Author Query Form Journal Code: AMRE Transmittal No.: AMRE1442

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6 53 7 Genetic diversity and gene ﬂow decline with elevation in the near 54 8 eastern ﬁre salamander (Salamandra infraimmaculata) at Mount 55 9 ∗ 56 10 Hermon, Golan Heights 57 11 58

12 59 1,∗∗ 2 1 3 4 13 Kathleen Prei§ler , Eliane Küpfer , Fabian Löfﬂer , Arlo Hinckley , Leon Blaustein , 60 14 Sebastian Steinfartz1 61 15 62

16 Abstract. The Near Eastern fire salamander (Salamandra infraimmaculata) reaches its southern distribution range in Israel. 63 Although the population structure has been analysed in central Israel and at the southern distribution limit, we lack knowledge 17 64 on populations in the northern area, such as along Mount Hermon. S. infraimmaculata occurs at Mt. Hermon along an 18 altitudinal gradient and appears to be fragmented by urban and agricultural landscape. We studied the genetic structure of 65 19 four populations based on microsatellite loci and the mitochondrial D-loop to determine the genetic diversity and connectivity 66 between populations. We observed moderate gene flow at lower parts, i.e. from Tel Dan and Nimrod Castle to Banias 20 67 indicating extant but limited connectivity. Genetic diversity and gene flow declined along the altitudinal gradient at Mt. 21 Hermon, reaching rock-bottom levels in the highest located population of Nimrod Pool. The observed isolation-by-elevation 68 22 gradient might induce a higher extinction risk for the highland populations of S. infraimmaculata. 69

23 70 Keywords: D-loop, genetic ﬁtness, Israel, microsatellites, migration barrier. 24 71

25 72 The Near Eastern ﬁre salamander (Salamandra suitable habitat and breeding sites it is classi- 26 73 infraimmaculata) is one of the southernmost oc- 27 ﬁed as “Near Threatened” by IUCN criteria (Pa- 74 curring Salamandra species. Its patchy distribu- 28 penfuss et al., 2009; Bogaerts et al., 2013). The 75 tion encompasses parts of Turkey, Syria, Iran, 29 76 Iraq, Lebanon, and Israel. Due to the loss of species is reaching the southern periphery of its 30 77

31 distribution range in Northern Israel where it 78 32 is restricted to three peripheral population clus- 79 33 1 - Institute for Biology, Molecular Evolution and Sys- ters: Galilee, Mt. Carmel and Mt. Hermon (see 80 34 tematics of Animals, University Leipzig, Talstra§e 33, 81 ﬁg. 1) and is considered endangered (Dolev, 35 04103 Leipzig, Germany 82 2 - Zoological Institute, Evolutionary Biology, Technische 36 Perevolotsky, and Lachman, 2004). Recent ge- 83 Universität Braunschweig, Mendelssohnstra§e 4, 38106 37 84 Braunschweig, Germany netic population studies revealed a restriction of 38 85 3 - Conservation and Evolutionary Genetics Group, connectivity between populations in Galilee and 39 Estación Biológica de Doñana (EBD-CSIC), Avda. 86 40 UNCORRECTEDAmerico Vespucio, 26, 41092 Sevilla, Spain Mt. Carmel, possibly PROOF driven by land use change, 87 41 4 - Department of Evolutionary and Environmental such as settlements and agriculture, and a de- 88 42 Ecology, University of Haifa, Haifa, Israel 89 ∗ crease in genetic diversity towards the southern 43 This contribution is dedicated to Leon Blaustein who 90 passed away on June 23rd, 2020 to remember him as an 44 distribution limit (Blank et al., 2013; Sinai et 91 expert on Israeli ﬁre salamanders. 45 al., 2019). But the dispersal and migration be- 92 ∗∗Corresponding author; 46 e-mail: [email protected] haviour of S. infraimmaculata is also strongly 93 47 94

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20 67 Figure 1. Population structure analysis of four S. infraimmaculata sites located in northern Israel. Map not to scale. (A) 21 Distribution of sampling sites Tel Dan (TD), Banias (Ba), Nimrod Castle (NC) and Nimrod Pool (NP) across area and 68 22 elevation. (B) Median joining network of mitochondrial haplotypes in comparison with individuals from Galilee and Mt. 69 23 Carmel. Hatch marks indicate the number of mutations. (C) Genetic structure assuming K = 2-4 as inferred by STRUCTURE. 70 Bar plot depicts individual proportions of assignment to each cluster. (D) Principal component analysis with the first two 24 71 components displayed. Each dot represents an individual. Populations are color-coded according to STRUCTURE K = 3but 25 note that Banias is here closer to Tel Dan. An inertia ellipse is spanning the individuals of each population. 72 26 73 27 restricted by low elevation and steep slope (Ker- In 2013/2014, We took tail clips from fire 74 28 shenbaum et al., 2014; Sinai et al., 2019). Popu- salamander larvae as described elsewhere 75 29 lations along the southern foothills of Mt. Her- (Segev et al., 2015) from four sites: Tel Dan 76 30 mon, such as the Tel Dan population are sepa- Nature Reserve (“TD”), Banias (“Ba”), Nimrod 77 31 rated from the Galilee by the Hula valley and Castle (“NC”) and Nimrod Pool (“NP”). See ta- 78 32 appear to be fragmented by urban and agricul- ble 1 for detailed sample information. We stored 79 33 tural landscapes. Moreover, populations follow the tissue separately in 1.5 ml Eppendorf tubes 80 34 filled with 96% EtOH at −20¡C until sample 81 an altitudinal gradient along the range of Mt. 35 processing and extracted genomic DNA follow- 82 Hermon. 36 ing a salt extraction protocol. 83 We aim to evaluate the genetic population 37 The mitochondrial D-loop was amplified and 84 structure and connectivity of S. infraimmacu- 38 sequenced using published primer combinations 85 lata populations at Mt. Hermon based on nu- 39 (L-Pro-ML 5 -GGCACCCAAGGCCAAAATT 86 40 clearUNCORRECTED (microsatellite loci) and mitochondrial (D- CT-3; H-12S1-ML PROOF 5-CAAGGCCAGGACCA 87 41 loop) haplotype variation, with a special focus AACCTTTA-3) (Steinfartz, Veith, and Tautz, 88 42 on the effect of elevation. Our data will provide 2000). We checked, edited, and aligned the 89 43 information on the degree of genetic isolation D-loop sequences using CodonCode Aligner 90 44 between populations and its causes, thus being v8.0.2 (CodonCode Corporation, www. 91 45 informative for local conservation of this threat- codoncode.com). For the detailed PCR pro- 92 46 ened species. tocol, see supplementary material. In order 93 47 94 AMRE (brill2x v1.31) amre1442.tex 2020/11/19 14:21 [other] p. 3/7

Elevation limits gene ﬂow 3

1 Table 1. Estimates of genetic diversity parameters for each S. infraimmaculata population: TD-Tel Dan, B-Banias, NC- 48 Nimrod Castle, NP-Nimrod Pool located at different elevations. N: number of individuals analysed per marker system, PA: 2 49 private alleles, Ar: allelic richness, Ho: observed heterozygosity, He: expected heterozygosity, Fis: inbreeding coefﬁcient and 3 95% conﬁdence interval. 50

4 Site TD Ba NC NP 51 5 52 Coordinates Latitude 33¡1453N 33¡1438N 33¡1508N 33¡1456N 6 53 Longitude 35¡3910E 35¡4115E 35¡4245E 35¡4510E 7 54 Elevation [m.a.s.l.] 192 328 743 1022 8 55

9 N D-loop 22 0 15 35 56 10 Msats 25 7 21 46 57 11 A 4642433858 PA 532 7 12 59 Ar 3.14 3.42 2.92 2.55 13 Ho 0.54 0.62 0.51 0.39 60 14 He 0.56 0.62 0.53 0.42 61 F 0.039 −0.001 0.026 0.07 15 is 62

16 Fis 95% CI low −0.069 −0.201 −0.064 0.0056 63 high 0.1368 0.1309 0.1132 0.1309 17 64

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20 to present the full Israeli haplotype network, each sampling site using the R statistics (R Core 67

21 we incorporated S. infraimmaculata sequences Team, 2019) package “diveRsity” (Keenan et 68

22 from Galilee and Mt. Carmel sampled in a sep- al., 2013). We tested for isolation-by-distance 69 23 arate project (see sample information in supple- (IBD) and isolation-by-elevation (IBE) as a 70

24 mentary table S1). The alignment was checked correlation between genetic distance (Fst, pair- 71 25 and exported in MEGA7 (Kumar, Stecher, and wise differences) and geographic distance (eu- 72 26 Tamura, 2016). The median-joining haplotype clidean) or elevational distances (pairwise) us- 73 27 network was created in PopArt v1.7 (Leigh and ing the Mantel test implemented in Arlequin 74 28 Bryant, 2015). v3.5.2.2. Population structure was estimated in 75 29 We used 14 polymorphic microsatellite loci STRUCTURE v2.3.4 (Pritchard, Stephens, and 76 30 (Sal E8, Sal E7, Sal E6, Sal 3, Sal E14, Donnelly, 2000) by running an admixture model 77 31 Sal E11, G6, SST-A6-I, SST-A6-II, SST-E11, with LOCPRIOR assuming correlated allele fre- 78 32 SST-B11, SST-C3, SST-C2, SST-F10) as de- quencies for 10 iterations of K = 1-5 with a 79 33 scribed in Steinfartz et al. (2004) and Hen- burn-in of 100,000 and an MCMC of 500,000. 80 34 drix et al. (2010) to infer nuclear-based popu- STRUCTURE HARVESTER (Earl and vonHoldt, 81 35 lation structure. For the detailed PCR proto- 2012) identified the best fitting K to explain 82 36 col, see supplementary material. We genotyped the population structure based on Delta K and 83 37 the microsatellite loci with GeneMarker v3.0.0 Mean LnP (K) (Pritchard, Stephens, and Don- 84 38 (SoftGenetics¨). We discarded individuals with nelly, 2000; Evanno, Regnaut, and Goudet, 85 39 more than 15% missing data and loci with more 2005). We used CLUMPP v1.1.2 (Jakobsson and 86 40 thanUNCORRECTED 7% of missing alleles from further anal- Rosenberg, 2007) andPROOF distruct v1.1 (Rosenberg, 87 41 ysis. Alleles were checked for scoring errors, 2003) to visualize the population’s probabil- 88 42 allele dropouts, and null alleles with MICRO- ity of assignment to each cluster. STRUCTURE 89 43 90 CHECKER v2.2.3. Allelic richness (AR), the population clustering was corroborated by a 44 91 number of alleles per locus (Na), the inbreed- principal component analysis (PCA, R pack- 45 92 ing coefficient (Fis), and observed (Ho) and ex- age “adegenet” (Jombart, 2008)). A hierarchical 46 93 pected (He) heterozygosities were calculated for analysis of molecular variance between/within 47 94 AMRE (brill2x v1.31) amre1442.tex 2020/11/19 14:21 [other] p. 4/7

4 K. Prei§ler et al.

1 strata (AMOVA, 1000 permutations) performed did not include zero). The presence of more pri- 48 2 in Arlequin simulated the impact of STRUC- vate alleles (N = 7) at NP than in the other 49 3 TURE predicted clustering on the genetic vari- populations (table 1) supported the isolation of 50 4 51 ance. this population. Although we used a rare-faction 5 The analysis of the mitochondrial D-loop re- 52 method, the low sample size analyzed from Ba 6 vealed two haplotypes in the sampled Mt. Her- 53 could still have biased the results as indicated 7 mon populations and the reference sites from 54 8 Mt. Carmel and Galilee. Haplotype I is only by the higher diversity estimates compared to 55 9 shared by individuals from Galilee (n = 6) and TD which is occurring at a lower elevation. 56 10 Carmel (n = 31) whereas haplotype lineage II Population structure analysis suggested a 57 11 is found in all sampled populations (Galilee n = clustering of sampled individuals in two or three 58 12 = = = 59 37, Carmel n 10, TD n 22, NC n 15, NP distinct genetic groups (K = 2: mean Ln P(K) = 13 = 60 n 35, fig. 1A). Two mutational steps separate −2091.83 SD 0.59, delta K = 485.64; K = 3: 14 the two haplotypes (T-C and C-T). Sequencing 61 mean Ln P(K) =−2027.87 SD 2.71, delta K = 15 failed for all samples from Banias. 62 12.75, K = 4: mean Ln P(K) =−1998.57 SD 16 PCR failed for microsatellite loci Sal E7, 63 = = = 17 Sal E14 and Sal G6 resulting in their omis- 7.6, delta K 7.02, K 5: mean Ln P(K) 64 18 sion from the dataset. The analysis with MICRO- −2022.60 SD 28.88; fig. 1A, supplementary fig. 65 19 CHECKER indicated that loci C3, B11, F10 and S2). Delta K peaked at K = 2, suggesting ab- 66 20 E11 show signs of null alleles due to homozy- sent gene flow between sites TD, B, NC to NP 67 21 gote excess. However, this is not consistent (fig. 1B). However, the standard deviation of the 68 22 69 across populations. Indicated errors due to stut- mean Ln P(K) was smallest at K = 3 before it 23 tering were re-checked and validated. Devia- 70 rapidly increased at K = 4 to 5. This assump- 24 tions from Hardy-Weinberg Equilibrium were 71 25 not consistent across loci or populations (sup- tion marks Ba a hybrid zone between TD and 72 26 plementary table S2). NC; NP remains isolated (fig. 1B). The strong 73 27 Population differentiation was greatest be- genetic divergence of NP is corroborated by the 74 28 tween NP and all other sites. Ba-TD (pairwise PCA (PC1 = 22.9%, PC2 = 7.57%; fig. 1C). By 75 29 76 Fst = 0.069, P < 0.01), Ba-NC (Fst = 0.076, P < clustering the sampling sites into K = 2 and 3, 30 = 77 0.01), Ba-NP (Fst 0.244, P < 0.01), TD-NC the AMOVA derived molecular variance among 31 = = 78 (Fst 0.103, P < 0.01), TD-NP (Fst 0.229, sites drops from 17.86% (K = 1) to 8.17% and 32 P < 0.01, NC-NP (F = 0.191, P < 0.01). 79 st 6.62% respectively (supplementary table S3); 33 This genetic divergence is mainly driven by ge- 80 34 ographic distance as well as by elevation as indi- confirming an extant population structure. The 81 = 35 cated by significant patterns of IBD (Pearson’s simulation of K 4 indicates a higher gene flow 82 36 r = 0.67, P = 0.037) and IBE (Pearson’s r = fromNCtoBathanfromTDtoBa(fig.1B). 83 37 0.58, P = 0.049, supplementary fig. S1). More- Further splitting (K = 5) confirms this pattern 84 38 over, elevation had a strong effect on genetic di- (supplementary fig. S3). 85 39 =− = 86 versity (AR: Pearson’s r 0.88, P 0.11; Ho: Our results indicate a substantial population 40 UNCORRECTED=− = =− = PROOF 87 r 0.84, P 0.15; He: r 0.86, P 0.13). structure of S. infraimmaculata at the southern 41 This negative correlation, however, was not sig- 88 foothills of Mt. Hermon. Moreover, elevation 42 nificant due to the low size of sampled locations 89 43 (N = 4). Located at the highest elevation, in- seems to have a strong impact on population 90 44 dividuals at NP show the lowest genetic diver- structure as here the genetic isolation from other 91 45 sity and experience significant inbreeding (table populations results in an increase of inbreeding 92 46 1, the 95% CI of the difference between means and lower genetic diversity indices. 93 47 94 AMRE (brill2x v1.31) amre1442.tex 2020/11/19 14:21 [other] p. 5/7

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1 The sharing of the common D-loop haplo- toads (Bufo boreas). The high number of pri- 48 2 type I by Mt. Hermon populations and popu- vate alleles present in NP are most likely the re- 49 3 lations from the Galilee and Mt. Carmel indi- sult of strong genetic drift which is typical for 50 4 cate historical gene flow and/or initial coloniza- small and highly isolated populations. In how 51 5 tion of S. infraimmaculata at its southern dis- far the evolution of these new neutral alleles are 52 6 tribution range. The presence of a private hap- a proxy for environmental adaptation at higher 53 7 lotype in the Galilee and Mt. Carmel, however, elevations remains unclear. 54 8 shows that this connection has been interrupted Climate change and the increasing landscape 55 9 and that populations of these areas follow their fragmentation in Israel are likely to further drive 56 10 own evolutionary trajectories already for a sig- the process of isolation of S. infraimmaculata 57 11 nificant amount of time. Populations at the low, populations if open migration corridors with 58 12 medium and high elevation range of the Mt. suitable steppingstone habitats are not present. 59 13 Low genetic diversity and presumably small 60 Hermon region display different patterns of ge- 14 population size render the studied Mt. Hermon 61 netic structure. Low (TD, Ba) and medium (NC) 15 populations prone to extinction in case of ex- 62 elevation sites show signs of genetic coherence 16 treme events, such as drought or infectious dis- 63 with moderate degrees of Fst differentiation and 17 eases (Bar-David et al., 2007; Markert et al., 64 gene flow. While these populations form a sin- 18 2010; Martel et al., 2014). This high extinc- 65 gle genetic unit compared to the high elevation 19 tion pattern is common among peripheral popu- 66 site (NP) for K = 2, a further splitting of K sup- 20 lations due to suboptimal habitat e.g. in ani- 67 ports the existence of separate genetic groups 21 mals (bustard (Otis tarda) in Africa (Palacín et 68 with substantial intermixing between sites (fig. 22 al., 2016), great crested newt (Triturus crista- 69 1). Thus, it seems that migration and disper- 23 tus) in Great Britain (Miró et al., 2017)) and 70 sal of salamanders is possible through human- 24 plants (forest trees (Fady et al., 2016)) alike. 71 25 mediated landscape structures at low and medi- However, peripheral populations are important 72 26 um elevations. Our local findings contradict the in evolutionary terms as high selective pressures 73 27 view that dispersal of S. infraimmaculata is lim- lead to significant evolutionary changes (Fady 74 28 ited through low elevations as found in the range et al., 2016). To conclusively determine the de- 75 29 of Galilee (Kershenbaum et al., 2014; Sinai et gree of isolation and potential threat, detailed 76 30 al., 2019). estimates about population sizes and migration 77 31 The high elevation population NP (>1000 m) rates based on genome-wide loci analysis are 78 32 displays for all types of analyses a strong ge- crucial. Our study lines up in a number of publi- 79 33 netic differentiation with regard to low and me- cations demonstrating the isolation, low genetic 80 34 dium elevation sites. We would like to em- diversity, and potential small population sizes of 81 35 phasize that the geographic distance between the peripheral S. infraimmaculata populations 82 36 NC and NP is in the same range as for NC in Israel amplifying the need for conservation 83 37 and TD. Elevation gradients are common phys- measures. 84 38 ical barriers to migration in other animals (Po- 85 39 lato et al., 2017; Valbuena-Ureña et al., 2018) 86 Acknowledgements. We feel deep grief at the loss of our 40 andUNCORRECTED plants (Reis et al., 2015) due to associated PROOF 87 colleague Leon Blaustein, a great scientist and wonderful 41 high energetic costs and higher predation risk person. We would like to thank Rabea Dabous, Eitan Nisim 88 42 (Blank et al., 2013). We observed that the in- and the Rangers from the Tel Dan Nature Reserve for pro- 89 viding access to the reserve. Special thanks to Sergé Boe- 43 creasing genetic isolation manifested in declin- garts for helpful discussions. We thank Meike Kondermann 90 44 ing allelic richness and heterozygosity along the and Christian Wartenberg for conducting laboratory work 91 45 and Daniel J. Goedbloed for helping in fieldwork. We are 92 gradient entailing increased inbreeding. Addis grateful for assistance and permits issued by the Israeli Na- 46 et al. (2015) found the same pattern in boreal ture and Parks Authority (INPA, 2013/40174, 2013/40284 93 47 94 AMRE (brill2x v1.31) amre1442.tex 2020/11/19 14:21 [other] p. 6/7

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1 and 2014/40672). The German-Israel Project-cooperation Hendrix, R., Hauswaldt, S.J., Veith, M., Steinfartz, S. 48 2 (DIP, DFG reference number BL 1271/1-1, STE 1130/8-1) (2010): Strong correlation between cross-amplification 49 funded the study. success and genetic distance across all members of 3 50 Author contribution. EK, SS, LB developed the concept. ‘True Salamanders’ (Amphibia: Salamandridae) re- 4 EK and AH conducted the fieldwork. KP and FL performed vealed by Salamandra salamandra-specific microsatel- 51 5 the statistical analyses. KP wrote the manuscript with input lite loci. Mol. Ecol. Resour. 10: 1038-1047. 52 6 by SS. Jakobsson, M., Rosenberg, N.A. (2007): CLUMPP: a clus- 53 ter matching and permutation program for dealing with 7 54 label switching and multimodality in analysis of popula- 8 tion structure. Bioinformatics 23: 1801-1806. 55 Supplementary material. Supplementary material is avail- 9 Jombart, T. (2008): adegenet: a R package for the multi- 56 able online at: variate analysis of genetic markers. Bioinformatics 24: 10 https://doi.org/10.6084/m9.figshare.13221008 57 1403-1405. 11 58 Keenan, K., McGinnity, P., Cross, T.F., Crozier, W.W., 12 Prodöhl, P.A. (2013): diveRsity: an R package for the es- 59 13 References timation and exploration of population genetics param- 60 14 eters and their associated errors. Methods Ecol. Evol. 4: 61 Addis, B.R., Lowe, W.H., Hossack, B.R., Allendorf, F.W. 782-788. 15 62 (2015): Population genetic structure and disease in mon- Kershenbaum, A., Blank, L., Sinai, I., Merilä, J., Blaustein, 16 tane boreal toads: more heterozygous individuals are L., Templeton, A.R. (2014): Landscape influences on 63 17 more likely to be infected with amphibian chytrid. Con- dispersal behaviour: a theoretical model and empirical 64 18 serv. 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1 Polato, N.R., Gray, M.M., Gill, B.A., Becker, C.G., Casner, (2019): The role of landscape and history on the genetic 48 2 K.L., Flecker, A.S., Kondratieff, B.C., Encalada, A.C., structure of peripheral populations of the near eastern 49 Poff, N.L., Funk, W.C., Zamudio, K.R. (2017): Genetic 3 fire salamander, Salamandra infraimmaculata, in north- 50 diversity and gene flow decline with elevation in mon- ern Israel. Conserv. Genet. 20: 875-889. 4 51 tane mayflies. Heredity (Edinb). 119: 107-116. Steinfartz, S., Küsters, D., Tautz, D. (2004): Isolation 5 Pritchard, J.K., Stephens, M., Donnelly, P. (2000): Inference and characterization of polymorphic tetranucleotide mi- 52 of population structure using multilocus genotype data. 6 crosatellite loci in the fire salamander Salamandra sala- 53 Genetics 155: 945-959. mandra (Amphibia: Caudata). Mol. Ecol. Notes 4: 626- 7 R Core Team (2019): R: a language and environment for 54 628. 8 statistical computing. 55 Steinfartz, S., Veith, M., Tautz, D. (2000): Mitochondrial se- 9 Reis, T.S., Ciampi-Guillardi, M., Bajay, M.M., Souza, A.P. 56 de, Santos, F.A.M. dos (2015): Elevation as a barrier: quence analysis of Salamandra taxa suggests old splits 10 57 genetic structure for an Atlantic rain forest tree (Bathysa of major lineages and postglacial recolonizations of cen- 11 australis) in the Serra do Mar mountain range, SE tral Europe from distinct source populations of Salaman- 58 12 Brazil. Ecol. Evol. 5: 1919-1931. dra salamandra.Mol.Ecol.9: 397-410. 59 Rosenberg, N.A. (2003): distruct: a program for the graph- Valbuena-Ureña, E., Oromi, N., Soler-Membrives, A., Car- 13 60 ical display of population structure. Mol. Ecol. Notes 4: ranza, S., Amat, F., Camarasa, S., Denoël, M., Guil- 14 137-138. laume, O., Sanuy, D., Loyau, A., Schmeller, D.S., Ste- 61 15 Segev, O., Polevikove, A., Blank, L., Goedbloed, D., infartz, S. (2018): Jailed in the mountains: genetic diver- 62 16 Küpfer, E., Gershberg, A., Koplovich, A., Blaustein, L. sity and structure of an endemic newt species across the 63 (2015): Effects of tail clipping on larval performance Pyrenees. PLoS One 13: e0200214. 17 and tail regeneration rates in the near eastern fire sala- 64 18 mander, Salamandra infraimmaculata.PLoSOne10: 65 Submitted: September 7, 2020. Final revision received: 19 e0128077. 66 Sinai, I., Segev, O., Weil, G., Oron, T., Merilä, J., Tem- November 8, 2020. Accepted: November 10, 2020. 20 67 pleton, A.R., Blaustein, L., Greenbaum, G., Blank, L. Associate Editor: Inigo Martínez-Solano. 21 68

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