Genetics of speciation in Mimulus
Todd Vision, Department of Biology, University of North Carolina at Chapel Hill, tjv@email.unc.edu
Overview
The plant genus Mimulus (monkeyflower) has been intensively studied by
evolutionary geneticists interested in species differences for over 50 years
(Vickery 1951). Recently, Quantitative Trait Locus (QTL) mapping studies
have identified loci underlying reproductive isolation in two different sections
of the genus. We are part of a research consortium that is working to identify
the genes that underlie these QTL using the tools of comparative genomics
and population genetics, and to understand the evolutionary history and
ecological significance of these loci.
Reproductive isolation via pollinator discrimination
Mimulus lewisii and M. cardinalis are closely related and highly interfertile
sister species with adjoining geographic distributions in the Sierra Nevada
Mountains of California. They show strong differences in pollination
syndrome and habitat preference, and hybrids are rare. M. lewisii is found at
higher elevations and is almost exclusively bee-pollinated. M. cardinalis is at
lower elevations and is pollinated by hummingbirds. Field studies in an area
of sympatry at Yosemite on segregating F2 progeny (Figure 1) have shown
that pollinators discriminate on the basis of petal size, pigmentation and
nectar volume (Schemske and Bradshaw 1999).
An F2 linkage map was generated using RAPD markers. QTL mapping
revealed that major genes underlie the majority of flower traits that distinguish
these species (Bradshaw et al. 1995, 1998). Substitution of the major
anthocyanin QTL (yup) from M. cardinalis into M. lewisii reduced bee
visitation by 80%. Similarly, an allelic substitution from M. lewisii into M.
cardinalis at the major
nectar QTL (NEC1) reduced
hummingbird visitation
by 50%. Thus, these two
loci together account for
much of the reproductive
isolation between the two
species. Nearly isogenic
lines (NIL) are being created
that “Mendelize” these loci
and make them amenable
to positional cloning.
Fig 1.M. lewisii (A), an F1 hybrid
(B), M. cardinalis (C), and
examples of variation in floral traits
found in F2 hybrids (D-L). From
Schemske and Bradshaw (1999).
Fig 3. QTL map for 7 floral traits in
526 M. guttatus x nasutus F2
progeny. Dashed line = 0.05
significance threshold. 24 minor QTL
were found on the 14 linkage groups.
From Fishman and Willis (2002).
Reproductive isolation via hybrid sterility
Species in the M. guttatus complex are exceptionally diverse in
habitat and mating system. Fishman and Willis (2001) have
mapped the QTL underlying phenotypic divergence and intrinsic
reproductive isolation between M. guttatus (the most common
species in the genus and a showy-flowered outcrosser) and M.
nasutus (a selfing species with small, often cleistogamous, flowers,
Figure 2) using AFLP and microsatellite markers. Differences in
floral morphology are underlain by many minor QTL. However, one
pair of epistatically interacting nuclear loci cause complete pollen
sterility in 1/8 of the hybrid F2 offspring, consistent with the classic
speciation model of Dobzhansky and Muller. An additional
cytonuclear incompatibility system causes complete male sterility in
¼ of offspring carrying the M. guttatus cytoplasm. Both
Dobzhansky-Muller factors and the nuclear restorer locus of the
cytonuclear incompatibility are being isolated in NIL.
Fig 2. M. nasutus growing in a mixed clump
with M. guttatus in Shirley Creek, Calaveras
Co, California. The red lines indicate M.
nasutus flowers (photo: Mark MacNair)
Genomic tools for positional cloning
Positional cloning of the QTL in these two species pairs will be
facilitated by generating genetic and physical maps that are anchored
by comparative mapping markers (see next section).
30,000 Expressed Sequence Tags (EST) are being generated from M.
guttatus. Alignment of these sequences against genomic DNA from
Arabidopsis thaliana allows us to design PCR primers that flank introns
in Mimulus. The resulting length and sequence polymorphisms are
being used to score 1000 markers (using fluorescent capillary
electrophoresis) in two mapping populations: 500 clonally propgated
F2 progeny from M. guttatus x nasutus and 500 Recombinant Inbred
Lines (RIL) from M. lewisii x cardinalis. From these data, we will
obtain high-resolution linkage maps (Vision et al 2000) that can be
aligned with physical maps for these species.
For physical mapping, two 11X Bacterial Artificial Chromosomes (BAC)
libraries have been constructed for M. guttatus (38K BAC with an
average insert size of 120kb) and a similar library is being constructed
for M. lewisii. These are being fingerprinted to generate a minimal
tiling path, the clones of which will then be end-sequenced. The
markers on the genetic map will then be localized to tiling path clones
using a pooled overgo mapping strategy. Clones found to contain QTL
will be shotgun sequenced in their entirety. In addition, transformation
protocols are being developed to allow transgenic testing of candidate
QTL.
Comparative mapping
The markers used to align the physical and genetic maps are homologous
to known genes, and so may be used to align the maps of Mimulus and
other genetic model species (particularly Arabidopsis thaliana and the more
closely related Lycopersicon esculentum, or tomato), for which genome
sequence is now or will soon be available. By comparison of conserved
chromosomal segments among these species using the FISH software
package and the Phytome database, both developed in our laboratory
(Calabrese et al. 2003), the gene content of a given region of Mimulus
chromosome can be predicted (Ku et al. 2000). These predictions will be
used to infer candidate genes and to design markers for fine-mapping.
Population genetic history of QTL regions
One currently popular strategy for fine-mapping QTL is to survey the
pattern of genetic variability and linkage disequilibrium along the
chromosomal region of interest (Schlotterer 1999). Population genetic
theory tells us that if a locus has been under directional selection, genetic
variability will be reduced and linkage disequilibrium will be locally strong
in the neighborhood of the causative polymorphism. From such data, we
may also infer of the strength of selection, the age of the mutation, and
the biogeographic history of the alleles, providing insight into “why” and
“how” such speciation genes arise. To obtain these data in Mimulus, we
will survey variable microsatellites obtained from BAC end-sequencing.
Literature Cited
Bradshaw HD, Wilbert SM, Otto KG, Schemske DW (1995) Nature 376:762-765
Bradshaw HD, Otto KG, Frewen BE, McKay JK, Schemske DW (1998) Genetics 149:367-382
Calabrese PP, Chakravarty S, Vision TJ (2003) Bioinformatics 19:i74-i80
Fishman L, Willis JH (2001) Evolution 55:1932-1942
Fishman L, Willis JH (2002) Evolution 56: 2138–2155
Ku H-M, Vision TJ, Liu J, Tanksley SD. (2000) PNAS 97:9121–9126
Schemske DW, Bradshaw HD (1999) PNAS 96: 11910-11915
Schlotterer C (2003) Trends in Genetics 19, 32-38
Vickery RK (1951) Carnegie Inst Wash. Yearb. 50:118-119
Vision TJ, Brown DG, Shmoys DB, Durrett RT, Tanksley SD (2000) Genetics155:407-420
Project Participants
Clemson University Genome Institute: Fred Tompkins
Duke University: John Willis, Fred Dietrich
Michigan State University: Doug Schemske
University of Montana: Lila Fishman
University of North Carolina at Chapel Hill: Todd Vision
University of Washington: Toby Bradshaw
Supported by the National Science Foundation (Frontiers in
Integrative Biological Research program), DBI-022731