Wei Wang

Xiao-Guo Xiang

Kun-Li Xiang

Rosa Del C Ortiz

Florian Jabbour

Zhi-Duan Chen

A dated phylogeny of Lardizabalaceae reveals an unusual long-distance dispersal across the Pacific Ocean and the rapid rise of East Asian subtropical evergreen broadleaved forests in the late Miocene

Temperate South American-Asian disjunct distribution is the most unusual in organisms, and challenging to explain. Here, we address the origin of this unusual disjunction in Lardizabalaceae using explicit models and molecular data. The family (approximately 40 species distributed in ten genera) also provides an opportunity to explore the historical assembly of East Asian subtropical evergreen broadleaved forests, a typical and luxuriant vegetation in East Asia. DNA sequences of five plastid loci of 42 accessions representing 23 species of Lardizabalaceae (approximately 57.5% of estimated species diversity), and 19 species from all other six families of Ranunculales, were used to perform phylogenetic analyses. By dating the branching events and reconstructing ancestral ranges, we infer that extant Lardizabalaceae dated to the late Cretaceous of East Asia, and that the temperate South American lineage might have split from its East Asian sister-group at c. 24.4 Ma. A trans-Pacific dispersal possibly by birds from East Asia to South America is plausible to explain the establishment of the temperate South American-East Asian disjunction in Lardizabalaceae. Diversification rate analyses indicate that net diversification rates of Lardizabalaceae experienced a significant increase around c. 7.5 Ma. Our findings suggest that the rapid rise of East Asian subtropical evergreen broadleaved forests occurred in the late Miocene, associated with the uplift of the Tibetan Plateau and the intensified East Asian monsoon, as well as the higher winter temperature and atmospheric CO 2 levels.

Introduction

Intercontinental disjunctions of organisms have fascinated biologists for over a century [START_REF] Raven | Angiosperm biogeography and past continental movement[END_REF], and understanding how these disjunctions were shaped is a central theme in biogeography [START_REF] Lomolino | Biogeography[END_REF]. Trans-equatorial Andean-Asian disjunction in which closely related terrestrial organisms are separated by the entire width of the Pacific Ocean is relatively rare. [START_REF] Maekawa | Floristic relation of the Andes to eastern Asia with special reference to the Trans-Palaeo-Equatorial distribution[END_REF] listed 16 angiosperm groups. Molecular phylogenetic analyses added three, i.e. Gleditsia (Fabaceae; [START_REF] Schnabel | Cladistic biogeography of Gleditsia (Leguminosae) based on ndhF and rpl16 chloroplast gene sequences[END_REF], Chrysosplenium (Saxifragaceae; [START_REF] Soltis | Phylogenetic relationships and evolution in Chrysosplenium (Saxifragaceae) based on matK sequence data[END_REF], and Gaultheria (Ericaceae; [START_REF] Lu | Why is fruit colour so variable? Phylogenetic analyses reveal relationships between fruit-colour evolution, biogeography and diversification[END_REF]. The extreme trans-equatorial Andean-Asian distribution is presented by the organisms occurring in temperate South America and Asia. [START_REF] Thorne | Major disjunctions in the ranges of seed plants[END_REF] considered that this disjunct type was the most unusual and difficult to explain among his sixteen categories of disjunct ranges of intercontinental magnitude.

Lardizabalaceae presents a striking case of temperate South American-Asian disjunct distribution (Fig. 1; [START_REF] Thorne | Major disjunctions in the ranges of seed plants[END_REF]. The family encompasses ten genera and approximately 40 species [START_REF] Qin | A taxonomic revision of the Lardizabalaceae[END_REF][START_REF] Wang | Phylogeny and classification of Ranunculales: Evidence from four molecular loci and morphological data[END_REF]. Two monospecific genera, Lardizabala and Boquila, are endemic to southwestern South America, while the remaining genera and species are restricted to eastern Asia from the Himalayas to Korea and Japan (Fig. 1; [START_REF] Qin | A taxonomic revision of the Lardizabalaceae[END_REF]. Several competing hypotheses have been proposed to explain the current distribution pattern of Lardizabalaceae (Fig. 1). ( 1) "Vicariance" hypothesis, in which the disjunction was caused by the separation of Laurasia and Gondwanaland [START_REF] Maekawa | Floristic relation of the Andes to eastern Asia with special reference to the Trans-Palaeo-Equatorial distribution[END_REF][START_REF] Qin | A taxonomic revision of the Lardizabalaceae[END_REF]. This hypothesis implies that Lardizabalaceae originated and was broadly distributed across the supercontinent before it broke up completely (c. 150 Ma; [START_REF] Mao | Distribution of living Cupressaceae reflects the breakup of Pangea[END_REF], and the African lineage(s) underwent extinction [START_REF] Raven | Angiosperm biogeography and past continental movement[END_REF]. ( 2) "Migration" hypothesis, according to which the family was originally Laurasian and then extended to South America through North America and Central America [START_REF] Raven | Angiosperm biogeography and past continental movement[END_REF][START_REF] Schuster | Plate tectonics and its bearing on the geographical origin and dispersal of angiosperms[END_REF][START_REF] Tiffney | Fruits and seeds of the Tertiary Brandon Lignite. VII. Sargentodoxa (Sargentodoxaceae)[END_REF], or through the East Indies, Australia and Antarctica [START_REF] Tiffney | Fruits and seeds of the Tertiary Brandon Lignite. VII. Sargentodoxa (Sargentodoxaceae)[END_REF]. This implies that fossils or other evidence for occurrences of this family could be found in geographically intermediate areas. The fossils of Sargentodoxa found in the early Miocene and middle Eocene of North America [START_REF] Tiffney | Re-evaluation of the age of the Brandon Lignite (Vermont, USA) based on plant megafossils[END_REF][START_REF] Manchester | Biogeographical relationships of North American Tertiary floras[END_REF] seem to support the former migration route.

However, this will depend on how Sargentodoxa is related to other Lardizabalaceae, especially to the South American genera Lardizabala and Boquila [START_REF] Tiffney | Fruits and seeds of the Tertiary Brandon Lignite. VII. Sargentodoxa (Sargentodoxaceae)[END_REF]. ( 3) "Rafting" hypothesis, based on which the family originated in Gondwanaland and dispersed to Laurasia through the Indian plate as a vector [START_REF] Schuster | Plate tectonics and its bearing on the geographical origin and dispersal of angiosperms[END_REF]. This means that the South American taxa would be early divergent in the family. ( 4) "Long-distance dispersal (LDD)" hypothesis, according to which the current distribution of the family in southern South America is a result of LDD in more recent times [START_REF] Christenhusz | An overview of Lardizabalaceae[END_REF]. However, [START_REF] Thorne | Major disjunctions in the ranges of seed plants[END_REF] suggested that direct LDD between Chile and eastern Asia was rather unlikely given the vast land discontinuity.

These competing hypotheses remain to be tested in a phylogenetic context using explicit models and the absolute age of the temperate South American-Asian disjunction estimated by molecular data.

The overwhelming majority of Lardizabalaceae species are restricted to the East Asian subtropical evergreen broadleaved forests (EBLFs), which mainly occur in c.

23 to 39° N and 97 to 141° E [START_REF] Song | Evergreen broad-leaved forest of East Asia[END_REF], extending westward to 95° E within Tibet (e.g., Chayu and Motuo counties in the Yaluzangbujiang region; [START_REF] Tang | The subtropical vegetation of southwestern China: Plant distribution, diversity and ecology[END_REF]. This range is highly congruent with the distribution area of East Asian Lardizabalaceae (Fig. 1). With the exception of the monospecific Decaisnea, which is an erect shrub, the remaining Lardizabalaceae plants are lianas (woody climbers) [START_REF] Qin | A taxonomic revision of the Lardizabalaceae[END_REF]. Lardizabalaceae is one of the three most dominant liana families in the East Asian subtropical EBLFs [START_REF] Cai | Diversity of vines in subtropical zone of East China[END_REF], which contribute substantially to the local abundance of woody plants [START_REF] Cai | Diversity of vines in subtropical zone of East China[END_REF][START_REF] Tang | The subtropical vegetation of southwestern China: Plant distribution, diversity and ecology[END_REF]. The South American Lardizabala and Boquila also occur in EBLFs [START_REF] Qin | A taxonomic revision of the Lardizabalaceae[END_REF]. Lianas are an important physiognomic and structural component of tropical and subtropical forests [START_REF] Gentry | The distribution and evolution of climbing plants[END_REF][START_REF] Song | Evergreen broad-leaved forest of East Asia[END_REF] and can thereby be regarded as an indicator of both biomes [START_REF] Gentry | The distribution and evolution of climbing plants[END_REF][START_REF] Wang | Menispermaceae and the diversification of tropical rainforests near the Cretaceous-Paleogene boundary[END_REF]. Lardizabalaceae thus offers a good opportunity to investigate the historical assembly of East Asian subtropical EBLFs.

East Asian subtropical EBLF is recognized as a zonal vegetation type or a climatic climax of the central part of the subtropical zone, which developed under the monsoon climate conditions of East Asia [START_REF] Wu | Vegetation of China[END_REF][START_REF] Song | Evergreen broad-leaved forest of East Asia[END_REF]. This vegetation covers large areas of East Asia and harbors high biodiversity and endemism compared to the same latitudinal zones elsewhere on the planet, which are mostly arid and semiarid lands [START_REF] Tang | The subtropical vegetation of southwestern China: Plant distribution, diversity and ecology[END_REF]. East Asian subtropical EBLFs comprise 2600 genera and approximately 14600 species of seed plants, of which more than 50% are endemic [START_REF] Wu | Vegetation of China[END_REF]. Owing to human activities, primary subtropical EBLFs have been largely destroyed in East Asia, particularly in eastern China [START_REF] Tang | The subtropical vegetation of southwestern China: Plant distribution, diversity and ecology[END_REF].

Paleovegetational reconstructions and molecular phylogenetics have contributed to our understanding of the origin of East Asian subtropical EBLFs. Some paleovegetations of Yunnan in southwestern China assigned to the Miocene were suggested as reminiscence of subtropical EBLFs [START_REF] Jacques | Reconstruction of Neogene zonal vegetation in South China using the Integrated Plant Record (IPR) analysis[END_REF]. Nevertheless, recent U-Pb zircon dates show that these vegetations in southwestern China might have been established by the early Oligocene, c. 33 Ma [START_REF] Linnemann | New U/Pb dates show a Paleogene origin for the modern Asian biodiversity hot spots[END_REF]. Dated phylogeny of Dendrobium (Orchidaceae), an epiphytic element of Asian EBLFs, has also supported the view that the EBLFs have existed in mainland Asia at least since the beginning of the Oligocene [START_REF] Xiang | Biogeographical diversification of mainland Asian Dendrobium (Orchidaceae) and its implications for the historical dynamics of evergreen broad-leaved forests[END_REF]. Using Theaceae as a proxy, [START_REF] Yu | Insights into the historical assembly of East Asian subtropical evergreen broadleaved forests revealed by the temporal history of the tea family[END_REF] suggested that the progressive development of the subtropical EBLFs in East Asia occurred around the Oligocene-Miocene boundary and was driven by the intensification of East Asian monsoon (EAM). [START_REF] Yu | Insights into the historical assembly of East Asian subtropical evergreen broadleaved forests revealed by the temporal history of the tea family[END_REF] also detected an increase of diversification rate in the Theaceae that occurred in the latest Miocene (5.4-5.7 Ma; crown of Schima).

During the Miocene, the climate and vegetation of East Asia, as well as other regions on the planet, were undoubtedly altered dramatically, in association with the Indo-Eurasian collision-induced tectonic activities from the Neogene onward [START_REF] Zachos | Trends, rhythms, and aberrations in global climate 65 Ma to present[END_REF][START_REF] Guo | Onset of Asian desertification by 22 Myr ago inferred from loess deposits in China[END_REF][START_REF] Liu | Where were the monsoon regions and arid zones in Asia prior to the Tibetan Plateau uplift?[END_REF]. In particular, the significant enhancements of the EAM occurred in the Miocene [START_REF] Guo | Onset of Asian desertification by 22 Myr ago inferred from loess deposits in China[END_REF]. However, the historical assembly of East Asian subtropical EBLFs in the Miocene remains unclear.

Here, we reconstruct the most comprehensive phylogenetic tree for Lardizabalaceae to date based on five plastid DNA regions. By integrating phylogenetic, molecular dating and biogeographic methods, we then test the aforementioned four hypotheses explaining of the temperate South American-East Asian disjunct distribution. Finally, we use Lardizabalaceae as a proxy to investigate the assembly history of East Asian subtropical EBLFs, namely we attempt to evaluate whether East Asian subtropical EBLFs might have experienced a rapid rise during the Miocene, associated with the intensified EAM.

Materials and methods

Taxon sampling and DNA sequencing

We sampled 23 species from 40 of Lardizabalaceae (Ranunculales), representing all ten genera recognized in the family [START_REF] Qin | A taxonomic revision of the Lardizabalaceae[END_REF][START_REF] Wang | Phylogeny and classification of Ranunculales: Evidence from four molecular loci and morphological data[END_REF]. Outgroups included nineteen species covering all other six families of Ranunculales (Table S1).

The samples were collected from the field or from herbarium specimens. Based on previous studies [START_REF] Wang | Phylogeny and classification of Ranunculales: Evidence from four molecular loci and morphological data[END_REF][START_REF] Sun | Complete plastome sequencing of both living species of Circaeasteraceae (Ranunculales) reveals unusual rearrangements and the loss of the ndh gene family[END_REF], the tree was rooted with Euptelea polyandra (Eupteleaceae), the earliest-diverging lineage in the order. Five plastid loci were used: rbcL, matK, and ndhF genes, trnL-F regions (trnL intron, and trnL [UAA] 3' exon-trnF [GAA] intergenic spacer), and rpS16 intron. Each species was sequenced for the five DNA loci except for Kingdonia uniflora, whose ndhF gene is absent [START_REF] Sun | Complete plastome sequencing of both living species of Circaeasteraceae (Ranunculales) reveals unusual rearrangements and the loss of the ndh gene family[END_REF]. Voucher information and GenBank accession numbers are listed in Table S1.

Total genomic DNA was extracted from silica-gel-dried leaves or herbarium material using the NuClean PlantGen DNA Kit (CWBIO, Beijing, China). The primers used in this study are listed in Table S2. Sequencing reactions were conducted using the ABI Prism Bigdye Terminator Cycle Sequencing Kit (Applied Biosystems, ABI) and analyzed using ABI 3730xl automated DNA sequencers.

Phylogenetic analyses

Sequences were aligned using CLUSTAL X [START_REF] Thompson | The Clustal X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools[END_REF], and further adjusted manually in BioEdit v5.0.9 [START_REF] Hall | BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. Five difficult-to-align regions in trnL-F (289 sites) and two difficult-to-align regions in rpS16 (36 sites) were removed from the analyses. The aligned lengths of the rbcL, matK, ndhF, trnL-F, and rpS16 sequences were 1395, 1263, 2136, 1232, and 1236 bp, respectively, resulting in a concatenated 42-taxon alignment of 7262 bp. We first conducted maximum likelihood (ML) searches using RAxML v8.0 [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF] for each of the five loci. As there were no significantly conflicting nodes (> 85% bootstrap support), all subsequent analyses were carried out for the concatenated dataset only. Phylogenetic analyses for the concatenated dataset were performed using ML, Bayesian inference (BI), and maximum parsimony (MP) methodologies in RAxML v8.0 [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF], MrBayes v3.2.5 [START_REF] Ronquist | MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space[END_REF], and PAUP* v4.0b10 [START_REF] Swofford | PAUP*: Phylogenetic analysis using parsimony (*and other methods)[END_REF], respectively. For ML analysis, the GTR + Γ substitution model was assigned for each region. For BI analysis, each region was assigned its own model of nucleotide substitution, as determined by the Akaike Information Criterion (AIC) in jModeltest v2.1.4 [START_REF] Posada | jModelTest: phylogenetic model averaging[END_REF]. The detailed descriptions of these three analytical methods can be found in [START_REF] Wang | New insights into the phylogeny of Burasaieae (Menispermaceae) with the recognition of a new genus and emphasis on the southern Taiwanese and mainland Chinese disjunction[END_REF].

Divergence time estimation

Divergence times were estimated using BEAST v2.4.0 [START_REF] Bouckaert | Beast 2: a software platform for Bayesian evolutionary analysis[END_REF].

To convert stratigraphic ages into absolute ages, we used the geological time scale of Cohen et al. (2013, updated). Four fossils confidently placed in our tree were selected as minimum-age constraints (Fig. 2). ( 1) Decaisnea bornensis, seeds from the late Oligocene of Germany, corresponds to the extant Decaisnea in morphology, including placement and size of the hilum, raphe and micropylar point and in anatomy of the seed coat [START_REF] Mai | Die oligozänen und untermiozänen Floren NW-Sachsens und des Bitterfelder Raumes[END_REF]. The stem group age of Decaisnea was given a lower bound of 23.03 Ma (C 1 ). ( 2) Sargentodoxa globosa from the middle Eocene of Oregon belongs to the extant Sargentodoxa based on its ovoid seeds with an oblique hilar facet and a thick seed coat and the placement of the raphe, microphyle, and chalaza [START_REF] Manchester | Biogeographical relationships of North American Tertiary floras[END_REF]. The stem group node of Sargentodoxa was given a lower bound of 41.2 Ma (C 2 ). (3) A fossil flower, Kajanthus lusitanicus, was reported from the Early Cretaceous (late Aptian-early Albian, 110 Ma) in the Chicalhão site, western Portugal [START_REF] Mendes | Fossil Kajanthus lusitanicus gen. et sp. nov. from Portugal: floral evidence for Early Cretaceous Lardizabalaceae (Ranunculales, basal eudicot)[END_REF]. Phylogenetic analysis suggested that Kajanthus was close to Sinofranchetia, but given that the fossil could be connected as well to any deeper branch of Lardizabalaceae without increasing the number of reconstructed character changes [START_REF] Mendes | Fossil Kajanthus lusitanicus gen. et sp. nov. from Portugal: floral evidence for Early Cretaceous Lardizabalaceae (Ranunculales, basal eudicot)[END_REF], we used this fossil to constrain the stem group age of Lardizabalaceae (C 3 ). (4) Leefructus mirus described from the Early Cretaceous Yixian Formation in China (122.6 Ma;[START_REF] Sun | A eudicot from the Early Cretaceous of China[END_REF]. Based on a phylogenetic analysis, Leefructus was placed as sister to the Ranunculaceae [START_REF] Wang | Accelerated evolution of early angiosperms: Evidence from ranunculalean phylogeny by integrating living and fossil data[END_REF] and was used to constrain the stem group age of the family (C 4 ). Given phylogenetic uncertainty of fossil calibrations, we conservatively enforced all four fossil calibrations with a uniform prior distribution [START_REF] Mao | Distribution of living Cupressaceae reflects the breakup of Pangea[END_REF]. To avoid overestimation of root age, we set a 131.7 Ma (95% highest posterior density (HPD): 129.7-133.4 Ma) maximum age for the root (C 5 ), which is the estimated crown group age of eudicots [START_REF] Magallón | A meta-calibrated timetree documents the early rise of flowering plant phylogenetic diversity[END_REF], with a normal distribution (Fig. 2: analysis 1).

For comparison, we also constrained the root age to be 153 Ma (138-172 Ma) (Fig. 2: analysis 2) based on [START_REF] Smith | An uncorrelated relaxed-clock analysis suggests an earlier origin for flowering plants[END_REF]. This age is highly congruent with those estimated by [START_REF] Harris | A complete fossil-calibrated phylogeny of seed plant families as a tool for comparative analyses: Testing the 'Time for Speciation' hypothesis[END_REF] and [START_REF] Salomo | The emergence of earliest angiosperms may be earlier than fossil evidence indicates[END_REF] without the oldest tricolpate pollen calibration. To evaluate the impact of the Early Cretaceous Kajanthus on divergence times of Lardizabalaceae, dating analyses were also run with the exclusion of this calibration (Fig. 2: analyses 3-4). To minimize rate heterogeneity for the dating analysis, we removed two taxa with long branches (Fig. S1), Circaeaster agrestis and Ranunculus macranthus, and then conducted the same four dating analyses using the 40-taxon data matrix (Fig. 2: analyses 5-8). We also estimated divergence times by excluding the Early Cretaceous Leefructus (analyses 9-12).

All dating analyses were performed under an uncorrelated lognormal clock model, a birth-death prior process for incomplete sampling, and the GTR + I + Γ model for each region separately. Two distinct runs were conducted with 50 million generations and trees sampled every 5,000 th generations. Convergence and ESS > 200 were assessed using Tracer v1.6 [START_REF] Rambaut | Tracer v1.6[END_REF]. The maximum clade credibility (MCC) tree, median ages and their 95% HPD were generated using TreeAnnotator v1.8.2 (part of the BEAST package). Among these twelve dating analyses, divergence times for Lardizabalaceae are highly congruent (Figs. 2 andS4; see below Results).

Biogeographic analyses

Lardizabalaceae once occurred in regions outside its present-day distribution range (Fig. 1; Table S3). For example, Sargentodoxa was found in North America and Europe [START_REF] Tiffney | Fruits and seeds of the Tertiary Brandon Lignite. VII. Sargentodoxa (Sargentodoxaceae)[END_REF][START_REF] Wilde | The Middle Eocene plant taphocoenosis from Eckfeld (Eifel, Germany)[END_REF][START_REF] Manchester | Biogeographical relationships of North American Tertiary floras[END_REF][START_REF] Mai | Die mittelmiozaenen und obermiozaenen Floren aus der Meuroer und Raunoer Folge in der Lausitz[END_REF] and Decaisnea was discovered in Europe [START_REF] Mai | Zur Bedeutung von Relikten in der Florengeschichte[END_REF]. Akebia parvisemina was reported by [START_REF] Mai | Die mittelmiozaenen und obermiozaenen Floren aus der Meuroer und Raunoer Folge in der Lausitz[END_REF] from the late Miocene of Germany, but it lacks anatomical details and diagnostic features for extant Akebia seeds, therefore its identity is uncertain [START_REF] Manchester | Eastern Asian endemic seed plant genera and their paleogeographic history throughout the Northern Hemisphere[END_REF]. Four geographical areas were coded to cover distributions of all extant and fossil taxa of Lardizabalaceae (Fig. 1): (A) East Asia, (B) South America, (C) North America, and (D) Europe. Due to the challenges in scoring other under-sampled families for geographic areas, we restricted biogeographic analysis to Lardizabalaceae and its sister Circaeasteraceae (this study; [START_REF] Wang | Phylogeny and classification of Ranunculales: Evidence from four molecular loci and morphological data[END_REF][START_REF] Sun | Complete plastome sequencing of both living species of Circaeasteraceae (Ranunculales) reveals unusual rearrangements and the loss of the ndh gene family[END_REF].

To infer the location of the disjunction between eastern Asian and South American lineages, ancestral areas were reconstructed using the R package BioGeoBEARS v0.2.1 [START_REF] Matzke | Probabilistic historical biogeography: new models for founder-event speciation, imperfect detection, and fossils allow improved accuracy and model-testing[END_REF]. We tested the three models (DEC, DIVALIKE, and BAYAREALIKE) including or not a "jump dispersal" parameter J that permits founder-event speciation in the species's history. Founder-event speciation is the result of a LDD event of individual lineages founding a population outside the ancestral range [START_REF] Matzke | Model selection in historical biogeography reveals that founder-event speciation is a crucial process in island clades[END_REF]. The fit for the different models was evaluated using the AIC values. The seeds of Lardizabalaceae are embedded in a sweet, gelatinous, white pulp and can be dispersed by birds and other animals [START_REF] Qin | A taxonomic revision of the Lardizabalaceae[END_REF], so we did not constrain the availability of connections between areas.

Diversification rate analyses

We used two R packages with the BEAST MCC tree from which all outgroups were pruned to investigate the temporal dynamics of diversification rates of Lardizabalaceae. First, we employed Bayesian analysis of macro-evolutionary mixture (BAMM) implemented in BAMM v2.5 [START_REF] Rabosky | Automatic detection of key innovations, rate shifts, and diversity-dependence on phylogenetic trees[END_REF] to infer speciation and extinction rates, as well as the possible shifts of speciation across the phylogeny.

Missing taxa were taken into account by using a fraction of total species in a clade (see details in Fig. S1). We performed two independent runs on the MCC tree with a reversible jump MCMC run of 10 million generations, sampling parameters every 1000 th generations. Following the suggestion of [START_REF] Rabosky | BAMMtools: an R package for the analysis of evolutionary dynamics on phylogenetic trees[END_REF] for small trees (< 500 tips), we used a Poisson prior value of 1.0 for the 'ExpectedNumberofShifts' that implies a null hypothesis of zero rate shifts across the phylogeny. ESS values were calculated in the R package CODA (> 200). The BAMM output files were analyzed by discarding 15% burn-in samples using the R package BAMMtools v2.0.2 [START_REF] Rabosky | BAMMtools: an R package for the analysis of evolutionary dynamics on phylogenetic trees[END_REF]. The posterior distribution of the BAMM analysis was used to estimate the distinct rate shift configurations within the 95% credible set. We also used the plotRateThroughTime function in BAMMtools to plot speciation, extinction and net diversification rates.

Second, we used MEDUSA [START_REF] Alfaro | Nine exceptional radiations plus high turnover explain species diversity in jawed vertebrates[END_REF] implemented in Geiger v1.3-1 [START_REF] Harmon | GEIGER: investigating evolutionary radiations[END_REF] to infer rate shifts. As in the BAMM analysis, the missing taxa were assigned to monophyletic terminal clades (Fig. S1). Because the set of a cut-off value for a significant change in ∆AIC can influence the inferred number of shifts [START_REF] Alfaro | Nine exceptional radiations plus high turnover explain species diversity in jawed vertebrates[END_REF], we selected the model with the lowest AIC score.

Results

Phylogenetic analyses

Three phylogenetic analyses (ML, BI and MP) recovered nearly identical topologies with overall strong node support in particular at the backbone (Fig. S1).

Circaeasteraceae containing Circaeaster and Kingdonia is strongly supported as sister to Lardizabalaceae (MP-BS = 96%, ML-BS = 99%, PP = 1.0). The monophyletic Lardizabalaceae is strongly supported (MP-BS = 100%, ML-BS = 100%, PP = 1.0). Within Lardizabalaceae, Sargentodoxa is sister to the remaining taxa of the family, followed by Decaisnea, and then Sinofranchetia. Each of Lardizabaleae and Akebieae is supported as monophyletic. Akebia is monophyletic with strong support (MP-BS = 92%, ML-BS = 97%, PP = 1.0), whereas Holboellia, Parvatia and Stauntonia are not monophyletic.

Divergence time estimates and biogeographic analyses

Divergence time estimates based on the different ages of the root are highly consistent (analyses 1 vs. 2, 3 vs. 4, 5 vs. 6, and 7 vs. 8). The results with and without the Early Cretaceous Kajanthus as a calibration point are congruent (analyses 1 vs. 3, 2 vs. 4, 5 vs. 7, and 6 vs. 8). The inclusion of two long-branch taxa did not alter the results significantly (analyses 1 vs. 5, 2 vs. 6, 3 vs. 7, and 4 vs. 8). A comparison of the time estimates for selected nodes using the eight different analyses (1-8) is shown in Fig. 2. The exclusion of the Early Cretaceous Leefructus did not alter the results significantly (analyses 1 vs. 9, 2 vs. 10, 5 vs. 11, and 6 vs. 12). Detailed comparisons among divergence times estimated in analysis 2 and analyses 9-12 are shown in Table S4.

Results from the BEAST analysis 2 with the root set as 153 Ma, three calibrating fossils, and 42 taxa are here reported and were used for biogeographical and diversification rate analyses. The chronogram resulting from the analysis 2 is shown in Fig. 2. Ancestral area reconstructions using the DEC and DIVALIKE models including or not a parameter J generated congruent results, although DIVA LIKE was identified as the best-fitting model for our data (Table S5). Results from the ancestral area reconstructions conducted for Lardizabalaceae under the DIVALIKE model are indicated in Fig. 3a. The crown age of Lardizabalaceae was estimated at 76.9 Ma (95% HPD: 45.98-109.3). The most recent common ancestor of the family was most likely distributed in East Asia. A dispersal event was inferred from East Asia to South America (nodes 1/2). The age of node 1 was estimated at 40.61 Ma (95% HPD: 21.62-63.98). The age for the split of the South American Lardizabaleae containing Boquila and Lardizabala and its East Asian sister group (Akebieae) was estimated at 24.4 Ma (95% HPD: 12.23-41.31; node 2). The crown age of Akebieae was estimated at 7.49 Ma (95% HPD: 3.76-13.77; node 3).

Diversification rate analyses

The phylorate plot of the speciation rate obtained from BAMM analysis indicated diversification heterogeneity within Lardizabalaceae (Fig. 3b). One acceleration of diversification rates was detected at the crown of Akebieae (c. 7.5 Ma) by sampling the maximum a posteriori configuration with the highest frequency (f = 0.97; Fig. 3b), and its net diversification rate is 0.24 species per million years (myr), whereas the background net rate is 0.0446. The rate-through-time plots suggested that the global speciation and net diversification rates of Lardizabalaceae significantly accelerated at the same time (Fig. 4). Comparing pure-birth versus birth-death models, MEDUSA favored the pure-birth model (AICc = 78.66). Under this model, MEDUSA detected one shift in net diversification rates (Fig. 3a) relative to background level in Lardizabalaceae (r = 0.0179), i.e. a shift at the crown of Akebieae (r = 0.3607).

Discussion

Phylogeny and divergence times of Lardizabalaceae

Our analyses supported Circaeasteraceae as the sister group of Lardizabalaceae, in agreement with previous phylogenetic studies (e.g., [START_REF] Wang | Phylogeny and classification of Ranunculales: Evidence from four molecular loci and morphological data[END_REF][START_REF] Sun | Complete plastome sequencing of both living species of Circaeasteraceae (Ranunculales) reveals unusual rearrangements and the loss of the ndh gene family[END_REF]. Our results suggested that Sargentodoxa was sister to all the other Lardizabalaceae, followed by Decaisnea, and the South American Lardizabaleae is sister to the East Asian Akebieae. These relationships are in agreement with previous results [START_REF] Hoot | The utility of atpB genes sequences in resolving phylogenetic relationships: Comparison with rbcL and 18S ribosomal DNA sequences in the Lardizabalaceae[END_REF][START_REF] Wang | Molecular phylogeny of the Lardizabalaceae based on trnL-F sequences and combined chloroplast data[END_REF][START_REF] Wang | Phylogeny and classification of Ranunculales: Evidence from four molecular loci and morphological data[END_REF], but are usually resolved here with greater support.

Our twelve dating analyses generated highly congruent time estimates for Lardizabalaceae (Figs. 2 andS4). Analysis 2 suggests a crown group age of 76.9 Ma (95% HPD: 45.98-109.3; node 1) for the family, which is older than the estimate of [START_REF] Wefferling | Dated phylogeography of western North American subalpine marshmarigolds (Caltha spp., Ranunculaceae): Miocene-Pliocene divergence of hexaploids, multiple origins of allododecaploids during the Pleistocene, and repeated recolonization of Last Glacial Maximum glaciated regions[END_REF]44.88 Ma), but our credibility interval overlapped with the assumptions of [START_REF] Anderson | Dating phylogenetically basal eudicots using rbcL sequences and multiple fossil reference points[END_REF], 66 Ma and 95 Ma inferred by penalized likelihood and nonparametric rate smoothing methods, respectively. [START_REF] Wefferling | Dated phylogeography of western North American subalpine marshmarigolds (Caltha spp., Ranunculaceae): Miocene-Pliocene divergence of hexaploids, multiple origins of allododecaploids during the Pleistocene, and repeated recolonization of Last Glacial Maximum glaciated regions[END_REF] estimated the split time between Lardizabaleae and Akebieae and the crown age of Akebieae at 18.63 Ma and 7.86 Ma, respectively, which are in agreement with our results: 24.4 Ma (95% HPD: 12.23-41.31; node 2) and 7.49 Ma (95% HPD: 3.76-13.77; node 3). There is no a temporal conflict between the South American lineage and the Tertiary wood record from Patagonia [START_REF] Schönfeld | Über eine fossile Liane aus Patagonien[END_REF], although the exact age of the fossil remains unclear.

A plausible explanation of the temperate South American-East Asian disjunction in

Lardizabalaceae

Our study suggests a late Oligocene origin of the temperate South American-East Asian disjunct distribution in Lardizabalaceae, and eastern Asia as the source for colonization of temperate South America. Several proposed hypotheses that might explain the cause of the extreme trans-pacific disjunction in the family are discussed below:

1. One possibility is that Lardizabalaceae was distributed across the supercontinent before the complete separation of Laurasia and Gondwanaland [START_REF] Maekawa | Floristic relation of the Andes to eastern Asia with special reference to the Trans-Palaeo-Equatorial distribution[END_REF][START_REF] Qin | A taxonomic revision of the Lardizabalaceae[END_REF]. Our estimated crown group age for the family (c. 76.9 Ma) is obviously younger than the timing of their separation (c. 150 Ma; [START_REF] Mao | Distribution of living Cupressaceae reflects the breakup of Pangea[END_REF]. We find no data in the published fossil records indicating that Lardizabalaceae ever occurred in Africa, as would be expected if its current distribution was to be attributed to vicariance event between Laurasia and Gondwanaland [START_REF] Raven | Angiosperm biogeography and past continental movement[END_REF]. Thus, the vicariance hypothesis between Laurasia and Gondwanaland could be rejected.

2. The present-day distribution of Lardizabalaceae can come into existence if the family originated in Gondwanaland and then spread to Laurasia through the Indian plate [START_REF] Schuster | Plate tectonics and its bearing on the geographical origin and dispersal of angiosperms[END_REF]. However, our biogeographical analyses show that Lardizabalaceae originated in eastern Asia (Fig. 3a), in agreement with most authors' views (e.g., [START_REF] Raven | Angiosperm biogeography and past continental movement[END_REF][START_REF] Tiffney | Fruits and seeds of the Tertiary Brandon Lignite. VII. Sargentodoxa (Sargentodoxaceae)[END_REF][START_REF] Wu | The families and genera of angiosperms in China: A comprehensive analysis[END_REF]. Moreover, our phylogenetic analyses indicate that the South American Lardizabaleae is nested within the eastern Asian taxa (Fig. S1). Additionally, the estimated age of 24.4 Ma for the divergence of Lardizabaleae from the East Asian ancestor also makes it impossible to invoke the Indian plate as a vector for this group, which would have required an estimated date of divergence at least as early as 65 Ma [START_REF] Mcloughlin | The breakup history of Gondwana and its impact on pre-Cenozoic floristic provincialism[END_REF]).

Another potential explanation for the present-day distribution of

Lardizabalaceae has to do with two likely migration routes. One is the migration through the boreotropical route to North America and then southward to South America [START_REF] Raven | Angiosperm biogeography and past continental movement[END_REF][START_REF] Schuster | Plate tectonics and its bearing on the geographical origin and dispersal of angiosperms[END_REF][START_REF] Tiffney | Fruits and seeds of the Tertiary Brandon Lignite. VII. Sargentodoxa (Sargentodoxaceae)[END_REF]. Our dating analysis indicates that the split between the South American lineage and its eastern Asian group occurred at 24.4 Ma, which is younger than the timing of disruption of the boreotropical flora, c. 34 Ma [START_REF] Collinson | Vegetational and floristic changes around the Eocene/Oligocene boundary in western and central Europe[END_REF]. Moreover, no fossils or other evidence for occurrences of Lardizabalaceae were found in Central America and northern South America. Thus, this northern migration route seems just unlikely.

An alternative migration route was through the East Indies, Australia, and Antarctica to South America. The split time between the South American and East Asian groups was estimated at 24.4 Ma, which is slightly younger than the opening of the Drake Passage between South America and Antarctica (ca. 30-35 Ma;[START_REF] Mcloughlin | The breakup history of Gondwana and its impact on pre-Cenozoic floristic provincialism[END_REF]. Since the late Eocene, global climate was cooling [START_REF] Zachos | Trends, rhythms, and aberrations in global climate 65 Ma to present[END_REF], thermophilic taxa could therefore not migrate through Antarctica [START_REF] Lian | A multi-locus phylogeny for the Neotropical Anomospermeae (Menispermaceae): Implications for taxonomy and biogeography[END_REF]. Furthermore, no fossil representative of this family was found in the geographically intermediate areas. This route also appears unlikely.

4. The direct trans-pacific LDD from East Asia to temperate South America (Fig. 3a) seems to be more parsimonious and plausible than the other competing hypotheses.

Fruits of Lardizabalaceae are berries or fleshy follicles filled with whitish and sweet gelatinous pulp (Fig. 1), which are highly favored by birds and other animals [START_REF] Qin | A taxonomic revision of the Lardizabalaceae[END_REF]. The edible fruits of Stauntonia, Akebia and Lardizabala are consumed by humans and even sold on local markets [START_REF] Christenhusz | An overview of Lardizabalaceae[END_REF]. In the family, seeds with hard seed coat are usually small (< 1 cm; [START_REF] Qin | A taxonomic revision of the Lardizabalaceae[END_REF], and can therefore be carried for long distances by animals. Recently, a mid-Miocene LDD event from East Asia to temperate South America was found in Gaultheria (13.57-15.11 Ma;Ericaceae;[START_REF] Lu | Why is fruit colour so variable? Phylogenetic analyses reveal relationships between fruit-colour evolution, biogeography and diversification[END_REF], whose fruits are also favored by birds. By using miniature archival geolocating tags, [START_REF] Shaffer | Migratory shearwaters integrate oceanic resources across the Pacific Ocean in an endless summer[END_REF] found that a small seabird, the sooty shearwater (Ardenna grisea, Procellariidae), can fly across the entire Pacific Ocean.

Bird-mediated LDD has been shown to be the best explanation for the establishment of the bipolar disjunction, another huge geographical discontinuity (e.g., [START_REF] Popp | A single Mid-Pleistocene long-distance dispersal by a bird can explain the extreme bipolar disjunction in crowberries (Empetrum)[END_REF][START_REF] Lewis | Direct long-distance dispersal shapes a New World amphitropical disjunction in the dispersal-limited dung moss Tetraplodon (Bryopsida: Splachnaceae)[END_REF].

Diversification of East Asian subtropical EBLFs in the late Miocene

Our analyses show that extant Lardizabalaceae date to the Late Cretaceous of East Asia (Fig. 2), but a significant increase of net diversification rates of the family occurred relatively recently, beginning in the late Miocene of East Asia, c. 7.5 Ma (95% HPD: 3.76-13.77; Fig. 3). The timing of the rapid diversification within Lardizabalaceae is similar to those of other representative plant groups in East Asian subtropical EBLFs, such as woody bamboos (c. 7.2 Ma; Arundinarieae, Poaceae; [START_REF] Zhang | Multi-locus plastid phylogenetic biogeography supports the Asian hypothesis of the temperate woody bamboos (Poaceae: Bambusoideae)[END_REF] and Quercus sect. Cyclobalanopsis (c. 8.3 Ma;Fagaceae;Deng et al., 2017). Additionally, several elements of East Asian subtropical EBLFs, such as Camellia (c. 6.8 Ma; Theaceae; [START_REF] Yu | Insights into the historical assembly of East Asian subtropical evergreen broadleaved forests revealed by the temporal history of the tea family[END_REF]; Coptis (c. 6.5 Ma; Ranunculaceae; Xiang et al., 2018), and Sabia (c. 7.9 Ma;Sabiaceae;[START_REF] Yang | Boreotropical range expansion and long-distance dispersal explain two amphi-Pacific tropical disjunctions in Sabiaceae[END_REF], also appear to have diverged during the same period. We therefore suggest that the late Miocene was a relevant period for the assembly and evolution of modern East Asian subtropical EBLFs.

Several potential factors might have been responsible for the diversification burst of East Asian subtropical EBLFs during the late Miocene (Fig. 4). The rise of East Asian subtropical EBLFs has been attributed to intensified EAM [START_REF] Yu | Insights into the historical assembly of East Asian subtropical evergreen broadleaved forests revealed by the temporal history of the tea family[END_REF], which was in turn related to the uplift of the Tibetan Plateau [START_REF] Liu | Where were the monsoon regions and arid zones in Asia prior to the Tibetan Plateau uplift?[END_REF]. The accelerated uplift of the Plateau, especially its northeastward expansion could have enhanced the EAM, as well as the aridification of inland Asia [START_REF] An | Changes of the monsoon-arid environment in China and growth of the Tibetan Plateau since the Miocene[END_REF].

Thermochronometry and magnetostratigraphy studies revealed that the northeastern Tibetan Plateau experienced apparent tectonic activities in 8-7 Ma [START_REF] Shen | History of Asian eolian input to the Sea of Japan since 15 Ma: Links to Tibetan uplift or global cooling? Earth Planet[END_REF][START_REF] Fang | Cenozoic magnetostratigraphy of the Xining Basin, NE Tibetan Plateau, and its constraints on paleontological, sedimentological and tectonomorphological evolution[END_REF], in agreement with the intensification of the EAM inferred from dust accumulation rate in northern China [START_REF] Guo | Onset of Asian desertification by 22 Myr ago inferred from loess deposits in China[END_REF], that can bring high precipitation in summer. The χ fd /HIRM (frequency-dependent magnetic susceptibility/hard isothermal remanent magnetization) record also suggests intensified East Asian summer monsoon precipitation during c. 8.5 to 7 Ma [START_REF] Nie | Dominant 100,000-year precipitation cyclicity in a late Miocene lake from northeast Tibet[END_REF]. The relationship between rainfall and diversification rates of plant groups in East Asian subtropical EBLFs remains unknown, but based on the distribution maps of 11,405 woody species in China, [START_REF] Wang | Patterns, determinants and models of woody plant diversity in China[END_REF] indicated that annual rainfall is the best single water-related predictor for the species richness of woody plants in China.

The major limiting factor for the distribution of East Asian subtropical EBLFs is low temperature in the winter [START_REF] Song | Evergreen broad-leaved forest of East Asia[END_REF]. Although cooling has been a general trend from the mid-Miocene Climatic Optimum onwards [START_REF] Zachos | Trends, rhythms, and aberrations in global climate 65 Ma to present[END_REF], palynological data in Southwest China indicate that the temperature, especially the mean temperature of the coldest month, has gradually increased since the Middle Miocene [START_REF] Zhang | Vegetation and climatic changes of SW China in response to the uplift of Tibetan Plateau[END_REF]. Similar to the temperature, atmospheric CO 2 levels have an effect on biodiversity in humid, warm forests [START_REF] Royer | Diversity in neotropical wet forests during the Cenozoic is linked more to atmospheric CO 2 than temperature[END_REF][START_REF] Lian | A multi-locus phylogeny for the Neotropical Anomospermeae (Menispermaceae): Implications for taxonomy and biogeography[END_REF]. High atmospheric CO 2 levels occurred in the late Miocene [START_REF] Tripati | Coupling of CO 2 and ice sheet stability over major climate transitions of the last 20 million years[END_REF], which might also have driven the rapid rise of East Asian subtropical EBLFs during that period.

Conclusions

Molecular dating, together with phylogenetic and biogeographical analyses, indicates a late Oligocene origin of the temperate South American-East Asian disjunct distribution in Lardizabalaceae. Our findings suggest that a bird-mediated LDD event across the Pacific is the most plausible explanation for the present-day disjunct distribution of Lardizabalaceae between temperate South America and East Asia, and possibly also in other plant groups exhibiting a temperate South American-East Asian disjunction. In addition, our data suggest that a burst of diversification of East Asian subtropical EBLFs occurred in the late Miocene, promoted by the intensified EAM and other environmental factors. This study may provide new insights into the historical assembly of East Asian subtropical EBLFs. S3. The dashed lines indicate the main hypothesized colonization routes towards South America (see details in Text).

Image of berries full of whitish and sweet gelatinous pulp of Akebia quinata is presented in the low left, photograph by W Wang. [START_REF] Shen | History of Asian eolian input to the Sea of Japan since 15 Ma: Links to Tibetan uplift or global cooling? Earth Planet[END_REF], global temperature inferred from δ 18 O levels in benthic foraminifera [START_REF] Zachos | Trends, rhythms, and aberrations in global climate 65 Ma to present[END_REF], the mean temperature of the coldest month (MCMT) reconstructed from palynological data in Yunnan, southwest China [START_REF] Zhang | Vegetation and climatic changes of SW China in response to the uplift of Tibetan Plateau[END_REF], intensification of the East Asian monsoon (EAM) inferred from dust accumulation rate (DAR) in northern China [START_REF] Guo | Onset of Asian desertification by 22 Myr ago inferred from loess deposits in China[END_REF], and atmospheric CO 2 measured from boron/calcium ratios of surface-dwelling foraminifera compared with climate records [START_REF] Tripati | Coupling of CO 2 and ice sheet stability over major climate transitions of the last 20 million years[END_REF]. For Lardizabalaceae, speciation, extinction and net diversification rates through time were obtained from the BAMM analysis carried out under the 'ExpectedNumberofShifts' as 1 based on the maximum clade credibility (MCC) tree of scenario 2 of BEAST analyses. Color density shading denotes confidence in rate reconstructions.

Fig. 1 .

 1 Fig. 1. Modern (grey areas) and fossil (numbers) distribution of Lardizabalaceae. 1-6

Fig. 2 .

 2 Fig. 2. Time-calibrated phylogeny of Lardizabalaceae using all four fossil calibration

Fig. 3 .

 3 Fig. 3. Combined biogeographic and diversification rate analyses of Lardizabalaceae.

Fig. 4 .

 4 Fig. 4. Potential factors responsible for the diversification burst of East Asian

Acknowledgements

We sincerely thank Hai-Ning Qin for identification of plant materials and discussion of the fossils used in this study. We also thank An-Min Lu for identifying plant materials and reading an earlier version of this manuscript. This research was partially funded by the Strategic Priority Research Program of Chinese Academy of Sciences (XDB31000000) and the National Natural Science Foundation of China (31770231, 31770233 and 31470315).


Lian Lian

Rosa Del C Ortiz

Florian Jabbour

Cai-Fei Zhang

Xiao-Guo Xiang

Andrey S Erst

Tian-Gang Gao

Zhi-Duan Chen

Wei Wang

Rosa Del

C Ortiz

Phylogeny and biogeography of Pachygoneae (Menispermaceae), with consideration of the boreotropical flora hypothesis and resurrection of the genera Cebatha and Nephroia

Keywords: Amphi-Pacific disjunction, Biogeography, Boreotropics, Cocculus, Menispermaceae, Phylogeny

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Understanding how intercontinental disjunct distributions of organisms have been shaped is a fundamental question in biogeography [START_REF] Raven | Plant species disjunctions: a summary[END_REF][START_REF] Lomolino | Biogeography[END_REF]. The amphi-Pacific tropical disjunction has fascinated biogeographers and palaeoecologists since van Steenis and Thorne (van Steenis, 1962;[START_REF] Thorne | Major disjunctions in the geographic ranges of seed plants[END_REF].

Taxa with such a disjunction are found in tropical and subtropical regions on both sides of the Pacific Ocean with the highest biodiversity on Earth [START_REF] Wu | On the significance of Pacific intercontinental discontinuity[END_REF][START_REF] Raven | Tropical floristics tomorrow[END_REF][START_REF] Van Damme | Tropical amphi-Pacific disjunctions in the Cladocera (Crustacea: Branchiopoda)[END_REF]. In plants, the disjunct distributions between eastern Asia and North America have been extensively studied in temperate elements [START_REF] Donoghue | Patterns in the assembly of temperate forests around the Northern Hemisphere[END_REF][START_REF] Wen | Timing and modes of evolution of eastern Asian-North American biogeographic disjunctions in seed plants[END_REF][START_REF] Wen | Intercontinental disjunctions between eastern Asia and western North America in vascular plants highlight the biogeographic importance of the Bering land bridge from late Cretaceous to Neogene[END_REF]. Few studies have been devoted to investigate the amphi-Pacific tropical disjunction [START_REF] Li | Phylogeny and biogeography of Dendropanax (Araliaceae), an amphi-Pacific disjunct genus between tropical/subtropical Asia and the Neotropics[END_REF][START_REF] Huang | Origins and evolution of cinnamon and camphor: A phylogenetic and historical biogeographical analysis of the Cinnamomum group (Lauraceae)[END_REF]. [START_REF] Thorne | Major disjunctions in the geographic ranges of seed plants[END_REF] listed 89 genera, 4 tribes or subtribes, 3 or 4 subfamilies, and 8 to 11 families of angiosperms that exhibit the amphi-Pacific tropical distribution. How these taxa achieved their present-day amphi-Pacific disjunct ranges is still poorly understood. [START_REF] Wolfe | Some aspects of plant geography of the Northern Hemisphere during the late Cretaceous and Tertiary[END_REF] proposed the "boreotropical flora" hypothesis to explain this pattern. This hypothesis postulates that during climatically warm periods of the Paleogene, the boreotropical flora with many tropical and subtropical elements spanned the mid-latitudes of the Northern Hemisphere and populations could exchange between areas via land bridges, then owing to the climatic deterioration during the late Eocene, boreotropical elements migrated southwards following the shrinking of subtropical and tropical zones of Asia and America, finally resulting in the amphi-Pacific tropical disjunction pattern [START_REF] Wolfe | Some aspects of plant geography of the Northern Hemisphere during the late Cretaceous and Tertiary[END_REF]Tiffney 1985a). When the estimated ages for the splits of the tropical lineages in both continents are during the middle to late Eocene, the boreotropical flora hypothesis is usually adopted as the most likely explanation (e.g., [START_REF] Nie | Phylogenetic and biogeographic complexity of Magnoliaceae in the Northern Hemisphere inferred from three nuclear data sets[END_REF][START_REF] Li | Molecular phylogenetic analysis of the Persea group (Lauraceae) and its biogeographic implications on the evolution of tropical and subtropical amphi-Pacific disjunctions[END_REF][START_REF] Li | Phylogeny and biogeography of Dendropanax (Araliaceae), an amphi-Pacific disjunct genus between tropical/subtropical Asia and the Neotropics[END_REF][START_REF] Huang | Origins and evolution of cinnamon and camphor: A phylogenetic and historical biogeographical analysis of the Cinnamomum group (Lauraceae)[END_REF][START_REF] Liu | Phylogeny of the Ampelocissus-Vitis clade in Vitaceae supports the New World origin of the grape genus[END_REF][START_REF] Yang | Boreotropical range expansion and long-distance dispersal explain two amphi-Pacific tropical disjunctions in Sabiaceae[END_REF]. Based on this hypothesis, as the period of climate cooling in the late Eocene may have caused the thermophilic lineages to move to lower latitudes, the tropical lineages should have separated earlier than the subtropical and temperate. However, this scenario has never been tested in a phylogenetic framework by including closely related elements spanning the tropical and subtropical zones.

The tribe Pachygoneae (Menispermaceae) was re-delimited by [START_REF] Ortiz | Phylogeny and a revised tribal classification of Menispermaceae based on molecular and morphological data[END_REF] based on molecular and morphological data. This re-delimited tribe comprises four genera and about 40 species (Fig. 1; [START_REF] Ortiz | Phylogeny and a revised tribal classification of Menispermaceae based on molecular and morphological data[END_REF]. Haematocarpus, containing 2 species, is endemic to Southeast Asia. Pachygone, consisting of 12 species, is primarily distributed in Southeast Asia, as well as in northeastern Australia and adjacent islands. Hyperbaena contains about 22 species, which are entirely restricted to the tropical America. Cocculus currently contains about 8 species, among which three species are found in tropical and subtropical Asia and eastern Asia, two in subtropical North America, one in tropical Africa, and two from tropical Asia to Africa (note that Cocculus madagascariensis, apparently from Madagascar was based on a mislabeled collection that was actually from Central America, likely from Mexico; Forman, 1974a). Thus, the distribution patterns of this tribe provide an opportunity to test the boreotropical flora hypothesis.

Of the four genera in Pachygoneae, only the circumscription of Cocculus has been controversial. Based on the plastid (matK and trnL-F) and nuclear (ITS) sequences, [START_REF] Wang | Phylogeny and morphological evolution of tribe Menispermeae (Menispermaceae) inferred from chloroplast and nuclear sequences[END_REF] found that Pachygone was nested in Cocculus. In the same year, [START_REF] Ortiz | Molecular phylogeny of the moonseed family (Menispermaceae): Implications for morphological diversification[END_REF] analyzed the plastid ndhF data and also found a non-monophyletic Cocculus, in which the sampled four species of the genus were distributed in three clades. Similar results were found in subsequent studies [START_REF] Hoot | Phylogenetic patterns in Menispermaceae based on multiple chloroplast sequence data[END_REF][START_REF] Jacques | Integrating fossils in a molecular-based phylogeny and testing them as calibration points for divergence time estimates in Menispermaceae[END_REF][START_REF] Wang | Menispermaceae and the diversification of tropical rainforests near the Cretaceous-Paleogene boundary[END_REF][START_REF] Ortiz | Phylogeny and a revised tribal classification of Menispermaceae based on molecular and morphological data[END_REF], although [START_REF] Wefferling | Phylogeny and fruit evolution in Menispermaceae[END_REF] found that Bayesian inference (BI) supported Cocculus as monophyletic, whereas maximum parsimony (MP) and maximum likelihood (ML) analyses recognized it to be polyphyletic. No more than four species of the genus were included in each of the aforementioned phylogenetic analyses. Importantly, the type species of Cocculus, C. hirsutus, has not been sampled in any molecular study, which has prevented us from updating the classification of this genus.

In this study, our objectives are (1) to reconstruct phylogenetic relationships within Pachygoneae using six plastid and nuclear DNA regions with a focus on Cocculus, hence with a more comprehensive sampling than in any previous study, (2) to clarify the circumscription of Cocculus, and (3) to investigate the historical biogeography of the tribe in the context of the boreotropical flora hypothesis.

Materials and methods

Taxon sampling and DNA sequencing

We sampled 35 taxa, representing all four of the currently recognized genera of Pachygoneae (Fig. 1; Table S1). Our sampling encompasses all eight species of Cocculus, among which its type species, C. hirsutus, was included in a molecular phylogenetic analysis for the first time. Thirty additional species were sampled as outgroups, representing all other six tribes in the Menispermoideae [START_REF] Ortiz | Phylogeny and a revised tribal classification of Menispermaceae based on molecular and morphological data[END_REF].

Menispermeae were used to root the tree based on the results of [START_REF] Wang | Menispermaceae and the diversification of tropical rainforests near the Cretaceous-Paleogene boundary[END_REF] and [START_REF] Ortiz | Phylogeny and a revised tribal classification of Menispermaceae based on molecular and morphological data[END_REF].

Six DNA regions, including plastid (rbcL, atpB, matK, ndhF, trnL-F) and nuclear (26S rDNA) loci, were used in this study. Genomic DNA extraction, amplification, sequencing, and alignments followed [START_REF] Wang | Menispermaceae and the diversification of tropical rainforests near the Cretaceous-Paleogene boundary[END_REF][START_REF] Wang | The rise of angiosperm-dominated herbaceous floras: Insights from Ranunculaceae[END_REF].

Voucher information and GenBank accession numbers are listed in Table S1.

Phylogenetic analyses

Phylogenetic analyses of the cpDNA (rbcL, atpB, matK, ndhF, trnL-F), 26S rDNA and combined cpDNA and 26S rDNA datasets were conducted using ML and BI methods in RAxML 8.0 [START_REF] Stamatakis | RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF] and MrBayes 3.2.5 [START_REF] Ronquist | MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space[END_REF], respectively. RAxML was performed with the GTR + Γ substitution model for each DNA region, and nodal support was assessed using the fast bootstrap option with 1000 replicates. For BI analyses, each DNA region was assigned its own model of nucleotide substitution, as determined by the Akaike Information Criterion (AIC) in jModeltest 2.1.4 [START_REF] Posada | jModelTest: phylogenetic model averaging[END_REF]. Two independent runs, each comprising four Markov Chain Monte Carlo (MCMC) chains, were conducted, sampling one tree every 1000 generations for 50 million generations, starting with a random tree. Stationarity was determined in Tracer 1.6 (Rambaut et al., 2014). Majority rule (> 50%) consensus trees were constructed after removing the burn-in period samples (the first 25% of sampled trees). Data homogeneity between the cpDNA and 26S rDNA was examined using the incongruence length difference (ILD) test [START_REF] Farris | Testing significance of incongruence[END_REF], implemented in PAUP* 4.0b10 [START_REF] Swofford | PAUP*: Phylogenetic Analysis Using Parsimony (*and other methods)[END_REF]. This test employed 1000 replicates, each with 10 random additions using tree-bisection-reconnection branch swapping.

Divergence time estimation

Divergence times were estimated using the combined six locus data in BEAST 2.1.3 [START_REF] Drummond | Bayesian phylogenetics with BEAUti and the BEAST 1.7[END_REF]. We used a reduced dataset of 50 taxa of Menispermoideae that kept one accession per species. Phylogenetic analyses [START_REF] Ortiz | Molecular phylogeny of the moonseed family (Menispermaceae): Implications for morphological diversification[END_REF](Ortiz et al., , 2016;;[START_REF] Wang | Menispermaceae and the diversification of tropical rainforests near the Cretaceous-Paleogene boundary[END_REF]this study) indicate that the species of Cocculus are distributed in four clades. Thus, we did not use Cocculus lottii from the early Middle Eocene of Germany [START_REF] Collinson | Fossil Fruits and Seeds of the Middle Eocene Messel biota, Germany[END_REF] as a calibration point. Here we selected six fossils that can be confidently placed in our tree (see Table S2 for the detailed justifications of their placements). Following the suggestion of [START_REF] Parham | Best practices for justifying fossil calibrations[END_REF], we used the uniform prior distribution for these six calibrating fossils. The maximum age of the root was set at 100 Ma, which is the estimated crown age of the Menispermoideae [START_REF] Wang | Menispermaceae and the diversification of tropical rainforests near the Cretaceous-Paleogene boundary[END_REF], with a normal distribution and standard deviation of three.

We first used Bayes factors [START_REF] Kass | Bayes Factors[END_REF] calculated by marginal likelihoods derived from path sampling [START_REF] Baele | Accurate model selection of relaxed molecular clocks in Bayesian phylogenetics[END_REF] to test four clock models (strict, exponential, lognormal, and random), and then to test two distinct tree speciation priors (Yule and birth-death, BD). Under the best-fit clock model and tree prior, the BEAST analysis was run relied on the GTR + I + Γ model for each DNA partition separately. The MCMC chains were run for 50 million generations, sampling every 5000 generations. Convergence and the adequate effective sample size values (> 200) were checked in Tracer 1.6 (Rambaut et al., 2014). The maximum clade credibility (MCC) tree with median branch lengths and 95% highest posterior density (HPD) intervals on nodes was then built using TreeAnnotator 2.1.3 (part of the BEAST package) with the initial 25% of trees discarded as burn-in. For comparative purpose, we also estimated divergence times using the six-locus dataset with all samplings (65 taxa). This analysis was conducted as described above.

Biogeographical analyses

Ree and Sanmartín (2018) suggested that the dispersal-extinction-cladogenesis (DEC) model with + J parameter is invalid because of errors in estimation of likelihoods. Here ancestral areas at internal nodes within the phylogenetic tree were inferred using the DEC model, implemented in Lagrange 2.0.1 [START_REF] Ree | Maximum likelihood inference of geographic range evolution by dispersal, local extinction, and cladogenesis[END_REF].

The species-level MCC tree generated from the BEAST analysis was used as the input tree. Four areas of endemism were defined according to the distribution of the Pachygoneae: (A) tropical and subtropical Asia, (B) tropical and subtropical America, (C) Africa, and (D) Australasia. Referring to [START_REF] Mao | Distribution of living Cupressaceae reflects the breakup of Pangea[END_REF], we specified dispersal probabilities between pairs of areas for four separate time slices. The maximum number of areas in the ancestral ranges was constrained to two, as no extant species of Pachygoneae is distributed in more than two areas of endemism.

Results

Phylogenetic analyses

The aligned cpDNA dataset included 7244 characters: rbcL, 1385 bp; atpB, 1406 bp; matK, 1250 bp; ndhF, 2060 bp; and trnL-F, 1143 bp. The aligned 26S rDNA sequences were 1328 bp nucleotides in length. The ILD test value was P = 0.34, indicating that the cpDNA and 26S rDNA datasets are not significantly different from random partitions of the combined dataset. Except for the presence of several nodes with weak support (BS < 70% and PP < 0.95), the topology of Menispermoideae phylogeny generated based on the cpDNA dataset (Fig. S1) was highly congruent with the 26S rDNA tree (Fig. S2).

The combined cpDNA and 26S rDNA dataset comprised 8572 characters. ML and BI analyses resulted in identical trees with strong support for most nodes (Fig. 2).

Divergence time estimation and biogeographical reconstruction

For the 50-taxon and 60-taxon data, the lognormal clock model and BD tree prior were identified as best-fit (Tables S3 andS4). Divergence time estimates based on the two different sampling strategies are highly congruent (Figs. 3 andS3). The species-level age estimation is here reported and was used for biogeographical analysis. Ancestral area inference is indicated in Figs. 4 and S4 and

Discussion

Phylogenetic relationships

This study presents the most comprehensive phylogenetic analysis of Menispermoideae to date, based on six DNA regions from the plastid and nuclear genomes (Fig. 2). The inter-tribal relationships within Menispermoideae recovered here are highly congruent with the results of previous studies [START_REF] Wang | Menispermaceae and the diversification of tropical rainforests near the Cretaceous-Paleogene boundary[END_REF][START_REF] Ortiz | Phylogeny and a revised tribal classification of Menispermaceae based on molecular and morphological data[END_REF]. In agreement with other studies [START_REF] Wang | Phylogeny and morphological evolution of tribe Menispermeae (Menispermaceae) inferred from chloroplast and nuclear sequences[END_REF][START_REF] Wang | Menispermaceae and the diversification of tropical rainforests near the Cretaceous-Paleogene boundary[END_REF][START_REF] Hoot | Phylogenetic patterns in Menispermaceae based on multiple chloroplast sequence data[END_REF][START_REF] Jacques | Integrating fossils in a molecular-based phylogeny and testing them as calibration points for divergence time estimates in Menispermaceae[END_REF][START_REF] Ortiz | Phylogeny and a revised tribal classification of Menispermaceae based on molecular and morphological data[END_REF], our results also support a non-monophyletic Cocculus. For the first time, our phylogeny includes all eight of the currently recognized species of Cocculus, which are distributed in four different clades (Fig. 2). Another important finding is that the newly sampled C. trilobus Mayuzumi 3826 and C. trilobus Tomoko Fukuda s.n. formed a clade nested in C. orbiculatus, whereas C. trilobus Hong YP H310 is nested in Pachygone and sister to P. sinica. By carefully checking the morphology of the three accessions, along with examination of the herbarium records, we found that C. trilobus Hong YP H310 has characteristics of P. sinica and is a misidentified accession. Thus, previous phylogenies where C. trilobus was a member of Pachygone [START_REF] Wang | Menispermaceae and the diversification of tropical rainforests near the Cretaceous-Paleogene boundary[END_REF][START_REF] Ortiz | Phylogeny and a revised tribal classification of Menispermaceae based on molecular and morphological data[END_REF] were erroneous due to a misidentified sample of P. sinica.

Biogeography of Pachygoneae

Our two dating analyses generated largely congruent time estimates for Pachygoneae (Figs. 3 andS3). The species-level age estimation suggests a crown group age of 47.65 Ma (95% HPD: 33.33-61.44) for the tribe, which coincides with previous time estimates [START_REF] Wang | Menispermaceae and the diversification of tropical rainforests near the Cretaceous-Paleogene boundary[END_REF][START_REF] Wefferling | Dated phylogeography of western North American subalpine marshmarigolds (Caltha spp., Ranunculaceae): Miocene-Pliocene divergence of hexaploids, multiple origins of allododecaploids during the Pleistocene, and repeated recolonization of Last Glacial Maximum glaciated regions[END_REF]. The stem group age of Hyperbaena is estimated to be 40.07 Ma (95% HPD: 30.20-52.08), which is highly congruent with the result of [START_REF] Wang | Menispermaceae and the diversification of tropical rainforests near the Cretaceous-Paleogene boundary[END_REF]33.5-54.7 Ma), yet it is much younger than that of [START_REF] Jacques | Integrating fossils in a molecular-based phylogeny and testing them as calibration points for divergence time estimates in Menispermaceae[END_REF]64.6 Ma). However, Jacques et al.

(2011) only used two plastid loci (rbcL and atpB) and did not retrieve the monophyly of Pachygoneae. In this study, we included more taxa of Pachygoneae and more characters than in previous studies, which are expected to be able generate more accurate estimates [START_REF] Yang | Comparison of likelihood and Bayesian methods for estimating divergence times using multiple gene loci and calibration points, with application to a radiation of cute-looking mouse lemur species[END_REF][START_REF] Jian | Resolving an ancient, rapid radiation in Saxifragales[END_REF]. Based on endocarp characters, the extinct species C. lottii from the Middle Eocene of the Messel Formation, Germany, is close to C. carolinus, C. orbiculatus, and C. hirsutus [START_REF] Collinson | Fossil Fruits and Seeds of the Middle Eocene Messel biota, Germany[END_REF]. This fossil record is in accordance with our time estimate for the most recent common ancestor of these three species (c. 44 Ma). These render support to the ages estimated here for Pachygoneae. 33.46-56.74; node 4). In the early Cenozoic, the floristic exchange between Eurasia and the New World might have occurred through either the North Atlantic land bridge (NALB) or the Bering Land Bridge (BLB) [START_REF] Wolfe | Some aspects of plant geography of the Northern Hemisphere during the late Cretaceous and Tertiary[END_REF]Tiffney, 1985b;[START_REF] Tiffney | The use of geological and paleontological evidence in evaluating plant phylogeographic hypotheses in the Northern Hemisphere Tertiary[END_REF]. The latitude of the NALB was ca. 15° lower than at present and is thought to have maintained sufficient warmth, light, and humidity for thermophilic plants to thrive, whereas the BLB was less likely for thermophilic taxa to disperse across during most of the Cenozoic owing to its high latitude (between 69° to 75° N), limited light availability, and lower temperatures [START_REF] Tiffney | The use of geological and paleontological evidence in evaluating plant phylogeographic hypotheses in the Northern Hemisphere Tertiary[END_REF].

After the cooling period near the early to middle Eocene boundary, the middle Eocene witnessed a warm interval (~43-46 Ma), but this warm interval was not as warm as in the early Eocene (Fig. 4; [START_REF] Wolfe | Relations of environmental change to angiosperm evolution during the late Cretaceous and Tertiary[END_REF][START_REF] Zachos | An early Cenozoic perspective on greenhouse warming and carbon-cycle dynamics[END_REF]. The fossil C. lottii, endocarps from Messel in Germany, suggest that Pachygoneae was distributed in Europe in the middle Eocene, although its exact systematic position in side Cocculus s.l. is unclear (Fig. 1; [START_REF] Collinson | Fossil Fruits and Seeds of the Middle Eocene Messel biota, Germany[END_REF]. Considering the age estimate and the fossil distribution in Europe, Pachygoneae most likely migrated into the New World via the NALB. Biogeographical analyses indicated that many thermophilic plant lineages, such as Dendropanax (Araliaceae; [START_REF] Li | Phylogeny and biogeography of Dendropanax (Araliaceae), an amphi-Pacific disjunct genus between tropical/subtropical Asia and the Neotropics[END_REF], Cinnamomum (Lauraceae; [START_REF] Huang | Origins and evolution of cinnamon and camphor: A phylogenetic and historical biogeographical analysis of the Cinnamomum group (Lauraceae)[END_REF], and Meliosma (Sabiaceae; [START_REF] Yang | Boreotropical range expansion and long-distance dispersal explain two amphi-Pacific tropical disjunctions in Sabiaceae[END_REF], might have expanded their ranges during the Eocene via the NALB.

Our divergence time estimates indicate that the clade containing the tropical

American Hyperbaena originated at about 40 Ma (node 5 in Fig. 4), which is congruent with a cooling interval in the late Eocene (~38-42 Ma; [START_REF] Wolfe | Relations of environmental change to angiosperm evolution during the late Cretaceous and Tertiary[END_REF][START_REF] Zachos | An early Cenozoic perspective on greenhouse warming and carbon-cycle dynamics[END_REF]. [START_REF] Li | Phylogeny and biogeography of Dendropanax (Araliaceae), an amphi-Pacific disjunct genus between tropical/subtropical Asia and the Neotropics[END_REF] suggested that the split of the Neotropical Dendropanax and its Asian sister group occurred at the same period (41.83 Ma). The clade now represented by the subtropical North American C. carolinus and C. diversifolius diverged from its Asian sister clade later, at about 32 Ma (node 6 in Fig. 4), a period of drastical climatic deterioration [START_REF] Wolfe | Relations of environmental change to angiosperm evolution during the late Cretaceous and Tertiary[END_REF][START_REF] Zachos | An early Cenozoic perspective on greenhouse warming and carbon-cycle dynamics[END_REF]. A similar split time was found in Magnoliaceae (30.22 Ma, 95% HPD: 22.20-38.43;[START_REF] Nie | Phylogenetic and biogeographic complexity of Magnoliaceae in the Northern Hemisphere inferred from three nuclear data sets[END_REF] and Diplazium (31.2 Ma, 95% HPD: 24.7-38.4; Athyriaceae; [START_REF] Wei | Eurasian origin, boreotropical migration and transoceanic dispersal in the pantropical fern genus Diplazium (Athyriaceae)[END_REF]. As the climate cooled, thermophilic lineages may have moved to lower latitudes, which could have resulted in the disjunction of the distribution of tropical lineages first, followed by subtropical and temperate lineages, as in Pachygoneae.

Thus, our study provides the new evidence for supporting the boreotropical flora hypothesis, in addition to the micro-and macrofossil evidence from the early Eocene branched or inconspicuous) (Forman, 1974b). Cocculus pendulus and C. balfourii also differ from remaining Asian/American Cocculus (Cocculus 3 and 4; Fig. 2) in many morphological characters, such as entire petals (vs. bifid or deeply emarginate), a gynoecium consisting of three carpels (vs. six), and endocarps lacking or with a few transverse ridges (vs. numerous) [START_REF] Diels | Menispermaceae[END_REF]Forman, 1974b). Therefore, our data support the resurrection of Cebatha to embrace two species, C. pendula and C. balfouriana.

Our phylogenetic analyses indicate that Cocculus 1 is in an unresolved position in relation to Pachygoneae, Tiliacoreae, Spirospermeae, and Cissampelideae (Fig. 2).

These five lineages diverged rapidly over a period shorter than 6 million years (Myr), and perhaps in as short as 1 Myr (Fig. 3). It is not reasonable to include Cebatha in Lianas, dioecious. Leaves ovate, trapezoid or spine-like. Flowers minute. Male flowers: sepals 6, ovate-elliptic, fleshy or membranous; petals 6, ovate-obovate, glabrous; stamens 6. Sepals and petals are the same as male flowers. Drupe reddish when fresh but turning black when dried, pericarp of dried fruit easily rubbed off.

Endocarp with no lateral ridges or no more than 15, not perforated in the center.

The reinstatement of Nephroia. In 1790, Loureiro established the genus Nephroia to include the one species Nephroia sarmentosa. [START_REF] Diels | Menispermaceae[END_REF] which has a distant relationship with C. hirsutus (Fig. 2). Cocculus 3 markedly differ from C. hirsutus in having glabrous sepals (vs. pubescent), six carpels (vs. three), and endocarps without a dorsal ridge (vs. a sharp dorsal ridge) [START_REF] Diels | Menispermaceae[END_REF]Forman, 1974b). In addition, the species in Cocculus 3 are restricted to forests, whereas Cocculus 1 and 2 clades inhabit dry and open areas. Thus, we resurrected Nephroia as a distinct genus and assigned N. orbiculata as its type. Our analyses further show that C. trilobus is embedded within C. orbiculatus, which supports the synonymy of C. trilobus under C. orbiculatus [START_REF] Forman | The Menispermaceae of Malesia: V. Tribe Cocculeae Hook. F. & Thoms[END_REF][START_REF] Luo | Menispermaceae[END_REF]. Thus, the resurrected Nephroia contains three species, N. orbiculata, N. carolina and N. diversifolia. The necessary taxonomic changes are proposed here.

Nephroia orbiculata (L.) Lian Lian & Wei Wang, comb. nov The inclusion of C. laurifolius in Pachygone. Cocculus laurifolius was first described by de Candolle (1818). However, de Candolle (1818) also noted that this species was unique in that the base of the petal does not embrace the filament. In agreement with the results of previous studies [START_REF] Wang | Phylogeny and morphological evolution of tribe Menispermeae (Menispermaceae) inferred from chloroplast and nuclear sequences[END_REF][START_REF] Wang | Menispermaceae and the diversification of tropical rainforests near the Cretaceous-Paleogene boundary[END_REF][START_REF] Ortiz | Phylogeny and a revised tribal classification of Menispermaceae based on molecular and morphological data[END_REF], our analyses also indicate that C. laurifolius is nested in Pachygone and is sister to P. valida. Cocculus laurifolius shares a few morphological similarities with Pachygone, including a gyneocium with three carpels and an endocarp lacking transverse ridges [START_REF] Diels | Menispermaceae[END_REF][START_REF] Jacques | Survey of the Menispermaceae endocarps[END_REF]. Thus, our results suggest that C.

  Cocculus pendulus and C. balfourii formed a clade (Cocculus 1; BS = 80%, PP = 0.99), which is in an unresolved position in relation to Pachygoneae, Tiliacoreae, Spirospermeae, and Cissampelideae. Within Pachygoneae, Haematocarpus is sister to the remaining taxa of the tribe (BS = 66%, PP = 0.94), followed by Cocculus hirsutus (Cocculus 2; BS = 87%, PP = 1.0), then Hyperbaena (BS = 86%, PP = 0.95). Hyperbaena is strongly supported as monophyletic (BS = 99%, PP = 1.0). Cocculus 3 clade contains Cocculus carolinus, C. diversifolius, and C. orbiculatus (including C. trilobus Mayuzumi 3826 and C. trilobus Tomoko Fukuda s.n.) (BS = 93%, PP = 1.0). Cocculus laurifolius (Cocculus 4) is embedded within Pachygone and is sister to P. valida (BS = 92%, PP = 1.0). Similarly, C. trilobus Hong YP H310 is nested in Pachygone and forms a clade with P. sinica (BS = 100%, PP = 1.0).

  Our molecular dating and ancestral area reconstruction analyses indicate that the most recent common ancestor of Pachygoneae was likely present in tropical Asia around the early-middle Eocene boundary (47.65 Ma, 95% HPD: 33.33-61.44; node 3), and subsequently dispersed into the New World by c. 44 Ma (95% HPD:

  any of the already recognized tribe, thus we propose a new tribal name for the genus: Cebatheae Lian Lian & Wei Wang, trib. nov. TYPE: Cebatha Forsk. Fl. Aegypt. -Arab.: 171. 1775.

  1818. ≡ Wendlandia caroliniana (L.) Nutt., Gen. N. Amer. Pl. 1: 241. 1818. ≡ Cebatha carolina (L.) Britton, Mem. Torrey Bot. Club 5: 162. 1894. ≡ Epibaterium carolinum (L.) Britton, Ill. Fl. N. U.S. (Britton & Brown), ed. 2, 2: 131. 1913. TYPE: U.S.A. South Carolina, Williamsburg Co., 5 miles southeast of Gourdin, 10 Jul 1939, R.K. Godfrey & R.M. Tryon 373 (BH, neotype designated by Reveal & Jarvis 2009: 980, not seen; isoneotype, GH-00257737, not seen). Nephroia diversifolia (DC.) Lian Lian & Wei Wang, comb. nov. ≡ Cocculus diversifolius DC. in Syst. I: 523. 1818. ≡ Cebatha diversifolia (DC.) Kuntze, Revis. Gen. Pl. 1: 9. 1891. ≡ Epibaterium diversifolium (DC.) Tidestr. , Proc. Biol. Soc. Washington 48: 39. 1935. TYPE: DC. plate 10 deposited in G, including two drawings copied from Plates 1490 and 1519, Torner Collection of Sessé and Mociño Biological Illustrations (lectotype designated by McVaugh 2000: 391).

  laurifolius is a member of Pachygone. A new combination is made here. Pachygone laurifolia (DC.) Lian Lian & Wei Wang, comb. nov. ≡ Cocculus laurifolius DC. in Syst. I: 530. 1818. ≡ Menispermum laurifolium (DC.) Roxb., Fl. Ind. 3: 815. 1832. ≡ Nephroica laurifolia Miers, Ann. Mag. Nat. Hist. ser. 2, 7(37): 42. 1851. ≡ Holopeira laurifolia (DC.) Miers , Ann. Mag. Nat. Hist. ser. 3, 19(109): 29. 1867. ≡ Cebatha laurifolia (DC.) Kuntze , Revis. Gen. Pl. 1: 9. 1891. TYPE: India (holotype, G-00201635 [image!]).

Fig. 1 .

 1 Fig. 1. Geographic distribution of Pachygoneae. The number of species sampled in

Fig. 2 .

 2 Fig. 2. ML tree inferred from the combined six plastid and nuclear dataset. Numbers

Fig. 3 .

 3 Fig. 3. Chronogram of Menispermoideae with 50 taxa based on the six-locus dataset

  for details). Nodes of interests were marked as 1-8. The grey area shows that the five tribes have diversified rapidly over a period of < 6 million years. Pal = Paleocene; Oli = Oligocene; Pl = Pliocene.

Fig. 4 .Fig. 5 .

 45 Fig. 4. Ancestral range reconstruction (ARR) using Lagrange, modified from Fig. S4.

Table S5 .

 S5 The five Menispermoideae lineages (Pachygoneae, Cocculus 1-i.e., Cebatheae, Tiliacoreae, Spirospermeae, and Cissampelideae) started to diverge rapidly at 72.69 Ma (95% HPD: 63.88-83.05; node 1). Cocculus 1 diverged from Pachygoneae at 66.92 Ma (95% HPD: 54.43-79.59; node 2). The most recent common ancestor of Pachygoneae was likely present in tropical Asia at 47.65 Ma (95% HPD: 33.33-61.44; node 3). One dispersal event was inferred from Asia to the New World (between nodes 3 and 4). The age of node 4 was estimated at 44 Ma (95% HPD: 33.46-56.74). Two American lineages, Hyperbaena and C. carolinus-C. diversifolius, originated at 40.07 Ma (95% HPD: 30.20-52.08; node 5) and 32.49 Ma (95% HPD: 20.80-44.63; node 6), respectively. The dispersal from Asia into Australia took place at c. 20.24-8.11 Ma (between nodes 7 and 8).

  reduced Nephroia to Cocculus, i.e. Cocculus sarmentosus, and he also thought that C. orbiculatus was a synonym of C. trilobus. Forman (1962) accepted C. trilobus as including C. sarmentosus and C. orbiculatus. However, Forman (1968) suggest that C. orbiculatus is the valid name, instead of C. trilobus. Our phylogenetic analyses indicate that C. orbiculatus, C. trilobus, C. carolinus and C. diversifolius form a clade (Cocculus 3),

  . ≡ Menispermum orbiculatum L. in Sp. Pl. 1: 341. 1753. ≡ Menispermum trilobum Thunb. in Fl. Jap. 194. 1784. ≡ Cocculus orbiculatus (L.) DC. in Syst. I: 523. 1817. TYPE: "Cocculi Orientalis Frutex, convolvulaceus, orbiculatis foliis, prona parte villosis, ex una de Insulis Crocodilorum" in Plukenet, Amalth. Bot., 61, t. 384, f. 6, 1705 (lectotype designated by Troupin in Bull. Jard. Bot. État Bruxelles 25: 141. 1955). = Nephroia sarmentosa Lour. in Fl. Cochinch. 2: 692. 1790. TYPE: Cochinchina, Loureiro 565 (holotype, BM-000554339, [image!]) Nephroia carolina (L.) Lian Lian & Wei Wang, comb. nov. ≡ Menispermum carolinum L. in Sp. Pl. 1: 340. 1753. ≡ Cocculus carolinus (L.) DC. in Syst. I: 524.

Acknowledgements

We sincerely thank Frédéric M.B. Jacques, Ya-Ping Hong and Hui Ren for collecting materials and Xiang-Yun Zhu for his help with nomenclature. Peter Stevens kindly read an earlier version of the manuscript. This research was funded by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB31030000) and the National Natural Science Foundation of China (31770231, 31470315 and 31770233). References Bacon, C.D., Baker, W.J., Simmons, M.P., 2012. Miocene dispersal drives island

Molecular and morphological evidence indicates the need to reinstate Cebatha and Nephroia, establish a new tribe for Cebatha, and transfer C. laurifolius to Pachygone. Summary of the key taxonomic revisions in this study is shown in Fig. 5. The geographic distribution of the new delimited Pachygoneae is shown in Fig. S5. Further details about the required taxonomic changes are presented below. The reinstatement of Cebatha and proposal for a new tribe Cebatheae. In 1775, Forsskål (1775) established two genera Cebatha and Leaeba, but did not place any species in them. One year later, Forster and Forster (1776) established the genus Epibaterium to include the sole species E. pendulum, which was reduced to Cocculus by de Candolle (1818), i.e. C. pendulus. Kuntze (1891) reduced Leaeba, Epibaterium,

and Cocculus to Cebatha. However, Diels (1910) used Cocculus as the generic name, which was accepted by subsequent authors (e.g., Troupin, 1962; Forman, 1974b;Kessler, 1993;Luo et al., 2008). Our phylogenetic analyses indicate that C. pendulus and C. balfourii (Cocculus 1) do not cluster with other Cocculus species (Fig. 2).

These two species are markedly different from Cocculus 2 (C. hirsutus, the type of Cocculus) in having the pericarp of dried fruit being easily removed (vs. hard to remove), glabrous and entire petals (vs. pubescent and bifid or deeply emarginate), and imperforate condyle (vs. perforate) (Diels, 1910;Troupin, 1962; Forman, 1974b;Jacques, 2009). In addition, endocarps of C. pendulus lack a sharp dorsal ridge, whereas endocarps of C. balfourii and C. hirsutus have a wing-like dorsal ridge, but endocarps of C. balfourii and C. hirsutus are different in lateral ridges (unbranched vs.