Molecular cytogenetics and phylogenetic modeling to study chromosome evolution in the araceae and sex chromosomes in the cucurbitaceae

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.