Origin and divergence of Hawaiian katydids Xuhua Xia xxia@uottawa.ca http://dambe.bio.uottawa.ca

Key questions Where do they come from? America Asia Mixed Arrived multiple times or just once? Position of Hawaiian Islands relative to continents. Slide 2 Xuhua Xia

~3800 km from Continental America Hawaiian Islands with inferred approximate time on their volcanic origin in million years (Clague 1996;Clague and Sherrod 2014) Nihoa: 7.3 Kauai 5.8 ~3800 km from Continental America Oahu 3.9 Molokaʻi 2.1 Maui 1.3 Lanai 1.3 Kahoʻolawe 1.3 Hawaiian Islands with inferred approximate time on their volcanic origin in million years (Clague 1996;Clague and Sherrod 2014) Kohala 1.1 Mauna Kea 0.4 Hualālai 0.5 More than 5000 km from Asia Mauna loa 0.4 Kīlauea 0.3 Slide 3 Hawaii Xuhua Xia

MP tree from Shapiro et al, 2006MPE Implicit assumption: the katydids are from America (America) Hawaiian katydids in genus Banza are flightless Mitochondrial COX1 and CytB genes from 23 specimens in 13 speices. Bremer support: Consider the MP tree with N1 steps) and and one particular node and its descendents. If we move that node and its descendents else where, what is the shortest tree we can get? If this shortest tree requires N2 steps, then Bremer support is N1 - N2. MP tree with bootstrap support above branch and Bremer support below Xuhua Xia

Key questions Where do they come from? Which ancestral lineage? America Asia Mixed Which ancestral lineage? Neoconocephalus (America) Euconocephalus (Old world) Ruspolia (Old world) Arrived multiple times or just once? Slide 5 Xuhua Xia

root W (A) A Nihoa: 7.3 X Ancestral lineages B Kauai 5.8 Y C D Oahu 3.9 E Z Molokaʻi 2.1 Importance of sufficient taxon sampling (misinterpretation if W, X, Y in (A) were not sampled F Maui 1.3 Lanai 1.3 root Kahoʻolawe 1.3 If W, X and Y are not sampled in (A), then we may conclude that a single colonization leads to the present -day Banza species. Kohala 1.1 Mauna Kea 0.4 Hualālai 0.5 Mauna loa 0.4 Kīlauea 0.3 Ancestral lineages tip Slide 6 Hawaii Xuhua Xia (B)

Euconocephalus indica L28A JQ793675,JQ793734 658 592 Asia Species ACCN(1) LCOI LCytB Distribution Banza nihoa_A DQ649491,DQ649515 1233 729 Nihoa B. nihoa_B DQ649492,DQ649516 1255 B. kauaiensis_A DQ649483,DQ649507 Kauai B. kauaiensis_B DQ649484,DQ649508 B. unica_A DQ649501,DQ649525 Oahu B. unica_B DQ649502,DQ649526 1117 B. parvula_A DQ649497,DQ649521 748 B. parvula_B DQ649498,DQ649522 1254 B. molokaiensis_A DQ649487,DQ649511 695 Molokai B. molokaiensis_B DQ649488,DQ649512 659 B. deplanata_A DQ649481,DQ649505 686 Lanai B. deplanata_B DQ649482,DQ649506 B. brunnea_A DQ649479,DQ649503 West Maui B. brunnea_B DQ649480,DQ649504 747 B. mauiensis_A DQ649485,DQ649509 744 B. mauiensis_B DQ649486,DQ649510 B. pilimauiensis_A DQ649499,DQ649523 East Maui B. pilimauiensis_B DQ649500,DQ649524 B. nitida_A DQ649493,DQ649517 Hawaii B. nitida_B DQ649495,DQ649519 1222 705 B. nitida_C DQ649494,DQ649518 690 R. lineosa NC_033991 1534 1137 East Asia R. dubia NC_009876 1537 Neoconocephalus sp DQ649489,DQ649513 America Euconocephalus indica L28A JQ793675,JQ793734 658 592 Asia E. indicus L28B JQ793676,JQ793735 Conanalus pieli KX057724 Conocephalus maculatus KM244677 1540 1140 Asia/Africa Conocephalus melaenus KX057725 1134 Ducetia japonica KU885974 Asia/Australia Pseudokuzicus pieli KX057712 Only one of the six Euconocephalus indica specimens was shown.

Methods Sequence alignment Phylogenetic reconstruction MAFFT: LINSI option (‘–localpair’ and ‘–maxiterate = 1000’) MUSCLE: default is optimized Phylogenetic reconstruction Substitution model: GTR+ Maximum likelihood phylogenetics PhyML RAxML Dating: DAMBE/BEAST Geophylogeny: PGT Slide 8 Xuhua Xia

Conclusion: the ancestor of Banza species is from the Old World Conanalus_pieli Conocephalus_melaenus Conocephalus_maculatus 84 99 Neoconocephalus_sp R_lineosa E_indicus_L28B E_indicus_L28A 86 E_indicus_L73B E_indicus_L82B 95 E_indicus_L73A E_indicus_L82A 85 100 R_dubia B_unica_A B_unica_B B_kauaiensis_A B_kauaiensis_B B_parvula_A B_parvula_B B_deplanata_A B_deplanata_B B_molokaiensis_A B_molokaiensis_B 36 B_nitida_C B_nitida_A B_nitida_B 92 B_brunnea_A B_brunnea_B B_mauiensis_A B_mauiensis_B 97 B_pilimauiensis_A B_pilimauiensis_B 96 68 52 B_nihoa_A B_nihoa_B 21 78 Pseudokuzicus_pieli Ducetia_japonica The bootstrap values are low concerning the ancestors of Banza katydids. However, if we swap the position of Newconocephalus_sp with any or all of the Old World katydids (Ruspulia and Euconocephalus species), then the tree becomes significantly worse. Conclusion: the ancestor of Banza species is from the Old World The tree with Ruspolia dubia and the Banza nihoa swapped is almost significantly worse Conclusion: Banza species is polyphyletic, resulting from two colonization events. PhyML: GTR+Gamma, All sites (B_nihoa_A:0.01,B_nihoa_B:0.04,(((R_lineosa:0.06964124,(((E_indicus_L28B:0.235550,E_indicus_L28A:0.659532)0.8640:0.161793,(E_indicus_L73B:0.06,E_indicus_L82B:0.6522)0.9480:0.397727)0.:0.07,(E_indicus_L73A:0.558078,E_indicus_L82A:0.313838)0.8490:0.140528)0.9490:0.02261116)1.:0.06891932,(Neoconocephalus_sp:0.15457102,((Conanalus_pieli:0.16021299,(Conocephalus_melaenus:0.16121604,Conocephalus_maculatus:0.13053579)0.84:0.03743520)0.9950:0.05871648,(Pseudokuzicus_pieli:0.15749201,Ducetia_japonica:0.16795585)1.:0.07356446)0.9950:0.06657992)0.7790:0.03012536)0.21:0.778311,(R_dubia:0.10850578,((B_unica_A:0.01524050,B_unica_B:0.02408028)0.9970:0.03188430,((((B_kauaiensis_A:0.586313,B_kauaiensis_B:0.349128)0.9990:0.01814360,(B_parvula_A:0.890657,B_parvula_B:0.523292)1.:0.01923868)0.9890:0.01673299,((B_deplanata_A:0.01,B_deplanata_B:0.046262)1.:0.03989517,(B_molokaiensis_A:0.04,B_molokaiensis_B:0.046722)1.:0.02706206)0.3650:0.716351)0.9980:0.02763518,((B_nitida_C:0.03526642,(B_nitida_A:0.01017195,B_nitida_B:0.584193)0.9990:0.01497834)0.9150:0.642806,((B_brunnea_A:0.115384,B_brunnea_B:0.195426)1.:0.02335712,((B_mauiensis_A:0.05,B_mauiensis_B:0.044468)0.9680:0.405846,(B_pilimauiensis_A:0.183159,B_pilimauiensis_B:0.088263)0.9590:0.384203)1.:0.019737)0.6840:0.108278)1.:0.02468725)0.52:0.01404262)1.:0.06248301)0.92:0.02021360)1.:0.11672649); Misinterpretations if missing Ruspolia and Euconocephalus: Banza monophyly One colonization event Ancestor: Neoconocephalus

four Calibration points Ducetia_japonica 8.345±0.501 Pseudokuzicus_pieli Conocephalus_melaenus 8.174±0.492 Conocephalus_maculatus 8.461±0.445 9.536±0.405 Conanalus_pieli Neoconocephalus_sp R_lineosa 9.556±0.388 E_indicus_L28B 0.423±0.127 E_indicus_L28A 4.048±0.407 0.473±0.117 E_indicus_L82A 7.915±0.391 0.422±0.131 E_indicus_L73A 0.537±0.114 E_indicus_L82B 0.315±0.101 E_indicus_L73B B_nihoa_B 7.244±0.435 0.000±0.000 B_nihoa_A R_dubia B_kauaiensis_B 0.444±0.097 B_kauaiensis_A 1.893±0.194 7.3 B_parvula_B 0.561±0.111 B_parvula_A 3.042±0.188 B_deplanata_A 0.022±0.023 B_deplanata_B 2.832±0.252 B_molokaiensis_A Our results suggest two successful colonization events that resulted in the extrant Banza species. The first colonization event occurred about 7.3 million years ago by an ancestor closely related to Ruspolia lineosa, and gave rise to B. nihoa. The second colonization event occurred about 3.9 million years ago by an ancestor closely related to Ruspolia dubia and gave rise to all other Banza species. As both R. lineosa and R. dubia inhabit eastern and southeastern Asia, including the coastal regions, the ancestral lineage of Banza species is inferred to be in Asia instead of America. 6.914±0.320 0.023±0.022 B_molokaiensis_B B_brunnea_B four Calibration points 4.059±0.193 0.149±0.060 B_brunnea_A 1.3 B_pilimauiensis_A 0.131±0.052 B_pilimauiensis_B 0.443±0.090 B_mauiensis_A 3.9 2.509±0.168 0.023±0.022 B_mauiensis_B B_nitida_A 0.742±0.136 B_nitida_B 1.1 B_nitida_C B_unica_A Least-squares dating 1.753±0.212 B_unica_B

H0: as good as Best Tree: p = 0.056 Pseudokuzicus_pieli Ducetia_japonica Conanalus_pieli Conocephalus_maculatus Conocephalus_melaenus Neoconocephalus_sp E_indicus_L73B E_indicus_L82B E_indicus_L73A E_indicus_L82A E_indicus_L28A E_indicus_L28B R_lineosa B. unica_B B. unica_A B. nitida_C B. nitida_B B. nitida_A B. mauiensis_B B. mauiensis_A B. pilimauiensis_B B. pilimauiensis_A B. brunnea_A B. brunnea_B B. molokaiensis_B B. molokaiensis_A B. deplanata_B B. deplanata_A B. parvula_A B. parvula_B B. kauaiensis_A B. kauaiensis_B R_dubia B. nihoa_B B. nihoa_A Pseudokuzicus_pieli Ducetia_japonica Conanalus_pieli Conocephalus_maculatus Conocephalus_melaenus Neoconocephalus_sp E_indicus_L73B E_indicus_L82B E_indicus_L73A E_indicus_L82A E_indicus_L28A E_indicus_L28B R_lineosa R_dubia B. unica_B B. unica_A B. nitida_C B. nitida_B B. nitida_A B. mauiensis_B B. mauiensis_A B. pilimauiensis_B B. pilimauiensis_A B. brunnea_A B. brunnea_B B. molokaiensis_B B. molokaiensis_A B. deplanata_B B. deplanata_A B. parvula_A B. parvula_B B. kauaiensis_A B. kauaiensis_B B. nihoa_B B. nihoa_A Pseudokuzicus_pieli Ducetia_japonica Conanalus_pieli Conocephalus_maculatus Conocephalus_melaenus Neoconocephalus_sp E_indicus_L73B E_indicus_L82B E_indicus_L73A E_indicus_L82A E_indicus_L28A E_indicus_L28B R_lineosa R_dubia B. unica_B B. unica_A B. nitida_C B. nitida_B B. nitida_A B. mauiensis_B B. mauiensis_A B. pilimauiensis_B B. pilimauiensis_A B. brunnea_A B. brunnea_B B. molokaiensis_B B. molokaiensis_A B. deplanata_B B. deplanata_A B. parvula_A B. parvula_B B. kauaiensis_A B. kauaiensis_B B. nihoa_B B. nihoa_A tree li Dli +- SE pKH pSH pRELL 1* -16988.077 0.000 0.000 -1.000 -1.000 0.949 2 -16994.721 -6.644 4.172 0.056 0.362 0.051 3 -17034.430 -46.353 14.325 0.001 0.001 0.000 Best Tree H0: as good as Best Tree: p = 0.056 H0: as good as Best Tree: p = 0.001

Second colonization from East Asia R. lineosa R. dubia Euconocephalus sp. (A) B_unica_B B_unica_A B_nitida_C B_nitida_B B_nitida_A B_mauiensis_B B_mauiensis_A B_pilimauiensis_B B_pilimauiensis_A B_brunnea_A B_brunnea_B B_molokaiensis_B B_molokaiensis_A B_deplanata_B B_deplanata_A B_parvula_A B_parvula_B B_kauaiensis_A B_kauaiensis_B R_dubia B_nihoa_B B_nihoa_A R_lineosa+Euconocephalus B. nihoa B. kauaiensis B. parvula B. unica B. molokaiensis B. deplanata B. brunnea B. mauiensis B. nitida B. pilimauiensis (B) Second colonization from East Asia