Maximum Likelihood Tree of 11949529 Base Pair Exome Alignment

Home , Abyssinian hare, Alaskan hare, Broom hare, Cape hare, Corsican hare, Ethiopian highland hare

LCF_TUC_2060 (6) Black-tailed jackrabbit LCF_CAL_1954

LAM_Allab3_Bo (10) LAM_NBerg67_Rockies (12) Snowshoe hare LAM_A0604_WA (5) (14) LAM_A0965_WA

LER_VIE_1639 (48) European hare LER_PYR_1546

LST_STA65 (9) (60) (47) Ethiopian highland hare 90 LST_STA89 (59) LHB_HAB35 (63) Abyssinian hare (51) LHB_HAB68

LCP_SAF_1903 (52) Cape hare LCP_TAN_3023 (53) (17) LFG_FAG114 (54) Ethiopian hare LFG_FAG96

LCR_ITA_1957 (20) Corsican hare LCR_ITA_1958 (19) (3) LCS_1894 (23) Broom hare LCS_1891 (18) 62 LGR_SEV_1163 (27) Iberian hare LGR_CRE_2553

LMS_PRI_2461 (26) (31) Manchurian hare LMS_PRI_2460

LTW_JRRK_3 (30) (35) White-tailed jackrabbit (2) LTW_MTA_3280

(34) LTM_CAT_2012 (39) LTM_AFR_3108 Mountain hare

(38) LTM_MAG_1862

LOT_2110 (44) Alaskan hare LOT_2109

LCL_1956 White-sided jackrabbit

(66) European rabbit Pygmy rabbit

0.002 Figure S1 – Maximum likelihood tree of 11,949,529 base pair exome alignment (positions with no missing data for any individual), generated using a simultaneous maximum likelihood (ML) search in RAxML and rapid bootstrapping run under the GTR+G model of sequence evolution (autoMRE option), and setting the European and pygmy rabbits as outgroups. We show branch supports for branches with support smaller than 100 in bold. Node labels in parenthesis correspond to node labels in the ancestral reconstruction output in Supplementary Table S6.

OryCun European rabbit 31 a) BID_RC_444 Pygmy rabbit LCL_1956 White-sided jackrabbit LCF_CAL_1954 1: 0.69; 1.00 Black-tailed jackrabbit LCF_TUC_2060 31: 1.00; 1.00 LAM_Allab3_Bo 4: 0.77; 1.00 LAM_NBerg67_Rockies Snowshoe hare 3: 0.81; 1.00 LAM_A0965_WA 2: 0.81; 1.00 30: 0.43; 1.00 LAM_A0604_WA LER_PYR_1546 5: 0.71; 1.00 European hare LER_VIE_1639 LCP_SAF_1903 Cape hare 13: 0.41; 1.00 8: 0.51; 1.00 LCP_TAN_3023 29: 0.38; 1.00 7: 0.43; 1.00 LFG_FAG114 6: 0.84; 1.00 Ethiopian hare 12: 0.55; 1.00 LFG_FAG96 LST_STA65 9: 0.88; 1.00 Ethiopian highland hare LST_STA89 11: 0.60; 1.00 LHB_HAB35 10: 0.71; 1.00 Abyssinian hare LHB_HAB68 28: 0.61; 1.00 LMS_PRI_2461 14: 0.92; 1.00 Manchurian hare LMS_PRI_2460 LGR_CRE_2553 23: 0.46; 1.00 15: 0.87; 1.00 Iberian hare LGR_SEV_1163

22: 0.39; 0.89 LTW_JRRK_3 16: 0.64; 1.00 White-tailed jackrabbit LTW_MTA_3280 21: 0.67; 1.00 LTM_AFR_3108 27: 0.41; 1.00 20: 0.70; 1.00 LTM_CAT_2012 Mountain hare 19: 0.35; 0.79 LTM_MAG_1862 18: 0.35; 0.96 LOT_2109 17: 0.57; 1.00 Alaskan hare LOT_2110 LCS_1894 Broom hare 26: 0.92; 1.00 LCS_1891 25: 0.42; 1.00 LCR_ITA_1957 24: 0.42; 1.00 Corsican hare LCR_ITA_1958

2.0

White-sided jackrabbit b) Black-tailed jackrabbit 8: 1.00; 1.00 Snowshoe hare

9: 0.43; 1.00 Broom hare 2: 0.94; 1.00 Corsican hare

7: 0.41; 1.00 10: 0.38; 1.00 Manchurian hare

6: 0.46; 1.00 Iberian hare

5: 0.39; 0.89 White-tailed jackrabbit

4: 0.67; 1.00 11: 0.61; 1.00 Mountain hare 3: 0.71; 1.00 Alaskan hare

European hare

Ethiopian hare 12: 0.41; 1.00 1: 0.53; 1.00 Cape hare 13: 0.55; 1.00 Ethiopian highland hare 14: 0.71; 1.00 Abyssinian hare

European rabbit 8: 1.00; 1.00 Pygmy rabbit

1.0

Figure S2 – ASTRAL species tree obtained with 8889 gene trees without a) or with b) assigning individuals to species. Nodes in both trees are numbered and annotated with quartet scores and posterior probabilities. Further information for each node can be found in Supplementary Tables S7 and S8.

a) BID_RC_444 Pygmy rabbit OryCun European rabbit LAM_A0604_WA LAM_A0965_WA Snowshoe hare LAM_NBerg67_Rockies LAM_Allab3_Bo LCP_SAF_1903 Cape hare LCP_TAN_3023 99,9 LFG_FAG114 Ethiopian hare LFG_FAG96 LHB_HAB35 Abyssinian hare LHB_HAB68 79,7 LST_STA65 Ethiopian highland hare LST_STA89 LER_PYR_1546 98,7 European hare LER_VIE_1639 LCR_ITA_1957 Corsican hare LCR_ITA_1958 LCS_1891 98,6 Broom hare LCS_1894 LGR_CRE_2553 Iberian hare 99,5 LGR_SEV_1163 81,1 LOT_2109 Alaskan hare LOT_2110 60,3 53,8 48,9 LTM_MAG_1862 LTM_CAT_2012 Mountain hare LTM_AFR_3108 LTW_JRRK_3 White-tailed jackrabbit LTW_MTA_3280 LMS_PRI_2460 Manchurian hare LMS_PRI_2461 LCF_CAL_1954 Black-tailed jackrabbit LCF_TUC_2060 LCL_1956 White-sided jackrabbit

2.0

b) Pygmy rabbit

European rabbit

Cape hare

Ethiopian hare

Abyssinian hare

49,5 Ethiopian highland hare

European hare

Corsican hare

Broom hare

Iberian hare 97,7

65,2 Alaskan hare 99,8 Mountain hare

White-tailed jackrabbit

Manchurian hare

Snowshoe hare

Black-tailed jackrabbit

White-sided jackrabbit

1.1

Figure S3 – SVDquartets species tree generated with 45,779 unlinked SNPs without a) or with b) assigning individuals to species. For both trees, we show branch supports for branches with support lower than 100% of 1000 bootstrap rounds.

27 Mountain hare and Alaskan hare 22 26 White-tailed jackrabbit 21 20 25 Iberian hare

19 24 Manchurian hare

23 Corsican hare and Broom hare

18 13 Abyssinian hare 9 12 Ethiopian highland hare 10 17 11 8 Cape hare and Ethiopian hare 7 European hare 16 6 Snowshoe hare 15 5 Black-tailed jackrabbit

2 4 White-sided jackrabbit

3 Pygmy rabbit

2 European rabbit

9 10 11 15 1.00

0.75

0.50

0.25

0.00 11,7 | 8,9 11,8 | 7,9 11,9 | 7,8 16,4 | 2,3 16,3 | 2,4 16,2 | 3,4 10,8 | 12,13 10,13 | 12,8 10,12 | 13,8 10,7 | 18,19 10,18 | 19,7 10,19 | 18,7

16 17 18 19 1.00 Topology 0.75 t1

e freq. 0.50 v t2 0.25

relati t3 0.00 15,4 | 17,5 15,17 | 4,5 15,5 | 17,4 16,5 | 18,6 16,18 | 5,6 16,6 | 18,5 11,19 | 17,6 11,17 | 19,6 11,6 | 17,19 11,18 | 20,23 11,23 | 18,20 11,20 | 18,23

20 21 22 1.00

0.75

0.50

0.25

0.00 21,25 | 26,27 21,26 | 25,27 21,27 | 25,26 19,23 | 21,24 19,24 | 21,23 19,21 | 23,24 20,24 | 22,25 20,25 | 22,24 20,22 | 24,25 Figure S4 – Frequencies of the three main topologies around focal branches of the ASTRAL species tree (Fig. 1b). For each internal branch of the tree in (a), the frequency of the three possible topologies connecting the four neighboring branches is shown in (b). The title of each bar plot in (b) corresponds to labeled internal branches in the tree in (a). The most frequent topology is shown in red, and the two alternative topologies are shown in blue. On the x-axis of each bar plot, the topology of each quartet is represented using the neighboring branch labels. The branches leading to Mountain hares and Alaskan hares, Cape and Ethiopian hares, and Corsican and Broom hares were collapsed given the non-monophyly of the individuals of these species (Supplementary Figs. S2a and S3a).

Figure S5 - Divergence time tree of Lepus, estimated from alignments of 9015 protein coding ortholog genes (10,863,822 bp alignment), with three partitions (1st, 2nd and 3rd codon positions) using (a) molecular-based dates extrapolated from deep fossil calibrations in the lagomorphs or (b) fossil calibrations for the divergence of the genus Lepus and the Lepus species (see Materials and Methods). Node labels in bold black are divergence time estimations in millions of years. Numbers in parenthesis in each node represent node numbers in Supplementary Table S5.

Distance to species tree 1.0 0.8 0.6 ed RF distance z 0.4 mali r No 0.2 0.0 Chromosome Autosomes Gene tree X pairwise distance

Figure S6 – Box plots of the normalized Robinson-Foulds distance between chromosome X and autosomes gene trees and the genome-wide species tree (Supplementary Fig. S2a), and pairwise distance among all gene trees.

BID_RC_444 Pygmy rabbit [0.99,0.99] OryCun European rabbit LCL_1956 White-sided jackrabbit LTM_MAG_1862 Mountain hare [0.00,0.00] LOT_2109 [0.34,0.22] Alaskan hare [0.59,0.59] LOT_2110 LTM_CAT_2012 [0.00,0.00] Mountain hare [0.49,0.49] LTM_AFR_3108 LTW_JRRK_3 [0.00,0.06] [0.42,0.40] White-tailed jackrabbit LTW_MTA_3280 LMS_PRI_2461 [0.02,0.02] [0.91,0.91] Manchurian hare LMS_PRI_2460 LGR_CRE_2553 [0.89,0.89] Iberian hare [0.00,0.00] LGR_SEV_1163 LCS_1894 Broom hare [0.99,0.99] [0.94,0.94] LCS_1891 [0.01,0.05] LCR_ITA_1957 [0.06,0.11] Corsican hare LCR_ITA_1958 LCP_SAF_1903 [0.51,0.51] Cape hare [0.15,0.15] LCP_TAN_3023 [0.02,0.06] LFG_FAG114 [0.75,0.75] Ethiopian hare [0.13,0.13] LFG_FAG96 LST_STA65 [0.77,0.77] Abyssinian hare LST_STA89 [0.00,0.04] [0.34,0.34] [0.03,0.03] LHB_HAB35 [0.46,0.40] Ethiopian highland hare LHB_HAB68 LER_PYR_1546 [0.70,0.70] European hare LER_VIE_1639 [0.06,0.11] LAM_Allab3_Bo [0.73,0.73] LAM_NBerg67_Rockies Snowshoe hare [0.56,0.50] LAM_A0965_WA [0.54,0.47] LAM_A0604_WA LCF_CAL_1954 [0.51,0.51] Black-tailed jackrabbit LCF_TUC_2060

2.0 Figure S7 – Majority Rule Consensus Tree constructed from 8889 maximum likelihood gene trees. Tree nodes are annotated with the Internode Certainty (IC) and Internode Certainty All (ICA).

a) White-sided jackrabbit Black-tailed jackrabbit 7 8 Snowshoe hare

Manchurian hare

6 Iberian hare 9

5 White-tailed jackrabbit

4 Mountain hare 3 10 Alaskan hare

Broom hare 2 Corsican hare

11 European hare

Ethiopian hare 12 1 Cape hare 13 Ethiopian highland hare 14 Abyssinian hare

European rabbit 8 Pygmy rabbit

0.7 Pygmy rabbit b) 94,1 European rabbit Snowshoe hare Cape hare 79,8 Ethiopian hare 60,5 European hare 94,1 56,3 Abyssinian hare 62,9 100 Ethiopian highland hare 70,2 Manchurian hare Corsican hare 100 100 Broom hare 99,4 Iberian hare

98,4 Alaskan hare 100 Mountain hare 94,1 99,9 White-tailed jackrabbit Black-tailed jackrabbit White-sided jackrabbit

1.1

Figure S8 – Chromosome X species trees. (a) ASTRAL species tree obtained from 181 chromosome X gene trees and (b) SVDquartets species obtained from 1473 unlinked SNPs localized in the X chromosome. Node labels in (a) correspond to node labels in Supplementary Table S9 and (b) 1000 bootstrap support.

Figure S9 – Minimum D-statistic (Dmin; Malinksy et al 2018, Nat. Ecol. Evol.) between all possible trios of taxa in the species tree plotted against their z-score. Significant values are highlighted in orange. The number of significant and non-significant Dmin values is given above the graph.

Abyssinian hare Pygmy rabbit White-sided jackrabbit Black-tailed jackrabbit Snowshoe hare Ethiopian highland hare Cape hare Ethiopian hare European hare Broom hare Corsican hare Manchurian hare Iberian hare White-tailed jackrabbit Alaskan hare Mountain hare

European rabbit Abyssinian hare Alaskan hare Pygmy rabbit White-sided jackrabbit Black-tailed jackrabbit Snowshoe hare Ethiopian highland hare Cape hare Ethiopian hare European hare Corsican hare Broom hare Manchurian hare Iberian hare Mountain hare White-tailed jackrabbit

European rabbit 0.38 0.58 0.95 0.57 0.36 2.53 0.97 / 1 / 1 0.13 0.71 0.30 0.18 0.26 0.27 0.001 0.34 2.46 0.06 0.61 0.11 0.001 0.19 / 0.001 0.31 0.50 0.08 0.13 0.03 / 5.93 0.001 0.08 0.20 0.23 0.41 0.47 0.15 0.73 0.71 0.19 5.9 5.93 1 reticulations log(likelihood) =-35053284 BIC = 70106904 AICc = 70106642 3 reticulations log(likelihood) = -34947779 BIC = 69895986 AICc = 69895653 b) d) Abyssinian hare Alaskan hare Alaskan hare Pygmy rabbit White-sided jackrabbit Snowshoe hare Black-tailed jackrabbit Cape hare Ethiopian hare Ethiopian highland hare European hare Corsican hare Broom hare Manchurian hare Iberian hare White-tailed jackrabbit Mountain hare Abyssinian hare Pygmy rabbit White-sided jackrabbit Black-tailed jackrabbit Cape hare Ethiopian hare Ethiopian highland hare Iberian hare Manchurian hare European hare Broom hare Corsican hare Snowshoe hare White-tailed jackrabbit Mountain hare

European rabbit

European rabbit Alaskan hare Abyssinian hare Pygmy rabbit White-sided jackrabbit Black-tailed jackrabbit Snowshoe hare Cape hare Ethiopian hare Ethiopian highland hare Manchurian hare Iberian hare White-tailed jackrabbit Mountain hare European hare Broom hare Corsican hare

European rabbit / 1 0.35 2.53 0.95 0.95 2.53 0.07 0.57 0.36 / 1 0.57 0.08 2.46 0.36 0.96 0.65 0.91 0.52 0.42 0.68 0.19 0.001 0.19 0.001 0.44 0.71 0.73 0.21 / 0.001 0.001 0.001 0.001

/ 0.06 0.17 0.001 0.50 0.03 0.48 0.32 0.06 0.40 0.19 0.08 / 0.21 0.02 / 5.94 0.76 0.001 0.27 0.08 0.16 0.001 0.60 0.11 0.50 0.30 0.64 0.45 0.76 0.09 0.24 5.33 5.97 5.90 0 reticulations log(likelihood) = -35200370 BIC = 70401031 AICc = 70400804 2 reticulations log(likelihood) = -34988194 BIC = 69976770 AICc = 69976473 4 reticulations log(likelihood) = -34944764 BIC = 69890000 AICc = 69889632 c) a) e)

Figure S10 – Rooted pseudo-maximum likelihood networks for Lepus, with 0 to 4 reticulations inferred from 8889 gene trees with PhyloNet. Red branches represent reticulations, and blue branches represent variations to the species tree topology in Figure 1b. Branch lengths are represented in black and inheritance probabilities are represented in red. Bellow each network, we report the log likelihood, Bayesian information criteria (BIC) and Akaike information criteria corrected for small sample sizes (AICc). LCL LCL LCF LCR LAM LCS LGR LCP LMS LFG LTW LST LOT LHB Migration Migration weight LTM weight LER 0.5 LCS 0.5 LGR LCR LMS LST LOT 0 LHB 0 LTM LFG LTW LCP LAM 10 s.e. 10 s.e. LER LCF

0.00 0.02 0.04 0.06 0.08 0.00 0.02 0.04 0.06 0.08

Drift parameter Drift parameter

LCL LCS LMS LCR LTW LCP LFG

LOT LHB LTM LST LGR LER Migration LCS weight LGR 0.5 LCR Migration weight LMS LCP 0.5

LFG LTW LST 0 LHB LTM LER 0 LOT

LAM LAM 10 s.e. LCF LCF 10 s.e. LCL 0.00 0.02 0.04 0.06 0.08 0.00 0.02 0.04 0.06 0.08

Drift parameter Drift parameter

LCS LMS

LCR LOT LCP LTM LFG LTW LHB LER

LST LGR LCS LGR Migration Migration LCR weight LMS weight 0.5 0.5 LHB LOT

LTM LST

LTW LFG 0 0 LER LCP

LAM LAM

LCF LCF 10 s.e. 10 s.e. LCL LCL

0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.00 0.01 0.02 0.03 0.04 0.05 0.06

Drift parameter Drift parameter

LCL LCL

LAM LAM

LGR LGR

LCS LST LCR LHB LST LFG LCP LHB

Migration Migration LCR weight LFG weight 0.5 0.5 LCP LCS

LOT LER LOT LTM 0 0 LTM LTW

LMS LTW LER LMS 10 s.e. 10 s.e. LCF LCF

0.00 0.01 0.02 0.03 0.04 0.05 0.00 0.01 0.02 0.03 0.04 0.05

Drift parameter Drift parameter

LCL LCL

LCF LCF

LGR LGR

LCR LST

LCS LHB

LST LCP

LFG Migration LHB Migration weight weight LCR 0.5 LCP 0.5 LFG LCS LER LER

LOT LTW

0 0 LTM LOT

LTW LTM

LMS LMS 10 s.e. 10 s.e. LAM LAM

0.00 0.01 0.02 0.03 0.04 0.05 0.00 0.01 0.02 0.03 0.04 0.05

Drift parameter Drift parameter

Figure S11 Figure S11 (continued) – TreeMix inference of splits and admixture using 30,709 biallelic SNPs for no migration events (m=0) to nine migration events (m=9). The white-sided jackrabbit (LCL) was used as outgroup. LAM – Snowshoe hare; LCF – Black-tailed jackrabbit; LGR – Iberian hare; LCR – Corsican hare; LCS – Broom hare; LST – Ethiopian highland hare; LHB – Abyssinian hare; LCP – Cape hare; LFG – Ethiopian hare; LER – European hare; LOT – Alaskan hare; LTM – Mountain hare; LTW – White-tailed jackrabbit; LMS – Manchurian hare.

m=0 m=1 m=2

LAM LAM LAM LCF LCF LCF LCL LCL LCL LCP LCP LCP LFG LFG LFG LHB LHB LHB LST LST LST LER LER LER LCR LCR LCR LCS LCS LCS LGR LGR LGR LMS LMS LMS LOT LOT LOT LTM LTM LTM LTW LTW LTW T T T O O O TM TM TM TW TW TW LST LCL LCL LST LST LCL LCF LCF LCF L L L L L L LCP LCS LCP LCP LFG LER LFG LHB LER LCS LCS LFG LHB LER LHB LCR LCR LCR L L L LGR LAM LGR LMS LAM LGR LMS LAM LMS

m=3 m=4 m=5

LAM LAM LAM

LCF LCF LCF

LCL LCL LCL

LCP LCP LCP

LFG LFG LFG

LHB LHB LHB

LST LST LST

LER LER LER

LCR LCR LCR

LCS LCS LCS

LGR LGR LGR

LMS LMS LMS

LOT LOT LOT

LTM LTM LTM

LTW LTW LTW T T T O O O TM TM TM TW TW TW LCL LCL LCL LST LST LST LCF LCF LCF L L L L L L LCP LFG LHB LCS LCP LFG LER LCS LCP LFG LHB LER LCS LHB LER LCR LCR LCR L L L LAM LMS LAM LGR LMS LGR LMS LGR LAM

m=6 m=7 m=8

LAM LAM LAM

LCF LCF LCF

LCL LCL LCL

LCP LCP LCP

LFG LFG LFG

LHB LHB LHB

LST LST LST

LER LER LER

LCR LCR LCR

LCS LCS LCS

LGR LGR LGR

LMS LMS LMS

LOT LOT LOT

LTM LTM LTM

LTW LTW LTW T T T O O O TM TM TM TW TW TW LCL LST LCL LST LCL LST LCF LCF LCF L L L L L L LCP LCP LFG LHB LER LCS LCP LFG LHB LER LCS LFG LHB LER LCS LCR LCR LCR L L L LAM LAM LGR LMS LAM LGR LMS LGR LMS

m=9

LAM

LCF

LCL

LCP

LFG

LHB

LST

LER

LCR

LCS

LGR

LMS

LOT

LTM

LTW T O TM TW LCL LST LCF L L LFG LCS LCP LHB LER LCR L LMS LAM LGR

Figure S12 – Residuals for the TreeMix networks of Supplementary Figure S11, from 0 to 9 migration events. The white-sided jackrabbit (LCL) was used as outgroup. LAM – Snowshoe hare; LCF – Black-tailed jackrabbit; LGR – Iberian hare; LCR – Corsican hare; LCS – Broom hare; LST – Ethiopian highland hare; LHB – Abyssinian hare; LCP – Cape hare; LFG – Ethiopian hare; LER – European hare; LOT – Alaskan hare; LTM – Mountain hare; LTW – White-tailed jackrabbit; LMS – Manchurian hare.

a) Snowshoe hare vs Alaskan hare

6000 TCF4 average Dxy (1 windows) ARNTL2 average Dxy (1 windows) EBF2 average Dxy (3 windows) 4000 Frequency 2000 0

0.00 0.01 0.02 0.03 0.04 0.05

Dxy

b) Snowshoe hare vs Mountain hare

6000 TCF4 average Dxy (9 windows) ARNTL2 average Dxy (1 windows) EBF2 average Dxy (3 windows) 4000 Frequency 2000 0

0.00 0.01 0.02 0.03 0.04 0.05

Dxy

c) Snowshoe hare vs White-tailed jackrabbit 7000

6000 TCF4 average Dxy (6 windows) ARNTL2 average Dxy (2 windows) EBF2 average Dxy (4 windows) 5000 4000 Frequency 3000 2000 1000 0

0.00 0.01 0.02 0.03 0.04 0.05

Dxy

Figure S13 – Exome-wide observed genetic divergence (gray distribution) and average genetic divergence (colored lines) for fraction of admixture (fd) outlier windows (Supplementary Table S12) between snowshoe hares and (a) Alaskan hare, (b) Mountain hare and (c) white-tailed jackrabbits. The number of outlier windows used to estimate dxy is reported above the graph, and in dark gray we highlight the lower 1% percentile of the distribution.