Systematics and Evolution of Amelanchier (Rosaceae)

Polyploidy, which is tightly linked to apomixis, leads to taxonomic complexity in Amelanchier. To understand this complexity requires discovery and systematic study of ploidy-level variation and reproductive mode (sexuality or apomixis). We used flow cytometry to estimate genome size for 581 plants covering a broad taxonomic diversity within the genus. To infer reproductive mode, we used flow cytometric DNA measurements of the embryo and endosperm of mature seeds. We documented diploidy in 199 plants, including the first diploid estimates for six species and two entities currently not formally named. For diploid plants we recorded a diploid embryo and triploid endosperm in all seeds except two, which had a 2x embryo and 8x endosperm. We infer that this endosperm indicates apomixis, the first report of such at the diploid level in the genus. We documented 44 triploids and the first apomictic triploids in Amelanchier, with 3x embryos and 8x, l0x, or 12x endosperm. We recovered four potential pathways for the triploid bridge, suggesting an important role for triploids in polyploid evolution in Amelanchier. We report tetraploidy in 338 plants, including the first tetraploid estimates for numerous taxa. All tetraploids had 4x embryos, and nearly all had 12x endosperm. This endosperm ploidy is consistent with apomixis and fertilization of the central cell by two sperm. In four seeds, endosperm was 6x, the expected ploidy of sexual reproduction in tetraploids. The average frequency of apomixis in diploid, triploid, and tetraploid plants was 1.89%, 80.43%, and 98.63%, respectively. We estimated the average frequency of apomixis of two plants of A. laevis to be of 99.09%. These two plants are siblings derived from selfing one individual in which we estimated a frequency of apomixis of 99.2% based on RAPD analysis of a progeny study. We found no evidence for genome downsizing in polyploids, perhaps because apomixis in Amelanchier prevents genome downsizing. It has long been appreciated that understanding diploids creates a context for untangling the evolution and systematics of polyploid agamic complexes like Amelanchier. We sampled 10 diploids, five of which are eastern North America, two western North America, two western Eurasia, and one eastern Asia. DNA sequences from nrDNA ETS and ITS and the nuclear gene LEAFY were used for phylogeny reconstruction. Morphometric, multivariate analyses tested the null hypothesis of no discrete groups. Diploid Amelanchier species are morphologically, ecogeographically, and (in almost all cases) genetically cohesive. Genetic data support a well resolved diploid phylogeny congruent with morphological results. We report five diploid hybrids, four of which we found as single individuals. We confirm the presence of LEAFY paralogs, AFL1 and AFL2, previously reported in closely related Malus. In six species, we found strongly divergent AFL2 sequence variants, some of which we infer to be gene duplications. In stark contrast to taxonomically complex polyploid Amelanchier species, our morphological and genetic data reveal that diploid species form discrete taxa that would be accepted as species by most species concepts. Amelanchier diploid species hybrids may be important as first steps toward polyploidy. Polyploid apomicts in Amelanchier frequently form taxa that are weakly differentiated, narrowly distributed, and referred to as microspecies. To develop a tractable taxonomy of the polyploid apomicts in the Amelanchier sanguinea complex requires an appreciation of their morphology, geographic distribution, and evolutionary history. We used a phylogeny of eastern North American diploid Amelanchier species nrDNA ETS and ITS sequences as a framework for exploring evolutionary relationships of four tetraploid apomicts in the A. sanguinea complex. Morphometric, multivariate analyses tested the null hypothesis of no discrete groups. Each polyploid apomict contains nrDNA ribotypes that nest within two clades of diploid species that are strongly divergent genetically and morphologically. Morphological data provide limited resolution and do not recover taxa within the A. sanguinea complex as discrete groups. The polyploids studied here have complex hybrid origins and form a nearly continuous gradient of morphological variation. Apomixis and the attendant reduction in genetic recombination may have maintained some of the evolutionary history of these polyploids in their DNA sequences