Abstract
Asp vipers (Vipera aspis) and adders (Vipera berus) coexist in the Loire-Atlantique department in France where the two species reach their respective range limits. This contact zone is of special interest since hybridization has been recently discovered there. We carried out extensive sampling to further investigate the hybrid status of morphologically typical individuals and to evaluate the proportion of genetically admixed individuals in this area. Using microsatellite markers, no hybrids with typical morphological traits of either asp vipers or adders were detected. All recently investigated individuals with intermediate morphological traits were shown to be hybrids. A rather low proportion of genetically admixed individuals (1.5-3%) and a very small number of supposed second-generation hybrids suggest reduced fertility of first-generation hybrids or low viability of their progeny. The investigation of mtDNA of newly sampled hybrids support the finding that hybridization only occurs between female V. aspis and male V. berus. Several possible explanations for the unidirectional hybridization are discussed and consequent future studies suggested.
Introduction
Studying contact zones is essential for understanding hybridization processes (Tarroso et al., 2014). Natural hybridization can be observed in about 10% of species in major faunal groups, while it is even more frequent in plants (Mallet, 2008). While hybridization is mainly detected in closely related taxa due to the lack of (or limited) reproduction barriers, it can sometimes also be observed in well-defined species (Mallet, 2008). Reproductive barriers can be either pre- or postzygotic (Rundle and Nosil, 2005). Prezygotic barriers to hybridization include spatial and temporal isolation, natural selection against immigrants, sexual isolation and post-mating, prezygotic isolation (Rundle and Nosil, 2005). In the absence of prezygotic reproductive barriers, postzygotic barriers can still impede hybridization as F1 hybrids are often sterile, while F2 hybrids are often not viable or exhibit reduced fitness (Stebbins, 1958). These latter barriers can be related to either intrinsic or ecologically-induced postzygotic isolation, while sexual selection against hybrids may represent another postzygotic barrier (Rundle and Nosil, 2005). If reproductive barriers do not fully isolate two species, introgression can result in reticulated phylogenies, adaptations and speciation (Mallet, Besansky and Hahn, 2016). Introgression does not always affect both species equally and asymmetric introgression is frequent (Currat et al., 2008).
In snakes, it has been shown that some valid species are hybridizing in the wild (e.g., Mebert, 2008; Mebert et al., 2011), but it was mostly observed for phylogenetically closely related ones. However, introgressive hybridization is still a rarely observed phenomenon in snakes (e.g., Sanders, Rasmussen and Guinea, 2014; Kindler et al., 2017; Schultze et al., 2019). In the genus Vipera, the speciation of most taxa is quite recent (about 17 Myr ago for the oldest one, but less than 2 Myr for the youngest; Freitas et al., 2020), leading to several contact zones with F1 and later-generation hybrids, with variable levels of introgression, mainly dependent on the time since the species have been separated. For instance, in the contact zone between Vipera aspis, V. latastei and V. seoanei in Spain, frequent hybridization between V. aspis and V. latastei occurrs (two closely related species), whereas hybrids between V. latastei and V. seoanei (belonging to two different subgenera) are only rarely present (Tarroso et al., 2014). Natural hybrids of viper species are also found e.g., between V. berus and V. ammodytes (Mihalca et al., 2003; Czirják et al., 2015), V. aspis and V. ammodytes (Lapini, 1988; Bagnoli, Capula and Luiselli, 2014) and between V. berus and V. renardi (Pavlov et al., 2011). Additionally, hybrids of viper species were repeatedly bred in captivity (Mertens, 1964; Saint Girons, 1977; Faoro, 1986; Saint Girons, 1990; Schweiger, 2009). In the Vipera genus, many species show parapatric distributions (Chamorro et al., 2020) and frequently a lack of total genetic isolation (e.g., Czirják et al., 2015) with evidences for incomplete reproductive barriers (Pavlov et al., 2011; Tarroso et al., 2014). However, not all contact zones result in hybridization between the parapatric species (e.g., Luiselli, Filippi and Lena, 2009).
The para-Mediterranean Vipera aspis (asp viper, subgenus Vipera) and the Euro-Siberian Vipera berus (adder, subgenus Pelias) diverged about 10 to 15 Mya (Szyndlar and Rage, 2002; Freitas et al., 2020). While the warm-adapted V. aspis occurs throughout northeastern Spain, the southern half of France, Italy, northwestern Slovenia and the Black Forest, the cold-adapted V. berus is distributed throughout a large Eurosiberian range (Saint Girons, 1980a). Both species can easily be distinguished by their morphology, including dorsal pattern, head scalation and the presence (V. aspis) or absence (V. berus) of an upturned snout (Saint Girons, 1980a). While V. aspis has 42 chromosomes, V. berus has only 36 (Saint Girons, 1977). The two species usually occupy contrasted climatic niches and habitats (Saint Girons, 1975; Scali et al., 2011) as they differ in physiology and water requirements (Guillon et al., 2013). Still, contact zones exist in Switzerland (Monney, 1996), Italy (Scali et al., 2011), Slovenia (Mebert et al., 2015) and France (Saint Girons, 1975; Naulleau, 1986). Even if the ecological niches are different between both species, there is always some slight overlap at the margin of both niches, where the species usually occupy contrasted microhabitats (Guillon et al., 2013), and where supposed hybrids were found in different parts of France: one individual with mixed morphological traits was photographed in Auvergne (Geniez, 2015), a few individuals were found and morphologically identified as hybrids by Viaud-Grand-Marais (1869) in the Deux-Sèvres department (V. berus population currently extinct) and by Saint Girons (1975) in the Loire-Atlantique department. These supposed hybrids showed intermediate morphological traits, specifically head scalation, dorsal pattern, or eye colour, not typical for either V. aspis or V. berus. The existence of hybrids in the Loire-Atlantique department was later genetically confirmed by Guiller, Lourdais and Ursenbacher (2017).
In the study of Guiller, Lourdais and Ursenbacher (2017), ten individuals from the contact zone showing mixed morphological traits were genetically analysed and compared to reference individuals of each parental species (
- (i) What is the hybrid status of morphologically intermediate individuals? We predict that mainly F1 and only rarely post-F1 lineages occur.
- (ii) Are there hybrids with typical morphological traits of one of the parental species? Since we predicted a rare presence of post-F1 lineages, we expect at most a few hybrids with typical morphological traits.
- (iii) What is the proportion of genetically admixed individuals in the contact zone? By analysing randomly sampled individuals using microsatellite markers we expect a low proportion of genetically admixed individuals.
- (iv) Is gene flow directional? By investigating the mtDNA as a marker of the maternal lineage, we expect a unidirectional hybridization with V. aspis as the maternal parent as it was the case for all hybrids analysed in the study of Guiller, Lourdais and Ursenbacher (2017).
Materials and methods
Species ecology in the study area
The Loire-Atlantique department constitutes the northern limit of the asp viper distribution, while it represents the southern limit of the adder distribution (Naulleau, 1986). In this area, V. berus is growing faster, reach sexual maturity earlier and give birth to a higher number of viable offspring than V. aspis (Saint Girons, 1975; Guiller, 2012; Guiller, Legentilhomme and Lourdais, 2012). The main mating season of both species is in spring, but V. aspis can also occasionally mate in autumn (Saint Girons, 1975). The spermiogenesis of V. berus takes place in spring and a pre-mating ecdysis is required before the onset of reproduction. In V. aspis, spermiogenesis mainly occurs in summer and males are ready to mate soon after their emergence from hibernation (Saint Girons, 1975). While male V. aspis are ready to mate earlier than male V. berus, in contrary, the ovulation of female V. aspis takes place a bit later than this of female V. berus (Saint Girons, 1975).
Sampling
A total of 170 V. aspis, 212 V. berus and 11 individuals with intermediate morphological traits were sampled randomly since 2010 in one particular location of the contact zone (municipality of Blain in the Loire-Atlantique department). More details about the extent (1.35 km2) and structure of the study area can be found in Guiller et al. (2018). Of those 11 morphologically intermediate individuals, seven were specifically chosen to investigate their hybrid status in Guiller, Lourdais and Ursenbacher (2017). The other four individuals with intermediate morphological traits were newly sampled following the previous study and included in the present one. To assess the proportion of genetically admixed individuals of V. aspis and V. berus in the study site and to detect possible hybrids lacking intermediate morphological traits, 112 of the remaining 386 non-analysed individuals were chosen randomly for genetic analyses (selecting the first 112 samples collected, checking for a similar proportion of both species and both sexes: 30 male and 28 female V. aspis, 24 male and 28 female V. berus and two females with intermediate morphological traits).
Furthermore, the two morphologically intermediate individuals which were not included in the random sample as well as three additional morphologically intermediate individuals newly sampled in other parts of the contact zone (in municipalities of Le Gâvre and Marsac sur Don) were also investigated. These last three samples were only considered to assess if individuals with intermediate morphological traits are hybrids and if hybridization is directional.
Since in the previous study (Guiller, Lourdais and Ursenbacher, 2017), microsatellite primers, which could only be amplified in one of the two focal species, were also included, and as Structure and NewHybrids (see methods below) could not deal with a large number of missing data, it made sense to re-analyse the samples using only microsatellite markers working for both species. Therefore, six of the seven samples collected after 2010 (mentioned above) as well as two of three additional samples with intermediate morphological traits collected during a non-random sampling before 2010 were re-analysed with the same microsatellite markers as in the present study (see below). One of these latter two re-analysed individuals was showing intermediate morphological traits but was genetically identified as pure V. aspis by Guiller, Lourdais and Ursenbacher (2017). Two samples could not be re-analysed due to a lack of extant DNA. For the previous study, reference samples of individuals showing typical morphological traits (20 V. aspis and 20 V. berus) were collected about 10 km away from the contact zone and were also re-analysed. Consequently, the complete dataset for the genetic analysis regrouped a total of 165 samples (120 collected after 2010 in the contact zone of which 112 randomly chosen to assess proportion of genetically mixed individuals; 43 from different locations).
DNA extraction and microsatellite amplification
DNA of 165 individuals was extracted from clipped ventral scales using the DNeasy Blood and Tissue Kit (Qiagen, Hombrechtikon, Switzerland). Polymerase chain reactions (PCR) of microsatellite markers were initially conducted using 21 fluorescent-labelled primers developed for V. aspis (Geser et al., 2013) and V. berus (Ursenbacher, Monney and Fumagalli, 2009) in five different multiplex PCR. Multiplex PCRs were performed in 10 μl reactions with the Type-it Microsatellite PCR Kit (Qiagen) following manufacturer’s instructions (32 cycles using 2 μl template DNA and primers with a concentration between 0.1 and 0.3 μM after preliminary tests) in an Eppendorf Mastercycler Gradient (Vaudaux-Eppendorf AG, Schönenbuch, Switzerland). PCR products were visualized with an ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA) and lengths of the alleles were assessed using Peak Scanner™ Software v2.0 (Applied Biosystems). Due to poor performance of the multiplex PCR, a lack of cross-species amplification or a lack of polymorphism, 14 primers were removed after first tests. Finally, only seven microsatellite markers (Va-P8, Va-P20, Va-P25, Va-P26, Va-P29, Va-P51 and Vb-A8) could be amplified and were polymorphic in both species and were kept for further analyses.
Assessment of population structure and hybridization level
To detect possible hybrids and to see if the results with seven microsatellites are consistent with the previous results, a Bayesian cluster analysis was performed using Structure 2.3.4 (Pritchard, Stephens and Donnelly, 2000). Structure was run three times with different settings. First, correlated allele frequencies were used and no prior information was entered into the software (Falush, Stephens and Pritchard, 2003). Second, the same analysis was performed using independent allele frequencies (as the involved species diverged > 10 Mya ago). Finally, a third analysis was performed using prior population information to test for hybrids (reference samples were denoted as pure using USEPOPINFO = 1 with POPFLAG = 1 for V. aspis and POPFLAG = 2 for V. berus, and USEPOPINFO = 0 for individuals of the contact zone; Hubisz et al., 2009). All the three analyses were performed using the admixture model which can “deal with hybrid zones in a natural way” as mentioned in Pritchard (2009). Ten replicates per K ranging from 1 to 10 were ran, each with 400 000 MCMC iterations after a burn-in of 200 000 iterations. As the results were similar with all the three methods, only the outcome of the first analysis is shown hereinafter.
The most probable number of clusters K was assessed using the mean log-likelihood L(K) as well as the ΔK method (Evanno, Regnaut and Goudet, 2005), both implemented in StructureHarvester Web 0.6.94 (Earl and vonHoldt, 2012). The ten runs corresponding to the most probable number K were pooled using the pipeline CLUMPAK (Kopelman et al., 2015).
To assess the level of hybridization of individuals, NewHybrids 1.1 beta (Anderson and Thompson, 2002) was used allowing to calculate the probability of an individual being assigned to one of the following genotypes: pure V. aspis (PVa), pure V. berus (PVb), F1, F2, first-generation backcross with V. aspis (F1 × V. aspis; BcVa) and first-generation backcross with V. berus (F1 × V. berus; BcVb). The analysis was performed twice with a burn-in of 100 000 followed by 1 000 000 sweeps, once using “Jeffreys” priors and once using “Uniform” priors, both without placing priors on mixing proportions and allele frequencies. As the two methods yielded similar results and as assignments of the simulated data were more efficient and accurate using Jeffreys priors, only results of the analysis with Jeffreys priors are shown below. The 20 V. aspis and 20 V. berus reference individuals from Guiller, Lourdais and Ursenbacher (2017) were included in the analysis denoted as pure individuals collected from another location (using the z and s option of the program). The three additional individuals with intermediate morphological traits were also denoted as sampled in another location (using the s option of the program).
To assess the statistical power of Structure and NewHybrids for correctly identifying individuals as purebreds or hybrids, 500 individuals of each of the following levels were simulated with HybridLab 1.0 (Nielsen, Bach and Kotlicki, 2006; Vähä and Primmer, 2006): pure V. aspis (PVa), pure V. berus (PVb), F1, F2, first-generation backcross with V. aspis (F1 × V. aspis; BcVa) and first-generation backcross with V. berus (F1 × V. berus; BcVb). To simulate the 500 individuals of each parental species, only individuals which were assigned ⩾ 99% to one of the species with Structure (= 110 individuals in total) were used as input. Using those simulated 1000 individuals as input, 500 F1 and subsequently, 500 F2 and 1000 backcrosses (500 BcVa and 500 BcVb) were simulated. With those simulated 3000 individuals, the analyses with Structure and NewHybrids were repeated with the same settings stated above.
A Principal Coordinates Analysis (PCoA) was performed on a matrix of genetic distances using GenAlEx 6.51b2 (Peakall and Smouse, 2006; Peakall and Smouse, 2012) to visualize the genetic differences between the species and to assess a supposed intermediate status of hybrids.
The proportion of genetically admixed individuals was calculated in two different ways. First, only the 112 randomly chosen individuals were taken into account for the calculation. Second, all the individuals sampled from 2010 onwards were included in the calculation: on one hand, all morphologically intermediate individuals were genetically analysed (sampled after 2010:
Assessment of the maternal lineage
To assign the maternal lineage, a portion of the cytochrome b of the putative hybrids was amplified following the protocol mentioned in Ursenbacher et al. (2006), or, when only short DNA fragments could be replicated, a small part of the cytochrome b (but sufficient to discriminate between V. aspis and V. berus) was amplified using the new primers L15192Vb (5′-CACACATTGCACGAGGC-3′) and H15301Vb (5′-AACATAGCCGAAGAAGGC-3′) and similar PCR conditions were applied. The sequences were later compared to already published sequences of V. berus and V. aspis with Nucleotide BLAST in order to determine the maternal lineage.
Structure analysis (K = 2) of V. aspis and V. berus samples collected in the Loire Atlantique department (France). Morphologically intermediate individuals include individual 4.14 which was genetically assigned to V. aspis. The hybridization level of individuals 14 and 71 could not be clearly assigned.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
Structure analysis (K = 2) of V. aspis and V. berus samples collected in the Loire Atlantique department (France). Morphologically intermediate individuals include individual 4.14 which was genetically assigned to V. aspis. The hybridization level of individuals 14 and 71 could not be clearly assigned.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
Structure analysis (K = 2) of V. aspis and V. berus samples collected in the Loire Atlantique department (France). Morphologically intermediate individuals include individual 4.14 which was genetically assigned to V. aspis. The hybridization level of individuals 14 and 71 could not be clearly assigned.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
Genetic assignment of morphological intermediate individuals (putative hybrids between Vipera aspis and V. berus) in the Loire-Atlantique department (France) using Structure (K = 2) and NewHybrids. Probabilities of classification using NewHybrids > 0.6 are highlighted in bold. The individuals 70, 71 and 106 are not from exactly the same location but were found about 1 km apart. Individuals in italics were non-randomly sampled before 2010, while the other individuals were randomly sampled after 2010. PVa = pure Vipera aspis; PVb = pure Vipera berus; BcVa = F1 × Vipera aspis; BcVb = F1 × Vipera berus.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
Genetic assignment of morphological intermediate individuals (putative hybrids between Vipera aspis and V. berus) in the Loire-Atlantique department (France) using Structure (K = 2) and NewHybrids. Probabilities of classification using NewHybrids > 0.6 are highlighted in bold. The individuals 70, 71 and 106 are not from exactly the same location but were found about 1 km apart. Individuals in italics were non-randomly sampled before 2010, while the other individuals were randomly sampled after 2010. PVa = pure Vipera aspis; PVb = pure Vipera berus; BcVa = F1 × Vipera aspis; BcVb = F1 × Vipera berus.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
Genetic assignment of morphological intermediate individuals (putative hybrids between Vipera aspis and V. berus) in the Loire-Atlantique department (France) using Structure (K = 2) and NewHybrids. Probabilities of classification using NewHybrids > 0.6 are highlighted in bold. The individuals 70, 71 and 106 are not from exactly the same location but were found about 1 km apart. Individuals in italics were non-randomly sampled before 2010, while the other individuals were randomly sampled after 2010. PVa = pure Vipera aspis; PVb = pure Vipera berus; BcVa = F1 × Vipera aspis; BcVb = F1 × Vipera berus.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
Results
Assignment analyses
Among the 165 genetically analysed individuals, 14 could be identified as hybrids, all of them having intermediate traits. One morphologically intermediate individual (4.14) was genetically identified as pure V. aspis, as in Guiller, Lourdais and Ursenbacher (2017). All the morphologically typical individuals were identified as purebred (78 individuals as pure V. aspis and 72 individuals as pure V. berus).
Assignment and classification of the individuals of V. aspis (PVa) and V. berus (PVb) analysed in the Loire Atlantique department (France). On the left, results of Structure (K = 2) are shown followed by the percentage of correctly identified individuals using two different thresholds (0.9 and 0.95, respectively). On the right, the percentage of correctly identified individuals using two different thresholds (0.9 and 0.6, respectively) in NewHybrids.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
Assignment and classification of the individuals of V. aspis (PVa) and V. berus (PVb) analysed in the Loire Atlantique department (France). On the left, results of Structure (K = 2) are shown followed by the percentage of correctly identified individuals using two different thresholds (0.9 and 0.95, respectively). On the right, the percentage of correctly identified individuals using two different thresholds (0.9 and 0.6, respectively) in NewHybrids.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
Assignment and classification of the individuals of V. aspis (PVa) and V. berus (PVb) analysed in the Loire Atlantique department (France). On the left, results of Structure (K = 2) are shown followed by the percentage of correctly identified individuals using two different thresholds (0.9 and 0.95, respectively). On the right, the percentage of correctly identified individuals using two different thresholds (0.9 and 0.6, respectively) in NewHybrids.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
StructureHarvester identified the most probable number of clusters K = 2 (fig. 1) using the mean log-likelihood L(K) (supplementary fig. S1) as well as the ΔK method (supplementary fig. S2). Structure assigned all the 14 morphologically intermediate individuals (except for 4.14) on average 50.8% to V. aspis (max value: 76.3%, individual 71; table 1), while the morphologically typical individuals were genetically assigned to their according species by at least 85.1% to V. aspis and 94.2% to V. berus and on average 98.6% to V. aspis and 99.1% to V. berus (tables 1 and 2).
As NewHybrids performed better in identifying later generation hybrids using Jeffreys priors, only the output of this analysis is shown (tables 1 and 2 and fig. 2). NewHybrids confirmed the result of Structure and none of the morphologically intermediate individuals (except for 4.14) could be assigned to either one of the parental forms with a probability of more than 22.7%. Twelve of the 14 morphologically intermediate individuals (except for 4.14) were considered to be F1 with a probability of at least 81.3%, while one (71) was assigned as F1 with a probability of 45.3% and the last individual (14) as a BcVa (47.9%) or less likely as F2 (44.2%).
NewHybrids results using Jeffreys priors of V. aspis and V. berus samples collected in the Loire Atlantique department (France). Morphologically intermediate individuals include individual 4.14 which was genetically assigned to V. aspis. The hybridization level of individuals 14 and 71 could not be clearly assigned. BcVa = F1 × Vipera aspis; BcVb = F1 × Vipera berus.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
NewHybrids results using Jeffreys priors of V. aspis and V. berus samples collected in the Loire Atlantique department (France). Morphologically intermediate individuals include individual 4.14 which was genetically assigned to V. aspis. The hybridization level of individuals 14 and 71 could not be clearly assigned. BcVa = F1 × Vipera aspis; BcVb = F1 × Vipera berus.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
NewHybrids results using Jeffreys priors of V. aspis and V. berus samples collected in the Loire Atlantique department (France). Morphologically intermediate individuals include individual 4.14 which was genetically assigned to V. aspis. The hybridization level of individuals 14 and 71 could not be clearly assigned. BcVa = F1 × Vipera aspis; BcVb = F1 × Vipera berus.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
Assignment and classification of the simulated individuals with different hybridization levels based on V. aspis and V. berus samples collected in the Loire Atlantique department (France). On the left, the percentage of correctly identified simulated pure individuals using two different thresholds (0.9 and 0.95, respectively) in the Structure analysis (K = 2). On the right, the percentage of correctly identified simulated individuals of the different hybridization levels using two different thresholds (0.9 and 0.6, respectively) in NewHybrids. PVa = pure Vipera aspis; PVb = pure Vipera berus; BcVa = F1 × Vipera aspis; BcVb = F1 × Vipera berus.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
Assignment and classification of the simulated individuals with different hybridization levels based on V. aspis and V. berus samples collected in the Loire Atlantique department (France). On the left, the percentage of correctly identified simulated pure individuals using two different thresholds (0.9 and 0.95, respectively) in the Structure analysis (K = 2). On the right, the percentage of correctly identified simulated individuals of the different hybridization levels using two different thresholds (0.9 and 0.6, respectively) in NewHybrids. PVa = pure Vipera aspis; PVb = pure Vipera berus; BcVa = F1 × Vipera aspis; BcVb = F1 × Vipera berus.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
Assignment and classification of the simulated individuals with different hybridization levels based on V. aspis and V. berus samples collected in the Loire Atlantique department (France). On the left, the percentage of correctly identified simulated pure individuals using two different thresholds (0.9 and 0.95, respectively) in the Structure analysis (K = 2). On the right, the percentage of correctly identified simulated individuals of the different hybridization levels using two different thresholds (0.9 and 0.6, respectively) in NewHybrids. PVa = pure Vipera aspis; PVb = pure Vipera berus; BcVa = F1 × Vipera aspis; BcVb = F1 × Vipera berus.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
Evaluation of statistical power
The different methods performed relatively well in assigning and classifying simulated pure individuals of both species (table 3). Structure was able to correctly identify 90.4% of the simulated pure V. aspis and 97.8% of the simulated pure V. berus when using a threshold of 0.9. However, using a threshold of 0.95, only 18.6% of the simulated pure V. aspis and 31.4% of the simulated pure V. berus could be correctly identified. Similarly, NewHybrids was able to correctly assign 76.2% of simulated pure V. aspis and 96.6% of simulated pure V. berus using a threshold of 0.9. Structure as well as NewHybrids performed better in identifying pure V. berus than pure V. aspis. As hybrids are usually identified with lower statistical power, a second threshold for the assignment probabilities of NewHybrids was set to 0.6 as it was also done by do Prado et al. (2017). Using this threshold of 0.6, NewHybrids was able to correctly classify 90.2% of simulated F1, 51.2% of simulated F2, 72.2% of simulated BcVa (F1 × Vipera aspis) and 77.2% of simulated BcVb (F1 × Vipera berus). NewHybrids performed badly in identifying hybrids using a threshold of 0.9 and could only classify 18-34.6% of the individuals correctly. The application of the different thresholds on our real data demonstrates the reliability of our results: using a threshold of 0.9, Structure and NewHybrids yielded exactly the same outcome and could correctly assign 98.72% of the pure V. aspis and 100% of the pure V. berus (table 2). Among the 15 individuals with intermediate morphological traits 12 could be classified as F1 with NewHybrids using a threshold of 0.6 (lowest value 0.813 for individual 4.28, table 1). Two individuals with intermediate morphological traits (14 and 71) could not be clearly assigned to any hybrid category with NewHybrids using a threshold of 0.6 (highest values 0.479 and 0.453, respectively, table 1). Only one individual was assigned to one of the species with Structure using a threshold of 0.9 (highest value 0.763 for individual 71, table 1).
Principal Coordinates Analysis (PCoA) based on 10 microsatellite markers showing the distribution of morphologically intermediate individuals in between the pure V. aspis and pure V. berus individuals in the Loire Atlantique department (France). Individual 4.14 was morphologically intermediate but genetically assigned to pure V. aspis.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
Principal Coordinates Analysis (PCoA) based on 10 microsatellite markers showing the distribution of morphologically intermediate individuals in between the pure V. aspis and pure V. berus individuals in the Loire Atlantique department (France). Individual 4.14 was morphologically intermediate but genetically assigned to pure V. aspis.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
Principal Coordinates Analysis (PCoA) based on 10 microsatellite markers showing the distribution of morphologically intermediate individuals in between the pure V. aspis and pure V. berus individuals in the Loire Atlantique department (France). Individual 4.14 was morphologically intermediate but genetically assigned to pure V. aspis.
Citation: Amphibia-Reptilia 43, 2 (2022) ; 10.1163/15685381-bja10091
Principal coordinate analysis (PCoA)
The PCoA showed an intermediate position of the hybrids between the two parental species (fig. 3). The first coordinate axis of the PCoA explained 21.4% of the variation, the second coordinate axis 5.5%, respectively. The genetically pure V. aspis with intermediate morphological traits (4.14) was situated in between other genetically pure V. aspis even though it was placed at an edge position. The morphologically intermediate individual 71, which was assigned with a probability of 76.3% to belong to V. berus, was situated among the other putative hybrids yet nearer to pure V. berus samples than to pure V. aspis samples. Similarly, the morphologically intermediate individual 14, which was assigned to V. aspis with a probability of 62.4% in Structure and which could not be clearly assigned with NewHybrids, was situated in between the pure V. aspis and the hybrids.
Proportion of genetically admixed individuals and direction of gene flow
Two of the 112 newly analysed and randomly selected individuals were identified as hybrids what results in a proportion of genetically admixed individuals of 1.79%. Using the second evaluation method, we estimated a proportion of genetically admixed individuals of 2.80% (
Discussion
Our study demonstrates that hybridization between the two species occurs but is rare and we confirm unidirectional crossing. All analysed individuals showing typical morphological traits of one species could be properly assigned to their supposed species suggesting that hybrids can be already identified based on their intermediate morphology. We discuss our findings below.
Hybridization mainly to F1 level
In contrast to the previous study by Guiller, Lourdais and Ursenbacher (2017), most of the hybrids (except of individuals 14 and 71) could be assigned to F1. The differences can be explained by the use of markers that amplified well in both species and likely provide more reliable results.
Only one individual (14) could be most likely assigned as a backcross (F1 × parental) or F2. We can assume that F1 hybrids (and rare F2 or backcrosses) have a low fitness. Even if F1 hybrids seem to have the same survival rate as their parental species (several F1 individuals from this study were observed in the field over several years by GG), a similar fecundity would result in a higher proportion of F2 and backcrosses. Low viability of second-generation hybrids was observed in litters of wild F1 females that gave birth in captivity. These litters (
Low proportion of genetically admixed individuals
Interspecific hybridization rates in natural populations are usually 0.1% or less per generation (Mallet, Besansky and Hahn, 2016). Species are alleged to be able to hybridize more often if their divergence time is less than 15 Mya (Bolnick and Near, 2005). This is supported by our study, where the occurrence of hybridization is confirmed with a divergence time of 13-14 Mya between the two focal species (Freitas et al., 2020). In the Loire-Atlantique department, the proportion of genetically admixed individuals is still quite low (between 1.5 and 3%) and could be considered comparable to the contact zone occurring in Slovenia (one single individual with some signs of ancient hybridization among 28 genetically tested V. berus and V. aspis individuals; Mebert et al., 2015) or in northern Spain, where hybrids between V. aspis and V. seoanei (two species separated by the same time span as V. aspis and V. berus; Freitas et al., 2020) were not detected (but the habitat is not considered optimal for V. seoanei) and only one between V. latastei and V. seoanei was found (Tarroso et al., 2014). Even if V. seoanei possesses a similar reproductive cycle as the asp viper, with an additional mating period in autumn (in opposite to V. berus; Saint Girons 1980b), the differentiation between these species belonging to two different subgenera (Pelias and Vipera 1) seems to be sufficient to imply a limited gene flow. Similar observations were conducted between Vipera 1 and Vipera 2 groups (following Nilson and Andrén, 1997) as no hybrids were detected in Slovenia between V. aspis and V. ammodytes (Mebert et al., 2015). In contrast, hybridization within the same subgenus (e.g., within Vipera 1 or Pelias) is more common, as locally observed between V. latastei and V. aspis (Tarroso et al., 2014) or related to old gene flow between V. kazankovi and V. renardi (Zinenko et al., 2016) or V. renardi and V. berus (Pavlov et al., 2011). The old split between the 3 subgenera (about 10 Myr) is perhaps sufficient to strongly reduce reproductive compatibility to the formation of only a few F1 and backcross individuals, whereas the more recent speciation within the subgenus (< 10 Mya; see Freitas et al., 2020) permits more important gene exchanges, which is still possible to detect long time after introgression (Zinenko et al., 2016; Pavlov et al., 2011).
Because no hybrids have been found in the Alps (Monney, 1996; Scali et al., 2011), ecological factors could explain the occurrence of hybrids at lower altitudes like in the studied area. Even if habitat segregation was demonstrated in high altitudes (Monney, 1996; Scali et al., 2011; Mebert et al., 2015), as well as in our study area, where V. berus occupies basking sites with higher humidity and colder microconditions (Guillon et al., 2013), recent anthropogenic disturbance induced by the intensified agricultural farmland could be a possible explanation for an increased contact between the species and consequently the occurrence of hybrids in our study area (Grabenstein and Taylor, 2018). Furthermore, a higher homogeneity of habitats in the degraded farmland compared to the Alps could possibly reduce the effect of differentiated microhabitat selection by both species, leading to increased encounters between the two species and thus more frequent hybridization events. Additionally, differences in phenology probably pose the most likely explanation for the absence of hybrids in higher altitudes as different mating periods were observed in the Alps (Monney, 1996) while overlapping mating periods were demonstrated in our lowland study area (Guillon et al., 2013; Guiller et al., 2014).
Evidences for directional hybridization
In all hybrid individuals (
(1) Prezygotic barriers: as V. berus is cold-adapted in opposite to V. aspis, one can hypothesise that males of V. berus are emerging earlier from hibernation and thus the likelihood for a female asp viper to encounter a male adder during the early mating season is bigger. However, in the study site, it has been shown that male V. aspis and male V. berus emerge at about the same time from hibernation (Guiller, Legentilhomme and Lourdais, 2014). Additionally, only male V. berus need to terminate their spermiogenesis (Prestt, 1971; Nilson, 1980) and undergo a pre-mating ecdysis after hibernation (Saint Girons, 1980b). This implies that male adders likely start their search for females later in the year than male V. aspis. Because of year-to-year variation in temperature and precipitation, the probability of an interspecific mating is likely fluctuating.
Moreover, both species use specific pheromones (Saint Girons, 1980a) and mating experiments conducted by Saint Girons (1975) demonstrated that single specimens of different viper species do not interbreed when placed in the same artificial enclosure. But when he formed an “artificial population” out of different species, interspecific copulations occurred. This observation could be explained by a blending of pheromones within the enclosure which impedes males from identifying their conspecific females. As those observations would explain interspecific copulation in a habitat where both species are abundant, an impact on the unidirectional hybridization remains questionable. But we can hypothesize that male asp vipers perform better in recognizing their conspecific females in mixed populations than adders, what would prevent them from mating with female adders; or alternatively that adders are also attracted to asp viper pheromones. Differences in courtship behaviour could also explain unidirectional hybridization if V. aspis females respond to the courtship behaviour of V. berus males, but V. berus females not to that of V. aspis males.
Another possibility for a pre-zygotic reproductive barrier could be an incompatibility of V. aspis hemipenes with V. berus cloaca since hemipenes of V. aspis are longer with more basal spines than those of V. berus (Zuffi, 2002). Because copulation between a male asp viper and a female adder was observed by Saint Girons (1975), this explanation is not supported.
As multipaternity is known from both species (V. aspis: S. Nanni personal communication; V. berus: Ursenbacher, Erny and Fumagalli, 2009), it is likely that some females are mating with males of both species. Sperm competition could lead to the survival of V. berus sperms only what would explain the unidirectional hybridization. But if this would be the only reason, the lack of matings of female V. berus with only male V. aspis is improbable.
(2) Postzygotic barriers: copulation may be possible in both directions, but the genomes of the two species may form a viable zygote only in one direction resulting in asymmetric viability. As asp vipers have 42 chromosomes (2n), while adders have 36 chromosomes (2n), we can hypothesise that hybridization is only possible between females with 42 chromosomes and males with 36 chromosomes. In fishes, it has been shown that hybrids can survive if the number of maternal chromosomes is greater than or equal to the number of paternal chromosomes, whereas a hybridization hardly produces living progeny if the chromosomal number of the maternal fish is lower than that of the paternal fish (Liu, 2010). However, a crossing between a female V. seoanei (2n = 36 as for V. berus) and a male V. aspis (2n = 42) resulted in viable individuals with 39 chromosomes (2n; Saint Girons, 1977). But still, other genetic incompatibilities between V. berus females and V. aspis males are likely, e.g., due to cytoplasmic issues (Ferree and Barbash, 2009) or sex chromosomes (Matute and Gavin-Smyth, 2014).
Differential survival represents another postzygotic barrier which could explain the observed results. If there is embryonic survival, the hybrid neonates mothered by V. berus possibly do not survive in the wild. Considering the extensive sampling in the area by one of the authors (GG), it seems very unlikely that all hybrids between female V. berus and male V. aspis older than one year would have been overseen if there would be any.
The hypotheses mentioned before could be tested in captivity or semi-captivity. Monitoring of the hybrids in the field should give a better insight into the fitness, survival and fecundity of the hybrids. In fact, during the writing of this article three additional adult hybrids were detected and a female V. aspis produced a litter of four young, one typical V. aspis and three with intermediate morphological traits. By comparing the reproduction rates of the parental species as well as of the hybrids, a better understanding of the unidirectional hybridization could be achieved.
Corresponding author; e-mail: valerie.zwahlen@hotmail.com
Acknowledgements
We would like to thank K. Mebert and L. Oeschger for their helpful inputs and two anonymous reviewers as well as the associate editor for comments on the manuscript. Furthermore, we are grateful to the vipers that had to leave a scale for this study. The genetic analyses were conducted with financial support from the University of Basel. This study also received financial and technical support from the Centre National de la Recherche Scientifique (CNRS). Samples were taken with the permission of local authorities.
Supplementarymaterial
Supplementary material is available online at: https//doi.org/10.6084/m9.figshare.19619778
References
Anderson, E.C., Thompson, E.A. (2002): A model-based method for identifying species hybrids using multilocus genetic data. Genetics 160: 1217-1229.
Bagnoli, C., Capula, C., Luiselli, L. (2014): Preliminary data on natural hybridization between Vipera ammodytes and Vipera aspis in a contact zone of northeastern Italy. In: Scripta Herpetologica – Studies on Amphibians and Reptiles in Honour of Benedetto Lanza, p. 31-38. Capula, M., Corti, C., Eds, Edizioni Belvedere, Latina.
Boissinot, A., Guiller, G., Legentilhomme, J., Grillet, P., Lourdais, O. (2015): Déclin alarmant des reptiles dans les bocages de l’Ouest de la France. Le Courrier de la Nature 289: 35-41.
Bolnick, D.I., Near, T.J. (2005): Tempo of hybrid inviability in Centrarchid fishes (Teleostei: Centrarchidae). Evolution 59: 1754-1767.
Chamorro, D., Martínez-Freiría, F., Real, R., Muñoz, A.-R. (2021): Understanding parapatry: how do environment and competitive interactions shape Iberian vipers’ distributions? Journal of Biogeography 48: 1322-1335.
Currat, M., Ruedi, M., Petit, R.J., Excoffier, L. (2008): The hidden side of invasions: massive introgression by local genes. Evolution 62: 1908-1920.
Czirják, G.Á., Köbölkuti, L.B., Tenk, M., Szakács, A., Kelemen, A., Spînu, M. (2015): Hemorrhagic stomatitis in a natural hybrid of Vipera ammodytes × Vipera berus due to inappropriate substrate in terrarium. J. Vet. Med. Sci. 77: 701-703.
do Prado, F.D., Fernandez-Cebrian, R., Hashimoto, D.T., Senhorini, J.A., Foresti, F., Martinez, P., Porto-Foresti, F. (2017): Hybridization and genetic introgression patterns between two South American catfish along their sympatric distribution range. Hydrobiologia 1: 319-343.
Duguy, R., Saint Girons, H. (1990): Vipera berus en Vendée. Ann. Soc. Sci. Nat. Charente-Marit 7: 1005-1007.
Earl, D.A., vonHoldt, B.M. (2012): STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv. Genet. Resour. 4: 359-361.
Evanno, G., Regnaut, S., Goudet, J. (2005): Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol. Ecol. 14: 2611-2620.
Falush, D., Stephens, M., Pritchard, J.K. (2003): Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 164: 1567-1587.
Faoro, G. (1986): Bemerkungen zur Verbastardisierung von Vipera ammodytes ammodytes mit Vipera aspis atra. Herpetofauna (Ludwigsburg) 8: 6-7.
Ferree, P.M., Barbash, D.A. (2009): Species-specific heterochromatin prevents mitotic chromosome segregation to cause hybrid lethality in Drosophila. PLOS Biol. 7: e1000234.
Freitas, I., Ursenbacher, S., Mebert, K., Zinenko, O., Schweiger, S., Wüster, W., Brito, J.C., Crnobrnja-Isailović, J., Halpern, B., Fahd, S., Santos, X., Pleguezuelos, J.M., Joger, U., Orlov, N., Mizsei, E., Lourdais, O., Zuffi, M.A.L., Strugariu, A., Zamfirescu, Ş.R., Martínez-Solano, Í., Velo-Antón, G., Kaliontzopoulou, A., Martínez-Freiría, F. (2020): Evaluating taxonomic inflation: towards evidence-based species delimitation in Eurasian vipers (Serpentes: Viperinae). Amphibia-Reptilia 41: 1-27.
Geniez, P. (2015): Serpents d’Europe, d’Afrique du Nord et du Moyen-Orient, 1st Edition. Delachaux et Niestlé SA, Paris.
Geser, S., Kaiser, L., Zwahlen, V., Ursenbacher, S. (2013): Development of polymorphic microsatellite loci markers for the Asp viper (Vipera aspis) using high-throughput sequencing and their use for other European vipers. Amphibia-Reptilia 34: 109-113.
Grabensteiner, K.C., Taylor, S.A. (2018): Breaking barriers: causes, consequences, and experimental utility of human-mediated hybridization. Trends Ecol. Evol. 33: 198-212.
Guiller, G. (2012): Comparaison in natura de la croissance et de l’âge de la maturité sexuelle chez Vipera berus (Linnaeus, 1758) et Vipera aspis (Linnaeus, 1758) dans le département de la Loire-Atlantique. Bull. Soc. sci. nat. Ouest Fr. 34: 186-199.
Guiller, G., Legentilhomme, J., Lourdais, O. (2012): Stratégies reproductrices de trois espèces de serpents vivipares en situation de sympatrie en Loire-Atlantique (44): Vipera berus (Linnaeus, 1758), Vipera aspis (Linnaeus, 1758) et Coronella austriaca (Laurenti, 1768). Bull. Soc. Herp. Fr. 142-143: 123-143.
Guiller, G., Legentilhomme, J., Lourdais, O. (2014): Phénologie d’activité d’une communauté de squamates au nord de la Loire-Atlantique. Bull. Soc. Herp. Fr. 150: 25-39.
Guiller, G., Lourdais, O., Ursenbacher, S. (2017): Hybridization between a Euro-Siberian (Vipera berus) and a Para-Mediterranean viper (V. aspis) at their contact zone in western France. J. Zool. 302: 138-147.
Guiller, G., Delemarre, J.-L., Charles, M., Legentilhomme, J. (2018): Actualisation de la zone de contact entre Vipera berus et Vipera aspis dans le département de la Loire-Atlantique (France). Bull. Soc. Herp. Fr. 165: 3-14.
Guillon, M., Guiller, G., DeNardo, D.F., Lourdais, O. (2013): Microclimate preferences correlate with contrasted evaporative water loss in parapatric vipers at their contact zone. Can. J. Zool. 92: 81-86.
Hubisz, M.J., Falush, D., Stephens, M., Pritchard, J.K. (2009): Inferring weak population structure with the assistance of sample group information. Mol. Ecol. Resour. 9: 1322-1332.
Kindler, C., Chèvre, M., Ursenbacher, S., Böhme, W., Hille, A., Jablonski, D., Vamberger, M., Fritz, U. (2017): Hybridization patterns in two contact zones of grass snakes reveal a new Central European snake species. Sci. Rep. 7: 7378.
Kopelman, N.M., Mayzel, J., Jakobsson, M., Rosenberg, N.A., Mayrose, I. (2015): Clumpak: a program for identifying clustering modes and packaging population structure inferences across K. Mol. Ecol. Resour. 15: 1179-1191.
Lapini, L. (1988): Osservazioni sulla fauna erpetologica (Amphibia, Reptilia) dell’alta Val Torre (Italia nord-orientale, Prealpi Giulie). Gortania 9 (1987): 201-222.
Liu, S. (2010): Distant hybridization leads to different ploidy fishes. Sci. China Life Sci. 53: 416-425.
Luiselli, L., Filippi, E., Lena, E. (2009): Ecological relationships between sympatric Vipera aspis and Vipera ursinii in high-altitude habitats of Central Italy. J. Herpetol. 41: 378-384.
Mallet, J. (2008): Hybridization, ecological races and the nature of species: empirical evidence for the ease of speciation. Philos. Trans. R. Soc., B 363: 2971-2986.
Mallet, J., Besansky, N., Hahn, M.W. (2016): How reticulated are species? BioEssays 38: 140-149.
Matute, D.R., Gavin-Smyth, J. (2014): Fine mapping of dominant X-linked incompatibility alleles in Drosophila hybrids. PLoS Genet. 10: e1004270.
Mebert, K. (2008): Good species despite massive hybridization: genetic research on the contact zone between the watersnakes Nerodia sipedon and N. fasciata in the Carolinas, USA. Mol. Ecol. 17: 1918-1929.
Mebert, K., Trapp, B., Asta, A., Velensky, P., Böhme, W. (2011): Hybrids between Natrix tessellata and N. natrix/maura. Mertensiella 18: 154-156.
Mebert, K., Jagar, T., Grželj, R., Cafuta, V., Luiselli, L., Ostanek, E., Golay, P., Dubey, S., Golay, J., Ursenbacher, S. (2015): The dynamics of coexistence: habitat sharing versus segregation patterns among three sympatric montane vipers. Biol. J. Linn. Soc. 116: 364-376.
Mertens, R. (1964): Über Reptilienbastarde, III. Senck. biol. 45: 33-49.
Mihalca, A., Catoi, C., Gherman, C., Ghira, I. (2003): Presence of Caryospora simplex Léger, 1904 (Apicomplexa: Eimeriidae) in natural hybrids of Vipera ammodytes and Vipera berus (Serpentes: Viperidae) from Romania. Bulletin UASVM, Veterinary Medicine 60: 264-267.
Monney, J.-C. (1996): Biologie comparée de Vipera aspis L. et de Vipera berus L. (Reptilia, Ophidia, Viperidae) dans une station des Préalpes bernoises. Faculté des Sciences, p. 174. Université de Neuchâtel, Neuchâtel.
Naulleau, G. (1986): Répartition de Vipera spis et de Vipera berus (Reptilia, Viperidae) dans l’Ouest de la France (Loire-Atlantique). Bull. Soc. Herp. Fr. 39: 16-19.
Nielsen, E.E.G., Bach, L.A., Kotlicki, P. (2006): hybridlab (version 1.0): a program for generating simulated hybrids from population samples. Mol. Ecol. Notes 6: 971-973.
Nilson, G. (1980): Male reproductive cycle of the European adder, Vipera berus, and its relation to annual activity periods. Copeia 1980: 729-737.
Nilson, G., Andrén, C. (1997): Evolution, systematics and biogeography of Palearctic vipers. Symp. Zool. Soc. Lond. 70: 31-42.
Pavlov, A.V., Zinenko, O., Joger, U., Stümpel, N., Petrova, I.V., Malenyov, A.L., Zaitseva, O.V., Shurshina, I.V., Bakiev, A.G. (2011): Natural hybridization of the eastern steppe viper Vipera renardi and the common adder V. berus. Proc. Samara Sci. Center Russ. Acad. Sci. 13: 172-178.
Peakall, R., Smouse, P.E. (2006): GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol. Ecol. Notes 6: 288-295.
Peakall, R., Smouse, P.E. (2012): GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research – an update. Bioinformatics 28: 2537-2539.
Prestt, I. (1971): An ecological study of the viper Vipera berus in southern Britain. J. Zool. 164: 373-418.
Pritchard, J.K., Stephens, M., Donnelly, P. (2000): Inference of population structure using multilocus genotype data. Genetics 155: 945-959.
Pritchard, J.K. (2009): Documentation for STURCTURE software: version 2.3. http://pritch.bsd.uchicago.edu/structure.html.
Rohde, K., Hau, Y., Weyer, J., Hochkirch, A. (2015): Wide prevalence of hybridization in two sympatric grasshopper species may be shaped by their relative abundances. BMC Evol. Biol. 15: 191.
Rundle, H.D., Nosil, P. (2005): Ecological speciation. Ecol. Lett. 8: 336-352.
Saint Girons, H. (1975): Coexistence de Vipera aspis et de Vipera berus en Loire-Atlantique: un problème de compétition interspécifique. Rev. Ecol. – Terre Vie 29: 590-613.
Saint Girons, H. (1977): Caryotypes et évolution des Vipères Européennes (Reptilia, Viperidae). Bull. Soc. Zool. Fr. 102: 39-49.
Saint Girons, H. (1980a): Biogéographie et évolution des vipères européennes. C. R. Soc. Biogéogr. 496: 146-172.
Saint Girons, H. (1980b): Le cycle des mues chez les vipères européennes. Bull. Soc. Zool. Fr. 105: 551-559.
Saint Girons, H. (1990): Croissance, maturité sexuelle et variations ontogéniques des périodes d’alimentation et des mues chez deux vipères hybrides (Vipera aspis × Vipera seoanei) dans des conditions semi-naturelles. Bull. Soc. Herp. Fr. 54: 62-68.
Sanders, K.L., Rasmussen, A.R., Guinea, M.L. (2014): High rates of hybridisation reveal fragile reproductive barriers between endangered Australian sea snakes. Biol. Conserv. 171: 200-208.
Scali, S., Mangiacotti, M., Sacchi, R., Gentilli, A. (2011): A tribute to Hubert Saint Girons: niche separation between Vipera aspis and V. berus on the basis of distribution models. Amphibia-Reptilia 32: 223-233.
Schultze, N., Laufer, H., Kindler, C., Fritz, U. (2019): Distribution and hybridisation of barred and common grass snakes (Natrix helvetica, N. natrix) in Baden-Württemberg, South-western Germany. Herpetozoa 32: 229-236.
Schweiger, M. (2009): Vipera ammodytes: von abnormen Vipern und Hybriden. Draco 39: 22-30.
Stebbins, G.L. (1958): The inviability, weakness, and sterility of interspecific hybrids. In: Advances in Genetics, p. 147-215. Demerec, M., Ed., Academic Press, New York.
Szyndlar, Z., Rage, J.-C. (2002): Fossil record of the true vipers. In: Biology of the Vipers, p. 419-444. Schuett, G.W., Höggren, M., Douglas, M.E., Greene, H.W., Eds, Eagle Mountain Publishing, Eagle Mountain, UT.
Tarroso, P., Pereira, R.J., Martínez-Freiría, F., Godinho, R., Brito, J.C. (2014): Hybridization at an ecotone: ecological and genetic barriers between three Iberian vipers. Mol. Ecol. 23: 1108-1123.
UICN France, MNHN, SHF (2015): La Liste rouge des espèces menacées en France – Chapitre Reptiles et Amphibiens de France métropolitaine. Paris.
Ursenbacher, S., Carlsson, M., Helfer, V., Tegelström, H., Fumagalli, L. (2006): Phylogeography and Pleistocene refugia of the adder (Vipera berus) as inferred from mitochondrial DNA sequence data. Mol. Ecol. 15: 3425-3437.
Ursenbacher, S., Erny, C., Fumagalli, L. (2009): Male reproductive success and multiple paternity in wild, low-density populations of the adder (Vipera berus). J. Hered. 100: 365-370.
Ursenbacher, S., Monney, J.C., Fumagalli, L. (2009): Limited genetic diversity and high differentiation among the remnant adder (Vipera berus) populations in the Swiss and French Jura Mountains. Conserv. Genet. 10: 303-315.
Vähä, J.-P., Primmer, C.R. (2006): Efficiency of model-based Bayesian methods for detecting hybrid individuals under different hybridization scenarios and with different numbers of loci. Mol. Ecol. 15: 63-72.
Viaud-Grand-Marais, A. (1867): Études médicales sur les serpents de la Vendée et de la Loire-Inférieure. Del’Imprimerie L. Toinon, Saint-Germain.
Zinenko, O., Sovic, M., Joger, U., Gibbs, H.L. (2016): Hybrid origin of European vipers (Vipera magnifica and Vipera orlovi) from the Caucasus determined using genomic scale DNA markers. BMC Evol. Biol. 16: 76.
Zuffi, M.A.L. (2002): A critique of the systematic position of the asp viper subspecies Vipera aspis aspis (Linnaeus, 1758), Vipera aspis atra Meisner, 1820, Vipera aspis francisciredi Laurenti, 1768, Vipera aspis hugyi Schinz, 1833 and Vipera aspis zinnikeri Kramer, 1958. Amphibia-Reptilia 23: 191-213.
Footnotes
Associate Editor: Fernando Martínez-Freiría