Next Article in Journal
Beyond the Spectrum: Unleashing the Potential of Infrared Radiation in Poultry Industry Advancements
Previous Article in Journal
Surveillance of Antimicrobial Resistance of Escherichia coli Isolates from Intestinal Contents of Dairy and Veal Calves in the Veneto Region, Northeaster Italy
Previous Article in Special Issue
A Cryptic Subterranean Mammal Species, the Lesser Blind Mole Rat (Nannospalax leucodon syrmiensis)—Retreated but Not Extinct
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Animals
Volume 14
Issue 10
10.3390/ani14101430
animals-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Phylogeographic Substructuring in the Southernmost Refugium of the European Common Frog Rana temporaria

by
Marija Ilić
1,
Vanja Bugarski-Stanojević
2,†,
Bogdan Jovanović
3,
Gorana Stamenković
2,
Katarina Zorić
1,
Momir Paunović
1 and
Jelka Crnobrnja-Isailović
3,4,*,†
1
Department of Hydroecology and Water Protection, Institute for Biological Research “Siniša Stanković”, National Institute of Republic of Serbia, University of Belgrade, 11108 Belgrade, Serbia
2
Department of Genetic Research, Institute for Biological Research “Siniša Stanković”, National Institute of Republic of Serbia, University of Belgrade, 11108 Belgrade, Serbia
3
Department of Evolutionary Biology, Institute for Biological Research “Siniša Stanković”, National Institute of Republic of Serbia, University of Belgrade, 11108 Belgrade, Serbia
4
Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niš, 18000 Niš, Serbia
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Animals 2024, 14(10), 1430; https://doi.org/10.3390/ani14101430
Submission received: 8 March 2024 / Revised: 25 April 2024 / Accepted: 1 May 2024 / Published: 10 May 2024
(This article belongs to the Special Issue Wildlife Genetic Diversity)
Browse Figures
Versions Notes

Abstract

:

Simple Summary

Rana temporaria is one of the most widespread Palearctic brown frogs, with two main genetic clades in Europe, geographic spatial pattern of which is insufficiently known. We analyzed samples from the understudied western and central Balkans to evaluate the haplotype diversity of widely used 16SrRNA and MT-CYTB mitochondrial gene sequences and to recognize the contour of a possible contact zone between the main clades. The results revealed a suture zone between the Western and Eastern Clades in the central part of the Balkan Peninsula. Overall, haplotype diversity in the western and central Balkans sample is high. Harboring both main genetic clades of R. temporaria qualifies the Balkan Peninsula as another important center of species’ genetic diversity, as well as rich in unique haplotypes.

Abstract

Rana temporaria is one of the most widespread Palearctic brown frogs. We aimed to clarify distribution pattern of two main genetic clades in the understudied Balkan peninsula by using 16SrRNA and MT-CYTB sequences, already widely applied in analyses of populations from other parts of Europe, while focusing on the broad area along the Morava river (central Balkans) as a known gap in the species distribution. Additionally, we were interested in revealing the extent of haplotype diversity within the main genetic clades in the Balkans, particularly around the supposed suture zone. The results revealed a suture zone between the Western and Eastern Clades in the central part of the Balkan Peninsula. This indicated the existence of a historical barrier between the Balkan Mountain Belt and geographically close mountains surrounding the Vlasina Plateau (Rhodope/Serbian–Macedonian Massif). The overall observed haplotype diversity in populations of R. temporaria from the Balkan Peninsula seems high. Harboring both main genetic clades of R. temporaria qualifies the Balkan Peninsula as another important center of species’ genetic diversity, as well as rich in unique haplotypes. This points out the necessity of applying conservation measures focused on the common European frog populations and habitats in this part of the species’ distribution area.

1. Introduction

Rana temporaria (“the common frog” or “the European Common frog”) is one of the most widespread and abundant amphibians in Europe [1], except in the southern parts of the continent, where its distribution is apparently fragmented [2]. It spreads from northern Spain, through western, north, and central Europe, to eastern Europe and further eastward to northern Asia [3]. In the South of Europe, it also inhabits northern and central parts of Italian Peninsula and the higher mountain parts of the Balkan Peninsula [1,2,4]. Some European populations are recorded at very high elevations (up to 2800 m) in the Alps and Pyrenees [5,6,7]. The common European frog has the highest genetic variation of all western Palearctic brown frogs [8]. Although designated as “Least Concern” [3], it could be one of the most fragile anurans in southern Europe, regarding the impact of projected climate change [9,10].
European brown frogs’ lineage separated from their central Asian ancestor about 22.2 million years (Mya later in the text) ago, in the Early Miocene [11]. Radiation over Europe and speciation were later influenced by climatic oscillations between warmer and colder periods, particularly in the Pleistocene. Cold periods would initiate retreat in refugia, followed by genetic divergence as a consequence of spatiotemporal isolation, while, during warmer periods, species would expand their area. The major refugial regions for European flora and fauna during the time of Pleistocene were the three southern ones (the Iberian, Apennine, and Balkan peninsulas) and one in southwestern Russia [12]. Somewhere at the boundary between the Early and Late Pliocene, about 3 Mya, the R. temporaria species group split into five lineages, one of them being R. temporaria (including the recently proclaimed species R. parvipalmata) [6].
The first molecular genetic study on R. temporaria was undertaken by Pidancier et al. [13], using the cytochrome b mitochondrial (mt later in the text) desoxyribonucleic acid (DNA later in the text) sequence (MT-CYTB); two distinct haplotype groups were recorded, based on samples from northern Spain, the United Kingdom, France, northern Italy, coastal parts of Slovenia and Croatia, and Montenegro (Western Clade), as well as those from southeastern Serbia, Romania, Germany, Denmark, the Scandinavian Peninsula, and Russia (Eastern Clade). The results suggested that the postglacial recolonization of Europe would have occurred from two major lineages: one westward from Italy and one eastward from the Balkans, with a current suture zone between them localized somewhere between Germany and the Balkans.
Analysis of R. temporaria MT-CYTB data [14] further confirmed the existence of two distinct clades, which split some 700 thousand years (kya later in the text) ago at the onset of the Ice Age. The eastern European and Fennoscandian populations were grouped into the Eastern Clade, while the Western Clade consisted of populations from Britain, Germany, Switzerland, and Spain. Palo et al. [14] assumed that populations from eastern Europe spread from one main glacial refugium because of their genetic uniformity, while western populations might have had more ‘cryptic’ refugia in central Europe. Schmeller et al. [15] recognized the secondary contact zone of two main clades between the rivers Weser and Elbe in northern Germany. Teacher et al. [16] further contoured the western border of the Eastern Clade through northeast Spain, southeast France, northern Italy, Switzerland, and Austria, with some overlaps between two groups detected in France, Spain, and Switzerland. The authors supposed that the refugia of the Eastern Clade were probably settled in the Apennine or Balkan peninsulas, with routes of expansion directed north, into eastern Europe, and west, along the Mediterranean coast. Later, Stefani et al. [17] questioned the existence of Eastern Clade haplotypes in Italy and on the Iberian Peninsula and considered the entire Apennine Peninsula as a refugium of the Western Clade; meanwhile, Van Rensburg et al. [18] further studied genetic differentiation within the most divergent subspecies, R. t. parvipalmata, and found low differentiation among all analyzed individuals. This taxon was recently proclaimed as a separate species—R. parvipalmata [19]. The Iberian Peninsula was then indicated as the area of geographic origin of R. temporaria [20]. From there, the species could spread, early on, into most of Europe, where its populations persisted in multiple refugia. Later, it was concluded that R. temporaria recolonized western Europe almost exclusively from multiple refugia in the Italian Alps [10]. Recently, the hypothesis that the part of the secondary contact zone between two main clades occurs in Switzerland was rejected, and it was suggested that it is positioned more to the east [21].
Genetic identification of R. temporaria from the Balkan Peninsula has been partially carried out in these comprehensive phylogeographic studies. Despite the patchy distribution of this species in the Balkans and, therefore, its “insular” population organization [22], no unique haplotypes were detected in this geographic area ([20] and references therein). There is a huge distribution gap in the Balkan part of Serbia and in North Macedonia from North to South, i.e., along the Morava and later the Vardar river valleys, as well as along their tributaries; in the Pannonian part of Serbia (Vojvodina Autonomous Province), the European Common frog has been recorded only in the hills of the east–southeast region [23,24]. Results of previous studies suggested that the supposed suture zone between the Western and Eastern Clades in the Balkans could follow the direction of the Morava and Vardar river valleys. Therefore, our aim here is to clarify the distribution patterns of the common European frog’s main clades in the Balkan Peninsula, focusing mainly on the broad area around the Morava river disjunction. To this end, we compare the nucleotide polymorphism of two mitochondrial gene sequences (MT-CYTB and 16S ribosomal ribonucleic acid—16SrRNA later in the text) from our sample and a set of sequences imported from GenBank from a wide European distribution area. Here, we also analyze the extent of haplotype diversity within the main clades of this species in the Balkans—predominantly around supposed suture zone—compared to the rest of Europe.

2. Materials and Methods

We analyzed the amount of nucleotide polymorphism in two mitochondrial gene sequences—16SrRNA and MT-CYTB—which have been broadly studied in this genus [20,21]. These gene sequences are functionally different and could have experienced differential mutational rates and selective constraints during evolution. 16SrRNA gene sequences have moderately well-conserved secondary structures among distantly related taxa [25] and are usually applied for distinguishing between well-resolved species and establishing relations between genera [26].

2.1. Sample Collection

Overall, 27 tissue samples (eggs or fingertips of adult individuals), originating from 14 different localities (Figure 1 and Figure 2), were collected within two periods: 2013–2017 (territory of the Republic of Serbia), and 1986–2007 (Batrachological collection of the Institute for Biological Research “Siniša Stanković” National Institute of the Republic of Serbia, University of Belgrade—for details, see [27]). All samples are listed in Table 1.

2.2. DNA Extraction, Amplification, and Sequencing

The tissue samples were preserved in 96% ethanol and were used for the extraction of total DNA with the AccuPrep Genomic DNA Extraction kit (Bioneer Corporation, Daejeon, Republic of Korea). The quantity of DNA extracts was examined by NanoPhotometer N60/N50 (Impplen, GmbH) and the quality was visualized by 1% agarose gel electrophoresis. Polymerase chain reactions (PCRs) for the amplification of 16SrRNA mtDNA gene fragments were set using universal primers: 16Sar: 5′-CGCCTGTTTATCAAAAACAT-3′ and 16Sbr: 5′-CCGGTCTGAACTCAGATCACGT-3′ described in [6], and, for MT-CYTB fragments, from [20]: RCytb-F 5′-TTAGTAATAGCCACAGCTTTTGTAGGC-3′ and RCytb-R 5′-AGGGAACGAAGTTTGGAGGTGTGG-3′. The reactions were set in a GeneAmp PCR System 2700 (Applied Biosystems, Waltham, MA, USA). PCR for both genes was performed with 5x Colorless GoTaq® Reaction Buffer (Promega Corporation, Madison, WI, USA), 2.5 mM MgCl2, 0.4 mM of each of the dNTPs, 0.5 µM of each amplimer, 1 U of GoTaq® Hot Start DNA Polymerase (Promega), and 100 ng of genomic DNA, in a final volume of 50 µL. The temperature profiles were as described in [6] and [20] (for 16SrRNA mtDNA and MT-CYTB, respectively). Since universal primers for 16SrRNA primer were used, we paid special attention to maintaining sterile conditions, in order to avoid contamination. Sequences were provided in both directions by a third party (Macrogen, Amsterdam, The Netherlands).

2.3. Phylogenetic Analyses and Haplotype Diversity

All collected sequences were individually inspected using software FinchTV 1.4.0 chromatogram viewer (Geospiza Inc., Seattle, WA, USA), compared and analyzed with BioEdit Ver. 7.2.5 [28]. The occurrence of chimeric sequences and stop codons was examined and compared with available sequences in GenBank using Basic Local Alignment Search Tool (BLAST) analysis. Sequences were then aligned using ClustalW employed in MEGA Ver. X software [29,30]. Genetic diversity parameters (h—the number of haplotypes, Hd—haplotype (gene) diversity, and Pi—nucleotide diversity) were estimated using DNA Sequence Polymorphism Analysis of Large Datasets Version 6 - DnaSP Ver. 6 later in the text [31]. The assessment of evolutionary divergence between different sequence groups was calculated in the MEGA X software using the Kimura 2-parameter model—K2p [32]. The rate variation among sites was modelled with a gamma distribution (shape parameter = 1). Codon positions included were 1st + 2nd + 3rd + Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary divergence rates were calculated between different predominantly “Serbian” haplotypes and, separately, between the overall European sample and the western and central Balkans sample.
Phylogeny was reconstructed for 16SrRNA and MT-CYTB genes separately. For both genes, phylogenetic analysis was carried out using two different methods to confirm the strength of the tree topology: The Maximum Likelihood (ML) in MEGA X and the Bayesian analysis in MrBayes [33]. A best-fit substitution model in aligned sequences was surveyed by JModelTest v.2.1.4 [34]. The trees were created using FigTree Ver. 1.3.1 [35] (accessed on 8 August 2023) and MEGA X. The Bayesian (BI later in the text) analyses originated with random starting trees and were run for 1 × 106 generations, sampling every 100th generation, with the burn-in value set to 500. Combined trees of the various runs created a 50% majority rule consensus tree with the Bayesian posterior probability values of the relevant branches.
The haplotype network is calculated and graphically presented using Phylogenetic Network Software 10.2.0.0. (NETWORK 10.2.0.0. Software later in the text) [35] as a Median Joining Network, consisting of nodes and links (nucleotide differences), which connect the nodes. The nodes are either sequences from the dataset, or median vectors (mv)—a hypothesized, often ancestral, sequence required to connect existing sequences within the network with maximum parsimony. The evolutionary history of 16SrRNA and MT-CYTB gene nucleotide sequences was inferred by using the Maximum Likelihood method and the Hasegawa–Kishino–Yano model [36] and Tamura–Nei model [37], respectively, as proposed by analysis conducted in JModelTest v.2.1.4.

3. Results

3.1. 16SrRNA Gene Nucleotide Sequence Comparison

Sequences from eight sampled tissues were submitted to the GenBank database under accession numbers PP648216–PP648224 (Table 1). The final multiple alignment of 16SrRNA gene was 429 nt long and included 90 sequences: 8 from the field-collected samples and 82 from GenBank, including 19 sequences from our previous publication (Table S1). The total number of sites (excluding sites with gaps/missing data) was 357, and the G + C content was 0.441. There were 331 invariable (monomorphic) sites and 26 variable (polymorphic) sites, out of which 11 were found to be parsimony-informative. The overall number of R. temporaria haplotypes (h) was 21, while, in the western and central Balkans sample, h was 6. Their distribution is presented in Figure 1. The overall haplotype diversity (Hd) was 0.8689 ± 0.022, and the nucleotide diversity (per site) (Pi) was 0.01027 ± 0.00045.
Nucleotide diversity between six predominantly “Serbian” haplotypes (Table 2), calculated using DnaSP, demonstrated the lowest values (shown in bold), i.e., the highest similarity, between h5 (Jagodnja, Lučani, Goč Mt., Kopaonik Mt., and Prokletije Mt.), h19 (Oštrozub Mt. and Vlasina Plateau), and h21 (Šar Mt.) in the Western predominantly “Serbian” Clade. Similarly, haplotype h2 (Đerdap, Grza, Bigar, and Stara Mt.) was the most like h20 (Bela Crkva) in the Eastern “Serbian” Clade (Table 2). The highest nucleotide divergence was inferred between the newly discovered “Serbian” haplotypes—western h21 (Šar Mt.) and eastern h20 (Bela Crkva). Intragroup nucleotide diversity was much higher in the Western predominantly “Serbian” Clade (Pi = 0.00207) than in the Eastern “Serbian” Clade (Pi = 0.00042). Intergroup nucleotide diversity among these two clades (27 sequences) was Pi = 0.00791 and, of the total analyzed common European frog sample without outgroup (88 sequences), Pi = 0.01027.
Evolutionary analyses conducted in MEGA X involved 90 16SrRNA nucleotide sequences and a total of 445 positions in the final dataset. Evolutionary divergences were estimated between all 21 haplotypes generated from the total sample from Serbia and Montenegro and imported from the GenBank. Evolutionary divergence, calculated between the two clades, i.e., the Western Clade (h1, h3–8, h10–19, and h21) and the Eastern Clade (h2, h9, and h20), was 0.0138, and the standard error (SE later in the text) was 0.0051) (Table 3). Correspondingly, comparison between the predominantly “Serbian” haplotypes, i.e., the Western predominantly “Serbian” Clade (h3, h5, h19, and h21) and the Eastern “Serbian” Clade (h2 and h20), produced an almost identical evolutionary divergence of 0.0137 (SE 0.0056).

3.2. 16SrRNA Gene Phylogeny

Both phylogenetic methods produced trees with similar topology. Here, we present ML phylogenetic tree in Figure 3, with support values inferred from both methods. The tree was rooted by the outgroup, phylogenetically closer to R. temporaria than R. iberica and the outgroup, which was phylogenetically more distant, such as B. bufo. ML analysis involved 90 nucleotide sequences and there was a total of 445 positions in the final dataset. The tree with the highest log likelihood (−1258.02) is shown. The evolutionary history was inferred by using the Maximum Likelihood method and the Hasegawa–Kishino–Yano model [36]. A discrete Gamma distribution was used to model the evolutionary rate differences among sites (five categories (+G, parameter = 0.7515)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 40.27% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.
At the base of the ML tree is the Western Clade—a heterogeneous group of haplotypes containing fourteen different haplotypes, four of which were found in the central and western Balkans (Serbia and Montenegro). Most haplotypes of the Western Clade were detected within sample group from Spain. They were divided into two substantially diverged clades: the first was gathered around haplotypes h3 and h8 and the second around haplotype h6. The haplotype h3 was widespread: in addition to Serbia, it was also found in France and Spain. We found it in Serbia at two sampling sites, Zlatibor Mt. and Lučani. This haplotype had only one nucleotide change, related to h5, which was prevalent in western, southwestern, and central Serbia, and northeastern Montenegro (Jagodnja, Lučani, Goč Mt., Kopaonik Mt., and Prokletije Mt.). The haplotype h21 from the Šar Mt. recently separated from h5. In addition to the central and western Balkans, h5 was also detected in Croatia and Italy. The most distant subclade was the one containing haplotype h7, which was most common in western Europe (Ireland, Switzerland, Germany, France, and Belgium). The haplotypes h16 and h17, which were detected in Germany, were directly derived from h7. There was also haplotype h18, from Switzerland, that deviated most strongly from the Western group (five nucleotide changes).
The Eastern Clade comprised only three haplotypes, two of which were found in Serbia. The haplotype h2 from the first subclade was detected in the largest geographical area: from Denmark and Sweden in the northern European region, across Russia, Ukraine, and the Czech Republic in the east, to the central Balkans in the south. In Serbia, it was found in four localities in the eastern/southeastern part of the country: Đerdap, Grza, Bigar, and Stara Mt. A sample from Bela Crkva in southeastern Vojvodina (h20) differed from h2 by only one nucleotide change (same as h9 in Russia). It was placed outside the distributional range reported in the IUCN distributional map for R. temporaria. This haplotype, as well as h6, found in the Spanish subclade, were linked to haplotypes of the Western Clade by hypothetical evolutionary intermediaries, i.e., median vectors.

3.3. MT-CYTB Gene Nucleotide Sequence Comparison

MT-CYTB sequences from sampled tissues were submitted to the GenBank database under accession numbers PP695247–PP695273 (Table 1). The final MT-CYTB gene multiple alignment comprised 114 sequences: 27 from our field-collected samples and 87 from GenBank (Table S2). The total number of sites (excluding sites with gaps/missing data) was 273, and the G + C content was 0.423. There were 248 invariable (monomorphic) sites and 25 variable (polymorphic) sites, out of which 17 were found to be parsimony-informative. The total number of mutations was 27. The overall number of common European frog haplotypes (h) was 28, and, among samples from the western and central Balkans, h = 8. Their distribution is presented in Figure 2. The overall haplotype diversity, Hd, was 0.903 ± 0.017; nucleotide diversity (per site), Pi, was 0.01781 ± 0.00140.
Nucleotide diversity (Pi) among eight predominantly “Serbian” haplotypes (Table 4) demonstrated the highest similarity (lowest values) between h1 (Jagodnja, Zlatibor Mt., Goč Mt., and Oštrozub Mt.), h23 (Lučani), h25 (Vlasina Plateau), h26 (Kopaonik Mt. and Prokletije Mt.), and h28 (Šar Mt.) in the Western predominantly “Serbian” group, as well as between h22 (Grza, Đerdap, Bela Crkva, and Bigar), h24 (Grza), and h27 (Stara Mt.) in the Eastern “Serbian” group. The highest Pi was calculated between h25 (Vlasina Plateau) and h27 (Stara Mt.). Intragroup nucleotide diversity for the Western predominantly “Serbian” Clade and Eastern Serbian Clade was 0.00223 and 0.00197, respectively, while, between those two groups, it was 0.01505 (27 sequences).
Evolutionary analyses conducted in MEGA X involved 114 MT-CYTB nucleotide sequences and a total of 569 positions in the final dataset. Evolutionary divergences were estimated between all 28 haplotypes and between two groups: the Western and Eastern Clade. Evolutionary divergence between the Western Clade (h1–h4, h8–h21, h23, h25, h26, and h28) and Eastern Clade (h5, h22, h24, and h27) was 0.0282 (SE 0.0083). In comparison to this value, two groups of predominantly “Serbian” haplotypes—Western predominantly “Serbian” group (h1, h23, h25, h26, and h28) and Eastern “Serbian” group (h22, h24, and h27)—showed a higher evolutionary divergence of 0.0344 (SE 0.0082) (Table 5).

3.4. MT-CYTB Gene Phylogeny

Both phylogenetic methods produced trees with similar topologies. Here, we present ML phylogenetic tree in Figure 4. ML analysis involved 119 nucleotide sequences, and there was a total of 569 positions in the final dataset. The tree with the highest log likelihood (−1951.63) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbour-Join and BioNJ algorithms to a matrix of estimated pairwise distances.
The evolutionary history was inferred on a dataset that involved 114 nucleotide sequences and a total of 569 positions, by using the Maximum Likelihood method. The tree with the highest log likelihood (−1820.16) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbour-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura–Nei model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories (+G, parameter = 0.9418)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 34.89% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.
As with the previous one, this tree was rooted by the outgroup—R. iberica and B. bufo. In the base of the phylogenetic tree (Figure 4) is the Western Clade, which was much more diversified, composed of 24 different haplotypes. Samples from the western and central Balkans were composed of five haplotypes: four new and one (h1) also detected in Greece. This clade was the most closely related to Croatian haplotypes, as well as those collected in Switzerland. Haplotype h4 was the most frequent in Europe from the Western Clade, found in Ireland, Switzerland, Germany, and Belgium. The Eastern Clade was composed of only four haplotypes, out of which three were found exclusively among Serbian samples. Haplotype h22, found in Serbia, was connected to the most widespread Eastern Clade haplotype (h5) by only two nucleotide changes. It extended from north-eastern Europe to certain localities in Germany and France.

4. Discussion

Our results represent a complement to the previous studies on R. temporaria phylogeography in Europe ([10,18,38] and references therein), by filling the gap in knowledge on phylogenetic relations among populations situated in the less studied area of the Balkan Peninsula. It has been suggested that postglacial recolonization of European fauna would have occurred from three major southern refugial zones [39]; in the case of R. temporaria, the Balkan Peninsula was supposed to be a part of the historical contact zone between Western and Eastern lineages [13]. Indeed, our results confirmed that haplotypes detected in the central Balkans belonged to both main genetic clades of the common European frog; additionally, two unique 16SrRNA haplotypes of the Western Clade, as well as five and two unique MT-CYTB haplotypes of the Western and Eastern Clade, respectively, were detected in the Balkans. What was not completely known previously was the exact position of the suture zone between the two main clades in this part of the species area, and this is partially clarified in this study.
The major genetic distinction was recognized between eastern–southeastern population samples (the Eastern “Serbian” Clade) and the southeastern–southwestern—western ones (the Western predominantly “Serbian” Clade). The border zone between the two was contoured by the Morava/southern Morava river valley, towards the confluence of the Nišava river, and then by the Nišava river valley to the east. Distribution of haplotypes and their relatedness reflect historical routes connecting populations at the western edge of the Rhodope (or Serbian–Macedonian) Massif (for the explanation see [40] and references therein) to those distributed throughout the Scardo-Pindus and Dinaric mountain ranges on the southwest and west of the central Balkans. All these populations belong to the Western Clade. The fact that populations situated in the westernmost part of the Stara (Balkan) Mountains are nested within the Eastern Clade indicates that some historical barrier separated common European frogs of the Balkan Mountain Belt from those inhabiting geographically close mountains around Vlasina Plateau (Rhodope/Serbian–Macedonian Massif). The split of two main lineages could have happened as far back as between 0.215 Mya and 0.71 Mya, as proposed by [18]. These authors supposed that separation of the two main clades may have occurred much earlier than estimated.
The distribution pattern of the two major haplotype groups within R. temporaria remind one, on thinking of spatial genetic structuring, of viviparous populations of another boreal zoogeographic element—the European common lizard Zootoca vivipara [41]. Viviparity in this species evolved during/after the Pleistocene glaciations and rapidly spread among the southeastern populations, somewhere between the Balkan Peninsula and the south of Russia [42]. It is supposed that newly evolved viviparous populations would have (re)colonized northeastern and northwestern countries during interglacial periods (including the Holocene) [43]. Recently, Horreo et al. [44] have shown that (similar to the phylogeographic substructuring of the common European frog), haplotypes of European common lizard populations from eastern Europe (Russia, Belarus, Poland, Ukraine, and Romania), the Baltic countries, Finland, and neighboring parts of Sweden cluster together; the remaining northern, western, and central European populations cluster with those of the western (Montenegro, central Serbia) and central Balkans (Pirin Mt. in western Bulgaria). The authors supposed that dry Mediterranean biota extended in lowlands during the warmer postglacial period of the late Pleistocene, which could have become barriers to the spreading of elements of boreal fauna, including R. temporaria, in certain directions from certain refugia [44].
The presence of unique haplotypes and the structuring of the Western Clade in the area of the central and western Balkan Peninsula suggests the existence of more than two smaller refugia for the common European frog within this large southeastern European refugium. It further supports the view that the influence of different processes shaped the phylogeography of this species in a quite discontinuous landscape and “across a diverse topography” [38]. The distribution of haplotypes presented in this study showed that populations of the Western Clade comprised a higher overall number of unique haplotypes than those belonging to the Eastern Clade. Zeissett and Beebee [45] previously offered an explanation that there was only one cryptic refugium for R. temporaria in the climatically more monotonous eastern part of the continent, in comparison to multiple ones in western Europe. Our study suggests that multiple refugia could also have existed in the western and central Balkans (within the distribution areas of both main clades). However, the possible impact of insufficient sampling on the obtained lower haplotype diversity within the Eastern Clade could not be neglected, as this study detected new haplotypes in the previously unsampled areas of northeastern, eastern, and southeastern Serbia. The detection of additional haplotypes on the Carpathian–Balkan mountain belts could happen in the future, which would contribute to the increase in overall haplotype diversity in the Eastern Clade. Unique haplotypes discovered in the southernmost part of the distribution area of the mainly homogeneous Eastern Clade are important findings because they reflect dynamic geological and/or climatic history of that part of the central Balkans.
In comparison with the overall haplotype diversity of R. temporaria in Europe, a relatively high diversity of the two analyzed mtDNA sequences, especially of MT-CYTB sequence, was detected in the limited territory of the Balkan Peninsula. The detected level of evolutionary divergence in these two gene sequences was similar between the Western predominantly “Serbian” Clade and the Eastern “Serbian” Clade, in comparison to those recorded between Eastern and Western Clades of the entire sample analyzed here. The Pleistocene glaciations were relatively recent historical events, which caused the establishment of many temporary refugial areas in Europe [39]. The difference between the Balkan Peninsula and the rest of Europe, regarding the comparison of the extent of evolutionary divergence between the two main clades of R. temporaria, is quite interesting when a fast-evolving gene sequence (i.e., MT-CYTB) is analyzed: the higher haplotype divergence recorded in the southeast of Europe, in comparison to its central and western parts, indicates the longer and stronger isolation of local populations.
The Balkan Peninsula undoubtedly was an important Pleistocene refugium for European herpetofauna; for a number of analyzed species, populations from the Balkans are genetically closer to western than eastern conspecifics, thus reflecting the possible direction of recolonization routes [46]. Regarding the common European frog, this study revealed a relatively high number of unique haplotypes in populations distributed in the western and central Balkans. These results suggest that the Balkan Peninsula could be qualified as another “hot spot” on the map of haplotype richness of this species. Recognizing and mapping rare and/or unique haplotypes, as well as applying appropriate conservation measures in a timely manner is of utmost importance for the conservation of the species’ genetic diversity in the area of concern. For that purpose, more samples per locality are needed for the appropriate approximation of the population’s genetic structure using microsatellite analysis, which is planned to be undertaken in the future.

5. Conclusions

The Balkan Peninsula was an important Pleistocene refugium for the common European frog. This study recognized that one part of the suture zone between the two main genetic clades in Europe occurs in the central part of the Balkans. Moreover, populations from the western and central Balkans are characterized by a relatively high number of unique haplotypes. Recognizing and mapping rare and/or unique haplotypes, as well as applying appropriate conservation measures in a timely manner is of utmost importance for the conservation of the species’ genetic diversity in the area of concern.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ani14101430/s1. Table S1: imported sequences of Rana temporaria 16SrRNA gene fragment; Table S2: imported sequences of Rana temporaria MT-CYTB gene fragment.

Author Contributions

Conceptualization, M.I. and J.C.-I.; methodology, M.I., V.B.-S. and G.S.; software, V.B.-S.; validation, M.I., V.B.-S. and G.S.; formal analysis, M.I., V.B.-S., G.S. and K.Z.; investigation, B.J. and J.C.-I.; resources, M.I. and J.C.-I.; data curation, M.I.; writing—original draft preparation, M.I.; writing—review and editing, M.I., V.B.-S., B.J., K.Z., G.S., M.P. and J.C.-I.; visualization, V.B.-S.; supervision, M.P.; project administration, M.P.; funding acquisition, M.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Ministry of Science, Technological Development and Innovation of the Republic of Serbia, grant number 451-03-66/2024-03/200007. J.C.-I. was also funded by Ministry of Science, Technological Development and Innovation of the Republic of Serbia, grant number 451-03-65/2024-03/200124.

Institutional Review Board Statement

Collection permits were issued by the Ministry of Energetics, Development and Nature Protection of the Republic of Serbia No. 353-01-312/2014-08 and No. 353-01-42/2014, as well as by the Ministry of Agriculture and Nature Protection of the Republic of Serbia No. 53-01-42/2014, No. 353-01-170/2016-17 and No.353-01- 2666/2016-17.

Informed Consent Statement

Not applicable.

Data Availability Statement

DNA nucleotide sequences were deposited in the NCBI GenBank, and can be assessed at https://www.ncbi.nlm.nih.gov/genbank, accessed on 5 March 2024.

Acknowledgments

The authors are grateful to the Institute for Biological Research “Siniša Stanković”, National Institute of the Republic of Serbia, University of Belgrade, for permission to use samples from the batrachological collection and to Milena Cvijanović for assistance.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Speybroeck, J.; Beukema, W.; Bok, B.; Van Der Voort, J.; Velikov, I. Field Guide to the Amphibians and Reptiles of Britain and Europe; Bloomsbury Publishing: London, UK, 2016; pp. 166–169. [Google Scholar]
  2. Sillero, N.; Campos, J.; Bonardi, A.; Corti, C.; Creemers, R.; Crochet, P.-A.; Crnobrnja-Isailović, J.; Denoël, M.; Ficetola, G.F.; Gonçalves, J.; et al. Updated distribution and biogeography of amphibians and reptiles of Europe. Amphib.-Reptil. 2014, 35, 1–31. [Google Scholar] [CrossRef]
  3. IUCN SSC Amphibian Specialist Group. Rana Temporaria. The IUCN Red List of Threatened Species 2022; IUCN SSC Amphibian Specialist Group: London, UK, 2022; p. e.T173101508A89705642. [Google Scholar] [CrossRef]
  4. Di Toro, F.; Minuti, G.; Coppari, L.; De Albentiis, M.; Laghi, P.; Scaravelli, D.; Ricciardi, V.; Bruni, G. Rana temporaria on Monti della Laga (Central Italy): Isolated population or wide distribution? First record in Abruzzo and Marche. Acta Herpetol. 2021, 16, 45–51. [Google Scholar] [CrossRef]
  5. Vences, M.; Grossenbacher, K.; Puente, M.; Palanca, A.; Vieites, D.R. The Cambalès fairy tale: Elevational limits of Rana temporaria (Amphibia: Ranidae) and other European amphibians revisited. Folia Zool. 2003, 52, 189–202. [Google Scholar]
  6. Veith, M.; Kosuch, J.; Vences, M. Climatic oscillations triggered post-Messinian speciation of Western Palearctic brown frogs (Amphibia, Ranidae). Mol. Phylogenetics Evol. 2003, 26, 310–327. [Google Scholar] [CrossRef] [PubMed]
  7. Tiberti, R.; von Hardenberg, A. Impact of introduced fish on Common frog (Rana temporaria) close to its altitudinal limit in alpine lakes. Amphib.-Reptil. 2012, 33, 303–307. [Google Scholar] [CrossRef]
  8. Reh, W.; Seitz, A. The influence of land-use on the genetic structure of populations of the common frog Rana temporaria. Biol. Conserv. 1990, 54, 239–249. [Google Scholar] [CrossRef]
  9. Maiorano, L.; Amori, G.; Capula, M.; Falcucci, A.; Masi, M.; Montemaggiori, A.; Pottier, J.; Psomas, A.; Rondinini, C.; Russo, D.; et al. Threats from climate change to terrestrial vertebrate hotspots in Europe. PLoS ONE 2013, 8, e74989. [Google Scholar] [CrossRef]
  10. Marchesini, A.; Ficetola, G.F.; Corneti, L.; Battisti, A.; Vernesi, C. Fine-scale phylogeography of Rana temporaria (Anura: Ranidae) in a putative secondary contact zone in the southern Alps. Biol. J. Linn. Soc. 2017, 122, 824–837. [Google Scholar] [CrossRef]
  11. Najibzadeh, M.; Veith, M.; Gharzi, A.; Rastegar-Pouyani, N.; Rastegar-Pouyani, E.; Kieren, S.; Pesarakloo, A. Molecular phylogenetic relationships among Anatolian-Hyrcanian brown frog taxa (Ranidae: Rana). Amphib.-Reptil. 2017, 38, 339–350. [Google Scholar] [CrossRef]
  12. Hewitt, G.M. Speciation, hybrid zones and phylogeography- or seeing genes in space and time. Mol. Ecol. 2001, 10, 537–549. [Google Scholar] [CrossRef]
  13. Pidancier, N.; Miaud, C.; Taberlet, P. Premiers résultats sur la biogéographie de la Grenouille rousse Rana temporaria (Amphibiens, Anoures). Bull. Soc. Herpetol. Fr. 2001, 107, 27–34. [Google Scholar]
  14. Palo, J.U.; Schmeller, D.S.; Laurila, A.; Primmer, C.R.; Kuzmin, S.L.; Merilä, J. High degree of population subdivision in a widespread amphibian. Mol. Ecol. 2004, 13, 2631–2644. [Google Scholar] [CrossRef] [PubMed]
  15. Schmeller, D.S.; Palo, J.U.; Merillä, J. A contact zone between two distinct Rana temporaria lineages in Northern Germany. Alytes 2008, 25, 93–98. [Google Scholar]
  16. Teacher, A.G.F.; Garner, T.W.J.; Nichols, R.A. European phylogeography of the common frog (Rana temporaria): Routes of postglacial colonization into the British Isles, and evidence for an Irish glacial refugium. Heredity 2009, 102, 490–496. [Google Scholar] [CrossRef] [PubMed]
  17. Stefani, F.; Gentilli, A.; Sacchi, R.; Razzetti, E.; Pellitteri-Rosa, D.; Pupin, F.; Galli, P. Refugia within refugia as a key to disentangle the genetic pattern of a highly variable species: The case of Rana temporaria Linnaeus, 1758 (Anura, Ranidae). Mol. Phylogenetics Evol. 2012, 65, 718–726. [Google Scholar] [CrossRef] [PubMed]
  18. Van Rensburg, A.J.; Robin, M.; Phillips, B.; Van Buskirk, J. European common frog (Rana temporaria) recolonized Switzerland from multiple glacial refugia in northern Italy via trans-and circum-Alpine routes. Ecol. Evol. 2021, 11, 15984–15994. [Google Scholar] [CrossRef] [PubMed]
  19. Galán, P.; Ludewig, A.-K.; Kmiec, J.; Hauswaldt, S.; Cabana, M.; Ferreiro, R.; Vences, M. Low mitochondrial divergence of rediscovered southern relict populations of Rana temporaria parvipalmata in Spain. Amphib.-Reptil. 2010, 31, 144–148. [Google Scholar]
  20. Dufresnes, C.; Nicieza, A.G.; Litvinchuk, S.N.; Rodrigues, N.; Jeffries, D.L.; Vences, M.; Perrin, N.; Martínez-Solano, I. Are glacial refugia hotspots of speciation and cytonuclear discordances? Answers from the genomic phylogeography of Spanish common frogs. Mol. Ecol. 2020, 29, 986–1000. [Google Scholar] [CrossRef]
  21. Vences, M.; Hauswaldt, J.S.; Steinfartz, S.; Rupp, O.; Goesmann, A.; Künzel, S.; Wengel, P.O.; Vieites, D.R.; Nieto-Roman, S.; Haas, S.; et al. Radically different phylogeographies and patterns of genetic variation in two European brown frogs, genus Rana. Mol. Phylogenetics Evol. 2013, 68, 657–670. [Google Scholar] [CrossRef]
  22. Wright, S. Isolation by distance. Genetics 1943, 28, 114–138. [Google Scholar] [CrossRef]
  23. Vukov, T.; Kalezić, M.L.; Tomović, L.J.; Krizmanić, I.; Jović, D.; Labus, N.; Džukić, G. Amphibians in Serbia—Distribution and diversity patterns. Bull. Nat. Hist. Mus. Belgrade 2013, 6, 90–112. [Google Scholar] [CrossRef]
  24. Urošević, A.; Tomović, L.J.; Krizmanić, I.; Anđelković, M.; Golubović, A.; Maričić, M.; Ajtić, R.; Ćorović, J.; Čubrić, T.; Tomašević-Kolarov, N.; et al. Distribution and Diversity of Brown Frogs (Rana spp., Anura, Amphibia) in Serbia. Bull. Nat. Hist. Mus. Belgrade 2018, 11, 227–245. [Google Scholar] [CrossRef]
  25. Caetano-Anollés, G. Tracing the evolution of RNA structure in ribosomes. Nucleic Acids Res. 2002, 30, 2575–2587. [Google Scholar] [CrossRef] [PubMed]
  26. Fox, G.E.; Wisotzkey, J.D.; Jurtshuk, P., Jr. How Close Is Close: 16s RNA Sequence identity may not be sufficient to guarantee species identity. Int. J. Syst. Evol. Microbiol. 1992, 42, 166–170. [Google Scholar] [CrossRef] [PubMed]
  27. Džukić, G.; Cvijanović, M.; Urošević, A.; Vukov, T.; Tomašević Kolarov, N.; Ajduković, M.; Ivanović, A.; Kalezić, M. The Batrachological Collection of the Institute for Biological Research” Siniša Stanković”, University of Belgrade. Bull. Nat. Hist. Mus. 2015, 8, 118–167. [Google Scholar]
  28. Hall, T.A. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 1999, 41, 95–98. [Google Scholar]
  29. Thompson, J.D.; Higgins, D.G.; Gibson, T.J. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22, 4673–4680. [Google Scholar] [CrossRef] [PubMed]
  30. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef] [PubMed]
  31. Rozas, J.; Ferrer-Mata, A.; Sanchez-DelBarrio, J.C.; Guirao-Rico, S.; Librado, P.; Ramos-Onsins, S.E.; Sánchez-Gracia, A. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol. Biol. Evol. 2017, 34, 3299–3302. [Google Scholar] [CrossRef]
  32. Kimura, M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 1980, 16, 111–120. [Google Scholar] [CrossRef]
  33. Ronquist, F.; Huelsenbeck, J.P. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 2003, 19, 1572–1574. [Google Scholar] [CrossRef] [PubMed]
  34. Darriba, D.; Taboada, G.L.; Doallo, R.; Posada, D. JModelTest 2: More models, new heuristics and parallel computing. Nat. Methods 2012, 9, 772. [Google Scholar] [CrossRef] [PubMed]
  35. Bandelt, H.-J.; Forster, P.; Röhl, A. Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol. 1999, 16, 37–48. [Google Scholar] [CrossRef] [PubMed]
  36. Hasegawa, M.; Kishino, H.; Yano, T. Dating the human-ape split by a molecular clock of mitochondrial DNA. J. Mol. Evol. 1985, 22, 160–174. [Google Scholar] [CrossRef] [PubMed]
  37. Tamura, K.; Nei, M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 1993, 10, 512–526. [Google Scholar] [PubMed]
  38. Vences, M.; Sarasola-Puente, V.; Sanchez, E.; Amat, F.; Hauswaldt, S.J. Diversity and distribution of deep mitochondrial lineages of the common frog, Rana temporaria, in northern Spain. Salamandra 2017, 53, 25–33. [Google Scholar]
  39. Veiss, S.; Ferrand, N. Current perspectives in phylogeography and the significance of South European refugia in the creation and maintenance of European biodiversity. In Phylogeography of Southern European Refugia; Weiss, S., Ferrand, N., Eds.; Springer: Dordrecht, The Netherlands, 2007; pp. 341–357. [Google Scholar]
  40. Crnobrnja-Isailović, J.; Dinov, J.; Isailović, O.; Ranđelović, V. Westernmost record of Zootoca vivipara (Lichtenstein, 1823), in the Rhodope Massif, Serbia. Herpetozoa 2015, 27, 162–165. [Google Scholar]
  41. Dely, O.G.; Böhme, W. Lacerta vivipara Jacquin 1787—Waldeidechse. In Handbuch der Reptilien und Amphibien Europas; Böhme, W., Ed.; Aula: Wiesbaden, Germany, 1984; Volume 2/I, pp. 362–393. [Google Scholar]
  42. Surget Groba, Y.; Heulin, B.; Guillaume, C.-P.; Thorpe, R.S.; Kyprianova, L.; Vogrin, N.; Maslak, R.; Mazzoti, S.; Venczel, M.; Ghira, I.; et al. Intraspecific phylogeography of Lacerta vivipara and the evolution of viviparity. Mol. Phylogenetics Evol. 2001, 18, 449–459. [Google Scholar] [CrossRef] [PubMed]
  43. Heulin, B.; Guillaume, C.P.; Bea, A.; Arrayago, M.J. Interpretation biogeographique de la bimodalite’ de reproduction du lezard Lacerta vivipara: Un modele pour l’etude de l’evolution de la viviparite. Biogeographica 1993, 69, 1–11. [Google Scholar]
  44. Horreo, J.L.; Pelaez, M.L.; Suarez, T.; Breedveld, M.C.; Heulin, B.; Surget-Groba, Y.; Tuula, A.; Oksanen, T.A.; Fitze, P.S. Phylogeography, evolutionary history and effects of glaciations in a species (Zootoca vivipara) inhabiting multiple biogeographic regions. J. Biogeogr. 2018, 45, 1616–1627. [Google Scholar] [CrossRef]
  45. Zeissett, I.; Beebee, T.J.C. Amphibian phylogeography: A model for understanding historical aspects of species distributions. Heredity 2008, 101, 109–119. [Google Scholar] [CrossRef] [PubMed]
  46. Crnobrnja-Isailović, J. Cross-section of a refugium: Genetic diversity of amphibian and reptile populations in the Balkans. In Phylogeography of Southern European Refugia; Weiss, S., Ferrand, N., Eds.; Springer: Dordrecht, The Netherlands, 2007; pp. 327–337. [Google Scholar]
Animals 14 01430 g001
Figure 1. Distribution of 21 haplotypes of R. temporaria generated from 16SrRNA dataset, including those imported from the GenBank. Left: European haplotypes involved in this study, with the most widespread highlighted, namely blue/green triangles h3, h5, and h7 (Western Clade) and red triangles h2 (Eastern Clade). Right: haplotypes and sampling localities in the western and central Balkans (predominantly in Serbia). “Western predominantly Serbian Clade” is represented with green/blue triangle symbols, and the “Eastern Serbian Clade” with red. (Source of the map: IUCN (International Union for Conservation of Nature) and Conservation International 2021. Rana temporaria. The IUCN Red List of Threatened Species. Version 2023-1. Accessed on 23 April 2024.).
Figure 1. Distribution of 21 haplotypes of R. temporaria generated from 16SrRNA dataset, including those imported from the GenBank. Left: European haplotypes involved in this study, with the most widespread highlighted, namely blue/green triangles h3, h5, and h7 (Western Clade) and red triangles h2 (Eastern Clade). Right: haplotypes and sampling localities in the western and central Balkans (predominantly in Serbia). “Western predominantly Serbian Clade” is represented with green/blue triangle symbols, and the “Eastern Serbian Clade” with red. (Source of the map: IUCN (International Union for Conservation of Nature) and Conservation International 2021. Rana temporaria. The IUCN Red List of Threatened Species. Version 2023-1. Accessed on 23 April 2024.).
Animals 14 01430 g001
Animals 14 01430 g002
Figure 2. Distribution of 28 haplotypes of R. temporaria generated from MT-CYTB dataset, including those imported from the GenBank. Left: European haplotypes included in this study, with the most widespread highlighted, namely h4 - yellow triangles (Western Clade) and h5 – violet triangles (Eastern Clade). Right: haplotypes and sampling localities predominantly from Serbia, newly sampled in this study. “Western predominantly Serbian Clade” is represented with green symbols, and the “Eastern Serbian Clade” with red. (Source of the map: IUCN (International Union for Conservation of Nature) and Conservation International 2021. Rana temporaria. The IUCN Red List of Threatened Species. Version 2023-1. Accessed on 23 April 2024.).
Figure 2. Distribution of 28 haplotypes of R. temporaria generated from MT-CYTB dataset, including those imported from the GenBank. Left: European haplotypes included in this study, with the most widespread highlighted, namely h4 - yellow triangles (Western Clade) and h5 – violet triangles (Eastern Clade). Right: haplotypes and sampling localities predominantly from Serbia, newly sampled in this study. “Western predominantly Serbian Clade” is represented with green symbols, and the “Eastern Serbian Clade” with red. (Source of the map: IUCN (International Union for Conservation of Nature) and Conservation International 2021. Rana temporaria. The IUCN Red List of Threatened Species. Version 2023-1. Accessed on 23 April 2024.).
Animals 14 01430 g002
Animals 14 01430 g003
Figure 3. Phylogenetic relationship inferred from 16SrRNA nucleotide comparison, presented with two trees. (A): Median Joining Network tree represents connections among 21 haplotypes of R. temporaria from 16SrRNA dataset. Node size corresponds to a sample number of a specific haplotype; mv—a hypothesized, often ancestral, sequence required to connect existing sequences within the network with maximum parsimony. Transverse lines represent one nucleotide change. (B): ML phylogenetic tree. Some sample sequences belonging to the same haplotype have been condensed into haplotype groups for better visibility, and their color corresponds to the color of the nodes in the MJ network tree. Support values of both methods (ML and BI) are placed at the nodes in that order. The dash indicates branch support <50. Red square—Eastern Clade; green square—Western Clade.
Figure 3. Phylogenetic relationship inferred from 16SrRNA nucleotide comparison, presented with two trees. (A): Median Joining Network tree represents connections among 21 haplotypes of R. temporaria from 16SrRNA dataset. Node size corresponds to a sample number of a specific haplotype; mv—a hypothesized, often ancestral, sequence required to connect existing sequences within the network with maximum parsimony. Transverse lines represent one nucleotide change. (B): ML phylogenetic tree. Some sample sequences belonging to the same haplotype have been condensed into haplotype groups for better visibility, and their color corresponds to the color of the nodes in the MJ network tree. Support values of both methods (ML and BI) are placed at the nodes in that order. The dash indicates branch support <50. Red square—Eastern Clade; green square—Western Clade.
Animals 14 01430 g003
Animals 14 01430 g004
Figure 4. Phylogenetic trees inferred from MT-CYTB nucleotide comparison. (A): Median Network tree generated using NETWORK software represents connections among all 28 haplotypes from MT-CYTB dataset. Node size corresponds to a sample number of a specific haplotype; mv—a hypothesized, often ancestral, sequence required to connect existing sequences within the network with maximum parsimony. Transverse lines represent one nucleotide change. (B): ML phylogenetic tree with bootstrap values presented beside the nodes. Some sample sequences belonging to the same haplotype have been condensed into haplotype groups for better visibility and their color corresponds to the color of the nodes in the MJ network tree. Support values of both methods (ML and BI) are placed at the nodes in that order. The dash indicates branch support <50. Red square—Eastern Clade; green square—Western Clade.
Figure 4. Phylogenetic trees inferred from MT-CYTB nucleotide comparison. (A): Median Network tree generated using NETWORK software represents connections among all 28 haplotypes from MT-CYTB dataset. Node size corresponds to a sample number of a specific haplotype; mv—a hypothesized, often ancestral, sequence required to connect existing sequences within the network with maximum parsimony. Transverse lines represent one nucleotide change. (B): ML phylogenetic tree with bootstrap values presented beside the nodes. Some sample sequences belonging to the same haplotype have been condensed into haplotype groups for better visibility and their color corresponds to the color of the nodes in the MJ network tree. Support values of both methods (ML and BI) are placed at the nodes in that order. The dash indicates branch support <50. Red square—Eastern Clade; green square—Western Clade.
Animals 14 01430 g004
Table 1. List of samples with names of geographic localities and information on type of sequence analyzed with the GenBank accession numbers and a haplotype. (SRB—Serbia; MNE—Montenegro).
Table 1. List of samples with names of geographic localities and information on type of sequence analyzed with the GenBank accession numbers and a haplotype. (SRB—Serbia; MNE—Montenegro).
Laboratory No.16SrRNA
GenBank No./Haplotype		MT-CYTB GenBank No./HaplotypeSampling LocalityCoordinates
1RT_3_M_RS/PP695247/h22Bigar, SRB44.2238, 21.8722
2RT_6_M_RS/PP695248/h22Bigar, SRB44.2238, 21.8722
3RT_7_M_RS/PP695249/h22Bigar, SRB44.2238, 21.8722
4RT_1-20_RS/PP695250/h1Lučani, SRB43.8600, 20.1689
5RT_M14_RS/PP695251/h22Đerdap, SRB44.3935, 22.1743
6RT_M28_RS/PP695252/h23Lučani, SRB43.8600, 20.1689
7RT_M29_RS/PP695253/h23Lučani, SRB43.8600, 20.1689
8RT_M30_RS/PP695254/h1Lučani, SRB43.8600, 20.1689
9RT_M31_RS/PP695255/h22Grza, SRB44.8964, 21.6459
10RT_M32_RS/PP695256/h22Grza, SRB44.8964, 21.6459
11RT_M33_RS/PP695257/h22Grza, SRB44.8964, 21.6459
12RT_M43_RS/PP695258/h24Grza, SRB44.8964, 21.6459
13RT_M44_RS/PP695259/h24Grza, SRB44.8964, 21.6459
14RT_M45_RS/PP695260/h24Grza, SRB44.8964, 21.6459
15RT_M46_RS/PP695261/h1Lučani, SRB43.8600, 20.1689
16RT_M47_RS/PP695262/h1Lučani, SRB43.8600, 20.1689
17RT_M48_RS/PP695263/h1Lučani, SRB43.8600, 20.1689
18RT_M74_RS/PP695264/h25Vlasina,
Cvetkova reka, SRB		42.7526, 22.3263
19RT_M91_RSPP648216/h5PP695265/h26Mt. Kopaonik,
Jablanova Ravan, SRB		43.2909, 20.7844
20RT_M92_RSPP648217/h2PP695266/h27Mt. Stara Planina,
Kopren, SRB		43.3057, 22.8162
21RT_M93_RSPP648218/h20PP695267/h22Bela Crkva,
Jaruga Stream, SRB		44.8732, 21.4127
22RT_M94_RSPP648219/h5PP695268/h1Mt. Goč,
Stanišinci, SRB		43.5279, 20.8954
23RT_M95_RSPP648220/h5PP695269/h1Jagodnja,
Mačkov kamen, SRB		44.3299, 19.2926
24RT_M96_RSPP648221/h21PP695270/h28Mt. Šar planina, SRB42.0957, 20.8257
25RT_M97_RSPP648222/h19PP695271/h1Mt. Oštrozub,
Zeleniče, SRB		42.8673, 22.2235
26RT_M99_RSPP648223/h5PP695272/h26Prokletije, Vusanje, MNE42.5243, 19.8431
27RT_M103_RSPP648224/h3PP695273/h1Zlatibor, SRB43.7003, 19.6714
Table 2. Nucleotide diversity (Pi) calculated from 16SrRNA dataset analysis between each of six predominantly “Serbian” haplotypes (the lowest values are underlined and the highest are presented in red color).
Table 2. Nucleotide diversity (Pi) calculated from 16SrRNA dataset analysis between each of six predominantly “Serbian” haplotypes (the lowest values are underlined and the highest are presented in red color).
h2h3h5h19h20
h30.00598
h50.007820.00124
h190.003870.002220.00086
h200.000830.004660.003760.00932
h210.002480.001550.000540.003110.01399
Table 3. Evolutionary divergences calculated in MEGA X, calculated from 90 16SrRNA nucleotide sequences including outgroup, estimated between the Western and Eastern Clades from the total European sample analyzed here, as well as Western predominantly “Serbian” and Eastern “Serbian” Clades. Standard error estimates are shown above the diagonal.
Table 3. Evolutionary divergences calculated in MEGA X, calculated from 90 16SrRNA nucleotide sequences including outgroup, estimated between the Western and Eastern Clades from the total European sample analyzed here, as well as Western predominantly “Serbian” and Eastern “Serbian” Clades. Standard error estimates are shown above the diagonal.
Western CladeEastern CladeOutgroup
Western Clade 0.00510.0288
Eastern Clade0.0138 0.0298
Outgroup0.20930.2160
Eastern “Serbian” CladeWestern
Predominantly “Serbian” Clade
0.0056
Western Predominantly “Serbian” Clade0.0137
Table 4. Nucleotide diversity calculated from MT-CYTB dataset analysis between each of eight predominantly “Serbian” haplotypes (the lowest values are underlined and the highest are presented in red color).
Table 4. Nucleotide diversity calculated from MT-CYTB dataset analysis between each of eight predominantly “Serbian” haplotypes (the lowest values are underlined and the highest are presented in red color).
h1h22h23h24h25h26h27
h220.01742
h230.001050.01165
h240.012520.001520.01822
h250.001020.007820.004090.01420
h260.001400.013250.004090.019320.00239
h270.006590.001650.020450.002840.030470.02270
h280.001750.008640.006820.016100.007160.004780.03405
Table 5. Evolutionary divergences conducted in MEGA X calculated from 119 MT-CYTB nucleotide sequences, estimated between the Western and Eastern Clades from the total European sample analyzed here, as well as Western predominantly “Serbian” and Eastern “Serbian” Clades. Standard error estimates are shown above the diagonal.
Table 5. Evolutionary divergences conducted in MEGA X calculated from 119 MT-CYTB nucleotide sequences, estimated between the Western and Eastern Clades from the total European sample analyzed here, as well as Western predominantly “Serbian” and Eastern “Serbian” Clades. Standard error estimates are shown above the diagonal.
Western Clade Eastern CladeOutgroup
Western Clade 0.00830.0310
Eastern Clade0.0282 0.0300
Outgroup0.26110.2481
Eastern “Serbian” CladeWestern Predominantly
“Serbian” Clade
0.0082
Western
Predominantly
“Serbian” Clade		0.0344
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Ilić, M.; Bugarski-Stanojević, V.; Jovanović, B.; Stamenković, G.; Zorić, K.; Paunović, M.; Crnobrnja-Isailović, J. Phylogeographic Substructuring in the Southernmost Refugium of the European Common Frog Rana temporaria. Animals 2024, 14, 1430. https://doi.org/10.3390/ani14101430

AMA Style

Ilić M, Bugarski-Stanojević V, Jovanović B, Stamenković G, Zorić K, Paunović M, Crnobrnja-Isailović J. Phylogeographic Substructuring in the Southernmost Refugium of the European Common Frog Rana temporaria. Animals. 2024; 14(10):1430. https://doi.org/10.3390/ani14101430

Chicago/Turabian Style

Ilić, Marija, Vanja Bugarski-Stanojević, Bogdan Jovanović, Gorana Stamenković, Katarina Zorić, Momir Paunović, and Jelka Crnobrnja-Isailović. 2024. "Phylogeographic Substructuring in the Southernmost Refugium of the European Common Frog Rana temporaria" Animals 14, no. 10: 1430. https://doi.org/10.3390/ani14101430

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop