A large-scale systematical investigation of the influence of Pleistocene climate oscillation on mangrove population dynamics could enrich our knowledge about the evolutionary history during times of historical climate change, which in turn may provide important information for their conservation. In this study, phylogeography of a mangrove tree Sonneratia alba was studied by sequencing three chloroplast fragments and seven nuclear genes. A low level of genetic diversity at the population level was detected across its range, especially at the range margins, which was mainly attributed to the steep sea-level drop and associated climate fluctuations during the Pleistocene glacial periods. Extremely small effective population size (Ne) was inferred in populations from both eastern and western Malay Peninsula (44 and 396, respectively), mirroring the fragility of mangrove plants and their paucity of robustness against future climate perturbations and human activity. Two major genetic lineages of high divergence were identified in the two mangrove biodiversity centres: the Indo-Malesia and Australasia regions. The estimated splitting time between these two lineages was 3.153 million year ago (MYA), suggesting a role for pre-Pleistocene events in shaping the major diversity patterns of mangrove species. Within the Indo-Malesia region, a subdivision was implicated between the South China Sea (SCS) and the remaining area with a divergence time of 1.874 MYA, corresponding to glacial vicariance when the emerged Sunda Shelf halted genetic exchange between the western and eastern coasts of the Malay Peninsula during Pleistocene sea-level drops. Notably, genetic admixture was observed in populations at the boundary regions, especially in the two populations near the Malacca Strait, indicating secondary contact between divergent lineages during interglacial periods. These interregional genetic exchanges provided ample opportunity for the re-use of standing genetic variation, which could facilitate mangrove establishment and adaptation in new habitats, especially in the context of global climate changes. Phylogeogrpahic analysis in this study reveal that Pleistocene sea-level fluctuations had profound influence on population differentiation of the mangrove tree S. alba. Our study highlights the fragility of mangrove plants and offers a guide for the conservation of coastal mangrove communities experiencing ongoing changes in sea-level.

Scyphiphora hydrophyllacea Gaertn. f. (Rubiaceae) is an endangered mangrove species found in China, and its only known location is in Hainan Island. Previous studies conducted on S. hydrophyllaceae have mainly focused on its location, biological characteristics, and medical effects. However, to date, there has been no published report regarding the genetics or genome of this endangered mangrove species. In this study, we developed valuable chloroplast genome-related molecular resources of S. hydrophyllaceae by comparing with it related Rubiaceae species. The chloroplast genome of S. hydrophyllaceae was found to be a circular molecule with a total size of 155,132 bp, and it is observed to have a quadripartite structure. The whole chloroplast genome contains 132 genes, of which 88 and 36 are protein-coding and transfer RNA genes, respectively; it also contains four ribosomal RNA genes with an overall GC content of 37.60%. A total of 52 microsatellites were detected in the S. hydrophyllacea chloroplast genome, and microsatellite marker detection identified A/T mononucleotides as majority simple sequence repeats in all nine Rubiaceae chloroplast genomes. Comparative analyses of these nine chloroplast genomes revealed variable regions, including matK, rps16, and atpF. All nine species shared 13 RNA-editing sites distributed across eight coding genes. Phylogenetic analyses based on the complete sequences of the chloroplast genomes revealed that the position of S. hydrophyllaceae is closer to the Coffeeae genus than to Cinchoneae, Naucleeae, Morindeae, and Rubieae in the Rubiaceae family. The genome information reported in this study could find further application in the evolution and population genetic studies, and it helps improve our understanding of the endangered mechanism and the development of conservation strategies of this endangered mangrove plant.

Background: As the error rate is high and the distribution of errors across sites is non-uniform in next generation sequencing (NGS) data, it has been a challenge to estimate DNA polymorphism (θ) accurately from NGS data. Results: By computer simulations, we compare the two methods of data acquisition - sequencing each diploid individual separately and sequencing the pooled sample. Under the current NGS error rate, sequencing each individual separately offers little advantage unless the coverage per individual is high (>20X). We hence propose a new method for estimating θ from pooled samples that have been subjected to two separate rounds of DNA sequencing. Since errors from the two sequencing applications are usually non-overlapping, it is possible to separate low frequency polymorphisms from sequencing errors. Simulation results show that the dual applications method is reliable even when the error rate is high and θ is low. Conclusions: In studies of natural populations where the sequencing coverage is usually modest (~2X per individual), the dual applications method on pooled samples should be a reasonable choice.