Exploring the speciation continuum of slow worms: Location and extent of the Anguis fragilis/veronensis hybrid zone in southeastern France

Abstract

With five currently recognized species that form several secondary contact zones, slow worms (Anguidae: Anguis) offer a valuable model to study the fate of evolutionary lineages in the face of hybridization and genetic introgression. The relationships between the Western Slow Worm Anguis fragilis and the Italian Slow Worm Anguis veronensis are particularly puzzling. Their respective distributions remain poorly known on the edges of their parapatric ranges, as both species lack external differentiation. Contra earlier mitochondrial phylogenies, new phylogenomic inferences have shown that A. fragilis and A. veronensis are sister taxa, thus casting doubts on their specific status. In this study, we analyze the A. fragilis/veronensis transition in southeastern France, based on one mitochondrial (ND2) and two nuclear (PRLR and HA1) genetic markers in 81 specimens from 61 localities. The ranges of A. fragilis and A. veronensis roughly extends northwest and southeast of the Rhône-Durance valleys, respectively, with clear signs of introgressive hybridization in the areas of contact (notably the eastern parts of the low Rhône valley). Based on the three molecular markers analyzed, gene flow does not seem to reach outside the narrow hybrid zone, which likely indicates (incomplete) intrinsic reproductive isolation. Hence, we provisionally suggest maintaining A. veronensis as a separate species from A. fragilis. More generally, patterns of genetic divergence, external differentiation, and hybridization (both historical and contemporary) in Anguis ssp. supports a speciation continuum spanning from cryptic, genetically compatible alloparapatric lineages to phenotypically distinct, deeply diverged and fully reproductively isolated taxa able to coexist in sympatry.

Full text

Amphibia-Reptilia (2023) DOI:10.1163/15685381-bja10123 brill.com/amre
Short Note
Exploring the speciation continuum of slow worms: location and
extent of the Anguis fragilis/veronensis hybrid zone in southeastern
France
Christophe Dufresnes1,∗, Patricia Sourrouille2, Anthony Olivier3, Jean-Marie Ballouard4,
Marie-France Leccia5,RémiTiné
6, Marc Cheylan7, Maxime Le Henanff8, Jean Nicolas9,
Sébastien Caron4, Grégoire Massez10, Alexandre Cluchier8, Romain Levasseur4,11, Fabien Pille4,12,
Olivier Peyre13, Marc Thibault3, Angelica Crottini14,15,16, Nicolas Fuento17 , Pierre-André Crochet2
1 - Laboratory for Amphibian Systematic & Evolutionary Research, College of Biology & the Environment,
Nanjing Forestry University, Nanjing, 210037, People’s Republic of China
2 - CEFE, CNRS, Univ. Montpellier, EPHE, IRD, 34293 Montpellier, France
3 - Tour du Valat, Research Institute for the Conservation of Mediterranean Wetlands, 13200 Arles, France
4 - CRCC – Centre for Research and Conservation of Chelonians, SOPTOM, Var, 1065 routes du Luc, 83660
Carnoules, France
5 - Parc National du Mercantour, 06000 Nice, France
6 - Syndicat Mixte Camargue Gardoise, Centre du Scamandre, Route des Iscles, Gallician, 30600 Vauvert,
France
7 - Biogéographie et Ecologie des Vertébrés, CEFE, EPHE-PSL University, Univ. Montpellier, CNRS, IRD,
34293 Montpellier, France
8 - ECO-MED – Ecologie & Médiation, Résidence Atrium, 113 rue Raymond Recouly, 34070 Montpellier,
France
9 - 7, rue du Labro, 34260 Camplong, France
10 - Les Amis des Marais du Vigueirat, Les Marais du Vigueirat, Chemin de l’Etourneau, 13104 Mas Thibert,
France
11 - 474 rue des Comtes de Provence, 83143 Le Val, France
12 - Laboratory of Ecology and Conservation of Amphibians (LECA), Freshwater and Oceanic Science Unit
of Research (FOCUS), University of Liège, 4020 Liege, Belgium
13 - PANDORA, 20 rue Durrell, BP61261, 84922 Avignon cédex 9, France
14 - CIBIO/InBio, Centro de Investigação em Biodiversidade e Recursos Genéticos Campus Agrário de
Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, Portugal
15 - Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, 4099-002 Porto, Portugal
16 - BIOPOLIS Program in Genomics, Biodiversity and Land Planning, CIBIO, Campus de Vairão, 4485-661
Vairão, Portugal
17 - LPO PACA, 6 Av. Jean Jaurès, 83400 Hyères, France
*Corresponding author; e-mail: christophe.dufresnes@hotmail.fr
ORCID iD: Dufresnes: 0000-0002-8497-8908
Received 19 August 2022; final revision received 30 November 2022; accepted 4 January 2023;
published online 20 January 2023
Associate Editor: Jiri Smid
©Koninklijke Brill NV, Leiden, 2023. DOI:10.1163/15685381-bja10123
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2C. Dufresnes et al.
Abstract. With five currently recognized species that form several secondary contact zones, slow worms (Anguidae: Anguis)
offer a valuable model to study the fate of evolutionary lineages in the face of hybridization and genetic introgression. The
relationships between the Western Slow Worm Anguis fragilis and the Italian Slow Worm Anguis veronensis are particularly
puzzling. Their respective distributions remain poorly known on the edges of their parapatric ranges, as both species lack
external differentiation. Contra earlier mitochondrial phylogenies, new phylogenomic inferences have shown that A. fragilis
and A. veronensis are sister taxa, thus casting doubts on their specific status. In this study, we analyze the A. fragilis/veronensis
transition in southeastern France, based on one mitochondrial (ND2) and two nuclear (PRLR and HA1) genetic markers in
81 specimens from 61 localities. The ranges of A. fragilis and A. veronensis roughly extends northwest and southeast of
the Rhône-Durance valleys, respectively, with clear signs of introgressive hybridization in the areas of contact (notably the
eastern parts of the lower Rhône valley). Based on the three molecular markers analyzed, gene flow does not seem to reach
outside the narrow hybrid zone, which likely indicates (incomplete) intrinsic reproductive isolation. Hence, we provisionally
suggest maintaining A. veronensis as a separate species from A. fragilis. More generally, patterns of genetic divergence,
external differentiation, and hybridization (both historical and contemporary) in Anguis ssp. supports a speciation continuum
spanning from cryptic, genetically compatible alloparapatric lineages to phenotypically distinct, deeply diverged and fully
reproductively isolated taxa able to coexist in sympatry.
Keywords: hybridization, multilocus barcoding, phylogeography, reptiles, taxonomy.
Introduction
With the discovery and the taxonomic recog-
nition of species-level lineages that bear little
external differences to the human eye, genetic
barcoding has become the right-hand tool of
herpetologists (Vences et al., 2005; Padial et
al., 2010; Chenuil et al., 2019). Reliable knowl-
edge of geographic distributions is a prerequi-
site for the conservation of such cryptic taxa,
and to get insight into their evolutionary history,
but major sampling gaps remain, even for the
well-studied European diversity (Sillero et al.,
2014). Of particular interest is the characteriza-
tion of secondary contact zones, both to identify
shared biogeographic drivers of species distri-
butions (e.g., Beddek et al., 2018; Dufresnes
and Litvinchuk, 2022), and to assess how repro-
ductive isolation evolves between diverging lin-
eages in respect to their genetic, phenotypic and
ecological differentiation (Barton and Hewitt,
1985; Hewitt, 1988; Payseur, 2010), with appli-
cations for species delimitation (Dufresnes et
al., 2020b, 2021). While mitochondrial DNA
(mtDNA) barcoding has initially been advo-
cated as a fast, cost-effective and reliable tool
for both specimen identification and species
discovery (Hebert et al., 2003), mtDNA is
prone to several limitations (Rubinoff and Hol-
land, 2005), especially when applied to closely
related taxa that still hybridize. MtDNA barcod-
ing is notably insufficient for the study of hybrid
zones, where its haploid nature and clonal inher-
itance precludes the identification of hybrids
and intergrades. A growing body of literature
thus recommends the development of multilo-
cus barcoding tools to assess the boundaries
between cryptic species (Rubinoff et al., 2006;
Dufour et al., 2017; Liu et al., 2017; Dufresnes
and Jablonski, 2022).
The slow worm species complex (Anguis
spp.) is a promising system to explore these
topics. Substantial molecular and phenotypic
divergences have led to raise the number of
species from two to five within the last decade
(Gvoždík et al., 2010, 2013, 2023; Jablonski
et al., 2016), and current taxonomic authorities
now recognize A. graeca and A. cephallonica in
the Balkan Peninsula, A. fragilis and A. colchica
across Western and Eastern Europe, respec-
tively, and A. veronensis in the Italian Peninsula
(Speybroeck et al., 2020; fig. 1). With the excep-
tion of A. cephallonica (Thanou et al., 2014,
2021), all share relatively similar morphologies,
and mapping their respective ranges requires
to associate occurrence datasets with barcoding
results (Jablonski et al., 2017, 2021). At present,
the parapatric areas of four pairs of species
could be approximately located (fig. 1) and
hybrid zone studies have already targeted two of
them. In the Peloponese, Thanou et al. (2021)
demonstrated full isolation between A. graeca
and A. cephallonica, whereas in Central Europe,
the less differentiated A. fragilis and A. colchica
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Genetic barcoding of a slow worm hybrid zone 3
Figure 1. Simplified phylogeny (top left) and distribution of the five recognized species of slow worms (top right), with
genetic barcoding of the A. fragilis/veronensis contact zone in southeastern France (bottom panels). The tree (top left) was
adapted from the phylogenomic results of Gvoždík et al. (2023); arrows illustrate past and present hybridization between
some investigated species pairs (dash lines) but complete isolation between others (disrupted line). The European map (top
right) was built from the species occurrence dataset published by Jablonski et al. (2021). The bottom maps show the average
proportions of A. fragilis (red) and A. veronensis (yellow) alleles, combining all three markers (ND2,PRLR,HA1). Sampled
administrative departments are abbreviated by their two-digits codes, as follow. 04: Alpes-de-Haute-Provence; 05: Haute-
Alpes; 06: Alpes-Maritimes; 13: Bouches-du-Rhône; 30: Gard; 34: Hérault; 83: Var; 84: Vaucluse. Photo: A. veronensis (C.
Dufresnes).
form hybrid populations across the Danube val-
ley (Szabó and Vöros, 2014; Benkovský et al.,
2021). These contrasted patterns fit the gen-
eral expectation that hybridizability is reduced
with increased molecular and phenotypic diver-
gence (Singhal and Moritz, 2013; Dufresnes et
al., 2021), setting grounds for future compara-
tive speciation studies in the genus.
Here we focus on a third Anguis species
pairs: the Western European A. fragilis and the
Italian A. veronensis. Of particular interest is
their rather puzzling phylogenetic relationships.
Analyses of mitochondrial sequences had sug-
gested that A. veronensis is the sister species
of the morphologically distinct A. cephallonica
(Gvoždík et al., 2013; Jablonski et al., 2016),
but recent phylogenomic data revealed that this
was caused by a mitochondrial capture during
a Messinian event of hybridization, and that A.
veronensis is in fact the sister species of A. frag-
ilis (Gvoždík et al., 2023; fig. 1), which is in
line with their mostly similar ecology and mor-
phology – no reliable diagnostic characters exist
for their identification (Gvoždík et al., 2013). In
turn, however, it begs the question whether A.
veronensis should still be considered as a dis-
tinct species from A. fragilis, since this status
essentially relied on its phylogenetic position in
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4C. Dufresnes et al.
the misleading mitochondrial tree (Speybroeck
et al., 2020).
One way to assess the validity of A. vero-
nensis as a biological species, and more gen-
erally to understand how genetic cohesion is
maintained along the Anguis speciation contin-
uum, is to characterize its contact zone with
A. fragilis. Their transition putatively extends
along the Alpine Arc from the Mediterranean
to the Adriatic coasts (Jablonski et al., 2021),
and in its eastern margins, hybridization has
been detected in two scattered localities from
northeastern Italy and Slovenia (Gvoždík et al.,
2013).
In this study, we report on mitochondrial and
nuclear barcoding analyses from southeastern
France, where both A. fragilis and A. veronen-
sis had been previously detected (Gvoždík et al.,
2013; Renet et al., 2018). Our results allow to
accurately locate the transition and provide pre-
liminary insights on hybridization, gene flow,
and landscape barriers that mediate their contact
zone.
Methods
A total of 81 tissue samples were obtained
from 61 southeastern France localities, namely
between the Cevennes (southeastern part of
Massif Central) and the Southern Alps at the
border with Italy. Samples consisted of tail tips
from live specimens (stored in 96% ethanol),
muscle from frozen dead specimens, or var-
ious tissues from dried dead specimens. An
additional 14 samples from pure populations
of A. fragilis (France, Sweden) and A. vero-
nensis (Italy) were also included as references.
A few were previously analyzed by Gvoždík et
al. (2013). Origins of specimen are detailed in
table 1. DNA was extracted using the Qiagen
DNAeasy blood & tissue kit, following the sup-
plier’s procedure.
All individuals were barcoded with the mito-
chondrial gene NADH dehydrogenase subunit
2(ND2), which discriminates A. fragilis and
A. veronensis by as much as 9.2% of sequence
divergence (Gvoždík et al., 2013). PCRs were
carried out in 20 μl reaction volumes with 0.5
μL of each primer (sequences given in supple-
mentary table S1), 10 μL of Taq Sigma PCR
mix, and 2–4 μl of DNA template, under the fol-
lowing thermocycling conditions: 94°C for 1,
40 cycles of 94°C for 30, 60°C for 40 and
72°C for 1, and 72°C for 10. PCR products
were checked on a 1% Agarose gel and Sanger-
sequenced in one or two directions. Sequences
were quality-checked, manually aligned and
trimmed (1,038bp) in MEGA X (Kumar et al.,
2018), and assigned to the A. fragilis or the A.
veronensis ND2 lineages.
For nuclear barcoding, we initially attempted
to analyze four candidate genes with Anguis
sequences available on GenBank: prolactin
receptor (PRLR), hard acidic keratin exon 1
(HA1), oocyte maturation factor gene (C-mos),
and recombination-activating gene exon 1
(RAG1). For each gene, the reference specimens
were amplified and sequenced using the primers
listed in supplementary table S1, and a similar
protocol as ND2 (see above). Sequences were
aligned and trimmed in MEGA X and screened
for diagnostic mutations. However, only PRLR
and HA1 successfully amplified and featured
private alleles between taxa (see also Gvoždík
et al., 2013). For the PLRL alignment (557bp),
fragilis/veronensis diagnostic SNPs segregate
at 37bp (T/G), 50bp (A/G), 363bp (A/G) and
548bp (T/C). For the HA1 alignment (965bp,
plus a 7bp insertion in a few samples), frag-
ilis/veronensis diagnostic SNPs segregate at
251bp (A/T), 437bp (G/A), 811bp (A/G) and
836bp (T/C). We thus subsequently barcoded
these two genes in all 81 southern France indi-
viduals. For genotype scoring, sequence chro-
matograms were screened visually, and het-
erozygotes were identified by confirming dou-
ble peaks at the diagnostic sites. Finally, the
ND2 and PLRL datasets were complemented
with the information of six specimens collected
on Saint-Marguerite Island and barcoded by
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Genetic barcoding of a slow worm hybrid zone 5
Tab l e 1. Details on the specimens included in this study. For each genetic marker, A. fragilis (F) and A. veronensis (V) alleles are indicated. French administrative departments are abbreviated
by their two-digits codes, as follow: 04: Alpes-de-Haute-Provence; 05: Haute-Alpes; 06: Alpes-Maritimes; 13: Bouches-du-Rhône; 30: Gard; 34: Hérault; 64: Pyrénées-Atlantiques; 66:
Pyrénées-Atlantiques; 83: Var; 84: Vaucluse; 88: Vosges; 93: Seine-Saint-Denis. Geographic coordinates are given alongside their precision (Prec. in meters).
Sample/voucher ND2 PRLR HA1 Locality Latitude Longitude Prec Ref.
Southeastern France
T11411 F OP255875 FF OP209530 FF OP209625 34: Montarnaud, Bois de la Rouvière 43.6693 3.6669 30 1
T11412 F OP255876 FF OP209531 FF OP209626 34: Montarnaud, Bois de la Rouvière 43.6693 3.6669 30 1
T11678 F OP255877 FF OP209532 FF OP209627 30: Scamandre 43.60511 4.34377 100 1
T11679 F OP255878 FF OP209533 FF OP209628 30: Scamandre 43.60511 4.34377 100 1
T11680 F OP255879 FF OP209534 FF OP209629 30: Scamandre 43.60511 4.34377 100 1
T11681 F OP255880 FF OP209535 FF OP209630 30: Scamandre 43.60511 4.34377 100 1
T11682 F OP255881 FF OP209536 FF OP209631 30: Scamandre 43.60511 4.34377 100 1
T11683 F OP255882 FF OP209537 FF OP209632 30: Scamandre 43.60511 4.34377 100 1
T11684 F OP255883 FF OP209538 FF OP209633 30: Gargattes 43.58881 4.34605 100 1
T10658 F OP255884 FF OP209539 FF OP209634 30: Salagosse, Ruisseau Le Souls 44.01925 3.55538 20 1
T10659 F OP255885 FF OP209540 FF OP209635 30: Salagosse, Ruisseau Le Souls 44.01925 3.55538 20 1
T616/BEV.9248 F OP255886 FF OP209541 FF OP209636 30: Campis 43.9802 3.62444 10 1, 2
T11961 F OP255887 FF OP209542 VV OP209637 84: Velleron 43.95908 5.04113 5 1
T11962 F OP255888 VV OP209543 FF OP209638 84: Velleron 43.95908 5.04113 5 1
T12438 F OP255889 FF OP209544 FF OP209639 84: Veaux 44.21109 5.21002 5 1
T12439/BEV.15166 F OP255890 FF OP209545 FF OP209640 84: Avignon, La Motte 43.99518 4.84297 50 1
T13515 V OP255891 VV OP209546 FV OP209641 84: Mérindol 43.74239 5.20102 10 1
T13519 F OP255892 FF OP209547 FF OP209642 05: La Motte-en-Champsaur, Molines-en-Champsaur 44.74537 6.12134 10 1
T13520 F OP255893 FF OP209548 FF OP209643 05: La Motte-en-Champsaur, Molines-en-Champsaur 44.7454 6.12098 10 1
T13344 V OP255894 VV OP209549 VV OP209644 04: Malikai, Saint Florent 44.02807 6.07974 20 1
T4204/BEV.8149 F OP255895 VV OP209550 FV OP209645 04: Saint-Étienne-les-Orgue, Station de Lure 44.1128 5.7817 300 1
T11416/BEV.14686 F OP255896 FF OP209551 FF OP209646 13: Tour du Valat 43.5087 4.6678 200 1
T11418/BEV.14688 F OP255897 FF OP209552 FF OP209647 13: Tour du Valat 43.5087 4.6678 200 1
T10050/BEV.14692 F OP255898 FF OP209553 FF OP209648 13: Tour du Valat 43.5087 4.6678 999 1
T11417/BEV.14687 F OP255899 FV OP209554 FF OP209649 13: Tour du Valat 43.5087 4.6678 200 1
T13097 F OP255900 FF OP209555 FF OP209650 13: Tour du Valat 43.5091 4.6687 200 1
T9542 F OP255901 FF OP209556 FF OP209651 13: Villeneuve 43.5738 4.6238 10 1
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6C. Dufresnes et al.
Tab l e 1. (Continued.)
Sample/voucher ND2 PRLR HA1 Locality Latitude Longitude Prec Ref.
T11973/BEV.10349 V OP255902 VV OP209557 VV OP209652 13: Meyreuil 43.4748 5.5003 300 1
T13345 F OP255903 FV OP209558 FV OP209653 13: Arles 43.63089 4.74755 20 1
T12197/BEV.14877 V OP255904 VV OP209559 FV OP209654 13: Arles, Tourades 43.6820 4.6800 250 1
T12498 F OP255905 FF OP209560 FV OP209655 13: Arles, Bois de la Commanderie 43.55719 4.69987 10 1
T543/BEV.15111 F OP255906 FF OP209561 FF OP209656 13: Mas Thibert, Marais du Vigueirat 43.52316 4.76307 100 1
T13657/BEV.15522 F OP255907 FV OP209562 FV OP209657 13: Mas Thibert, la Volpelière 43.58000 4.72083 10 1
T13291 V OP255908 VV OP209563 FV OP209658 13: Fontvieille, Vallon de la Lèque 43.75626 4.73766 100 1
T13338 F OP255909 VV OP209564 VV OP209659 13: Entressens 43.5901 4.9553 500 1
T13339 V OP255910 VV OP209565 VV OP209660 13: Istres, Domaine de Sulauze 43.5527 4.9958 500 1
T10364/BEV.14104 F OP255911 VV OP209566 FF OP209661 13: Raphèle-lès-Arles, Moulès 43.6574 4.7582 20 1
T13343 F OP255912 FV OP209567 FF OP209662 13: Raphèle-lès-Arles, Moulès 43.65188 4.74360 20 1
T13654/BEV.15519 F OP255913 FF OP209568 FF OP209663 13: Raphèle-les-Arles, Marais des Chanoines 43.62424 4.7138 200 1
T13655/BEV.15520 F OP255914 FV OP209569 VV OP209664 13: Raphèle-les-Arles, Marais des Chanoines 43.62424 4.7138 200 1
T13512/BEV.7657 F OP255915 FF OP209570 VV OP209665 13: Le Paradou 43.72324 4.7945 15 1
T13516 V OP255916 VV OP209571 FV OP209666 13: Istres, Parc de l’Olivier 43.53031 4.9783 10 1
T13517 V OP255917 VV OP209572 VV OP209667 13: La Roque-d’Anthéron, Le Dévens 43.74379 5.28197 10 1
T13518 V OP255918 VV OP209573 VV OP209668 13: Lambesc 43.65388 5.25635 10 1
T13521 V OP255919 VV OP209574 VV OP209669 13: Trets 43.47233 5.69251 10 1
T13522 V OP255920 VV OP209575 VV OP209670 13: Trets 43.47233 5.69251 10 1
T13523 V OP255921 VV OP209576 VV OP209671 13: La Barben, Touloubre 43.62593 5.23186 10 1
T13656/BEV.15521 F OP255922 VV OP209577 FV OP209672 13: Barbegal 43.69642 4.73289 20 1
T11429 V OP255923 VV OP209578 VV OP209673 83: Gonfaron 43.3194 6.3180 100 1
T11432 V OP255924 VV OP209579 VV OP209674 83: Gonfaron 43.3194 6.3180 100 1
T11424 V OP255925 VV OP209580 VV OP209675 83: Gonfaron 43.3216 6.2911 100 1
T11425 V OP255926 VV OP209581 VV OP209676 83: Gonfaron 43.3216 6.2911 100 1
T11433 V OP255927 VV OP209582 VV OP209677 83: Gonfaron 43.3216 6.2911 100 1
T11431 V OP255928 VV OP209583 VV OP209678 83: Pignans 43.28671 6.22292 100 1
T11434 V OP255929 VV OP209584 VV OP209679 83: Le Cannet-des-Maures 43.3251 6.4046 100 1
T11430 V OP255930 VV OP209585 VV OP209680 83: Fréjus 43.4680 6.8372 100 1
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Genetic barcoding of a slow worm hybrid zone 7
Tab l e 1. (Continued.)
Sample/voucher ND2 PRLR HA1 Locality Latitude Longitude Prec Ref.
T11426 V OP255931 VV OP209586 VV OP209681 83: Vidauban 43.4021 6.4237 100 1
T11427 V OP255932 VV OP209587 VV OP209682 83: Vidauban 43.4021 6.4237 100 1
T11428 V OP255933 VV OP209588 VV OP209683 83: Vidauban 43.4021 6.4237 100 1
T10661 V OP255934 VV OP209589 VV OP209684 83: La Garde-Freinet, Les Mouleyrettes 43.33492 6.4260 20 1
T4193/BEV.11033 V OP255935 VV OP209590 VV OP209685 83: Montagne de Malay 43.70465 6.66207 999 1
T4209/BEV.10323 V OP255936 VV OP209591 VV OP209686 83: Vallon de Saint-Daumas 43.31916 6.40703 500 1
T4210/BEV.10334 V OP255937 VV OP209592 VV OP209687 83: Les Blaïs 43.4020 6.3890 800 1
T4211/BEV.10381 V OP255938 VV OP209593 VV OP209688 83: Les Mayons, Les Aires Vertes 43.3207 6.36138 400 1, 2
T8102/BEV.13539 V OP255939 VV OP209594 VV OP209689 83: Les Mayons 43.3179 6.36026 200 1
T11419 V OP255940 VV OP209595 VV OP209690 83: Carcès 43.4807 6.1861 100 1
T11420 V OP255941 VV OP209596 VV OP209691 83: Carcès 43.4807 6.1861 100 1
T11421 V OP255942 VV OP209597 VV OP209692 83: Carcès 43.4807 6.1861 100 1
T11422 V OP255943 VV OP209598 VV OP209693 83: Carcès 43.4807 6.1861 100 1
T11423 V OP255944 VV OP209599 VV OP209694 83: Carcès 43.4807 6.1861 100 1
T11671 V OP255945 VV OP209600 VV OP209695 83: Massif de la Sainte-Baume 43.3275 5.7609 10 1
T13290 V OP255946 VV OP209601 VV OP209696 83: Porquerolles 42.99636 6.19559 10 1
T12260 V OP255947 VV OP209602 VV OP209697 06: Breil-sur-Roya, Col de Brouis 43.92162 7.47718 20 1
T4202/BEV.8147 V OP255948 VV OP209603 VV OP209698 06: Villars-sur-Var 43.93618 7.0935 10 1, 2
T4203/BEV.8148 V OP255949 VV OP209604 VV OP209699 06: Andon 43.78671 6.8642 10 1, 2
T12250/BEV.14903 V OP255950 VV OP209605 VV OP209700 06: Breil-sur-Roya, Mont Gros 43.91423 7.47944 20 1
T12252/BEV.14905 V OP255951 VV OP209606 VV OP209701 06: Saint-Martin-Vésubie, Col de Salèse 44.13734 7.23544 20 1
T12257/BEV.14910 V OP255952 VV OP209607 VV OP209702 06: Isola 44.19173 7.05826 20 1
T12258/BEV.14911 V OP255953 VV OP209608 VV OP209703 06: Morignole 44.08741 7.64778 20 1
T12259/BEV.14912 V OP255954 VV OP209609 VV OP209704 06: Saorge, Maurion 43.9986 7.51668 20 1
T13530/BEV.7704 V OP255955 VV OP209610 VV OP209705 06: Tende 44.0818 7.5932 20 1
ISM01 V MH316865 VV MH316862 – – 06: Ile Sainte-Marguerite 43.52361 7.04528 200 3
ISM02 V MH316865 VV MH316862 – – 06: Ile Sainte-Marguerite 43.52361 7.04528 200 3
ISM03 V MH316865 VV MH316862 – – 06: Ile Sainte-Marguerite 43.52361 7.04528 200 3
ISM04 V MH316865 VV MH316862 – – 06: Ile Sainte-Marguerite 43.52361 7.04528 200 3
ISM05 V MH316865 VV MH316862/3 – – 06: Ile Sainte-Marguerite 43.52361 7.04528 200 3
ISM06 V MH316865 VV MH316862/4 – – 06: Ile Sainte-Marguerite 43.52361 7.04528 200 3
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8C. Dufresnes et al.
Tab l e 1. (Continued.)
Sample/voucher ND2 PRLR HA1 Locality Latitude Longitude Prec Ref.
Reference samples
T4150/BEV.11018 F OP255956 FF OP209611 FF OP209706 France, 88: Le Tholy, Bouvacot 48.07781 6.75729 100 1, 2
T10363 F OP255957 FF OP209612 FF OP209707 France, 64: Cette-Eygun 42.93997 −0.60098 20 1
T10660 F OP255958 FF OP209613 FF OP209708 France, 66: Coustouges 42.36494 2.65377 10 1
T4201 F OP255959 FF OP209614 FF OP209709 France, 66: Nohèdes, Col de Portus 42.62954 2.27295 500 1
T4206 F OP255960 FF OP209615 FF OP209710 France, 66: Mont-Louis 42.5206 2.0976 300 1
T11685/MIRZC0970 F OP255961 FF OP209616 FF OP209711 France, 93: Montreuil, Les murs à pêches 48.86351 2.45639 100 1
T11686/MIRZC09995 F OP255962 FF OP209617 FF OP209712 France, 93: Montreuil, Les murs à pêches 48.86351 2.45639 100 1
T4025 F OP255963 FF OP209618 FF OP209713 Sweden, Mörby gård 59.14513 18.20671 100 1
T4026 F OP255964 FF OP209619 FF OP209714 Sweden, Mörby gård 59.14513 18.20671 100 1
T11687/ACZC3209 V OP255965 VV OP209620 VV OP209715 Italy, Lazio, Suso 41.50639 13.08083 ? 1, 2
T11688/ACZC3216 V OP255966 VV OP209621 VV OP209716 Italy, Firenze, Barberino di Mugello 44.02497 11.22419 ? 1
T11689/ACZC3482 V OP255967 VV OP209622 VV OP209717 Italy, Basilicata, Cropani 40.03729 16.10146 ? 1, 2
T11691/ACZC5171 V OP255968 VV OP209623 VV OP209718 Italy, Grosseto, Prata 43.0943 10.98525 ? 1
T11691/ACZC5386 V OP255969 VV OP209624 VV OP209719 Italy, Grosseto, Prata 43.0829 10.9854 ? 1
1This study; 2Gvoždík et al. (2013); 3Renet et al. (2018).
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Genetic barcoding of a slow worm hybrid zone 9
Renet et al. (2018) (table 1). In each popula-
tion, the proportions of fragilis/veronensis alle-
les were computed for each marker separately,
and averaged over the three markers, as a hybrid
index.
Results and discussion
Genetic barcoding of one mitochondrial (ND2)
and two nuclear genes (PRLR and HA1)re-
vealed consistent patterns regarding the loca-
tion of the transition between the slow worm
taxa A. fragilis and A. veronensis in southeastern
France. Figure 1 shows the population hybrid
index averaged over markers. Results from each
marker separately are presented in supplemen-
tary fig. S1. All sequences were archived on
GenBank (table 1).
The respective distributions of veronensis and
fragilis alleles roughly follow the Rhône and
the Durance rivers, with evidence for hybridiza-
tion (fig. 1 and supplementary fig. S1). In
the lower Rhône valley, particularly between
Etang de Berre and the Grand Rhône (the
main eastern branch of the Rhône River in its
delta), both mitotypes occur and many indi-
viduals shared the nuclear alleles of the two
species. Admixed individuals were also identi-
fied in the Grande Camargue, i.e., on the west
bank of the Grand Rhône (samples T12498
and T11417/BEV.14687, see table 1). In the
Southern Alps, traces of admixture were further
detected in the Durance valley. Nearly all pos-
sible hybrid genotypes were represented among
our samples (table 1). Allele sharing was thus
not limited to F1s, but involved gene flow and
introgression via F2s and/or backcrosses. The
hybrid zone yet appears to be rather narrow:
only 30 km separates pure A. veronensis in
Etang de Berre (sample T13339) from pure A.
fragilis in the west bank of the Grand Rhône
(fig. 1).
In the administrative departments of south-
eastern France, Anguis populations should thus
be considered as follow. Alpes-Maritimes (06),
Var (83) and the eastern and central parts of
Bouches-du-Rhône (13) are inhabited by pure
A. veronensis. The western parts of Bouches-
du-Rhône (13) are populated by A. veronen-
sis/fragilis hybrids. Gard (30) and Hérault (34)
host pure A. fragilis. Further north, large parts
of the Alps remain unsampled, but we can pre-
dict the hybrid zone to run along southern Vau-
cluse (84), Alpes-de-Haute-Provence (04) and
possibly in northern Alpes-Maritimes (06) and
southern Hautes-Alpes (05). Our results should
be useful to local wildlife managers, and hope-
fully motivate future barcoding efforts to fill the
sampling gaps.
Anguis fragilis and A. veronensis were recent-
ly shown to be Pliocene sister lineages (Gvoždík
et al., 2023), and are thus more closely related
than previously assumed from mitochondrial
phylogenies (Gvoždík et al., 2013). While the
morphological and genetic similarities with A.
fragilis raise concern on the taxonomic sta-
tus of A. veronensis, their genetic cohesiveness
seems to be maintained despite local hybridiza-
tion across an extensive area of parapatry (from
the Mediterranean coast to the Italian Alps).
Both species were reported in close proxim-
ity in southern Switzerland (Ursenbacher and
Zwahlen, 2015), again with limited admixture
(S. Ursenbacher, unpubl. microsatellite data),
and instances of hybridization were locally
documented in Slovenia and northeastern Italy
(Gvoždík et al., 2013). Estimating admixture
with only two nuclear loci, as we have done
here, should obviously be considered prelim-
inary, and assessing how far foreign alleles
truly diffuse into the taxa’s respective ranges
will require the analysis of additional markers.
Among future research, hybrid zone analyses of
samples collected along pre-defined geographic
transects at the identified local transitions (e.g.,
eastern side of the Rhône valley) would allow
estimating population genetic parameters rele-
vant to understand the dynamics of the hybrid
zone, for instance, by fitting clines on allele fre-
quencies (e.g., Dufresnes et al., 2020c). For the
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10 C. Dufresnes et al.
time being, the fact that gene flow appears geo-
graphically restricted despite many opportuni-
ties for introgression suggests that some intrin-
sic post-zygotic isolation (genetic incompatibil-
ities) efficiently prevents merging of the A. frag-
ilis and A. veronensis genomes, and that A. vero-
nensis merits to keep its species status.
In the light of the most up-to-date species tree
(Gvoždík et al., 2023), hybridization patterns in
slow worms fit the empirical rule that the degree
of genetic divergence negatively correlates with
the extent of gene flow across secondary con-
tact zones in reptiles (Singhal and Moritz, 2013)
and amphibians (Dufresnes et al., 2021). While
the most basal and morphologically differen-
tiated species A. cephallonica achieves sym-
patry and complete genetic isolation with A.
graeca (∼11My of divergence), the younger
A. graeca and A. veronensis both continue to
hybridize with A. fragilis nowadays (respec-
tively ∼6My and ∼4My of divergence). In addi-
tion, the past mitochondrial capture in A. vero-
nensis demonstrates that A. cephallonica was
still able to admix with the A. fragilis/veronensis
ancestor ∼5Mya, at which time their respec-
tive branches had diverged by ∼6My (Gvoždík
et al., 2023). Taken together, past and present
patterns of divergence, hybridization, distribu-
tion and external differentiation in slow worms
support the trend that species complexes are
primarily initiated in alloparapatry (Rasolon-
jatovo et al., 2020), and that a substantial
amount of divergence is needed to evolve effec-
tive reproductive barriers and phenotypic dis-
tinctiveness that enables sympatry without the
risks of despeciation and competitive exclusion.
These hypotheses should be further explored by
extending investigations to additional species
pairs, notably A. graeca and A. fragilis in the
Balkans. Moreover, the case of Anguis calls for
caution when interpreting the speciation contin-
uum in the light of mitochondrial phylogenies,
which appear misleading in an increasing num-
ber of animal taxa (e.g., Hinojosa et al., 2019;
Bernardo et al., 2019; Dufresnes et al., 2020a;
Dufresnes and Jablonski, 2022).
Finally, the respective ranges of A. frag-
ilis and A. veronensis emphasize the Rhône
River as a contemporary biogeographic barrier
for some of the French herpetofauna. Like A.
fragilis, the river corresponds to the eastern
range limit of the newt Triturus marmoratus
and of the lizards Podarcis liolepis and Psam-
modromus algirus (Lescure and de Massary,
2012). The barrier may be primarily geographic
rather than ecological: Mediterranean species
of similar ecological affinities thrive on either
side of the Rhône (e.g., Pelobates cultripes,
Psammodromus edwarsianus,Malpolon mon-
spessulanus,Zamenis scalaris). Moreover, the
admixed nature of the Anguis populations on
both sides of the Rhône illustrates its perme-
ability throughout the Holocene. We hypothe-
size that this was facilitated by the fluctuating
fluvial dynamics of the river’s network in the
delta within the last millenaries (Bruneton et
al., 2001; Arnaud-Fassetta, 2003; Bravard and
Gaydou, 2015), combined with the connectivity
offered by the riverine forest that grows directly
on the banks and promotes widespread occur-
rence and high density of slow worms (AO,
pers. obs.). In a similar fashion, upstream sec-
tions of the Rhône roughly delineate species
boundaries between the hybridizing toads Bufo
bufo and B. spinosus (Arntzen et al., 2020), yet
with local exceptions and long-range allele dif-
fusion on either side (Dufresnes et al., 2020c).
The downstream sections may have also influ-
enced the ancient ranges of these species, but
B. spinosus has since expanded eastward and
genetically assimilated B. bufo all the way to
Italy (Arntzen et al., 2017). While examples
of (porous) species boundaries mediated by the
Rhône remain scarce overall, the river may
more likely cause intraspecific genetic struc-
ture, which would be worthy of consideration
in a conservation framework (e.g., management
units). Hybrid zones between Iberian and extra-
Iberian amphibian and reptile lineages are typi-
cally located further west in meridional France,
for instance along the dry Garonne (e.g., Hyla,
Dufresnes et al., 2020d) and Ariège valleys
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Genetic barcoding of a slow worm hybrid zone 11
(e.g., Natrix, Asztalos et al., 2020). Despite vast
and geographically complex territories, France
generally remains under-represented in phylo-
geographic studies of European amphibians and
reptiles (e.g., Gvoždík et al., 2013), thus hinder-
ing our appreciation of its genetic diversity.
Acknowledgements. We are grateful to F. Arnaboldi, D.
Arsovski, J.-M. Ballouard / SOPTOM, H. Brosius, F.
Chapelur, S. Combeaud, P. Coursier-Fournial, V. Danias,
B. Delahaie, F. Dhermain, E. Didner, J. Dublon, G. Gillet,
C. Jakob / Ecole domaine du possible, T. Lebard, I.
Lhommedet, A. Lucas, J.-L. Lucchesi, A. Lyet, L. Malth-
ieux, P. Migaud, A. Miralles, P. Ormea, B. Schatz, T.
Schwartz, A. Rocha, L. Tillion, F. Veyrunes, M. Voronkoff,
A. Westerström, L. Westrin and L. Zimmerman for their
help with samples collection and to P. Geniez for his
help with the samples curation. CD was supported by
the Research Found for International Scientists (RFIS)
of the National Science Foundation of China (NSFC)
(N°3211101356), as well as the Taxon-Omics Priority Pro-
gramme (SPP1991) of the Deutsche Forschungsgemein-
schaft (DFG) (grant number VE247/19-1). AC was sup-
ported by the Portuguese National Funds through FCT–
Fundação para a CiênciaeaTecnologia(researchcontract
2020.00823.CEECIND).
Supplementary material. Supplementary material is avail-
able online at:
https://doi.org/10.6084/m9.figshare.21820509
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