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Macaranga (Euphorbiaceae) includes about 280 species with a palaeotropic distribution. The genus not only comprises some of the most prominent pioneer tree species in Southeast Asian lowland dipterocarp forests, it also exhibits a substantial radiation of ant-plants (myrmecophytes). Obligate ant-plant mutualisms are formed by about 30 Macaranga species and 13 ant species of the genera Crematogaster or Camponotus. To improve our understanding of the co-evolution of the ants and their host plants, we aim at reconstructing comparative organellar phylogeographies of both partners across their distributional range. Preliminary evidence indicated that chloroplast DNA introgression among closely related Macaranga species might occur. We therefore constructed a comprehensive chloroplast genealogy based on DNA sequence data from the noncoding ccmp2, ccmp6, and atpB-rbcL regions for 144 individuals from 41 Macaranga species, covering all major evolutionary lineages within the three sections that contain myrmecophytes. A total of 88 chloroplast haplotypes were identified, and grouped into a statistical parsimony network that clearly distinguished sections and well-defined subsectional groups. Within these groups, the arrangement of haplotypes followed geographical rather than taxonomical criteria. Thus, up to six chloroplast haplotypes were found within single species, and up to seven species shared a single haplotype. The spatial distribution of the chloroplast types revealed several dispersals between the Malay Peninsula and Borneo, and a deep split between Sabah and the remainder of Borneo. Our large-scale chloroplast genealogy highlights the complex history of migration, hybridization, and speciation in the myrmecophytes of the genus Macaranga. It will serve as a guideline for adequate sampling and data interpretation in phylogeographic studies of individual Macaranga species and species groups.

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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.

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