherche Scientifique et
Technologique, CENAREST (permit #AR0010/13, AR0024/14), Ministère des
Forêts et de la Faune, MINFOF (permit #153/AO/MINFOF/PNCM,
008/A/MINFOF/R), and Ministère de la Recherche Scientifique et de
l’Innovation, MINRESI, as well as all our valuable field guides for
helping organize field collections and processing samples for
exportation. We also thank University of California, Berkeley’s Vincent
J. Coates Genomic Sequencing Laboratory (GSL), for sequencing services.
This research was supported by National Science Foundation grant OISE
1243524. Finally, we would like to thank the two anonymous reviewers for
their extensive suggestions and comments which led to the improvement of
this manuscript.
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