Research Article |
Corresponding author: Uwe Fritz ( uwe.fritz@senckenberg.de ) Academic editor: Ralf Britz
© 2023 Uwe Fritz, L. Lee Grismer, Marika Asztalos.
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:
Fritz U, Grismer LL, Asztalos M (2023) 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. Vertebrate Zoology 73: 383-395. https://doi.org/10.3897/vz.73.e103319
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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.
Austria, Colubridae, cline analyses, distribution, Germany, Italy, Natricidae
Grass snakes belong to the most abundant and most widely distributed Palearctic species of snakes (
Based on genetics, N. natrix and N. helvetica hybridize in three distinct contact zones: (1) the Rhine region, (2) north of the Alps in Tyrol and southernmost Bavaria, and (3) south of the Alps in northeastern Italy (
We compared previously published genetic data for approximately 1000 Natrix natrix, N. helvetica and their hybrids (mtDNA data for 1062 grass snakes, microsatellite data for 952 grass snakes;
To enlarge the sample size of pure N. natrix and N. helvetica for our morphological comparisons, we harvested all usable iNaturalist data for grass snakes from Germany and Austria. For Italy, we restricted our investigation to the northern regions Piemonte, Liguria, Valle d’Aosta (Vallée d’Aosta), Lombardia, Trentino-Alto Adige (Trentino-Südtirol), Friuli-Venezia Giulia, Veneto, and Emilia-Romagna. We had to disregard Switzerland because exact geographic coordinates for iNaturalist records were mostly not accessible for this country.
iNaturalist records were identified either as pure representatives of N. natrix, N. helvetica, or as hybrids (Supplementary Table S1) using six putatively species-diagnostic coloration and pattern traits (see also
As far as possible, the following traits were scored for each iNaturalist snake:
(1) Number of occipital spots and (2) shape of posterior dark occipital spot:
Natrix helvetica and N. natrix have a characteristic head pattern consisting either of a light-colored closed collar (N. helvetica) or of two separate light-colored crescents (N. natrix, N. helvetica). In N. helvetica, only one posterior dark-colored or black spot flanks the collar on each side, so that a bipartite pattern is present. This dark spot is typically elongated backwards. In N. natrix, an additional dark-colored or black spot is developed in front of the crescents, so that a tripartite pattern is present. The posterior dark spot in N. natrix is typically narrow (Fig.
Differences in head pattern of (A) Natrix natrix (photo: Henrik Bringsøe) and (B) N. helvetica (photo: Wolfgang Wüster). (a) Anterior dark occipital spot, (c) light occipital crescent/collar, (p) posterior dark occipital spot. Note the presence of (a) in N. natrix and its absence in N. helvetica and that (p) is narrow in N. natrix and wide in N. helvetica. This figure was inspired by a similar one in
These character states were translated for each individual into a simple coding system consisting of three numbers separated by slashes. The coding accounts for presence/absence and intensity, with values ranging from 0 to 3. Here are four examples illustrating our coding system:
0/0/1 = no anterior dark spot present/no light collar or crescent present (i.e., this area on the occiput has the same color as the body)/narrow posterior dark spot present
0/1/2 = no anterior dark spot present/ light collar or crescent present/elongated posterior dark spot present
0/1/3 = no anterior dark spot present/light collar or crescent present/greatly elongated posterior dark spot present
1/1/1 = anterior dark spot present/light collar or crescent present/narrow posterior dark spot present.
For statistical analysis, the recorded character states were simplified. It was distinguished between grass snakes with or without an anterior dark spot, i.e., between a bipartite and a tripartite head pattern. In addition, the size of the posterior dark spot was evaluated separately.
(3) Shape of light occipital markings:
Natrix natrix typically has widely separated light-colored occipital crescents, while the light elements in N. helvetica are frequently more medially extended, so that they form either a closed collar or that the tips of the extended light elements are meeting dorsally. In aged N. helvetica, the collar may be completely faded out, so that it has the same coloration as the body.
Coded character states:
0 = crescents widely separated
1 = tips of crescents meet nearly
2 = tips meet
3 = closed collar
Body = marking faded.
(4) Color of light occipital spots or collar:
Natrix helvetica is thought to have paler light markings compared to N. natrix.
Coded character states:
0 = white to light yellow
1 = yellow
2 = orange
Body = color faded.
(5) Presence/absence of side bars:
Side bars on the body flanks are diagnostic for N. helvetica; their absence is diagnostic for N. natrix.
Coded character states:
0 = side bars absent
1 = side bars present
2 = pronounced side bars present
(6) Presence/absence of back stripes:
Dorsal stripes are known only in southeastern populations of N. natrix and unknown from other populations of this species or from N. helvetica. Thus, the presence of back stripes in the southeast of our study region is either diagnostic for N. natrix or indicates, in combination with traits of N. helvetica, hybridization.
Coded character states:
0 = back stripes absent
1 = back stripes present.
When a character state was intermediate or invisible, it was recorded as such. Some grass snakes could not be assigned to any of the character states. This included melanistic individuals (n = 25) and representatives of the so-called picturata morph (black snakes with small light speckles;
To infer the transition between the two grass snake species in their contact zones, hybrid clines were separately examined for phenotypes, mtDNA and microsatellite data using the R package hzar (
(1) A 600-km-long Rhine transect from the Eifel region, North Rhine-Westphalia, Germany to the Bavarian Upper Palatinate region, Germany;
(2) a 300-km-long Alpine transect in northern direction across the Alps from South Tyrol, Italy to the region of Munich, Germany;
(3) a 625-km-long Italian transect in west-east direction across northeastern Italy.
Each transect begins within the distribution range of N. helvetica and runs across one of the hybrid zones into the distribution range of N. natrix. Thus, each transect allows to examine the character transitions across each hybrid zone. The Italian transect is the same as used in
Using qgis 3.4.5 samples were pooled for each transect into sections of 25 km length, including samples within 50 km (Rhine and Alpine transects) or 55 km (Italian transect, phenotypic data only) distance on both sides of the transect.
For microsatellite data, the mean proportion Q of cluster membership (as inferred by structure;
Mitochondrial identity of genetically studied samples (n = 1062). A Entire study range, B Rhine transect, C Alpine transect, D Italian transect. In cline analyses, the transition between mtDNA lineages corresponding to Natrix helvetica (mtDNA lineages C and E) or N. natrix (mtDNA lineages 3 and 4) were used. Data are from
The individual datasets (mtDNA, microsatellites, and morphology for each contact zone) were processed independently, except for the Italian transect, for which only the phenotypic data were processed. Using a burn-in of 10,000 iterations, followed by additional 90,000 iterations, the 15 implemented models were fitted to the mean proportions of cluster membership, haplotype frequency, or phenotypic identity. Then, the best cline model was selected based on the lowest AIC score (Supplementary Table S2), and the corresponding Maximum Likelihood cline was plotted. Observed frequency data were plotted over the associated fuzzy cline regions (95% credible cline regions).
Results for microsatellites and mtDNA of the Italian transect were taken from
To understand which phenotypic trait is most informative for discriminating between N. natrix and N. helvetica, we compared the percentages of each character state of our iNaturalist data for each species and hybrids. Since back stripes are restricted to individuals from the southeast of our study region (i.e., the distribution range of the subspecies N. n. vulgaris;
The distribution of morphologically determined grass snakes largely matches the distribution pattern revealed by genetic analyses (Figs
However, there are a few exceptions. In western Germany, a few morphologically intermediate individuals and a few grass snakes identified as N. natrix were recorded far away from the hybrid zone, inside the distribution range of N. helvetica (Fig.
In northwestern Germany, where grass snakes are rare and where we had a gap for DNA samples, the few photo records available on iNaturalist suggest that the contact zone of the two species is close to the Dutch border in the region of the Ems River and extends from there southeastwards to the region of Höxter.
The genetic and phenotypic transition between N. natrix and N. helvetica was examined along three transects. For each transect, the clines for the two genetic markers (mtDNA, microsatellites) and morphology were steep. For the Rhine transect (Fig.
Cline analyses for microsatellite data, mitochondrial DNA, and morphology for the Rhine contact zone of Natrix helvetica and N. natrix. Map: cline centers for microsatellites in red, for mtDNA in yellow, and for morphology in orange. Other diagrams: Maximum Likelihood clines for microsatellites (Q values of cluster membership), mtDNA identity, and morphological identity; fuzzy cline regions (95% credibility intervals) in grey.
For the Alpine transect (Fig.
Cline analyses for microsatellite data, mitochondrial DNA, and morphology for the Alpine contact zone of Natrix helvetica and N. natrix. Map: cline centers for microsatellites in red, for mtDNA in yellow, and for morphology in orange. Other diagrams: Maximum Likelihood clines for microsatellites (Q values of cluster membership), mtDNA identity, and morphological identity; fuzzy cline regions (95% credibility intervals) in grey.
The cline center for microsatellites of the Italian transect (Fig.
Cline analyses for microsatellite data, mitochondrial DNA, and morphology for the Italian contact zone of Natrix helvetica and N. natrix. Map: cline centers for microsatellites in red, for mtDNA in yellow, and for morphology in orange. Other diagrams: Maximum Likelihood clines for microsatellites (Q values of cluster membership), mtDNA identity, and morphological identity; fuzzy cline regions (95% credibility intervals) in grey. Clines for mtDNA and microsatellites redrawn from
For six traits the occurrences of individual character states were compared for grass snakes from iNaturalist photos identified as N. natrix, N. helvetica, or hybrids (Fig.
Coloration and pattern variation in barred grass snakes. A Typical Natrix helvetica helvetica with pronounced side bars and contrasting head pattern; Röttgen near Bonn, North Rhine-Westphalia, Germany (photo from iNaturalist, Santiago Jaume-Schinkel). B Aged N. h. helvetica with faded light occipital collar and weak side bars; Hofheim am Taunus, Hesse, Germany (photo from iNaturalist, pfadfinder). C N. h. helvetica with uniform body coloration and small side bars; Gengenbach, Baden-Württemberg, Germany (photo from iNaturalist, Corinna Herr). D N. h. sicula with faded light occipital collar, small side bars and spotted body pattern; Bichlersee, Bavaria, Germany (photo Frank Glaw).
Natrix natrix and N. helvetica differed regarding the frequencies and percentages of character states in all studied traits (Fig.
Percentages of character states in traits of morphologically identified Natrix natrix, N. helvetica and their putative hybrids (iNaturalist data). Melanistic individuals and grass snakes representing the picturata morph were excluded. The sample for back stripes was restricted to snakes from Austria and northeastern Italy.
Hybrids of the two Natrix species often displayed intermediate percentages between the parental species (Fig.
The present study is another example indicating that georeferenced photos from iNaturalist and similar Citizen Science platforms can provide valuable morphological data for research and may also enhance our knowledge of the distribution of the depicted taxa or hybrid zones (e.g.,
A focus of our present investigation was the comparison of coloration and pattern data derived from iNaturalist photos and previously published genetic data to examine whether the morphological transition between N. natrix and N. helvetica parallels the genetic transition across their contact zones (
With respect to the present study, it is important to keep in mind that morphological and genetic information represent independent datasets, i.e., the morphological data come from the same geographic areas as the genetic data, but not from the same individuals. Therefore, we expect that we misidentified some hybrids and backcrosses as pure N. natrix or N. helvetica, or vice versa, especially in the contact zones of the two species, because morphological characters do not always strictly correlate with genetic identity.
Keeping this caveat in mind, the agreement of the morphological and genetic datasets is intriguing. With respect to distribution and cline analyses, the three datasets (mtDNA, microsatellites, coloration and pattern) yielded geographically concordant patterns (Figs
Given the putative difficulties to morphologically identify these two species of grass snake, we compared the percentages of character states of several traits to determine which are most reliable for differentiating N. natrix and N. helvetica. During our study it became apparent that differentiation is complicated because some representatives of the northern subspecies N. h. helvetica (western Germany) differ subtly in coloration and pattern from the southern subspecies N. h. sicula (from southern Bavaria across the Alps southwards; Fig.
Despite this, the eponymous presence of side bars, as weak as they may be, remains one of the best characters for differentiating the barred grass snake N. helvetica from N. natrix. Two other useful traits are the number of occipital spots (three in N. natrix, two in N. helvetica) and the shape of the posterior dark occipital spot (narrow in N. natrix, wide in N. helvetica). The discriminatory power of these traits even increases when they are combined with one another and with the remaining traits.
These remaining species-specific traits show considerably more variability (Fig.
Finally, it is noteworthy that some cline widths revealed in the present study are wider than those reported in previous studies (
Despite these differences, the clines of the morphological and the two genetic datasets are steep and concordant for all three studied contact zones (Figs
The Italian contact zone is also peculiar with respect to one morphological trait. In this region, a southern subspecies of the common grass snake (N. n. vulgaris; see
Our investigation is another example demonstrating that valuable morphological and distributional data can be extracted from iNaturalist. Using a large dataset of georeferenced photos of grass snakes from iNaturalist, we could show that common grass snakes (N. natrix) and barred grass snakes (N. helvetica) can be reliably differentiated using coloration and pattern traits. Three characters are particularly useful for species differentiation (presence/absence of side bars, number of occipital spots, shape of posterior dark occipital spot). Distributional data derived from iNaturalist records agree well with the genetically inferred distribution of the two species. For northwestern Germany, a region corresponding to a sampling gap for genetics, iNaturalist records suggest that the contact zone of the two species is near the Ems River and extends from there southeastwards to the region of Höxter, North Rhine-Westphalia. Cline analyses using previously published genetic data and morphological data from iNaturalist recovered geographically concordant steep clines for the transition between the two species in their hybrid zones. The contact zone in northeastern Italy should be examined genetically in more detail. Coloration and pattern traits suggest a unimodal hybrid zone in the southern part of this region, in contrast to the bimodal hybrid zones in the Rhine and Alpine regions and farther north in Italy.
Harald Ahnelt (Vienna), Ralf Britz (Dresden), and Wolfgang Wüster (Bangor) made helpful comments on a previous version of this study. Frank Glaw (Munich) discussed some aspects of an earlier manuscript and provided a photo of a Bavarian N. helvetica. Additional photos were donated by Henrik Bringsøe (Copenhagen) and Wolfgang Wüster (Bangor).
Table S1
Data type: .xlsx
Explanation note: Studied iNaturalist material and genetic data from previous studies.
Table S2
Data type: .pdf
Explanation note: Akaike Information Criterion (AIC) scores for fitted clines under different models using the R package hzar (