BackgroundWith high quantity and quality data production and low cost, next generation sequencing has the potential to provide new opportunities for plant phylogeographic studies on single and multiple species. Here we present an approach for in silicio chloroplast DNA assembly and single nucleotide polymorphism detection from short-read shotgun sequencing. The approach is simple and effective and can be implemented using standard bioinformatic tools.ResultsThe chloroplast genome of Toona ciliata (Meliaceae), 159,514 base pairs long, was assembled from shotgun sequencing on the Illumina platform using de novo assembly of contigs. To evaluate its practicality, value and quality, we compared the short read assembly with an assembly completed using 454 data obtained after chloroplast DNA isolation. Sanger sequence verifications indicated that the Illumina dataset outperformed the longer read 454 data. Pooling of several individuals during preparation of the shotgun library enabled detection of informative chloroplast SNP markers. Following validation, we used the identified SNPs for a preliminary phylogeographic study of T. ciliata in Australia and to confirm low diversity across the distribution.ConclusionsOur approach provides a simple method for construction of whole chloroplast genomes from shotgun sequencing of whole genomic DNA using short-read data and no available closely related reference genome (e.g. from the same species or genus). The high coverage of Illumina sequence data also renders this method appropriate for multiplexing and SNP discovery and therefore a useful approach for landscape level studies of evolutionary ecology.

Aim We take advantage of next generation sequencing-based technology to assess how landscape-level dynamics, biogeographical history and functional factors shape the distribution of genetic diversity in rain forest trees. To achieve this, we explore chloroplast genomic diversity and divergence patterns across multiple, co-distributed species from three major centres of rain forest diversity.Location Subtropical rain forests in south-eastern Australia: Nightcap-Border Ranges, Dorrigo and Washpool.Methods We assembled chloroplast genomic data from whole-genome shotgun libraries for 71 species collected across three major centres of rain forest diversity. Chloroplast single nucleotide polymorphisms were used to measure genetic variation within and between multiple populations, species and locations, and we used this information to identify patterns related to landscapelevel processes, biogeographical origins and functional characteristics.Results We identified Nightcap-Border Ranges and Dorrigo as containing multiple refugia and Washpool as primarily a recolonized area. We found that rapidly expanding lineages of Indo-Malesian origin exhibit significantly lower levels of genetic diversity than Gondwanan lineages with longer local histories. Functional traits related to persistence and dispersal were the most important in regulating the distribution of genetic diversity. Main conclusionsThe distribution and assembly of species reflect interactions and competition between different floristic elements at different stages of continental occupation. Additionally, given the multiple evolutionary origins and histories of the Australian rain forest flora, it is important to avoid treating rain forest communities as single management units.

Standardized phylogeographic studies across codistributed taxa can identify important refugia and biogeographic barriers, and potentially uncover how changes in adaptive constraints through space and time impact on the distribution of genetic diversity. The combination of next-generation sequencing and methodologies that enable uncomplicated analysis of the full chloroplast genome may provide an invaluable resource for such studies. Here, we assess the potential of a shotgun-based method across twelve nonmodel rainforest trees sampled from two evolutionary distinct regions. Whole genomic shotgun sequencing libraries consisting of pooled individuals were used to assemble species-specific chloroplast references (in silicio). For each species, the pooled libraries allowed for the detection of variation within and between data sets (each representing a geographic region). The potential use of nuclear rDNA as an additional marker from the NGS libraries was investigated by mapping reads against available references. We successfully obtained phylogeographically informative sequence data from a range of previously unstudied rainforest trees. Greater levels of diversity were found in northern refugial rainforests than in southern expansion areas. The genetic signatures of varying evolutionary histories were detected, and interesting associative patterns between functional characteristics and genetic diversity were identified. This approach can suit a wide range of landscape-level studies. As the key laboratory-based steps do not require prior species-specific knowledge and can be easily outsourced, the techniques described here are even suitable for researchers without access to wet-laboratory facilities, making evolutionary ecology questions increasingly accessible to the research community.

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