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Sousa dos Santos, Aretuza (2014): Molecular cytogenetics and phylogenetic modeling to study chromosome evolution in the araceae and sex chromosomes in the cucurbitaceae. Dissertation, LMU München: Faculty of Biology



This study involved the combination of molecular-cytogenetic data and phylogenetic approaches to infer pathways by which chromosome numbers and sizes may have changed during the course of evolution. The two systems for which I generated new data are the monocot plant family Araceae and Coccinia, a genus of Cucurbitaceae. Araceae have about 3800 species in 118 genera, and chromosome numbers range from 2n = 168 to 2n = 8, the latter the lowest number so far and newly reported in my study. The small genus Coccinia includes C. grandis, with the largest known Y chromosome in plants, as documented in my work. The thesis comprises four published or submitted papers. The first paper reports the result of phylogenetic modeling of chromosome number change along a phylogeny for the Araceae with 113 genera represented. I used a maximum likelihood approach to find the most likely combination of events explaining today’s chromosome numbers in the 113 genera. The permitted events were chromosome gains (i.e. breaks), losses (i.e. fusions), doubling (polyploidization), or fusion of gametes with different ploidy. The best-fitting model inferred an ancestral haploid number of 16 or 18, higher than previously suggested numbers, a large role for chromosome fusion, and a limited role of polyploidization. The sparse taxon sampling and deep age (at least 120 Ma) of the events near the root of Araceae caution against placing too much weight on “ancestral” numbers, but inferred events in more closely related species can be tested with cytogenetic methods, which I did in two further studies (papers 2 and 3). I selected Typhonium, with 50-60 species, a range of 2n = 8 to 2n = 65 chromosomes. The family-wide study had suggested a reduction from a = 14 to 13 by fusion in Typhonium, but had included relatively few of its species. I built a phylogeny that included 96 species and subspecies sequenced for a nuclear and two chloroplast markers, and then selected 10 species with 2n = 8 to 24 on which to perform fluorescence in situ hybridization (FISH) with three chromosomal probes (5S rDNA, 45S rDNA, and Arabidopsis-like telomeres; paper 2). The results supported chromosome fusion in two species where I found interstitially located telomere repeats (ITRs), which can be a signal of end-to-end fusions, and polyploidization in one species where I found multiple rDNA sites. I then extended my cytological work to other lineages of Araceae, selecting 14 species from 11 genera in key positions in the family phylogeny, which I enlarged to 174 species, adding new chromosome counts and FISH data for 14 species with 2n = 14 to 2n = 60 (paper 3). With the new data, I confirmed descending dysploidy as common in the Araceae, and I found no correlation between the number of rDNA sites and ploidy level (which would have pointed to recent polyploidy). I detected ITRs in three further species, all with 2n = 30. I also discovered gymnosperms-like massive repeat amplification in Anthurium. Similar ITRs are only known from Pinus species. Paper 4 presents molecular-cytogenetic data for Coccinia grandis, one of a handful of angiosperms with heteromorphic sex chromosomes. The male/female C-value difference in this species is 0.09 pg or 10% of the total genome. My FISH and GISH results revealed that the Y chromosome is heterochromatic, similar to the Y chromosomes of Rumex acetosa, but different from the euchromatic Y chromosome of Silene latifolia; it is more than 2x larger than the largest other chromosome in the genome, making C. grandis an ideal system for sequencing and studying the molecular steps of sex chromosome differentiation in plants.