Nothofagus cunninghamii (Hook.) Oerst, or myrtle beech, is a long-lived, evergreen, monoecious, wind-pollinated tree reaching 50 m in height (Curtis, 1967), but at the altitudinal maximum of its range may be reduced to a compact shrub under 50 cm. The species is remarkably uniform in morphology across its range apart from variation in leaf size, which is strongly correlated with summer temperatures (Jordan & Hill, 1994). Seed is gravity-dispersed, generally one tree height from the mother tree (Howard, 1973; Hickey et al., 1982; Tabor et al., 2007), but may be dispersed downstream in water courses (Howard, 1973). The species’ distribution is broken by some major dry land and sea barriers (Howard & Ashton, 1973; Fig. 1). Across its range, this species is confined to cool, humid climates where rainfall exceeds c. 1000 mm yr−1 with at least 50 mm rainfall during the driest month (Jackson, 1965; Busby, 1986; Lindenmayer et al., 2000). The small populations outside this climatic range (e.g. Yarlington Tier; Fig. 1) are all special topographic sites with precipitation supplemented by ground water and/or cloud stripping (Harle et al., 1993). Nothofagus cunninghamii does not currently co-occur with any other species of its subgenus, Lophozonia. Its sister species, N. moorei, is restricted to mountain ranges of northern New South Wales and southern Queensland (Busby, 1986), c. 780 km north of the northernmost population of N. cunninghamii. The western Tasmanian endemic, N. gunnii (subgenus Fuscospora), sometimes co-occurs with N. cunninghamii but the two species do not hybridize (Hill & Read, 1991).
Fresh leaves were collected from 342 adult trees (327 stands) of N. cunninghamii in natural populations, including nearly all known parts of the species’ distribution, apart from some remote parts of western Tasmania. Only one tree was sampled from most stands, but up to three widely spaced individuals were sampled at some locations, including some very isolated populations (see Supporting Information, Tables S1 and S2, for list of sample locations). Latitude, longitude and altitude information were recorded for each sample collected. Tree form was noted. Five individuals of N. moorei were also sampled from the northern (Lamington National Park, Springbrook National Park and Bar Mountain) and southern extremes of this species’ range (two samples from Barrington Tops National Park c. 490 km south of the northern populations of this species). For use as outgroups, leaf samples of the New Zealand endemic N. menziesii and the Chilean species N. glauca were obtained from the Royal Tasmanian Botanical Gardens, Hobart, Tasmania. Phylogenies based on morphology, nuclear DNA and chloroplast DNA each indicate that N. moorei is the sister of N. cunninghamii, that N. menziesii is sister of this clade and that the resulting clade is sister to a small clade containing N. glauca Manos (1997). These samples were analysed in two groups: a range-wide survey whereby a haplotype phylogeny was created from chloroplast sequence and PCR-RFLP data using 213 N. cunninghamii samples from across the distribution of the species and all outgroups; and a fine-scale study of haplotype distribution that used three PCR-RFLP characters and screened 149 samples from the northeast highlands of Tasmania (including 20 samples used in the previous study). For this study, individual trees were sampled a minimum of c. 1 km apart.
Total genomic DNA was extracted from 1 g of adult leaves, following the CTAB protocol of Doyle & Doyle (1990). DNA concentration and purity were assessed using agarose gel electrophoresis with ethidium bromide staining and comparison with a standard molecular weight marker (Lambda HindIII). DNA concentration was standardized at 5 ng µl−1.
Sixteen regions of cpDNA were amplified using universal primers (trnD–trnT, trnS–trnfM, trnK–trnQ, rpoC1–trnC, trnV–rbcL, rpl23–psbA3, atpH–atpI, atpI–rpoC2, rpoC2-f–rpoC2-r, orf184–petA, petA-f–psbE-r, clpp–psbB, psbB–petB, petB–petD, trnH–trnK and trnK–trnK) (Demesure et al., 1995; Dumolin-Lapégue et al., 1997b; Grivet et al., 2001). All PCR reactions were performed in a total volume of 25 µl containing 2.5 mm MgCl2; 100 µg ml−1 bovine serum albumin; 80 µm each of dATP, dCTP, dGTP and dTTP; 5 pm of each primer; 1 × PCR buffer (67 mm Tris-HCl, 16.6 mm (NH4)2 SO4, 0.5% Triton X-100 and 5 µg of gelatin); two units of Taq DNA polymerase; and c. 10 ng of genomic DNA. PCR amplification was performed by a MJ Research PTC-225 Tetrad thermocycler (GMI, Inc., Ramsey, MN, USA) as follows: an initial melt of 4 min at 94°C; 30 cycles of 45 s at 92°C, 45 s at annealing temperature (see Table S3 for details of annealing temperatures of each fragment), 4 min at 72°C; and a final extension for 10 min at 72°C. PCR products were digested with a range of restriction enzymes in a total reaction volume of 20 µl containing 5–10 µl of PCR product. The products of the restriction digests were size-fractionated in a 2.2% agarose gel in TBE at 100 V for 90 min. Restriction fragment length polymorphisms (RFLPs) were identified visually by comparing restriction fragment patterns between samples.
Screening of eight N. cunninghamii samples (representing all major parts of the species range) and 150 fragment/restriction endonuclease combinations (using TaqI, HinfI, AluI, DpnII, HaeIII, Hinp1I, RsaI, DdeI, MspI, HphI, NcoI, SspI, AseI, StyI, NciI, DraI, ClaI, BstUI, EcoRV) revealed one RFLP. Screening of 48 or 72 samples and 16 fragment/restriction endonuclease combinations detected two additional RFLPs. All samples for the range-wide survey were screened for the three fragment/restriction endonuclease combinations that detected polymorphisms.
Owing to the paucity of cpDNA variation detected using PCR/RFLP, cpDNA fragments of all samples for the range-wide survey were sequenced. These were partial sequences of the intergenic spacer regions petN1–psbM2R, psbM2–trnD (Lee & Wen, 2004), trnS–trnfM (Demesure et al., 1995), and trnL–trnF (Taberlet et al., 1991) and the intron between rps16/1 F–rps16/1 R (Nishizawa & Watano, 2000). PCR conditions were as follows: petN1–psbM2R and psbM2–trnD, denaturation for 4 min at 94°C, followed by 35 cycles of 1 min at 94°C, 2 min at 50°C, and 2 min at 72°C; trnS–trnfM, denaturation for 4 min at 94°C, followed by 30 cycles of 45 s at 92°C, 45 s at 62°C, and 4 min at 72°C; trnL–trnF, denaturation for 1 min at 95°C, followed by 35 cycles of 1 min at 95°C, 1 min at 50°C and 45 s at 72°C; rsp16/1 F–rsp16/1 R, denaturation for 3 min at 95°C, followed by 25 cycles of 1 min at 94°C, 1 min at 55°C and 1 min at 72°C. All had a final extension for 10 min at 72°C, except trnL–trnF, which had a final extension of 7 min at 72°C. Before sequencing, PCR products were purified using the Qia-Quick PCR purification kit (Qiagen Pty Ltd, Doncaster, Victoria, Australia). Sequencing reactions were performed using a Beckman Coulter Quick Start Kit following a modified protocol using 0.64 µl of 5 µm primer, and 6 µl of purified PCR product in a final volume of 10 µl. Sequence reactions were analysed using a Beckman Coulter CEQ 2000 automated sequencer (Beckman Coulter, Inc., Fullerton,CA, USA). Polymorphisms detected in only one sequencing reaction were checked by repeating the PCR and the sequencing reaction. In all cases where unexpected haplotype distributions were found (e.g. N. moorei), sequences and PCR-RFLP analyses were repeated, and samples rechecked.
For the fine-scale study of northeast Tasmanian haplotypes, restriction endonucleases that would enable the quick and easy identification of individuals carrying either the C1 or NE1 haplotypes (see later) were identified using NEBcutter V2.0 (http://tools.neb.com/NEBcutter2/index.php). The restriction endonuclease HaeIII cut the trnL–trnF fragment of C1 individuals once, and zero times in NE1 individuals as a result of a 17 base pair (bp) deletion. The endonuclease Hpy188III was found to distinguish the psbM2–trnD fragment of individuals carrying NE1 haplotype as a result of an extra restriction site in this haplotype. For this analysis a new internal reverse primer was developed (5′ ... CCGGGACTCGTCTTTATCATACTTC ... 3′) that amplified a cpDNA fragment c. 540 bp in length compared with the original c. 1200 bp fragment. This allowed better separation of the polymorphic fragments that differed by 77 bp in length between C1 and NE1 haplotypes. All 149 samples were screened with these two new endonuclease/fragment combinations and the previously identified atpI–rpoC2 fragment/TaqI combination.
Phylogenetic relationships of haplotypes
Evolutionary relationships between haplotypes (including N. glauca, N. menziesii and N. moorei) were investigated by maximum parsimony analysis undertaken using the program PAUP* version 4.0b10 (Swofford, 2000). PCR-RFLP polymorphisms, single nucleotide polymorphisms and insertions/deletions were scored as binary characters, except for two indel variants (characters 1 and 2; Table 1), which were scored as a multi-state character with three states. In addition, parallel variation was seen in two adjacent base pairs (a doublet). This was treated as a single character (character 20, Table 1). Parsimony analysis was undertaken using a heuristic search with 1000 replicates of stepwise, random branch swapping addition sequence followed by tree-bisection-reconnection (TBR). As a result of significant levels of homoplasy of some characters (in particular, characters 19 and 20; Table 1), all characters were reweighted iteratively by the maximum value of their rescaled consistency index (Farris, 1969). A strict consensus of all the shortest trees found using this procedure was constructed. Branch support was assessed by bootstrap analysis (Felsenstein, 1985) with 1000 heuristic search pseudo-replicates using the same search parameters as those in the parsimony analysis. Nothofagus glauca and N. menziesii were used as outgroups in all searches based on the topologies identified by (Manos, 1997).
Table 1. Nothofagus cunninghamii haplotypes (and the single N. moorei haplotype) with cpDNA sequence and PCR/RFLP characters shown in comparison to the N. cunninghamii C1 haplotype
Spatial clustering and regional haplotype diversity
Within the 213 samples collected across the range of N. cunninghamii, the spatial structuring of haplotypes was investigated. The single nearest geographic neighbour for each sample was determined using a specially written macro in SAS 9.1 (SAS Institute Inc., Cary, NC, USA). This program also performed a permutation test (Manly, 1997) with 10 000 randomized repeats testing whether the nearest neighbour of each sample was more often of the same haplotype than expected by chance (a proxy of spatial structure). This was carried out across all samples and within each region (Victoria, eastern Tasmania and western Tasmania). This procedure was also applied to the 149-sample set from northeast Tasmania in the fine-scale study.
Haplotype diversity was also compared across these regions through rarefaction analysis (Simberloff, 1979). The three regions were randomly subsampled to the size of the least sampled region (N = 26) 10 000 times. Differences in haplotype richness were then tested using a permutation test (Manly, 1997) with 10 000 randomized repeats, programmed in SAS 9.1 (SAS Institute Inc.). Similar analyses were performed comparing low-, medium- and high-altitude samples in western Tasmania, rarefying each to 43 samples. Low altitude was defined as < 150 m above sea level (m asl), the estimated treeline during the Last Glacial Maximum (Colhoun, 1985; Gibson et al., 1987). High altitude was defined as > 500 m asl, which divided the remaining samples approximately evenly and is far above any plausible estimate of LGM tree lines given that all palaeoclimatic estimates show LGM temperatures at least 5°C lower than present (Galloway, 1965; Colhoun, 1985, 2000).