Research Article |
Corresponding author: Daniel Jablonski ( daniel.jablonski@uniba.sk ) Academic editor: Uwe Fritz
© 2021 Oleg Kukushkin, Oleg Ermakov, Iulian Gherghel, Svetlana Lukonina, Anton Svinin, Igor Doronin, Evgeniy Simonov, Daniel Jablonski.
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:
Kukushkin O, Ermakov O, Gherghel I, Lukonina S, Svinin A, Doronin I, Simonov E, Jablonski D (2021) The mitochondrial phylogeography of the Crimean endemic lizard Darevskia lindholmi (Sauria, Lacertidae): Hidden diversity in an isolated mountain system. Vertebrate Zoology 71: 559-576. https://doi.org/10.3897/vz.71.e62729
|
Abstract
The Lindholm rock lizard, Darevskia lindholmi, is the only member of the genus Darevskia whose range is restricted solely to Europe, representing a local endemism found only in the Crimean Mountains. In our study, we investigated the cytochrome b gene (mtDNA) of 101 D. lindholmi sequences from 65 Crimean localities, representing its entire range. We found that D. lindholmi is highly genetically structured, and its range is divided into populations belonging to three mitochondrial lineages. The Lindholm rock lizard populations inhabiting the middle part of the Crimean Mountains (further referred to as the Central lineage) are sharply differentiated from the other two lineages (the Common and the Southwestern lineages), which are present in most of the species range. The genetic distance between the Central lineage and the other two taken together is 4.6%, according to our results, suggesting that the divergence occurred during the Early Pleistocene. The narrowly distributed Southwestern lineage and the widespread Common lineage, on the other hand, are differentiated by 1%. Field observations on the representatives of the main evolutionary groups show that their ecology is also different: the Central lineage is a mesophilic and cold-resistant form, while the other two closely related lineages are more xerophilic and thermophilic. Results of the potential ranges modeling and ecological niche analysis confirm that the genetic lineages occupy different niches of the Crimea. Furthermore, the area of inhabitation of the Central lineage splits the western and eastern parts of the Common lineage range, while the Southwestern lineage is restricted along the coast of the southwestern coast of the peninsula. The long-term co-existence of deeply divergent sister mitochondrial lineages in a relatively small (circa 7,000 km2) isolated mountain system serves as a mesocosm for understanding the speciation process. Our data suggest that the Central lineage warrants further taxonomic investigation.
Caucasus, Crimea, cryptic lineage, endemism, Lacertidae, Quaternary, speciation
The genus Darevskia Arribas, 1999 includes more than 30 species of medium-sized lizards with ranges localized mainly in the Caucasus, Anatolia, and neighboring regions, including southeastern Europe and northern Iran (
Darevskia lindholmi currently inhabits an isolated mountain system of the Crimean Peninsula, without any geographical connection to the Caucasus that is expected as the radiation center of the genus (
In our study, we thus investigated the mitochondrial (mtDNA) phylogeography and the niche divergence of D. lindholmi, particularly i) studying intraspecific genetic variability, and ii) assessing the spatial distribution and ecological niches of the detected mtDNA lineages to provide a biogeographical and taxonomic context.
Tissue samples were collected mainly by O.K. between 2011 and 2019 throughout the entire species’ range (Fig.
Distribution of mitochondrial DNA haplogroups in Darevskia lindholmi. Circles indicate the sampling localities: dark-blue – Southwestern lineage, blue – Common lineage; yellow – Central lineage. The type locality of the species is indicated by a star. Geographic position of the localities are given in Table S1. The location of the Crimean Peninsula in the Northern Black Sea region and the position of the Crimean Mountains are shown on the inset maps.
The cyt b gene (1131 bp) was amplified using a primer pair LgLu (5’-AAC CRC YGT TGT MTT CAA CTA-3’) and RtHr (5’-GGY TTA CAA GAC CAG YGC TTT-3’;
The sequences were aligned and edited manually using BioEdit v. 7.0 software (
Various lizard groups were used as “external” calibration age constraints in our analysis (Tables S1, S2). We used the previously published six calibration points (Mya, millions of years ago ± standard deviation) widely applied in Lacertidae phylogenetic studies (e.g.
The potential distribution estimates for the main evolutionary lineages of D. lindholmi were calculated using eight algorithms (Classification Tree Analysis, Multivariate Adaptive Regression Splines, General Additive Models, Generalized Linear Models, Artificial Neural Networks, MaxEnt, Generalized Boosted Models, and Random Forests) by performing 10 replicates using a bootstrap approach in the “SSDM” R package (
To model the potential distribution of each detected lineage as well as the species, we used the WorldClim 2 database at 1 km resolution (www.worldclim.org; Fick et al. 2017). The WorldClim 2 database comprises 19 climatic variables that are a combination of temperature and precipitation factors previously shown to be highly important in shaping species distributions. The first step in selecting the predictors for our models was to ensure that the variables were not highly correlated and hence to eliminate the collinearity among them by calculating Pearsons’s correlation coefficients for all pairs of variables using SDMToolbox in ArcGIS 10 (
To further test the unicity of the ecological niches occupied by the main D. lindholmi mtDNA lineages (see Results) we performed two niche overlap metrics to assess the degree of observed ecological overlap between each lineage by performing Schoener’s D (
Among 101 sequences of D. lindholmi, three different mitochondrial lineages were identified: The Common, inhabiting most of the species range; the Southwestern, localized in the extreme south of the Crimean Peninsula, and the strongly differentiated Central lineage, confined to the middle part of the Crimean Mountains. The phylogenetic reconstructions, based on the ML and BI, had similar topologies.
When comparing D. lindholmi with other members of the D. saxicola complex (Table
The average genetic difference (p-distantce, %) between cyt b sequences per site (below the diagonal) and standard deviation (above the diagonal) within the Darevskia saxicola complex.
Species/lineage | D. lindholmi/ Common | D. lindholmi/ Southwestern | D. lindholmi/ Central | D. saxicola | D. brauneri | D. szczerbaki |
D. lindholmi/ Common | – | 0.3 | 0.6 | 0.6 | 0.6 | 0.6 |
D. lindholmi/ Southwestern | 0.9 | – | 0.6 | 0.6 | 0.6 | 0.6 |
D. lindholmi/ Central | 4.4 | 4.1 | – | 0.7 | 0.6 | 0.6 |
D. saxicola | 4.9 | 4.7 | 5.2 | – | 0.7 | 0.5 |
D. brauneri | 4.7 | 4.4 | 5.5 | 5.2 | – | 0.6 |
D. szczerbaki | 4.0 | 4.0 | 4.7 | 2.7 | 4.9 | – |
The average value of uncorrected pairwise genetic distances in the analyzed dataset is 1.6±0.2%. The genetic distance of 4.4±0.6% was found between the Central and the Common lineages, 4.1±0.6% between the Central and Southwestern lineages, and 0.9±0.3% between the Southwestern and Common lineages (Table
Due to the length of the different sequences of the final dataset, we analyzed the haplotype networks based on 92 sequences of the species where we found 35 unique haplotypes. The Common lineage (67 samples from 42 localities) comprises most of the analyzed samples and represents the highest number of haplotypes (27 out of 35; Table
Bayesian tree reconstructed with 1131 bp cytochrome b gene sequences. Numbers show posterior probabilities/ bootstrap support (values above 50% are given); **indicates 1.0 or 100% support, while *indicates 0.95–0.99 or 80–99% support. Haplogroup colors reflect the identified mtDNA lineages of Darevskia lindholmi. The numbers on the terminal branches of the tree correspond to the localities on Fig.
The haplotype network for Darevskia lindholmi based on cytochrome b. Haplogroup colors correspond with the identified mtDNA lineages. The numbers of transverse strokes on the branches (in bold) correspond to the number of nucleotide substitutions. The numbers on network elements correspond to the localities on Fig.
Genetic diversity in Darevskia lindholmi: n – sample size; H – number of haplotypes; h – haplotype diversity; π – nucleotide diversity (per site); S – total number of polymorphic positions; k – average number of nucleotide substitutions; Fu’s Fs is the Fs test value; Tajima D.
Lineage | n | H | h±SD | π±SD (%) | S | k | Fu’s Fs | Tajima D |
Common | 67 | 27 | 0.915±0.021 | 0.40±0.02 | 33 | 4.53 | –11.61* | –1.11 |
Southwestern | 9 | 4 | 0.583±0.183 | 0.08±0.03 | 4 | 0.88 | –1.28 | –1.49 |
Central | 16 | 4 | 0.442±0.145 | 0.07±0.03 | 5 | 0.83 | –0.68 | –1.61 |
D. lindholmi in the whole | 92 | 35 | 0.935±0.013 | 1.60±0.19 | 81 | 18.14 | – | – |
*P support |
Darevskia lindholmi divergence from other members of the genus is estimated at 2.1 Mya (95% HPD: 1.6–2.7 Mya). The data suggests that the Central lineage became isolated 1.8 Mya (95% HPD: 1.2–2.3 Mya). The split of the Southwestern and the Common lineages occurred only 0.5 Mya (95% HPD: 0.2–0.8 Mya). The mean (as well as the 95% HPD interval) estimated divergence times inside the D. saxicola complex and main lineages within D. lindholmi are presented in Fig.
Maximum credibility chronogram (MCC) inferred in BEAST v. 1.10.4 with bars, showing the 95% HPDI of divergence time estimates. The vertical gray line marks the Messinian Salinity Crisis (MSC; 5.96–5.33 Mya). Red circles at the nodes indicate used calibration points, white circles indicate posterior probabilities above 0.95, and black circles denote groups of taxa forced to be monophyletic in the analysis.
Our models performed very well in estimating the potential distributions and ecological niches of D. lindholmi, as demonstrated by both the AUC and Kappa metrics [Central: AUC= 0.96 (±0.04), Kappa= 0.65 (±0.24); Common: AUC= 0.97 (±0.02), Kappa= 0.7 (±0.26); Southwestern: AUC= 0.99 (±0.01), Kappa= 0.8 (±0.2)]. This suggests that the ecological niches estimated are highly reliable for all genetic lineages modeled. The potential distribution of the lineages is also well estimated as shown by the low omission scores [Central lineage omission error = 0.05 (±0.06), Common omission error = 0.01 (±0.01), Southwestern omission error = 0.01 (±0.01)]. The intermodel variability also shows a high degree of stability in our ensamble among all eight algorithms used (Fig. S1), which also suggests that the models are highly reliable. The predictors with the highest power for predicting the ranges and ecological niches for all of the main evolutionary lineages of the Crimean Darevskia are the mean temperature of the wettest quarter of the year (bio8), temperature annual range (bio7), and the amount of precipitation during the driest month (bio14). The response curves of the three lineages to these three environmental predictors are very strong as well, and cumulatively are capable of explaining 57.2% of the variation from the potential distribution estimates across all lineages. To disentangle the direction of the relationship between the probability of distributions of each lineage and environemntal variables, we found positive correlations between the probability of presence of the lineages and isothermality (Common lineage vs. bio3: r=0.274; Central lineage vs. bio3: r=0.406; Southwestern lineage vs. bio3: r=0.209), the mean temperature of driest quarter of the year (Common lineage vs. bio9: r=0.232; Central lineage vs. bio9: r=0.29; Southwestern lineage vs. bio9: r=0.31), mean temperature of coldest quarter of the year (Common lineage vs. bio11: r=0.588; Central lineage vs. bio11: r=0.582; Southwestern lineage vs. bio11: r=0.625) and the amount of precipitation during the driest month (Common lineage vs. bio14: r=0.468; Central lineage vs. bio14: r=0.719; Southwestern lineage vs. bio14: r=0.537), while we found negative correlations between the temperature annual range (Common lineage vs. bio7: r= –0.743; Central lineage vs. bio7: r= –0.82; Southwestern lineage vs. bio7: r= –0.77), the mean temperature of wettest quarter of the year (Common lineage vs. bio8: r= –0.847; Central lineage vs. bio8: r= –0.913; Southwestern lineage vs. bio8: r= –0.869) and the precipitation’s seasonality (Common lineage vs. bio15: r= –0.196; Central lineage vs. bio15: r= –0.128; Southwestern lineage vs. bio15: r= –0.018). The potential ranges based on the fundamental niches of the Common, Southwestern, and Central lineages suggests the possibility of some potential geographic overlap in areas where the lineages enter in contact, however, this was not observed based on the omission error of the models which suggests that likely the lineages do not cohabit the same localities (Fig.
Potential distribution of Darevskia lindholmi evolutionary lineages: Central, Common, and the Southwestern. The intensity of the colour is related with the degree of suitability for the species presence. The black polygon represents the minimum convex polygon presence localities for each lineage.
To test the unicity of the niches modeled in relation to the available environmental conditions we performed a background analysis where we found significant differences between the niches occupied by the three lineages. The observed niche overlap between the lineages was very high (Central vs. Common: Schoener’s D=0.82, Hellinger’s-based I=0.97; Central vs. Southwestern: Schoener’s D=0.80, Hellinger’s-based I=0.97; Common vs. Southwestern: Schoener’s D=0.90, Hellinger’s-based I=0.98). However, due to small gradients, these overlaps were still shown to represent different niches based on the identity background analysis (t-test, df =99, P<0.05). The identity test indicated that the null-hypothesis regarding niche overlap can be rejected and that Central lineage and Common lineage (DH0 is 0.85±0.02 vs. DH1 = 0.82, and IH0 is 0.98±0.007 vs. IH1 = 0.97) occupy different niches, and the Central lineage and Southwestern lineage (DH0 is 0.90±0.06 vs. DH1 = 0.80, and IH0 is 0.98±0.02 vs. IH1 = 0.97) are marginally, but significantly, differentiated in their respective niches. The identity background test also shows that the Common lineage and the Southwestern lineage do not occupy different niches and a high degree of overlap exists (DH0 is 0.88±0.06 vs. DH1 = 0.90, and IH0 is 0.88±0.07 vs. IH1 = 0.98), which is in congruence with the observed genetic relationships described above (Fig.
To reconstruct the origin of D. lindholmi in Crimea is challenging due to the controversial paleogeography of the North Black Sea region as well as the existence of some gaps on the local orogeny, especially from later geological periods (
Our data also shows intraspecific genetic divergence in D. lindholmi, although it is less diverged in comparison with some Caucasian populations of the D. saxicola complex (
Present-day deep-water parts of the Black Sea were formed at the end of the Pliocene and beginning of the Pleistocene (
The level (less than 1%) and time (0.2–0.8 Mya; Fig.
The range dynamics of D. lindholmi distribution pattern could be dependent on the dynamics of the forest massif boundaries. Our models on the range dynamics of D. lindholmi based on climatic niche modeling suggest that during the Last Glacial Maximum (21 Kya) the species inhabited the Crimean Mountains (Fig.
Distribution ranges of the lizard genetic lineages do not show any significant overlap (Fig.
Based on observations made in the field, D. lindholmi can be characterized as a mesophilic cold-tolerant species. Its distribution at low altitudes is limited by insufficient humidity, while in the highlands by the average temperature of the summer months (
Our models show significant variation in the response of each lineage to the environmental variables used to model their distributions. In general, three variables accounted for almost two-thirds of the variation (Table
The percentage contribution (%) of selected bioclimatic predictors to estimating the potential distributions and ecological niches of Darevskia lindholmi lineages.
Bioclimatic variables | Code | Central | Common | Southwestern |
Isothermality | bio3 | 8.87 | 10.25 | 12.27 |
Temperature annual range | bio7 | 23.65 | 27.77 | 16.45 |
Mean temperature of wettest quarter | bio8 | 16.26 | 18.67 | 19.67 |
Mean temperature of driest quarter | bio9 | 9.33 | 10.07 | 12.52 |
Mean temperature of coldest quarter | bio11 | 11.55 | 7.20 | 10.86 |
Precipitation of driest month | bio14 | 21.14 | 16.06 | 13.17 |
Precipitation seasonality | bio15 | 9.21 | 9.98 | 15.06 |
The uncorrected p-distance and time of divergence of the Central lineage (Tab.
We would like to express sincere gratitude to the following persons for the assistance during the field works, the providing of photos, maps, and other materials (alphabetically): Vitaly Giragosov, Roman Gorelov, Marina Khramova, Marina Khrisanova, Yuliya Krasylenko, Spartak Litvinchuk, Konstantin Milto, Anton Nadolnyi, Pavel Oksinenko, Ekaterina Polyakova, Elena Sviridenko, Oksana Tishchenko, Sergey Tokarev, Aleksandr Trofimov, Ilya Turbanov, and Larisa Znamenskaya. We also thank to Riley Tedrow, for valuable feedback and improving the English of the manuscript at its final stages. We thanks the anonymous reviewers for their attention to our work and criticism, which contributed to the improvement of the manuscript quality. The work of O.K. and I.D. was carried out within the framework of the State assignments Nos 121032300023-7 and АААА-А19-119020590095-9, respectively. O.K. and O.E. conducted research at personal expenses without involving additional sources of funding. The participation of I.D. was funded from the research grant No. 19-04-00514 of the Russian Foundation of Basic Research. The work of D.J. was supported by the Slovak Research and Development Agency under contract No. APVV-19-0076.
Figures S1–S6
Data type: .pdf
Explanation note: Supplementary illustrative materials.
Tables S1–S3
Data type: .xls
Explanation note: Table S1. A list of Darevskia tissue samples and cytochrome b sequences used in analysis. — Table S2. GenBank accession numbers of cytochrome b sequences used for the divergence time estimates and chronogram construction. — Table S3. Pearson correlation table shows high correlated variables (│r│> 0.70)