Molecular Markers Show How Pollen and
Seed Dispersal Affect Population Genetic
Structure in Coast Live Oak (Quercus
agrifolia Née)1
Richard S. Dodd,2 Zara Afzal-Rafii,2 and Wasima Mayer2
Abstract
Coast live oak (Quercus agrifolia Née) occurs in the coast range mountains from southern
Mendocino County, California, to the Sierra San Pedro Martir, Baja California, Mexico. In
Northern and Central California, coast live oak is suffering heavy mortality as a result of
infection by Phytophthora ramorum. Earlier studies indicated biochemical differentiation of
central coast populations from those of Northern and Southern California, an important hybrid
zone in Northern California. A possible differential response to inoculation between
populations from Northern and Southern California has also been reported. Here, we report
results of a rangewide genetic diversity study of this species using chloroplast and nuclear
microsatellite markers. The chloroplast genome is inherited maternally and its dispersal is
limited because of the relatively large heavy seeds of oaks. We analyzed chloroplast
microsatellites in more than 500 individuals from 41 populations and nuclear microsatellites
in about 500 individuals from 28 populations of coast live oak from throughout its range.
Based upon the chloroplast haplotype distributions, at least four biogeographic groups were
detected. Nuclear microsatellite markers revealed reduced levels of genetic structure as a
result of pollen dispersal. We discuss the roles of seed and pollen dispersal in the evolution of
populations of coast live oak and how this information may help in developing strategies for
studies of resistance to the sudden oak death disease.
Keywords: Biogeography, chloroplast DNA, genetic structure, pollen, Quercus, seed.
Introduction
Post-European settlement of California has brought substantial and rapid changes to
the ecology of oak woodlands. Early Spanish settlement introduced large-scale
ranching that reduced continuous stands to pockets of residual woodland, particularly
in the inland valleys. Subsequent human population expansion has resulted in urban
and suburban development and conversion of woodland for agriculture that have
furthered the process of fragmentation of oak woodland. A theoretical consequence
of fragmentation and decreased population sizes is reduced genetic diversity as
populations become increasingly isolated and suffer from the stochastic
consequences of genetic drift (Hedrick 2005). Genetic diversity has been linked to
fitness traits (Frankham 2003, Charpentier and others 2005) and may enhance
ecosystem recovery following environmental extremes (Reusch and others 2005).
Therefore, the future management for healthy oak woodlands requires an
understanding of the level and distribution of genetic variation, so that maximum
1
Abbreviated version of this paper was presented at the Sixth California Oak Symposium: Today’s
Challenges, Tomorrow’s Opportunities, October 9-12, 2006, Rohnert Park, California.
2
Department of Environmental Science Policy and Management, 137 Mulford Hall, University of
California, Berkeley, CA 94720.
485
GENERAL TECHNICAL REPORT PSW-GTR-217
genetic variation can be conserved and appropriate genetic resources can be selected
for restoration.
The traditional approach for forest tree species was to establish replicated
provenance studies to detect genetic variations in adaptive traits (Morgenstern 1996).
However, provenance studies are costly and require a long time to obtain results.
Molecular markers, such as microsatellites, permit the estimation of neutral genetic
variance and its partition as a result of the balance between gene flow and genetic
drift. By comparing genetic structure of nuclear DNA with that of chloroplast DNA
(cpDNA), the effects of gene flow by seed can be distinguished from that of pollen.
Ennos (1994) estimated gene flow by pollen to be about 200 times more important
than gene flow by seed in European oaks. Therefore, it is to be expected that a
stronger pattern of genetic structure would be discerned for cpDNA than for nuclear
DNA. Also, the two molecular systems may provide different information on
population structure. The chloroplast genome is particularly useful for inferring the
migration of lineages over evolutionary time because of its slower mutation rates and
its smaller effective population size (Petit and others 1997). Whereas, the more
rapidly evolving nuclear genome is ideal for studying rates and direction of gene flow
(Beerli and Felsenstein 1999).
Coast live oak (Quercus agrifolia Née) occupies the coastal mountain ranges
from the southern Ukiah Valley, Mendocino County in Northern California, to the
Sierra San Pedro Martir in Baja California, Mexico. Urban development has heavily
impacted populations in Southern California, and ranching has fragmented
populations in the interior valleys. In the last 10 years, sudden oak death has brought
heavy mortality in some areas of Northern California. coast live oak occupies areas
that are likely to see substantial climatic change over the next century, with
increasing humidity in the southwest and increasing aridity in the north (Hayhoe and
others 2004). The habitat envelope that is defined by a Mediterranean climate under
the influence of summer fog may become more limiting in the future.
Here, we present data on a study of cpDNA and nuclear DNA diversity
throughout the range of coast live oak to detect possible geographic structure that
would be useful in conservation of genetic resources.
Methods
Leaf samples were collected from 505 individuals from 38 populations for
chloroplast DNA analysis and from 499 individuals from 28 populations for nuclear
DNA analysis. Populations were selected to cover the geographic range of the species
(table 1) and included the type variety agrifolia and var. oxyadenia from San Diego
County and from Baja California, Mexico. Variety oxyadenia was identified by the
dense tomentose undersurface of the leaf. The leaves were stored in plastic zipper-loc
bags at -20°C.
Total genomic DNA was extracted from the leaf samples using a simplified
CTAB (cetyltrimethyl ammonium bromide) method (Cullings 1992).
Chloroplast Microsatellites
Five pairs of primers developed for the amplification of chloroplast
microsatellite loci (μdt1, μdt3, μdt4, μcd4, μdt5) in Q. petraea and Q. robur
(Deguilloux and others 2003) were chosen to amplify chloroplast DNA in coast live
486
Molecular Markers Show How Pollen and Seed Dispersal Affect Population Genetic Structure in
Coast Live Oak (Quercus agrifolia Née)—Dodd
oak. The PCR reaction solution (10μl) contained four dNTPs (0.2mM each), 2.5 mM
of MgCl2, 0.2μM of each primer, 10x reaction buffer, 25ng DNA and 1 unit of
Amplitaq polymerase (Applied Biosystems, Foster City, CA). Amplifications were
performed on a Techne Genius thermal cycler with the following profile; 5 min
denaturing at 95°C, followed by 25 cycles of 1 min denaturing at 94°C, 1 min
annealing at the primer Tm (see Table 1 in Deguilloux and others 2003) and 1 min
extension at 72°C, with a final extension of 72°C for 8 min. The PCR product (0.75
μL) was mixed with a solution of 8 μL of formamide and 0.5 μL of 350 ROX size
standard (Applied Biosystems, Foster City, CA) and electrophoresed on an ABI 3100
automated sequencer (Applied Biosystems, Foster City, CA). Results were analyzed
with GENESCAN 3.7 and GENOTYPER 3.7 software (Applied Biosystems, Foster City,
CA).
Nuclear Microsatellites
Six pairs of primers developed for the amplification of nuclear microsatellite loci
(quru-GA-0A01, quru-GA-0C11, quru-GA-0C19, quru-GA-1C08, quru-GA-1F02,
quru-GA-2F05) in Quercus rubra (Aldrich and others 2002) were used to amplify
nuclear DNA in coast live oak. Amplifications were performed in a standard
polymerase chain reaction (PCR) mixture containing a buffer of 2.5mM Tris-HCl
(pH 8.0), 12.5 μM EDTA, 125 μM DTT. We added 2.5 mM MgCl2, 2.5mM each of
the amplification primers, 2.5μM of each dUTP, 250μg/mL BSA and 0.0375 units/μL
of Taq DNA Polymerase (Invitrogen). To facilitate PCR multiplexing, we used a
touchdown program to optimize for differences in annealing temperature. The PCR
reaction began with one activation cycle at 95oC for 10 min and then used the
following cycle parameters: a denaturation phase of one minute at 94°C, one minute
at 60°C and 35 seconds at 70°C for two cycles. The second phase followed for 18
cycles: 45 seconds at 93°C, 45 seconds at 59°C (reducing the annealing temperature
by 0.5°C each cycle) and 45 seconds at 70°C. Following this phase were 20 cycles of
30 seconds denaturing at 92oC, 30 seconds at 50oC and 1 min extension at 70oC. This
was followed by a final extension phase of 5 min at 72oC.
We used fluorescently labeled primers to visualize amplified PCR products on
an Applied Biosystems 3100 automated sequencer. A two uL aliquot of PCR product
was suspended in 8uL of formamide and 0.5uL of ROX 350 size standard (Applied
Biosystems) and denatured for 4 minutes at 93°C. Genotypes were scored by length
in base pairs using GENESCAN 3.7 and GENOTYPER 3.7 software (Applied
Biosystems, Foster City, CA) and recorded in a Microsoft Excel spreadsheet.
Data Analysis
Because the chloroplast genome is inherited clonally, we combined the five
microsatellite loci into a single haplotype for each individual. Nuclear microsatellites
were treated as independent loci with two alleles. We ran global tests of population
differentiation and analysis of molecular variance using the Weir & Cockerham
(1984) estimate of θ for small population size as implemented in FSTAT (Goudet
2001). For nuclear microsatellites, we evaluated allelic richness (A) and expected
heterozygosity (He) as measures of population genetic diversity using Fstat vers.
2.9.3 (Goudet 2001). A is highly dependent on population size, therefore we used a
rarefaction procedure recommended by El Mousadik & Petit (1996), as implemented
in FSTAT. FSTAT estimates the number of alleles in a sample corrected to the smallest
population sample-size, for all populations.
487
GENERAL TECHNICAL REPORT PSW-GTR-217
Table 1—Sampling localities and sample size (Nc –chloroplast, Nn -nuclear) for coast live oak
(Quercus agrifolia). Genetic diversity indices (A – allelic diversity and He – heterozygosity)
for nuclear microsatellites shown.
Population location
Hopland
Yorkville
Cloverdale
Geysers Rd
Forestville
Monticello Rd
Rockville Hills
Park,
Cordelia
Novato
Nicasio
China Camp State
Park
Lafayette
Sunol
Morgan State Park
Huddart Park
Saratoga Pass
Hwy92/Hwy 280
Soquel
UC Santa Cruz
campus
San Juan
Pacheco State Park
Hwy 152
Monterey
Bottchers Gap
Palo Colorado
Canyon
Molera State Park
San Miguel
Parkfield
York Mtn Rd
Chorro Creek
Ojai
Lebec
San Gabriel
El Cariso
Morettis Jnctn
Santa Ysabel
Julian
Peutz Vlly
La Mission
St. Thomas
San Pedro Martir
488
County
Mendocino
Mendocino
Sonoma
Sonoma
Sonoma
Napa
Solano
Latitude
38.9874
38.8705
38.8152
38.8262
38.4723
38.3557
38.3070
Longitude
123.0826
123.0826
122.9385
122.9153
122.9201
122.2086
122.1291
Nc
18
6
11
9
10
13
9
Solano
Marin
Marin
Marin
38.2167
38.0496
38.0662
38.0057
122.1145
122.5363
122.7055
122.4827
10
43
9
68
Contra Costa
Alameda
Contra Costa
San Mateo
San Mateo
San Mateo
Santa Cruz
Santa Cruz
37.9304
37.6070
37.8264
37.4263
37.3726
37.5111
37.1128
36.9938
122.1664
121.8739
121.8008
122.3114
122.2580
122.3496
121.9098
122.0627
12
10
San Benito
Merced
Merced
Monterey
Monterey
Monterey
36.8054
37.0666
37.0346
36.5699
36.3316
36.3940
121.5819
121.2009
121.2018
121.9007
121.7961
121.8785
12
22
Monterey
Monterey
Monterey
San Luis
Obispo
San Luis
Obispo
Ventura
Kern
Los Angeles
Riverside
San Diego
San Diego
San Diego
San Diego
Baja Norte
Baja Norte
Baja Norte
36.2874
35.8352
35.9079
35.5395
Nn
31
A
0.820
He
4.06
9
9
10
0.785
0.817
0.838
3.92
4.01
4.19
47
9
77
0.582
0.604
0.581
2.91
2.96
2.96
52
0.617
3.00
7
0.703
3.55
7
10
32
0.380
0.562
0.646
2.10
2.74
3.10
12
11
9
8
0.646
3.02
121.8467
120.6270
120.5518
120.8029
10
15
9
17
34
10
9
16
0.662
0.664
0.694
0.643
3.19
3.28
3.50
3.16
35.3492
120.7972
12
11
0.676
3.34
34.4293
34.8779
34.2980
33.6478
33.2055
33.1479
33.0984
32.8502
31.9840
31.5806
30.9727
119.1210
118.8975
117.8412
117.4170
116.7251
116.6754
116.8042
116.8042
116.7243
116.4179
115.7695
14
4
6
12
10
11
13
12
10
11
14
11
9
0.646
0.618
3.15
2.93
12
9
11
13
11
10
11
14
0.606
0.689
0.616
0.679
0.749
0.604
0.629
0.602
3.06
3.36
3.04
3.43
3.81
3.00
2.95
2.87
7
10
9
5
Molecular Markers Show How Pollen and Seed Dispersal Affect Population Genetic Structure in
Coast Live Oak (Quercus agrifolia Née)—Dodd
By simultaneously combining spatial and genetic distance data, hypotheses
concerning geographical pattern and genetic differentiation can be developed. We
investigated geographical locations where genetic barriers among populations might
occur using the software BARRIER vers. 2.2 (Manni and others 2004). This analytical
method makes use of Monmomier’s maximum difference algorithm (Monmonier
1973) to find edges associated with the highest rates of change in a distance (genetic
distance) matrix. The algorithm is applied to a network of geographic distances
among populations using Delaunay triangulation (Brassel & Reif 1979). Barriers are
placed perpendicular to edges that correspond to the largest genetic distances and are
continued across adjacent edges in order of maximum genetic distance until the
barrier reaches the limit of the network space, or a previously determined barrier. We
chose BARRIER over other programs, such as STRUCTURE (Pritchard and others 2000)
and GENELAND (Guillot and others 2005), because the algorithm is not dependent on
underlying genetic properties such as Hardy-Weinberg equilibrium within groups and
linkage equilibrium among loci as are STRUCTURE and GENELAND. The chloroplast
genome is clonally inherited and so, loci are fully linked. For the nuclear genome, we
used Slatkin’s linearized Fst (Slatkin 1995) genetic distances among populations for
input into BARRIER. For the chloroplast genome, we treated the haplotype (5 linked
loci) as a locus and each different haplotype as an allele. Analysis of molecular
variance was used to generate a genetic distance matrix of Φst (an analog of Fst) that
was input into BARRIER. Re-sampling random subsets of individuals within
populations provided 100 bootstrap replicate distance matrices to obtain statistical
confidence for the predicted barriers.
Results
Chloroplast DNA
A total of 31 haplotypes were detected. Analysis of molecular variance showed that
94 percent (95 percent confidence limits 90.9 to 97.1 percent) of haplotype variance
was among populations and only 6 percent (2.8 to 9.3 percent) was attributable to
trees within populations. The distribution of haplotypes in California suggested about
four major groups (fig. 1). 1) The San Francisco Bay Area and northwards,
haplotypes 1, 6 and 30 are common. Of these, only haplotype 6 is found outside of
this region at York Mountain Road in San Luis Obispo County and at Ojai in Ventura
County. 2) In the Monterey-Big Sur region, haplotype 17 is common and 16 and 21
are also present. These haplotypes are not found elsewhere. 3) In the coastal
mountains of San Luis Obispo County, haplotypes 2 and 28 are unique, haplotype 6
is shared with the north and haplotype 27 is shared with more interior populations
near Parkfield. 4) In extreme Southern California, six haplotypes were detected with
only one (haplotype 8) being detected outside of this region at Ojai and Lebec in
Ventura County. The populations from Baja California, Mexico, did not share any
haplotypes with sample sites further north. Extreme interior populations at Pacheco
State Park and Cordelia had unique haplotypes, suggesting that recent gene flow by
seed from more coastal populations has not penetrated these areas.
489
GENERAL TECHNICAL REPORT PSW-GTR-217
Figure 1—Distribution of 31 chloroplast haplotypes of coast live oak. Sequence of
numbers does not represent haplotype evolution.
The BARRIER analysis detected five major barriers to gene flow (fig. 2). The first
and most important of these divides populations into a southern and a northern group
along a line between Ojai to Lebec and the San Gabriel Mountains. The second most
important barrier separated interior population Parkfield. The third and fifth barriers
occurred in Baja California, Mexico, indicating relatively high differentiation among
these most southerly populations. The fourth barrier separated populations from the
central California coast from those further north and east.
490
Molecular Markers Show How Pollen and Seed Dispersal Affect Population Genetic Structure in
Coast Live Oak (Quercus agrifolia Née)—Dodd
Figure 2—Barrier analysis for coast live oak based on chloroplast microsatellites.
Barriers shown as thick lines and numbered in order of importance are derived by
combining a genetic distance matrix with Delaunay triangulations (fine lines) derived
from Voronoi tessellations (polygons) around sampled populations (dots). Bootstrap
support by re-sampling 100 subsets within populations shown for each barrier
segment.
Nuclear DNA
Analysis of molecular variance revealed a much lower level of population
differentiation for nuclear DNA than for chloroplast DNA: 23 percent (95 percent
confidence limit 17.9 to 28.6 percent) among populations and 77 percent (69.8 to
85.1 percent) within populations.
Seven significant barriers were detected among the 28 populations (fig. 3). The
first, most significant of these was in Northern California, separating populations
north of northern Marin County from all populations further south. The second
barrier separated interior population Pacheco, which was concordant with the
analysis of chloroplast DNA. The third barrier separated the central California coast
population from Big Sur. Although this was also generally concordant with the
chloroplast structure, more limited sampling for nuclear DNA along the coast
northwards to the San Francisco Peninsula precludes inference of the extent of this
partition based on nuclear DNA. The fourth and fifth barriers were also in Northern
California, separating the northernmost population from Cloverdale and the interior
population of Morgan respectively. The sixth barrier separated Lebec from Ojai and
populations further south. The final significant barrier occurred in Southern
California between populations from San Diego County and El Cariso on the
Cleveland National Forest in Riverside County.
491
GENERAL TECHNICAL REPORT PSW-GTR-217
Discussion
Overall population differentiation based on chloroplast markers was high in coast live
oak, consistent with a maternally inherited marker and suggesting that the heavy
seeds of coast live oak are dispersed over relatively short distances. The distribution
of chloroplast haplotypes in Figure 1 suggests four major biogeographic groups. The
barrier analysis indicates a most significant north-south split between the southernmost limit of the outer south coast ranges (sensu Hickman 1993) and the San Gabriel
Mountains of the Transverse Ranges. However, populations from Ojai and Lebec
shared one haplotype with some populations from Northern California and another
with populations from Southern California, suggesting a possible transitional region
of admixture of northern and southern groups. It will be interesting to sample more
intensively in this region to detect more precisely where the cpDNA break occurs.
The other cpDNA biogeographic regions supported by barriers included central coast
populations, populations from the San Francisco Bay and northern coast ranges,
interior populations and the extreme southern populations from Baja California,
Mexico. Topography may explain these breaks, but more intense sampling to confirm
this is necessary. For example, the haplotype map suggests a central coast group
extending through the Santa Lucia Mountains to the Monterey Bay, whereas the
barrier analysis extends this group north to the San Francisco Peninsula. It is
therefore unclear whether the Salinas Valley, or the San Francisco Bay is the
effective barrier to the dispersal of seed in this region. Our sampling was more
intensive in Northern California, and we intend to sample more populations in the
southern range to confirm our preliminary data.
Figure 3—Barrier analysis for coast live oak based on nuclear microsatellites.
Barriers shown as thick lines and numbered (roman numerals) in order of importance
are derived by combining a genetic distance matrix with Delaunay triangulations (fine
lines) derived from Voronoi tessellations (polygons) around sampled populations
(dots). Bootstrap support by re-sampling 100 subsets within populations shown for
each barrier segment.
As expected for an outcrossing, wind-pollinated species, the level of population
differentiation based on nuclear DNA was much lower than that of cpDNA. The
partition of population genetic structure based on nuclear DNA was only partially
492
Molecular Markers Show How Pollen and Seed Dispersal Affect Population Genetic Structure in
Coast Live Oak (Quercus agrifolia Née)—Dodd
concordant with that based on cpDNA. It has often been noted that these two marker
systems show conflicting patterns in oaks, with cpDNA variation among sympatric
species or populations depending more on geography than on phylogenetic
relationships (Whittemore and Schaal 1991, Dumolin-Lapégue and others 1999). The
nuclear DNA results supported the cpDNA data in identifying the Central Coast and
the interior populations as forming distinct groups. However, contrary to the cpDNA
results, there was more intense population differentiation based on nuclear DNA in
Northern California than in the south. Genetic diversity as measured by allelic
richness was also highest in two of the northernmost populations in California.
Earlier studies have shown that hybridization between coast live oak and interior live
oak is more prevalent in Northern California (Dodd and others 1993, 2002, Dodd and
A.-Rafii 2003) and this may partially explain the discordant patterns of population
differentiation based on the two marker systems. Elsewhere, we have shown that the
most common chloroplast haplotype for coast live oak in Northern California is
likely to be a haplotype of interior live oak (Quercus wislizeni A. DC.) and it can be
detected in populations of this latter species throughout Northern California beyond
the range of coast live oak (Dodd and others 2005). It would appear that pollen
swamping from coast live oak results in hybrid progeny that have captured the
maternally inherited cpDNA of interior live oak. Subsequent backcrossing of these
hybrid progeny to coast live oak results in coast live oak phenotypes with interior live
oak chloroplast genomes. In populations of interior live oak over much of northwestern California, the haplotype appears to be fixed, so that coast live oak resulting
from hybridization through pollen would also carry a fixed haplotype. In contrast to
the chloroplast genome, hybridization should result in the incorporation of the
nuclear genome of both species. Whereas backcrossing may dilute the genome of one
parent, it will nevertheless persist over many generations. Thus in a region of pollen
swamping, nuclear diversity should be relatively high. Similar effects of pollen
swamping have been found in Eucalyptus (Potts and Reid 1988, Potts and others
2003) and in European oaks (Petit and others 1997, Belahbib and others 2001).
Hybrids between coast live oak and Shreve oak have been detected from multilocus
genotypes (Dodd and Rafii 2003) and chloroplast sharing between these two species
in central coastal California was also detected here, but we were unable to determine
directionality.
Compared with other oak species, we have detected relatively high genetic
structure among populations based on chloroplast and nuclear DNA. It would be
most interesting to evaluate whether this translates equally to transcribed genes that
control traits of ecological importance. It is encouraging that hybridization with
interior live oak appears to be facile in Northern California where summer
temperatures are predicted to increase more than in the southwest (Hayhoe and others
2004). Whereas coast live oak is adapted to a Mediterranean climate under the
influence of summer fog, interior live oak is better adapted to more continental
conditions of drought and extreme temperatures. Hybrid products between these two
species may offer genotypes that will be well-adapted to the new ecological
conditions. Concerning disease resistance, our earlier studies were equivocal
regarding possible population variation in lesion size after inoculation with
Phytophthora ramorum. In spring inoculations, we detected significantly smaller
lesions in branch cuttings from populations from southern California compared with
Northern California, but this difference was not replicated in summer inoculations
(Dodd and others 2005). We are currently testing whether this could be a seasonal
effect.
493
GENERAL TECHNICAL REPORT PSW-GTR-217
Acknowledgements
The project was funded by the USDA-Forest Service, Pacific Southwest Research
Station USDA Forest Service, through research agreements 01-JV-11272135-173 and
01-JV-11272135 and by the Betty and Gordon Moore Foundation.
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