We sampled fresh leaf material for cpDNA analyses from 330 individuals of 200 populations of the five extant species of the subgenus Nothofagus (N. antarctica, N. betuloides, N. dombeyi, N. nitida and N. pumilio) along their entire range (Table 1, Fig. 3). We collected nine other Nothofagus species belonging to the subgenera Lophozonia and Fuscospora, as well as one individual of Betula pendula, to be used as outgroups. Voucher specimens were deposited in the herbarium of Centro Regional Universitario Bariloche, Universidad Nacional del Comahue, Argentina (BCRU). DNA extraction was performed using the DNeasy Plant Mini Kit (Qiagen) following the manufacturer’s instructions, and concentrations were assessed by electrophoresis on agarose gels and comparisons with a 1-kb DNA ladder (Fermentas). Reactions with 1–2 μl of DNA extract (c. 10 ng) and 4–6 μl of GeneReleaser® (BioVentures, Murfreesboro, Tennessee, USA) were performed prior to PCR, which segregate the inhibitors released during lysis, together with preservation agents that may interfere with amplification to facilitate DNA release (conditions: 15 min at 85°C, hold at 32°C). Noncoding regions of cpDNA were amplified by PCR using three universal primer pairs: psbB–psbH (BH) (Hamilton, 1999), trnL–trnF (LF) (Taberlet et al., 1991) and trnH–psbA (HA) (Hamilton, 1999). The PCR mix contained 10 ng DNA, 5 μl 5 × Green GoTaq® Reaction Buffer (Promega, Madison, WI, USA), 0.25 mM of each deoxynucleoside triphosphate (dNTP), 0.3 μM of each primer and 1.25 U of GoTaq® DNA polymerase (Promega) in a total volume of 25 μl (conditions: 4 min at 95°C; 35 cycles of 1 min at 94°C, 1 min annealing at 57°C for BH, 54°C for LF and 56°C for HA, and 1.5 min at 72°C; and a final 6 min at 72°C). In addition, the complete ITS region, including the 5.8S rRNA gene, was amplified using the primers CY1 and CY3 (Wright et al., 2006). We sequenced only one individual from each species for the ITS region as a result of the highly conserved DNA sequences of this nuclear region for any given species (Acosta & Premoli, 2010). The PCR mix contained 1 μl of template DNA (10 ng), 0.625 U GoTaq® DNA polymerase (Promega), 5 μl 5 × Green GoTaq® Reaction Buffer (Promega), 0.25 mM of each dNTP and 0.3 μM of each primer in a total volume of 25 μl. The PCR cycling scheme was 4 min at 95°C; 30 cycles of 30 s at 94°C, 1 min at 56°C and 2 min at 72°C; a 10-min extension at 72°C; and a final hold at 15°C. The PCR products were purified using 2.5 U of Exonuclease I (USB) and 0.25 U of shrimp alkaline phosphatase (USB, Santa Clara, California, USA) for 10 μl of PCR product (conditions: 15 min at 37°C, 15 min at 85°C). The amplified DNA was sequenced with an ABI PRISM 3100 Avant Genetic Analyzer (Applied Biosystems) at Universidad Nacional del Comahue using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The cycle sequencing reactions were performed following the manufacturer’s protocols. The amplified regions were sequenced in both directions for at least one individual from each population and/or species. Sequences of each sampled cpDNA haplotype and nuclear ITS ribotype were deposited in GenBank (Accession Numbers: BH: GQ863274–GQ863275, GQ863285–GQ863286, GQ863367–GQ863370, GQ863397–GQ863399, GQ863401–GQ863402, GQ863405, GU152886–GU152887, GU152889, GU152891–GU152893, JN247414–JN247418; LF: GQ863302–GQ863303, GQ863313–GQ863314, GQ863371–GQ863374, GQ863379–GQ863381, GQ863383–GQ863384, GQ863387, GU152870–GU152871, GU152873, GU152875–GU152877, JN247419–JN247423; HA: GQ863330–GQ863331, GQ863341–GQ863342, GQ863375–GQ863378, GQ863388–GQ863390, GQ863392–GQ863393, GQ863396, GU152878–GU152879, GU152881, GU152883–GU152885, JN247424–JN247428; ITS: GQ863229–GQ863232, GQ863237–GQ863238, GQ863240, GQ863265–GQ863268, JN247411–JN247413). Sequences for ITS of N. alessandri were obtained from GenBank (Accession Number: U96854). Sequencing data for all regions were aligned with MEGA4 (Tamura et al., 2007) with gap additions where needed, and concatenated manually into a single combined dataset for the analyses. The cpDNA analysis resulted in a total of 15 haplotypes which were shared between at least two of the five species within subgenus Nothofagus.