Comparative chloroplast DNA phylogeography of two tropical pioneer trees, Macaranga gigantea and Macaranga pearsonii (Euphorbiaceae)

Macaranga (Euphorbiaceae) has received much ecological and evolutionary research attention as a genus that includes some of the most conspicuous pioneer trees of Southeast Asian tropical rainforests and because of its manifold associations with ants, including about 30 species that are obligate ant-plants (myrmecophytes). We used sequence data from three chloroplast DNA loci (ccmp5, ccmp6, atpB-rbcL) to assess phylogeographical patterns in species of Macaranga, section Pruinosae, sampled from various regions of Borneo and the Malay Peninsula. Forty-nine chloroplast DNA haplotypes (HT) were identified among 768 specimens from five species, Macaranga gigantea (N = 329; 23 HT), Macaranga pearsonii (N = 347; 21 HT), Macaranga puberula (N = 24; 4 HT), Macaranga hosei (N = 48; 6 HT), and Macaranga pruinosa (N = 20; 5 HT). Forty-one haplotypes were species-specific, whereas eight haplotypes were shared by two, three, or four species and occupied internal positions in a parsimony network. Population genetic parameters based on haplotype frequencies proved to be in a similar range in the non-myrmecophytic M. gigantea and in the ant-associated M. pearsonii, which have overlapping distributions in northern and eastern Borneo. A comparison of G _ST and N _ST values revealed a strong phylogeographic structure in both species, whereas colonization pathways suggested by the network topology were different. Both species exhibited similar levels of haplotypic diversity and moderate to high levels of population differentiation. There were no obvious indications for an influence of the symbiotic ant partners on the population structure of their host plants.     

A chloroplast genealogy of hordeum (poaceae): Long-term persisting haplotypes, incomplete lineage sorting, regional extinction, and the consequences for phylogenetic inference

To analyze reasons for inconclusive results of earlier chloroplast phylogenies in the grass genus Hordeum, we established a genealogy of chloroplast haplotypes by sequencing the trnL-trnF region in 875 individuals, covering all 31 species of the genus. Although the outcomes of phenetic and parsimony analyses of 88 haplotypes were ambiguous, a network approach showed that in Hordeum ancient chloroplast types co-occur with their descendants. Moreover, we found up to 18 different chloroplast haplotypes within a single species and up to 6 species sharing single haplotypes. Persisting polymorphisms together with incomplete lineage sorting occurred preferentially in the rapidly speciating New World taxa of the genus, where ancient chloroplast types have survived for at least 4 Myr. Lineages-through-time plots and a high number of missing chloroplast haplotypes indicated far-reaching extinction of chloroplast lineages in Europe and particularly the Mediterranean. Survival of these lineages in East Asia and North America resulted in chloroplast relationships that markedly differed from nuclear estimations of species relationships. Thus, even for the deepest splits in the genus, reaching back more than 9 Myr, no safe phylogenetic inference from chloroplast data is possible in Hordeum. The chloroplast genealogy, however, revealed biogeographic patterns and indicated processes involved in speciation in Hordeum. We conclude that the described phenomena are not restricted to Hordeum and that the knowledge of the chloroplast relationships within a genus is indispensable to prevent misinterpretation of phylogeographic data within single species.     

Molecular analysis of phylogenetic relationships among Myrmecophytic macaranga species (Euphorbiaceae)

Many species of the paleotropical pioneer tree genus Macaranga Thou. (Euphorbiaceae) live in association with ants. Various types of mutualistic interactions exist, ranging from the attraction of unspecific ant visitors to obligate myrmecophytism. In the latter, nesting space and food bodies are exchanged for protection by highly specific ant partners (mainly species of the myrmicine genus Crematogaster). As a first step toward elucidating the coevolution of ant-plant interactions in the Macaranga-Crematogaster system, we have initiated a molecular investigation of the plant partners' phylogeny. Nuclear ribosomal DNA internal transcribed spacer (ITS) sequences were analyzed for 73 accessions from 47 Macaranga species, representing 17 sections or informally described species groups. Three accessions from the putative sister taxon Mallotus Lour, were included as outgroups. Cladograms of the ITS data revealed Macaranga to be nested within Mallotus. ITS sequences are highly similar within section Pachystemon s.str., suggesting a relatively recent and rapid radiation of obligate myrmecophytes within this section. Forty-three accessions, mainly of ant-inhabited species, were additionally investigated by random amplified polymorphic DNA (RAPD) and microsatellite-primed PCR (MP-PCR) techniques. Phenetic analysis of RAPD and MP-PCR banding profiles generally confirmed the ITS results. Best resolutions for individual clades were obtained when ITS and RAPD/MP-PCR data were combined into a single matrix and analyzed phenetically. The combined analysis suggests multiple (four) rather than a single evolutionary origin of myrmecophytism, at least one reversal from obligate myrmecophytism to nonmyrmecophytism, and one loss of mutualistic specifity.     

Diversity, evolutionary specialization and geographic distribution of a mutualistic ant-plant complex: Macaranga and Crematogaster in South East Asia

The most conspicuous and species-rich ant-plant mutualism in the Malesian region is found in the important pioneer tree genus Macaranga , yet little is known about the identities or community ecology of the species involved. Our studies have revealed a far more complex system than previously thought. This paper presents the first extensive investigation in the whole distribution area of myrmecophytic Macaranga. All ant-inhabited species were restricted to the moister parts of SE Asia: Peninsular Malaysia, South and East Thailand, Sumatra and Borneo. We found a rather strict and similar altitudinal zonation of myrmecophytic Macaranga species in all regions. Here we focus on the majority of the 19 Macaranga species obligatorily associated with ants of the genus Crematogaster. We identified a total of 2163 ant queens which belonged to at least eight (morpho)species of the small subgenus Decacrema as well as to one non-Decacrema (probably from Atopogyne ). The ant species were not randomly distributed among the Macaranga species but distinct patterns of associations emerged. Despite common sympatric distribution of Macaranga species, in most cases a surprisingly high specificity of ant colonization was maintained which was, however, often not species-specific but groups of certain plant species with identical ant partners could be found. These colonization patterns usually but not always mirror existing taxonomic sections within the genus Macaranga. Possible mechanisms of specificity are discussed. The results are compared with other ant-plant mutualisms.     

Development and characterization of chloroplast microsatellite markers in Macaranga (Euphorbiaceae).

As part of our study on the phylogeography of the ant-plant genus Macaranga, we have screened for polymorphic regions in the chloroplast genome. Initially, ten universal PCR primer pairs targeted at chloroplast microsatellite loci were applied to a small set of specimens, covering various taxonomic levels from intrafamilial to intraspecific. Eight primer pairs produced PCR fragments that behaved as single and discrete bands on agarose gels. The five most promising candidate pairs were further analysed with an extended set of DNA templates, and PCR products were separated on sequencing gels. The number of size variants per locus varied from two to eight, combining into 17 haplotypes among 29 Macaranga accessions from 10 species. Comparative sequencing demonstrated that microsatellites were responsible for the observed size variation at three of five loci, whereas variation at the other loci was caused by larger insertions and (or) deletions (indels). In addition to poly(A) and poly(T) repeats, which are typically found in chloroplast DNA, we also identified a variable (CT)n repeat, with n = 4 to n = 8. Sequencing revealed three examples of size homoplasy, one of which was caused by a single base substitution that raised the actual number of haplotypes to 18. Relationships between haplotypes were assessed by phenetic analyses of size variants and by constructing a parsimony network based on sequence variation. For both types of analysis, the distribution of haplotypes correlated with geographically circumscribed regions rather than with taxonomic boundaries.     

Chloroplast DNA variation and reticulate evolution in sexual and apomictic sections of dandelions

Sequencing of the trnL-trnF intergenic spacer in chloroplast DNA (cpDNA) from 237 sexual and apomictic species of dandelions (genus Taraxacum) from Europe, Asia and arctic North America revealed 46 haplotypes, which differed mainly by a variable number of polymorphic tRNA pseudogenes next to the trnF gene. The haplotypes could be divided into 20 cpDNA lineages, but independent duplications and deletions of the pseudogene copies made it difficult to further reconstruct the phylogeny. Intraspecific cpDNA variation was found in the primitive sexual T. serotinum. However, in contrast to a recent study, no cpDNA variation was detected within 12 apomictic species representing a variety of haplotypes. The cpDNA haplotype may therefore help to define these critical apomicts. On the other hand, the genetic variation may easily be overestimated, if the clones are not correctly identified, because some morphologically similar microspecies carried very different haplotypes. In all, 36 sections of the genus were sampled. Four primitive, mainly sexual, sections only displayed a group of ancient haplotypes, whereas morphologically more advanced sections often exhibited many different haplotypes from up to seven cpDNA lineages. In the latter cases, the lineages were rarely unique to a certain section. For example, the two most widespread haplotypes, belonging to different lineages, were found together in nine sections. This suggests that significant gene flow has occurred among the advanced sections, although sexual reproduction is not currently known in several of them. The result is consistent with the reticulate distribution of morphological characters among the sections.     

Slippery ant-plants and skilful climbers: selection and protection of specific ant partners by epicuticular wax blooms in Macaranga (Euphorbiaceae)

 In many ant-plant species of the genus Macaranga in South-East Asia, conspicuous blooms of epicuticular wax crystals cover the stem surface. We found that many ant species were unable to walk on these surfaces. Only the specific ant partners of glaucous Macaranga host plants were capable of moving on the slippery stems without difficulty. Therefore, the epicuticular coatings of Macaranga myrmecophytes appear to have a selective function and protect the associated ants against competitors. The epicuticular aggregates function as a physical barrier; no evidence of chemical repellence was found. The extent to which ”foreign” ant species are excluded from a tree strongly depends on inclination, diameter and length of the glaucous stem sections. The particular growth form of some glaucous Macaranga ant-plants enhances the influence of the wax barriers. The ant associates of glaucous and glossy Macaranga ant-plants (genera Crematogaster and Camponotus) differ strongly in their capacity to adhere to the glaucous stems. For this reason, the wax blooms in Macaranga can act as an ecological isolation mechanism for the sympiotic ants. Within the genus Macaranga, we find a high correspondence between the occurrence of glaucousness and obligatory ant association (50% in ant-plants; 6.7% in non-myrmecophytes). The genus Macaranga thus represents one of the few cases known so far where epicuticular wax crystals are likely to have evolved in relation to insects. Received: 2 January 1997 / Accepted: 9 June 1997     

Chemical contents of Macaranga food bodies: adaptations to their role in ant attraction and nutrition

1. Macaranga (Euphorbiaceae) is a paleotropical tree genus comprising myrmecophytic and non-myrmecophytic species. All species are presumed to possess food bodies (FBs) to maintain or attract ants as anti-herbivore defence.
2. The hypothesis was tested that Macaranga species differing in their mode of association with ants would produce FBs differing in their chemical composition. We investigated contents of carbohydrates, proteins and lipids in FBs of four myrmecophytic and one non-myrmecophytic Macaranga as well as one Parthenocissus (Vitaceae) species.
3. On a dry weight basis, FBs of myrmecophytes contained relatively higher amounts of proteins compared to carbohydrates than those of non-myrmecophytes. Soluble carbohydrates showed species-specific patterns and were found in especially high amounts in both non-myrmecophytes. Furthermore, Parthenocissus FBs contained higher amounts of soluble compared to polymerous substances not only in carbohydrates but also in proteins.
4. FBs seem to be specifically adapted to their respective role in ant attraction and nutrition, with myrmecophytes providing ants with high amounts of lipids and proteins and non-myrmecophytes mainly offering carbohydrates in the form of common soluble sugars.     

Molekulare Studien an Ameisenbäumen

Molecular systematic studies in Southeast Asian ant‐plants Myrmecophytes are plants that are permanently inhabited by ants. They provide nesting space and feed their partners, whereas the ants protect their hosts from herbivores and competitors such as climbers and lianas. The manifold relationships between tropical pioneer trees of the genus Macaranga and their partner ants of the genus Crematogaster constitute the most important and species‐rich mutualistic ant‐plant relationship of the Paleotropics. We use comparative DNA sequencing and molecular marker technologies to evaluate phylogenetic relationships among the about 30 myrmecophytic Macaranga species as well as their co‐evolution with ants. We also study the population genetics and historical biogeography of particular Macaranga species groups. Patterns of genetic diversity and differentiation in these pioneer tree species show interesting correlations with their reproductive biology and with characteristics of the increasingly fragmented pioneer habitats in the rainforests of Sundaland.     

The naming of <Emphasis Type="Italic">Phragmites</Emphasis> haplotypes

The genus Phragmites includes several species, of which only Phragmites australis has a worldwide distribution. It has been several decades since the last formal taxonomic examination of the genus and a number of recent genetic studies have revealed novel diversity and unique lineages within the genus. In my initial work on genetic variation in Phragmites (Saltonstall in Proc Nat Acad Sci 99:2445–2449, 2002), I came up with a naming scheme for identifying chloroplast DNA haplotypes which combined unique sequences at two loci, designated by numbers, to form haplotypes, designated by letters. Here I describe this naming system in more detail, explain how it has evolved over time as more genetic data has become available, provide a summary of all haplotypes currently available on GenBank, and address some common misunderstandings about how the haplotypes are named.     

Molecular phylogeny of Crematogaster subgenus Decacrema ants (Hymenoptera: Formicidae) and the colonization of Macaranga (Euphorbiaceae) trees

To elucidate the evolution of one of the most species-rich ant-plant symbiotic systems, the association between Crematogaster (Myrmicinae) and Macaranga (Euphorbiaceae) in South-East Asia, we conducted a phylogenetic analysis of the ant partners. For the phylogenetic analysis partial mitochondrial cytochrome oxidase I and II were sequenced and Maximum Parsimony analysis was performed. The analyzed Crematogaster of the subgenus Decacrema fell into three distinct clades which are also characterized by specific morphological and ecological traits (queen morphology, host-plants, and colony structure). Our results supported the validity of our currently used morphospecies concept for Peninsula Malaysia. However, on a wider geographic range (including North and North-East Borneo) some morphospecies turned out to be species complexes with genetically quite distinct taxa. Our phylogenetic analysis and host association studies do not indicate strict cocladogenesis between the subgenus Decacrema and their Macaranga host-plants because multiple ant taxa occur on quite distinct host-plants belonging to different clades within in the genus Macaranga. These results support the view that host-shifting or host-expansion is common in the ants colonizing Macaranga. Additionally, the considerable geographic substructuring found in the phylogenetic trees of the ants suggests that allopatric speciation has also played a role in the diversification and the current distribution of the Decacrema ants.     

The rare silver gum, Eucalyptus cordata, is leaving its trace in the organellar gene pool of Eucalyptus globulus

The process of genetic assimilation of rare species by hybridizing congeners has been documented in a number of plant genera. This raises the possibility that some of the genetic diversity found in phylogeographical studies of widespread species has been acquired through hybridization with species that are now rare or extinct. In this fine-scale phylogeographical analysis, we show that a rare eucalypt species is leaving its trace in the chloroplast genome of a more abundant congener. The heart-leafed silver gum, Eucalyptus cordata, is a rare endemic of south-eastern Tasmania. Its populations are scattered amidst populations of more abundant related species, including the Tasmanian blue gum, Eucalyptus globulus. Using 339 samples from across the full range of both species, we compared chloroplast (cp) DNA haplotype phylogeography in E. globulus and E. cordata. The genealogy and distribution of chloroplast haplotypes suggest that E. globulus has acquired cpDNA from E. cordata in at least four different mixed populations. Shared haplotypes are highest in E. globulus sampled within 2 km of known E. cordata populations and drop to zero at a distance of 25 km from the nearest known E. cordata population. Localized haplotype sharing occurs in the absence of obvious hybrid zones or locally shared nuclear ribosomal DNA sequences. Given that the future loss of E. cordata from some mixed populations is likely, these findings indicate that phylogeographical analyses of organellar DNA should consider the possibility of introgression, even from species that have been eliminated from the sites of interest.     

Phylogeny and genetic diversity of D-genome species of Aegilops and Triticum (Triticeae, Poaceae) from Iran based on microsatellites, ITS, and trnL-F

Cereal species of the grass tribe Triticeae are economically important and provide staple food for large parts of the human population. The Fertile Crescent of Southwest Asia harbors high genetic and morphological diversity of these species. In this study, we analyzed genetic diversity and phylogenetic relationships among D genome-bearing species of the wheat relatives of the genus Aegilops from Iran and adjacent areas using allelic diversity at 25 nuclear microsatellite loci, nuclear rDNA ITS, and chloroplast trnL-F sequences. Our analyses revealed high microsatellite diversity in Aegilops tauschii and the D genomes of Triticum aestivum and Ae. ventricosa, low genetic diversity in Ae. cylindrica, two different Ae. tauschii gene pools, and a close relationship among Ae. crassa, Ae. juvenalis, and Ae. vavilovii. In the latter species group, cloned sequences revealed high diversity at the ITS region, while in most other polyploids, homogenization of the ITS region towards one parental type seems to have taken place. The chloroplast genealogy of the trnL-F haplotypes showed close relationships within the D genome Aegilops species and T. aestivum, the presence of shared haplotypes in up to three species, and up to three different haplotypes within single species, and indicates chloroplast capture from an unidentified species in Ae. markgrafii. The ITS phylogeny revealed Triticum as monophyletic and Aegilops as monophyletic when Amblyopyrum muticum is included.     

Carbon and nitrogen contents of food bodies in three myrmecophytic species of Macaranga: implications for antiherbivore defense mechanisms

In Macaranga myrmecophytes, differences in the production of the food bodies (FBs), on which symbiont ants feed, may relate to the intensity of antiherbivore defense by the ants. Interspecific comparisons among Macaranga species on such a mutualistic cost give important information on their strategies and evolution of antiherbivore defense. In this study, the carbon and nitrogen contents of FBs as well as the production rate of FBs were measured in three Macaranga species, M. winkleri, M. trachyphylla, and M. beccariana. There were significant differences in the production rates of FBs among species; the investment in FBs was greater in the Macaranga species in which ant defenses were more intensive. The carbon and nitrogen contents of FBs were significantly different among the three species, although they did not match the intensity of ant defense; the nitrogen content, especially, was greatest in the species of least intensive ant defense. It is suggested that Macaranga plants may have differentiated in the dependence on ant defense by controlling the total amount of nitrogen of FBs, not simply by nitrogen content.     

Mitochondrial DNA variation and reticulate evolution of the genus Abies

The sequences of three regions of mitochondrial DNA (mtDNA) of a total length of 5226 bp were used to study the phylogeography of the genus Abies. The mtDNA haplotype network, comprising 36 studied Abies taxa, consisted of two branches; the first represented all American species plus two Asian, and the second included the remaining Eurasian species. Within these clusters, the haplotypes formed nine major groups, generally corresponding to the clades of the previously obtained phylogeny based on chloroplast DNA (cpDNA), but the relationships of these groups were significantly different; species assignment to the particular mtDNA haplotype group was more in line with its geographical distribution. In addition, the mtDNA haplotype network contains cycles indicating the recombination. It is assumed that the incongruence of cpDNA and mtDNA phylogenies is caused by the introgression capture of alien mtDNA during species hybridization and thus contains information about past migrations. The cases of incongruence of mitochondrial and chloroplast DNA suggesting a migration of Abies between Asia and North America are discussed.     

Multiple origins of polyploidy and the geographic structure of Heuchera grossulariifolia

Multiple origins of polyploidy from an ancestral diploid plant species were investigated using restriction site polymorphism and sequence variation in the chloroplast DNA (cpDNA) of Heuchera grossulariifolia (Saxifragaceae). Phylogenetic analysis indicated that autopolyploidy has arisen at least twice in the evolutionary history of this species and potentially up to as many as seven times. These results suggest a greater range of independent polyploid origins as compared to a previous study of H. grossulariifolia using cpDNA restriction sites that indicated a minimum of three independent origins. Moreover, most polyploid populations did not contain cpDNA haplotypes from a single origin, but rather combined haplotypes from at least two polyploid origins. Past migration among polyploid populations of independent origin or localized polyploid formation may explain the distribution of polyploid haplotypes within and among populations. The analysis also revealed a discrepancy between relatedness and geographical location. In nearly all sympatric populations of diploids and polyploids, polyploids had the same cpDNA haplotypes as diploids from a geographically remote population. This geographical discordance has several possible explanations, including small sample sizes, extinction of parental diploid haplotypes, chloroplast introgression, and homoplasy in the cpDNA sequence data. We conclude that the recurrent formation of polyploids is an important evolutionary mechanism in the diversification of H. grossulariifolia .     

DNA Barcoding is not enough: mismatch of taxonomy and genealogy in New Zealand grasshoppers (Orthoptera: Acrididae).

DNA barcoding has been touted as a program that will efficiently and relatively cheaply inform on biological diversity; yet many exemplars purporting to demonstrate the efficacy of the method have been undertaken by its principal proponents. Critics of DNA barcoding identify insufficient within-taxon sampling coupled with the knowledge that levels of haplotypic paraphyly are rather high as key reasons to be sceptical of the value of an exclusively DNA-based taxonomic. Here I applied a DNA barcoding approach using mtDNA sequences from the cytochrome oxidase I gene to examine diversity in a group of endemic New Zealand grasshoppers belonging to the genus Sigaus . The mtDNA data revealed high genetic distances among individuals of a single morpho-species, but this diversity was geographically partitioned. Phylogenetic analysis supported at least four haplogroups within one species ( Sigaus australis ) but paraphyly of this species with respect to several others. In some instances two morphologically and ecologically distinct species shared identical mtDNA haplotypes. The mismatch of genealogy and taxonomy revealed in the Sigaus australis complex indicates that, if used in isolation, DNA barcoding data can be highly misleading about biodiversity. Furthermore, failure to take into account evidence from natural history and morphology when utilizing DNA barcoding will tend to conceal the underlying evolutionary processes associated with speciation.
© The Willi Hennig Society 2007.     

Macaranga ant-plants hide food from intruders: correlation of food presentation and presence of wax barriers analysed using phylogenetically independent contrasts

Many tropical ant-plants provide specialized ant partners with food, which may attract foreign ants parasitizing the mutualism. We present evidence for the ant-plant genus Macaranga , showing that ant competition has forced host plants to hide food resources and restrict access to the mutualists. In Macaranga myrmecophytes, the influence of ant competition strongly depends on the presence of slippery 'wax barriers'. Of all Macaranga ant-plant species, 50% have waxy stems that can be climbed only by the specific ant partners and not by other ant species. We compared the presentation of food (food bodies and extrafloral nectar) between waxy and non-waxy Macaranga host plants using traditional and phylogenetic comparative methods. Consistent with the hypothesized effect of ant competition, wax-free Macaranga host species had fewer extrafloral nectaries and more often produced food bodies under recurved or tubular stipules inaccessible to other ants; closed stipules were less persistent in waxy hosts. Several traits showed phylogenetic signal, but our finding of a more promiscuous food presentation in waxy Macaranga hosts was still supported by phylogenetic comparative analyses. We conclude that competition among ants is an important factor in the evolution of myrmecophytism, and that it has given rise to traits acting as protective filter mechanisms.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 177–193.     

Phylogeny of the genus Lophozia (Dumort.) Dumort. s. str. inferred from nuclear and chloroplast sequences ITS1-2 and TRNL-F

Maximum parsimony and maximum likelihood phylogenetic trees were constructed for 21 taxa of Lophozia s. str. and the related genera, Schistochilopsis (5 species), Protolophozia elongate, and Obtusifolium obtusum based on pooled nuclear ITS 1-2 and chloroplast trnL-F DNA sequences. The trees were characterized by similar topology. It was demonstrated that the genus Lophozia s. str. was monophyletic, excluding L. sudetica, which deserved isolation into a distinct cryptic genus. The species distribution among the clades disagreed with the sections distinguished based on anatomical and morphological data. The relationships within the genus Schistochilopsis were consistent with the sectioning of the genus, based on morphological characters. Analysis of molecular data provided more precise definition of the systematic position of a number of taxa. Small genetic divergence of geographically distant forms was demonstrated.     

Phylogeography of the Southeast Asian stone oaks (Lithocarpus)

Abstract Aim To describe current geographical patterns of genetic diversity and infer the historical population dynamics of the stone oaks (Lithocarpus) in Southeast Asia. Location We sampled three populations in Indochina: (1) Yunnan province, China; (2) Pyin Oo Lwin area, Myanmar and (3) north‐western Vietnam; two in western Borneo: (1) South‐western Sarawak and (2) West Kalimantan, Indonesia; two in central Borneo: (1) north coastal Sarawak and (2) north‐eastern Sarawak, Malaysia; and two in northern Borneo: (1) Central Sabah and (2) Northern Sabah, Malaysia. Methods A phylogenetic reconstruction of chloroplast DNA sequence variation from numerous individuals of multiple species was used to determine geographical distribution of genetic diversity. A resampling scheme was used to determine the significance of these patterns at different hierarchical levels of the phylogeny. Results were compared with a previously published set of nuclear DNA sequence data. Results A high level of chloroplast sequence variation was found, which was divided equally between two major clades separated by four non‐homoplasious changes. One clade was confined to the island of Borneo, while the other was widespread. Strong geographical structure was observed in the chloroplast sequence variation. The Indo‐chinese populations were much more closely related than expected, comparable with the highly endemic and isolated population on the western coast of Borneo. Conversely, individuals from the Kelabit Highlands were found to be more distantly related than expected. The highest levels of genetic endemism were observed in western Borneo. More geographical structure was observed in the Bornean clade than in the Widespread clade, because of limited genetic diversity in the Widespread clade. Relatively weak geographical structure was found in the nuclear sequence variation: only populations in southern China and central Sabah were significantly related. Conclusions The high levels of chloroplast genetic diversity and the persistence of an ancestral haplotype that is a single step away from a haplotype found in Castanopsis indicates the continuous presence of tropical rain forest in Southeast Asia throughout the evolutionary history of the genus (c. 40 Myr). This conclusion is supported by the high frequency of numerous endemic types observed in every population and the relatively few number of ‘missing’ haplotypes. This situation suggests both limited migration and limited extinction. In contrast, the nuclear genetic diversity contained less geographical structure, indicating that our taxonomic sampling among populations was unbiased and that gene flow mediated through pollen is less geographically restricted and contains less geographical structure than purely seed‐mediated (chloroplast) gene flow. The most likely scenario suggested by the evidence involves four major patterns: (1) the widespread presence of an ancestral haplotype; (2) the large degree of separation (four non‐homoplasious base pairs) between the types found in the two major clades; (3) the concentration of derived types from both major clades found in central and northern Borneo; and (4) the molecular endemism found in each location. These patterns suggest four primary things about the population dynamics of Lithocarpus since the late Eocene: (1) populations have either spanned the entire region throughout much of the evolutionary history of the genus or substantial populations have persisted in both Indochina and Borneo with limited migration between them; (2) significant fragmentation has occurred subsequently between the Asian mainland and the Melasian island archipelago, leading to independent genetic diversification in both regions; (3) several locations possessing significant independent histories, have experienced little migration and have never gone completely extinct; and (4) that the central highlands of Borneo have been re‐invaded from the north and the west. The timing of these events is difficult to ascertain but probably predate the Quaternary Period, suggesting that although the recent ice ages might have affected the overall distribution of rain forest in Southeast Asia, it managed to persist in most regions even through the most dramatic drying events.