Content uploaded by Daniele Canestrelli
Author content
All content in this area was uploaded by Daniele Canestrelli on Mar 26, 2017
Content may be subject to copyright.
Available via license: CC BY 4.0
Content may be subject to copyright.
Climate change promotes hybridisation
between deeply divergent species
Daniele Canestrelli
1
, Roberta Bisconti
1
, Andrea Chiocchio
1
,
Luigi Maiorano
2,3
, Mauro Zampiglia
1
and Giuseppe Nascetti
1
1Department of Ecological and Biological Science, Universita
`degli Studi della Tuscia, Viterbo,
Italy
2Department of Biology and Biotechnology ‘Charles Darwin’, University of Roma ‘La Sapienza’,
Rome, Italy
3Department of Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, Naples, Italy
ABSTRACT
Rare hybridisations between deeply divergent animal species have been reported for
decades in a wide range of taxa, but have often remained unexplained, mainly
considered chance events and reported as anecdotal. Here, we combine field
observations with long-term data concerning natural hybridisations, climate,
land-use, and field-validated species distribution models for two deeply divergent
and naturally sympatric toad species in Europe (Bufo bufo and Bufotes viridis species
groups). We show that climate warming and seasonal extreme temperatures are
conspiring to set the scene for these maladaptive hybridisations, by differentially
affecting life-history traits of both species. Our results identify and provide evidence
of an ultimate cause for such events, and reveal that the potential influence of
climate change on interspecific hybridisations goes far beyond closely related species.
Furthermore, climate projections suggest that the chances for these events will
steadily increase in the near future.
Subjects Ecology, Evolutionary Studies, Zoology
Keywords Climate change, Hybridisation, Pre-mating reproductive barriers, Life-history traits
INTRODUCTION
Hybridisation is a widespread phenomenon in nature (Mallet, 2005). However, its
frequency, diversity of outcomes, underlying mechanisms, its role in the evolutionary
process, and how to deal with it in conservation biology have been controversial topics for
more than a century (Arnold, 2006;Schwenk, Brede & Streit, 2008). Much of our
knowledge about the link between hybridisation dynamics in animals and climate
changes, comes from studies of hybrid zones (Hewitt, 2011), where the reshuffling of
species’ ranges in response to changing climates brought into contact closely related and
previously allopatric species. Pre-mating reproductive barriers could be incomplete
between these species, and their genomes could still be porous to introgression, with
several far reaching implications (Mallet, 2005;Arnold, 2006;Schwenk, Brede & Streit,
2008;Hewitt, 2011). Not surprisingly, species of ancient divergence and with a long-lasting
history of sympatry have contributed the least to this body of knowledge (Mallet, 2005;
Schwenk, Brede & Streit, 2008). These species have had ample opportunity to evolve strong
pre-mating reproductive barriers, either as a by-product of a longer allopatric divergence
How to cite this article Canestrelli et al. (2017), Climate change promotes hybridisation between deeply divergent species. PeerJ 5:e3072;
DOI 10.7717/peerj.3072
Submitted 9 December 2016
Accepted 7 February 2017
Published 23 March 2017
Corresponding author
Roberta Bisconti, bisconti@unitus.it
Academic editor
David Roberts
Additional Information and
Declarations can be found on
page 12
DOI 10.7717/peerj.3072
Copyright
2017 Canestrelli et al.
Distributed under
Creative Commons CC-BY 4.0
or because of character displacement in response to natural selection (Coyne & Orr, 2004;
Pfennig & Pfennig, 2009). Consequently, hybridisation events are extremely improbable
between these species (e.g. Proietti et al., 2014), and their observation incidental in
the wild.
We witnessed to one such event in Southern Italy (on 10 May 2014) between two
toad species, the common toad Bufo bufo and the green toad Bufotes balearicus (Fig. 1).
They belong to the Bufo bufo and Bufotes viridis species groups, whose divergence has
been estimated to the Oligocene (around 20–30 million years ago; Maxson, 1981;
Garcia-Porta et al., 2012), and which have largely overlapping distributions in Central,
Eastern, and Southern Europe (Sillero et al., 2014). Although syntopy is not uncommon,
especially in lowland areas, they show distinct spatio-temporal patterns of habitat use
(reviewed in Lanza, Nistri & Vanni, 2006), making hybridisation at least three-times
unexpected. First, they show markedly different breeding phenologies: Bufo bufo is an
early and explosive breeder, while Bufotes balearicus is a late and prolonged breeder
(Lanza, Nistri & Vanni, 2006). In Italy, breeding activities of Bufo bufo begin earlier in the
year (early winter to early spring, with variation among sites at different altitude and
latitude) and usually last one to two weeks. Bufotes balearicus starts breeding later
(middle to late spring), and this activity may last two to three months. Furthermore,
breeding activities in syntopic areas have been systematically reported as asynchronous,
regardless of intraspecific differences between sites (Lanza, Nistri & Vanni, 2006).
Second, Bufo bufo and Bufotes balearicus display differences in their altitudinal
distribution. Bufo bufo breeding sites commonly occur from 0-2,000 metres above sea level
(MASL), while Bufotes balearicus shows marked preferences for sites in lowland areas,
rarely being observed above 1,000 MASL (Romano, De Cicco & Utzeri, 2003;Spilinga et al.,
2007). Third, in spite of the largely polytopic habits of these two species, differences exist
in habitat and breeding site preferences. Bufotes balearicus favours open areas and
bushlands and breeds in temporary shallow waters, and Bufo bufo most commonly
inhabits forested habitats while using slow-running or deeper, wider standing waters as
breeding sites.
Remarkably, based on previous assessments (Carpino & Capasso, 2008), all these
differences applied to toad populations at our study site as well. This site is a high-altitude
pond located at the margins of a forested area (latitude: 40.9429N; longitude: 14.7096E;
altitude: 1,330 MASL; Fig. 2). Bufo bufo was reported to breed at this site on February,
whereas Bufotes balearicus was absent here and in neighbouring areas above 800 MASL
at least until the year 2007, the year of the last herpetological assessment (Carpino &
Capasso, 2008).
Here, we combine our field observation with data from previous reports of similar
hybridisation events for the Bufo bufo and Bufotes viridis groups in Europe along the
last century, in order to study the causation of these ‘improbable’ events. To this end,
we examined the contribution of multiple factors, including all those commonly
invoked to explain novel interspecific hybridisations among animal species in
the wild.
Canestrelli et al. (2017), PeerJ, DOI 10.7717/peerj.3072 2/16
Figure 1 Interspecific hybridisation between the common toad (Bufo bufo) and the green toad (Bufotes balearicus) in the wild. The hybrid pair
(A) was found spawning (B) on 10 May 2014, at Lake Campo Maggiore, a high-elevation pond within the Partenio Regional Park, Southern Italy
(latitude: 40.9429N; longitude: 14.7096E; altitude: 1,330 MASL). The majority of tadpoles from the hybrid egg-string reared under standard
laboratory conditions were heavily malformed (inset), and none survived until metamorphosis; this pattern was not observed for control tadpoles
from con-specific matings. Photos: M. Zampiglia.
Canestrelli et al. (2017), PeerJ, DOI 10.7717/peerj.3072 3/16
MATERIALS AND METHODS
Assessing natural hybridisation
We sampled and carried to the laboratory a strip, approximately 1.5 m in length from
the clutch laid by the hybrid pair on 10 May 2014. We visually searched for additional
clues of hybridisation at the breeding pond for the subsequent 10 days. Although no
further heterospecific pairs were observed, we collected and carried to the laboratory
two additional egg-strings newly laid by unobserved parents. Fieldwork was approved
Figure 2 Land-use change detection analysis. (A) Location of the study area in Italy. (B) Map of the land-use in 2006, as obtained from
direct interpretation of an aerial photo collected on 31 October 2006; in the same map, the exact location where the hybridisation event has
been registered is indicated, as well as all areas where land-use was different when compared to a second aerial photo collected on 9 October 2014.
Both aerial photos were obtained from Google Earth Pro 7.1.5.1557 (Google Inc., Mountain View, CA, USA). (C) Average percent change (range of
percent change in parenthesis) in land-use classes from 2006 to 2014; total square kilometre area for each land-cover class in 2006 is provided in the
last column. Aerial photos of the breeding site and its neighbourhoods collected on 31 October 2006 (D), and 9 October 2014 (E).
Canestrelli et al. (2017), PeerJ, DOI 10.7717/peerj.3072 4/16
by the Italian Ministry of Environment (permission number: 0042634, dated
7 August 2013).
In order to confirm the hybrid nature of the egg-string laid by the heterospecific pair
(against the hypotheses of unfertilised eggs and of undetected homospecific paternity)
and to address the parental species of the other egg-strings, we monitored egg and tadpole
development under laboratory conditions and analysed the pattern of variation of
individual larvae at diagnostic genetic markers. Tadpoles were reared under standardised
light and food conditions, in plastic boxes (0.8 0.5 0.2 m) filled with oxygenated tap
water. Larval mortality was checked twice daily, from hatching to metamorphosis.
Tadpoles of Bufo bufo and Bufotes balearicus can be distinguished by larval morphology
(Ambrogio & Mezzadri, 2014) while hybrid tadpoles are usually heavily malformed
(Montalenti, 1932,1933). However, in order to achieve correct identification and to
verify the absence of backcrosses between hybrids and parental individuals, we analysed
genetic variation at the following allozyme loci: (i) malate dehydrogenase (Mdh-1 and
Mdh-2; EC 1.1.1.37); (ii) isocitrate dehydrogenase (Icdh-1 and Icdh-2; EC 1.1.1.42); (iii)
and malate dehydrogenase NADP+-dependent (Mdhp-1; EC 1.1.1.40). Fifty tadpoles from
each egg-string were euthanised using a 200 mg/L solution of MS222, 10 days after hatching,
and stored at -80 C until subsequent analyses. The diagnostic value of each allozyme locus
was verified through preliminary analyses of 20 individuals per species, sampled from two
sites in neighbouring areas, where no evidence of potential hybridisation had been observed
(Bufo bufo: 41.1737N, 14.5834E; Bufotes balearicus: 40.8866N, 14.9318E). Standard
horizontal starch gel electrophoresis and zymogram visualisation were carried out,
following previously published standard protocols (Harris & Hopkinson, 1976).
Quantifying anthropogenic habitat change
In order to assess anthropogenic habitat change as a possible explanation for the
hybridisation event we registered, we performed a land-use change analysis considering an
area of 119 km
2
, inside the Partenio Regional Park (Regione Campania, Italy). The
clearing where the hybridisation event was registered is located in the middle of the study
area, approximately 15.6 km from the northern boundary and 2 km from the south-eastern
boundary (Fig. 2). We obtained a map of the area from Google Earth Pro 7.1.5.1557
(Google Inc., Mountain View, CA, USA). By using the ‘historical imagery’ tool, and
keeping the extent and resolution of the map constant, we selected two images: one from
10 October 2014, five months after the hybridisation event, and one from 31 October
2006, a few months before the last assessment that confirmed the absence of Bufotes
balearicus from the site (Carpino & Capasso, 2008). The two images were imported into
ArcGIS 10.3.1 (ESRI©), with a resolution of 4.5 m per pixel, and were georeferenced using
administrative boundaries as reference points (RMS error = 6.23 m for the 2014 image;
RMS error = 4.48 m for the 2006 image). The images were interpreted using direct
recognition (Campbell, 1978), considering five discrete land-use classes that hold a
clear ecological importance for both toad species: agriculture, forests, natural clearings,
natural vegetation (other than forests), and artificial areas. For both images, a vector layer
(format shapefile, ESRI©) was produced at a 1:25,000 scale. To perform the land-cover
Canestrelli et al. (2017), PeerJ, DOI 10.7717/peerj.3072 5/16
change analysis, following Falcucci, Maiorano & Boitani (2007), each vector layer was
transformed into a raster layer using four different pixel resolutions: 25, 50, 75, and 100 m.
The change detection analysis was performed for each pixel resolution, resulting in an
average percentage change for every land-cover class.
Climate influence on altitudinal distribution pattern
Based on historical records of occurrence (Carpino & Capasso, 2008), and considering the
species’ altitudinal distributions in peninsular Italy (Lanza et al., 2007;Guarino et al.,
2012), the presence of a Bufo bufo population at the study site was expected, whereas the
presence of Bufotes balearicus was not expected, either at this site or within neighbouring,
high-altitude areas. Therefore, we focused the following analyses on the latter.
To address the plausibility of climate forcing on recent altitudinal distribution changes
for Bufotes balearicus, we calibrated a correlative species distribution model in peninsular
Italy considering the presence of the species across the 21st century. Then we projected the
distribution model to the current climate (average over 2007–2013) and to the future
(average 2070–2100). The model was calibrated considering six bioclimatic variables
theoretically important for the presence of the species: temperature seasonality, mean
temperature of the warmest quarter, mean temperature of the coldest quarter,
temperature annual range, precipitation seasonality, and precipitation of the coldest
quarter. We obtained all climate variables at 1 km resolution from WORLDCLIM
(Hijmans et al., 2005), which provides climate layers representative of the 1950–2000 time
frame. To obtain the corresponding climate variables for the 2007–2013 time frame, we
followed the procedure presented in Maiorano et al. (2013) and considered monthly
temperature and precipitation values (spatial resolution equal to 50 50 km) from
the Climatic Research Unit of the University of East Anglia (database: CRU TS3.22;
Harris et al., 2014). To downscale the CRU database to the 1 km
2
of the WORLDCLIM
database, we first calculated climate anomalies by contrasting monthly temperature and
precipitation values for 2007–2013 against the 1950–2000 climate data, as obtained from
the same CRU TS3.22 database. Anomalies were calculated as absolute temperature
difference (C) and relative precipitation differences (% change). By using bilinear
resampling, we downscaled the anomalies to 0.0083of spatial resolution (≈1 km). Then,
in order to obtain monthly maps of temperature and precipitation for 2007–2013,
we applied the anomaly corrections to the WORLDCLIM climate layers. Finally,
we calculated all the derived climate maps mentioned above.
The bioclimatic layers for 2070–2100 were obtained directly at the resolution of 1 km
2
from the WORLDCLIM database considering three emission scenarios (A1B, A2, and B1),
and many different global circulation models (24 GCMs for the A1B emission scenario,
19 GCMs for A2, and 18 GCMs for B1) developed under the 4th assessment report of
the Intergovernmental Panel on Climate Change (2007).
To calibrate the models, we used the ensemble forecasting approach (Arau
´jo & New,
2007) implemented in BIOMOD, a bioclimatic niche modelling package for the
Renvironment (Thuiller et al., 2009). We used the following eight models: (i) generalised
linear models; (ii) generalised additive models; (iii) classification tree analysis;
Canestrelli et al. (2017), PeerJ, DOI 10.7717/peerj.3072 6/16
(iv) artificial neural networks; (v) generalised boosted models; (vi) random forests;
(vii) flexible discriminant analysis; and (viii) multivariate adaptive regression spline.
All models were calibrated over the entirety of peninsular Italy south of the Po river
(212,460 km
2
), with 350 points of presence for Bufotes balearicus collected before 2000,
plus 10,000 background points (see Supplemental Information 1). All models were
evaluated using a repeated split-plot procedure (70% of the data used for calibration,
30% left apart for evaluation; the entire procedure repeated 10 times for each model;
Thuiller et al., 2009), and by measuring the area under the receiver operating characteristic
(ROC) curve (AUC) (Swets, 1988). All models with AUC values greater than 0.7
(Swets, 1988) were projected over the entire study area using the 1950–2000, the
2007–2013, and the 2070–2100 climate layers. We measured the minimum probability of
presence obtained in correspondence of the available points of presence for 1950–2000,
and we used this threshold to define areas of species presence (all areas above this
minimum threshold of probability) in all periods considered. Moreover, considering
100 m wide elevation classes, we calculated the elevation-specific average probability of
presence for all three periods, and obtained a model of the probability of presence for
Bufotes balearicus along the elevation range in peninsular Italy (Fig. 3).
We further investigated the plausibility of a link between climate change and altitudinal
shifts by turning the model prediction into a working hypothesis. Based on the model
results, we selected a geographic area close to our study site, and carried out field searches
for further, unknown sites of occurrence of Bufotes balearicus, above 1,200 MASL. To select
the geographic area, we adopted the following criteria: (i) location on a mountain massif,
as close as possible to our study site; (ii) presence of potential breeding sites (e.g. ponds)
at altitudes 1,200 MASL; (iii) presence of Bufotes balearicus populations at lower
elevations along the same mountain; and (iv) absence of obvious anthropogenic habitat
discontinuities between low and high altitude areas. Accordingly, we identified the Picentini
Mountains (within the Picentini Mountains Regional Park, roughly located 25 km south-
east of our study site) as an area of best fit for our criteria. Field searches began on 2 May
2015, and lasted until the first evidence of Bufotes balearicus in the area was found (21 May).
The rationale underlying this experimental integration was as follows: although failure to
identify new high-altitude sites of occurrence would not be strong evidence against a role of
climate change in promoting altitudinal shifts at Lake Campo Maggiore or elsewhere, a
positive result would provide support for the model prediction, and therefore support the
hypothesis that our initial finding belongs to a suite of events promoted by climate change.
Climate influence on breeding phenology
Our observation of the hybrid pair in May 2014 suggests delayed breeding activity of
Bufo bufo causing an overlap with the normal breeding period of Bufotes balearicus
(Lanza, Nistri & Vanni, 2006;Carpino & Capasso, 2008;Guarino et al., 2012). Therefore,
subsequent analyses were focused on Bufo bufo. Notably, while there is strong evidence
for a link between the breeding phenology of Bufo bufo and annual temperature cycles
(Reading, 1998,2003;Tryjanowski, Rybacki & Sparks, 2003), the same does not hold true
for species of the Bufotes viridis group (including Bufotes balearicus).
Canestrelli et al. (2017), PeerJ, DOI 10.7717/peerj.3072 7/16
A search of academic and grey literature revealed five additional observations of
hybrid pairs among representatives of the two species groups in four different locations
(Fig. 3): two sites located in the Czech Republic (Vlc
ˇek, 1995,1997;Zavadil & Roth, 1997),
one in Sweden (Lang, 1926), and one in Austria (Duda, 2008).
The annual activity cycle of Bufo bufo populations can be affected by several
environmental features, including climate, and the five sites (including our observation)
span a wide range of latitudes. Thus, rather than considering average winter temperatures,
we based our analysis on the period when this species begins its breeding activity in
each area, according to regional atlases and databases (Gilsen & Kauri, 1959;Cabela,
Grillitsch & Tiedemann, 2001;Nec
ˇas, Modry
´& Zavadil, 1997;Guarino et al., 2012).
In addition, previous studies suggested that the beginning of this activity is linked to the
average temperatures of the preceding one to two months (Reading, 1998). Therefore, in
Figure 3 Climate correlates of the interspecific hybridisation events observed in the wild between species of the common toad (Bufo bufo) and
the green toad (Bufotes viridis) species groups. Bar plots showing frequency distribution (%) of bimonthly mean temperature deviations (T
m
)
from the 1961–1990 average, compared to the two months preceding the breeding activity at each geographic region: (A) December to January
(Italy, this study), (B) February to March (Sweden, Lang, 1926), and (C–E) January to February (Czech Republic, Vlc
ˇek, 1995,1997,Zavadil & Roth,
1997, respectively). Values for the years when hybrid mates were observed are marked using red arrows. Optimal bar width was computed for each
climatic series following the Freedman–Diaconis rule. (F) Average probability of presence vs elevation at sea level (m) as modelled for the pre-2000
climate (red line), the 2007–2013 climate (solid black line), and the 2070–2010 climate (blue line); the black dotted line indicates the minimum
plausible level of probability of presence, above which the species can be considered present, while below is considered absent. (G) Mean tem-
perature data for each site, and year of observation of interspecific mates. CsL, climatic series length, in years, before the observed event; b-MT,
bimonthly mean temperature; R, rank over the entire climatic series (1 = mildest); T
m
, deviation from the 1961–1990 average temperature (C);
T
10y
, deviation from the preceding 10 year average temperature (C).
Canestrelli et al. (2017), PeerJ, DOI 10.7717/peerj.3072 8/16
our testing for a link between hybridisation events and climate anomalies, we set the
period of interest to the two months preceding the usual start of the breeding activity, for
each geographic area. Accordingly, we analysed date ranges covering December to January
for the site in south-central Italy, January to February for the sites in Czech Republic and
Austria, and February to March for the site in Sweden.
Long-term climate data for our study site were provided, by the Montevergine
Observatory (40.9360N; 14.7288E), as monthly averages since the year 1884. In order to
gain climate data for the four sites of past hybridisation, we searched the NOAA database
(http://gis.ncdc.noaa.gov) of monthly observational data using the following two criteria:
(i) climate station closest to the site of interest; and (ii) time series of at least 40 years
before the year of the observed hybridisation event. The following stations best matched
these search criteria: København Landbohøjskolen, Denmark (Id: DA000030380; latitude:
55.683N; longitude: 12.533E); Praha Klementinum, Czech Republic (Id: EZE00100082;
latitude: 50.090N; longitude: 14.419E); Wien, Austria (Id: AU000005901; latitude:
48.233N; longitude: 16.35E); Oravska
´Lesna
´(Id: LOE00116364; latitude: 49.366N;
longitude: 19.166E).
For each climatic series retrieved, we analysed bimonthly average temperatures
considering the entire temporal series, and the 10 years preceding the hybridisation event,
i.e. a time-lapse approximating the average lifetime of a toad in the wild (Lanza, Nistri &
Vanni, 2006).
To test the null hypothesis that an association between hybridisation events and
temperature anomalies was due to chance alone, we carried out binomial probability tests.
We set the probability threshold of a single event to 0.02, based on the highest value
calculated for the ratio between year rank (mildest = first rank) and climatic series length
(i.e. the first out of 47 available years from the climatic station DA000030380). Since
hybridisation events were both spatially and temporally distant, data independence was
assumed. However, to err on the side of caution, we carried out the analyses considering
the two observations in eastern Czech Republic, as both independent and fully dependent
(i.e. as a single observation); then we took the highest value as the confidence level for
accepting/rejecting the null hypothesis stated above.
Finally, the paucity of hybridisation events recorded qualifies these events as rare, and
testifies to the strength of the pre-mating isolation mechanisms. On the other hand, given
such rareness, we cannot exclude the occurrence of potentially unobserved, unreported, or
undetected hybridisation events. Thus, we explored how potentially unknown events
could affect the significance of our test. To this aim, we carried out additional binomial
probability tests by progressively increasing the number of events while leaving the
number of ‘successes’ unchanged. The null hypotheses of no association was rejected at
the nominal probability threshold a= 0.05.
RESULTS AND DISCUSSION
At the time of our observation (10 May 2014), we counted nine males, three females, and
eight juveniles (22–26 mm long; presumably one year old) of Bufotes balearicus, plus two
female Bufo bufo, and various newly spawned egg-strings.
Canestrelli et al. (2017), PeerJ, DOI 10.7717/peerj.3072 9/16
All tadpoles from the putatively hybrid egg-string were identified as first-generation
hybrids by their heterozygote status at all loci analysed. In line with previous findings
(Montalenti, 1932,1933), most of them were heavily malformed (see Fig. 1), and none
reached the metamorphosis. Instead, at all the loci analysed, tadpoles from the additional
two clutches sampled at the breeding site were homozygotic for Bufotes balearicus
diagnostic alleles, and were thus identified as belonging to this parental species. As
expected, they did not show abnormalities, neither in the external morphology nor in the
ontogenetic pathway.
Despite their wide sympatry, ease of observation, and more than a century-old
knowledge of hybridisation in laboratory crosses, our literature searches for previous
reports of interspecific breeding pairs in the wild, identified just five additional
observations within a 94 year time span (Sweden, Lang, 1926; Czech Republic, Vlc
ˇek, 1995,
1997;Zavadil & Roth, 1997; Austria, Duda, 2008).
Three main hypotheses have been invoked to explain recently established interspecific
hybridisations among animal species, and may have played a role in the present case by
promoting syntopy and breeding season overlap (Crispo et al., 2011;Chunco, 2014):
species translocations, anthropogenic habitat degradation (a derivation of the Anderson’s
‘hybridisation of the habitat’ model; Anderson, 1948) and climate changes.
In the case of Bufo bufo and Bufotes viridis, a species translocation can be firmly
excluded in all the reported cases, based on the extensive knowledge of their natural
geographic distributions (Lanza, Nistri & Vanni, 2006;Sillero et al., 2014), as well as on the
fossil data of both species in Europe (Martı
´n & Sanchiz, 2011).
Anthropogenic habitat degradation has been proposed as a main causative agent in
some case (Duda, 2008). By reducing the diversity and number of potential breeding sites
in a given area, physical alterations of habitat could promote syntopy of previously
allotopic populations. Although plausibly contributing, this hypothesis cannot explain the
entire pattern, and it does not apply to all cases. Our study site (but see also Zavadil &
Roth, 1997) is located within a protected area established in 1993, and an analysis of
contemporary and historical aerial photos of this site and neighbouring areas clearly show
the absence of any physical alterations of potential relevance for the two species (Fig. 2).
Moreover, habitat degradation could not explain the overlap of the two breeding seasons.
Climate changes, however, significantly improve our ability to explain the occurrence of
hybridisation events between these species.
By promoting a recent altitudinal migration of Bufotes balearicus from neighbouring,
lower altitude sites, the ongoing climate warming engendered the unexpected syntopy at
our study site. Support to this argument (the only alternative to recent translocation),
comes from our models of the distribution of Bufotes balearicus in peninsular Italy, based
on a set of known occurrences collected before year 2000, and projected to the average
climate over the period 2007–2013. Indeed, our models indicated that the species’
presence above 1,200 MASL was highly improbable under pre-2000 climate, but became
plausible during 2007–2013 (Fig. 3). Furthermore, projecting the models under future
climate projections for the time period 2070–2100 under different emission scenarios the
general pattern remains unchanged (Fig. 3). The reliability of the models was clearly
Canestrelli et al. (2017), PeerJ, DOI 10.7717/peerj.3072 10/16
confirmed by the field-validation procedure (see Materials and Methods). Indeed, our
field searches of Bufotes balearicus at high-altitude sites of predicted presence in post-2000
projections were successful. We found a previously unreported site of occurrence within
the Picentini Mountains (latitude: 40.8251N; longitude: 14.9864E; roughly 25 km
south-east of the study site), thus confirming that upward migrations of Bufotes balearicus
are ongoing, as predicted by our bioclimatic model (see also Zavadil & Roth, 1997).
Besides being a co-factor for syntopy, climate changes also contributed to the
hybridisation events by promoting an overlap of the breeding activities. Analysing long-
term climate series, we found that the years when hybridisation events were recorded in
Europe (including our observation) ranked first or second hottest on record at most sites,
over time series from 47 to 214 years long. Moreover, bimonthly mean temperatures at
these sites were 2.4 C to 5.5 C above the 1961–1990 averages, and 1.9 C to 4.3 C above
the preceding 10 years averages (Fig. 3). Binomial probability tests allowed us to reject the
null hypothesis of random association between hybridisation events and extremely mild
winters, with very high confidence (binomial probability: P2.3 10
6
). Also, additional
binomial probability tests, carried out in order to explore how unrecorded events could
affect the significance of our test, indicated that the null hypothesis of random association
was rejected (at a= 0.05) until the number of events was 77, while leaving unchanged
the number of ‘successes’.
Although Bufo bufo is expected to bring forward its breeding activity after mild winters
(Reading, 1998,2003;Tryjanowski, Rybacki & Sparks, 2003), at least three lines of support
may help explaining this apparently counter-intuitive pattern. In years, when the breeding
season begins earlier (after a mild winter), breeding has been observed to last longer
(Gittins, Parker & Slater, 1980). Furthermore, a second and lower peak of breeding activity
has been often observed later in the season (Pages, 1984;Reading, 1998), especially after
mild winters (Reading, 1998). Finally, extensive ecophysiological investigations on
bufonid toads, including Bufo bufo, indicate that increased temperatures during
hibernation lead to significant alterations of several processes affecting the breeding
activity, including body size condition, annual ovarian cycle, and seasonal
synchronisation of breeding (Jørgensen, 1992).
Our analyses do not indicate climate change as the single explanatory factor. The
environmental contexts in which interspecific interactions occur, the diverse forms of
habitat disturbance, or behavioural changes during altitudinal migrations (Canestrelli,
Bisconti & Carere, 2016;Canestrelli et al., 2016), might be locally influential. Nonetheless,
these analyses clearly show that climate changes played a fundamental part in promoting
hybridisation events. In light of the direction of these changes (Intergovernmental Panel on
Climate Change, 2014), and of the results of our modelling exercise, we hypothesise that
these events will become progressively more common in the near future. Most
importantly, our results reveal a wider potential influence of climate changes on
interspecific reproductive interactions, particularly in the many instances where climate-
driven asynchrony and/or allotopy are integral components of the reproductive isolating
barriers.
Canestrelli et al. (2017), PeerJ, DOI 10.7717/peerj.3072 11/16
Hybridisation events among non-closely related species are generally believed to yield
events that are transient, and potentially affecting local population demography at most,
because strongly maladaptive (Rhymer & Simberloff, 1996;Malone & Fontenot, 2008).
Nevertheless, there may be exceptions, whereby the effects of maladaptive processes
propagate from population to community level (Farkas et al., 2015). Moreover, as revealed
by years of investigation on the hybridisation process in several animal taxa, including
amphibians, new evolutionary pathways have been sometime opened by such rare and
maladaptive events (Arnold, 2006).
ACKNOWLEDGEMENTS
We thank Maurizio Severini and Graziano Crasta for statistical advice, Vincenzo Capozzi
for providing climatic data for the Montevergine Observatory, and Paola Arduino for
providing support during laboratory procedures.
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
This research was supported by the Italian Ministry of Education, University and Research
(PRIN project 2012FRHYRA). The funders had no role in study design, data collection,
and analysis, decision to publish, or preparation of the manuscript.
Grant Disclosures
The following grant information was disclosed by the authors:
Italian Ministry of Education, University and Research, PRIN project: 2012FRHYRA.
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Daniele Canestrelli conceived and designed the experiments, performed the
experiments, analysed the data, contributed reagents/materials/analysis tools, wrote the
paper, prepared figures and/or tables, reviewed drafts of the paper.
Roberta Bisconti performed the experiments, analysed the data, prepared figures and/or
tables, reviewed drafts of the paper.
Andrea Chiocchio performed the experiments, analysed the data, prepared figures and/or
tables, reviewed drafts of the paper.
Luigi Maiorano analysed the data, contributed reagents/materials/analysis tools, wrote
the paper, prepared figures and/or tables, reviewed drafts of the paper.
Mauro Zampiglia performed the experiments, analysed the data, prepared figures and/or
tables, reviewed drafts of the paper.
Giuseppe Nascetti conceived and designed the experiments, performed the experiments,
contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Canestrelli et al. (2017), PeerJ, DOI 10.7717/peerj.3072 12/16
Animal Ethics
The following information was supplied relating to ethical approvals (i.e. approving body
and any reference numbers):
Observational study: Field experiments were approved by the Italian Ministry of
Environment (permission number: 0042634 dated 7 August 2013).
Data Deposition
The following information was supplied regarding data availability:
The raw data has been supplied as Supplemental Dataset Files.
Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/
10.7717/peerj.3072#supplemental-information.
REFERENCES
Ambrogio A, Mezzadri S. 2014. Girini d’Italia: Tadpoles of Italy. Piacenza: Gavia Edizioni.
Anderson E. 1948. Hybridization of the habitat. Evolution 2(1):1–9 DOI 10.2307/2405610.
Arau
´jo MB, New M. 2007. Ensemble forecasting of species distributions. Trends in Ecology and
Evolution 22(1):42–47 DOI 10.1016/j.tree.2006.09.010.
Arnold ML. 2006. Evolution Through Genetic Exchange. Oxford: Oxford University Press.
Cabela A, Grillitsch H, Tiedemann F. 2001. Atlas zur Verbreitung und O
¨kologie der Amphibien und
Reptilien in O
¨sterreic. Wien: Naturhistorischen Museums.
Campbell JB. 1978. A geographical analysis of image interpretation methods. Professional
Geographer 30(3):264–269 DOI 10.1111/j.0033-0124.1978.00264.x.
Canestrelli D, Bisconti R, Carere C. 2016. Bolder takes all? The behavioral dimension of
biogeography. Trends in Ecology and Evolution 31(1):35–43 DOI 10.1016/j.tree.2015.11.004.
Canestrelli D, Porretta D, Lowe WH, Bisconti R, Carere C, Nascetti G. 2016. The tangled
evolutionary legacies of range expansion and hybridization. Trends in Ecology and Evolution
31(9):677–688 DOI 10.1016/j.tree.2016.06.010.
Carpino F, Capasso S. 2008. I vertebrati terrestri del Parco regionale del Partenio. Napoli: Edizioni
Scientifiche Italiane.
Chunco AJ. 2014. Hybridization in a warmer world. Ecology and Evolution 4(10):2019–2031
DOI 10.1002/ece3.1052.
Coyne JA, Orr HA. 2004. Speciation. Sunderland: Sinauer Associates.
Crispo E, Moore J-S, Lee-Yaw JA, Gray SM, Haller BC. 2011. Broken barriers: human-induced
changes to gene flow and introgression in animals. BioEssays 33(7):508–518
DOI 10.1002/bies.201000154.
Duda M. 2008. First record of a natural male hybrid of Bufo (Pseudepidalea)viridis Laurenti, 1768
and Bufo (Bufo)bufo Linnaeus, 1758 from Austria. Herpetozoa 20:184–186.
Falcucci A, Maiorano L, Boitani L. 2007. Changes in land-use/land-cover patterns in Italy and
their implications for biodiversity conservation. Landscape Ecology 22(4):617–631
DOI 10.007/s10980-006-9056-4.
Farkas TE, Hendry AP, Nosil P, Beckerman AP. 2015. How maladaptation can structure
biodiversity: eco-evolutionary island biogeography. Trends in Ecology and Evolution
30(3):154–160 DOI 10.1016/j.tree.2015.01.002.
Canestrelli et al. (2017), PeerJ, DOI 10.7717/peerj.3072 13/16
Garcia-Porta AJ, Litvinchuk SN, Crochet PA, Romano A, Geniez PH, Lo-Valvo M,
Lymberakis P, Carranza S. 2012. Molecular phylogenetics and historical biogeography of
the west-palearctic common toads (Bufo bufo species complex). Molecular Phylogenetics and
Evolution 63(1):113–130 DOI 10.1016/j.ympev.2011.12.019.
Gilsen T, Kauri H. 1959. Zoogeography of the Swedish amphibians and reptiles. Acta Vertebratica
1:191–397.
Gittins SP, Parker AG, Slater FM. 1980. Population characteristics of the common toad
(Bufo bufo) visiting a breeding site in mid-Wales. Journal of Animal Ecology 49(1):161–173
DOI 10.2307/4281.
Guarino FM, Aprea G, Caputo V, Maio N, Odierna G, Picariello O. 2012. Atlante degli Anfibi e dei
Rettili della Campania. Napoli: Massa Editore.
Harris H, Hopkinson DA. 1976. Handbook of Enzyme Electrophoresis in Human Genetics.
New York: Elsevier/North-Holland.
Harris I, Jones PD, Osborn TJ, Lister TH. 2014. Updated high resolution grids of monthly
climatic observation: the CRU TS3.1 dataset. International Journal of Climatology
34(3):623–642 DOI 10.1002/joc.3711.
Hewitt GM. 2011. Quaternary phylogeography: the roots of hybrid zones. Genetica
139(5):617–638 DOI 10.1007/s10709-011-9547-3.
Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A. 2005. Very high resolution interpolated
climate surfaces for global land areas. International Journal of Climatology 25(15):1965–1978
DOI 10.1002/joc.1276.
Intergovernmental Panel on Climate Change (IPCC). 2007. Climate Change 2007: The Physical
Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the IPCC.
Cambridge: Cambridge University Press.
Intergovernmental Panel on Climate Change (IPCC). 2014. Climate change 2014: synthesis
report. In: Core Writing Team, Pachauri RK, Meyer LA, eds. Contribution of Working Groups I, II
and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Geneva:
IPCC, 151.
Jørgensen CB. 1992. Growth and reproduction. In: Feder M, Burggren W, eds. Environmental
Physiology of the Amphibians. Chicago: University of Chicago Press, 439–466.
Lang K. 1926. Groddjurens och kraldjurens utbredning inom Bara harad (Malmohuslan).
Fauna och Flora Uppsala 21:145–150.
Lanza B, Andreone F, Bologna MA, Corti C, Razzetti E. 2007. Amphibia—Fauna d’Italia.
Vol. XLII. Bologna: Calderini Editore.
Lanza B, Nistri A, Vanni S. 2006. Iconography of Italian Amphibia. Modena: Grandi e Grandi
editori.
Maiorano L, Cheddadi R, Zimmermann NE, Pellissier L, Petitpierre B, Pottier J,
Laborde H, Hurdu BI, Pearman PB, Psomas A, Singarayer JS, Broennimann O,
Vittoz P, Dubuis A, Edwards ME, Binney HA, Guisan A. 2013. Building the niche
through time: using 13,000 years of data to predict the effects of climate change on
three tree species in Europe. Global Ecology and Biogeography 22(3):302–317
DOI 10.1111/j.1466-8238.2012.00767.x.
Mallet J. 2005. Hybridization as an invasion of the genome. Trends in Ecology and Evolution
20(5):229–237 DOI 10.1016/j.tree.2005.02.010.
Malone JH, Fontenot BE. 2008. Patterns of reproductive isolation in toads. PLoS ONE 3(12):e3900
DOI 10.1371/journal.pone.0003900.
Canestrelli et al. (2017), PeerJ, DOI 10.7717/peerj.3072 14/16
Martı
´n C, Sanchiz B. 2011. Lisanfos KMS. Version 1.2. Madrid: Museo Nacional de Ciencias
Naturales.
Maxson LR. 1981. Albumin evolution and its phylogenetic implications in toads of the genus Bufo.
II. Relationships among Eurasian Bufo. Copeia 3:579–583 DOI 10.2307/1444561.
Montalenti G. 1932. Sull’embriogenesi degli ibridi di Bufo vulgaris eBufo viridis.Rendiconti
dell’Accademia Nazionale dei Lincei, Classe di Scienze Fisiche, Matematiche e Naturali, Roma
15:994–1000.
Montalenti G. 1933. L’ontogenesi degli ibridi fra Bufo vulgaris eBufo viridis.Physiological Zoology
6(3):329–395 DOI 10.1086/physzool.6.3.30151364.
Nec
ˇas P, Modry
´D, Zavadil V. 1997. Czech Recent and Fossil Amphibians and Reptiles: Atlas and
Field Guide. Frankfurt Am Main: Edition Chimaira.
Pages JM. 1984. Ecologie des amphibiens du sud-ouest de la Montagne Noire. Alytes 3:56–69.
Pfennig KS, Pfennig DW. 2009. Character displacement: ecological and reproductive responses
to a common evolutionary problem. Quarterly Review of Biology 84(3):253–276
DOI 10.1086/605079.
Proietti MC, Reisser J, Marins LF, Marcovaldi MA, Soares LS, Monteiro DS, Wijeratne S,
Pattiaratchi C, Secchi ER. 2014. Hawksbill loggerhead sea turtle hybrids at Bahia, Brazil:
where do their offspring go? PeerJ 2:e255 DOI 10.7717/peerj.255.
Reading CJ. 1998. The effect of winter temperatures on the timing of breeding activity in the
common toad Bufo bufo.Oecologia 117(4):469–475 DOI 10.1007/s004420050682.
Reading CJ. 2003. The effects of variation in climatic temperature (1980–2001) on breeding
activity and tadpole stage duration in the common toad, Bufo bufo.Science of the Total
Environment 310(1–3):231–236 DOI 10.1016/S0048-9697(02)00643-5.
Rhymer JM, Simberloff D. 1996. Extinction by hybridization and introgression.
Annual Review of Ecology, Evolution, and Systematics 27(1):83–109
DOI 10.1146/annurev.ecolsys.27.1.83.
Romano A, De Cicco M, Utzeri C. 2003. Bufo viridis Laurenti, 1768 in Monti Simbruini Regional
Park: altitude record for Peninsular Italy. Herpetozoa 16:91–93.
Schwenk K, Brede N, Streit B. 2008. Introduction. Extent, processes and evolutionary impact of
interspecific hybridization in animals. Philosophical Transactions of the Royal Society B:
Biological Sciences 363(1505):2805–2811 DOI 10.1098/rstb.2008.0055.
Sillero N, Campos J, Bonardi A, Corti C, Creemers R, Crochet PA, Isailovic JC, Denoe
¨lM,
Ficetola GF, Gonc¸alves J, Kuzmin S, Lymberakis P, de Pous P, Rodrı
´guez A, Sindaco R,
Speybroeck J, Toxopeus B, Vieites DR, Vences M. 2014. Updated distribution and
biogeography of amphibians and reptiles of Europe. Amphibia-Reptilia 35(1):1–31
DOI 10.1163/15685381-00002935.
Spilinga C, Esposito C, Forcina G, Romano A. 2007. Nuovi dati sulla distribuzione di alcuni
anfibi nel Lazio. Aldrovandia 3:125–126.
Swets JA. 1988. Measuring the accuracy of diagnostic systems. Science 240(4857):1285–1293
DOI 10.1126/science.3287615.
Thuiller W, Lafourcade B, Engler R, Araujo MB. 2009. BIOMOD: a platform for
ensemble forecasting of species distributions. Ecography 32(3):369–373
DOI 10.1111/j.1600-0587.2008.05742.x.
Tryjanowski P, Rybacki M, Sparks T. 2003. Changes in the first spawning dates of common frogs
and common toads in western Poland in 1978–2002. Annales Zoologici Fennici 40:459–464.
Vlc
ˇek P. 1995. Mexidruhovy amplexus ropuch. Z
ˇiva 43:83.
Canestrelli et al. (2017), PeerJ, DOI 10.7717/peerj.3072 15/16
Vlc
ˇek P. 1997. Poznatky z k
rı
´z
ˇenı
´ropuchy obecne
´s ropuchou zelenou. Z
ˇiva 45:132–133.
Zavadil V, Roth P. 1997. Natural hybridisation between Bufo viridis and Bufo bufo in the
Douposke
´hory hills (northwest Bohemia, Czech Republic) with general comments on
hybridisation of European green and common toads. In: Bo
¨hme W, Bischoff W, Ziegler T, eds.
Herpetologia Bonnensis: Proceedings of the 8th Ordinary General Meeting of the Societas Europaea
Herpetologica,Bonn.
Canestrelli et al. (2017), PeerJ, DOI 10.7717/peerj.3072 16/16
