Research Article |
Corresponding author: Uwe Fritz ( uwe.fritz@senckenberg.de ) Corresponding author: Sylvain Ursenbacher ( sylvain.ursenbacher@infofauna.ch ) Academic editor: Robert Jadin
© 2024 Andreas Schild, Hannes Baur, Stefan T. Hertwig, Uwe Fritz, Sylvain Ursenbacher.
This 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.
Citation:
Schild A, Baur H, Hertwig ST, Fritz U, Ursenbacher S (2024) Genetic identification, morphology and distribution of Natrix helvetica subspecies in southern and western Switzerland (Reptilia, Squamata, Serpentes). ZooKeys 1205: 223-238. https://doi.org/10.3897/zookeys.1205.123911
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Most of Switzerland is inhabited by the nominotypical subspecies of the barred grass snake (Natrix helvetica helvetica), which is characterized by mitochondrial DNA lineage E. Only in the northeast of the country, the common grass snake (N. natrix) occurs and hybridizes with N. h. helvetica in a narrow contact zone. However, we discovered that in southern and western Switzerland barred grass snakes representing another mtDNA lineage (lineage C) are widely distributed. Lineage C is typical for Alpine populations of the southern subspecies N. h. sicula. Our microsatellite analyses of the Swiss samples revealed differences between the two subspecies and also a substructure with two clusters in each subspecies. Furthermore, we discovered a contact and hybrid zone of N. h. helvetica and N. h. sicula along the northern shore of Lake Geneva and also confirm that interbreeding with alien common grass snakes (N. n. moreotica, mtDNA lineage 7) occurs there. This finding is of concern for nature conservation and measures should be taken to prevent further genetic pollution. Using morphometrics, we found no differences between the two subspecies of N. helvetica, while N. natrix was slightly distinct from N. helvetica.
Microsatellites, mitochondrial DNA, morphometrics, nuclear DNA, taxonomy
Grass snakes constitute a complex of three species which were regarded as conspecific for many decades (
There are many regions across the distribution range of grass snakes where non-native individuals were introduced (France:
The aim of the present study is to determine the natural distribution of N. h. sicula in Switzerland. To do so, we collected DNA samples from wild snakes and museum specimens to determine their mtDNA lineage. In addition, we used nuclear DNA markers (microsatellites) for Bayesian cluster analyses to estimate the amount of admixture between snakes corresponding to different mitochondrial lineages. In accordance with the concept of integrative taxonomy, we also used various characters of pholidosis and colour pattern as well as morphometric measurements to identify possible external morphological differences in our sample.
The immediate study area was limited to southern and western Switzerland. It comprised the cantons of Vaud, Valais and Ticino (Fig.
Distribution of mitochondrial lineages of grass snakes in southern and western Switzerland (a) and microsatellite clusters according to our STRUCTURE analyses (b). Symbols and colours of mitochondrial lineages correspond to
Samples were incubated with ATL buffer and proteinase K (Qiagen) in a heat block for 16–20 h at 56 °C. Scales were previously placed in water for 24 h to remove alcohol. After digestion, liquid from swab tips was extracted using a centrifuge. The DNA was purified following the protocol “Purification of total DNA from Animal Tissues (Spin-Column Protocol)” of the DNeasy Blood and Tissue Kit (Qiagen) using a Qiagen robot.
To determine the mtDNA lineage, the cytochrome b gene (cyt b) and the NADH dehydrogenase subunit 4 gene with adjacent regions coding for tRNAs (ND4) were used, as in previous studies on grass snakes (e.g.,
The same thirteen microsatellite loci were used as in
For inferring the nuclear genomic identity of the 73 successfully processed samples, the Bayesian clustering approach based on the Monte Carlo Markov chain (MCMC) algorithm implemented in the software STRUCTURE ver. 2.3.4 (
Only snakes exceeding 50 cm in total length and with a genotypic cluster assignment ≥ 80% were used for morphological examinations. Following
To obtain landmark data, standardized pictures were used showing the right and dorsal sides of the head of adult snakes. Fixing the focal length and manual focus of the camera ensured that the scale of the pictures was identical, which was double-checked using a ruler in the pictures. Photographs were taken twice to calculate mean landmark coordinates, which reduces potential inaccuracies due to slight shifts in photographing and placing landmarks. M. Chèvre provided only a single photograph per snake, for which landmark coordinates were produced twice to account for imprecise landmark placing. Mostly easily identifiable junctions of scales were chosen as landmarks to facilitate the workflow (Suppl. material
Some landmarks were removed for analysis because the sample size was too small for 27×2 coordinate variables. Landmark 8 was removed because the temporal scale was sometimes divided and/or small, so it did not reach the 7th supralabial scale. Landmarks 12, 14, 16 and 18 were excluded as they all have other landmarks in close proximity. Lastly, landmarks 21, 24 and 27 were removed because they are located at the edge and might already be influenced by the curvature of the head. Therefore, only nineteen landmarks (1–7, 9–11, 13, 15, 17, 19, 20, 22, 23, 25, 26) were finally used (Suppl. material
Landmarks were placed in the software TPSDIG2 ver. 2.30 (
All analyses of the mean landmark coordinates were performed in MORPHOJ ver. 1.07a (
Distance measurements (SVL, TL, HL and HW; Suppl. material
Scale counts (VS, SCS, PTS and GS; Suppl. material
Distance measurements, scale counts, measures for colour markings and three additional variables (RelRedPos, TPOS and BW; Suppl. material
The distribution of mtDNA lineages in southern Switzerland is shown in Fig.
According to
Fig.
Genetic differentiation (FST) values are similar between clusters, except for the slightly lower values for E-GE/E-ML and for E-ML/natrix, whereas the highest value was observed between C-VS/E-GE (Suppl. material
For morphological and landmark analyses, snakes representing the clusters E-ML and E-GE were merged in one cluster E, while C-VS and C-TI were kept separate. C-VS and C-TI are geographically divided by mountainous regions difficult to cross for grass snakes, whereas E-ML and E-GE are in contact and admixing. Additionally, the number of samples for E-GE is very limited.
Mahalanobis and Procrustes permutation tests revealed significant morphological differences between all clusters (Table
Statistical test results of Canonical Variate Analysis (CVA) of lateral and dorsal landmarks (Fig.
Lateral landmarks | |||||||
Mahalanobis distances among groups | Procrustes distances among groups | ||||||
natrix | E | C-VS | natrix | E | C-VS | ||
E | 4.8305 | E | 0.0308 | ||||
C-VS | 8.2776 | 5.6166 | C-VS | 0.0380 | 0.0285 | ||
C-TI | 6.5247 | 4.5882 | 5.2595 | C-TI | 0.0431 | 0.0324 | 0.0237 |
P values from permutation tests | P values from permutation tests | ||||||
natrix | E | C-VS | natrix | E | C-VS | ||
E | < 0.001 | E | < 0.001 | ||||
C-VS | < 0.001 | < 0.001 | C-VS | < 0.001 | < 0.001 | ||
C-TI | < 0.001 | < 0.001 | < 0.001 | C-TI | < 0.001 | < 0.001 | 0.0783 |
Dorsal landmarks | |||||||
Mahalanobis distances among groups | Procrustes distances among groups | ||||||
natrix | E | C-VS | natrix | E | C-VS | ||
E | 2.7561 | E | 0.0232 | ||||
C-VS | 3.5488 | 3.7053 | C-VS | 0.0236 | 0.0268 | ||
C-TI | 4.4217 | 4.1624 | 2.9620 | C-TI | 0.0375 | 0.0389 | 0.0264 |
P values from permutation tests | P values from permutation tests | ||||||
natrix | E | C-VS | natrix | E | C-VS | ||
E | < 0.001 | E | < 0.001 | ||||
C-VS | < 0.001 | < 0.001 | C-VS | < 0.001 | < 0.001 | ||
C-TI | < 0.001 | < 0.001 | < 0.001 | C-TI | < 0.001 | < 0.05 | < 0.001 |
Canonical Variate Analysis (CVA) of dorsal (a) and lateral (b) landmark coordinates for grass snakes assigned to the microsatellite clusters of Natrix helvetica (E, C-VS, C-TI) and N. natrix (N. n. vulgaris, mtDNA lineage 3, from northeastern Switzerland). Only individuals with a genotypic cluster assignment ≥ 80% are included; circles represent 95% confidence ellipses.
Shape PCA and standard PCAs show no differentiation of mtDNA lineages E, C-VS and C-TI of N. helvetica (Fig.
Shape PCA of distance measurements (a), standard PCA of scale counts (b) and standard PCA of colour marking measurements (c) for grass snakes assigned to the microsatellite clusters of Natrix helvetica (C-TI, C-VS, E) and N. natrix (N. n. vulgaris, mtDNA lineage 3, from northeastern Switzerland). Only individuals with a genotypic cluster assignment ≥ 80% are included.
After the discovery of two putatively alien mtDNA lineages of grass snake around Lausanne (lineages C and 7 of
Our study shows that N. h. sicula is widely distributed in the cantons of Ticino and Valais and ranges to the canton of Vaud, along Lake Geneva, where it hybridizes with N. h. helvetica, as evidenced by microsatellite genotypes. Therefore, N. h. sicula should no longer be considered as non-native around Lausanne, as supposed by
The two microsatellite clusters of N. h. helvetica and N. h. sicula correspond to local population structure. It is possible that our cluster E-GE (Fig.
North of Lake Geneva, we were not only able to detect the natural contact and hybridization zone of the two subspecies of N. helvetica. Our microsatellite data (Fig.
The distribution and hybrid zone of N. h. helvetica and N. h. sicula in Switzerland can be explained in a biogeographical context.
In this context, two other northern records of grass snakes with lineage C are difficult to interpret (Langnau im Emmental, canton Bern, and Muotathal, canton Schwyz, directly at the border to canton Uri). For the snake from Muotathal no genotype is available, but the snake from Langnau im Emmental is, according to its microsatellite genotype, a pure representative of the cluster E-ML. Also, two other snakes from the canton Bern have an admixed genotype (Fig.
In contrast to genetic data, our morphological analyses revealed only a weak differentiation among the studied grass snakes. Only the two species N. helvetica and N. natrix could be morphologically discriminated with some confidence, while the used traits were not helpful in discriminating the two subspecies of individual genetic clusters within N. helvetica. This does not contradict the validity of the involved two subspecies because morphological traits that can be distinguished by humans are neither necessarily biologically relevant nor a prerequisite for taxonomic distinctness (compare, for instance,
We thank M. Chèvre for advice, data, photos and his lab protocols and V. Zwahlen for assistance with processing microsatellites. Special thanks go to all researchers and institutions that provided samples: M. Chèvre, S. Dubey, E. Gallice, T. Gil, G. Meier, B. Meister, D. Muri, the Musée de la nature Sion (S. Gerber), the Museo cantonale di storia naturale Lugano (N. Zambelli), and the Naturhistorisches Museum Bern (R. Hagmann). We thank all authorities of the cantons of Vaud, Valais and Ticino for the necessary permits. The figures were enhanced by M. H. Fischer. An anonymous reviewer, P. Mikulíček and the editor R. Jadin provided helpful comments on the manuscript.
The authors have declared that no competing interests exist.
Captures in the wild were conducted according to the local authorities’ requirements (veterinary authorisation number GR_2013_15kü).
This study was funded by the Swiss Bundesamt für Umwelt (BAFU).
Conceptualization: SU. Data curation: AS, SU. Formal analysis: AS, HB. Funding acquisition: SU. Investigation: AS. Methodology: SU. Project administration: SU. Resources: HB, STH. Supervision: SU. Visualization: AS, UF. Writing – original draft: AS, UF. Writing – review and editing: AS, HB, STH, SU, UF.
Hannes Baur https://orcid.org/0000-0003-1360-3487
Stefan T. Hertwig https://orcid.org/0000-0003-1617-8180
Uwe Fritz https://orcid.org/0000-0002-6740-7214
Sylvain Ursenbacher https://orcid.org/0000-0001-5093-6403
All data that support the findings of this study are available in the main text or the Suppl. material. No new haplotypes were identified in the present study, which is why no sequences were uploaded to the European Nucleotide Archive (ENA).
Supplementary information
Data type: docx
Explanation note: fig. S1. Landmark positions. fig. S2. STRUCTURE HARVESTER results indicating the optimal number of microsatellite clusters (K). fig. S3. Wireframe graph showing shape changes along the canonical variate 1 (CV1) for dorsal landmarks. fig. S4. Wireframe graph showing the shape changes along the canonical variate 1 (CV1) for lateral landmarks. table S2. Morphological variables measured. Illustrations from