Mid-Tertiary dispersal, not Gondwanan vicariance explains distribution patterns in the wax palm subfamily (Ceroxyloideae: Arecaceae)

Molecular Phylogenetics and Evolution 45 (2007) 272–288 www.elsevier.com/locate/ympev

Mid-Tertiary dispersal, not Gondwanan vicariance explains distribution patterns in the wax palm subfamily (Ceroxyloideae: Arecaceae) Philipp Tre´nel a, Mats H.G. Gustafsson a, William J. Baker b, Conny B. Asmussen-Lange c, John Dransfield b, Finn Borchsenius a,* a

c

Department of Biological Sciences, University of Aarhus, Ny Munkegade, Building 1540, DK-8000 Aarhus C, Denmark b Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK Department of Ecology, Royal Veterinary and Agricultural University Copenhagen, Rolighedsvej 21, DK-1958 Frederiksberg, Denmark Received 7 December 2006; revised 17 March 2007; accepted 22 March 2007 Available online 4 April 2007

Abstract The Ceroxyloideae is a small but heterogeneous subfamily of palms (Arecaceae, Palmae). It includes a Caribbean lineage (tribe Cyclospathae), a southern hemisphere disjunction (tribe Ceroxyleae), and an amphi-Andean element (tribe Phytelepheae), until recently considered a distinct subfamily (Phytelephantoideae) due to its highly derived morphology. A variety of hypotheses have been proposed to account for the biogeography of the subfamily, involving Gondwanan vicariance, austral interplate dispersal from South America to Australia via Antarctica, Andean orogeny, and Pleistocene refuges. We assessed the systematic classification and biogeography of the group based on a densely sampled phylogeny using >5.5 kb of DNA sequences from three plastid and two nuclear genomic regions. The subfamily and each of its three tribes were resolved as monophyletic with high support. Divergence time estimates based on penalized likelihood and Bayesian dating methods indicate that Gondwanan vicariance is highly unlikely as an explanation for basic disjunctions in tribe Ceroxyleae. Alternative explanations include a mid-Tertiary trans-Atlantic/trans-African dispersal track and the ‘‘lemurian stepping stones’’ hypothesis. Austral interplate dispersal of Oraniopsis to Australia could have occurred, but apparently only in the midEocene/early Oligocene interval after global cooling had begun. Our data do not support Pleistocene climatic changes as drivers for speciation in the Andean-centered Phytelepheae as previously proposed. Radiation in this tribe coincides largely with the major uplift of the Andes, favoring Andean orogeny over Pleistocene climatic changes as a possible speciation-promoting factor in this tribe. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Arecaceae; Palmae; Ceroxyleae; Phytelepheae; Cyclospatheae; Molecular systematics; Molecular dating; PRK; RPB2; matK; ndhF; trnD–trnT; Dispersal; Vicariance; Gondwana; Pleistocene refuges; Andean orogeny; Austral interplate dispersal; Antarctica

1. Introduction The Ceroxyloideae is one of five subfamilies in the palm family (Arecaceae, Palmae) and comprises 8 genera and 42 species (Dranfield et al., 2005; Govaerts and Dransfield, 2005). Bringing together a small Caribbean lineage (tribe Cyclospatheae), a Gondwanan disjunction (tribe Ceroxyleae), and a morphologically highly derived, amphi*

Corresponding author. Fax: +45 89422722. E-mail address: fi[email protected] (F. Borchsenius).

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Andean lineage (tribe Phytelepheae) previously known as subfamily Phytelephantoideae (sensu Uhl and Dransfield, 1987), the Ceroxyloideae is one of the most heterogeneous assemblages in the family, both in terms of morphology and biogeography. Furthermore, it is ecologically outstanding. In spite of its limited number of taxa, it spans a wide range of latitudes (25°N–33°S) and altitudes including the highest elevation record of any palm (Ceroxylon parvifrons, 3500 m above sea level; Borchsenius et al., 1998). It occupies a diverse array of habitats including seasonally dry forests (Cyclospatheae, Ceroxyleae), lowland

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rainforests (Phytelepheae, Ceroxyleae), and mountain forests (Ceroxyleae). It even includes the world’s only aquatic palm species that is fully submerged during early stages of its life cycle (Ravenea musicalis; Dransfield and Beentje, 1995). Molecular phylogenetic studies over the last decade have indicated that the Phytelepheae is related to the remaining two tribes of Ceroxyloideae, but do not agree on the precise nature of the relationships. Phytelepheae has been found to be sister to either Cyclospatheae (Asmussen and Chase, 2001; Hahn, 2002a,b) or Ceroxyleae (Hahn, 2002a,b; Lewis and Doyle, 2001; Uhl et al., 1995). All these studies, however, were based on a limited taxon sample for the Ceroxyloideae and generally reported low support values for the inferred relationships. The most comprehensive family level phylogeny so far published (Asmussen et al., 2006) analyzed 7102 characters from four plastid regions for a total of 178 palm species. It found Cyclospatheae to be sister to a Ceroxyleae–Phytelepheae clade. Bootstrap support for the monophyly of the Ceroxyloideae was, however, only 63%, rendering it the most poorly supported subfamily in the Palmae. Recent developments in molecular divergence time estimation (reviewed in Renner, 2005, and Rutschmann, 2006) offers means for revisiting existing biogeographic hypotheses by investigating whether both pattern and timing of cladogenesis is in concordance with a given biogeographic scenario. Following the general acceptance of Wegener’s continental drift theory in the late 1960s vicariance became accepted as the predominant mechanism in historical biogeography due to its ability to offer a unifying explanation for parallel disjunctions in unrelated groups (Willey, 1988). Recent research, however, has revived the importance of dispersal (reviewed in de Queiroz, 2005), although this position has not been unchallenged (Heads, 2005). Several studies have shown that known geological processes believed to have caused vicariance, such as the break-up of Gondwana land, fail to fit expected patterns (e.g., Sanmartı´n and Ronquist, 2004) and/or expected timing of cladogenesis (e.g., Knapp et al., 2005; Richardson et al., 2004; Renner, 2004a), or that mixed scenarios involving both vicariance and dispersal needed to be invoked (e.g., Heinrichs et al., 2006; Sytsma et al., 2004). Morley (2000, 2003) stressed Tertiary interplate dispersal routes as important alternatives to vicariance, and Renner (2004b) pointed to the role of sea currents and prevailing wind directions as unifying explanations for repeated trans-oceanic dispersals. The diverse distribution patterns in Ceroxyloideae (Fig. 1) have received much attention in the study of palm biogeography (e.g., Moore, 1973; Uhl and Dransfield, 1987). Tribe Cyclospatheae, with only one genus (Pseudophoenix, 4 spp), is distributed in the Caribbean region. Three species are restricted to the interior parts of the island of Hispaniola, while the fourth (Ps. sargentii) has a wider range in coastal areas of the Florida Keys, Bahamas, Greater Antilles, Mexico and Belize (Zona, 2002). Pseudophoenix is easily recognized by its solitary habit, promi-

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nently ringed, often swollen stems, the presence of a crownshaft, otherwise not present in the subfamily, and solitary, hermaphroditic, and stalked (pseudopedicellate) flowers on many-branched inflorescences (Henderson et al., 1995). Tribe Ceroxyleae (4 genera, 30 spp.) is distributed across former Gondwanan landmasses of the southern Hemisphere (Dransfield et al., 1985): Ravenea occurs in Madagascar (15 spp.) and the adjacent Comoros Islands (2 spp.), Oraniopsis in northeastern Australia (1 sp.), Juania on the Juan Ferna´ndez archipelago some 600 km west of Chile (1 sp.), and Ceroxylon in the tropical part of the Andes mountains (11 spp.). Ceroxyleae shares a number of characters with Cyclospatheae such as solitary, generally tall-stemmed habit, several peduncular bracts, solitary flowers on multi-branched inflorescences, and triovulate gynoecia (Uhl and Dransfield, 1987). Both groups have small to medium-sized, generally reddish fruits, presumably dispersed by birds. Ceroxyleae differs from Cyclospatheae in its dioecious breeding system, in the lack of a crownshaft, and in their flowers borne on true pedicels (Uhl and Dransfield, 1987). Besides holding the highest elevation record of any palm (C. parvifrons), the tribe is remarkable in comprising the world’s tallest palm species (C. quindiuense, 60 m tall; Henderson et al., 1995). Finally, tribe Phytelepheae (3 genera, 8 spp.) occurs in the amphiAndean lowlands of NW South America and adjacent Panama (Barfod, 1991). One species (Ammandra decasperma) occurs on both sides of the Andes, while the remaining seven are restricted to one of three centers of endemism separated by the Andean cordilleras: (1) the western Amazon, (2) the inter-Andean valleys of the Cauca and Magdalena rivers, and (3) the Pacific Choco´ biogeographic region stretching from Panama to Ecuador. It constitutes a morphologically highly distinct lineage within the palm family differing from most other palms in having four-parted rather than three-parted female flowers, polyandrous male flowers with up to 1200 stamens per flower (a record among all angiosperms) and woody, multi-carpellate fruits arranged in head-like clusters (Barfod, 1991). Due to its unusual morphology, botanists of the 19th century placed members of Phytelepheae in various monocot families such as the Cyclanthaceae, Typhaceae, Pandanaceae, and the Arecaceae. Phytelepheae has only recently been subsumed into the Ceroxyloideae on the basis of molecular evidence (Asmussen et al., 2006; Dranfield et al., 2005). Four main hypotheses have been put forward to explain the distribution patterns found among members of Ceroxyloideae: (1) Dransfield et al. (1985) and Uhl and Dransfield (1987) suggested for tribe Ceroxyleae an ancient origin on Gondwanaland during late Cretaceous times followed by rafting on fragments of land destined to become present day Madagascar, Australia and South America, with subsequent dispersion of Juania to the volcanic and much younger (4 million years; Stuessy et al., 1984) Robinson Crusoe Island of the Juan Ferna´ndez archipelago (Gondwanan vicariance hypothesis). (2) Uhl and Dransfield (1987) presented a modified version of this hypothesis, stating that

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Fig. 1. Distribution of palm subfamily Ceroxyloideae and 5 Eocene fossil floras mentioned in the text (+): (1) Laguna del Hunco and Rı´o Pichileufu´, Patagonia, mid-Eocene palm fossils, proxy mean annual temperature (MAT): 16.6 ± 2.0 °C (Wilf et al., 2005); (2) Seymour Island, Antarctic Peninsula, Eocene/Oligocene palmoid pollen (Cranwell, 1959), Eocene–earliest Oligocene MAT: 7–15 °C (Thorn and DeConto, 2006); (3) McMurdo Sound, Antarctica, Eocene/Oligocene palmoid pollen (Cranwell et al., 1960), mid–late-Eocene MAT: <13 °C (Thorn and DeConto, 2006); (4) Maslin Bay, Australia, mid-Eocene palm fossils (Greenwood and Conran, 2000); (5) Hotham heights, Australia, early Eocene MAT: 14–20 °C (Carpenter et al., 2004).

rather than an early rafting on the Australian plate during the Late Cretaceous, Australian Oraniopsis may have reached its present day position by migration via an Antarctic route during the early Tertiary, when Antarctic climate was probably suitable for megathermal taxa (austral interplate dispersal hypothesis). (3) Moore (1973) noted that the distribution of tribe Phytelepheae coincides well with three postulated forest refuges (sensu Haffer, 1970) that are believed to have functioned as areas where rainforest taxa retreated during Pleistocene glacial periods (Pleistocene refuge hypothesis). (4) In contrast, Prance (1982), followed by Barfod (1991) and Morley (2000), concluded that the amphi-Andean disjunctions in Phytelepheae were best explained by the Andean up-lift history from Late Miocene/Pliocene times onward, when the northern Andes probably reached elevations high enough to prevent dispersal across the cordilleras (Andean vicariance hypothesis). Here, we present a densely sampled phylogeny of palm subfamily Ceroxyloideae based on more than 5.5 kb of nuclear and plastid DNA sequence data, including 85% of its accepted species and all its genera. The purpose of the study was (1) to readdress the monophyly of Ceroxyloideae and clarify relationships within in the group; and (2) to evaluate the existing hypotheses concerning the biogeographic history of the Ceroxyloideae. More specifically, we tested whether topology and timing of cladogenesis, using molecular dating techniques, are in concordance with (a) the Gondwanan vicariance hypothesis, (b) the austral interplate dispersal hypothesis, (c) the Pleistocene refuge hypothesis, and (d) the Andean vicariance hypothesis. 2. Material and methods 2.1. Taxon sampling, DNA sequencing, and alignment A total of 49 accessions belonging to 35 of the currently recognized 41 species of palm subfamily Ceroxyloideae,

representing all eight genera of the subfamily, were included in the analysis (Supplementary Table 1). Ten outgroup taxa were selected among five tribes of subfamily Arecoideae, which is sister to Ceroxyloideae (Asmussen et al., 2006). Sequences were obtained for plastid regions matK (GenBank Accession Nos. EF128228–EF128265), 30 ndhF (EF128266–EF128314), and trnD–trnT (EF128315–EF128361), as well as for the nuclear regions PRK intron 4 (EF128362–EF128396) and RPB2 intron 23 (EF128397–EF128433). Voucher information and Genbank number for each accession is provided in Supplementary Table 1. Total genomic DNA was extracted from silica gel dried plant material, fresh material or herbarium specimens using the DNeasyÒ Plant Mini Kit (QIAGEN, Crawley, West Sussex, UK). For 19 accessions raw genomic DNA was obtained from the DNA Bank at the Royal Botanic Gardens, Kew, UK. Polymerase chain reactions (PCR) were set up using the Ampliqon DNA Polymerase Master ˚ byhøj, Denmark). Final Mix kit (Bie & Berntsen A-S, A PCR reaction volumes of 25 ll were prepared using the following reagent volumes: 5 ll of 5 M betaine, 1 ll of each primer (10 lM), 1 ll total DNA, 13 ll Taq Master Mix, and 4 ll autoclaved H2O. PCR amplifications were conducted on a DNA Engine PTC 200 (MJ Research, Inc., Waltham, MA, USA). Primers were for the PRK region: PRK717F and PRK969R of Lewis and Doyle (2002), for the RPB2 region: RPB2-M11R of Roncall et al. (2005) and RPB2Forward of Thomas et al. (2006), and for the 30 ndhF region: 1101F of Olmstead and Sweere (1994) and 2110R of Terry et al. (1997). PCR and sequencing of the regions matK and trnD–trnT were according to the protocols described in Asmussen et al. (2006) and Demesure et al. (1995), respectively. Thermal cycling conditions for each region are listed in Supplementary Table 2. PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN). Purified PCR products were

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Model recommended by AIC in MrModeltest. No. of ingroup nodes with Boostrap support >90%. a

trnD–trnT 3 ndhF

47+9 913–919 902 83 (9.2%) 42 (4.7%) GTR+I+C 38 113 0.81 0.94 3 47+10 1686–1910 1789 134 (7.5%) 63 (3.5%) GTR+I+C 24 167 0.83 0.91 2

matK RPB2 PRK

42+10 454–980 1106 281 (25.4%) 148 (13.4%) GTR+C >25,000 413 0.83 0.92 8

0

cpDNA nDNA

44+10 586–805 940 339 (36.1%) 180 (19.2%) GTR+C >25,000 462 0.86 0.94 9 b

2.2.2. Tree search and clade support Data were analyzed both under the parsimony optimality criterion and using Bayesian methods. In all analyses, gaps

Table 1 Data and tree statistics

2.2.1. Data congruence and conflict assessment Congruence of data partitions was investigated by pairwise (cf. Baker and DeSalle, 1997) as well as simultaneous partition homogeneity tests (PHT) as implemented in PAUP*4.0b10 (Swofford, 2002) by running 1000 permutation cycles with each cycle consisting of a heuristic maximum parsimony (MP) search of 10 random sequence addition replicates with TBR swapping and holding no more than five trees each step. Uninformative characters were excluded prior to analysis (Lee, 2001). Initial pairwise PHTs showed no phylogenetic incongruence for any of the three plastid regions (p > 0.43), and since each of these contributed only a limited number of informative characters (Table 1), they were combined in all further analyses (cpDNA). The amount of conflict among the three data partitions (cpDNA, PRK, and RPB2) in the combined analysis was quantified using the partitioned branch support (PBS; Baker and DeSalle, 1997; Gatesy et al., 1999) as implemented in TreeRot.v2 (Sorenson, 1999). The PBS provides a measure of the relative contribution from each locus to the Bremer support (i.e., decay index) in the combined analysis. A negative locus contribution is interpreted as a phylogenetic signal in conflict with the remaining loci (Gatesy et al., 1999). Bremer support and PBS was calculated by subjecting each of the constrained trees produced by TreeRot.v2 to 1000 heuristic MP searches of 10 random sequence addition replicates with TBR swapping and holding no more than five trees each step.

No. of ingroup + outgroup taxa Sequence length (bp) No. of aligned characters No. of variable characters No. of parsimony informative characters Substitution modela Maximum parsimony trees Tree length Ensemble Consistency Index Ensemble Retention Index BS > 90%b

2.2. Phylogenetic analysis

47+7 849–915 832 56 (6.7%) 24 (2.9%) GTR+C 31 67 0.82 0.92 1

47+10 — 3523 273 (7.8%) 129 (3.7%) GTR+I+C >25,000 359 0.79 0.91 7

Combined cpDNA

47+10 — 5569 893 (16.0%) 587 (10.5%) GTR+I+C 5471 1253 0.82 0.92 17

Combined cp+nDNA

sequenced by Macrogen, Inc., Seoul, South Korea. All regions were sequenced in both directions and sequences were edited and assembled in contigs using Sequencher 3.1.1 (Gene Codes Corporation, Ann Arbor, MI, USA). An initial automated alignment was obtained using the internet version of Dialign-T (Subramanian et al., 2005) with default settings for nucleotide data (http://dialignt.gobics.de/submission?type=dna). Dialign-T employs an improved version of the segment-based alignment approach (Morgenstern, 1999) and has been shown to produce alignments comparable to ClustalW for globally related sequences, while it outperforms that program for locally related sequences (Subramanian et al., 2005). In our study, Dialign-T produced alignments highly similar to those produced by ClustalW (as implemented in BioEdit; Hall, 1999) for the sequences of the generally more conservative plastid regions, but produced alignments with significantly fewer gaps for the non-coding, variable nuclear sequences. Alignments were finally refined by eye using BioEdit. The analyzed matrix is available at TreeBase (www.treebase.org) or on request from the first author.

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were treated as missing characters. All maximum parsimony (MP) searches were carried out in PAUP*4.0b10, with constant characters excluded and default settings otherwise. To adequately explore the tree space a two-step search strategy was employed for all data sets and combined analyses. Initially, 1000 replicate searches with random sequence addition followed by TBR branch-swapping and saving no more than 20 shortest trees each step were conducted. Subsequently, a second round of TBR swapping was performed on the trees collected during the initial 1000 replicates but this time saving up to 25,000 optimal trees, with all trees swapped to completion. To avoid the potential effects of a known bug in PAUP*4.0b10, MP-trees found were filtered (filter = best) to ensure that only the shortest trees were retained. Clade support was calculated using the bootstrap (BS; Felsenstein, 1985) by conducting 1000 re-sampling searches, each consisting of 10 random searches with TBR branch swapping and holding no more than five trees each step. Bayesian analysis of the combined data set was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). Prior to analysis, each data set was submitted to a series of hierarchical likelihood ratio tests using MrModeltest (Nylander, 2004) and an appropriate substitution model was chosen using the Akaike information criterion (Table 1). During the Metropolis-coupled Markov chain Monte Carlo (MCMC) tree search, all parameters were uncoupled across partitions. Prior settings were left at their default values (uniform priors). Parameter values for the Metropolisproposals (props settings) were set according to Nylander et al. (2004). The temperature parameter was set to 0.01. Two independent MCMC processes, each with four simultaneous chains of which three were heated chains, were initiated using random starting trees and run for 2,000,000 generations with a sampling frequency of one every 100th generation. Trees sampled prior to stabilisation of likelihood scores and parameter estimates for the two runs were omitted (burn-in; 800,000 generations). The remaining trees from the two runs (24,000 trees) were combined in a majority rule consensus tree summarizing information on topology and branch lengths (means of the posterior probability density) as implemented in MrBayes 3.1.2. 2.3. Biogeographic analysis 2.3.1. Divergence times estimation Initial likelihood ratio tests for rate constancy were conducted for each partition in PAUP*4.0b10. In all cases, the null-hypothesis of a clock-like evolution was rejected (p < 0.001). Two methods that relax the strict molecular clock by assuming auto-correlated rates between parent and daughter branches were used to estimate divergence dates: Sanderson’s (2002) semiparametric Penalized Likelihood method (PL); and the Bayesian relaxed clock (BRC) approach of Thorne and Kishino (2002). All divergence date analyses were conducted using the combined data set, as this is expected to minimize the effect of potentially biased signals of single partitions (Bell and Donoghue,

2005; Renner, 2005). The tree resulting from Bayesian analysis was chosen as the input tree with taxa causing zerolength branches pruned out (Fig. 4). PL analysis was conducted using the program r8s1.71 (Sanderson, 2003) and the following settings: outgroup taxa pruned, four searches from random starts, all with optimization via the Truncated-Newton method, log-scaled rate penalty and default settings otherwise. Solutions for age estimates were subjected to a gradient check to control whether estimated ages actually represented peaks in the likelihood landscape and whether the active constraints (i.e., with the bound hit) were reached in an upward moving fashion on a slope in this landscape. An optimal smoothing parameter value of 320 was found by cross-validation (crossv = yes cvstart = 0 cvinc = 0.5 cvnum = 10) prior to divergence date estimation. 95%-confidence intervals were obtained from the curvature of the likelihood surface around the parameter estimate using the divtime command and a cut-off value of four (Cutler, 2000). BRC divergence date estimation was carried out using the multidistribute program package (available from J. Thorne, NC State University, USA) and PAML (Yang, 2000) according to the protocol described in Rutschmann (2005). Data were treated as a single partition. MCMC approximated Bayesian posterior ages of branching events and 95% credibility intervals were calculated given a set of priors and user-specified time constraints. The MCMC run consisted of 1,000,000 cycles of burn-in, followed by 2,000,000 generations during which parameter estimates were sampled every 100th generation. Following the recommendations in the program manual, mean, and standard deviation of the prior distribution for the time expected between tip and root, for the rate prior at the root, and for the Brownian motion prior were set to 1.0 (corresponding to 100 million years), to 0.01, and to 1.0, respectively. Two independent MCMC chains were run to ensure that parameter settings in the analysis resulted in stationary estimates. 2.3.2. Calibration and time constraints The fossil record for Ceroxyloideae is limited. However, Brown (1956) reported a sandstone cast fossil (Phytelephas olsonii) from late Miocene/early Pliocene substrates of Ecuador that remarkably resembles an ivory-nut. Based on the shape and size of this fossil Barfod (1991) concluded that there was ‘‘no doubt that this fossil species is . . . a Phytelephas’’. In addition, Harley and Baker (2001) noted a similarity between extant Ravenea pollen and the distinctly monoporate, spinose pollen fossil Echimonoporopollis grandiporus described by Saxena et al. (1992) from lower to middle Eocene sediments of India (Harley, 2006). Monoporate, spinose pollen is scarce amongst extant monocots, found elsewhere in modern and fossil species of the Pandanaceae (Hotton et al., 1994) and Araceae (Stockey et al., 1997). However, E. grandiporus pollen differs from that of these two families in being larger in size, having a larger pore diameter, and having larger spines. In these

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characters, E. grandiporus does show character state overlap with Ravenea (Ferguson et al., 1987). On the other hand, the exine of extant Ravenea appears to be thicker in relation to overall pollen size when compared to E. grandiporus, leading Harley (2006) to cast some doubt on the identity of this fossil. Fossils of other Ceroxyloideae have been reported, but are considered too unreliable (Barfod, 1991; Harley and Baker, 2001; Harley, 2006) to be used as calibration points. These include a fossilized vessel-less stem from the Miocene of the West Indies (Phytelephas sewardii; Kaul, 1943), the semitectate monosulcate fossil pollen Liliaciditus tritus known from near-shore habitats of Paleocene to lower Oligocene strata of south-eastern North America (resembling Pseudophoenix; Frederiksen, 1980; Melchior, 1998), the monoporate fossil pollen Jacobipollis from the Miocene of India (resembling Ammandra; Harley, 2006), the simple-tectate fossil pollen Palmaepites eocenica from the Eocene of India (resembling Juania; Biswas, 1962), and finally two coarsely reticulate pollen fossil types: Luminidites from the late Oligocene–early Miocene of New Zealand (Pocknall and Mildenhall, 1984) and Longapertitis from Paleocene strata of northern Pakistan (Frederiksen, 1994), late Cretaceous to late Eocene strata of Africa (Pan et al., 2006), and late Eocene strata of Colombia (Jaramillo and Dilcher, 2000), both resembling modern Ceroxylon (Harley and Baker, 2001; Harley, 2006). Other sources of calibrations available for this study include constraints based on secondary dating or geological age. Savolainen et al. (2006) estimated the age of the crown node of the Ceroxyloideae to approximately 60 million years (my) in a recent molecular dating analysis of the Arecoid line (i.e., Arecoideae–Ceroxyloideae clade). Estimates obtained in that study are well in line with the estimated crown node ages of the palm family (110 ± 16 my—Janssen and Bremer, 2004; 73 ± 5 my— Wikstro¨m et al., 2001), the Arecoid line (68 ± 6 my—Wikstro¨m et al., 2001), and with the oldest unequivocal palm fossils (e.g., Sabalites, 84 my—Harley, 2006). We therefore consider the estimate reliable. Finally, the age of the Comoros Islands can be used to enforce a maximum constraint on the age of the crown node of the Comoros endemic clade of Ravenea (R. moorei/R. hildebrandtii) recovered in the current study with high support (see results). The Comoros islands are part of a 60 my old volcanic lineament that has progressively been formed over the Grande Comore hot spot giving rise to the Seychelles, Northern Madagascar and finally the Comoros. Northern Madagascar passed this hot spot approximately 10 my ago, and the oldest of the Comoros islands (Mayotte) emerged around 5.7 my ago (Emerick and Duncan, 1982). Enforcement of this constraint assumes that the four Ravenea species not sampled for our phylogeny do not form part of the Comoros endemic clade, but we consider it highly unlikely that this should prove to be the case. Our final set of calibration points thus consisted of four constraints enforced in the following way (with reference

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to the nodes numbered in Fig. 4): (1) Node R (the crown node of the Ceroxyloideae) was fixed to 60 my based on the molecular dating study of the Arecoid line by Savolainen et al. (2006); (2) Node L (crown node of Ravenea moorei/R. hildebrandtii clade) was set to a maximum of 5.7 my based on the geological age of the Comoros Islands; (3) Node H (stem node of Phytelephas) was constrained to a minimum of 5.2 my based on the macrofossil Ph. olsonii; (4) Node C (stem node of Ravenea) was constrained with a minimum of 37 my based on the microfossil E. grandiporus. Due to the uncertainty associated with the latter microfossil record all analyses were carried out with this constraint included (PL-MI, BRC-MI) and excluded (PL-ME, BRC-ME) and results were compared. We also explored the effect of relaxing the fixed constraint on the crown node of Ceroxyloideae (constraint 1). Specifically, we tested how far back in time this node would have to be pushed in order to permit acceptance of (a) the Gondwanan vicariance hypothesis, and (b) the austral interplate dispersal hypothesis. In these analyses we fixed relevant nodes at the minimum age necessary to accept each hypothesis, relaxed the constraint on node R, and performed the analyses. The obtained confidence intervals for node R were then compared with the presumed mid-Cretaceous radiation of palms (Harley, 2006; Janssen and Bremer, 2004). To evaluate whether Savolainen et al.’s (2006) divergence age of node R might be an underestimate we also fixed this node to 126 my based on Janssen and Bremer’s (2004) 110 ± 16 my age for the crown node of the Palmae, and analyzed this under PL (smoothing = 100). This corresponds to a scenario where the Ceroxyloideae diverged shortly after the origin of the family. If the age of a fossil record was stated in the literature as a range due to imprecision in age estimation, we used the youngest age as a minimum constraint. All time estimates for nodes must be regarded as minimum ages (Heads, 2005). 3. Results 3.1. Phylogenetic analyses 3.1.1. cpDNA, PRK, and RPB2 analyses In concordance with what has previously been reported for palm cpDNA (e.g., Asmussen and Chase, 2001), each single plastid region contributed only a limited number of phylogenetically informative characters on a per site basis (Table 1). The two nuclear DNA (nDNA) regions were significantly more variable in both nucleotide composition and sequence length. This resulted in alignments containing numerous small and several large gaps for these regions (up to 544 bp in PRK; up to 274 bp in RPB2). Non-orthologous copies of PRK and RPB2 were not evident in the current study, although they have been reported in other studies of palms (Gunn, 2004; Lewis and Doyle, 2002; Norup et al., 2006; Thomas et al., 2006; but see Loo et al., 2006). Parsimony analyses of the cpDNA, PRK, and RPB2 data sets each resulted in >25,000 equally most parsimoni-

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ous trees (Table 1). Strict consensus trees from these regions were topologically highly congruent and displayed similar degrees of resolution and clade support (Fig. 2; Table 1). In the analyses of cpDNA, PRK, and RPB2 the Ceroxyloideae was resolved as monophyletic with BS support values of 82%, 60%, and 100%, respectively. In each of the three analyses, the three tribes Cyclospatheae, Ceroxyleae, and Phytelepheae received strong support (BS > 97%). The RPB2 consensus tree placed Phytelepheae as sister to Ceroxyleae (84% BS), while the PRK and cpDNA trees left inter-tribal relationships unresolved. The RPB2 data set supported a basal split in Phytelepheae with one clade consisting of Ammandra and Aphandra and the other clade corresponding to genus Phytelephas. None of the separate analyses resolved inter-generic relationships inside tribe Ceroxyleae, except the cpDNA data set that indicated Juania as sister to Ceroxylon. The position of Oraniopsis inside Ceroxyleae was not resolved in any of the analyses. Ravenea was consistently indicated to split basally into two clades. 3.1.2. Phylogenetic congruence and combined analysis The simultaneous PHT for the three partitions PRK, RPB2, and cpDNA did not demonstrate significant incongruence among the three datasets (p = 0.17), nor did any of the pair-wise PHTs among all separate partitions

(p = 0.16–0.87). The combined data set (cp+nDNA) consisted of a total of 5569 characters. MP-searches yielded 5471 trees, with a CI and RI of 0.82 and 0.92, respectively (Table 1). The strict consensus tree of the combined cp+nDNA data analysis (Fig. 3) had the highest resolution and nodal support of all MP analyses carried out, with 36 ingroup nodes resolved and 21 of those nodes receiving a BS of 70% or higher. PBS analysis revealed that the three partitions PRK, RPB2, and cpDNA contributed with 34%, 37%, and 29% to the MP-tree length, respectively. Summed over all nodes, each DNA region contributed positively to the phylogenetic signal of the combined tree, with the PRK, RPB2, and cpDNA data sets accounting for 23%, 42%, and 35% of the positive signal, respectively. Data set conflict was restricted to resolution of the outgroup, the Pseudophoenix clade, the Ammandra/Aphandra clade, Ravenea, and the branch supporting the Ceroxyleae/Phytelepheae sister relationship (Fig. 3). In general, the three partitions cpDNA, PRK, and RPB2 contributed rather differentially to the resolution of the combined analysis tree: Resolution of Phytelepheae relationships were dominated by the signal of RPB2 and PRK; Ceroxyleae relationships were resolved mostly by cpDNA and PRK; and Cyclospatheae relationships were solely defined by cpDNA and RPB2 characters. Fourteen of 23 nodes below the genus level were supported by cpDNA, although this is

Fig. 2. Strict consensus trees of >25,000 equally parsimonious trees resulting from Fitch parsimony analyses of combined cpDNA (matK+30 ndhF+trnD– trnT) data and separate nDNA PRK and RPB2 data sets. Only ingroup is shown. Bootstrap proportions >50% are given above the branches. (Am.) Ammandra, (Ap.) Aphandra, (C.) Ceroxylon, (J.) Juania, (O.) Oraniopsis, (Ph.) Phytelephas, (Ps.) Pseudophoenix, and (R.) Ravenea.

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279

Fig. 3. Strict consensus tree of 5471 equally most parsimonious trees of combined cp+nDNA data, a total of 5569 characters of matK, 30 ndhF, trnD–trnT, PRK, and RPB2 sequences for 35 species of palm subfamily Ceroxyloideae plus 10 outgroup taxa. Tribal taxonomy according to Dransfield et al. (2005) is indicated. Bootstrap >50% is shown above branches. Partitioned branch support (PBS) is given below branches in the following order: cpDNA+PRK+RPB2. Relationships that are corroborated by all three partitions are indicated by a bold branch; a stippled branch indicates data set conflict. (Am.) Ammandra, (Ap.) Aphandra, (C.) Ceroxylon, (J.) Juania, (O.) Oraniopsis, (Ph.) Phytelephas, (Ps.) Pseudophoenix, and (R.) Ravenea.

often stated to be uninformative at lower taxonomic levels, especially in palms (e.g., Loo et al., 2006; Roncall et al., 2005). MP analyses of the combined cp+nDNA data set strongly support the monophyly of subfamily Ceroxyloideae (100% BS), and each of its three tribes Ceroxyleae, Cyclospatheae, and Phytelepheae (all 100% BS; Fig. 3). Ceroxyleae and Phytelepheae formed a moderately supported clade (77% BS).

Relationships within Cyclospatheae were fully resolved but weakly supported. In tribe Phytelepheae, Ammandra and Aphandra formed a highly supported clade (99% JK) that in turn is sister to a monophyletic Phytelephas (99% BS). Intra-generic relationships of Phytelephas were highly resolved, albeit with generally weak BS support. Tribe Ceroxyleae fell into two clades, one consisting of Ravenea and one consisting of Juania, Oraniopsis, and Ceroxylon. The lat-

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Fig. 4. Summary chronogram of palm subfamily Ceroxyloideae. Chronogram topology is taken from the PL-MI analysis and bars indicate the minimummaximum range of obtained divergence time estimates under the PL-ME, PL-MI, BRC-ME, and BRC-MI analyses (see text), not to be confused with confidence intervals. These are given in Table 2. Bayesian posterior probabilities (PP) are shown in three categories. Bold branches: 1.00–0.99 PP; thin branches: 0.98–0.95 PP; and stippled branches: <0.95 PP. Insert shows topology with branch length information prior to ultrametrization in PL or BRC. Distributions are indicated. (j) inter-Andean region; (h) western Andean/Choco´ region; (r) eastern Andean/Amazonian region. (P) Pleistocene, (PA) Paleocene, (PLI) Pliocene; (Am.) Ammandra, (Ap.) Aphandra, (C.) Ceroxylon, (J.) Juania, (O.) Oraniopsis, (Ph.) Phytelephas, (Ps.) Pseudophoenix, and (R.) Ravenea.

ter clade had only weak support (61% BS) and a Bremer support of 1 (Fig. 3). For Ravenea, the basal split in two clades found in separate analyses re-appeared with high support. The two species endemic to the Comoros Islands (R. hildebrandtii and R. moorei) were highly supported sisters (100% BS). The Bayesian majority-rule consensus tree of the combined data set yielded a highly resolved topology of 38 ingroup nodes (Fig. 4) of which 27 received a posterior

probability (PP) > 0.95. The tree is fully congruent with the strict consensus tree of the combined cp+nDNA MP analysis. 3.2. Divergence times The estimated divergence times for the nodes of the phylogeny are summarized in Table 2 and Fig. 4. Generally, minimum age estimates were higher in BRC analyses than

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Table 2 Divergence time estimates and 95%-confidence/credibility intervals (in millions of years ago) in palm subfamily Ceroxyloideae; node numbering according to Fig. 4 Node (Crown node of. . .)

PL–MI

PL–ME

BRC–MI

BRC–ME

A (Ceroxyleae–Phytelepheae clade) B (Cyclospatheae) C (Ceroxyleae) D (Oraniopsis–Ceroxylon split) E (Ceroxylon–Juania split) F (Ceroxylon) G (Ravenea) H (Phytelepheae) I (W/E Andean split in Phytelephas) K (P. seemannii/P. schottii split)

55 (54, 56) 5 (4, 5) 37b 32 (31, 34) 26 (25, 28) 11 (10, 14) 25 (24, 26) 17 (16, 19) 6 (6, 7) 3 (3, 4)

53 (51, 54) 5 (4, 5) 24 (22, 26) 21 (19, 23) 17 (15, 19) 7 (6, 9) 16 (15, 18) 16 (15, 18) 6 (5, 7) 3a

58 (55, 60) 4 (2, 7) 41 (37, 48) 38 (31, 45) 34 (25, 42) 21 (12, 31) 29 (20, 40) 25 (15, 36) 14 (6, 24) 6 (1, 14)

58 (35, 58) 4 (2, 6) 39 (21, 44) 35 (19, 41) 32 (16, 38) 19 (7, 27) 27 (13, 35) 25 (11, 32) 13 (4, 20) 6 (1, 14)

PL, penalized likelihood; BRC, Bayesian relaxed clock; MI, microfossil-based constraint included; ME, microfossil-based constraint excluded. a Confidence search failed. b Active constraint.

in PL analyses, and 95%-credibility intervals of the BRC analyses were significantly wider than PL 95%-confidence. Exclusion of the microfossil E. grandiporus (node C) affected divergence times significantly only in PL analyses, where it resulted in more recent estimates for node C and subsequent nodes. Nodes outside the Ceroxyleae were largely unaffected by the exclusion of E. grandiporus. A bound constraint was hit only in a single case (PL-MI, node C). Otherwise, constraints were consistent with the age of the ingroup root node R. The 126 my-calibration scenario of node R resulted in a PL 95%-confidence interval of 54– 43 my for node C. Recalibrating the tree by forcing the age of the split between Ravenea and remaining Ceroxyleae to comply with the Gondwanan vicariance hypothesis by fixing node C at 80 my resulted in a 245–172 my confidence interval for node R. Fixing node D at 49 my (austral interplate dispersal scenario during the Paleocene/Eocene thermal maximum) gave a confidence interval of 175–119 my for node R. Divergence time estimates indicate that the Comoros Islands have only recently been reached by Ravenea (Table 2) and the constraint for node L (5.7 my) was not hit in any of the analyses. Juania australis appears to have reached the volcanic Juan Ferna´ndez islands by secondary dispersal, as earlier proposed by Skottsberg (1956), since the divergence time for this species (Table 2) significantly predates the age of the Robinson Crusoe island (4 my; Stuessy et al., 1984). Divergence estimates for Cyclospatheae are not older than the Pliocene (Table 2), indicating a recent radiation just after when modern geographical settings of the Caribbean were in place (Graham, 2003) 4. Discussion 4.1. Phylogeny and systematics of the Ceroxyloideae The data presented here strongly support the recognition of Ceroxyloideae in its current circumscription as a distinct subfamily of the palms. By increasing the taxon sample for Ceroxyloideae from ten species (Asmussen

et al., 2006) to 35 species (this study), we obtained an increase in support for the monophyly of the subfamily from 63% to 100% BS. Furthermore, all three tribes of Ceroxyloideae (Ceroxyleae, Cyclospatheae, and Phytelepheae) are highly supported (100% BS). The clade of Ceroxyleae and Phytelepheae, recovered by Asmussen et al. (2006) with a 67% BS, received in our study 77% BS. The current study stresses the morphologically, ecologically and biogeographically heterogeneous nature of Ceroxyloideae as a result of true evolutionary history. While obvious synapomorphies for Ceroxyloideae are still lacking, the sister relation between Ceroxyleae and the Phytelepheae provides a valuable comparative framework for the study of the highly derived nature of the Phytelepheae. Shared conditions between the two tribes include a dioecious breeding system and flowers that are open from early in development. Furthermore, Ceroxyleae, Ammandra, Phytelephas aequatorialis, and Ph. tumacana all have stalked (pedicellate) male flowers (Barfod and Uhl, 2001; Uhl and Dransfield, 1987). In some species of Ceroxylon, flowers are polyandrous and sometimes four-parted (Uhl and Moore, 1977), superficially resembling Phytelepheae. While Uhl and Moore (1980) demonstrated that the polyandrous condition in Ceroxylon differs ontogenetically from that in Phytelepheae, congenitally open flowers (Uhl et al., 1995) and the pedicellate condition of the male flower (Barfod et al., 1999) nevertheless may be true synapomorphies for that clade. It has been suggested that the derived nature of tribe Phytelepheae is the result of pollinator-driven (Barfod et al., 1999) reductions in floral structures (Barfod, 1991). Future studies, comparing phytelephoid and ceroxyloid floral development and pollination, may bring an answer to this question. The current study resolved intergeneric relationships within tribe Phytelepheae with high support. We found Aphandra natalia to be sister to Ammandra decasperma (98% BS), and this clade in turn being sister to Phytelephas (99% BS). Inter-generic relationships in Phytelepheae have previously been studied by Barfod (1991) and Barfod et al.

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(1999) on the basis of morphological, anatomical, and ecological characters. Due to problems of homology assessment between the ingroup and potential outgroups, however, these studies remained rather inconclusive. Barfod (1991) and Barfod et al. (1999) favoured tentatively an analysis where data were weighted for floral characters displaying evident transformation series. These analyses resolved Ammandra as sister to the rest of the Phytelepheae, while Aphandra was either sister to or nested inside Phytelephas. A similar relationship was found in the cpDNA study by Asmussen et al. (2006) where Phytelephas was paraphyletic. Our data strongly refute these findings. Ap. natalia, the sole species in its genus, was originally described as a species of Ammandra based on shared vegetative characters with Am. decasperma (e.g., pinnae with a prominent midrib and two abaxially submarginal lateral ribs; Balslev and Henderson, 1987). The numerous autapomorphic character states of Ap. natalia should, however, be sufficient to justify its continued recognition at the generic level (Barfod and Uhl, 2001). Resolution of the inter-generic relationships in tribe Ceroxyleae are crucial for interpretations of the biogeographical history of this tribe. In the current study, Ceroxyleae splits basally into two clades, one consisting of Ravenea and the other comprising Oraniopsis, Juania and Ceroxylon. Within the latter clade, Oraniopsis is sister to the remaining genera (Fig. 3). However, the position of Oraniopsis received only poor support (61% BS), and was defined by only a single character from the PRK partition (Fig. 3). Previous phylogenetic analyses including members of Ceroxyleae have produced conflicting results regarding the position of Oraniopsis. Plastid DNA studies by Asmussen and Chase (2001) and Asmussen et al. (2006) found Oraniopsis to be sister to Ravenea, whereas Uhl et al.’s (1995) study based on morphological and RFLP characters reported relationships main to the ones presented here. The extremely short branch found in the current study for the position of Oraniopsis might be suggestive of an ancient sweep stake radiation during a relatively short time span, but could also be due to the general lack of parsimony informative characters among closely related species in the DNA regions analyzed here. More characters may be needed to substantiate basal divergences in Ceroxyleae. A final remark regards the former genus Louvelia, included in Ravenea by Beentje (1995) based on the observation of inconsistent distribution of the characters used to distinguish the two genera. The only character Beentje found to differ consistently was a fleshy endocarp in Ravenea versus a rather sclerified endocarp in Louvelia. In the current study, Ravenea falls into two highly supported clades. The first clade contains the former Louvelia species R. lakatra and R. louvelii (=L. madagascariensis) plus R. xerophila, for which an affinity to R. louvelii was noted by Beentje (1995) based on the shared conditions of having a dense layer of sheath remnants covering the distal part of the trunk and petals connate by the filamental calli of the antesepalous stamens. The second clade contains the

remaining species of Ravanea s.s. plus R. albicans (=L. albicans). Final settlement of the generic delimitation must await the inclusion into the phylogeny of the two species R. nana and R. dransfieldii, whose intermediate morphology were the direct reason for merging the two genera (Beentje, 1995). 4.2. Historical biogeography and divergence dates 4.2.1. The Gondwanan vicariance hypothesis The pattern of cladogenesis in tribe Ceroxyleae as recovered in the current study (Fig. 3) matches the break-up sequence of Gondwanan landmasses as currently understood (Sanmartı´n and Ronquist, 2004; Wells, 2003), with (Ravenea, (Oraniopsis, (Ceroxylon, Juania))) corresponding to the area-relationships (Madagascar, (Australia, (South America, Juan Ferna´ndez Islands))). However, the timing of cladogenesis contradicts the Gondwanan vicariance hypothesis. Indo-Madagascar lost direct land connection to Antarctica and reached its modern position relative to Africa approximately at the Barremian/Aptian boundary (120 my; Wells, 2003). Madagascar stayed geographically relatively close to remaining Gondwanan landmasses probably until the end of the Albian (97 my; O’Neill et al., 2003; Wells, 2003). Hay et al. (1999) proposed a later break-up of Gondwana recognizing a land bridge connecting Indo-Madagascar with Antarctica via the Kerguelian Plateau until as late as the Santonian (88 my), a scenario corroborated by dated phylogenies of Malagasy reptiles (Noonan and Chippindale, 2006). The final break-up of Gondwana can thus be assumed to have occurred somewhere between 120 and 80 my ago. The divergence time of Ceroxyleae as estimated in the current study (node C in Fig. 4; 42–24 my) is significantly younger than this period. Gondwanan vicariance seems therefore not justified. However, Heads (2005) has on theoretical grounds pointed out that ages obtained from dating studies should be interpreted as minimum ages and, hence, can not be used to reject ancient biogeographic events per se. Experimental use of a 126 my-calibration scheme for node R based on Janssen and Bremer (2004) did not push the crown node of Ceroxyleae (node C) sufficiently far back in time to obtain consistency with Gondwanan vicariance (54– 43 my). Fixing node C to 80 my resulted in an age of the Ceroxyloideae (245–172 my) far too old prior to the presumed mid-Cretaceous origin of the palm family (Janssen and Bremer, 2004; Harley, 2006). Although PL smoothing tends to overestimate the age of the root when deep calibration points are lacking (Sanderson, 2004), we nevertheless consider the observed differences too large to be accounted for by this potential bias. Despite the presence of a Gondwanan vicariance pattern of cladogenesis, we must therefore reject this hypothesis for the disjunct distribution of tribe Ceroxyleae on the southern hemisphere. Dismissal of the Gondwana-vicariance hypothesis for tribe Ceroxyleae raises the question of how the group did reach its current distribution. Moore (1973) suggested for

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the Ravenea/Ceroxylon disjunction a possible role of Africa, with subsequent extinction of Ceroxyleae on this continent. Recent studies have renewed the focus on a South American/African dispersal link during the Tertiary (Pennington and Dick, 2004) involving dispersal via sea currents and prevailing winds (Renner, 2004b) or stepping stone dispersal along former Atlantic island chains (Walvis Ridge/Rio Grande Rise and Sierra Leone Ridges), which might sporadically have been above sea level until as late as the Oligocene (Morley, 2003). Pan et al. (2006), in a review of the fossil history of palms in Africa, found evidence for a formerly diverse palm flora of pre-Oligocene Africa similar in composition to the contemporary paleoflora of South America. Finally, sporadic land connections across the present-day Mozambique Channel from the mid-Eocene to upper Oligocene (45–26 my) have been suggested to have facilitated mammalian and floristic exchange between Africa and Madagascar (McCall, 1997; Yuan et al., 2003). The eastern orographic rainforests of Madagascar, today housing the great majority of Madagascar’s palm flora and 73% of the Malagasy species of Ravenea (Dransfield and Beentje, 2003), did probably not evolve before the Eocene, when Madagascar entered the trade-wind belt (Wells, 2003). In our study, estimates for the split between Ravenea and remaining Ceroxyleae fall in the range from the midEocene to the late Oligocene (41–24 my; Table 2). These dates are compatible with a trans-Atlantic/trans-African dispersal scenario during the mid-Tertiary and comfortably postdate the age of the orographic rainforests of Madagascar. Interestingly, the time estimates for basal divergences in Ceroxyleae overlaps with estimates for inter-continental divergences in other palm lineages (Cocoseae—Gunn, 2004; Savolainen et al., 2006; Chamaedoreae—Argelia Cuenca, pers. comm.). Gunn (2004) estimated an age of 42 my for the divergence between South American and African oil palms (Elaeis oleifera and E. guineensis); 38– 36 my for the divergence between the South African Jubaeopsis and its South American sister clade; and 42–39 my for the divergence of the Malagasy clade (Beccariophoenix and Voanioala) in tribe Cocoseae. Savolainen et al. (2006) reported a divergence age of 30 my for Beccariophoenix, approximately 10 my younger than that reported by Gunn (2004). These dates indicate that trans-Atlantic/trans-African dispersal paths may have been open for palms during the mid-Tertiary offering a unifying explanation for South American–African/Indian Ocean disjunctions in the palm family. However, since a robust fossil record for Ceroxyleae is missing in Africa, alternative explanations cannot be ruled out for the Ceroxyleae. One such alternative explanation involves the ‘‘lemurian stepping-stones’’ (Schatz, 1996), a mid-Tertiary interplate dispersal path towards Madagascar from a possible EastGondwanan source area. Due to dropping temperatures from the Eocene onward, sea levels declined, and portions of volcanic island lineaments of the Indian Ocean basin, including the Broken Ridge, the Kerguelian Plateau, the

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NinetyEast Ridge, and the Seychelles-Mascarene Ridge might have become emergent (Kemp and Harris, 1975; Morley, 2003; O’Neill et al., 2003). Facilitated by increasing oceanic and atmospheric circulation due to the opening of the Drake Passage at the Eocene/Oligocene boundary, these ridges could have served as stepping-stones for long distance interplate dispersal between Australia, India, and Madagascar (Kemp and Harris, 1975; Schatz, 1996). For instance, Kemp and Harris (1975) reported 26 pollen fossils, amongst them two palm-like types, from an Eocene/ Oligocene deep-sea drilling site positioned at the Ninety East Ridge, with the majority of this paleoflora showing an Australian floristic affinity. The possible existence of a trans-Indian Ocean dispersal link is furthermore corroborated by floristic affinities between Australia on one side and elements in the flora of the Mascarenes (e.g., Acacia; Bell and Evans, 1978) and Madagascar (e.g., Hibbertia; Schatz, 1996) on the other side. The ‘‘lemurian stepping stones’’ hypothesis offers an explanation for the occurrence of the Ravenea-like microfossil E. grandiporus in Eocene strata of India and would suggest true affinity of this fossil to Ceroxyleae. However, under this scenario, Oraniopsis would be expected to be sister to Ravenea, as found by Asmussen and Chase (2001) and Asmussen et al. (2006), rather than to a Ceroxylon–Juania clade, as found in the current study and by Uhl et al. (1995). Clearly, deep-level relationships in Ceroxyleae need further clarification, as does the identity of E. grandiporus (Harley, 2006), before a reasonably complete model of Ceroxyleae biogeography can be reached. 4.2.2. The austral interplate dispersal hypothesis Divergence time estimates for the split between Australian Oraniopsis and the Ceroxylon–Juania clade were in the current study concentrated around the time window between the mid-Eocene and early-Oligocene (Table 2). Fixing the split between Ceroxylon and Oraniopsis to the time of the Paleocene/Eocene thermal maximum resulted in an age interval most likely too old for the Ceroxyloideae (175–119 my), indicating that diversification probably took place after the proper early Eocene climatic maximum at around 49 my ago (Morley, 2000, 2003). According to paleontological data, warm, and equable climates persisted at high latitudes of the southern hemisphere during the warmer phases of the Paleocene and Eocene until at least the mid-Eocene (45 my), with cold month means of >10 °C reported for New Zealand midEocene floras at paleolatitudes of 45–55°S and estimated sea surface temperatures for the Antarctica between 8 and 12 °C (Greenwood and Wing, 1995). Palms were present in the paleoflora of mid-Eocene Patagonia (Wilf et al., 2005), and palm-like pollen has been reported from Eocene sediments of Antarctica (Cranwell, 1959; Cranwell et al., 1960) and South Australia (Greenwood and Conran, 2000; Fig. 1). Carpenter et al. (2004) reported a south Australian early Eocene paleoflora similar in composition to the modern flora of northeastern Queensland, indicating

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that climatic conditions would have been suitable for modern Ceroxyleae at high latitudes at that time. Subsequently, global temperatures followed a general downward trend forcing frost-intolerant taxa towards lower latitudes (Greenwood and Wing, 1995; Morley, 2000). Nevertheless, warm- to cool-temperate, precipitation-rich climates supporting evergreen, temperate rainforests have been inferred for Antarctica on the basis of the fossil record until the onset of the early Oligocene glacial period, with proxy mean annual temperatures of 5–15 °C and 12 to +15 °C for the Antarctic Peninsula and the whole of Antarctica, respectively (Thorn and DeConto, 2006). Morley (2000, 2003) regarded the Paleocene/Eocene thermal maximum, which ended at around 49 my ago, as the last chance for megathermal taxa to take this route, with subsequent dispersal only possible for taxa hardy enough to tolerate decreasing temperatures and the prolonged dark period during the winter. Growth experiments carried out by Read and Francis (1992) demonstrated that woody plants experience less physiological stress under prolonged darkness when temperatures are cool when compared to milder conditions, implying that cold-tolerance is reinforced by prolonged darkness. Tribe Ceroxyleae must be considered as a cool-hardy element in the palm family. The Andean genus Ceroxylon occurs in cool and moist montane forests from Venezuela to Bolivia (Henderson et al., 1995), with one species (C. parvifrons) reaching altitudes of more than 3500 m above sea level where it experiences minimum mean annual temperatures of 9.0 °C (Borchsenius and Skov, 1997). Juania australis from the Robinson Crusoe Island (Juan Ferna´ndez archipelago) occurs at 33°S latitude, well outside the tropical belt, where it experiences temperatures as low as 7.2 °C (Skottsberg, 1953). Ravenea contains one drought-adapted species (R. xerophila) and three montane species (R. nana, 1900 m; R. robustior, 2000 m; R. dransfieldii, 1700 m; Dransfield and Beentje, 1995, 2003). Finally, Oraniopsis grows in montane rain forests of northeastern Queensland. Its inland-most occurrence lies southwest of Atherton at elevations above 1100 m (Dransfield et al., 1985), where it experiences cold month minimum means of 10 °C (Commonwealth of Australia, 2006). Thus, if Eocene ancestors had similar ecological characteristics, late-Eocene austral interplate dispersal in Ceroxyleae is not ecologically implausible. 4.2.3. Andean vicariance versus Pleistocene refuges The great species richness centered along the foothills and slopes of the tropical Andes has variously been attributed to present-day ecological mechanisms and historical factors concerning climatic and geological properties of that region (e.g., Pirie et al., 2006). Among the historical explanations, unstable Pleistocene climates and the orogeny of the Andes have been proposed to have facilitated speciation, finally increasing diversity (Prance, 1982; Richardson et al., 2001). According to our results diversification of tribe Phytelepheae occurred before the Pleistocene epoch (Table 2, Fig. 4). Therefore, our data do not support Pleis-

tocene climatic fluctuations as a major driver for speciation in tribe Phytelepheae. An exception might be the split between Panamanian Phytelephas seemanni and north Colombian Ph. schotti, as its confidence interval enters the Pleistocene (14–1 my). Haffer (1970) suggested that after the uplift of the Andes, Pacific, and Amazonian lowlands remained in contact during favorable periods of the Pleistocene via a dispersal corridor along the Caribbean coastline. Differentiation of Ph. seemanni and Ph. schotti could have been caused by interruption of this connection. Furthermore, it is not clear whether the warmer or wetter phases of the Pleistocene may have facilitated range expansions and eventually secondary contact across the lower passes of the Andes, as suggested for the occurrence of a scattered population of Ammandra decasperma in the Magdalena valley (Bernal et al., 2001). In opposition to Pleistocene climatic changes, the orogeny of the Andes could indeed have promoted major diversification in Phytelepheae. Basal divergence in Phytelepheae is in the current study estimated to have occurred during the early Miocene (node H; 25–16 my). Subsequent diversifications occurred after the mid-Miocene (<14 my) and increased in frequency towards the Miocene/Pliocene interface (5 my; Fig. 4). In this, it coincides with the major orogeny of the Andes (GregoryWodzicki, 2000; Hoorn et al., 1995; Van der Hammen and Hooghiemstra, 2001). According to biostratigraphic data, hills of up to 1000 m existed already during the mid-Miocene; elevations increased to 1500 m around 7 my ago (Van der Hammen and Hooghiemstra, 2001); and at the early Pliocene (4 my) the northern Andes had attained 40% of present-day altitudes corresponding to more than 2000 m (Gregory-Wodzicki, 2000). Although conclusions are limited by wide confidence intervals in BRC analyses and the generally weakly supported inter-specific relationships in genus Phytelephas, pattern and timing (PL analyses) in this genus correspond surprisingly well to the sequential uplift of the cordilleras of the northern Andes as currently understood (Fig. 4): A primary east-west Andean split (node I) could have occurred due to uplift activity of the Ecuadorian and Colombian Cordilleras during the late-Miocene, followed secondly by an east-north Andean split (node J) during the Pliocene when the Eastern Cordillera largely exceeded 1500 m elevation, and finally dispersal into the northern Choco´ region and Panama following the closure of the isthmus of Panama at around 3 my ago (node K; Graham, 2003; Hoorn et al., 1995; Van der Hammen and Hooghiemstra, 2001). Phytelepheae is essentially a lowland group generally found below 500 m elevation and not exceeding 1500 m altitudes (Henderson et al., 1995). Seeds are rodent-dispersed and rather unsuited for long distance dispersal (Barfod, 1991; Bernal et al., 2001). Thus, we cannot reject the Andean orogeny as a possible driver for speciation in Phytelepheae. Similar patterns of divergence estimates have been reported from other Andean-centered groups of plants (e.g., Annonaceae–Pirie et al., 2006; Inga–Rich-

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ardson et al., 2001). The same applies for genus Ceroxylon under PL estimates (<11 my; Table 2). 5. Conclusions The current study confirms the monophyly of subfamily Ceroxyloideae and each of its three tribes. Although the recovered phylogenetic pattern is corroborating ancient Gondwanan vicariance, the results of divergence time analyses are incompatible with this hypothesis. Southern hemispheric disjunctions in tribe Ceroxyleae are estimated to have arisen during a relatively short time span in the mid-Tertiary, inevitably involving repeated trans-oceanic dispersals. Topological patterns are widely used as evidence to deduce biogeographical histories of single groups (e.g., Baker and Dransfield, 2000; Hahn, 2002b; Swenson et al., 2001) as well as of biomes (e.g., Sanmartı´n and Ronquist, 2004). Our study emphasizes the importance of incorporating branch length and temporal information into biogeographical analyses besides the classical approach of pattern analysis (Hunn and Upchurch, 2001). In this we are in line with an increasing amount of studies that found evidence for Tertiary dispersal rather than Cretaceous vicariance as drivers for South-hemispheric disjunct distribution patterns in plants (e.g., Renner, 2004a; Richardson et al., 2004; Sytsma et al., 2004; Zerega et al., 2005). Few dating studies have been published for palms so far. Nevertheless, their general congruence on the timing of repeated trans-oceanic dispersals is striking, suggesting the Eocene–Oligocene interval as a period of major importance for an understanding of the evolution and biogeography of palms. Acknowledgments Financial support for this study was provided by the Carlsberg Foundation through a grant to F.B. and by the Danish Natural Science Research Council (Grant 272-060476). Additional funding for P.T.’s field work was provided by Knud Højgaards Fond and Frimodt Heineke Fonden. We are most grateful for this support. Several individuals and institutions generously contributed DNA material or sequences for this study, including Scott Zona and Carl Lewis at Fairchild Tropical Garden, the Montgomery Botanical Centre, the Royal Botanic Gardens, Kew, the late David Robinson at Earlscliffs Garden, Dublin, Argelia Cuenca at the Royal Veterinary and Agricultural University, Copenhagen, and Maria Vibe Norup at the University of Copenhagen. Priscilla Muriel, Alvaro Pe´rez and Hugo Navarrete provided invaluable help and assistance during field work and Anni Sloth, Camilla Ha˚kansson and Jane Frydenberg assisted us in the lab. Special thanks go to Henrik Balslev, Jens-Christian Svenning, Anders Barfod, Jean-Christophe Pinteaud, Gloria Galeano, and Rodrigo Bernal for their advice and support throughout this study.

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