The effect of phylogeographic history on species boundaries: a comparative framework in Hyla tree frogs

Because it is indicative of reproductive isolation, the amount of genetic introgression across secondary contact zones is increasingly considered in species delimitation. However, patterns of admixture at range margins can be skewed by the regional dynamics of hybrid zones. In this context, we posit an important role for phylogeographic history: hybrid zones located within glacial refugia (putatively formed during the Late-Pleistocene) should be better defined than those located in post-glacial or introduced ranges (putatively formed during the Holocene and the Anthropocene). We test this hypothesis in a speciation continuum of tree frogs from the Western Palearctic (Hyla), featuring ten identified contacts between species spanning Plio-Pleistocene to Miocene divergences. We review the rich phylogeographic literature of this group and examine the overlooked transition between H. arborea and H. molleri in Western France using a multilocus dataset. Our comparative analysis supports a trend that contacts zones resulting from post-glacial expansions and human translocations feature more extensive introgression than those established within refugial areas. Integrating the biogeographic history of incipient species, i.e. their age since first contact together with their genetic divergence, thus appears timely to draw sound evolutionary and taxonomic inferences from patterns of introgression across hybrid zones.

File S1: Details on the populations analyzed to study the H. arborea / molleri hybrid zone.

Phylogeography and contact zones in W-Palearctic Hyla -a summary
The following briefly reports on patterns of diversity and distribution in each W-Palearctic tree frog, based on available phylogeographic evidence, with a particular focus on the extent of admixture at species transitions and the relative age of contacts.

Hyla arborea
PHYLOGEOGRAPHY IN BRIEF -Detailed multilocus accounts evidenced three Pleistocene refugia throughout the Balkan Peninsula, namely in southern Greece, the Pannonian Basin and the Adriatic coast [1][2]. Northern and Western European populations belong to the Pannonian clade, which exhibit signs of recent demographic and spatial expansions [1,3]. Mitochondrial and nuclear diversity significantly decreased with distance from the Balkans, as expected with founder effects during post-glacial recolonization [1][2].
LGM conditions were accordingly suitable in the Balkans, especially along the Adriatic and Greek coastlines (Fig. 4). Northeastern Europe was accordingly unhospitable, but the climatic models did feature high probabilities of occurrence in northwestern Europe. Yet, and while private mtDNA haplotypes were found in the latter [1], these are weakly differentiated from the Balkan populations, and thus do not support an additional northern refugium. To conclude, it is very likely that H. arborea survived the Quaternary glaciations in the Balkan Peninsula and colonized northern and western Europe after the LGM.
CONTACT ZONES -(1) H. arborea/perrini in N-Italy / Slovenia. Narrow transition partially mediated by the Izonso River at the border between Slovenia and Italy [4]. Considered as a Pleistocene contact given that the area overlaps putative refugial ranges of both species, according to SDM analyses (Fig. 4, see [5] for H. perrini, and below) and high levels of genetic diversity (see [1] for H. arborea). Verardi and colleagues [4] analyzed this contact using diagnostic mtDNA and allozyme markers and found mostly pure individuals, expect for a few that exhibited very limited admixture (<5%). (2) H. arborea/perrini in W-Switzerland. Anthropocene contact resulting from the introduction of H. perrini in the 1950s by wildlife enthusiasts, within the natural range of H. arborea [6]. Multilocus analyses revealed that both species massively admixed and formed a hybrid swarm, where no pure individuals remain [7]. (3) H. arborea/orientalis in the Balkans. Narrow transitions with geographically restricted admixture along the Aegean coast (northern Greece) as well as the Balkan and Rhodope slopes, despite no dispersal barriers (CD and A. Brelsford pers. obs.). Considered as Pleistocene contacts since they encompassed putative refugia of both species, according to genetic [1,8] and SDM evidence (Fig. 4, see also [8] for H. orientalis). Cline analyses along two transects inferred hybrid zone widths of 30km in southeastern Serbia (nearby Niš) and 32km in northeastern Greece (Thrace) for microsatellite loci [9]. Mitochondrial DNA had more variable widths (39km and 6km, respectively; [9]). (4) H. arborea/orientalis in N-Europe. In Poland, H. orientalis reaches the confluence of the Vistula and Notec rivers in the west, and nuclear and mitochondrial introgression was documented over about 200km along the Baltic coastline (Gdansk area, [10]). Several hybrid populations were also found in central (Lodz area) and southern Poland (Krakow area; see also [11]). Considered as an Holocene contact as northern Europe was recolonized by both species after the LGM, as supported by inferences of population expansions [1,3,8], low genetic diversity, and unsuitable conditions predicted by SDM analyses (Fig. 4, see also [8] for H. orientalis). (5) H. arborea/molleri. Wide hybrid zone spanning across W-France with admixture detected over 400km from the Garonne River to Normandy (Fig.  1). Considered as a Holocene contact given that H. arborea colonized the region from the Balkans after the LGM, which is supported by signature of demographic expansions and patterns of genetic diversity [1][2][3]. Cline analyses along the Atlantic coastline suggested widths of 98km for nuclear loci (microsatellites) and 42km for mitochondrial DNA (Fig. 2), centered between Bordeaux and Nantes, but with extensive admixture on the H. arborea side.