Phylogenetic reconstruction of fungal sexchromosome evolution in Microbotryum'

1
Phylogenetic reconstruction of fungal
sex-chromosome evolution in Microbotryum.
Michael E. Hood and Jessie L. AbbateDept. of
Biology, University of Virginiamichael.hood_at_virgi
nia.edu, http//www.people.virginia.edu/meh2s/web
site/MEHood.htm
Abstract The haploid-mating fungus Microbotryum
violaceum serves as a model for the evolution of
dimorphic sex chromosomes for comparison with
canonical patterns of X-Y divergence in
diploid-mating animals and plants1. In this
study, we reconstructed the phylogenetic
histories of the alternate fungal sex chromosomes
using a locus linked to the mating-types (A1 or
A2) in Microbotyryum from Silene latifolia. The
resulting phylogeny included 1) separate clades
where A1 and A2-linked sequences appear to have
ceased recombination and diverged 2) clades
where there is little to no difference between
the A1 and A2 sequences, indicative of continued
recombination and 3) a paradoxical clade, again
with no differentiation between the A1 and A2
sequences, but with identity among fungal samples
expected to be highly divergent. As recently
found in some diploid-mating organisms, these
results suggest that locus has come into linkage
with the non-recombining regions of the fungal
sex chromosomes more than once, and that
subsequent differentiation can occur
independently in related lineages.
Fig 3. Phylogeny of A1 and A2 Mating Types

• Methods
• Samples of the parasitic fungus Microbotryum
  were isolated from the host genera Silene,
  Saponaria, and Dianthus in Europe and the US
  (Figs. 1).
• Haploid meiotic products of contrasting mating
  types (A1 and A2) were obtained by the isolation
  of linear tetrads using micro-manipulation.
• Baseline relatedness among samples was estimated
  using nuclear ITS and gamma-tubulin sequences
  (Fig. 2).
• We obtained the mating-type linked sequence from
  a genomic shotgun survey with sex
  chromosome-specific DNA1. It had a BLAST match
  to a sex-inducing pheromone in Volvox. (ca. 400
  bp)

S. nutans FRANCE 9
S. vulgaris ITALY 3
66
S. vulgaris ITALY 3
55
79
S. flos cuculi UK 3
S. nutans FRANCE 9
66
S. flos cuculi UK 3
S. latifolia USA 3
S. latifolia UK 2
28
S. latifolia CZECH REP
A1
S. latifolia ITALY 4
S. latifolia ITALY 2
68
S. latifolia FRANCE 5
S. dioica UK 1
94
S. dioica FRANCE 1
S. latifolia USA 3

S. latifolia ITALY 2
S. dioica FRANCE 1
A2
81
S. latifolia ITALY 4
S. latifolia FRANCE 5

• Results
• Sex chromosome sequences of Microbotryum from S.
  latifolia and Dianthus spp. were in complete
  linkage disequilibrium with the A1 and A2 mating
  types.
• In contrast, for the other clades the sequences
  had much lower levels of differentiation by
  mating type.
• The amount of variation among populations within
  a clade (Fig 3) reflects that previously
  observed background relatedness (Fig 2).
• There is an unexpected identity for the
  sequences from the North American and some
  European lineages, which are known to be highly
  divergent based upon other studies.

Fig 1. Microbotryum on Diseased Hosts
S. latifolia UK 2
S. dioica UK 1
S. latifolia CZECH REP
43

S. acaulis FRANCE 6
S. acaulis FRANCE 6
93
95
S. acualis ITALY 1
20
S. acaulis FRANCE 7
A1 A2
58
S. acaulis ITALY 1
S. acaulis FRANCE 7
25
S. acaulis FRANCE 3
71
S. acualis FRANCE 3
S. maritima UK 1
S. maritima UK 1
S. vulgaris SWIZ 1
S. vulgaris SWIZ 1
S. vulgaris FRANCE 10
A1 A2
S. vulgaris FRANCE 10
S. rupestris FRANCE 8

• Discussion
• These results suggest that the sequenced locus
  has come into the non-recombining regions of the
  fungal sex chromosomes multiple times since the
  divergence of Microbotryum on different host
  genera. Also, subsequent differentiation can
  occur independently in sister lineages (i.e. the
  apparently non-recombining in samples from
  Dianthus hosts, but recombining from Saponaria
  hosts).
• These results are qualitatively similar to those
  for birds2 and mammals3, and suggest that
  non-recombining regions of sex chromosomes are
  very dynamic and may be subject to similar
  evolutionary forces across the broadest diversity
  of eukaryotes.
• In clades where A1 and A2 sequences are not
  differentiated, it is unclear whether the locus
  is recombining on the fungal sex chromosomes or
  resides on an autosome. To distinguish between
  these possibilities, we are sequencing flanking
  regions and probing karyotypes (i.e. whether the
  patterns of differentiation are determined by an
  expanding and contracting region of
  non-recombination on the sex chromosomes versus
  translocations involving autosomes).
• The incongruence between the expected baseline
  relatedness and mating type linked sequence for
  the clade containing samples native to the US
  (from S. virginica and S. caroliniana) cannot be
  explained by recombination, and may reflect an
  unusual degree of DNA sequence conservation.

S. rupestris FRANCE 8
99
A1 A2
S. caroliniana USA 3
S. caroliniana USA 3
S. virginica USA 2
S. virginica USA 2
S. virginica USA 1
S. virginica USA 1
Sap. officinalis FRANCE 4

Sap. ocymoides FRANCE 4
Sap. officinalis ITALY 7
Sap. ocymoides ITALY 7
A1 A2
Sap. ocymoides ITALY 6
Sap. officinalis ITALY 6
Sap. officinalis ITALY 5
Sap. ocymoides ITALY 5
D. monspessulanus FRANCE 10
D. neglectus ITALY 1
95
A2
D. alpinus ITALY 8
06
D. carthusianorum FRANCE 11
Fig 2. Nuclear ITS Sequence Data
98
D. sylvestris FRANCE 2
D. sylvestris ITALY 6
69
D. monspessulanus FRANCE 10
92
81
D. neglectus ITALY 1
D. alpinus ITALY 8
A1
96
D. sylvestris ITALY 6
0.01
D. carthusianorum FRANCE 11
99
D. sylvestris FRANCE 2
Figure Legends Fig. 1. Images of host flowers
showing anther-smut disease, caused by the fungus
Microbotryum. Fig. 2. ITS sequence phylogeny
(congruent with data from nuclear gamma-tubulin
and mitochondrial NADH1) includes outgroup.
Neighbor-joining method with 100 bootstrap
replication. Note native US samples distant from
European clade. Fig. 3. Phylogeny of sex
chromosome linked sequences for A1 (closed
squares, ) and A2 (open squares, ?) mating
types. Neighbor-joining method with 100 bootstrap
replication.
References 1. Hood, M. E., Antonovics, J., and
Koskella, B. 2004. Shared forces of sex
chromosome evolution in haploid-mating and
diploid-mating organisms. Genetics (in press). 2.
Ellegren, H. and Carmichael. A. 2001. Multiple
and independent cessation of recombination
between avian sex chromosomes. Genetics
158325-331. 3. Marais, G. and Galtier, N. 2003.
Sex chromosomes how X-Y recombination stops.
Current Biology 13R641-R643.