Systematic Entomology Page 2 of 55 1 1 Out of the Orient: Post-Tethyan transoceanic and trans-Arabian routes 2 fostered the spread of Baorini skippers in the Afrotropics 3 4 Running title: Historical biogeography of Baorini skippers 5 6 Authors: Emmanuel F.A. Toussaint1,2*, Roger Vila3, Masaya Yago4, Hideyuki Chiba5, Andrew 7 D. Warren2, Kwaku Aduse-Poku6,7, Caroline Storer2, Kelly M. Dexter2, Kiyoshi Maruyama8, 8 David J. Lohman6,9,10, Akito Y. Kawahara2 9 10 Affiliations: 11 1 Natural History Museum of Geneva, CP 6434, CH 1211 Geneva 6, Switzerland 12 2 Florida Museum of Natural History, University of Florida, Gainesville, Florida, 32611, U.S.A. 13 3 Institut de Biologia Evolutiva (CSIC-UPF), Passeig Marítim de la Barceloneta, 37, 08003 14 Barcelona, Spain 15 4 The University Museum, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan 16 5 B. P. Bishop Museum, 1525 Bernice Street, Honolulu, Hawaii, 96817-0916 U.S.A. 17 6 Biology Department, City College of New York, City University of New York, 160 Convent 18 Avenue, NY 10031, U.S.A. 19 7 Biology Department, University of Richmond, Richmond, Virginia, 23173, USA 20 8 9-7-106 Minami-Ôsawa 5 chome, Hachiôji-shi, Tokyo 192-0364, Japan 21 9 Ph.D. Program in Biology, Graduate Center, City University of New York, 365 Fifth Ave., New 22 York, NY 10016, U.S.A. 23 10 Entomology Section, National Museum of the Philippines, Manila 1000, Philippines 24 25 *To whom correspondence should be addressed: E-mail: [email protected] Page 3 of 55 Systematic Entomology 2 26 27 ABSTRACT 28 The origin of taxa presenting a disjunct distribution between Africa and Asia has puzzled 29 biogeographers for centuries. This biogeographic pattern has been hypothesized to be the result 30 of transoceanic long-distance dispersal, Oligocene dispersal through forested corridors, Miocene 31 dispersal through the Arabian Peninsula or passive dispersal on the rifting Indian plate. However, 32 it has often proven difficult to pinpoint the mechanisms at play. We investigate the biotic 33 exchange between the Afrotropics and the Oriental region during the Cenozoic, a period in 34 which geological changes altered landmass connectivity. We use Baorini skippers (Lepidoptera, 35 Hesperiidae) as a model, a widespread clade of butterflies in the Old-World tropics presenting a 36 disjunct distribution between the Afrotropics and the Oriental region. We use anchored 37 phylogenomics to infer a robust evolutionary tree for Baorini skippers and estimate divergence 38 times and ancestral ranges to test biogeographic hypotheses. Our phylogenomic tree recovers 39 strongly supported relationships for Baorini skippers and clarifies the systematics of the tribe. 40 Dating analyses suggest that these butterflies originated in the Oriental region, Greater Sunda 41 Islands, and Philippines in the early Miocene approximately 23 million years ago. Baorini 42 skippers dispersed from the Oriental region toward Africa at least five times in the past 20 43 million years. These butterflies colonized the Afrotropics primarily through trans-Arabian 44 geodispersal after the closure of the Tethyan seaway in the mid-Miocene. Range expansion from 45 the Oriental region toward the African continent likely occurred via the Gomphotherium land 46 bridge through the Arabian Peninsula. Alternative scenarios invoking long-distance dispersal and 47 vicariance are not supported. The Miocene climate change and biome shift from forested areas to 48 grasslands possibly facilitated geodispersal in this clade of skippers. 49 50 Systematic Entomology Page 4 of 55 3 51 52 53 KEYWORDS 54 Anchored phylogenomics; Arabotropical forest dispersal; butterfly evolution; Gomphotherium 55 land bridge; Hesperiidae; Lepidoptera phylogenomics; Neotethys Seaway closure; Old World 56 biogeography; transoceanic long-distance dispersal Page 5 of 55 Systematic Entomology 4 57 INTRODUCTION 58 The remarkable geological features of the Old-World tropics, such as the Indo-Australian 59 Archipelago (IAA), the Himalayan mountain range, and the Deccan traps are testaments to the 60 dynamic geological evolution that took place throughout the Cenozoic (Seton et al. 2012). The 61 collision of the Asian, Australian, and Pacific plates resulted in the assemblage of Wallacea (Hall 62 2013) and orogeny of New Guinea (Toussaint et al. 2014), allowing the Asian and Australian 63 biotas to connect through shallow marine straits and ephemeral land bridges between myriad 64 tropical islands (Voris 2000). The collision of the rifting Indian plate with the Asian plate in the 65 Paleocene (Hu et al. 2016), triggered the orogeny of the Himalayas (Zhang et al. 2012), and 66 connected the relic Gondwanan stock with the Laurasian biota. The closure of the Tethyan 67 Ocean in the Oligocene (Pirouz et al. 2017) triggered the orogeny of the Zagros mountain chain 68 along the Alpine-Himalayan orogenic belt, thereby connecting African and Asian biotas by a 69 land bridge (i.e., the Gomphotherium land bridge; Rögl 1998) through the Arabian Peninsula 70 (Harzhauser et al. 2007; Berra & Angiolini 2014). Disentangling the impact of these major 71 geological rearrangements on the evolution of the regional biota is necessary to understand the 72 mechanisms of lineage diversification in the Old-World tropics. 73 Of particular interest are clades of organisms disjunctly distributed in Asia and Africa, 74 because these allow testing hypotheses related to dispersal, vicariance, and extinction with 75 respect to the geology of the African, Arabian, Asian, and Indian plates. The distribution of such 76 clades has been explained by four biogeographic hypotheses. The first hypothesis, transoceanic 77 long-distance dispersal (LDD) between Asia and Africa, has been invoked to explain disjunct 78 distributions. Under this hypothesis (H1), the colonization of distant areas from a source 79 geographic range is accomplished directly and is not the result of range expansion followed by 80 widespread extinction/range contraction. A second hypothesis posits range expansion with trans- 81 Arabian dispersal (H2, also known as the “Gomphotherium land bridge hypothesis”, Harzhauser Systematic Entomology Page 6 of 55 5 82 et al. 2007) followed by regional extinction in the Arabian Peninsula. The Gomphotherium land 83 bridge hypothesis is based on geological evidence that the eastern part of the Tethys Ocean (i.e., 84 Neotethys) that once separated Africa and Asia closed as a result of the Arabian plate moving 85 northward and colliding with the Asian plate in the Oligocene ca. 27 million years ago (Ma), 86 engendering the orogeny of the Zagros mountain chain in the process (Pirouz et al. 2017). A 87 third hypothesis is derived from the “boreotropical dispersal hypothesis”. This latter postulate 88 suggests that lineages expanded their geographic ranges in higher latitudes through boreotropical 89 forests that existed in the Eocene and Oligocene, before climate cooling triggered the wane of 90 boreotropical forests, in turn promoting ecological vicariance (Wolfe 1975). Recent studies (e.g., 91 Couvreur et al. 2011; Sánchez-Ramírez et al. 2015) suggest that a similar process could be 92 invoked for Africa-Asia disjunctions, where range expansion could have happened after the 93 closure of the Tethys Ocean via now-vanished tropical forests in the Arabian Peninsula 94 (Ghazanfar & Fisher 1998; Griffin 2002). This hypothesis, which we coin “Arabotropical forest 95 dispersal” (H3), implies that range expansion would had to have happened between the 96 emergence of these habitats and their disappearance, triggered by the onset of a cooler climate in 97 the late Eocene and Oligocene (Zachos et al. 2001). As a result, this hypothesis predicts range 98 expansions resulting in ancestral widespread distributions, followed by vicariance triggered by 99 the aridification of the Arabian Peninsula and associated floristic reconfiguration (Ghazanfar & 100 Fisher 1998; Griffin 2002). The last hypothesis proposes that organisms rafted on the 101 Gondwanan Indian plate (“biotic ferry hypothesis” or “out-of-India hypothesis”; Datta-Roy & 102 Karanth 2009) and subsequently diversified in Asia. Under this hypothesis (H4), lineages 103 occurring in Gondwana during the Cretaceous and therefore in the landmass comprising Africa, 104 India, and Madagascar at the time, would have been transported via the rafting Indian plate 105 across the Indian Ocean, before docking in the Oriental region in the Eocene ca. 60 Ma (Hu et al. 106 2016). Although this hypothesis was initially supported by phylogenetic patterns, it has since Page 7 of 55 Systematic Entomology 6 107 been largely rejected by numerous studies of unrelated plants and animals that use molecular 108 dating approaches (Toussaint et al. 2016). A related mechanism is the use of the Indian rafting 109 plate as a “stepping stone” whereby taxa dispersed from the Oriental region to insular India 110 before its final docking and then dispersed toward other Gondwanan landmasses (Toussaint et al. 111 2018a). Both processes imply ancient African and Oriental divergences. Overall, there are few 112 empirical studies focusing on animal taxa that investigate the evolution of African-Asian 113 disjunctions in a phylogenetic framework with the aim to test these competing hypotheses. 114 Baorini grass skippers (Lepidoptera, Hesperiidae, Hesperiinae) are widespread butterflies 115 distributed from Madagascar, the Mascarenes, Africa and southern Europe, through the Arabian 116 Peninsula, and to the