Plastid phylogenomics clarifies broad-level relationships in Bulbophyllum (Orchidaceae) and provides insights into range evolution of Australasian section Adelopetalum

The hyper diverse orchid genus Bulbophyllum is the second largest genus of flowering plants and exhibits a pantropical distribution with a center of diversity in tropical Asia. The only Bulbophyllum section with a center of diversity in Australasia is sect. Adelopetalum. However, phylogenetic placement, interspecific relationships, and spatio-temporal evolution of the section have remained largely unclear. To infer broad-level relationships within Bulbophyllum and interspecific relationships within sect. Adelopetalum, a genome skimming dataset was generated for 89 samples, yielding 70 plastid coding regions and the nuclear ribosomal DNA cistron. For 18 additional samples, Sanger data from two plastid loci (matK, ycf1) and nuclear ITS were added using a supermatrix approach. The study provided new insights into broad-level relationships in Bulbophyllum, including phylogenetic evidence for the non-monophyly of sections Beccariana, Brachyantha, Brachypus, Cirrhopetaloides, Cirrhopetalum, Desmosanthes, Minutissima, Oxysepala, Polymeres and Sestochilos. Section Adelopetalum and sect. Minutissima s.s. formed a highly supported clade that was resolved in sister group position to the remainder of the genus. Divergence time estimations based on a relaxed molecular clock model placed the origin of Bulbophyllum in the early Oligocene (ca. 33.2 Ma) and of sect. Adelopetalum in the late Oligocene (ca. 23.6 Ma). Ancestral range estimations based on a BAYAREALIKE model identified the Australian continent as ancestral area of sect. Adelopetalum. The section underwent crown diversification during the mid-Miocene to the late Pleistocene, predominantly in continental Australia. At least two independent long-distance dispersal events were inferred eastwards from the Australian continent to New Zealand, and New Caledonia from the early Pliocene onwards, likely mediated by the predominantly westerly winds of the southern hemisphere. Retraction and fragmentation of eastern Australian rainforests from the early Miocene onwards are discussed as likely drivers of lineage divergence within sect. Adelopetalum, facilitating allopatric speciation.

The hyper diverse orchid genus Bulbophyllum is the second largest genus of flowering plants and 20 exhibits a pantropical distribution with a center of diversity in tropical Asia. The only Bulbophyllum 21 section with a center of diversity in Australasia is sect. Adelopetalum. However, phylogenetic 22 placement, interspecific relationships, and spatio-temporal evolution of the section have remained 23 largely unclear. To infer broad-level relationships within Bulbophyllum and interspecific 24 relationships within sect. Adelopetalum, a genome skimming dataset was generated for 89 samples, 25 yielding 70 plastid coding regions and the nuclear ribosomal DNA cistron. For 18 additional samples, 26 Sanger data from two plastid loci (matK, ycf1) and nuclear ITS were added using a supermatrix 27 approach. The study provided new insights into broad-level relationships in Bulbophyllum, including 28 phylogenetic evidence for the non-monophyly of sections Beccariana, Brachyantha,Brachypus,29 Cirrhopetaloides, Cirrhopetalum, Desmosanthes, Minutissima, Oxysepala, Polymeres and 30 Sestochilos. Section Adelopetalum and sect. Minutissima s.s. formed a highly supported clade that 31 was resolved in sister group position to the remainder of the genus. Divergence time estimations 32 based on a relaxed molecular clock model placed the origin of Bulbophyllum in the early Oligocene 33 (ca. 33.2 Ma) and of sect. Adelopetalum in the late Oligocene (ca. 23.6 Ma). Ancestral range 34 estimations based on a BAYAREALIKE model identified the Australian continent as ancestral area 35 of sect. Adelopetalum. The section underwent crown diversification during the mid-Miocene to the 36 late Pleistocene, predominantly in continental Australia. At least two independent long-distance 37 dispersal events were inferred eastwards from the Australian continent to New Zealand, and New 38 Caledonia from the early Pliocene onwards, likely mediated by the predominantly westerly winds of 39 1 Introduction 43 The hyper diverse orchid genus Bulbophyllum Thouars (Epidendroideae) is the second largest genus 44 of flowering plants with more than 2,100 species and exhibits exceptional morphological and 45 ecological diversity (Frodin, 2004;Pridgeon et al., 2014, WCSP 2022. Species of this predominantly 46 epiphytic genus occur in a wide range of tropical and subtropical habitats, from montane rainforests 47 to dry deciduous forests, savannah woodlands, and rocky fields with shrubby vegetation (Pridgeon et 48 al., 2014). Bulbophyllum is distributed pantropically, occupying all botanical continents defined by 49 Brummit (2001)  These studies revealed a strong biogeographic pattern within the genus with four main clades that 68 include species largely confined or endemic within one broader geographical area: 1) continental 69 Africa, 2) Madagascar and the Mascarene Islands, 3) Southern America, or 4) Asia (Fischer et  anchored by filamentous roots with small pseudobulbs that are crowded to widely spaced, and a 103 small single flat leaf arising from the apex of the pseudobulb. The inflorescence is single to few-104 flowered, with small white, cream or yellow flowers, sometimes with red or purple patterns. The 105 petals are smaller than the sepals but similar in shape, with the bases of the lateral sepals fused to the 106 column foot. The fleshly three-lobed labellum is firmly hinged to the apex of the column foot. 107 Previous cladistic analysis of sect. Adelopetalum based on morphological characters resolved two 108 main clades within the section, differentiated by the size and shape of the lower margin of the 109 stelidia: the filiform column appendages typical for most Bulbophyllum (Vermeulen, 1993

Phylogenetic relationships -Plastid data 248
The ML phylogeny inferred from the 70 loci plastid supermatrix provided strong support for the 249 monophyly of Bulbophyllum and its sister group relationship to Dendrobium (Fig. 1). Section 250 Adelopetalum and Minutissima s.s. formed a highly supported clade, here termed the 251 Adelopetalum/Minutissima clade, which was resolved in sister group position to the remainder of the 252 genus (ultrafast bootstrap support/UFBS 98) (Fig. 1, Fig. 2 were identified as polyphyletic or paraphyletic. Phylogenetic relationships described here based on 268 the plastid supermatrix (Fig.1, Fig. 2  The ML phylogeny based on the nuclear ribosomal DNA cistron was resolved with overall lower 294 support compared to analyses based on 70 plastid loci supermatrix (Fig. 3, Fig. 4)  Australian species are shown with an asterisk. 320

Divergence time estimation 321
The divergence time analysis based on a relaxed log normal clock and birth death prior with 322 speciation and extinction, which was identified as the model of best fit based on the Akaike 323 information criterion (Supplementary Material S4), is presented here (Fig. 5) with the Asian and 324 Afrotropical clades collapsed and the complete chronogram provided in Supplementary Material S7. 325 The divergence time analysis based on the plastid dataset was well resolved and highly supported 326 (Fig. 5, Supplementary Material S7  argyropus clade (Fig. 6)

Phylogenetic relationships 385
This study provided a broad plastid phylogenetic framework for Asian and Australasian sections of 386 Bulbophyllum and revealed a close relationship between sections Adelopetalum and Minutissima s.s., 387 that together form a highly supported early diverging lineage within the genus (Fig. 1, Fig. 2). 388 Relationships based on 70 plastid genes support a sister group relationship between the 389 Adelopetalum/Minutissima clade and the remainder of the genus (Asian + Afrotropical clades). 390 Within the Adelopetalum/Minutissima clade, analyses based on our 70 plastid loci supermatrix 391 showed a dichotomous split between the highly supported Minutissima s.s. and Adelopetalum clades. 392 Species were reconstructed in each of these clades according to their sectional placement except for 393 New results support morphological studies differentiating sect. Minutissima species from Australasia and 407 Asia (Jones and Clements, 2001) and show minute pseudobulbs are a trait that has evolved more than 408 once independently in the genus. 409 Within section Adelopetalum, phylogenetic analyses supported current species concepts, except for 410 species within the argyropus clade, which exhibited shallow genetic differentiation (Fig. 1, Fig. 3). 411 The species of the argyropus clade share morphological affinities and previous taxonomic treatments newportii, and the other uniting B. argyropus, B. bracteatum, and B. elisae (Vermeulen 1993). 425 Phylogenetic relationships based on plastid and nuclear markers found strong to moderate support for 426 the first clade recovered in the cladistic analysis, corresponding to the newportii clade in the present 427 analyses (Fig 1, Fig. 3). The second group found in the cladistics analysis included three species 428 placed in phylogenetic analyses within either the bracteatum clade (B. bracteatum, B. elisae) or the 429 argyropus clade (B. argyropus). However, relationships among these two lineages remained unclear 430 due to low support. The sister group relationship between B. bracteatum and B. argyropus recovered 431 in the cladistic analysis was not supported in phylogenetic reconstructions based on molecular data, 432 suggesting character states shared by these species may be homoplasious. Further studies using 433 ancestral character reconstruction are required to test the phylogenetic utility of morphological traits 434 utilised in prior studies. 435 While plastid phylogenomics has clarified major clades and intraspecific relationships within sect. 436 Adelopetalum in future studies. While assembling datasets with comprehensive species coverage within mega 448 diverse groups such as Bulbophyllum remains a challenge, the present study provides an example of 449 the use of a broad phylogenetic framework with targeted sampling within a section, to test the 450 monophyly and phylogenetic placement of groups of interest. 451

Spatio-temporal evolution of Bulbophyllum sect. Adelopetalum 452
Our divergence time analysis and ancestral range estimations showed that Bulbophyllum sect. 453 Adelopetalum represents an Australasian lineage that originated on the Australian continent during 454 the late Oligocene to early Miocene (Fig. 5, Fig. 6 Our phylogenetic analysis further resolved interspecific relationships in sect. Adelopetalum (Fig. 1). 478 Divergence time estimation showed that divergence among species occurred mainly during the 479 Miocene and Pliocene (Fig. 5) long distance dispersal events were inferred from the Australian continent eastward to the islands of 519 New Zealand and New Caledonia. 520

5
Conflict of Interest 521 The authors declare that the research was conducted in the absence of any commercial or financial 522 relationships that could be construed as a potential conflict of interest.