Range-Wide Nuclear Phylogeography of the Aesculapian Snake (Zamenis longissimus) and the Evolutionary Significance of a Northern Relict River-Valley Population System

Abstract

Background/Objectives: Relict populations can preserve genetic legacies that illuminate species history and inform conservation. Isolated northern populations of the Aesculapian snake (Zamenis longissimus) are considered remnants of a broader Holocene distribution, offering an opportunity to explore historical population connections and their conservation significance. We assessed diversity in Czech populations and provide the first range-wide phylogeographic perspective based on the nuclear marker SPTBN1. Methods: We analyzed mitochondrial DNA (mtDNA) and SPTBN1 in Czech populations, including the newly confirmed Běšický chochol locality (BCH), and assembled a range-wide SPTBN1 dataset. Results: Across Europe, SPTBN1 haplotypes s1 and s2 showed an east–west phylogeographic pattern that only partly corresponded to the major mitochondrial lineages; a previously unreported haplotype (s4) was identified in western Slovakia. In Bohemia (western Czech Republic), both SPTBN1 haplotypes co-occurred with Eastern and Western mitochondrial lineages at two lower Ohře River localities, Želinský meander (ZM) and BCH, whereas the upstream Stráž nad Ohří and introduced Central Bohemian populations contained only the Eastern mitochondrial lineage and nuclear haplotype s1. The Moravian population (eastern Czech Republic) carried both nuclear haplotypes but only Eastern-lineage mtDNA, demonstrating that nuclear haplotype coexistence alone is not diagnostic of contact between mitochondrial lineages. Conclusions: The genetic legacy of historical lineage contact extends beyond ZM to BCH. Together with the historical landscape configuration of the Ohře River valley, these findings support the interpretation that both localities are surviving fragments of a more extensive Holocene population system. Their retention of multiple components of evolutionary diversity highlights the conservation value of these northern relicts and warrants explicit consideration in future management.