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Keywords:

  • Australasia ;
  • dated phylogenetic tree ;
  • India ;
  • Pleistocene ;
  • Sapotoideae ;
  • South-East Asia ;
  • speciation ;
  • Wallace's Line

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Internal transcribed spacer (ITS) ribosomal DNA sequence data were generated for 80 of the c. 200 species of Isonandreae and were added to data from African and Neotropical representatives in subfamily Sapotoideae and outgroups in Sapotaceae. Bayesian dating and ancestral area reconstruction indicated that Isonandreae are derived from within an African grade. Multiple Australasian species or lineages are derived from Sundanian lineages in South-East Asia with stem ages originating from the late Oligocene. Sri Lankan and Indian lineages are also derived from Sundanian lineages. Our results are consistent with migration from Africa into Sundania followed by numerous over-water dispersal events across Wallace's Line into Australasia and migration from Sundania to the Indian subcontinent. Pleistocene speciation indicates that sea-level changes during that epoch could have been responsible for some species diversification in Sundania. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 130–140.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Tropical South-East Asia is one of the most species-rich regions on earth and is recognized as a biodiversity hotspot (Myers et al., 2000). Reconstructing the biogeographical history of taxa that occupy this region will enable a better understanding of how this biodiversity arose, and dated molecular trees allow us to determine whether evolutionary events coincide with historical tectonic or climatic events in a particular region (e.g. Donoghue & Moore, 2003; Pillon, 2012; Fay & Forest, 2013). A series of tectonic events have influenced diversification in the South-East Asian region. The Indian subcontinent separated from east Africa, Madagascar and Australia during the early Cretaceous at c. 120 Ma and collided with Eurasia in the Eocene from c. 50 Ma (Briggs, 2003). From the Oligocene (34–23 Ma) onward, the northward movement of the Australian Plate toward the Continental Asian Plate brought the emergent landmasses of these two continental shelves into close proximity (see Hall, 2009, for a review of South-East Asian tectonic history). These two plates converged around a line that was identified by Alfred Russel Wallace (1869) as a major biogeographical boundary between Sundania (largely Laurasian) and Australasia (largely Gondwanan) with a distinct fauna on either side. Plate convergence resulted in the meeting of major continental areas, but also had an impact on the emergence/submergence of islands in the archipelago. New Guinea is thought to have emerged as recently as 10 Ma (Hall, 2009), which is remarkable given the high species diversity on this largest of all tropical islands (see Takeuchi, 2005, for comparison of species diversity on different islands in the Malay Archipelago). To add to the complexity, changes in sea level during the Pleistocene (Voris, 2000) have led to successive isolation and reconnection of various island groups in either Sundania or Australasia, although there has never been a direct land connection between these two areas that are separated by a deep oceanic trench through the Makassar Straits. Given each of the above geological and climatic events we can test a series of hypotheses on the origins and patterns of diversification of lineages that occupy the region using dated molecular trees of these groups.

Tribe Isonandreae Hartog are one of five recognized in Sapotaceae by Pennington (1991) and were placed in subfamily Sapotoideae by Swenson & Anderberg (2005). They are distributed from India and Sri Lanka, through Indochina and South-East Asia to Australia and the Pacific Islands. This distribution makes them an ideal model group to study diversification patterns in South-East Asia.

Isonandreae are composed of seven genera (Palaquium Blanco, Aulandra H.J.Lam, Isonandra Wight, Madhuca Buch.-Ham. ex J.F.Gmel, Payena A.DC., Burckella Pierre and Diploknema Pierre) and are defined on the basis of a complex combination of floral characters. Harley (1991) surveyed the pollen morphology of Sapotaceae extensively and defined a series of types. Although the fossil pollen record for Sapotaceae is copious, there are no reports of tribe Isonandreae in South-East Asia. The earliest reports of fossil pollen of Sapotaceae are from the Senonian (89–65 Ma) of China (Song, Wang & Fei, 2004), although the affiliation of these fossils with Sapotaceae is doubtful (Harley, 1991). Sapotaceae from the Palaeocene (65–57 Ma) to Early Miocene (c. 23 Ma) have been recorded in Sarawak (Muller, 1968), although Harley (1991) was again unconvinced that these are sapotaceous. Pollen from the Late Cretaceous (71 Ma) Sapotaceoidaepollenites rotundus that could be related to Mimusopeae (M. M. Harley, pers. comm. 2009) has been found at Otway Basin off the coast of South Australia (Stoian, 2002), and an Australasian origin for Sapotaceae or lineages within the family is thus possible. Pollen grains from the mid Eocene of England (Gruas-Cavagnetto, 1976) are similar to morphological types IIA and VIA described by Harley (1991) that are characteristic of tribes Mimusopeae, Isonandreae and Sideroxyleae. Despite the reasonably extensive fossil pollen record, it is not complete enough and insufficiently well characterized to provide a complete picture of the biogeographical history of the family.

Given the geological history of South-East Asia we can postulate four hypotheses that would receive support through particular molecular tree topologies and node ages. (1) If Isonandreae had a Laurasian or Sundanian origin we would expect Gondwanan lineages (Indian or Australasian) to be derived from lineages currently occupying Laurasian land masses. Australasian lineages would have been increasingly likely to have migrated across Wallace's Line as the Asian and Australian Plates converged. Indian lineages would also be derived from Laurasian or Sundanian lineages if they had migrated into India as the subcontinent collided with Eurasia from the Eocene. (2) If Isonandreae had an Australasian origin, species from that region would form a basal grade from which Sundanian lineages would be derived, having diverged as a result of migration across Wallace's Line from the Miocene. (3) If Isonandreae had an Indian origin we would expect Indian/Sri Lankan species to form a basal grade from which Sundanian and Australasian species would be derived and that clades of Sundanian/Australasian species would diverge from their Indian ancestors when India collided with the Asian continent in the mid-Eocene. (4) If Isonandreae arrived as a result of a long-distance dispersal event from outside Sundania, India or Australasia it would be derived from a lineage that formed a grade of species that occupy the source area. Pleistocene sea-level changes (Voris, 2000) would have resulted in the successive isolation and reconnection of islands. This may have led to allopatric divergence and if so we would expect to see speciation events during the Pleistocene, although of course other events may have contributed to speciation during that time frame.

A Bayesian phylogenetic analysis of 46 nuclear (internal transcribed spacer, ITS) and plastid sequences of Sapotaceae [the trnH-psbA spacer, the trnC-trnD region (consisting of the trnC-petN spacer, the petN gene, the petN-psbM spacer, the psbM gene and the psbM-trnD spacer), the trnC-psbM region and the 3′ end of ndhF) including 20 representatives of Isonandreae (P. Wilkie, J. E. E. Smedmark, A. Anderberg, J. Tosh, K. Armstrong & J. E. Richardson, unpubl. data; Bayesian majority rule consensus tree available as online supplementary material as Supporting Information, Fig. S1) indicated strong support for the monophyly of the tribe. Separate analyses of the nuclear and plastid datasets revealed no examples of hard incongruence between trees (i.e. strongly supported topological differences with jackknife values > 95% or posterior probabilities > 0.99). The rDNA ITS was more variable than plastid genes, so this region was used to produce a phylogenetic tree with an expanded species sample. This study aims to produce a dated molecular tree and reconstruct node distributions of Isonandreae using the ITS sequence data that will allow testing of all of the above hypotheses so that we may determine patterns in their diversification throughout the Malesian region.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Data sets and taxon sampling

In total, 138 species of Sapotaceae were included in this study. At least 74 of the c. 200 species of Isonandreae were sequenced for ITS. Species of other tribes in subfamily Sapotoideae were included to reconstruct nodes that were used as calibration points to date the tree. As Sarcosperma Hook.f. has been shown to be the sister to all other Sapotaceae (Anderberg, Rydin & Källersjö, 2002; Anderberg & Swenson, 2003), Sarcosperma laurinum (Benth.) Hook.f. was used to root trees. To recover nodes that could be used to date the phylogenetic tree, sequences of a selection of taxa representing the major lineages in each of tribes Sideroxyleae and Mimusopeae were also utilized. Several species representing subfamily Chrysophylloideae (Anderberg & Swenson, 2003) were also included in the study. A list of accessions with voucher specimen details is provided online (Supporting Information, Table S1).

DNA extraction, amplification and purification

Total genomic DNA was extracted from leaves taken from herbarium specimens or dried in silica gel (Supporting Information, Table S1). About 20–25 mg of leaf tissue was ground using one or two 30-s cycles in a Mixer mill and subsequently treated with 1 mL 2% cetyltrimethylammonium bromide (CTAB) buffer (pre-heated at 65 °C) and 2 μL β-mercaptoethanol, following a modified version of the CTAB DNA protocol outlined by Doyle & Doyle (1990).

Amplifications of the ITS region were performed using the ITS5p/ITS8p primer pair (Möller & Cronk, 2001). PCR was carried out in 25-μL volume reactions containing 5.75 μL sterile distilled water, 0.5 μL genomic DNA, 2.5 μL 10× NH4 reaction buffer, 0.75 μL each primer, 1.25 μL 50 mm MgCl2, 2.5 μL 10 mm dNTPs, 10 μL 5 m betaine, 0.25 μL bovine serum albumin and 2.25 μL 5 u μL–1 Biotaq DNA polymerase buffer. The thermal cycling profile consisted of 5 min denaturation at 95 °C, followed by 35 cycles of 30 s at 95 °C for denaturation, 50 °C for 30 s for annealing and 72 °C for 30 s for extension, with a final extension period of 8 min at 72 °C. Extraction from herbarium specimens sometimes yields low quantities of degraded DNA. In these instances, a nested PCR was performed which involved using 1 μL of the first PCR product added to a second (nested) PCR reaction using the ITS1/ITS4 primer pair (White et al., 1990) with the same thermal cycling profile as above. Only four accessions, indicated in Supporting Information, Table S1, were sequenced using the nested PCR method. The PCR products were purified using the ExoSAP IT (GE Healthcare) enzymatic clean-up method following the manufacturer's instructions.

DNA sequencing, assembly and alignment

The purified DNA was used as a template for the sequencing reaction with ‘Big Dye’ terminators (v.3.1) and the Big Dye PCR profile. Sequencing was performed using 10 μm of the primers ITS5, ITS8, ITS1 and ITS4 in 10-μL reactions. Ten accessions did not produce clean sequences and were excluded from the analysis. Sequences of the complete ITS region (18S, ITS1, 5.8S, ITS2 and 26S) were assembled using Sequencher version 4.7. The sequences were then aligned using ClustalW in BioEdit (Hall, 1999) and exported as a Nexus file for phylogenetic analysis.

Phylogenetic analysis – maximum-parsimony (MP) and Bayesian inference

MP analysis was carried out using PAUP* version 4.0b10 (Swofford, 2002). All informative substitutions were used in the analyses, and indels were treated as missing data. The initial analysis consisted of an heuristic search of 10 000 replicates saving ten trees per replicate, with random stepwise addition, tree bisection-reconnection (TBR) branch swapping, MULTREES on, collapsing branches if maximum branch length is zero, and all characters unordered and equally weighted. All trees saved from this analysis were then used for a further heuristic search, activating steepest descent and MULTREES and saving a maximum of 10 000 trees. This strategy is considered to be sufficient to capture all topological variation (Sanderson & Doyle, 1992). No shorter trees were found in the additional analysis. A jackknife analysis as implemented in PAUP* ver. 4.0b10 (Swofford, 2002) was carried out that consisted of 10 000 replicates deleting 37% of characters, with TBR branch swapping and saving ten trees per replicate.

Bayesian analyses were conducted with MrBayes 3.1 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003), which uses the Markov chain Monte Carlo (MCMC) method (Larget & Simon, 1999) to search tree and parameter space. The Akaike information criterion (AIC) and hierarchical likelihood ratio test (hLRT) were used in MrModelTest (Nylander, 2004) to determine the most appropriate model for the data. Four MCMC chains with chain temperature set at default were run simultaneously for ten million generations, sampling trees and parameters every 1000 generations. Tracer version 1.5 (Rambaut & Drummond, 2007) was used to determine that a burn-in period of 10% (1000 trees) sufficed and the remaining trees were saved. LogCombiner was used to combine trees in a single file. TreeAnnotator (Drummond & Rambaut, 2007) was used to select the maximum clade credibility (MCC) tree that has the maximum sum of posterior probabilities on its internal nodes and summarizes the node height statistics in the posterior sample. Clade support was represented by posterior probability (PP) values, with PP values of more than 0.95 indicating strong support. The Bayesian MCC tree was used to define calibration points and other key nodes in the dating analysis.

Molecular dating

Type VIA pollen grains (Harley, 1991) characteristic of Sideroxyleae from the Ypresian of England (Gruas-Cavagnetto, 1976) were used as a fossil constraint. We assigned a log normal prior to the stem node of the Sideroxyleae clade [offset: 52.2 Ma (mean: 0.001)]. A mean of 0.001 was chosen so that 95% of the probability is contained in an interval between the mid-point (52.2 Ma) and the upper boundary of the upper Eocene (48.6–55.8 Ma). A log normal prior was chosen to bias in favour of older age estimates as suggested by Ho (2007).

An XML input file was generated in BEAUti version v.1.4.8 (Drummond & Rambaut, 2007). The best performing evolutionary model was identified under two different model selection criteria, the hLRT and the AIC as implemented in MrModelTest (Nylander, 2004). Both selection criteria indicated that a general time reversible (GTR) model with site heterogeneity being gamma distributed and with invariant sites was optimal. A relaxed clock, uncorrelated lognormal model was chosen based on the assumption of the absence of a molecular clock. To specify informative priors for all the parameters in the model, the Yule tree prior was used (recommended as being appropriate for species-level phylogenies, Ho, 2007). The XML file was manually modified to include a starting tree that had been generated in PAUP. Two runs were performed for the dating analysis each with the MCMC chain length set to 10 000 000, to screen every 10 000 and sample every 1000 trees. The XML file was run in BEAST software version v.1.4.8 (Drummond & Rambaut, 2007). LogCombiner was used to combine tree files and to remove the burn-in, which was again determined using Tracer version 1.5 (Rambaut & Drummond, 2007). A burn-in of 10% (1000 trees) sufficed, the remaining trees were saved and the MCC tree was constructed using TreeAnnotator. MCC files were visualized using FigTree version 1.3.1 (Rambaut, 2009).

Reconstruction of ancestral distributions

Two approaches were used to reconstruct ancestral distributions at nodes. Terminals were assigned to the following areas that were chosen based largely on tectonic history (see map inset, Fig. 2): Sundania (including part of mainland Asia to the south of the Isthmus of Kra and islands to the west of Wallace's Line), Asia (north of the Isthmus of Kra), Australasia (including islands to the east of Wallace's Line), Africa (including Madagascar), South Asia (India and Sri Lanka), North America, Hawai'i and the Seychelles.

The first approach for reconstructing ancestral distributions was performed using RASP (Reconstruct Ancestral State in Phylogenies; Yan, Harris & He, 2011, http://mnh.scu.edu.cn/soft/blog/RASP) software, which implements Bayesian binary MCMC (BBM) time-events curve analysis (Yan et al., 2011) that allows multiple states to be assigned to terminals. Terminals were assigned states based on the overall distribution of the species, many of which are distributed in multiple areas. BBM suggests possible ancestral ranges at each node and also calculates probabilities of each ancestral range at nodes. One analysis was performed with 5000 000 cycles, ten chains, sampling every 100, with a temperature of 0.1 and with the maximum number of areas set to four. The root node was custom defined as Asian that we believe to be the most likely state for the crown node of the family given that the Asian taxa Sarcosperma Hook.f and Eberhardtia Lecomte form a grade in which the rest of the family is nested.

A discrete phylogeographical analysis (DPA) using a standard continuous-time Markov chain (CTMC) was also performed to determine node distribution probabilities (Lemey et al., 2009). This approach incorporates branch length information and uncertainty in the tree topology. It does not allow terminals to have multiple states, so each sample in this analysis was coded according to the locality of the individual. As recommended by Lemey et al. (2009), priors for migration rates used a Γ distribution for the relative rate parameter (shape parameter = 1.0) and an exponential distribution (mean = 1.0) for the geosite model parameter. The XML file for the dating analysis was edited to specify the locality of each accession for the discrete phylogeographical analysis. The analysis required four runs in BEAST of 10 000 000 generations each before effective sample size (ESS) values > 200 for each parameter were achieved. Log files for the separate analyses were combined in LogCombiner and then imported into Tracer version 1.5 (Rambaut & Drummond, 2007) to check whether ESS values were adequate for each parameter (i.e. > 200). LogCombiner was then used to combine tree files and remove their burn-ins (first 1000 trees for each tree file) that were also determined using Tracer. TreeAnnotator was used to produce the MCC tree. MCC files were visualized using FigTree version 1.3.1 (Rambaut, 2009).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The ITS matrix contained 163 taxa and 1012 bp of sequence data of which 148 were variable but parsimony-uninformative and 407 were potentially parsimony-informative. Ten thousand trees were saved from the parsimony analysis with a length of 2279 steps. The trees from the MrBayes, parsimony and BEAST analyses were compared and there were no examples of hard incongruence. The dates obtained from BEAST analyses with or without ancestral state reconstructions were similar. Only the BEAST trees with ancestral state reconstructions are discussed but the Bayesian MCC tree and parsimony jackknife trees are provided as electronic supplementary information (Supporting Information, Figs S2, S3, respectively).

Figure 1 shows the MCC of the BEAST analysis with ancestral state reconstructions from the BBM analysis and indicates the relationships in Isonandreae and their relationships with other major clades. PP < 0.95 are indicated by dashed lines. Isonandreae are monophyletic with a PP of 1 and the tribe is nested in a grade of predominantly African taxa, most of which were placed in tribe Mimusopeae by Pennington (1991). Figure 2 shows the same MCC tree as in Figure 1, but with reconstructions based on the DPA analysis. PPs for geographical states of nodes for both analyses that are discussed in the text are indicated in Table 1.

figure

Figure 1. The maximum clade credibility (MCC) tree from the posterior sample generated from the BEAST analysis showing the relationships in Isonandreae and their relationships to other major clades. Calibration nodes and nodes discussed in the text are indicated. Broken lines indicate nodes with PP values < 0.95. Pie charts on nodes indicate ancestral state reconstructions based on the BBM.

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figure

Figure 2. The maximum clade credibility (MCC) tree from the posterior sample generated from the BEAST analysis showing the relationships in Isonandreae and their relationships to other major clades. Calibration nodes and nodes discussed in the text are indicated. Broken lines indicate nodes with PP values < 0.95. Lines are coloured based on the the results of the discrete phylogeographical analysis: purple, Asia; yellow, Africa; light blue, Australasia; red, India and Sri Lanka; dark blue, Sundania.

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Table 1. Calibration points and information on other key nodes
NodeMean node age (Ma)HPD interval (Ma)Most probable state BBM/DPA
  1. The mean minimum age estimates and their 95% highest posterior density intervals are indicated. NA, not available; MRCA, most recent common ancestor.

A. Sapotaceae crown84.567.1–105.0Asia 0.99/Africa 0.52
B. Sideroxyleae stem53.652.2–56.6Africa 0.66/0.93
C. Sideroxyleae crown44.538.3–50.3North America 0.89/0.91
D. Isonandreae stem40.532.6–48.3Africa 0.96/0.99
E. Mimusopeae crown38.8NAAfrica 1.0/0.99
F. Isonandreae crown36.529.0–43.6Sundania 0.69/0.94
G. Burckella stem node, west to east dispersal24.2NASundania 0.29/NA
H. Isonandra compta, Isonandra sp., stem, dispersal/migration to Indian subcontinent22.4NASundania or India 0.49/Sundania 0.95
I. Diploknema butyracea MRCA, dispersal/migration to Indian subcontinent19.812.8–26.7Asia 0.71/Sundania 0.99
J. Burckella crown node12.77.7–18.2Australasia 1.0/ Australasia 0.97
K. Madhuca leucodermis MRCA, west to east dispersal10.16.6–13.8Sundania 0.84/Sundania 0.97
L. Madhuca fulva, Madhuca microphylla, stem, dispersal/migration to Indian subcontinent7.7NAAustralasia 0.53/Sundania 0.92
M. Palaquium galactoxylum, Palaquium sp. 1 stem, west to east dispersal5.51.5–10.0Sundania or Australasia 0.70/Sundania 0.96
N. Palaquium lobbianum, Palaquium brassii stem, west to east dispersal4.92.9–7.2Sundania 0.34/Sundania 0.94
O. Palaquium sp. 2, west to east to dispersal1.30.1–2.9Sundania or Australasia 0.63/NA

Palaquium is not monophyletic with Aulandra longifolia H.J.Lam and Diploknema butyracea (Roxb.) H.J.Lam nested in it. Madhuca, Isonandra and Diploknema are polyphyletic. Burckella and Payena are strongly supported as monophyletic (PP 1). There is a strongly supported clade that contains most of the Madhuca spp. (PP 1). The 95% confidence intervals of the highest posterior densities (HPDs) at each node are indicated in Supporting Information, Figure S4.

The tree based on a subset of species but a larger number of genes (Supporting Information, Fig. S1) provided additional support for clades recovered in the ITS analysis. For example, the clade containing Isonandra compta Dubard, I. sp and I. perakensis King & Gamble had a PP of 1 in the combined analysis but had a PP < 0.5 in the BEAST analysis of ITS alone. There were no examples of hard incongruence between trees based on ITS compared with those from the combined analysis (Supporting Information, Fig. S1).

The stem age of Isonandreae is 40.5 Ma (HPD: 32.6–48.3 Ma; node D, Figs 1, 2) and the crown is estimated at 36.5 Ma (HPD: 29.0–43.6 Ma; node F, Figs 1, 2). The crown node of the clade that contains an African grade and Isonandreae has a probability of 1 (BBM) or 0.99 (DPA) of being from Africa. The stem node ages for lineages found to the east of Wallace's Line are shown in Table 1 at nodes G, K, N and O with mean ages ranging from 1.3 to 24.2 Ma. These lineages are all nested in a larger lineage composed of accessions that are found west of Wallace's Line. Sri Lankan lineages are also nested in otherwise Sundanian lineages at nodes H, I and L indicated in Table 1 with mean ages of 7.7, 19.8 and 22.4 Ma, respectively. The results indicate that the crown node of Sapotaceae is reconstructed as most probably Asian according to the BBM but most probably African according to the DPA, although the probability for the latter is low. The crown node of Isonandreae is reconstructed as being most probably Sundanian with a probability of being from that area of 0.69 (BBM) or 0.94 (DPA) and the most internal nodes in the tribe also have a higher probability of being from Sundania than from anywhere else: for example, crown of largest Madhuca clade (BBM 0.74 Sundania and 0.12 Sundania or Asia; DPA 0.96 Sundania), crown of Palaquium clade (BBM 0.56 Sundania and 0.41 Sundania or Asia; DPA 0.98 Sundania) and crown of Payena (BBM 0.84 Sundania or Asia; DPA 0.99 Sundania). Twenty-four species in Isonandreae evolved during the Pleistocene, i.e. from 2.58 Ma. That is 32% of the total of 74 sampled in this study.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Migration of Isonandreae

According to the BEAST analyses, Sapotaceae began to diversify from 84.5 Ma (HPD: 67.1–105.0 Ma; node A, Figs 1, 2) and the origin of the family is probably Asia as the rest of the family is derived in a grade made up of Sarcosperma and Eberhardtia that are Asian genera. Bell, Soltis & Soltis (2010) estimated the age of major clades of angiosperms using multiple fossils and found that the stem node of Sapotaceae was 74 (62–84) Ma using BEAST with fossils ages calibrated as exponential priors. In contrast Bremer, Friis & Bremer (2004) estimated a Sapotaceae stem node age of c. 102 Ma that is more consistent with the crown node age of the family estimated here. The age of the stem node of the Isonandreae/Mimusopeae clade is Mid Eocene, matching the age of type I and II pollen grains (Harley, 1991) characteristic of Mimusopeae and Isonandreae from the Mid Eocene of England (Gruas-Cavagnetto, 1976). The Isonandreae clade is nested in a grade, the early diverging lineages of which are African, consistent with a migration of this lineage from Africa into Asia from the time of the age of the stem node of the tribe at 40.5 Ma (HPD: 32.6–48.3 Ma; node D, Figs 1, 2). This migration could have occurred via Laurasia during this period as temperatures were high enough to support megathermal taxa that are now predominantly restricted to lower latitudes. The historical presence of Sapotaceae in Laurasia is demonstrated by pollen characteristic of Mimusopeae, Isonandreae and Sideroxyleae in the Eocene of England (Gruas-Cavagnetto, 1976; Harley, 1991).

The results are consistent with the hypothesis that Isonandreae began to diversify first in Laurasia or Sundania. Gondwanan (i.e. Indian or Australasian) Isonandreae are nested in a Sundanian lineage supporting the fact that early diversification in the tribe was in Sundania. Migration from Sundania into New Guinea/Australia, across Wallace's Line, occurred on five occasions between c. 24.2 and 1.3 Ma. The number of migrations may of course be increased by adding to our limited sampling to the east of Wallace's Line. This west to east migration is also evident in studies of Aglaieae (Meliaceae) by Muellner et al. (2008), Pseuduvaria Miquel (Annonaceae) by Su & Saunders (2009) and Begonia L. (Thomas et al., 2012). These results are consistent with previous hypotheses on the origin and patterns of migration of South-East Asian lineages (e.g. Good, 1960; Van Balgooy, 1976; Van Welzen & Slik, 2009), i.e. that migration of angiosperms across Wallace's Line was predominantly from west to east. This has recently been reinforced in a re-assessment of patterns of migration based on a review of phylogenetic studies for the region (Richardson, Costion & Muellner, 2012).

Migration could have occurred at any point between stem and crown nodes with the arrival time only being confirmed at the crown node by diversification. The age of the crown node of Burckella, 12.7 Ma (HPD: 7.7–18.2 Ma; node J, Figs 1, 2), indicated that it had definitely reached New Guinea by this time. New Guinea was believed to be submerged until the Mid Miocene (Hall, 2009), so the age of the onset of diversification of Burckella is consistent with dispersal onto the island following its emergence. The stem node of Burckella is c. 24.2 Ma (node G, Figs 1, 2) and migration from Sundania to New Guinea could have occurred at any time from that point onwards. It is possible that this lineage could have migrated to New Guinea via a now submerged island and/or that the sister lineage of extant Burckella may now be extinct. An extinct sister lineage would have decreased the age of the stem node of Burckella.

Many west-to-east migrations documented in the literature were accompanied by substantial radiation in the east (e.g. Muellner et al., 2008; Thomas et al., 2012). Extensive radiation has also occurred in Palaquium with approximately half of its 110 species occurring to the east of Wallace's Line. Smaller radiations have occurred in Madhuca, a genus of 100 species, which has only 14 species to the east of Wallace's Line and Payena with only one of 20 species to the east of the line. Burckella has about 14 species, all to the east of Wallace's Line in the Western Pacific from the Moluccas and New Guinea to Fiji, Samoa and Tonga. Our results indicate only one poorly supported example of a back migration from east of Wallace's Line to the west, although additional sampling may of course reveal more examples of this. The absence or low frequency of back-dispersal has been noted in Malesian fanged frogs (Limnonectes, Evans et al., 2003) and in Pseuduvaria (Annonaceae; Su & Saunders, 2009) and was also highlighted by Thomas et al. (2012) in his study of Begonia, who suggested that it may be due to niche pre-emption (Silvertown, 2004; Silvertown, Francisco-Ortega & Carine, 2005). Prior filling of niche space may inhibit later arrivals by close relatives. Niche space would have been filled in the more ancient terranes of Sundania in comparison with much of the area to the east of Wallace's Line that only emerged recently.

Despite this relative lack of back migration it is clear that Isonandreae have migrated over water across Wallace's Line on numerous occasions. One criticism of using fossil-calibrated dated trees has been that they only give minimum age estimates that may result in under-estimation of the true ages (Heads, 2005). If this were true, then many phylogenetic splits that have been attributed to trans-oceanic long-distance dispersal on the basis of fossil calibrated dated trees (e.g. Bartish et al., 2011) could in fact have been older and therefore may have been caused by tectonic events such as the break-up of Gondwanaland. If we are underestimating the ages of splits between Sundania and Australasia, then we could also be underestimating the ability of Sapotaceae to disperse across oceans because these events would have occurred earlier over an even wider expanse of ocean when these two regions were more geographically distant from one another. This is also true of any other tectonic convergence event such as that between the North and South American plates (see Cody et al., 2010; J. E. Richardson, G. A. Mondragon, J. Serrano, J. A. Hawkins, I. V. Bartish, U. Swenson, M. Gonzalez, J. Chave, J. Vieu & S. Madriñán, unpubl. data).

Our results show Sri Lankan lineages nested in Sundanian ones, indicating an origin in Sundania and either dispersal or overland migration to Sri Lanka. Migration occurred from Malesia into Sri Lanka (India) three times at c. 22.4 Ma (node H, Figs 1, 2), 19.8 Ma (node I, Figs 1, 2) and 7.7 Ma (node L, Figs 1, 2). More sampling, particularly of Indo-Chinese taxa, will be needed to determine whether Sri Lankan lineages arrived overland or by long-distance dispersal. An ‘Out of India’ scenario was suggested for Crypteroniaceae by Rutschmann et al. (2004) with overland migration from India occurring as the subcontinent collided with Laurasia. Based on the fossil record, Morley (1998) suggested an over water invasion of Sundania from India during the Mid Eocene when India and South-East Asia were positioned at similar latitudes and in the same climatic zone. In the Mid and Late Eocene evidence appeared for the presence in South-East Asia of pollen of taxa characteristic of the Palaeocene and Early Eocene of India, such as numerous palms, bombacoid Malvaceae, Sapindaceae, Thymeleaceae, Proteaceae, Linaceae, Olacaceae, Polygalaceae and Ctenolophonaceae (Morley, 1998, 2000; Lelono, 2000; Collinson & Hooker, 2003), suggesting dispersal of Indian elements to South-East Asia in these periods. Our results provide strong evidence to indicate that migration into sub-continental India and Sri Lanka from South-East Asia has also occurred during a different period of time than those migrations into Sundania indicated by the fossil record.

Pleistocene speciation

Pleistocene climatic events resulted in changes in land area (and therefore the potential area of occupation of terrestrial organisms) as a consequence of changing sea levels (Voris, 2000). Sea levels were much lower during glacial periods and consequently many present day islands, particularly in Sundania, would have been connected. Inter-glacial periods would have seen contraction of land areas into island ‘refugia’, and this contraction may have resulted in allopatric speciation. According to the mean ages of splits from a most recent common ancestor, 24 species evolved in Isonandreae during the Pleistocene, indicating that sea-level changes could have had resulted in substantial speciation, although it is, of course, possible that other events might have caused speciation during that epoch. In contrast, only three speciation events occurred in Neotropical Chrysophylloideae (J. E. Richardson, G. A. Mondragon, J. Serrano, J. A. Hawkins, I. V. Bartish, U. Swenson, M. Gonzalez, J. Chave, J. Vieu & S. Madriñán, unpubl. data) during the Pleistocene, although this was based on a sample of only 25% of species compared with the 40% of Isonandreae sampled here. If, on complete sampling, this pattern proves to be the case then it would indicate that for Sapotaceae, South-East Asia has been a more active laboratory of speciation than the Neotropics during the Pleistocene and that, despite Sapotaceae occupying similar biomes in both regions, speciation patterns and processes may differ between them.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Felix Forest and the DNA bank managers at the Royal Botanic Gardens, Kew, are thanked for supplying DNA aliquots and D. Ndiade Bourobou (CENAREST, Rougier and IRAF) is thanked for the DNA aliquot of Baillonella toxisperma. Charlie Danny Heatabun and the staff of the Indonesian Institute for Sciences (LIPI) are thanked for facilitating fieldwork in Indonesia.

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  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
boj12108-sup-0001-figures1.eps1137K

Figure S1. Bayesian majority rule consensus tree of an analysis of a subset of Isonandreae species based on ITS, the trnH-psbA spacer, the trnC-trnD region (consisting of the trnCpetN spacer, the petN gene, the petN-psbM spacer, the psbM gene and the psbM-trnD spacer), the trnC-psbM region and the 3′ end of ndhF). Bayesian support values are given above branches and bootstrap ones below.

boj12108-sup-0002-figures2.eps1349K

Figure S2. Parsimony jackknife consensus tree of ITS data with support values below branches.

boj12108-sup-0003-figures3.eps1294K

Figure S3. MCC tree of the Bayesian analysis of ITS data with support values indicated to the right of nodes.

boj12108-sup-0004-figures4.eps1333K

Figure S4. MCC tree from the BEAST analysis of ITS data with mean and HPDs of node ages.

boj12108-sup-0005-tables1.docx41K

Table S1. Voucher information and ITS GenBank numbers for accessions sampled. Nomenclature follows Pennington (1991); see Govaerts et al. (2001) for synonyms.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.