Acta Herpetologica 15(1): 47-54, 2020 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/a_h-7648 Genetic characteristics of an introduced population of Bombina bombina (Linnaeus, 1761) (Amphibia: Bombinatoridae) in Moselle, France Jean-Pierre Vacher1,2,*, Damien Aumaître3, Sylvain Ursenbacher2,4 1 Association BUFO, Musée d’Histoire naturelle et d’Ethnographie, 11 rue de Turenne, F-68000 Colmar, France 2 Section of Conservation Biology, Department of Environmental Sciences, University of Basel, St. Johanns-Vorstadt 10, CH-4056 Basel, Switzerland 3 Conservatoire d’espaces naturels de Lorraine, 3 rue Robert Schuman, 57400 Sarrebourg, France 4 info fauna – karch, University of Neuchâtel, avenue de Bellevaux 51, CH-2000 Neuchâtel, Switzerland * Corresponding author. Email: jpvacher@gmail.com Submitted on: 2019, 16th December; revised on: 2019, 21th December; accepted on: 2019, 22th December Editor: Adriana Bellati Abstract. The fire-bellied toad Bombina bombina has recently been introduced in Moselle, north-eastern France, in an area where the yellow-bellied toad Bombina variegata occurs naturally. Both species hybridize in a wide area through- out Europe where their distribution overlaps. Therefore, there is a risk of introgression regarding the Bombina varie- gata population in north-eastern France. In order to assess the status of the introduced population of Bombina bom- bina and its origin, we investigated its genetic characteristics and structure using both mitochondrial (cytochrome b) and nuclear DNA (microsatellites markers). The results demonstrated a lack of introgression in the Bombina var- iegata population. Though experiencing a bottleneck effect, the introduced Bombina bombina population displays a high genetic diversity. If a propensity for expansion is found within the introduced population of Bombina bombina, it could be considered as a potential invasive species in France, and thus threaten the native species. Keywords. Invasive species, population genetics, conservation, cytochrome b, microsatellites. INTRODUCTION Introduction of allochthonous species in natural habitats represents one of the aggravating factors of the current loss of biodiversity (Kats and Ferrer, 2003). Such introductions may induce numerous effects on native species, such as ecological competition, over predation, transmission and dispersal of pathogens, and genetic introgression (Mooney and Cleland, 2001; Strayer et al., 2006). In the context of conservation biology, it is impor- tant to understand the factors which enabled introduced species to adapt to their new environment, in order to define prevention, monitoring, and management plans for these species (Strayer et al., 2006). Amphibians are currently in the focus for conserva- tion biologists as they represent the most endangered group of vertebrates worldwide (Stuart et al., 2004; Stuart et al., 2008). Invasive alien species are a major threat to amphibians (Kats and Ferrer, 2003). Among them, other amphibian species can have an impact on native ones. For example, they can be an important source of disper- sal of the chytrid fungus Batrachochytrium dendrobatidis (Bd), a pathogen which affects many amphibian species around the globe and causes their decline (Ficetola et al., 2008; Fisher and Garner, 2007; Garner et al., 2006). Another possible threat linked with introduced species is genetic introgression, which can, in some cases, lead to local extinctions (Arntzen and Thorpe, 1999; Dufresnes et al., 2016; Rhymer and Simberloff, 1996). In France, at least six species of amphibians have been introduced: Triturus carnifex, Discoglossus pic- tus, Lithobates catesbeianus, Xenopus laevis, Pelophylax 48 Jean-Pierre Vacher et alii ridibundus, and P. bedriagae (Duguet and Melki, 2003; Lescure and de Massary, 2012). In 2009, an introduced population of the fire-bellied toad Bombina bombina has been discovered in the eastern part of the Moselle depart- ment, close to the Sarre valley (Vacher and Pichenot, 2012) (Fig. 1). In this region, the yellow-bellied toad Bombina variegata occurs naturally (Lescure et al., 2011; Lescure and de Massary, 2012; Thiriet and Vacher, 2010). This last species is considered as endangered in France and listed as ‘Vulnerable’ on the national red list pub- lished by the French Committee of the IUCN (UICN France et al., 2015). The occurrence of introduced Bom- bina bombina close to natural populations of B. variegata raises conservation concern as B. bombina could lead to the decline of B. variegata through introgression. Indeed, hybridization has already been demonstrated where both species are in contact in their native range (Gollmann et al., 1988; Szymura, 1976; Yanchukov et al., 2006). In order to characterize the status of this introduced popula- tion, and its possible interaction with the native Bombina variegata, we assessed if hybridization already occurred between the native and the introduced species using genetic markers as well as the putative origin of the intro- duced individuals. MATERIALS AND METHODS Sampling design and laboratory methods We collected 61 DNA samples from individuals morpho- logically assigned to Bombina bombina from three localities in Moselle (6.87°E, 48.92°N) and 64 samples from individu- als morphologically assigned to Bombina variegata from four neighbouring localities in Moselle and Bas-Rhin in 2011 and 2012. DNA samples were collected through buccal swabbing (Beebee, 2008; Pidancier et al., 2003). The two westernmost Bombina bombina localities were 1 km distant from each other, and the third was 5 km south-east from the others. DNA was extracted from the buccal swabs using the QIA- GEN DNeasy Blood & Tissue kit (QIAGEN®). As we suspected that all Bombina bombina individuals would originate from the same locality, we amplified by PCR a fragment of 1200 bp of the mitochondrial cytochrome b (cytb) using L16245 and H17444 primers (Hofman and Szymura, 2007) from only five out of the 61 samples. Amplifications were performed following Hofman and Szymura (2007) and sequencing were performed by Mac- rogen (Amsterdam, the Netherlands). The new sequences were deposited in GenBank (Table 1). Ten microsatellite loci specifically developed for the fire- bellied toad (BobomF2, Bobom5F, Bobom9H, Bobom1A, BobomF22, Bobom10F, Bobom8A, BobomD2, BobomB13 and Bobom11D) were amplified by PCR for all 125 samples, follow- ing the PCR conditions suggested by Hauswaldt et al. (2007) and Stuckas and Tiedemann (2006). Forward dyed primers were used in order to analyse them with an automatic sequencer (AB3130xl Applied Biosystem). Allele lengths were then read with the software PEAK SCANNER v.1.0 (Applied Biosystem). Data analysis We used cytb sequences from GenBank to confirm taxonomic assignation of our samples. The cytb sequences obtained were first aligned automatically using the software MAFFT (Katoh and Standley, 2013), and then the alignment was checked in MEGA (Tamura et al., 2011). We subsequent- ly grouped our sequences with other Bombina bombina cytb sequences published in a previous study on the phylogeography of the species (Fijarczyk et al., 2011). After inferring the best sequence evolutionary model in PartitionFinder v.1.1.1 (Lan- fear et al., 2012), using a BIC approach, we constructed a phy- logenetic tree with a Maximum Likelihood method in RAxML v.8 (Stamatakis, 2014) under the GTR+G model. We used one sequence of Bombina variegata as an outgroup to root our tree. After visualizing the position of our samples in the tree, we subsequently selected the sequences that were the closest to the samples from Moselle, and constructed a haplotype network using the software Hapview (Salzburger et al., 2011). Each microsatellite locus was first examined for null allele occurrence with MICRO-CHECKER v.2.2.3 (Van Oosterhout et al., 2004) for each population. Loci showing a high probability (P > 0.05) of null alleles were discarded from the dataset. For each retained locus, we estimated allele frequency, allelic rich- ness (AR), observed and expected heterozygosity (HO, HE), and intrapopulation structuration (FIS) with the packages adegenet (Jombart, 2008) and hierfstat (Goudet, 2005) implemented in R (R Development Core Team, 2016). Moreover, Hardy-Weinberg equilibrium was tested for each locus with allele randomizations (1000 permutations per test) with the package pegas (Paradis, 2010) implemented in R. In addition, we evaluated the num- ber of genetic clusters (K) using a Bayesian clustering approach implemented in the software STRUCTURE v.2.3.3 (Pritchard et Fig. 1. Global distribution of Bombina bombina and B. variegata and location of the newly introduced population in France. Map sources: Naturalearth/IUCN. 49Genetics of introduced Bombina bombina in France al., 2000). First, we conducted the analysis for the three puta- tive populations of B. bombina, and then for B. bombina and B. variegata populations together. We performed ten independ- ent runs for each K, and tested between 1 and 3 for B. bombina alone, and up to seven clusters for B. bombina and B. variegata grouped together, according to the number of localities we sam- pled. Each replicate was run for 400,000 iterations following a burn-in period of 200,000. As the three sampled populations of B. bombina were supposedly closely related, we used the admix- ture model with allele frequencies that were correlated among populations (Falush et al., 2003). In order to identify the most likely value of K, the logarithmic probability of the data [Ln P(D)] was estimated for each simulation. Additionally, the value of ΔK, representing the second order of change, was estimated (Evanno et al., 2005). Finally, we tested if a bottleneck effect (significant heterozygosity excess) was detected within the pop- ulation of Bombina bombina with the software BOTTLENECK v.1.2.02 (Cornuet and Luikart, 1996; Piry et al., 1999), using a Wilcoxon signed-rank test. Such an effect is expected after a strong reduction in population size (Hedrick et al., 1986), such as in recent introduced populations. RESULTS BLAST and haplotypes The BLAST results showed that the five samples matched with Bombina bombina cytb sequences deposit- ed in GenBank. Maximum Likelihood inference suggests that the samples from Lorraine are nested within a clade that originates from Austria and Czech Republic (Fig. 2). The haplotypes found in Moselle cluster within hap- logroup B3-1 (Fijarczyk et al., 2011), that contains speci- mens from southern Europe. More precisely, one indi- vidual of Moselle is identical to haplotype B14 (Fig. 3), that includes specimens from Czech Republic, Slovakia, Austria, Croatia, Serbia, Hungary, and Ukraine (Fijarc- zyk et al., 2011), and the four other haplotypes retrieved from the specimens from Moselle only differ from B14 by three to eight substitutions (Fig. 3). Genetic variation and diversity We detected an excess of homozygosity, thus the probable presence of null alleles for BobomF2, Bobo- mF22, and BobomD2 within B. bombina populations only. Therefore, these markers were discarded from the subsequent analyses for B. bombina, which were conse- quently conducted with seven microsatellites markers (Bobom5F, Bobom9H, Bobom1A, Bobom10F, Bobom8A, BobomB13, and Bobom11D). The number of alleles in B. bombina of Moselle varies from three (Bobom5F and Bobom1A) to ten (Bobom9H) Fig. 2. Best Maximum Likelihood inference obtained from RAxML using ~1000 bp of cytb mtDNA of Bombina bombina. Bootstrap values above 70 are given at each nodes. The tree is rooted on Bom- bina variegata (not shown). The GenBank accession numbers are provided, new sequences are indicated in bold. 50 Jean-Pierre Vacher et alii Fig. 3. Haplotype network based on a fragment of the mitochon- drial cytb in Bombina bombina. The haplogroups were defined in Fijarczyk et al. (2011). The five new haplotypes from the introduced specimens in Moselle (this study) are represented in red. Each line represents a single mutation, and the size of the circles represents the frequency of a haplotype. Table 1. GenBank accession numbers and additional information of the locality of the newly introduced Bombina bombina in France (in bold) and the samples from GenBank used in the genetic analysis. No Location Accession GenBank Reference 1 Moselle, France MN784157 This study 2 Moselle, France MN784158 This study 3 Moselle, France MN784159 This study 4 Moselle, France MN784160 This study 5 Moselle, France MN784161 This study 6 Hungary JF898363 Fijarczyk et al., 2011 7 Austria JF898357 Fijarczyk et al., 2011 8 Romania JF898366 Fijarczyk et al., 2011 9 Bulgaria JF898352 Fijarczyk et al., 2011 10 Hungary JF898356 Fijarczyk et al., 2011 11 Romania JF898353 Fijarczyk et al., 2011 12 Ukraine JF898347 Fijarczyk et al., 2011 13 Bulgaria JF898351 Fijarczyk et al., 2011 14 Bulgaria JF898350 Fijarczyk et al., 2011 15 Moldova JF898348 Fijarczyk et al., 2011 16 Hungary JF898364 Fijarczyk et al., 2011 17 Slovakia JF898345 Fijarczyk et al., 2011 18 Romania JF898346 Fijarczyk et al., 2011 19 Belarus JF898340 Fijarczyk et al., 2011 20 Turkey JF898362 Fijarczyk et al., 2011 21 Turkey JF898361 Fijarczyk et al., 2011 22 Ukraine JF898343 Fijarczyk et al. , 2011 23 Ukraine JF898342 Fijarczyk et al., 2011 24 Turkey JF898360 Fijarczyk et al., 2011 25 Moldova JF898349 Fijarczyk et al., 2011 26 Austria JF898358 Fijarczyk et al., 2011 No Location Accession GenBank Reference 27 Serbia JF898344 Fijarczyk et al., 2011 28 Romania JF898355 Fijarczyk et al., 2011 29 Moldova JF898354 Fijarczyk et al., 2011 30 Ukraine JF898339 Fijarczyk et al., 2011 31 Romania JF898365 Fijarczyk et al., 2011 32 Czech Republic JF898359 Fijarczyk et al., 2011 33 Ukraine JF898341 Fijarczyk et al., 2011 34 Belarus JF898338 Fijarczyk et al., 2011 35 Russia JF898330 Fijarczyk et al., 2011 36 Poland JF898321 Fijarczyk et al., 2011 37 Poland JF898326 Fijarczyk et al., 2011 38 Slovakia JF898325 Fijarczyk et al., 2011 39 Russia JF898334 Fijarczyk et al., 2011 40 Ukraine JF898333 Fijarczyk et al., 2011 41 Russia JF898332 Fijarczyk et al., 2011 42 Ukraine JF898329 Fijarczyk et al., 2011 43 Kazakhstan JF898328 Fijarczyk et al., 2011 44 Poland JF898324 Fijarczyk et al., 2011 45 Kazakhstan JF898327 Fijarczyk et al., 2011 46 Ukraine JF898337 Fijarczyk et al., 2011 47 Russia JF898335 Fijarczyk et al., 2011 48 Ukraine JF898331 Fijarczyk et al., 2011 49 Poland JF898322 Fijarczyk et al., 2011 50 Russia JF898336 Fijarczyk et al., 2011 51 Poland JF898320 Fijarczyk et al., 2011 52 Poland JF898323 Fijarczyk et al., 2011 Fig. 4. (A) Clusters from retrieved in the STRUCTURE analysis from seven microsatellite markers; (B) Distribution in space of the two nuclear DNA clusters. The geographic coordinates are not pro- vided on purpose. Map source: Global Forest Watch. 51Genetics of introduced Bombina bombina in France (Table 1). The mean AR was 4.23 (calculated from 61 dip- loid individuals). The mean HE value was 0.65, and the mean HO value was 0.66. There was no significant differ- ence between the overall values of HO and HE (Bartlett’s K-squared = 0.035, df = 1, P = 0.8). The overall FIS value was 0.01 and ranged from -0.05 for Bobom 11D to 0.09 for Bobom 5F (Table 1). Genetic structure of populations The analysis conducted with STRUCTURE did not reveal any population differentiation between the three sampling sites of Bombina bombina in Moselle. In the analysis conducted with all the samples of B. bombina and B. variegata, both species formed two well-differen- tiated clusters, indicating a complete lack of introgression (Fig. 4). Bottleneck The BOTTLENECK analysis revealed a bottle- neck effect within the Bombina bombina population of Moselle. Indeed, the Wilcoxon test showed an excess of heterozygotes compared to the expected equilibrium het- erozygosity under both the SMM and TPM models (Wil- coxon test: P = 0.007). DISCUSSION Globally, the genetic diversity observed in this intro- duced population of B. bombina is rather high, since the mean HE value of 0.65 for seven loci is similar to the ones found in 11 natural populations of Bombina bombina that occur in the core of the range of the species in Germany and that averages 0.70 [0.59-0.78] for six loci (Dolgener et al., 2012). This could be explained by the introduction of numerous individuals, maybe through different epi- sodes. As the biggest population was observed in a series of lakes that are used for fish farming, it is highly prob- able that the presence of Bombina bombina in this area resulted from the transport of tadpoles caught together with young fishes from one or several close localities in Central Europe. Such cases of translocations have been observed in Brandenburg and in Saxony (Berger, 1996; Dolgener et al., 2012), so it is very likely that the occur- rence of B. bombina in Moselle might result from a simi- lar event. As tadpoles are small organisms, it is possible that hundreds, or maybe thousands of them have been introduced, therefore maintaining a high genetic diversi- ty. Still it was expected to detect a bottleneck effect with- in this population as it is the case with recently intro- duced populations (Lee, 2002; Puillandre et al., 2008). In comparison with other amphibian species, the fire-bellied toad does not have a high fertility rate, with around 300- 400 eggs per female per year (Gollmann et al., 2011). Therefore, the high diversity and the high number of alleles observed in some markers indicate that introduc- tion of only a few founder individuals, as observed in other introduction of amphibians in France such as the bullfrog Lithobates catesbeianus (Ficetola et al., 2008), is unlikely. We could think that even though the primary source of the B. bombina population in Moselle resulted from numerous tadpoles, they still represent a small frac- tion of a broader population located in the core area of distribution and that though a bottleneck effect could be detected, it was not sufficient enough to affect the genetic diversity of this population. The discovery of Bombina bombina in Lorraine is recent, certainly dating back to 2009 (Vacher and Pichenot, 2012). Right now, its distribution is geographi- cally restricted and does not directly overlap with that of B. variegata (Fig. 4). As it can hybridize with B. var- iegata in the wild, a monitoring of B. bombina in the area should be set up to track the population dynam- ics, its dispersal behaviour, the evolution of its distribu- tion in the area, and to determine possible concurrence with B. variegata. However, both species seem to display contrasted ecological preferences in their native habi- tat: B. bombina is known to prefer ponds or swamps for reproduction compared to B. variegata that favours pud- dles (Barandun and Reyer, 1997; Gollmann B. et al., 2011; Gollmann G. et al., 2011; Kruuk and Gilchrist, 1997). Consequently, we might expect that ecological compe- tition should be limited. Moreover, B. bombina shows Table 2. Microsatellite loci used for the genetic analyses of 61 individuals of Bombina bombina introduced in Moselle, north- eastern France. The estimations were conducted with the adegenet and hierfstat packages implemented in R. bp: base pairs; AR: allelic richness; HO: observed heterozygosity; HE: expected heterozygo- sity; FIS: intrapopulation structure index. Microsatellite Length (bp) Alleles number AR HO HE FIS Bobom5F 126-146 3 3 0.54 0.64 0.09 Bobom9H 115-203 10 7.6 0.83 0.86 0.00 Bobom1A 341-353 3 2.95 0.57 0.54 -0.04 Bobom10F 207-223 4 3.97 0.75 0.73 -0.02 Bobom8A 275-315 5 3.77 0.61 0.63 -0.01 BobomB13 117-147 4 3.21 0.63 0.66 0.12 Bobom 11D 268-296 6 5.12 0.82 0.78 -0.05 52 Jean-Pierre Vacher et alii higher site fidelity, suggesting lower dispersal capacities (Gollmann et al., 2011). Still, B. bombina seems to dis- play a broader ecological tolerance by colonizing puddles in areas where ponds are scarce and where both species occur and hybridize (MacCallum et al., 1998). Therefore, a close attention to habitat components in the landscape (i.e., density of ponds and small lakes) should be inte- grated in a monitoring protocol to track the dynamics of this introduced population. The removal of a species at an early stage is normally the best method to avoid future competition with native species. We can consequently recommend to monitor the competition between both species of Bombina in the area, and perhaps also conduct some actions to reduce or remove this introduced popu- lation. Additionally, it would be necessary to scan for Bd and maybe other pathogens within this introduced pop- ulation, as they can represent a further threat on native amphibian populations that occur in the area such as the European tree frog Hyla arborea or the common frog Rana temporaria (Ohst et al., 2013). ACKNOWLEDGEMENTS We are grateful to Laurent Godé from the Parc naturel régional de Lorraine, Julie Lambrey from Conserv- atoire d’espaces naturels de Lorraine, and Julian Pichenot for their help on the field. We would like to thank Nico- las Boileau and Valerie Zwahlen for their help in the laboratory. A handling permit for Bombina variegata was delivered to Jean-Pierre Vacher by the Préfecture du département du Bas-Rhin and to Damien Aumaître by the Préfecture du département de la Moselle. This study was funded by the Direction régionale de l’environnement, de l’aménagement et du logement Lorraine (Grand Est) in the scope of the national action plan for Bombina variegata. We also thank two anonymous reviewers whose remarks helped enhance the manuscript. REFERENCES Arntzen, J.W., Thorpe, R.S. (1999): Italian crested newts (Triturus carnifex) in the basin of Geneva: Distribu- tion and genetic interactions with autochtonous spe- cies. Herpetologica 55: 423-433. Barandun, J., Reyer, H.U. 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