The genus Podocarpus (yellowwoods or mañíos) is the largest genus in the Podocarpaceae family, both in diversity and in distribution. It is divided into two monophyletic subgenera, Podocarpus and Foliolatus. The subgenus Podocarpus occurs in all continents of Gondwanan origin (i.e., South America, Africa, Oceania and Australia), while Foliolatus is found mainly in Asia . Biogeographically, this distribution is the product of vicariance and dispersal events that took place during the last 65 Mya [2, 3]. Historical reconstructions within Podocarpaceae combining fossil information with phylogenetic and phylogeographic tools have provided valuable information to test and disentangle biogeographic scenarios. However, early molecular phylogenies of Podocarpaceae were built using a handful chloroplast genes and a small number of nuclear DNA regions [4–7] and failed to agree on the intergeneric relationships within the family . More recent work using entire chloroplast genome sequences of species from 14 out of the 19 described genera of Podocarpaceae identified frequent rearrangements in gene order, mostly due to inversions and translocations; these changes are proposed to be driven by the presence of intermediate-size repeats located near the boundaries of colinear blocks [8, 9].
The chloroplast genome (or plastome) of terrestrial plants is approximately 120-220kb in size and consists of two copies of the inverted repeats (IRs) that separate the small and large single copy regions, SSC and LSC respectively [10, 11]. In gymnosperms, the plastome is highly variable in size and organization, and this variation is often used in phylogenetics to identify families, subgenera, and genera . Yet, certain families remain poorly sampled such as only eight of the 187 species within Podocarpaceae have been sequenced so far . Also, the loss of the large IR has been reported in several species, mainly conifers [14–16]. Furthermore, many rearrangements can be observed in these plastomes, some of which appear to have played an important role in their evolution [15–17]. A frequent change is the deletion of genes, as reported within the inverted repeat region in Cupressophyta (Araucariales and Cupressales) and Pinaceae . In Cupressoidaea, Sudianto et al.  suggest that added complexity results from the existence of isomeric plastomes. As in other species of Podocarpaceae [16, 17], the plastome of Retrophyllum piresii lacks one of the IRs. Similarly, the genus Lagostrobos, having the largest plastome described so far for the family (151.5 kb), lacks the IR region but hosts numerous intergenic spacer repeats. Wu and Chaw  concluded that in cupressophytes, mutation rates have a critical role in driving the evolution of plastomic size, while plastomic inversions evolve in a neutral manner. As repetitions play an important role in plastomic rearrangements [8, 21, 22], plastome sequencing and de novo assembly can be considered an efficient tool to understand phylogenetic relationships at different taxonomic levels, as well as to investigate the structural and functional evolution of plants .
A comparison of plastomes of two Podocarpus species, P. lambertii and P. totara, found differences in internal gene order due to four large inversions of about 20,000 bp in length each . Since P. lambertii occurs in Brazil, while P. totara occurs in New Zealand, and given the vicariant biogeographic history of the genus, it could be hypothesized that the large geographic distance between them correlates with genetic differences, reflected both as nucleotide substitutions and plastome rearrangements. Thus, geographic distance, evolutionary time and different adaptive traits could explain plastome structural differences found between these two congener species . Alternatively, these differences could have resulted from changes in a single lineage leading to either species rather than reflecting a trend for the whole genus. By comparing plastomes from only two species, these two alternatives cannot be distinguished.
More than half of the species of Podocarpus occur in South America. They are distributed in four clades (Quiroga et al. 2016) that are geographically structured into southern, tropical, and subtropical distributions, the latter of which is sister to the African subtropical clade and thus a good system to test the above hypotheses. Therefore, we sequenced the plastomes of four additional species of South American Podocarpus: P. nubigenus and P. salignus, found along the Patagonian temperate forests, P. parlatorei found at the southern Yungas rainforest, and P. sellowii, mainly distributed in the Atlantic rainforest, but also present in other Brazilian biomes in subtropical forests. We compared their plastomes with those available for P. lambertii from Mata Atlantica (southern Brazil) , P. latifolius and P. milanjianus from Africa , P. totara from New Zealand , and P. neriifolius from Southeast Asia , along with representatives from other genera within Podocarpaceae as outgroups. The aim of this work was to analyze stability (or lack thereof) of plastome organization within the genus Podocarpus, estimate the selective regimes affecting plastome protein-coding genes, and strengthen our current understanding of the phylogenetic relationships and biogeographic history of Podocarpus.