We studied genetic polymorphism and phylogeny using nuclear random amplified polymorphic DNA markers (RAPDs) and mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) in the three California Closed-Cone Pines: Pinus attenuata Lemm., P. muricata D. Don, and P. radiata D. Don. A total of 343 to 384 trees derived from 13 populations were analyzed using 13 mitochondria' gene probes and two restriction enzymes, and more than 90 RAPD loci generated by 22 primers. Southern hybridization was used to test homology among comigrating RAPD markers. Segregation analysis and Southern hybridization were carried out to distinguish between RAPD fragments of nuclear and organellar origin. Estimates of genetic diversity and population differentiation, and phylogenetic analyses based on RAPD and RFLP markers, were compared with those based on allozymes from a similar study. Twenty-eight distinct mtDNA haplotypes were detected among the three species. All three species showed limited variability within populations, but strong differentiation among populations. Based on haplotype frequencies, genetic diversity within populations (Hs) averaged 0.22, and population differentiation (GsT and 0) exceeded 0.78. Analysis of molecular variance (AMOVA) also revealed that more than 90% of the variation resided among populations. Species and populations could be readily distinguished by unique haplotypes, often using the combination of only a few probes. Twenty-eight of 30 (93%) comigrating RAPD fragments tested were homologous by Southern hybridization. Hybridization with enriched mtDNA, and chloroplast DNA (cpDNA) clones, identified one fragment as being of mtDNA origin and two as being of cpDNA origin, among 142 RAPD fragments surveyed. RAPD markers revealed moderately higher intrapopulation gene diversity and significantly higher total genetic diversity and population differentiation than did allozyme markers for each species. Simulation analysis to study effects of dominance on RAPD diversity suggested that dominance substantially depressed values of diversity within populations and inflated values of differentiation among populations. By comparison to our empirical analyses, we inferred that the underlying diversity of RAPD markers is substantially greater than that of allozymes. Results of phylogenetic analysis of RAPD markers were largely consistent with those from allozyme analysis, though they had many minor differences. Joint phylogenetic analysis of both the RAPD and allozyme markers strongly supported a common ancestor for P. radiata and P. attenuata, and south to north migration histories for all three species. Dendrograms based on mtDNA analysis, however, strongly disagreed with those based on allozymes, RAPDs, chloroplast DNA and morphological traits, suggesting convergent genome evolution. / Graduation date: 1999

Data from nuclear ribosomal internal transcribed spacer regions (nrITS) and chloroplast DNA (cpDNA) have failed to resolve phylogenetic relationships in Pinus. To provide greater interspecific resolution, five low-copy nuclear genes were developed from mapped conifer anchor loci. Four genes were sequenced from species representing all Pinus subsections. Individual loci do not uniformly support the nrITS or cpDNA hypotheses. Combined analysis of low-copy nuclear loci produces a well-supported subsectional topology. The phylogenetic positions of P. nelsonii and P. krempfii are of systematic interest. Results strongly support P. nelsonii as sister to sect. Parrya, and suggest a moderately well-supported position of P. krempfii as sister to the remaining sect. Quinquefoliae. The most informative locus, a Late Embryogenesis Abundant-like gene, was used to explore phylogenetic relationships among closely related species in subg. Strobus. Thirty-nine species were sequenced, with two or more alleles representing 33 species. Nineteen of 33 species exhibited allelic nonmonophyly in the strict consensus tree. Nucleotide diversity was strongly associated (P<0.0001) with the degree of species monophyly. While species nonmonophyly complicates phylogenetic interpretations, this locus offers greater topological support than cpDNA or nrITS. Lacking evidence for hybridization, recombination, or imperfect taxonomy, incomplete lineage sorting remains the best explanation for trans-species polymorphisms. The absence of allelic coalescence is a severe constraint in the application of phylogenetic methods in Pinus, and taxa sharing similar life history traits may show analogous patterns. While lack of coalescence may limit their utility in traditional phylogenetics, nuclear genes remain highly informative in describing speciation events. Pinus chiapensis is a threatened species originally described as a variety of P. strobus. Prior morphological work suggests P. chiapensis is a distinct species, but that taxonomy is not universally accepted. Multiple accessions of three probable progenitors were sequenced at three nuclear loci. No interspecific allele sharing occurs with P. chiapensis, and its alleles are monophyletic at two loci. Results demonstrate that P. chiapensis is a distinct species. However, determination of the sister species is complicated by lack of species monophyly and interlocus variability. Pinus ayacahuite is the least likely progenitor, but the relationship of P. chiapensis to P. monticola or P. strobus is unclear. / Graduation date: 2006