| Abstract |
- Plastid DNA restriction site and rearrangement mutations were used to study phylogenetic relationships among hard pines (subgenus Pinus). Total genomic DNA was cut with 19 restriction enzymes, blotted, and then probed mostly with 80% of the Douglas-Fir (Pseudotsuga menziesii) chioroplast genome (cpDNA) {initial study, using only 6bp recognition enzymes}; or, clones encompassing the entire chloroplast genome of Pinus contorta (Lidholm and
Gustafsson 1991) {final study, using only 4bp recognition enzymes}. A total of 202 mutations were recognized, 116 of which were phylogenetically informative, and subjected
to Wagner and Dollo bootstrap analyses. A number of clades were distinguished at the 95's significance level or above, revealing some important differences relative to the widely recognized taxonomy of Little and Critchfield (1969). The divisional differences are: 1) movement of subsection Sylvestres into the same monophyletic dade as
other Old World subsections (Pineae, Canariensis), subsection Sabiananae monophyletic with subsection Ponderosae, subsection Leiophylla in same monophyletic
dade as subsection Oocarpae, and, P. radiata in its own monotypic dade separate from subsection Oocarpae. Species reassignments include: P. teocote from subsection Ponderosae into Oocarpae dade, and, P. halepensis from subsection Sylvestres to be grouped monophyletically with P. pinea. Next, the 197 site mutations were used to
calculate percent sequence divergence among the species studied. Only results from the final study were discussed in detail, since the data from the initial study were
problematic. Overall rates of cpDNA evolution (1-2x10-10) in hard pines were approximately an order of magnitude lower than the value for angiosperms, but similar to values found in another long-lived woody plant taxa, Palms. Subsection Pineae {P. halepensis P. pinea} is hypothesized to be ancestral to the subgenus, arising sometime during the Late Jurassic-Early Cretaceous; while a Contortae-like progenitor is judged to be ancestral to New World taxa. In general, the divergence values give extensive insight into how major geological events shaped hard pine phylogeny. Compared to prior work, the use of restriction enzymes recognizing shorter sequences, thus
more frequently cutting, coupled with smaller, more homologous, probes have greatly increased resolution of the molecular phylogeny among hard pines. Future work in hard pine phylogeny should include more species, and the same probe-enzyme combinations, so that direct inferences can be made, while similar methods could be used to define relationships among the soft (subgenus Strobus) pines as well as other conifers. In any case, future studies
should attempt to obtain data in the form of concise mutations, and be expanded to include data from the nuclear and mitochondrial genomes. Also, chloroplast DNA (cpDNA) restriction maps were constructed for three hard (subgenus Pinus) pines: L
inea, P. sylvestres, and P. ponderosa; and compared with all other published Pinaceae cpDNA restriction maps. All restriction maps were approximately 120 Kb in size, and
lacking an inverted repeat (IR). Fragment patterns of hard pines versus soft (subgenus Strobus), and hard pines versus Douglas-Fir were very divergent. Gross structure among hard pines was well conserved, while the
microstructure (slO Kb) was much more variable. In particular, the fragment patterns from two separate events within the XbaI digest of P. sylvestres were reminiscent
of inversions, where a site gain/loss event replaces two medium sized fragments with one larger and one smaller band (total size is unaffected). This phylogenetic study
revealed 11 of these events, 9 of which occurred within probe X914 from P. contorta. To increase the robustness of our phylogenetic results and restriction mapping
studies, we decided to investigate the phenomenon within X914 more thoroughly by mapping the region with several enzymes. We uncovered three mini inversions of at least
1.8 Kb, 1.2 Kb and 0.9 Kb, that distinguish New World pines, subsection Contortae, and P. halepensis, respectively. Here the smallest known cpDNA inversions to date, in any species, and their phylogenetic implications in Pinus are reported.
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