Author Query Form Journal Code: AMRE Transmittal No.: AMRE1442
Total Page：16
File Type：pdf, Size：1020Kb
Author Query Form Journal code: AMRE Transmittal No.: AMRE1442 Dear Author, During the preparation of your manuscript for typesetting, some questions have arisen. They are listed below. Please check your proof carefully and mark all corrections at the appropriate place in the proof (e.g., by using on-screen annotation in the PDF file) or compile them in a separate list. Note: if you opt to annotate the file with software other than Adobe Reader then please also highlight the appropriate place in the PDF file. Any queries or remarks that have arisen during the processing of your manuscript are listed below and highlighted by flags in the proof. Location Query / Remark: Click on the Q link to find the query’s location in text in article Please insert your reply or correction at the corresponding line in the proof No queries Many thanks for your assistance. Page 1 of 1 AMRE (brill2x v1.31) amre1442.tex 2020/11/19 14:21 [other] p. 1/7 Amphibia-Reptilia 0 (2020): 1-7 brill.com/amre 1 48 2 Short Note 49 3 50 4 51 5 52 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 Preißler , 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, Talstraße 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, Mendelssohnstraße 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 © Koninklijke Brill NV, Leiden, 2020. DOI:10.1163/15685381-bja10038 AMRE (brill2x v1.31) amre1442.tex 2020/11/19 14:21 [other] p. 2/7 2 K. Preißler et al. 1 48 2 49 3 50 4 51 5 52 6 53 7 54 8 55 9 56 10 57 11 58 12 59 13 60 14 61 15 62 16 63 17 64 18 65 19 66 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 18 65 19 66 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).