Online Resource 1- PCR Conditions

Online Resources 1:7

Online Resource 1- PCR Conditions

Twenty ng or 50 ng for blood or fecal DNA, respectively, was used as a template in 20 L PCR reactions, along with 0.25 M of each primer, 500 M dNTPs, 2 mM MgCl2 and 1 U Taq DNA polymerase (Hylabs, Rehovot, Israel). For primer pairs d-loop1 and d-loop3, PCR conditions were: 5 min at 94°C followed by 15 sec at 94°C, 30 sec at annealing temp (starting at 60°C with 2°C decrements every three cycles until 50°C), 30 sec at 72°C, followed by 30 cycles of 15 sec at 94°C, 30 sec at 48°C, 30 sec at 72°C and a final elongation step of 10 min at 72°C. For Mt8, 12S1 and d-loop4 primers, PCR conditions were: 5 min at 94°C followed by 35 cycles of 15 sec at 94°C, 15 sec at 62°C (63.5°C for d-loop4) and 15 sec at 72°C, followed by a final elongation step of 10 min at 72°C. PCR products were electrophoresed on a 2% agarose gel and visualized by a G: Box camera (Syngene).

Online Resource 2- Restriction Enzymes

Differentiating the three mtDNA haplotypes using the following restriction enzymes:

Enzyme / Site / Locus / Fragments / Protocol
NcoI
(New England Biolabs, UK) / / 12S1 Allele 1 (Haplotype 1) / 53bp
and 90bp / 10 μl of PCR product was digested for two hours at 37°C
BfaI
(New England Biolabs, UK) / / 12S1 Allele 2 (Haplotype 2) / 107bp
and 35bp / 10 μl of PCR product was digested for two hours at 37°C

Online Resource 3- Haplotype and Nucleotide Diversity & Testing for Genetic Differences between Samples Collected in Different Years

Genetic diversity indices found in the founding population and the wild population.

Population / N / No.H / Hd (±SE) /  (±SE)
founding population / 30 / 3 / 0.55± 0.08 / 0.41± 0.22
Wild population / 393 / 3 / 0.60 ± 0.01 / 0.47 ± 0.25

N= sample size, No. H= number of haplotypes, Hd= haplotype diversity, = nucleotide diversity.

Genetic diversity indices found in the different subpopulations.

Population / N / No.H / Haplotype frequencies / Hd (±SE) /  (±SE)
Hap1 / Hap2 / Hap3
NH / 206 / 3 / 0.13 / 0.32 / 0.55 / 0.58± 0.02 / 0.47± 0.25
Paran / 38 / 3 / 0.16 / 0.24 / 0.60 / 0.57 ± 0.07 / 0.46 ± 0.25
Paran South / 30 / 3 / 0.10 / 0.23 / 0.67 / 0.50 ± 0.08 / 0.43 ± 0.23
Paran North / 29 / 3 / 0.21 / 0.31 / 0.48 / 0.65 ± 0.05 / 0.49 ± 0.27
Nekarot / 28 / 3 / 0.11 / 0.21 / 0.68 / 0.50 ± 0.09 / 0.42 ± 0.23
East Basins / 22 / 3 / 0.41 / 0.36 / 0.23 / 0.69 ± 0.04 / 0.42 ± 0.23

N= sample size, No. H= number of haplotypes, Hd= haplotype diversity, = nucleotide diversity.

Pairwise FST between the years using Arlequin v3.5(Excoffier & Lischer 2010), FST values showing no significant genetic divergences between samples collected in different years.

All samples / 2009 / 2010
2009 / 0.013, Pvalue=0.06
2010 / -0.004, Pvalue=0.52 / 0.003, Pvalue=0.55
2011 / -0.007, Pvalue=0.09 / 0.014, Pvalue=0.06 / -0.005, Pvalue=0.40

Online Resource 4- Genetic data on each of the females and males of the founding population

Sample Name / Male/
Female / Genetic marker allele / Haplotype
d-loop 1 / d-loop 3 / MT-8 / 12S1 / d-loop 4
F_ramon / Female / 1 / 3 / 1 / 2 / 2 / 2
F0 / Female / 2 / 2 / 2 / 1 / 1
F1 / Female / 1 / 3 / 1 / 2 / 2 / 2
F10 / Female / 1 / 3 / 1 / 2 / 2 / 2
F11 / Female / 1 / 1 / 1 / 3 / 2 / 3
F2 / Female / 1 / 3 / 1 / 2 / 2 / 2
F3 / Female / 1 / 3 / 1 / 2 / 2 / 2
F4 / Female / 2 / 2 / 1 / 3 / 3
F5 / Female / 2 / 2 / 2 / 1 / 1 / 1
F6 / Female / 2 / 2 / 2 / 1 / 1
F7 / Female / 2 / 2 / 2 / 1 / 1 / 1
F8 / Female / 1 / 3 / 1 / 2 / 2 / 2
F9 / Female / 1 / 3 / 1 / 2 / 2
M1 / Male / 2 / 2 / 2 / 1 / 1 / 1
M10 / Male / 1 / 3 / 1 / 2 / 2
M11 / Male / 2 / 2 / 2 / 1 / 1 / 1
M12 / Male / 2 / 2 / 2 / 1 / 1 / 1
M13 / Male / 1 / 3 / 1 / 2 / 2 / 2
M14 / Male / 1 / 3 / 1 / 2 / 2 / 2
M15 / Male / 1 / 3 / 1 / 2 / 2 / 2
M16 / Male / 1 / 1 / 1 / 3 / 2 / 3
M17 / Male / 1 / 1 / 1 / 3 / 2 / 3
M18 / Male / 1 / 3 / 1 / 2 / 2 / 2
M2 / Male / 1 / 3 / 1 / 2 / 2
M3 / Male / 1 / 3 / 1 / 2 / 2 / 2
M4 / Male / 2 / 2 / 1
M5 / Male / 1 / 3 / 1 / 2 / 2
M6 / Male / 1 / 3 / 1 / 2 / 2 / 2
M8 / Male / 1 / 3 / 2 / 2
M9 / Male / 1 / 3 / 1 / 2 / 2

Online Resource 5- Pairwise FST Between Subpopulations

Pairwise FST between subpopulations using Arlequin v3.5 (Excoffier & Lischer 2010), FST values showing a significant genetic divergences between the East Basins region and all other regions (except the Paran North region).

NH / Paran / Paran South / Paran North / Nekarot
Paran / -0.009
Paran South / 0.005 / -0.023
Paran North / -0.010 / -0.003 / 0.026
Nekarot / 0.009 / 0.021 / 0.035 / 0.034
East Basins / 0.100** / 0.105* / 0.157** / 0.023 / 0.162**

* = P <0.05, ** P<0.005

Online Resource 6- Bootstrap Analysis

East basins vs. NH: The frequency distributions of pairwise FSTvalues form 1000 bootstrapped datasets contrasted against the 'real' FST values.

East basins vs. AOS (all other subpopulations): The frequency distributions of pairwise FSTvalues form 1000 bootstrapped datasets contrasted against the 'real' FST values.

East basins vs. AOS (all other subpopulations): The frequency distributions of pairwise ΦSTvalues form 1000 bootstrapped datasets contrasted against the 'real' ΦST values.

No effect of differences in sample size on FST and ΦST values between the pairwise populations was found. In all cases the 'real' value falls inside the distribution.

Online Resource 7- Water Sourcesin the Study Area

Location of water sources map, which present three types of water sources: Water troughs maintained by the INPA (Trough); Natural water sources (e.g., springs, water-holes, NWS); Water pipe leakages (Tap). Each water source was classified as either permanent (blue circle) or temporary(blue square) water source.

Water sources are scarce in the Negev, and in the East region there is a permanent water source only at the north edge of the area.

Online Resource 8- Sensitivity Analysis

Sensitivity analysis flow chart:

Four main regions of equal size, North, East, South and West (a circle polygon of 5km/10km/15km radius), according to the main/sub basins in the study area (East basins, Nekarot, Paran North and Paran South):

Results of the different indices of each region (East/North/South/west), according to different region size (radius of 5km/10km/15km), and different connectivity maps (cells above 0.5/0.6/0.7/0.8/0.9), performed by Fragstats 4.1(McGarigal et al. 2002). The lower the East region index value (in terms of landscape connectivity), the highest it's ranked.

The East region is ranked first
The East region is ranked second
The East region is ranked third or fourth
Radius / Cells Above / Region / NP / PA / ENN / PROX / COHESION
5km / 0.5 / East / 129 / 88.8 / 247.03 / 1.11 / 58.89
5km / 0.5 / North / 430 / 529.8 / 153.17 / 4.52 / 74.26
5km / 0.5 / South / 192 / 208.5 / 205.45 / 4.04 / 81.5
5km / 0.5 / West / 372 / 401.8 / 163 / 3.07 / 71.63
5km / 0.6 / East / 67 / 42.5 / 270.53 / 1.16 / 54.03
5km / 0.6 / North / 350 / 261.8 / 166 / 1.9 / 60.09
5km / 0.6 / South / 78 / 110.5 / 208.09 / 6.02 / 87.59
5km / 0.6 / West / 299 / 237.3 / 185.61 / 1.61 / 63.51
5km / 0.7 / East / 49 / 22.5 / 195.46 / 0.63 / 36.06
5km / 0.7 / North / 177 / 95 / 227.22 / 0.83 / 46.33
5km / 0.7 / South / 49 / 54.5 / 264.81 / 3.51 / 77.05
5km / 0.7 / West / 155 / 106.5 / 241.97 / 1.19 / 59.68
5km / 0.8 / East / 20 / 7 / 317.16 / 0.16 / 23.11
5km / 0.8 / North / 66 / 23 / 324.64 / 0.33 / 22.9
5km / 0.8 / South / 26 / 28.5 / 165.36 / 2.48 / 81.04
5km / 0.8 / West / 63 / 41.8 / 199.21 / 0.89 / 59.34
5km / 0.9 / East / 1 / 0.3 / 0 / 0 / 0
5km / 0.9 / North / 6 / 2 / 1776.73 / 0.01 / 30.34
5km / 0.9 / South / 9 / 8.7 / 299.53 / 0.57 / 75.47
5km / 0.9 / West / 9 / 3.2 / 303.71 / 0.15 / 35.38
Radius / Cells Above / Region / NP / PA / ENN / PROX / COHESION
10km / 0.5 / East / 642 / 481 / 206.29 / 1.47 / 63.29
10km / 0.5 / North / 1484 / 2069 / 157.88 / 13.94 / 85.31
10km / 0.5 / South / 1109 / 1286.6 / 182.52 / 6.27 / 81.34
10km / 0.5 / West / 1328 / 1243.8 / 162.91 / 2.82 / 66.56
10km / 0.6 / East / 288 / 212.5 / 233.37 / 1.15 / 60.98
10km / 0.6 / North / 1200 / 1111.6 / 170.22 / 5.65 / 76.24
10km / 0.6 / South / 669 / 732.5 / 201.01 / 5.17 / 81.41
10km / 0.6 / West / 903 / 648.7 / 186.87 / 1.49 / 57.21
10km / 0.7 / East / 160 / 103.2 / 230.73 / 0.98 / 54.6
10km / 0.7 / North / 683 / 492 / 184.73 / 2.26 / 62.51
10km / 0.7 / South / 387 / 397 / 214.8 / 3.1 / 75.14
10km / 0.7 / West / 453 / 272.8 / 239.06 / 0.92 / 51.2
10km / 0.8 / East / 72 / 41.5 / 318 / 0.67 / 51.78
10km / 0.8 / North / 272 / 128 / 256.8 / 0.87 / 42.48
10km / 0.8 / South / 222 / 211 / 217.74 / 2.19 / 70.38
10km / 0.8 / West / 156 / 78 / 291.37 / 0.54 / 46.94
10km / 0.9 / East / 22 / 11 / 385.85 / 0.58 / 47.26
10km / 0.9 / North / 41 / 17.2 / 307.9 / 0.47 / 39.37
10km / 0.9 / South / 107 / 75.5 / 264.47 / 0.78 / 59.75
10km / 0.9 / West / 22 / 7.2 / 375.63 / 0.19 / 22.91
Radius / Cells Above / Region / NP / PA / ENN / PROX / COHESION
15Km / 0.5 / East / 1206 / 963.2 / 210.75 / 1.85 / 65.04
15Km / 0.5 / North / 2752 / 3572.1 / 165.3 / 11.53 / 83.29
15Km / 0.5 / South / 1979 / 2205.8 / 191.91 / 7.3 / 81.01
15Km / 0.5 / West / 2404 / 2538.9 / 171.68 / 3.85 / 73.16
15Km / 0.6 / East / 591 / 410.5 / 241.51 / 1.28 / 61.01
15Km / 0.6 / North / 2090 / 1878.5 / 178 / 5 / 73.67
15Km / 0.6 / South / 1233 / 1238.3 / 217.11 / 4.6 / 79.04
15Km / 0.6 / West / 1594 / 1345.2 / 187.26 / 2.1 / 64.85
15Km / 0.7 / East / 290 / 168.5 / 272.24 / 0.78 / 50.09
15Km / 0.7 / North / 1171 / 819.7 / 190.42 / 2.5 / 64.11
15Km / 0.7 / South / 719 / 671.8 / 230.13 / 2.39 / 72.71
15Km / 0.7 / West / 830 / 613.5 / 225.18 / 1.32 / 60.08
15Km / 0.8 / East / 91 / 51.8 / 471.01 / 0.64 / 50.07
15Km / 0.8 / North / 427 / 217.5 / 252.13 / 1.03 / 48.6
15Km / 0.8 / South / 412 / 329.5 / 258.25 / 1.65 / 66.41
15Km / 0.8 / West / 332 / 218 / 276.39 / 1 / 58.16
15Km / 0.9 / East / 28 / 14 / 340.59 / 0.51 / 45.5
15Km / 0.9 / North / 66 / 25.8 / 297.35 / 0.36 / 32.81
15Km / 0.9 / South / 173 / 103.7 / 271.12 / 0.62 / 53.39
15Km / 0.9 / West / 88 / 56.5 / 395.64 / 0.83 / 66.25

In most of the cases landscape connectivity within the East region is lower than in other regions, only in few cases the East region is ranked second and rarely third. Moreover, no other region seems to be as consistent in terms of low connectivity as the East region.

Result of a moving window analysis of the PROX index, using a 4km radius window in different connectivity maps (cells above 0.6, 0.7 and 0.8 probability of wild ass presence, shown from left to right)

Result of a moving window analysis of the PROX index on the basic connectivity map (cells above 0.7 probability of wild ass presence), using different window size (4km, 8km and 15km, shown from left to right).

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