GENETIC VARIATION IN EASTERN WASHINGTON POPULATIONS OF NAVARRETIA LEUCOCEPHALA (POLEMONIACEAE) A VERNAL POOL ENDEMIC

2005·7.3 MB·English

by  David L Boose

Madrono, Vol. 52, No. 2, pp. 99-106, 2005
GENETIC VARIATION IN EASTERN WASHINGTON POPULATIONS OF
NAVARRETIA LEUCOCEPHALA (POLEMONIACEAE)
A VERNAL POOL ENDEMIC
David L. Boose', Benjamin R. Harmeling, and Roger T. Turcotte
Department of Biology, Gonzaga University, 502 East Boone Avenue,
WA
Spokane, 99258
Abstract
We used Random Amplified Polymorphic DNA (RAPD) to investigate genetic variation in eight pop-
ulations of the vernal pool endemic Navarretia leucocepJuila Benth. in eastern Washington. Five of the
populations were subspecies minima, collected in Spokane County. The other three populations were
subspecies diffusa, a recently described taxon known only from adjacent Lincoln County in eastern
Washington. We also sampled a nearby vernal pool population ofA^. intertexta (Benth.) Hook., a closely
related species whose range overlaps with N. leucocephala. Distances among the sampled pools ranged
from a few tens of meters to 80 km. The populations differed in their levels of genetic variation, with
proportions ofpolymorphic loci ranging from 10% to 55% and mean gene diversities from 0.05 to 0.30.
Analysis of Molecular Variance (AMOVA) among the eight N. leucocephala populations showed signif-
icant spatial structuring, with 52% of the observed genetic variation due to differences between the
Spokane County and Lincoln County populations. Within each county, 10% — 34% ofthe genetic vari-
ation was due to differences among populations. Estimates of indicated significant population differ-
entiation among all populations ofTV. leucocephala (^st ~ 0.65), but the degree ofdifferentiation varied
with interpopulation distance. Populations 35-250 m apart were generally not differentiated. At adistance
of 1100-1800 m, some populations showed significant differentiation and others did not. Populations 80
km apart were significantly differentiated. On a distance-based phenogram, N. intertexta separatedclearly
from the N. leucocephala populations, which also grouped by subspecies, suggesting that the morpho-
logical features used to differentiate the taxa are paralleled by genetic differences.
Key Words: Columbia Plateau, genetic differentiation, RAPD, Navarretia, Polemoniaceae, vernal pools.
Plants inhabiting ephemeral wetlands known as a few hundred meters apart, separated from other
vernal pools are likely to show significant spatial such archipelagos by tens to hundreds ofkilometers
structure in their genetic variation. Natural selec- (Holland and Jain 1981). Most vernal pool plant
tion, restricted gene flow, and genetic drift can species do not occur in the intervening uplands, so
combine to create non-random distributions of ge- this structured distribution of habitat iinposes a
notypes at multiple spatial scales, with implications strongly patchy distribution of populations, among
for the evolutionary trajectories of vernal pool spe- which genetic variation may develop.
cies, and for efforts to preserve vernal pool biodi- Because vernal pools are filled primarily through
versity. Yet few experimental data exist on genetic precipitation, local soil and hydrologic factors have
variation within and among vernal pool populations a strong influence on the conditions experienced by
(Elam 1998). vernal pool plants (Keeley and Zedler 1998). These
Vernal pools occur in regions with cool, wet win- different chemical and hydrologic environments are
ters and hot, dry summers. They form when winter likely to select for different physiological and life-
rains fill depressions underlain by an impervious history characteristics, resulting in divergent selec-
layer, then dry out during the spring and early sum- tion pressures among pools. Many vernal pool plant
mer, remaining dry for several months (Keeley and species are endemic not only to vernal pools, but
Zedler 1998). While vernal pools are most numer- to specific subtypes ofpools, differentiated by local
ous in California, they also occur in the intermoun- climate factors, soil types, and topographic position
tain regions of several western states, including (Stone 1990; Alexander and Schlising 1998; Ban-
Oregon and Washington (Bjork 1997), as well as der and McMillan 1998).
Baja California, Mexico and areas ofArgentina and The potential for divergence among vernal pools
Chile (Keeley and Zedler 1998). Pools generally is enhanced by the restricted nature ofgene flow in
show a highly clustered distribution, occurring in many vernal pool plants. Leong et al. (1995) found
localized regions where the substrate and climate that pollinators foraged occasionally between ver-
conditions allow their formation. Within these re- nal pool patches 25 m apart, but rarely among
gions are "archipelagos" of pools a few meters to patches 80-100 m apart. Using fluorescent dyes to
track pollen movements in vernal pool Limuauthes
' Author for correspondence, email: boose©gonzaga. species. Thorp (1990) found that 97% of the dye
edu was distributed within 5 meters ofthe source plants.
MADRONO
100 [Vol. 52
with occasional dispersal up to 13 meters. In Lim- four subspecies, but with little bootstrap support
nanthes doiiglasii R. Br. subsp. rosea (Benth.) C.T. (Spencer 1997; Spencer and Porter 1997).
Mason, a vernal pool endemic, 85% of plants re- We used Random Amplified Polymorphic DNA
ceived pollen from donors within 10 cm, and fewer (RAPD) markers to investigate genetic variation
than 5% received pollen from as far away as 80 within and among populations of N. leiicocephala
meters (Kesseli and Jain 1982). Seed dispersal dis- from vernal pools in the Columbia Plateau of east-
tances in vernal pool plants are also expected to be ern Washington. RAPD markers (Williams et al.
highly skewed, with most seeds falling close to the 1990) are a PCR-based indicator of genetic varia-
maternal plant (Zedler 1990). For example, vernal tion commonly used in studies of population ge-
pool species in the genus Navarretia have indehis- netics. RAPD markers are dominant, so heterozy-
cent fruits, retaining their seeds within the capsule gotes cannot be directly distinguished from homo-
RAPD
until the pools fill the following winter or spring. zygotes at a particular locus. If populations
Swelling of the moistened seed coats bursts the are assumed to be in Hardy-Weinberg equilibrium,
membranous capsule, resulting in extremely local- then the frequency of the "null" allele can be es-
ized dispersal (Spencer and Spencer 2003). Seeds timated as the square root of the frequency of the
of vernal pool plants may spread through wind- negative phenotype. If the assumption of Hardy-
driven water movements, but transport among Weinberg equilibrium cannot bejustified, then mul-
pools is likely to be limited (Jain 1978). On the tilocus RAPD phenotypes can be treated as haplo-
other hand, some long-distance transport of seeds types, and genetic diversity and population differ-
obviously occurs, given the broad ranges of many entiation can be estimated based on pairwise dif-
vernal pool taxa, and the discontinuous nature of ferences between individual haplotypes (Weising et
their habitat (Schleidlinger 1981). al. 1995; Wolff and Morgan-Richards 1999).
The genus Navarretia includes 12 species and Specifically, we sought to address the following
two questions:
subspecies that are more or less restricted to vernal
pools and other ephemeral wetlands throughout the 1) What is the current distribution of genetic var-
western United States, and a single species found iation among populations ofA^. leiicocephala in
in similar habitats in Chile and Argentina (Spencer eastern Washington?
and Porter 1997; Bjork 2002; Spencer and Spencer 2) Is the distribution of genetic variation concor-
2003). Several taxa are widely distributed through- dant with the morphologically based subspecies
out the West. For example, N. leiicocephala Benth. designations of Bjork (2002)?
occurs from the San Joaquin Valley in California
north through Oregon and eastern Washington, and Methods
east into Idaho and Utah (Hitchcock et al. 1964;
Day 1993). A closely related species, N. iiitertexta In eastern Washington, vernal pools occur in
(Benth.) Hook., also occurs throughout much ofthe three distinct "tracts", running roughly from north-
east to southwest, following the paths of Pleisto-
range of A^. leiicocephala. In contrast to A^. leiico- cene floods that scoured off surface soils and ex-
cephala, N. intertexta is more of a habitat gener- posed basalt bedrock (Fig. 1). Outside of these
alist, occurring in vernal pools, but also inhabiting tracts, soils are too deep or too well-drained to sup-
moist uplands and meadows (Day 1993a). port vernal pool formation (Bjork 2002). We sam-
Day (1993b) identified five subspecies ofN. leii- pled eight populations ofN. leucocephala fromver-
cocephala based on morphological characteristics; nal pools in Spokane and Lincoln Counties. The
recently a sixth subspecies, N. leiicocephala subsp. three Lone Pine Road populations (L1-L3) are
dijfusa Bjork, was identified from vernal pools in subsp. diffusa, collected in the Swanson Lakes
t2h0e02C)o.lTumhbeimaoPsltatceaoummofoenasstuebrsnpWeacisehsinogftoN.n (lBeijiocrok- WinildtlhiefecAernteraali,n L"iDnacvoelnnpCoorutn"tyt(ra4c7t.6°{Ns,en1s1u8.B5j°oWr)k,
cephala in eastern Washington is N. leiicocephala 2002). The other five populations (SNl, SN2, SSl-
subsp. minima (Nutt.) Day, which is widely distrib- SS3) are subsp. minima, collected in the Turnbull
uted in the region. Subspecies diffusa and minima National Wildlife Refuge in Spokane County
both occur in similarhabitats in Lincoln county, but (47.4°N, 117.5°W), in the "Cheney-Palouse" tract
their ranges in the county do not overlap. Subspe- {sensu Bjork 2002). Pools within each group were
cies diffusa is not found elsewhere in the state 20-1600 m apart; the Lincoln County populations
(Bjork 2002). The degree to which the morpholog- are approximately 80 km distant from the Spokane
ical differences between these subspecies reflect County populations. To compare intraspecific vari-
molecular genetic variation is unknown. In a recent ation to interspecific variation, we also sampled a
phylogeny ofthe genus (Spencer 1997), an analysis population of N. intertexta occupying a small ver-
of22 morphological characters failed to distinguish nal pool located between the two SN populations.
among five subspecies of N. leucocephala (subsp. From six to ten entire plants were collected from
diffusa was not included). Adding sequence data each population, with no two collected individuals
from the ribosomal RNA internal transcribed spacer closer than two meters apart. DNA was extracted
(ITS) region separated N. I. minima from the other from approximately 50 mg of stem and leaf tissue
2005] BOOSE ET AL.: GENETIC VARIATION IN NAVARRETIA LEUCOCEPHALA 101
This matrix of individual distances was then used
to estimate measures of genetic structure through
an Analysis ofMolecular Variance (AMOVA; Weir
and Cockerham 1984). We partitioned total genetic
variance into three covariance components: be-
tween counties (Lincoln vs. Spokane), among pop-
ulations within a county, and within populations.
The significance of these partitions was evaluated
using permutation tests based on 1,000 randomi-
zations of the data for each level of partitioning
being tested (Schneider et al. 2000). We also ana-
lyzed each county's populations separately, parti-
tioning variation into among- and within-population
components.
The inter-individual genetic distances were also
used to calculate pairwise estimates of genetic dif-
ferentiation {'^st) among all nine sampled popula-
tions (Excoffier et al. 1992; Wright 1965; Weir and
Cockerham 1984). Significance of the values
was evaluated using permutation tests based on
3000 randomizations, and using Markov-based ex-
2X2
I act tests analogous to Fisher's exact test for
u tables (Raymond and Rousset 1995; Schneider et
Fig. 1. Map showing locations of the N. leucocephala al. 2000). Estimates of gene flow between popula-
populations sampled for this study. Shaded areas indicate tions (Nm) were calculated using the formula:
regions ofvernal pool occurrence in Eastern Washington.
Squares show locations of the sampled populations.
ANmd
+
using the Wizard® DNA extraction kit manufac- id - 1) 1
tured by Promega Corporation (Madison, Wiscon- where is the population size, m is the proportion
Gpsireni,nmoeUmrSisAc)(,ODpfeNorAlolnowwAialns,gmatAhm2Mep,limafainneuddfaAcwt1iu3t)rh,erut'hssrimenpegMrotR1o0Ec-oDbl-p. tdohfeemtehaecstpueaolxpucnlhuaatmnibgoiennrgmoifggrepanotepisunlga(,tSilaaontndksidnexisc19ht9ah1en)g.niunBmgebcgeaerunseoesf
Taq®mRMeadyMix (10 Tris-HCl, 50mM KCl, is unknown, we calculated the estimates with d =
1.5 MgCK, 0.001% gelatin, 0.2 dNTP, 2, as if the two populations being compared only
0.75 u Taq Polymerase; manufactured by Sigma- exchanged genes with one another. This is unlikely
Aldrich, St. Louis, Missouri, USA), with 100 ng to be true, but the gene flow estimates were meant
template DNA, and 25 pmol primer in a 25 uL to be for comparative purposes only, and not as
reaction. Reaction conditions consisted of 3 min at reliable measures of actual migration among pop-
95°C, followed by 45 cycles of 1 min at 95°C, 1 ulations.
min at 35°C, and 2 min at 72°C, ending with a three Pairwise (^^j values were also converted to esti-
minute final extension at 72°C. Amplification prod- mates of genetic distance using the formula D =
ucts were separated in 1.8% agarose gels and vi- 0„/(l - 0„) (Slatkin 1995), which produces a
sualized by staining with ethidium bromide. Gels measure proportional to divergence time between
were recorded using a digital camera with a Tiffen the populations. These estimates were used to cre-
deep yellow filter (#15). Each individual was am- ate a distance-based tree using the method of Fitch
plified at least three times with each primer, and and Margoliash (1967) in PHYLIP (Felsenstein
only strong bands appearing in at least two runs
1993).
were scored as present. The three primers produced
1 1 reliably scorable loci, all of which were poly- Results
morphic, and which produced 42 different multil-
ocus haploypes in the 77 individuals sampled. The RAPD markers showed significant genetic
Statistical analyses were carried out using the variation within and among populations of N. leu-
software package Arlequin 2.0 (Schneider et al. cocephala in eastern Washington. The 77 individ-
2000). For each population, we calculated the pro- uals sampled possessed 42 different multilocus hap-
portion of polymorphic loci, the mean number of lotypes, 32 of which were found in only a single
pairwise differences among individuals across loci, population. Across populations, the proportion of
and the average gene diversity (Nei 1987). Pairwise polymorphic loci ranged from 10% to 55%, and the
distances among all pairs ofindividuals (within and average numberofpairwise differences between in-
across populations) were computed as the number dividuals within a population ranged from 0.47 to
of different alleles between any two haplotypes. 3.33 (Table 1). Average gene diversity (the proba-
MADRONO
102 [Vol. 52
Table 1. Sample Size (n). Number of Multiloclis RAPD Haplotypes, PercentageofPolymorphic Loci (Pp)Mean
Pairwise Differences Among Individuals, and Mean Gene Diversity in Eight Populations of Navarretia leuco-
CEPHALA from EaSTERN WASHINGTON.
Haplotypes Mean pairwise Mean gene
Population n (number unique) Pp differences (±SD) diversity (±SD)
LI 6 5 (4) 36% 1.80 ± 1.20 0.16 ± 0.13
L2 6 6 (6) 55% 3.33 ± 1.99 0.30 ± 0.21
L3 8 7 (6) 60% 2.57 ± 1.54 0.25 ± 0.18
SNl 10 3 (0) 10% 0.47 ± 0.44 0.05 ± 0.05
SN3 9 7 (4) 45% 1.89 ± 1.19 0.17 ± 0.12
SSI 10 5 (2) 36% 1.47 ± 0.97 0.13 ± 0.1
SS2 10 5 (3) 36% 1.56 ± 1.01 0.14 ± 0.10
SS3 10 6 (4) 55% 2.38 ±1.41 0.21 ± 0.14
bility that any two individuals drawn from the pop- County populations was marginal (<I>^7 = 0.103, P
ulation will be different at a locus) ranged from = 0.077).
0.05 to 0.30. There was no geographic pattern to On a pairwise basis, values indicated signif-
the distribution of within-population variation. icant differentiation between most pairs of popula-
AMOVA
resuks indicated significant genetic dif- tions (Table 3). Permutation tests and exact tests
ferentiation in N. leucocephala at all three levels gave slightly different results for the significance of
analyzed (Table 2a). The majority of the variation these pairwise values. In particular, six O^y^val-
(ca. 53%) was between the two counties, with most ues found to be significant under the permutation
of the remainder (ca. 35%) found within popula- tests were not significant at the 0.05 level according
tions. When the populations were analyzed sepa- to the exact test (Table 3). Of these, the three in-
rately by county, the majority of the variation was volving population SS3 had p-values between
found to be within populations (Table 2b, c). In the 0.055 and 0.066 according to the exact test; the
Lincoln County populations, within-population var- three involving population SN3 had exact test p-
iation accounted for 89.76% of the total, while in values ranging from 0.11 to 0.26. When pairwise
Spokane County, it accounted for 66.35%. O^T^values were converted to estimates ofmigration
The overall value among all populations was rates, the vast majority were below 0.5 migrants per
0.652 (P < 0.001), indicating a high level of dif- year. Among the most similar populations, esti-
ferentiation. As in the AMOVA, much of this dif- mates ranged from 1 to 4 migrants per year (Table
ferentiation was due to differences between the two 3).
counties. When the two groups were analyzed sep- In the distance-based phenogram, populations
arately, the Spokane County populations still clustered together geographically, with the Lincoln
showed significant differentiation {^^r ^ 0.337, P County populations (L1-L3) grouped in a clade
< 0.001), while differentiation among the Lincoln separate from the Spokane County populations
Table 2. Results from Analysis of Molecular Variance (AMOVA) on Eight Populations of Navarretia leu-
cocephala FROM Eastern Washington. Results are based on 11 RAPD loci treated as multilocus haplotypes. a) All
populations combined, b) Five Spokane County populations only, c) Three Lincoln County populations only.
Source of variation df Sum of squares % of variation
a) All populations
Between counties 1 44.997 52.59 0.02
Among populations within counties 5 24.055 12.60 <0.01
Within populations 61 58.789 34.81
Total 68 127.841 100.00
b) Spokane County populations
Among populations 4 19.24 33.65 <0.01
Within populations 44 35.46 66.35
Total 48 54.69 100.00
c) Lincoln County populations
Among populations 2 4.82 10.26 0.08
Within populations 17 23.33 89.74
Total 19 28.15 100.00
2005] BOOSE ET AL.: GENETIC VARIATION IN NAVARRETIA LEUCOCEPHALA 103
Table 3. Pairwise Values (Above Diagonal) and Corresponding Estimated Number of Migrants Per Gen-
eration, NM (Below Diagonal), Among Eight Populations of Navarret/a leucocephala Based on 42 Multilocus
RAPD Haplotypes. Bold type indicates (b^rvalues significantly different from zero at P < 0.05. Asterisks indicate (i>sT
values found to be significant under permutation tests, but not under exact tests.
LI L2 L3 SNl SN3 SSI SS2 SS3
LI 0.175 0.094 0.841 0.735* 0.718 0.715 0.625
L2 1.18 0.058 0.742 0.633* 0.565 0.568 0.382*
L3 2.40 4.02 0.708 0.585* 0.542 0.552 0.423*
SNl 0.05 0.09 0.10 0.000 0.485 0.444 0.519
SN3 0.09 0.15 0.18 0.393 0.361 0.428*
SSI 0.10 0.19 0.21 0.27 0.39 0.067 0.085
SS2 0.10 0.19 0.20 0.31 0.44 3.47 0.220
SS3 0.15 0.40 0.34 0.23 0.33 2.70 0.89
(Fig. 2). Within the Spokane County populations, to be comparable to that for other annual plants in
populations SNl and SN3 are genetically indistin- general.
guishable, yet significantly differentiated from the Within-population genetic diversity will also de-
three SS populations. As expected, the population pend on the balance between opposing evolutionary
ofN. intertexta falls outside the clade that includes and ecological processes. On one hand, isolation
all eight A^. leucocephala populations, despite the and genetic drift could lead to reduced within-pop-
fact that it is less than 300 meters from population ulation diversity. In contrast, self-incompatibility
SN3, and less than 1700 ineters from the SS pop- and long-lived seed banks could work to maintain
ulations. significant diversity (Baker 1989; Hairston et al.
1996; Nunney 2002). All of the sampled N. leu-
Discussion cocephala populations are quite large, with popu-
Expectations for genetic variation in vernal pool lation sizes in the thousands of individuals. Thus,
plants will depend on the life-history ofthe species genetic drift is not expected to have a strong effect
involved (Elam 1998). Among plants in general, on within-population diversity, compared to other
annual species have been found to have lower with- evolutionary forces. While the mating system ofTV.
in-population genetic diversity than perennials, leucocephala is not known from experimental stud-
based on allozyme (Hamrick and Godt 1989) or ies, pollen:ovule ratios suggest that it is primarily
DNA markers (Nybom 2004). Summarizing 60 outcrossing (Cruden 1977; Plitmann and Levin
studies ofwild plant populations using RAPD anal- 1990; Spencer 1997). Information on the seed bank
ysis, Nybom (2004) found an overall mean gene is also unavailable, but the ephemeral, variable con-
diversity of 0.22 ± 0.12. Thus the amount of ge- ditions in vernal pools favor the development of
netic variation within populations of A^. leucoce- seed dormancy, and such dormancy is common
phala (mean gene diversity: 0.18 ± 0.08) appears among vernal pool plants (Elam 1998). These fac-
tors would tend to favor the maintenance of signif-
icant genetic diversity in populations of N. leuco-
N. intertexta cephala.
|SS3
The level of gene diversity we observed in N.
iTjssi leucocephala is comparable to that seen in RAPD
jSS2
JSN1 studies of plants with predominantly selfing or
SN3 mixed breeding systems. Nybom (2004) reported
L2 average gene diversities of 0.12 over ten predomi-
nantly selfing species, and 0.18 in eight species
L3 with mixed mating systems. Both of these values
were significantly lower than the average of 0.27
for 38 predominantly outcrossing species (Nybom
0.1 2004). Thus, while poUeniovule ratios may indicate
Fig. 2. Phenogram showing relationships among eight a tendency for outcrossing in N. leucocephala, lev-
populations of Navarretia leucocephala and one popula- els of genetic diversity suggest that selfing or mat-
tion of N. intertexta in eastern Washington. Populations ing among close relatives may be frequent in these
LI, L2, and L3 are subspeciesdiffusa from LincolnCoun- populations. Because of the limited seed dispersal
ctyi;espompiunliamtiaonfsroSmSIS,poSkSa2,neSSC3o,unStyN,laapnpdroSxNim3ataerleysu8b0spkem- distances in N. leucocephala, plants often occur in
distant. The population ofN. intertexta was collected ap- clumps of many individuals in very close proxim-
proximately 300 m from population SN3. Distances are ity. Members of these clumps are likely to be at
Slatkin's linearized distances, calculated from 11 RAPD least half-sibs, so pollinator movements among ad-
loci analyzed as multilocus haplotypes. jacent plants could result in significant inbreeding.
MADRONO
104 [Vol. 52
When all populations were considered together, populations, and pairwise comparisons among
N. leiicocephala showed levels of population dif- these pools show significant differentiation, with
ferentiation comparable to those found in RAPD Nm values from 0.39 to 0.52 (Table 3). No obvious
studies of other annual plant species. Nybom barriers to gene flow exist between the SN and SS
(2004) reported a mean (^^t value of 0.62 across populations, so the reason for this difference is un-
ten such studies, quite close to the value of 0.65 known. The Lincoln County populations are sepa-
we calculated forA^. leucocephala. But much ofthe rated from the Spokane County populations by a
differentiation we observed can be attributed to dif- distance of 80 km; not surprisingly, gene flow es-
ferences between the Spokane County and Lincoln timates between these regions are quite low.
County populations; separate analyses of these The pattern of genetic similarity depicted in Fig-
groups produce lower levels of differentiation. The ure 2 is consistent with Bjork's (2002) designation
Spokane County populations exhibit greater differ- of the Lincoln County populations of N. leucoce-
epnotpiualtaitoinon{s^^j{^^=t 0~.3307.)103t)h,anprthoebaLbilnycodluneCtoountthye pghraaplhaicasdaissteapnacreataensdubssupbescpieecsi.esUnfiodretntuintaytealrye, cgoeno--
smallernumber and closer proximity ofpopulations
founded in our sampling, so firm conclusions about
bisnaatsmeeprdlpeoodpnulianRtAiLoiPnnDcdsoilsntteaCnnodcuenstot(yi.NnycIrbneoagmseen2e0wr0ail4t,)h.<it>n^cjrveaalsuiensg graennteetdi.c Fsuoprpoerxtamfporlet,hewdeesihganvaetinoon aersetinmoatteyetofwatrh-e
The Spokane County populations exhibit a level amount of variation that might be observed be-
tween populations within a subspecies that are sep-
of differentiation similar to that seen in other
RAPD studies of plants with mixed mating sys- arated by 80-100 km. In addition, sampling ofdif-
tems. Nybom (2004) reported an average of fusa populations was quite localized, even within
0.40 for 18 such species, less than that seen for the restricted range of the subspecies. The samples
predominantly selfing species (mean O^y^ = 0.65; N used in this study were collected before the desig-
= 14), and greater than the average for outcrossing nation ofdiffusa as a separate subspecies, and were
species (mean <i>sj = 0.27; N = 73). Differentiation intended to sample N. leucocephala minima popu-
among the Spokane county populations is also lations separated by a range ofdistances. Sampling
higher than that found in most other studies of ver- of additional populations is hampered by the fact
nal pool plants, but this may be a result of the dif- that most vernal pools in eastern Washington occur
ferent markers used. Allozyme data from six vernal on private land, and landowners are generally re-
pool plant taxa, in two genera, showed Fsj values luctant to grant access.
ranging from 0.083 to 0.176 (Elam 1998). A sev- The genetic differentiation observed among N.
enth taxon, Limncmthesfloccosa Howell subsp. cal- leucocephala populations separated by relatively
ifomica Arroyo had an F^-, value of 0.963, indicat- short distances has implications for the conserva-
ing extreme genetic differentiation among popula- tion ofvernal pool habitats and theirassociated spe-
tions (Dole and Sun 1992; Elam 1998). The higher cies. Our SS and SN populations were separated by
(i>^j values we observed among A^. leucocephala less than 2 km, yet we detected significant genetic
populations may result in part from greater vari- differentiation among them. The large number of
ability of RAPD markers compared to allozymes. unique haplotypes found in each population also
vHaalmureiocfk0a.n3d57G±od0t.0(21498f9o)r arnenpuoarltepdlaanntapvoepurlaagteioFn^st suggests that no single pool is likely to be repre-
sentative of the genetic variation found across the
based on allozyme variation, compared to the av-
erage value of 0.62 reported by Nybom (2004) larger landscape. Efforts to preserve vernal pool di-
for annual plants using RAPD data. versity should therefore focus on protecting popu-
The pairwise F^j values observed in this study lations throughout the larger regions in which they
occur, rather than a few localized populations with
suggest that gene flow between pools a few hun-
significant numbers of individuals. This pattern in
dred meters apart is generally sufficient to prevent
differentiation. As a general rule, genetic drift can genetic variation is consistent with that seen at the
lead to differentiation between two populations if community level in vernal pool floras. Within the
the number of migrants between them {Nm) is less California Floristic Province over 100 species of
than one per generation (Slatkin 1987). The three plants are known to be endemic to, or primarily
SS populations are all less than 100 m from one associated with, vernal pools (Holland 1976; Kee-
another, and show low to insignificant levels ofdif- ley and Zedler 1998). Despite this diversity, indi-
ferentiation and estimated Nm values between 0.89 vidual pools generally contain only 15-20 plant
and 3.5 (Table 3). Similarly, the three Lincoln species, indicating a high degree of variation in
County populations are separated by 35-250 m, and species composition from pool to pool (Holland
show no significant differentiation. The effects of 1976; Keeley and Zedler 1998). Consequendy, ver-
intermediate distances on gene flow are equivocal. nal pool conservation efforts should be undertaken
Populations SNl and SN3 are 1300 m apart, yet over large spatial scales, so as to capture the great-
they show no differentiation. In contrast, the SS est amount ofbiological diversity, at the population
populations lie 1000-1800 m distant from the SN as well as the community level.
2005] BOOSE ET AL.: GENETIC VARIATION IN NAVARRETIA LEUCOCEPHALA 105
Acknowledgments Package) version 3.5c. Department ofGenetics, Uni-
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