4 , KUO-FANG CHUNG AND CHING-I PENG

AJB Advance Article published on May 21, 2015, as 10.3732/ajb.1400428. The latest version is at http://www.amjbot.org/cgi/doi/10.3732/ajb.1400428 RESEARCH ARTICLE

A M E R I C A N J O U R N A L O F B OTA N Y

THE MIOCENE TO PLEISTOCENE COLONIZATION OF THE PHILIPPINE ARCHIPELAGO BY BEGONIA SECT. BARYANDRA (BEGONIACEAE)1 MARK HUGHES2, ROSARIO RIVERA RUBITE3, PATRICK BLANC4, KUO-FANG CHUNG5, AND CHING-I PENG6,7 2Royal

Botanic Garden Edinburgh, 20a Inverleith Row, Edinburgh, UK, EH3 5LR; 3Department of Biology, College of Arts and Sciences, University of the Philippines Manila, Padre Faura, Manila, Philippines; 43CNRS, 3 rue Michel-Ange 75794 Paris, France; 5National Taiwan University, School of Forestry and Resource Conservation, Daan, Taipei 106, Taiwan; and 6Biodiversity Research Center, Academia Sinica, Nangang, Taipei 115, Taiwan • Premise of the study: One third of the species-rich Philippine flora is endemic, and most of the islands in the archipelago have never been connected to a continental region. We currently lack any well-sampled angiosperm phylogenies that span the archipelago, prohibiting the formation of informed hypotheses as to the evolution of this rich and highly endemic flora. • Methods: We produced time-calibrated phylogenetic trees from both nuclear (ITS) and chloroplast (ndhA intron, ndhF–rpl32 spacer, rpl32–trnL spacer, trnC–trnD spacer) regions of 41 species of Begonia sect. Baryandra, all except one endemic to the Philippines. Historical biogeography was reconstructed across the chloroplast phylogeny using a Bayesian binary method of character optimization. Comparison of phylogenies from the two genomes permitted insight into the prevalence of hybridization in the group. • Key results: The Philippine archipelago was colonized by Begonia sect. Baryandra in the late Miocene, via long-distance dispersal from western Malesia and a point of entry likely to be in the northwestern region of the archipelago. Palawan, Luzon, and Panay all bear early-branching lineages from this initial colonization. There have been Plio-Pleistocene dispersals from these islands into Borneo and Mindanao. Hybridization was common between species as evidenced by haplotype sharing and phylogenetic incongruence. • Conclusions: The phylogenies show a high degree of geographic structure, which millions of years of exposure to typhoons have not blurred, showing long-term species and population stability. The recent dispersals to Mindanao are congruent with the geologically recent arrival of the island at its current latitude in the southern Philippines. Key words: dispersal; historical biogeography; hybridization; phylogenetic incongruence.

The thousands of islands comprising the Philippine archipelago have differing continental, oceanic, and volcanic origins, with many of them having undergone recent rapid tectonic 1 Manuscript received 26 September 2014; revision accepted 24 April 2015. This research was supported by The University of the Philippines (System Enhanced Creative Work and Research Grant ECWRG 2014-09), Academia Sinica, Taiwan, National Science Council, Taiwan (NSC 101-2621-B-001-003-MY3 & NSC 98-2621-B-001-002-MY3), Royal Botanic Garden Edinburgh (supported by the Scottish Governments Rural and Environment Science and Analytical Services Division), the M. L. MacIntyre Begonia Trust and the Royal Society of Edinburgh Bilateral Program. The authors thank the Palawan Council for Sustainable Development, the Palawan Protected Areas Management Board, Palawan City Council, and the Department of Environment and Natural Resources for granting access and research permits, Annie Yang for support in the laboratory, Mr. Danilo N. Tandang of the Philippine National Herbarium for support in the field, and two reviewers for their insightful comments on the manuscript. 7 Author for correspondence (e-mail: [email protected])

doi:10.3732/ajb.1400428

movement (Hall, 2002). In combination with Pleistocene sea level fluctuations leading to changing island boundaries (Voris, 2000) and a very rich and highly endemic biota (Heaney et al., 1998; van Welzen et al., 2005), the archipelago is arguably the most geologically and biogeographically complex area of the Malesian region. To date, there are no well-sampled plant phylogenies covering the archipelago and hence a dearth of data on which to build hypotheses regarding colonization and speciation patterns of Philippine angiosperms. Four main routes have been proposed for the biotic colonization of the archipelago, namely from Taiwan in the north, from Borneo in the east via Palawan or the Sulu archipelago, or from Sulawesi in the south (Dickerson, 1928; Jones and Kennedy, 2008). These routes have been previously postulated as submerged land bridges (Dickerson, 1928) or “umbilici” (the Bornean routes; Diamond and Gilpin, 1983). However, the Philippines are mostly oceanic, and with the exception of Palawan are not thought to have experienced dry-land connections to the rest of the Malesian region (Heaney, 1986). The four colonization routes are best considered as stepping stone routes and also have to be considered in the light of ongoing massive tectonic

American Journal of Botany 102(5): 1–12, 2015; http://www.amjbot.org/ © 2015 Botanical Society of America

1 Copyright 2015 by the Botanical Society of America

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changes in the configuration of the archipelago, with Mindanao and the Visayas having moved from just south of the equator to their current position during the Miocene (Hall, 2002). The strongest evidence for a land bridge is between Borneo and Palawan, based on bathymetric data (Woodruff, 2010) and animal distributions (Heaney, 1986), though a connection of Palawan to Mindoro and Luzon is more doubtful. Phylogenetic analyses have highlighted animal dispersal routes into the archipelago, with bulbuls (Pycnonotidae) colonizing via the Sulu archipelago in the south and via the Batanes and Babuyan Islands in the north (Oliveros and Moyle, 2010), geckos (Gekko) colonizing from Palawan via the Buruanga Peninsula in Panay (Siler et al., 2012), and frogs (Rana) colonizing from Borneo via Palawan and Mindoro and via the Sulu archipelago (Brown and Guttman, 2002). Some islands within the archipelago will have been linked in the past due to Pleistocene sea level fall during glacial periods (Heaney, 1991; Heaney et al., 1998); these grouped islands have been termed Pleistocene Aggregate Island Complexes (PAICs; Esselstyn and Brown, 2009). Colonization by animals within the archipelago may have been facilitated through such PAIC formation (Heaney et al., 2005). Gecko dispersal appears to be have been facilitated this way through the PAIC composed of Panay, Negros, and Cebu, but contemporary island configuration better explains the distribution of gecko clades elsewhere in the archipelago (Siler et al., 2010). Similar results have been found for shrews (Crocidura), in which partitioning of genetic diversity could be explained in part by PAIC formation, but distributions were also affected by over-water dispersal among adjacent islands (Esselstyn and Brown, 2009). A number of angiosperm groups from the Philippines have been placed phylogenetically in a wider Malesian context using a small number of placeholders, i.e., with poor Philippine sampling. These give insight into routes and dates of entry into the Philippines, such as from the western Pacific islands during the Pleistocene for Aglaia (Grudinski et al., 2014), from Borneo during the Miocene for Alocasia (Nauheimer et al., 2012), from the Sunda Shelf, Sulawesi and New Guinea during the Miocene-Pliocene for Begonia (Thomas et al., 2012), and from Borneo during the Pleistocene for Cyrtandra (Atkins et al., 2001). However, in contrast to the considerable amount of data available for animal colonization patterns within the archipelago (Jones and Kennedy, 2008), there are no comparable studies for plants. To get beyond the broad picture of colonization routes and consider within-archipelago events, we need much denser sampling of clades within the Philippines. Begonia is an ideal genus for investigating biogeography; species tend to be narrowly endemic and poorly dispersed, with studies in South Africa (Hughes and Hollingsworth, 2008) and Mexico (Twyford et al., 2014) showing species in highly differentiated populations with low levels of gene flow from both pollen and seeds. This poor dispersal leads to geographically restricted monophyletic groups of species, which can be used to track deeper-time tectonic events and also permit the detection of recent long-distance dispersal events that give rise to phylogenetic/geographic discordance (de Wilde et al., 2011). This study reconstructs the colonization of the Philippine archipelago by Begonia sect. Baryandra using a dated molecular phylogeny and current distribution data. The section has 59 species of mostly lithophytic rhizomatous herbs, representing a monophyletic radiation with all except four species endemic to the Philippines (Rubite et al., 2013), and high levels of island endemism within the Philippines (Hughes, 2008). The fruits of sect. Baryandra are typical for the genus, being trilocular, winged,

dehiscent capsules suited for anemoballistic or rain-splash dispersal of the tiny seeds (de Wilde, 2011). The majority of species are found in midaltitude montane forests, with some species growing at lower altitudes near sea level, often on karst limestone (e.g., B. elnidoensis, B. hughesii, B. taraw, and B. wadei), and some at higher altitudes in cloud forests (e.g., B. angilogensis, B. klemmei, and B. oxysperma). The highly endemic nature of Begonia species has implications for the way in which the historical biogeography of the genus is reconstructed. The complex nature of the Philippine archipelago and paucity of previous cross-archipelago studies also influences the choice of appropriate analytical methods. The first formalization of historical biogeography in an analytical framework was built around a vicariance paradigm (Rosen, 1976, 1978), and this approach has fed into currently popular analytical methods. Dispersal–vicariance analysis (DIVA) (Ronquist, 1997), based on a fairly simple model that gives vicariance a lower cost than dispersal, became widely used following the rapid expansion of molecular phylogenetics. Such analysis of ancestral range reconstruction is most likely to be error-free only when all speciation is due to vicariance (Kodandaramaiah, 2009), which will not hold true for most groups. In the absence of a priori constraints, a DIVA analysis can produce everexpanding ancestral ranges that are meaningless. In Malesia, the ongoing accretion rather than divergence of land masses means a vicariance approach can be particularly misleading (Webb and Ree, 2012). Refinements have been made to the dispersal–extinction–cladogenesis model (DEC) of geographic range evolution by incorporating the fitting of a time-calibrated phylogeny into a scenario of area connections (Ree et al., 2005; Ree and Smith, 2008). This modification enables rates of dispersal and local extinction to be estimated by maximum likelihood. However, this model may also be a poor fit to island biogeography scenarios where we expect dispersal and lineage divergence events to coincide (Ree and Smith, 2008). In the absence of multiple studies to inform a dispersal and vicariance model in an area of highly complex geography with multiple factors influencing distributions, a model-free approach was used here, optimizing geographic distributions across a phylogeny as character states using a Bayesian binary method as implemented in RASP (Yu et al., 2012), allowing for a stochastic evolution of range akin to random dispersal. This approach gives a great deal of insight into the movement of Begonia sect. Baryandra across the Philippine archipelago following initial colonization. The results raise a number of hypotheses regarding the development of the Philippine endemic flora and are a first step toward larger meta-analyses of the plant biogeography of the region. MATERIALS AND METHODS Taxon sampling—A total of 42 species from Begonia sect. Baryandra was sampled from the Philippine archipelago (40 species) and Borneo (2 species). An additional seven taxa identified only to section were included from the Philippines (labeled sp.1–7 in Fig. 1). Voucher information is listed in Appendix 1. Accounting for samples identified to species level only, this sampling represents 41/59 (69%) of the species in Begonia sect. Baryandra as circumscribed in Rubite et al. (2013). Asian Begonia have been supported as monophyletic based on chloroplast sequence data (Thomas et al., 2012), leading us to choose one American and two African species as outgroups to root the phylogeny based on the same genome. An additional 15 Asian species from five other sections of the genus were added to represent phylogenetic branch length variation to ensure reliable dating estimates and to further confirm the monophyly of the ingroup. Alternative outgroups (Appendix 1) were used to root the phylogenies

HUGHES ET AL.—PHILIPPINE BEGONIA

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Fig. 1. Maximum clade credibility chronogram from an analysis of the chloroplast alignment in BEAST. Colours represent Bayesian MCMC optimization of geographic distributions on the phylogeny; black indicates 0.50); the BIC highlighted SYM+G as the most probable (BIC weight = 0.94), and this model was used for phylogenetic analysis. Bayesian phylogenetic analyses were carried out separately on the chloroplast and nuclear data sets using the program MrBayes 3.2.1 (Ronquist et al., 2012). Each data set was treated as a single partition, analyzed under the appropriate model of sequence evolution and the default parameters of two runs with four chains each, run for 10 000 000 generations with a sample tree taken every 10 000. The first 25% of sampled trees were discarded as burn-in, and the remainder summarized as a maximum clade credibility tree visualized using the program FigTree v.1.4.0 (Rambaut, 2009). The trees resulting from the two analyses were compared using the program Dendroscope 3 (Huson and Scornavacca, 2012). A time-calibrated phylogeny based on the aligned chloroplast matrix was constructed using an uncorrelated relaxed lognormal clock in the program BEAST v.1.7.5 (Drummond et al., 2012) and treating the data as a single partition. A secondary age calibration was necessary, as the only Begonia macrofossil known (Stults and Axsmith, 2011) is too young to provide a calibration point. Two calibration points were taken from the broadly sampled phylogeny in Thomas et al. (2012), one dating the age of all Asian Begonia (18.2 Ma) and one dating the origin of Malesian Begonia (13.0 Ma) both with a standard deviation of 3.57 Myr. Five separate runs were carried out, beginning with a random tree and run for 10 000 000 generations under a birth–death model of speciation, sampled every 10 000 generations. Plots of the logged parameters for each run were visualized using the program Tracer v.1.5. (Rambaut and Drummond, 2007) to confirm convergence between runs. The trees from each run were combined into one file using the program Logcombiner (Drummond et al., 2012) after a burn-in of 20% from each run, and were summarized into a single maximum clade credibility tree using the program TreeAnnotator v.1.7.5 (Drummond et al., 2012). Biogeographic analysis—The Philippine archipelago and neighboring islands were divided into nine regions (Fig. 1) based on the islands from which samples were available and current coastlines; an additional five areas (Africa+America, India, China, western Sunda Shelf [Peninsular Malaysia + Sumatra] and Sulawesi) were coded to accommodate the wider sampling.

Terminals on the tree were scored according to their collection locality; given the microendemism of most Begonia species, this scoring is an accurate reflection of biological reality. Three of the taxa sampled have ranges that cover more than one area: (1) Begonia longiscapa occurs in southern Luzon, Biliran, Leyte, Bohol, and Mindanao, a sample from Biliran was included in the analysis; (2) B. mindorensis is known from Palawan and Mindoro, no samples were taken from the latter island; (3) Begonia nigritarum was sampled from Luzon and Panay, with no samples from the tentative records in Mindoro, Bohol, and Mindanao (Hughes, 2008). Given the polyphyly of some species uncovered by this study, we adhere to the conservative approach of scoring only sampled localities, as we are not confident species occurrence records from other areas represent related genotypes. The historical biogeography of Begonia sect. Baryandra across the Philippine archipelago was reconstructed using a Bayesian binary method of character state optimization across the maximum clade credibility tree resulting from the BEAST analysis of the chloroplast sequence alignment described above. The chloroplast phylogeny was chosen for the biogeographic analysis to track seed dispersal across the archipelago rather than gene flow via pollen and to keep the results comparable with those of Thomas et al. (2012), which were also derived from a chloroplast phylogeny; chloroplasts in Begonia are maternally inherited (Peng and Chiang, 2000). The Bayesian binary analysis was implemented in the program RASP 2.1b (Yu et al., 2012), which uses Markov chain Monte Carlo (MCMC) source code from MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). The MCMC analysis was run for 50 000 generations using 10 chains, with an equal probability of dispersal between all regions, and a root distribution of Africa. The number of regions possible at each node was constrained to two.

RESULTS Sequence characteristics—The aligned sequence matrix for the nuclear data set was 894 bases long, with 354 (40%) being informative, and a further 119 (13%) being autapomorphic. The concatenated chloroplast sequence matrix was 7435 characters long after site exclusion, with 606 being informative (8%) and a further 646 (9%) being autapomorphic. The electropherograms for the nuclear ITS sequences obtained from B. hernandoides 1, B. taraw 1, Begonia sp. 2, and Begonia sp. 5 were polymorphic, whereas the sequences obtained from the chloroplast genome for those individuals were normal reads. Repeat PCR and sequencing reactions of the ITS gave the same result, confirming polymorphism in the individual plants sequenced. These sequences were not included in the phylogenetic analysis. Phylogenetic analyses and divergence time— The phylogenies based on chloroplast and nuclear data both show a strongly supported monophyletic Begonia sect. Baryandra (Fig. 2). Levels of support are generally quite high across the both analyses, with weakening support toward the base and the terminals of the trees. The chloroplast tree shows an early branching into four well-supported clades (A–D in Fig. 1; PP = 1.0 in each case), with the relationships between these clades given by unsupported short branches, best considered a four-way polytomy. The two different analyses of the chloroplast alignment (one Bayesian, Fig. 2; one time-calibrated Bayesian, Fig. 1) gave similar tree topologies, with only minor unsupported differences in resolution toward the terminals. The ITS phylogeny shows significant amounts of hard incongruence in comparison with the chloroplast phylogeny. Clades A, B, and D are present (labeled A′, B′, and D′ in Fig. 2), although the composition of clades A′ and D′ differs slightly from that found in the chloroplast analysis, and in addition, the internal topology differs significantly. Clade C from the chloroplast analysis is represented by two clades in the nuclear analysis C1′ and C2′, with clade C1′ being sister to clade both B′ and C′ with PP = 1.0. Of particular note in terms

Fig. 2.

Tanglegram based on a Bayesian analysis of the chloroplast alignment (left) and nrITS (right). Asterisks mark clades with PP 0.95 (Fig. 1). The ancestral area reconstruction at the stem of the Begonia sect. Baryandra clade is equivocal, and due to the lack of support at the base the clade, the point of entry into the Philippine archipelago is also uncertain. However, there is a clearer picture of the geographic range evolution of Begonia sect. Baryandra within the Philippines. Clade A has an ancestral area of Luzon, arriving ca. 8.5 Ma, and the 12 species sampled began to diverge ca. 4.2 Ma, in the early Pliocene. From this clade, there are three colonizations to other areas during the Pleistocene; northward to the Batan and Lanyu Islands (B. fenicis) and to Panay (B. copelandii) and southward to Mindanao (B. acuminatissima). Clade B has an ancestral area of Panay and has been present on the island with no dispersal since its arrival in the late Miocene. Clades C and D both have an ancestral area of Palawan, again with an origin in the late Miocene. Clade C has dispersed from Palawan to Mindanao (two events: one in the early Pliocene and another in the late Pleistocene), to Borneo (one event in the late Pliocene) and to Luzon (one event in the early Pliocene). Clade D has dispersed across the Philippine archipelago, including to the three relatively small volcanic islands Romblon, Biliran, and Camiguin, and to Panay, potentially in a single event in the Pleistocene that has led to rapid diversification into six species. The clade has also dispersed to Borneo (one event) and Luzon (at least two events) in the Pleistocene. The exact number of the dispersals to Panay and Luzon are somewhat uncertain due to the lack of support toward the terminals of clade D.

DISCUSSION Arrival of Begonia sect. Baryandra on the archipelago—The area of ancestry for Begonia sect. Baryandra remains enigmatic, as the clade is part of an unresolved four-way split including Begonia sect. Ridleyella (peninsular Malaysia), Begonia sect. Reichenhemia (western Sunda Shelf), and Begonia sect. Petermannia (Malesia) (Fig. 1). This lack of resolution was also found by Thomas et al. (2012) and indicates a large and rapid range expansion for the genus in the late Miocene from continental Asia across the Malesian region. Although the origin of the Begonia sect. Baryandra clade is somewhat obscure, some informed speculation regarding its entry into the Philippine archipelago is possible. First, of the four migration routes proposed by Dickerson (1928), none would seem to apply. Begonia sect. Baryandra is very species poor on Borneo (three species; Rubite et al. (2013)), and the species sampled here are highly nested in the phylogeny and represent dispersals westward to Borneo from Palawan rather than the other way round (Fig. 1). The only species in the section to occur north of the Philippines, B. fenicis, is also highly nested in the phylogeny and represents a northward dispersal from Luzon to Batan and Lanyu. There is no evidence for colonization from a southern route, as the four species sampled from Mindanao and environs are nested within clades with a Palawan or Luzon origin. As the Philippines is a center of diversity for the monophyletic Begonia sect. Baryandra, with samples from surrounding areas highly nested, the most likely scenario for its arrival on the archipelago is a long-distance dispersal event from western Malesia rather than a migration and subsequent extinction across the migration route. Further pinpointing the arrival locality of the ancestor of Begonia sect. Baryandra in the Philippines is hampered by the lack of resolution at the base of the clade, geographically speaking a three-way split between Luzon, Panay, and Palawan. This lack of resolution represents a rapid range expansion into those areas following dispersal to the Philippines. Panay and Luzon were contiguous at the time of arrival of Begonia sect. Baryandra in the late Miocene to ca. 5 Ma, with Luzon then continuing on a rapid northward trajectory relative to Panay, Mindoro, and Palawan, leaving Panay isolated in its current form as a large island in the western Visayas (Hall, 2002). The most likely scenario is that the long-distance dispersal event leading to the colonization of the Philippines between 11.2 and 8.5 Ma entered on one of the islands then comprising the near-contiguous northern western edge of the archipelago (Palawan, Panay, and Luzon), then rapidly dispersed to the other two. Colonization of the Philippines—Several factors will have influenced the way Begonia sect. Baryandra colonized the Philippines following the first successful dispersal to the northwest of the archipelago in the late Miocene: (1) lateral tectonic movement, (2) tectonic uplift and volcano building, (3) sea level changes, and (4) typhoon patterns. All of these elements interact with the biology of the species in Begonia sect. Baryandra. The geographic configuration of the archipelago has undergone huge change since the late Miocene, and the varying proximity of each island will have affected how likely it is to be colonized. The early branching of B. culasiensis is consistent with the lineage becoming isolated following the geological evolution of Panay and with its increasing separation from Luzon. The other six species on Panay are likely the result of two colonizations by dispersal from either Luzon or Palawan in the Pleistocene, followed by speciation in situ.


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Fig. 3. Maximum clade credibility chronogram from an analysis of the chloroplast alignment in BEAST. Node bars indicate 95% highest posterior density date ranges.


Mindanao was much farther south during the late Miocene, near the equator and hence out of the typhoon belt. The island reached its current latitude during the Pliocene-Pleistocene (Hall, 2002) and is now on the edge of the typhoon belt, and although it experiences typhoons, they are less frequent than in the Visayas and Luzon (Wu et al., 2014). The relatively late arrival of Mindanao into the typhoon belt is congruent with its Begonia flora being species poor and highly nested in the phylogeny. However, the island remains underexplored botanically relative to the rest of the Philippines and further work may change this view. The Mount St. Paul limestone ridge is the youngest geological formation in northern Palawan, being the result of uplift since the mid-Miocene (Piccini and Iandelli, 2011). It has three endemic Begonia species (B. taraw, B. hughesii, and B. tagbanua). The time of the split between these taxa (1.9–5.5 Ma) overlaps with the later phase of this uplift. The ongoing geological development of the Philippine archipelago, especially limestone karst uplift such as this, provides new habitats and the niche space for the evolution of new taxa. Sea level falls during the Pleistocene climatic fluctuations will have caused parts of the archipelago, which are currently islands, to become connected by dry land to form Pleistocene Aggregate Island Complexes (PAICs; Esselstyn and Brown, 2009). The extent to which these connections may have facilitated the dispersal of Begonia species across the aggregate islands is not likely to be large. For example, the proximity of Negros to Panay has not led to its colonization by Begonia sect. Baryandra, despite the linkage of the two in a PAIC along with Cebu and Masbate, which also lack any records for the section. The distribution of B. longiscapa in the southern Philippines conforms to the PAIC comprising Biliran, Leyte, Samar, and Mindanao. However, this is a midaltitude lithophytic species, and the type of lowland forest that would have colonized the soils of the exposed sea floor are unlikely to have formed suitable habitat. Another member of this clade, B. gitingensis, is endemic to the volcanic island of Sibuyan at altitudes of ca. 500 m a.s.l.; Romblon does not form part of a PAIC covering the distribution of other clade members, and hence the occurrence of B. gitingensis must be the result of over-sea dispersal. It is important to remember that for abiotically dispersed plants such as Begonia, their distribution is completely the result of dispersal over different scales, not migration: plants do not walk. Begonia populations are usually restricted to small areas of suitable microhabitat (Hughes and Hollingsworth, 2008; Hughes et al., 2011; Nakamura et al., 2013; Sang et al., 2013), and hence from a begonia’s “eye view” in the Philippines, we have a scenario of islands within islands. Chance events can play a disproportionately large role in determining the distributions of clades and species (Higgins et al., 2003; de Wilde et al., 2011). Genome incongruence— Comparison of the phylogenies derived from the nuclear and chloroplast data reveal considerable hard incongruence. The differing positions of B. wadei and B. elnidoensis (isolated and early branching in the chloroplast phylogeny, highly nested in the nuclear phylogeny) indicates hybridization. The position of the two species in the chloroplast phylogeny likely represents their true phylogenetic position, as they have an unusual fleshy-stemmed morphology and are restricted to coastal karst limestone in contrast with the morphology and ecology of the other species in the ITS phylogeny clade

D′, which are rhizomatous forest lithophytes. We conclude the ITS sequences in B. wadei and B. elnidoensis are likely to have been introgressed from the B. mindorensis lineage, which has an overlapping distribution in northern Palawan and the adjacent islands, with the hybridization event predating the split between B. wadei and B. elnidoensis. In the case of B. palawanensis, the position in the chloroplast phylogeny is incongruent with the taxonomy and morphology of the species and, hence, probably represents a case of chloroplast capture. In combination with the other incongruences and polyphyletic species observed in this study, there is considerable evidence for hybridization being fairly extensive in Begonia sect. Baryandra. For the ITS genotype in the nuclear genome of one species to become introgressed into the genome of another may indicate selection for genes linked to the nucleolar organizing regions where the nrITS reside. For chloroplast genotypes to become introgressed into another species may also indicate selection, either relative, i.e., due to genome incompatibilities leading to reduced male fitness in hybrids (Tsitrone et al., 2003), or absolute, i.e., the invading chloroplast genotype being fitter than the one it replaces leading to a selective sweep (Percy et al., 2014). If the latter, the ease with which Begonia species can swap chloroplast genomes would allow them access to a “plastid pool” of various genotypes from neighboring species. Some of the phylogenetic incongruence may also be due to incomplete lineage sorting. Begonia species have been reported to exist as long-term stable entities even through Pleistocene climatic cycles in Central America (Twyford et al., 2013) and the Ryuku Islands (Nakamura et al., 2014). Such isolated, long-lived populations with poor gene flow may make Begonia species prone to harboring between-population polymorphism and taking a relatively long time to reach monophyly. Influences on the origins of the Philippine flora—The arrival of the Philippines by Begonia sect. Baryandra by a single successful long-distance dispersal event and subsequent archipelago-wide radiation highlights the role of chance in shaping patterns of biodiversity. The colonization of much of the archipelago has been predominantly due to dispersal, although tectonic change may be responsible for early vicariance between Panay and Luzon. The predominantly young, isolated lineages of Begonia in Mindanao are consistent with the early, isolated position of the island to the south. Further investigations into the phylogenetic placement of Mindanao endemics would allow the testing of hypotheses as to the buildup of the Mindanao flora. If the tectonic position of the island has been a strong influence on floristic composition, we would expect older elements of the flora to be nested within clades from southern Malesia (the Moluccas and New Guinea) and younger elements nested with northern or western clades (the northern Philippines or Borneo). A pattern of younger clades being found in Mindanao than in Luzon has been reported for rodents (Jansa et al., 2006). No colonization of Palawan has been detected from other Philippine islands, which is contradictory to the prevailing typhoon track running east to west. Further, the phylogeny is highly geographically structured across the Philippines, and in that respect, comparable to that for Begonia in Sulawesi (Thomas et al., 2012), which is near the equator and hence out of the typhoon belt. This high degree of geographic structure is rather surprising and may indicate that typhoons have not had as much impact on Begonia distribution as one might expect given their dust-like seeds. The apparent lack


of typhoon-influenced dispersal on the Philippine flora needs to be further tested with groups having a similar dispersal syndrome to Begonia (e.g., Gesneriaceae) and differing dispersal syndromes (e.g., Asteraceae). PAIC formation has had a negligible influence on range expansion by Begonia sect. Baryandra, but this influence may be larger for other angiosperm groups. The influence of island linkage during sea-level fall on plant distributions is likely to depend heavily on the ecology of the plants in question and on their altitudinal range and soil preference. We would expect lowland and in particular coastal lowland species to be much more prone to range expansion via PAIC formation than montane species. Further studies are needed to test this hypothesis, either through comparative phylogenetic studies of genera with high endemism from different habitats, or through phylogeographic studies within species or species complexes. LITERATURE CITED ATKINS, H., J. PRESTON, AND Q. C. B. CRONK. 2001. A molecular test of Huxley’s line: Cyrtandra (Gesneriaceae) in Borneo and the Philippines. Biological Journal of the Linnean Society 72: 143–159. BROWN, R. M., AND S. I. GUTTMAN. 2002. Phylogenetic systematics of the Rana signata complex of Philippine and Bornean stream frogs: Reconsideration of Huxley’s modification of Wallace’s Line at the Oriental–Australian faunal zone interface. Biological Journal of the Linnean Society 76: 393–461. DE WILDE, J. J. F. E. 2011. Begoniaceae. In K. Kubitzki [ed.], The families and genera of vascular plants, 56–71. Springer, Berlin, Germany. DE WILDE, J. J. F. E., M. HUGHES, M. RODDA, AND D. C. THOMAS. 2011. Pliocene intercontinental dispersal from Africa to Southeast Asia highlighted by the new species Begonia afromigrata (Begoniaceae). Taxon 60: 1685–1692. DEMESURE, B., N. SODZI, AND R. J. PETIT. 1995. A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Molecular Ecology 4: 129–131. DIAMOND, J. M., AND M. E. GILPIN. 1983. Biogeographic umbilici and the origin of the Philippine avifauna. Oikos 41: 307–321. DICKERSON, R. E. 1928. Distribution of life in the Philippines. Monographs of the Bureau of Science, Manila 2: 1–322. DRUMMOND, A. J., M. A. SUCHARD, D. XIE, AND A. RAMBAUT. 2012. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular Biology and Evolution 29: 1969–1973. ESSELSTYN, J. A., AND R. M. BROWN. 2009. The role of repeated sea-level fluctuations in the generation of shrew (Soricidae: Crocidura) diversity in the Philippine Archipelago. Molecular Phylogenetics and Evolution 53: 171–181. GRUDINSKI, M., L. WANNTORP, C. M. PANNELL, AND A. N. MUELLNER-RIEHL. 2014. West to east dispersal in a widespread animal-dispersed woody angiosperm genus (Aglaia, Meliaceae) across the Indo-Australian Archipelago. Journal of Biogeography 41: 1149–1159. HALL, R. 2002. Cenozoic geological and plate tectonic evolution of SE Asia and the SW Pacific: Computer-based reconstructions, model and animations. Journal of Asian Earth Sciences 20: 353–431. HALL, T. A. 1999. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98 Available at http://www.mbio. ncsu.edu/bioedit/bioedit.html. HEANEY, L. 1991. A synopsis of climatic and vegetational change in Southeast Asia. Climatic Change 19: 53–61. HEANEY, L. R. 1986. Biogeography of mammals in SE Asia: Estimates of rates of colonization, extinction and speciation. Biological Journal of the Linnean Society 28: 127–165. HEANEY, L. R., D. S. BALETE, M. L. DOLAR, A. C. ALCALA, A. T. L. DANS, P. C. GONZALES, N. R. INGLE, ET AL. 1998. A synopsis of the mammalian fauna of the Philippine Islands. Fieldiana Zoology 88: 1–61.

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RAMBAUT, A., AND A. J. DRUMMOND. 2007. Tracer v1.5. Available at http:// beast.bio.ed.ac.uk/Tracer. REE, R. H., B. R. MOORE, C. O. WEBB, AND M. J. DONOGHUE. 2005. A likelihood framework for inferring the evolution of geographic range on phylogenetic trees. Evolution 59: 2299–2311. REE, R. H., AND S. A. SMITH. 2008. Maximum likelihood inference of geographic range evolution by dispersal, local extinction, and cladogenesis. Systematic Biology 57: 4–14. RONQUIST, F. 1997. Dispersal–vicariance analysis: A new approach to the quantification of historical biogeography. Systematic Biology 46: 195–203. RONQUIST, F., AND J. P. HUELSENBECK. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. RONQUIST, F., M. TESLENKO, P. VAN DER MARK, D. L. AYRES, A. DARLING, S. HÖHNA, B. LARGET, ET AL. 2012. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539–542. ROSEN, D. E. 1976. A vicariance model of Caribbean biogeography. Systematic Zoology 24: 431–464. ROSEN, D. E. 1978. Vicariant patterns and historical explanation in biogeography. Systematic Zoology 27: 159. RUBITE, R. R., M. HUGHES, G. J. ALEJANDRO, AND C.-I. PENG. 2013. Recircumscription of Begonia sect. Baryandra (Begoniaceae): evidence from molecular data. Botanical Studies (Taipei, Taiwan) 54: 38. SANG, J., R. KIEW, AND C. GERI. 2013. Revision of Begonia (Begoniaceae) from the Melinau Limestone in Gunung Mulu National Park and Gunung Buda National Park, Sarawak, Borneo, including thirteen new species. Phytotaxa 99: 1–34. SILER, C. D., AND J. R. OAKS, J. A. ESSELSTYN, A. C. DIESMOS, AND R. M. Brown. 2010. Phylogeny and biogeography of Philippine bent-toed geckos (Gekkonidae: Cyrtodactylus) contradict a prevailing model of Pleistocene diversification. Molecular Phylogenetics and Evolution 55: 699–710. SILER, C. D., J. R. OAKS, L. J. WELTON, C. W. LINKEM, J. C. SWAB, A. C. DIESMOS, AND R. M. BROWN. 2012. Did geckos ride the Palawan raft to the Philippines? Journal of Biogeography 39: 1217–1234. STULTS, D. Z., AND B. J. AXSMITH. 2011. First macrofossil record of Begonia (Begoniaceae). American Journal of Botany 98: 150–153. THOMAS, D. C., M. HUGHES, T. PHUTTHAI, W. H. ARDI, S. RAJBHANDARY, R. RUBITE, D. TWYFORD, AND J. E. RICHARDSON. 2012. West to east

dispersal and subsequent rapid diversification of the mega-diverse genus Begonia (Begoniaceae) in the Malesian archipelago. Journal of Biogeography 39: 98–113. THOMAS, D. C., M. HUGHES, T. PHUTTHAI, S. RAJBHANDARY, R. RUBITE, W. H. ARDI, AND J. E. RICHARDSON. 2011. A non-coding plastid DNA phylogeny of Asian Begonia (Begoniaceae): evidence for morphological homoplasy and sectional polyphyly. Molecular Phylogenetics and Evolution 60: 428–444. TSITRONE, A., M. KIRKPATRICK, AND D. A. LEVIN. 2003. A model for chloroplast capture. Evolution 57: 1776–1782. TWYFORD, A., C. KIDNER, AND R. ENNOS. 2014. Genetic differentiation and species cohesion in two widespread Central American Begonia species. Heredity 112: 382–390. TWYFORD, A., C. KIDNER, N. HARRISON, AND R. ENNOS. 2013. Population history and seed dispersal in widespread Central American Begonia species (Begoniaceae) inferred from plastome-derived microsatellite markers. Botanical Journal of the Linnean Society 171: 260–276. VAN WELZEN, P. C., J. W. F. SLIK, AND J. ALAHUHTA. 2005. Plant distribution patterns and plate tectonics in Malesia. Biologiske Skrifter 55: 199–217. Available at http://www.cabdirect.org/abstracts/20073082423.html. VORIS, H. K. 2000. Maps of Pleistocene sea levels in Southeast Asia: Shorelines, river systems and time durations. Journal of Biogeography 27: 1153–1167. WEBB, C. O., AND R. REE. 2012. Historical biogeography inference in Southeast Asia. In D. J. Gower, K. G. Johnson, J. E. Richardson, B. R. Rosen, L. Rüber, and S. T. Williams [eds.], Biotic evolution and environmental change in Southeast Asia, 191–215. Cambridge University Press, Cambridge, UK. WOODRUFF , D. S. 2010. Biogeography and conservation in Southeast Asia: How 2.7 million years of repeated environmental fluctuations affect today’s patterns and the future of the remaining refugial-phase biodiversity. Biodiversity and Conservation 19: 919–941. WU, L., C. CHOU, C.-T. CHEN, R. HUANG, T. R. KNUTSON, J. J. SIRUTIS, S. T. GARNER, ET AL. 2014. Simulations of the present and late-twentyfirst-century western North Pacific tropical cyclone activity using a regional model. Journal of Climate 27: 3405–3424. YU, Y., A. J. HARRIS, AND X. J. HE. 2012. RASP (Reconstruct Ancestral State in Phylogenies) 2.1b. Available at http://mnh.scu.edu.cn/soft/ blog/RASP.


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APPENDIX 1. Table of GenBank accession numbers and herbarium vouchers used in the molecular phylogenetic analysis (E, Royal Botanic Garden Edinburgh, Scotland; HAST, Biodiversity Research Center, Academia Sinica, Taiwan; PNH, National Museum, Manila, Philippines). Taxon

ndhA intron

ndhF–rpl32

rpl32–trnL

trnC–ycf6

ycf6–psbM

psbM–trnD

ITS

B. ×dinglensis ined. B. acuminatissima Merr. B. aff. gueritziana Gibbs B. albococcinea Hook. B. anisoptera Merr. B. biliranensis Merr. B. blancii M. Hughes & C.-I Peng B. bonthainensis Hemsl. B. calcicola Merr. B. camiguinensis Elmer B. castilloi Merr. B. chingipengii Rubite B. chloroneura P.Wilkie & Sands B. cleopatrae Coyle B. culasienesis ined. B. culasiensis ined. B. dipetala Graham B. dregei Otto & A.Dietr. B. elmeri Merr. B. elnidoensis ined. B. fenicis Merr. B. fenicis Merr. B. floccifera Bedd. B. forbesii King B. foxworthyii Burkill ex Ridl. B. gabaldonensis ined. B. gitingensis Elmer B. gueritziana Gibbs B. gueritziana Gibbs B. gutierrezii Coyle B. hernandioides Merr. B. hernandioides Merr. B. hughesii ined. B. hughesii ined. B. ignorata Irmsch. B. kingiana Irmsch. B. klemmei Merr. B. laruei M.Hughes B. longiscapa Warb. B. longiscapa Warb. B. longiscapa Warb. B. longiscapa Warb. B. manillensis A.DC. B. masoniana Irmsch. B. merrilliana ined. B. mindorensis Merr.

KR186444 KR186445 KR186446 KR186447 KR186448 KR186449 KR186450

KR186531 KR186532 KR186533 KR186534 KR186535 KR186536 KR186537

KR186705 KR186706 KR186707 KR186708 KR186709 KR186710 KR186711

KR186791 KR186792 KR186793 KR186794 KR186795 KR186796 KR186797

KR186878 KR186879 KR186880 KR186881 KR186882 KR186883 KR186884

KR186618 KR186619 KR186620 KR186621 KR186622 KR186623 KR186624

KR186970 KR186965 KR186976 no data KR186987 KR186997 KR187008

Peng P23859 (HAST) Rubite R321 (PNH) Peng P22344 (HAST) Peng P23302 (HAST) Rubite R479 (PNH) Rubite R311 (PNH) Peng P22545 (HAST)

KR186451 KR186452 KR186453 KR186454 KR186455 KR186456

KR186538 KR186539 KR186540 KR186541 KR186542 KR186543

KR186712 KR186713 KR186714 KR186715 KR186716 KR186717

KR186798 KR186799 KR186800 KR186801 KR186802 KR186803

KR186885 KR186886 KR186887 KR186888 KR186889 KR186890

KR186625 KR186626 KR186627 KR186628 KR186629 KR186630

no data JX656719 JX656702 no data JX656704 KR186966

Peng P22531 (HAST) Peng P20761 (HAST) Rubite R506 (PNH) Rubite R98 (PNH) Peng P23368 (HAST) Wilkie et al., 29015 (E)

KR186457 KR186458 KR186459 KR186460 KR186461 KR186462 KR186463 KR186464 KR186465 KR186466 KR186467 KR186468


KR186718 no data KR186719 KR186720 KR186721 KR186722 KR186723 KR186724 KR186725 KR186726 KR186727 KR186728




KR186967 KR186968 KR186969 no data no data KR186971 KR186972 KR186973 KR186974 no data JX656718 KF636487

Wilkie et al., 25373 (E) Peng P23793 (HAST) Rubite R234 (PNH) Peng P22520 (HAST) Peng P20868 (HAST) Rubite R319 (PNH) Peng P23508 (HAST) Peng P10794 (HAST) Unknown NK11979 (HAST) Peng P21216 (HAST) Peng P22685 (HAST) Peng P22721 (HAST)

KR186469 KR186470 KR186471 KR186472 KR186473 KR186474 KR186475 KR186476 KR186477 KR186478 KR186479 KR186480 KR186481 KR186482 KR186483 KR186484 KR186485 KR186486 KR186487 KR186488 KR186489






no data KR186975 KR186977 KR186978 KR186980 no data KR186981 KR186982 no data no data KR186983 no data KR186984 KR186985 KR186986 KR186988 KR186989 no data KR186990 KR186991

Peng P23356 (HAST) Rubite R255 (PNH) Peng P22311 (HAST) Peng P22342 (HAST) Blanc s.n. (E) Peng P21006 (HAST) Rubite R106 (PNH) Peng P23466 (HAST) Peng P23475 (HAST) Peng P22725 (HAST) Peng P21226 (HAST) Rubite R182 (PNH) Hughes et al. MH1398 (E) Rubite R298 (PNH) Rubite R309 (PNH) Rubite R316 (PNH) Rubite R420 (PNH) Rubite R256 (PNH) Peng P21411 (HAST) Peng P23765 (HAST) Rubite R326 (PNH)

KR186490 KR186491

KR186577 KR186578

KR186750 KR186751

KR186837 KR186838

KR186924 KR186925

KR186664 KR186665

KR187006 no data

Peng P23456 (HAST) Peng P20879 (HAST)

KR186492

KR186579

KR186752

KR186839

KR186926

KR186666

KR186995

Rubite R419 (PNH)

KR186493

KR186580

KR186753

KR186840

KR186927

KR186667

KR186996

Rubite R406 (PNH)

KR186494

KR186581

KR186754

KR186841

KR186928

KR186668

KR186998

Peng P23855 (HAST)

KR186495

KR186582

KR186755

KR186842

KR186929

KR186669

no data

Peng P23373 (HAST)

KR186496

KR186583

KR186756

KR186843

KR186930

KR186670

KR187000

Peng P23451 (HAST)

KR186497

KR186584

KR186757

KR186844

KR186931

KR186671

KR187001

Peng P23372 (HAST)

KR186498

KR186585

KR186758

KR186845

KR186932

KR186672

KR187002

Peng P23586 (HAST)

KR186499

KR186586

KR186759

KR186846

KR186933

KR186673

no data

Peng P23858 (HAST)

B. mindorensis Merr. B. nelumbifolia Schltdl. & Cham. B. nigritarum (Kamel) Steud. B. nigritarum (Kamel) Steud. B. nigritarum (Kamel) Steud. B. nigritarum (Kamel) Steud. B. nigritarum (Kamel) Steud. B. nigritarum (Kamel) Steud. B. nigritarum (Kamel) Steud. i (Kamel) Steud.

Voucher


APPENDIX 1.

Continued.

Taxon

ndhA intron

ndhF–rpl32

rpl32–trnL

trnC–ycf6

ycf6–psbM

psbM–trnD

ITS

B. ningmingensis D. Fang, Y. G. Wei & C.-I Peng B. obtusifolia Merr. B. oxysperma A.DC. B. oxysperma A.DC. B. palawanensis Merr. B. rubiteae M.Hughes B. rufipila Merr. B. sect. Reichenheimea

KR186500

KR186587

KR186760

KR186847

KR186934

KR186674

no data

Peng P20322 (HAST)

Voucher

KR186501 KR186502 KR186503 KR186504 KR186505 KR186506 KR186507

KR186588 KR186589 KR186590 KR186591 KR186592 KR186593 KR186594

KR186761 KR186762 KR186763 KR186764 KR186765 KR186766 KR186767

KR186848 KR186849 KR186850 KR186851 KR186852 KR186853 KR186854

KR186935 KR186936 KR186937 KR186938 KR186939 KR186940 KR186941

KR186675 KR186676 KR186677 KR186678 KR186679 KR186680 KR186681

KR186992 KR186993 KR186994 no data KR187004 KR187005 no data

B. sp. 1 B. sp. 2 B. sp. 3 B. sp. 4 B. sp. 5 B. sp. 6 B. sp. 7 B. sublobata Jack

KR186508 KR186509 KR186510 KR186511 KR186512 KR186513 KR186514 KR186515






KR187007 no data KR187009 KR187010 no data no data no data no data

B. subnummarifolia Merr. B. suborbiculata Merr. B. sutherlandii Hook.f. B. sykakiengii ined. B. sykakiengii ined. B. tagbanua ined. B. tagbanua ined. B. taraw ined. B. taraw ined. B. tayabensis Merr. B. tigrina Kiew B. trichocheila Warb. B. wadei Merr. & Quisumb. B. woodii Merr. B. woodii Merr.

KR186516 KR186517 KR186518 KR186519 KR186520 KR186521 KR186522 KR186523 KR186524 KR186525 KR186526 KR186527 KR186528 KR186529 KR186530






KR187012 KR187013 no data JX656720 no data JX656708 JX656721 KR186979 JX656714 JX656709 JX656716 JX656710 KF636465 JX656712 JX656722

Peng P23828 (HAST) Rubite R213 (PNH) Peng P23015 (HAST) Peng P23453 (HAST) Rubite R356 (PNH) Rubite R265 (PNH) Girmansyah et al. DEDEN1490 (E) Peng P23566 (HAST) Kokubagata GK71 (HAST) Rubite R136 (PNH) Rubite R290 (PNH) Peng P23452 (HAST) Peng P23418 (HAST) Rubite R238 (PNH) Girmansyah et al. DEDEN1486 (E) no voucher Rubite R353 (PNH) Jasper 1200-5 (HAST) Peng P23856 (HAST) Peng P23890 (HAST) Blanc s.n. (E) Peng P23472 (HAST) Blanc s.n. taraw2 (E) Blanc s.n. taraw1 (E) Rubite R360 (PNH) Peng P22720 (HAST) Peng P20764 (HAST) Rubite R699 (PNH) Peng P23479 (HAST) Peng P23496 (HAST)