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.

Nothofagus is the main component of southern South American temperate forests. Overexploitation in the past has led to the loss of 40% of the original distribution range. Genetic diversity as well as biological processes shaping the distribution of the genetic variation (e.g. gene flow) constitutes basic knowledge for the implementation of conservation measures and for the definition of Evolutionary Significant Units. Nuclear microsatellites are the marker of choice for gene flow and fine-scale genetic structure studies. We enlarged a previous set of microsatellites (SSRs) for South American Nothofagus species, with special concern to Nothofagus nervosa (Phil.) Dim. et Mil. Five new SSRs are presented with allele numbers up to 12 in a single population. The primers transferred well to five related species (N. obliqua (Mirb.) Oerst, N. glauca (Phil.) Krasser, N. dombeyi (Mirb.) Oerst , N. pumilio (Poepp et Endl.) Krasser and N. antarctica (G. Forster) Oerst, with allele numbers up to 11. The high level of polymorphism promises a sufficient power for gene flow and parentage analyses.

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The northernmost 'robles' of Argentina, located in Lagunas de Epulauquen (northwest Patagonia), are considered a population of Nothofagus obliqua, even though they differ in some respects from individuals of other populations of the species. In order to analyse a possible genetic basis of their distinctive character, we revised previous information and added new evidence based on biochemical (isozyme) and molecular (chloroplast DNA and nuclear microsatellite) markers, as well as quantitative trait variation (seed traits, leaf morphology, plant architecture and field performance). The comparison of Lagunas de Epulauquen specimens with specimens from other areas in Argentina demonstrated their genetic distinction. Glacial history, in conjunction with hybridisation processes, provides support for the main hypothesis to explain these results. Future research lines are proposed, aimed at identifying the taxonomic status of the Lagunas de Epulauquen population. The conservation value of these forests is highlighted.Keywords: Nothofagus obliqua; Taxonomy; Patagonia; Glacial history; Interspecific hybridisation ResumenLos robles más septentrionales de la Argentina, ubicados en Lagunas de Epulauquen (noroeste de Patagonia) son considerados una población de la especie Nothofagus obliqua, pero presentan características diferenciales respecto al resto de las poblaciones argentinas de esa especie. El presente trabajo reúne información generada previamente y otra original que demuestra el carácter genéticamente distintivo de esa población. Se presentan los resultados logrados a través de análisis genéticos con marcadores bioquímicos (isoenzimas) y moleculares (de ADN de cloroplasto y marcadores de microsatélites nucleares), con caracteres cuantitativos seminales, de morfología foliar, de arquitectura de las plantas y de aptitud a campo. La historia glacial de la región, en conjunción con procesos de hibridación pasados, se postula como la causa de esta diferenciación. Se propone la realización de nuevas líneas de investigación que permitan confirmar o re-definir el estatus taxonómico de los árboles de esta población. Adicionalmente se presenta una breve consideración sobre el valor de conservación de los bosques en Lagunas de Epulauquen, en relación a los resultados aquí presentados.