Hybrid zones of Natrix helvetica and N. natrix: Phenotype data from iNaturalist and genetics reveal concordant clines and the value of species-diagnostic morphological traits

Using georeferenced photographic records of 2944 grass snakes from Germany, Austria, and northern Italy as well as previously published mtDNA sequences (n = 1062) and microsatellite data (n = 952) for grass snakes from the same regions, we examined whether or not coloration and pattern reliably allow to differentiate between Natrix natrix and N. helvetica and if so, whether the distribution patterns revealed by phenotypes and genetics are congruent. Furthermore, we used cline analyses across hybrid zones to test whether the phenotypic transition from one species to the other parallels the steep clines unveiled by genetics. Our results suggest that the two species can be reliably differentiated using coloration and pattern. The most powerful diagnostic traits are the presence/absence of side bars on the body flanks, the number of occipital spots, and the shape of the posterior dark occipital spot. The distributions of morphologically identified N. natrix and N. helvetica match their genetically confirmed ranges. Single conflicting individuals morphologically identified as N. natrix or hybrids within the distribution range of N. helvetica either represent misidentifications or translocated snakes. For the genetic markers and phenotypes, our cline analyses revealed concordant steep clines across hybrid zones. However, the southern part of the hybrid zone in Italy, for which no sufficient genetic data are available, should be studied in more detail because the phenotypic data suggest a smooth cline in this region. The unexpected high percentage of putative hybrids with dorsal stripes in this region also calls for further research. For northwestern Germany, another region for which no genetically verified records are available, iNaturalist data suggest that the contact zone of N. natrix and N. helvetica is near the Ems River and extends from there southeastwards to the region of Höxter, North Rhine-Westphalia

The range-wide mitochondrial lineage of Natrix natrix scutata (Pallas, 1771) presented in the northern Zagros Mountains

Natrix natrix (Linnaeus, 1758) is a common species distributed from Central Europe to Central Asia. In this range, it forms four subspecies that include several mitochondrial evolutionary lineages. One of the lineages, the so-called mtDNA lineage 8, has a wide distribution from the Baltic area to Anatolia and Kazakhstan. In Anatolia, this lineage meets several others, however, their occurrence is unclear, especially in the south-eastern Türkiye where the species is uncommon. Obtaining one specimen from the poorly studied Hakkâri Province (Zagros part of Türkiye), we investigated its genetic affiliation (mitochondrial DNA) and basic morphology. The specimen represents a unique haplotype of the mtDNA lineage 8, closely related to populations from Georgia and northern and north-eastern Türkiye. It thus extends the occurrence of this mitochondrial lineage representing subspecies Natrix natrix scutata (Pallas, 1771) southward to the northern edge of the Zagros Mountains. Despite the phenotype polymorphism of this species, the morphological comparison also confirmed that selected characters are similar to other populations of the region

Genotyping the phenotypic diversity in Aegean Natrix natrix moreotica (Bedriaga, 1882) (Reptilia, Serpentes, Natricidae)

We examined the mitochondrial identity of Aegean Natrix natrix moreotica representing different morphotypes, with a focus on new material from Milos and Skyros. We found no correlation between distinct morphotypes and mitochondrial identity. Our results support that grass snake populations are polyphenetic and that southern subspecies, including island populations, show a higher variability than northern ones

Mitochondrial ghost lineages blur phylogeography and taxonomy of Natrix helvetica and N. natrix in Italy and Corsica

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Phenotyping in the era of genomics: MaTrics—a digital character matrix to document mammalian phenotypic traits

A new and uniquely structured matrix of mammalian phenotypes, MaTrics (Mammalian Traits for Comparative Genomics) in a digital form is presented. By focussing on mammalian species for which genome assemblies are available, MaTrics provides an interface between mammalogy and comparative genomics. MaTrics was developed within a project aimed to find genetic causes of phenotypic traits of mammals using Forward Genomics. This approach requires genomes and comprehensive and recorded information on homologous phenotypes that are coded as discrete categories in a matrix. MaTrics is an evolving online resource providing information on phenotypic traits in numeric code; traits are coded either as absent/present or with several states as multistate. The state record for each species is linked to at least one reference (e.g., literature, photographs, histological sections, CT scans, or museum specimens) and so MaTrics contributes to digitalization of museum collections. Currently, MaTrics covers 147 mammalian species and includes 231 characters related to structure, morphology, physiology, ecology, and ethology and available in a machine actionable NEXUS-format*. Filling MaTrics revealed substantial knowledge gaps, highlighting the need for phenotyping efforts. Studies based on selected data from MaTrics and using Forward Genomics identified associations between genes and certain phenotypes ranging from lifestyles (e.g., aquatic) to dietary specializations (e.g., herbivory, carnivory). These findings motivate the expansion of phenotyping in MaTrics by filling research gaps and by adding taxa and traits. Only databases like MaTrics will provide machine actionable information on phenotypic traits, an important limitation to genomics. MaTrics is available within the data repository Morph·D·Base (www.morphdbase.de)

Hybrid zones of Natrix helvetica and N. natrix: Phenotype data from iNaturalist and genetics reveal concordant clines and the value of species-diagnostic morphological traits

Using georeferenced photographic records of 2944 grass snakes from Germany, Austria, and northern Italy as well as previously published mtDNA sequences (n = 1062) and microsatellite data (n = 952) for grass snakes from the same regions, we examined whether or not coloration and pattern reliably allow to differentiate between Natrix natrix and N. helvetica and if so, whether the distribution patterns revealed by phenotypes and genetics are congruent. Furthermore, we used cline analyses across hybrid zones to test whether the phenotypic transition from one species to the other parallels the steep clines unveiled by genetics. Our results suggest that the two species can be reliably differentiated using coloration and pattern. The most powerful diagnostic traits are the presence/absence of side bars on the body flanks, the number of occipital spots, and the shape of the posterior dark occipital spot. The distributions of morphologically identified N. natrix and N. helvetica match their genetically confirmed ranges. Single conflicting individuals morphologically identified as N. natrix or hybrids within the distribution range of N. helvetica either represent misidentifications or translocated snakes. For the genetic markers and phenotypes, our cline analyses revealed concordant steep clines across hybrid zones. However, the southern part of the hybrid zone in Italy, for which no sufficient genetic data are available, should be studied in more detail because the phenotypic data suggest a smooth cline in this region. The unexpected high percentage of putative hybrids with dorsal stripes in this region also calls for further research. For northwestern Germany, another region for which no genetically verified records are available, iNaturalist data suggest that the contact zone of N. natrix and N. helvetica is near the Ems River and extends from there southeastwards to the region of Höxter, North Rhine-Westphalia

So different, yet so alike: North American slider turtles (Trachemys scripta)

We studied for the first time the molecular differentiation of all three currently recognized subspecies of Trachemys scripta, including the morphologically distinct western populations of T. s. elegans (‘western red-eared sliders’), using mitochondrial and nuclear DNA sequences (up to 3,236 bp and 2,738 bp, respectively) and 14 microsatellite loci. We found that only the quickly evolving microsatellite loci discriminated T. s. troostii and the western red-eared slider from the remaining two subspecies, while T. s. elegans and T. s. scripta were not distinct in any marker system. Our findings challenge the current intraspecific systematics of T. scripta and suggest that the conspicuous differences in coloration and pattern reflect population-specific, rather than taxonomic, differentiation. We abstain from synonymizing any subspecies because, for traditionalists and conservationists, abandoning the well-established and morphologically distinct subspecies of T. scripta is not desirable. However, if subspecies of T. scripta continue to be recognized, the current taxonomy with three subspecies is difficult to justify. Western red-eared sliders are morphologically distinct and differ from T. s. elegans and T. s. scripta, with respect to microsatellites, as much as T. s. troostii does. In view of this morphological and genetic evidence, subspecies status should be considered for western red-eared sliders

So different, yet so alike: North American slider turtles (Trachemys scripta)

We studied for the first time the molecular differentiation of all three currently recognized subspecies of Trachemys scripta, including the morphologically distinct western populations of T. s. elegans (‘western red-eared sliders’), using mitochondrial and nuclear DNA sequences (up to 3,236 bp and 2,738 bp, respectively) and 14 microsatellite loci. We found that only the quickly evolving microsatellite loci discriminated T. s. troostii and the western red-eared slider from the remaining two subspecies, while T. s. elegans and T. s. scripta were not distinct in any marker system. Our findings challenge the current intraspecific systematics of T. scripta and suggest that the conspicuous differences in coloration and pattern reflect population-specific, rather than taxonomic, differentiation. We abstain from synonymizing any subspecies because, for traditionalists and conservationists, abandoning the well-established and morphologically distinct subspecies of T. scripta is not desirable. However, if subspecies of T. scripta continue to be recognized, the current taxonomy with three subspecies is difficult to justify. Western red-eared sliders are morphologically distinct and differ from T. s. elegans and T. s. scripta, with respect to microsatellites, as much as T. s. troostii does. In view of this morphological and genetic evidence, subspecies status should be considered for western red-eared sliders

It takes two to tango - Phylogeography, taxonomy and hybridization in grass snakes and dice snakes (Serpentes: Natricidae: Natrix natrix, N. tessellata)

Using two mitochondrial DNA fragments and 13 microsatellite loci, we examined the phylogeographic structure and taxonomy of two codistributed snake species (Natrix natrix, N. tessellata) in their eastern distribution area, with a focus on Turkey. We found evidence for frequent interspecific hybridization, previously thought to be extremely rare, and for backcrosses. This underscores that closely related sympatric species should be studied together because otherwise the signal of hybridization will be missed. Furthermore, the phylogeographic patterns of the two species show many parallels, suggestive of a shared biogeographic history. In general, the phylogeographies follow the paradigm of southern richness to northern purity, but the dice snake has some additional lineages in the south and east in regions where grass snakes do not occur. For both species, the Balkan Peninsula and the Caucasus region served as glacial refugia, with several mitochondrial lineages occurring in close proximity. Our results show that the mitochondrial divergences in both species match nuclear genomic differentiation. Yet, in the former glacial refugia of grass snakes there are fewer nuclear clusters than mitochondrial lineages, suggesting that Holocene range expansions transformed the glacial hotspots in melting pots where only the mitochondrial lineages persisted, bearing witness of former diversity. On the other hand, the deep mitochondrial divergences in N. tessellata across its entire range indicate that more than one species could be involved, even though lacking micro satellite data outside of Turkey prevent firm conclusions. On the contrary, our microsatellite and mitochondrial data corroborate that N. megalocephala is invalid and not differentiated from sympatric populations of N. natrix. For Cypriot grass snakes, our analyses yielded conflicting results. A critical assessment of the available evidence suggests that N. natrix is a genetically impoverished recent invader on Cyprus and taxonomically not distinct from a subspecies also occurring in western Anatolia and the southern Balkans. Based on combined mitochondrial and nuclear genomic evidence we propose that for grass snakes the following subspecies should be recognized in our study region: (1) Natrix natrix vulgaris Laurenti, 1768, southeastern Central Europe and northern Balkans; (2) Natrix natrix moreoticus (Bedriaga, 1882), southern Balkans, western Anatolia, and Cyprus; and (3) Natrix natrix scutata (Pallas, 1771), eastern Anatolia, Caucasus region, Iran, northeastern distribution range (from eastern Poland and Finland to Kazakhstan and the Lake Baikal region). Thus, Natrix natrix cypriaca (Hecht, 1930) becomes a junior synonym ofN. n. moreoticus and Natrix natrix persa (Pallas, 1814) becomes a junior synonym ofN. n. scutata. Due to insufficient material, we could not resolve the status of Natrix natrix syriaca (Hecht, 1930) from the Gulf of Iskenderun, southeastern Turkey.Scientific and Technological Research Council of Turkey TuBTAK [116Z359]; Senckenberg; Slovak Research and Development Agency [APVV-19-0076]; Scientific Grant Agency of the Slovak Republic [VEGA 1/0242/21]The authors thank the Scientific and Technological Research Council of Turkey TuBTAK (project number 116Z359) for financial support. Marika Asztalos' work was funded by Senckenberg. Daniel Jablonski was supported by the Slovak Research and Development Agency under the contract APVV-19-0076 and by the grant VEGA 1/0242/21 of the Scientific Grant Agency of the Slovak Republic. All animal handling within the scope of the TuBTAK project was in accordance with wild-life husbandry standards approved by Decision 2016-18 of the Labora-tory Animals Ethical Committee at Ege University, Turkey. Suleyman lhan and Melodi Yenmi assisted with laboratory work. Frank Glaw and Ben Wielstra donated some samples. Henrik BringsOe allowed the use of two photos for a figure. Frank Glaw and an anonymous reviewer made helpful suggestions that improved an earlier version of this study. Flora Ihlow and Markward Herbert Fischer produced one map for us