2urn:lsid:arphahub.com:pub:45048D35-BB1D-5CE8-9668-537E44BD4C7Eurn:lsid:zoobank.org:pub:91BD42D4-90F1-4B45-9350-EEF175B1727AZooKeysZK1313-29891313-2970Pensoft Publishers10.3897/zookeys.1169.104594104594Short CommunicationAnimaliaChordataNatricidaeReptiliaSerpentesSquamataVertebrataBiogeographyEvolutionary biologyGeneticsMolecular systematicsSystematicsTaxonomyBalkansCyprusEuropeGreeceSouthern Europe and MediterraneanGenotyping the phenotypic diversity in Aegean Natrixnatrixmoreotica (Bedriaga, 1882) (Reptilia, Serpentes, Natricidae)JablonskiDanieldaniel.jablonski@uniba.skhttps://orcid.org/0000-0002-5394-01141TzorasElias2PanagiotopoulosAlexios3AsztalosMarikahttps://orcid.org/0000-0002-4090-11594FritzUwehttps://orcid.org/0000-0002-6740-72144Department of Zoology, Comenius University in Bratislava, Ilkovičova 6, Mlynská dolina, 842 15, Bratislava, SlovakiaComenius University in BratislavaBratislavaSlovakiaPatras, 264 42 Achaia, GreeceUnaffiliatedPatrasGreeceSection of Animal Biology, Department of Biology, University of Patras, 26500 Patras, GreeceUniversity of PatrasPatrasGreeceMuseum of Zoology (Museum für Tierkunde), Senckenberg Dresden, A. B. Meyer Building, 01109 Dresden, GermanyMuseum of ZoologyDresdenGermany
Corresponding author: Daniel Jablonski (daniel.jablonski@uniba.sk)
Academic editor: Robert Jadin
2023130720231169879489804F62-9E9C-57D7-91E8-6548A13031E1EB7F8E07-7EE9-4613-808F-09F6533E551181495580504202318062023Daniel Jablonski, Elias Tzoras, Alexios Panagiotopoulos, Marika Asztalos, Uwe FritzThis is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.http://zoobank.org/EB7F8E07-7EE9-4613-808F-09F6533E5511
We examined the mitochondrial identity of Aegean Natrixnatrixmoreotica 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.
Jablonski D, Tzoras E, Panagiotopoulos A, Asztalos M, Fritz U (2023) Genotyping the phenotypic diversity in Aegean Natrix natrix moreotica (Bedriaga, 1882) (Reptilia, Serpentes, Natricidae). ZooKeys 1169: 87–94. https://doi.org/10.3897/zookeys.1169.104594
Mediterranean islands are well known for their rich biodiversity with many endemic taxa. This diversity is particularly evident in the Aegean region, which has undergone a complex geological history shaping its biota (Poulakakis et al. 2015). Many Aegean islands have been isolated since the Miocene and, as a result, harbour endemic taxa. This includes, for example, the amphibians Lyciasalamandrahelverseni (Pieper, 1963) and Bufotesviridisdionysi Dufresnes, Probonas & Strachinis, 2020 and the reptiles Podarciscretensis (Wettstein, 1952), Mediodactylusbartoni (Štěpánek, 1934), and Macroviperaschweizeri (Werner, 1935). However, the diversity of the Mediterranean island herpetofauna has traditionally often been overestimated. During the 19th and the first half of the 20th century, many subspecies were described that reflect merely local phenotypes (Mertens and Wermuth 1960), which are not even necessarily restricted to the islands from where the taxa were described. Prime examples for this taxonomic inflation are the many invalid subspecies of island lizards (e.g., P.siculus, see Henle and Klaver 1986; Podnar et al. 2005). Also, some Aegean island populations of the grass snake Natrixnatrix (Linnaeus, 1758) have been described as endemic subspecies, e.g., Tropidonotusnatrixsyrae Hecht, 1930 from Syros or Tropidonotusnatrixdystiensis Hecht, 1930 from Euboea (Fig. 1). Only three decades ago, another subspecies, Natrixnatrixfusca Cattaneo, 1990, was erected from Kea (Cattaneo 1990). None of these subspecies is currently recognized and these taxa are understood to represent phenotypic variation (Fritz and Schmidtler 2020; Asztalos et al. 2021a).
A parsimony network for 54 ND4+tRNAs sequences of clade 3 (“yellow lineage” of Kindler et al. 2013, 2017) of Natrixnatrix. Photos show different grass snake phenotypes. Symbol size reflects haplotype frequency. Small black circles are missing node haplotypes; each line connecting two haplotypes corresponds to one mutation step, if not otherwise indicated by red numbers along lines. Haplotype terminology for N.natrix follows Kindler et al. (2013) (photos: D. Jablonski, E. Tzoras) B distribution of mitochondrial clades (from Kindler et al. 2013; Asztalos et al. 2021a) and of the picturata and schweizeri morphotypes in the the Aegean region and Cyprus (see details in Suppl. material 1: table S1). In addition, the type localities (see Fritz and Schmidtler 2020) of local taxa are indicated.
https://binary.pensoft.net/fig/877333
Milos, a small Aegean island with limited freshwater resources, is home to grass snakes that are less aquatic than elsewhere (Kratzer 1974). This population exhibits considerable phenotypic variation, and one of the local phenotypes led to the description of the subspecies Natrixnatrixschweizeri Müller, 1932, thought to be endemic to Milos and the neighbouring islands Kimolos and Polyaegos (Kabisch 1999). This taxon was originally erected for grass snakes bearing large dark blotches on the dorsal and lateral body (Müller 1932). Recent genetic studies by Kindler et al. (2013, 2017) and Asztalos et al. (2021a) have revealed that grass snakes from Milos possess mitochondrial haplotypes of the so-called “yellow lineage” (clade 3) of N.natrix, which is found throughout central-western Europe and parts of the Balkans. Following the subspecies concept and the criteria outlined in Kindler and Fritz (2018), Asztalos et al. (2021a) concluded that all grass snakes from the southern Balkans, western Turkey, and Cyprus represent the subspecies Natrixnatrixmoreotica (Bedriaga, 1882), which implicitly included all Aegean populations, i.e., also grass snakes from Milos. However, Asztalos et al. (2021a) did not provide information about the phenotypes of the genetically studied snakes.
Besides the “schweizeri morphotype,” melanistic grass snakes and the “picturata morphotype” are known to occur on Milos (Kreiner 2007). Representatives of the picturata morphotype are best described as melanistic grass snakes with many small light speckles (Kabisch 1999). All of these morphotypes have also been recorded outside of Milos, and they are known to coexist on other islands, such as Mykonos and Cyprus, in part together with additional phenotypes, like the striped “picturata morphotype” (Fig. 1; Wiedl and Böhme 1992; Covaciu-Marcov et al. 2006; Baier and Wiedl 2010; Cattaneo 2010; Bogaerts et al. 2018; Zotos et al. 2021; Fănaru et al. 2022; Fritz and Ihlow 2022).
In the subspecies N.n.moreotica, several mitochondrial lineages with many individual haplotypes occur in close geographic proximity or even together (Fig. 1; Kindler et al. 2013, 2017; Asztalos et al. 2021a). Until now, there was no attempt made to examine whether the different phenotypes are possibly correlated with distinct mitochondrial lineages. In the present study, we examine whether grass snakes of the different phenotypes bear one and the same or different mitochondrial lineages or haplotypes.
During fieldwork in the western Aegean, specifically on Milos (Cyclades) and Skyros (Sporades), we collected buccal swabs from three grass snakes each from Milos and Skyros representing different phenotypes (Table 1). Colouration and pattern of the sampled snakes is shown in Figs 1 and 2. The snakes were observed in various types of freshwater habitats (Suppl. material 1: fig. S1).
Morphotypes of Natrixnatrix from Milos and Skyros on natural ground Aschweizeri morphotype from Milos (11413) B melanistic morphotype from Milos (11402) Cpicturata morphotype from Milos (11401) Dpicturata morphotype from Skyros (11843) E melanistic picturata morphotype from Skyros (11842) Fpicturata morphotype from Skyros (11841). Photos: D. Jablonski (A–C), E. Tzoras (D–F).
https://binary.pensoft.net/fig/877334
Studied grass snake samples.
Field number
Sampled specimen
Collection site
Phenotype
Haplotype
11401
Adult female
Milos, 36.7078°N, 24.4897°E, 7 m a.s.l.
“picturata”
y15
11402
Subadult male
Milos, 36.7078°N, 24.4897°E, 7 m a.s.l.
melanistic
y15
11413
Juvenile; unknown sex
Milos, 36.7233°N, 24.5297°E, 51 m a.s.l.
“schweizeri”
y18
11841
Adult; unknown sex
Skyros, 38.9569°N, 24.5049°E, 10 m a.s.l.
“picturata”
y15
11842
Juvenile; unknown sex
Skyros, 38.9569°N, 24.5049°E, 10 m a.s.l.
melanistic “picturata”
y15
11843
Adult; unknown sex
Skyros, 38.9569°N, 24.5049°E, 10 m a.s.l.
regular “picturata”
y15
The buccal swabs were stored in 96% ethanol until analysis. Genomic DNA was extracted using standard DNA isolation kits and the partial ND4 gene plus adjacent DNA coding for tRNAs (tRNA-His, tRNA-Ser, tRNA-Leu) was amplified and sequenced following Kindler et al. (2013). The resulting mtDNA sequences (866 bp) were compared to the previously published data set of Kindler et al. (2013, 2017), Schultze et al. (2019), and Asztalos et al. (2021a, 2021b) using BioEdit (Hall 1999). For previously identified haplotypes of clade 3 (“yellow lineage”) and our new sequences, a parsimony network was drawn using TCS 1.21 (Clement et al. 2000) with gaps coded as fifth character state and a connection limit of 50 steps.
To obtain additional insights in the genetic and morphological variation of grass snakes in the Aegean region, we combined previously published genetic data (Kindler et al. 2013; Asztalos et al. 2021a) with literature and citizen-science data (mainly iNaturalist) for the distinct morphotypes (Suppl. material 1: table S1). Our definition of morphotypes follows Fritz and Schmidtler (2020) and Fritz and Ihlow (2022).
All previously published (Kindler et al. 2013) and our new mtDNA sequences of N.natrix from Milos and Skyros represent clade 3. The four previously published sequences and five of our six new sequences correspond to haplotype y15 (ENA accession number LT839118). Our subadult female from Milos with the schweizeri morphotype represents haplotype y18 (LT839121). The two haplotypes are similar and differ by five mutation steps (Fig. 1). Haplotype y15 is also known from Bulgaria, North Macedonia, Greece, and Serbia, whereas y18 was previously only recorded in Bulgaria. Closely related haplotypes are also found in the region, in particular y16 from Greece and y17 from Bulgaria and North Macedonia. These findings do not support that the different morphotypes correlate with different mitochondrial lineages or haplotypes.
The picturata and schweizeri morphotypes are not restricted to the Aegean region but also occur in other parts of the distribution range of N.natrix (Fritz and Ihlow 2022). The schweizeri morphotype is known for a long time from Milos, but also from Cyprus (Kabisch 1999; Baier et al. 2009). On the latter island occurs another mitochondrial lineage (clade 7; Fig. 1B). Using iNaturalist data, Fritz and Ihlow (2022) recorded the schweizeri morphotype also in continental Greece, on the Peloponnese, and on Ikaria (Dodecanese Islands), about 170 km northeast (airline) of Milos (Fig. 1B), i.e., in regions where mitochondrial clades 5 and 7 occur. The occurrence of the schweizeri morphotype in snakes harbouring different mitochondrial lineages implies that phenotypic and mitochondrial identities are decoupled. The same is true for the rare picturata morphotype, which sporadically occurs across the distribution ranges of N.natrix and N.helvetica (Fig. 1; Fritz and Schmidtler 2020; Bruni et al. 2022; Fritz and Ihlow 2022). Baier et al. (2009) reported for one site on Cyprus (Xyliatos Dam) the syntopic occurrence of regularly coloured grass snakes, melanistic individuals, and representatives of the picturata morphotype in almost equal frequencies, supporting that grass snake populations are polyphenetic. It seems that southern subspecies, including island populations, show a higher variability than northern ones (Fritz and Ihlow 2022).
It is likely that environmental and ecological conditions contribute to these local differences (see also Bury et al. 2020; Bruni et al. 2022), but a genetic component and selection are also likely to act in concert. For instance, the complete lack of the striped picturata morphotype both in N.helvetica and all northern populations of N.natrix (Fritz and Ihlow 2022; Fritz et al. 2023) would be hard to explain without a genetic component. To obtain a better understanding of the morphological variability of grass snakes, a comprehensive comparative study would be needed, examining how environmental variables, genetic identity, sex, and habitat are correlated with the different morphotypes.
Acknowledgments
We thank Kiriakos Galanis for assistance in the field, Jana Poláková and Martina Lawson for support in the lab, and Flora Ihlow for information.
Additional informationConflict of interest
The authors have declared that no competing interests exist.
Ethical statement
No ethical statement was reported.
Funding
This work was supported by the Scientific Grant Agency of the Slovak Republic VEGA 1/0242/21.
Author contributions
Daniel Jablonski: conceptualization, writing - original draft, writing - review and editing, data curation, formal analysis, funding acquisition, investigation, methodology. Elias Tzoras: writing - review and editing, data curation, investigation. Alexios Panagiotopoulos: writing - review and editing, data curation, investigation. Marika Asztalos: writing - review and editing, data curation, formal analysis, investigation, methodology. Uwe Fritz: conceptualization, writing - review and editing, data curation, formal analysis, investigation, methodology.
Author ORCIDs
Daniel Jablonski https://orcid.org/0000-0002-5394-0114
All of the data that support the findings of this study are available in the main text or Supplementary Information.
ReferencesAsztalosMAyazDBayrakcıYAfsarMTokCVKindlerCJablonskiDFritzU (2021a) It takes two to tango—phylogeography, taxonomy and hybridization in grass snakes and dice snakes (Serpentes: Natricidae: Natrixnatrix, N.tessellata).Vertebrate Zoology71: 813–834. https://doi.org/10.3897/vz.71.e76453AsztalosMGlawFFranzenMKindlerCFritzU (2021b) Transalpine dispersal: Italian barred grass snakes in southernmost Bavaria—this far but no further! Journal of Zoological Systematics and Evolutionary Research 59: 1136–1148. https://doi.org/10.1111/jzs.12471BaierFWiedlHJ (2010) The re-evaluated conservation status of the mountain populations of the highly endangered Cyprus grass snake, Natrixnatrixcypriaca (Hecht, 1930), with miscellaneous natural history notes.Salamandra46: 16–23.BaierFSparrowDJWiedlHJ (2009) The Amphibians and Reptiles of Cyprus.Edition Chimaira, Frankfurt am Main, 364 pp.BogaertsSPasmansFProtopapasDPafilisPLymberakisP (2018) Rediscovery of the grass snake (Natrixnatrix) on the island of Karpathos, Greece.Herpetology Notes11: 303–305.BruniGDi NicolaMRBanfiFFaraoneFP (2022) Distribution and characterization of melanism in grass snakes from Italy.Il Naturalista Siciliano46: 41–48. https://doi.org/10.5281/zenodo.6784679BurySMazgajsiTDNajbarBZającBKurekK (2020) Melanism, body size, and sex ratio in snakes—New data on the grass snake (Natrixnatrix) and synthesis. Naturwissenschaften 107: 22. https://doi.org/10.1007/s00114-020-01678-xCattaneoA (1990) I serpenti delle isole greche di Kythnos e Kea (Cicladi occidentali).Atti della Società Italiana di Scienze Naturali e del Museo Civico di Storia Naturale di Milano131: 209–219.CattaneoA (2010) Note eco-morfologiche su alcune specie ofidiche egee, con particolare riferimento alle popolazioni delle Cicladi centro-orientali (Reptilia).Il Naturalista Siciliano34: 319–350.ClementMPosadaDCrandallKA (2000) TCS: A computer program to estimate gene genealogies.Molecular Ecology9: 1657–1659. https://doi.org/10.1046/j.1365-294x.2000.01020.xCovaciu-MarcovSDGhiraICicort-LucaciuASSasIStrugariuABogdanH (2006) Contributions to knowledge regarding the geographical distribution of the herpetofauna of Dobrudja, Romania.North-Western Journal of Zoology2: 88–125.FănaruGTeleaAEGherghelIMelenciucR (2022) Melanism in the grass snake Natrixnatrix (Linnaeus, 1758) from the Danube Delta Biosphere Reserve, Romania.Herpetozoa35: 257–263. https://doi.org/10.3897/herpetozoa.35.e85310FritzUIhlowF (2022) Citizen Science, taxonomy and grass snakes: iNaturalist helps to clarify variation of coloration and pattern in Natrixnatrix subspecies.Vertebrate Zoology72: 533–549. https://doi.org/10.3897/vz.72.e87426FritzUSchmidtlerJF (2020) The Fifth Labour of Heracles: Cleaning the Linnean stable of names for grass snakes (Natrixastreptophora, N.helvetica, N.natrix sensu stricto).Vertebrate Zoology70: 621–665. https://doi.org/10.26049/VZ70-4-2020-07FritzUGrismerLLAsztalosM (2023) Hybrid zones of Natrixhelvetica and N.natrix: Phenotype data from iNaturalist and genetics reveal concordant clines and the value of species-diagnostic morphological traits.Vertebrate Zoology73: 383–395. https://doi.org/10.3897/vz.73.e103319HallTA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT.Nucleic Acids Symposium Series41: 95–98.HenleKKlaverCJJ (1986) Podarcissicula (Rafinesque-Schmaltz, 1810)—Ruineneidechse. In: BöhmeW (Ed.) Handbuch der Reptilien und Amphibien Europas, Band 2/II: Echsen III.Aula-Verlag, Wiesbaden, 254–342.KabischK (1999) Natrixnatrix (Linnaeus, 1758)—Ringelnatter. In: BöhmeW (Ed.) Handbuch der Reptilien und Amphibien Europas, Band 3/IIA: Schlangen II.Aula-Verlag, Wiebelsheim, 513–580.KindlerCFritzU (2018) Phylogeography and taxonomy of the barred grass snake (Natrixhelvetica), with a discussion of the subspecies category in zoology.Vertebrate Zoology68: 269–281. https://doi.org/10.3897/vz.68.e31615KindlerCBöhmeWCortiCGvoždíkVJablonskiDJandzikDMetallinouMŠirokýPFritzU (2013) Mitochondrial phylogeography, contact zones and taxonomy of grass snakes (Natrixnatrix, N.megalocephala).Zoologica Scripta42: 458–472. https://doi.org/10.1111/zsc.12018KindlerCChèvreMUrsenbacherSBöhmeWHilleAJablonskiDVambergerMFritzU (2017) Hybridization patterns in two contact zones of grass snakes reveal a new Central European snake species. Scientific Reports 7: 7378. https://doi.org/10.1038/s41598-017-07847-9KratzerH (1974) Beobachtungen über den Nahrungserwerb bei der Milos-Ringelnatter (Natrixnatrixschweizeri).Salamandra10: 49–54.KreinerG (2007) Die Schlangen Europas.Edition Chimaira, Frankfurt am Main, 317 pp.MertensRWermuthH (1960) Die Amphibien und Reptilien Europas (Dritte Liste, nach dem Stand vom 1. Januar 1960). Verlag Waldemar Kramer, Frankfurt am Main, XI + 264 pp.MüllerL (1932) [Untitled]. Blätter für Aquarien- und Terrarienkunde 43: 319.PodnarMMayerWTvrtkovićN (2005) Phylogeography of the Italian wall lizard, Podarcissicula, as revealed by mitochondrial DNA sequences.Molecular Ecology14: 575–588. https://doi.org/10.1111/j.1365-294X.2005.02427.xPoulakakisNKapliPLymberakisPTrichasAVardinoyiannisKSfenthourakisSMylonasM (2015) A review of phylogeographic analyses of animal taxa from the Aegean and surrounding regions.Journal of Zoological Systematics and Evolutionary Research53: 18–32. https://doi.org/10.1111/jzs.12071SchultzeNLauferHKindlerCFritzU (2019) Distribution and hybridisation of barred and common grass snakes (Natrixhelvetica, N.natrix) in Baden-Württemberg, south-western Germany.Herpetozoa32: 229–236. https://doi.org/10.3897/herpetozoa.32.e38897WiedlHBöhmeW (1992) Wiederentdeckung der Ringelnatter (Natrixnatrix ssp.?) auf Zypern—Vorläufiger Bericht.Herpetofauna80: 6–10.ZotosSStamatiouMNaziriAMeletiouSDemosthenousSPerikleousKErotokritouEXenophontosMZavrouDMichaelKSergidesL (2021) New evidence on the distribution of the highly endangered Natrixnatrixcypriaca and implications for its conservation.Animals11: 1–13. https://doi.org/10.3390/ani11041077Supplementary materials10.3897/zookeys.1169.104594.suppl181495501E5EE430-60B5-5A9C-93B0-5F36C824AFDA
Supplementary information
PDf file
figure S1. Habitats of grass snakes on Milos; table S1. Literature and citizen science records of the picturata and schweizeri morphotypes of Natrixnatrix in the Aegean region and Cyprus.
https://binary.pensoft.net/file/877335This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Daniel Jablonski, Elias Tzoras, Alexios Panagiotopoulos, Marika Asztalos, Uwe Fritz