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School of Plant Science and Cooperative Research Centre for Sustainable Production Forestry, University of Tasmania, GPO Box 252-55, Hobart, Tasmania 7001, Australia
Received for publication April 2, 1998. Accepted for publication November 10, 1998.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: chloroplast DNA • Eucalyptus • hybridization • Myrtaceae • reticulate evolution
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Hence, understanding eucalypt evolution is fundamental to understanding the evolution and ecology of a large component of the Australian biota. Eucalyptus is a complex genus of ancient origin (Ladiges, 1997
). While many taxa appear to be relictual, there is indication of recent speciation in certain groups (Pryor and Johnson, 1981
; Prober, Bell, and Moran, 1990
). Many species form complexes where extensive clinal variation between recognized taxa is common (Pryor and Johnson, 1971
; Potts and Wiltshire, 1997
) and reproductive barriers are weak (Potts and Wiltshire, 1997
). Whether this intergradation is a result of recent primary differentiation or due to secondary intergradation through hybridization is debatable (Pryor and Johnson, 1981
; Ladiges, 1997
). If hybridization (reticulate evolution) was a primary factor, then phylogenetic reconstruction may be questionable (McDade, 1992
; Rieseberg, 1995
), and this would challenge the foundation of current taxonomy. Chloroplast DNA variation has been useful for elucidating taxonomic relationships at higher taxonomic levels in the eucalypts (Sale et al., 1993
, 1996a
; Ladiges, Udovicic, and Drinnan, 1995
). In eucalypts, cpDNA is maternally inherited (Byrne, Moran, and Tibbits, 1993
). However, at lower taxonomic levels, a recent study of cpDNA in the subgenus Symphyomyrtus has shown marked discordance between cpDNA and species phylogeny (Steane et al., 1998
), suggestive of reticulate evolution.
The present study examines the utility of cpDNA in phylogenetic reconstruction in a group of closely related species in the subgenus Monocalyptus. Subgenus Monocalyptus contains more than 140 species that have been organized into one (Pryor and Johnson, 1971
) or two (Johnson, 1976
) sections, although at the morphological level, there are few obvious synapomorphic characters defining subgroups and no support for the two sections of Johnson (Ladiges, 1997
). This is clearly exemplified by instability in the taxonomic treatment of Monocalyptus species on the island of Tasmania (Table 1). Tasmania is a large island southeast of mainland Australia, but was linked by land bridges during Quaternary glacial epochs (Marginson and Ladiges, 1982
). Its flora displays a high level of endemism and relictual species (Kirkpatrick and Brown, 1984
), including eucalypts (Ladiges, Humphries, and Brooker, 1983
; Williams and Potts, 1996
). The six Tasmanian endemic species of subgenus Monocalyptus have been the subject of numerous evolutionary studies [reviewed in Williams and Potts (1996)
; Potts and Wiltshire (1997)
]. These species belong to the series Amygdalinae (Ladiges, Newnham, and Humphries, 1989
; Table 1), are often morphologically highly differentiated (Sale et al., 1996b
), but intergrade or hybridize in virtually all possible combinations (Williams and Potts, 1996
). The dynamics of hybridization between two of the most morphologically differentiated of these taxa, E. risdonii and E. amygdalina, has been extensively studied using morphological (Potts and Reid, 1988
; Whitham, Morrow, and Potts, 1994
) and RAPD (Sale et al., 1996b
) variation. The present study therefore aims to determine the level and pattern of variation in cpDNA within this group of endemic species and place it in a broader phylogenetic framework.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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showed that within subgenus Symphyomyrtus cpDNA can transgress species boundaries. Therefore, to be cautious, we choose outgroup taxa from other subgenera that were unlikely to share cpDNA with species of Monocalyptus. Specimens of E. globulus, 1 and 2, were identical to those used by Steane et al. (1998)
, in which they were designated GG1 and GG2, respectively. Specimens of E. lansdowneana and E. ceracea were identical to those used by Sale et al. (1996a)
, designated 1835B.K13 and 361/89, respectively. Care was taken to select individuals that were true to type.
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. Tissue (10 g) was ground to powder under liquid nitrogen using a mortar and pestle, then added to 50 mL extraction buffer (3.2% sorbitol, 5.0% polyethylene glycol 600, 0.05% bovine serum albumin, 0.05% spermine, 0.05% spermidine, 4% polyvinyl pyrrolidone 40, 0.05% 2-mercaptoethanol, 15 mmol/L EDTA, 50 mmol/L Tris, pH 8.0), on ice. The homogenate was filtered once through muslin, centrifuged (2000 g, 5 min), and the pellet was retained and suspended in 4 mL wash buffer (6.4% sorbitol, 0.1% 2-mercaptoethanol, 25 mmol/L EDTA, 50 mmol/L Tris, pH 8.0). The following reagents were added in order, with mixing: 1 mL of 5 mol/L NaCl, 0.8 mL of 8.6% hexadecyltrimethylammonium bromide/0.7 mol/L NaCl, and 1.6 mL of 5% N-laurylsarcosine. Samples were incubated at ambient temperature for 15 min, then at 55°C for 15 min, following which they were extracted twice with 8 mL chloroform : isoamyl alcohol (24:1), with mixing times of 30 min and 5 min, respectively, for the first and second extractions. The aqueous phase was separated from the organic phase by centrifugation (2000 g, 8 min). DNA was precipitated by addition of 6 mL ice-cold isopropanol and collected by centrifugation (2000 g, 5 min). The pellet was washed in 1 mL of 50% isopropanol/0.3 mol/L ammonium acetate for 30 min, air dried, resuspended in TE buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 7.4) containing RNase (10 µg/mL) and incubated at 37°C for 1 h. DNA was then reprecipitated with ethanol and 0.22 mol/L ammonium acetate, collected by spooling, washed in 70% ethanol, and stored at -20°C in TE buffer, pH 7.4.
DNA from each accession (1.5 µg per reaction) was digested with each of the following 19 restriction enzymes according to the manufacturers' instructions: Alu I, Ase I, BamH I, Ban II, Bcl I, Bgl II, BstN I, Dde I, Dra I, EcoR I, EcoR V, Eco0109 I, Hin d III, Hin f I, Nco I, Nsi I, Ssp I, Xho I, and Xmn I. Digested DNA was size fractionated by electrophoresis in 1.2% agarose for 
360 Vh, then transferred to nylon membrane by Southern blotting and cross-linking by exposure to UV radiation. Restriction fragments of phage lambda DNA cut with Hin d III were included as size markers. Nine chloroplast probes from Petunia (P1, P3, P4, P6, P10, P12, P14, P16, P20; Sytsma and Gottlieb, 1986
) and one from Nicotiana tabacum (pTBa1; Shinozaki et al., 1986
) were used. Probe DNA was labelled with 32P using random primers (T7 QuickPrime kit, AMRAD Pharmacia Biotech, Australia). Following prehybridization for up to 24 h in 1 L of hybridization solution (0.5% nonfat dried milk powder, 1% SDS, 0.6 mol/L NaCl, 0.06 mol/L trisodium citrate, pH 7.0), all blots were probed simultaneously for at least 18 h at 65°C in 300 mL of hybridization solution containing 400 ng cpDNA probe and 5 ng lambda probe. Blots were washed of excess probe with at least 750 mL of wash buffer (0.5% SDS, 0.3 mol/L NaCl, 0.03 mol/L trisodium citrate, pH 7.0) until background radiation dropped, and exposed to X-ray film (X-OMAT AR, Eastman Kodak Co., New York) at -80°C for up to 76 h with intensifying screens (Hyperscreen, Amersham, UK).
Data analysis
Autoradiographs were scored for presence or absence of restriction sites and for size mutations (where three or more enzymes indicated a conserved alteration in fragment size with a single probe). A data matrix was constructed comprising 78 characters across the 27 Eucalyptus accessions (Appendix). One percent of the matrix cells was scored as missing data. Where individuals had identical haplotypes, these were represented by a single individual to simplify cladistic analysis. Cladograms were generated by the parsimony software package PAUP version 3.1.1 (Swofford, 1993
) using Wagner parsimony. The condensed data matrix was analyzed using the exact branch-and-bound search option, with "Furthest" addition sequence, tree bisection-reconnection (TBR) branch swapping, and the save all minimal tree option (MULPARS) on. Bootstrap analysis was carried out using a heuristic search option, 1000 bootstrap replicates, TBR swapping, and 100 replicates of random addition sequence within each bootstrap replicate. Trees were rooted using E. ceracea from subgenus Eudesmia as the designated outgroup; replacing E. ceracea with various combinations of E. globulus, E. lansdowneana (both from subgenus Symphyomyrtus), and E. ceracea did not change the topology of the in-group (Monocalyptus). In addition, characters were mapped to the branches of an individual phylogenetic tree, enabling the identification of homoplasious characters.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and E. robertsonii (series Amygdalinae), both displayed a high degree of autapomorphy within this study. While single aberrations within the sampled individuals cannot be excluded, it seems more likely that these individuals represent monocalypt populations with distinctive cpDNA haplotypes. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1996a
). While some congruities between the cpDNA phylogeny and the morphologically based cladistic analysis of Ladiges, Humphries, and Brooker (1983)
were found at lower taxonomic levels, overall the cpDNA phylogeny within Monocalyptus was more indicative of the geographic proximity of the accessions than of published taxonomic relationships between species based on morphology (Table 1). Regardless of series, nine of the ten accessions of Monocalyptus sampled close to Hobart in southeastern Tasmania formed a clade comprising only two haplotypes (clade supported by character c5) for a total of seven species. This clade fell within a larger clade (clade 1) containing the remaining accession from the Hobart region (E. risdonii 2), four accessions from elsewhere in Tasmania, and two accessions from southwestern to southern central Victoria on the Australian mainland. The separation of clade 1 from clade 2, which contained only accessions from southeastern Victoria and New South Wales, also transcended taxonomic groupings. Overall, five out of seven species in which duplicate trees were sampled were polyphyletic in their cpDNA. This conforms exactly to the general pattern of variation in cpDNA observed by Steane et al. (1998)
in Symphyomyrtus series Viminales. There is clearly a high level of intraspecific variation in cpDNA in Eucalyptus species, and this is coupled with extensive sharing of related haplotypes by species in the same geographical area.
Little cpDNA variation was found in the Tasmanian Amygdalinae species compared to that found on the mainland of Australia. Recent divergence, whereby time since divergence is insufficient for numerous mutations to accumulate in the cpDNA, of some Tasmanian taxa may be part of the explanation. The Tasmanian Amygdalinae species are all endemic (Marginson and Ladiges, 1982
) and, despite marked morphological differentiation, speciation may not yet have occurred (Wiltshire, Potts, and Reid, 1992
). These results give little hope of finding species-specific cpDNA markers for the Tasmanian Amygdalinae. The Tasmanian Monocalyptus contained only cpDNA of clade 1, whereas mainland species had cpDNA of both clades 1 and 2. As E. dives from southern central Victoria appears to be basal to clade 1 (branch supported by one synapomorphic character), it is possible that the Tasmanian cpDNA haplotypes evolved from a common ancestral cpDNA found in that part of Victoria. This link may be indicative of the direction of colonization of Tasmania. The geographic separation of accessions with haplotypes from clades 1 and 2 and the lack of variation in the cpDNA of Tasmanian species may have been caused by a bottleneck event in Tasmania. This bottleneck could have occurred during a glacial event, when eucalypt forest cover was restricted in Tasmania (Kirkpatrick and Fowler, 1996
). Alternatively, there could have been a biogeographical barrier (e.g., geology, climate) across Victoria that prevented cpDNA of clade 2 from moving into western Victoria and Tasmania.
Three hypotheses may be presented to explain the overall lack of congruence between the cpDNA and species phylogenies: (1) convergent evolution of cpDNA or morphological species; (2) lineage sorting of cpDNA; and (3) hybridization and introgression (Soltis et al., 1991
; Steane et al., 1998
). Convergent evolution and lineage sorting could have played a role in the discordance between the species and cpDNA phylogenies, especially since there is evidence for recent divergence between species. Relatively recent speciation could result in morphologically different species possessing undifferentiated haplotypes. Likewise, haplotypes that differ by only a few characters may converge more easily than those differing by more characters. Recognition sites for restriction enzymes comprise several base pairs. Gain or loss of a restriction site can be achieved via changes in one or more bases of the recognition site. Thus, even if a restriction site is present or absent in two organisms, the process leading to the presence or absence may be different (convergent evolution) resulting in false homology. However, the potential for convergence in cpDNA decreases sharply as the number of character differences increases, hence this is unlikely to account for similarities between series. The observed results cannot be accounted for completely by lineage sorting and convergent evolution. For example, there is more divergence in cpDNA within E. risdonii (a highly localized endemic) than between E. risdonii and individuals from two different series (E. obliqua and E. delegatensis). These results are best explained by hybridization followed by introgression, possibly following the stepping stone model of Soltis et al. (1991)
. Furthermore, numerous characters separate samples of the two subspecies of E. willisii, a finding that is difficult to explain via the mechanisms of lineage sorting or convergent evolution of cpDNA, but that could be explained by introgression of cpDNA. However, the possibility that these subspecies may in fact be derived from two very divergent lineages that have converged in their adult morphology cannot be discounted. Certainly, the results of Newnham, Ladiges, and Whiffin (1986)
show as much differentiation in seedling morphology and volatile leaf oils between the two subspecies of E. willisii as they do between the two taxa E. willisii and E. pauciflora, from series Amygdalinae and series Psathroxyla, respectively.
The possibility of introgression of cpDNA from one lineage to another is partially supported by morphological evidence of hybridization. Within series, many species of Monocalyptus have been observed to hybridize and in some cases form extensive intergrade zones (Williams and Potts, 1996
; Wiltshire, Potts, and Reid, 1992
). It is therefore to be expected that species of the same series could share the same chloroplast genome. Although recorded, natural hybrids between series are much less common, and intergrade zones have not been observed (Potts and Reid, 1983
; Williams and Potts, 1996
). The observation that species from different series may share a common haplotype is therefore surprising. These data suggest that hybridization may be considerably more extensive and more significant in Eucalyptus than suspected previously. Further sampling will be necessary to confirm this indication. Interspecific hybridization and introgression were also strongly implicated in a recent study of cpDNA of species from subgenus Symphyomyrtus series Viminales (Steane et al., 1998
). These results led the authors to conclude that cpDNA may not be useful in phylogenetic reconstruction at low taxonomic levels within this subgenus. It is now possible to generalize this conclusion to Eucalyptus overall, since Symphyomyrtus and Monocalyptus together comprise the two most speciose subgenera within Eucalyptus. Nevertheless, as demonstrated in other plant species in Europe (Dumolin-Lapègue et al., 1997
) and North America (Soltis et al., 1997
), understanding the geographic pattern to cpDNA variation in Eucalyptus may be a useful source of information on past plant distributions in Australia.
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FOOTNOTES |
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2 Author for correspondence (e-mail: R.Vaillancourt@utas.edu.au).
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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