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. a) 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 b) 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.