Climatic oscillations during the Quaternary strongly affected the distribution of warm-temperate tree species, which experienced local restrictions into ice-free areas and posterior expansions. To evaluate the impact of these range movements on the genetic structure of populations, we performed a phylogeographical analysis of the species Nothofagus obliqua with chloroplast DNA markers. A total of 27 populations covering the whole natural distribution range were analyzed using polymerase chain reaction-restriction fragment length polymorphism. Diversity (h T , h S ), allelic richness (r g ), and differentiation among populations for unordered (G ST ) and ordered alleles (N ST ) were calculated. The relationships among haplotypes were evaluated by the construction of a minimum spanning network. The spatial distribution of the genetic variation was analyzed through a Mantel test and with a nested analysis of molecular variance to differentiate between geographic regions. The screening of 11 non-coding regions allowed the identification of 14 haplotypes. A high genetic differentiation was detected (N ST =0.875 and G ST = 0.824) with the existence of phylogeographic structure (p< 0.05). The distribution of the genetic variation was partially explained by the topography of the region when the populations were divided longitudinally into Coastal Mountains, Central Valley, and Andes Mountains ( RT = 0.093, p=0.001). In agreement with pollen records, our results support the hypothesis of Coastal refuges since the region harbors high diversity together with older and private haplotypes. Long-lasting persistence of some Coastal populations without contribution to recolonization is proposed. Additional refuges are also postulated along the Andes and Longitudinal Valley. Survival in multiple glacial refuges is discussed together with the possible migratory routes.

BackgroundNothofagus nervosa is one of the most emblematic native tree species of Patagonian temperate forests. Here, the shotgun RNA-sequencing (RNA-Seq) of the transcriptome of N. nervosa, including de novo assembly, functional annotation, and in silico discovery of potential molecular markers to support population and associations genetic studies, are described.ResultsPyrosequencing of a young leaf cDNA library generated a total of 111,814 high quality reads, with an average length of 447 bp. De novo assembly using Newbler resulted into 3,005 tentative isotigs (including alternative transcripts). The non-assembled sequences (singletons) were clustered with CD-HIT-454 to identify natural and artificial duplicates from pyrosequencing reads, leading to 21,881 unique singletons. 15,497 out of 24,886 non-redundant sequences or unigenes, were successfully annotated against a plant protein database. A substantial number of simple sequence repeat markers (SSRs) were discovered in the assembled and annotated sequences. More than 40% of the SSR sequences were inside ORF sequences. To confirm the validity of these predicted markers, a subset of 73 SSRs selected through functional annotation evidences were successfully amplified from six seedlings DNA samples, being 14 polymorphic.ConclusionsThis paper is the first report that shows a highly precise representation of the mRNAs diversity present in young leaves of a native South American tree, N. nervosa, as well as its in silico deduced putative functionality. The reported Nothofagus transcriptome sequences represent a unique resource for genetic studies and provide a tool to discover genes of interest and genetic markers that will greatly aid questions involving evolution, ecology, and conservation using genetic and genomic approaches in the genus.

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Summary11 newly discovered microsatellites were used to identify SSR markers for characterising South American Nothofagus species. This was carried out in six species. The sample sizes used were between four and six individuals per species. The cross-genera transferability of 34 Quercus SSRs was also essayed. Out of the 11 new microsatellite markers, three proved to be polymorphic (NnBIO 11, NgBIO 13 and NgBIO 14). The qualitative confirmation of the inheritance of these markers could also be verified. Polymorphism was also observed in five of the cross-genera transferred SSRs (QrBIO7, quru-GA-0A01, quru-GA-0C11, quru-GA-0I01, quru-GA-0M07). The number of alleles per locus found range between 1 and 6 per species. The eight polymorphic SSRs identified in this study will constitute a valuable tool in the gene flow studies that are currently being carried out in natural populations of South American Nothofagus species. The confirmation of crossspecies and cross-genera transferability opens the way for the use of SSRs as bridge markers in genetic mapping.