Evolution, 55(8), 2001, pp. 1569–1580
POPULATION SIZE AND FRAGMENTATION THRESHOLDS FOR THE MAINTENANCE
OF GENETIC DIVERSITY IN THE HERBACEOUS ENDEMIC
SCUTELLARIA MONTANA (LAMIACEAE)
MITCHELL B. CRUZAN
Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, Tennessee 37996
E-mail: [email protected]
Abstract. The level and distribution of genetic variation is thought to be affected primarily by the size of individual
populations and by gene flow among populations. Although the effects of population size have frequently been
examined, the contributions of regional gene flow to levels of genetic variation are less well known. Here I examine
the effects of population size and the number of neighboring populations (metapopulation density) on the distribution
and maintenance of genetic diversity in an endemic herbaceous perennial. Reductions in the proportion of polymorphic
loci and the effective number of alleles per locus were apparent for many populations with a census size of less than
100 individuals, but no effects of population size on levels of inbreeding were detected. I assess the effects of regional
population density on levels of diversity and inbreeding using stepwise regression analysis of metapopulation diameter
(i.e., the size of a circle within which population density is estimated). This procedure provides a spatially explicit
evaluation of the effects of metapopulation size on population genetic parameters and indicates the critical number
of neighboring populations (fragmentation threshold) for the regional maintenance of genetic diversity. Stepwise
regression analyses revealed fragmentation thresholds at two levels; at a scale of 2 km, where small metapopulations
resulted in greater levels of selfing or sibling mating, and at a scale of 8 km, where metapopulation size was positively
associated with higher levels of genetic diversity. I hypothesize that the smaller fragmentation threshold may reflect
higher levels of selfing in isolated populations because of the absence of pollinators. The larger threshold probably
indicates the maximum distance over which pollen dispersal rates are high enough to counteract genetic drift. This
study demonstrates that the regional distribution of populations can be an important factor for the long-term maintenance
of genetic variation.
Key words.
syndrome.
Dispersal, genetic erosion, inbreeding, metapopulation density, population size, specialized pollination
Received November 6, 2000.
The level of genetic variation within populations has received considerable attention because it is generally thought
to be indicative of overall species vitality and the potential
for evolutionary responses to environmental change (Templeton et al. 1990; Barrett and Kohn 1991; Ellstrand and Elam
1993; Frankel et al. 1995). Although it would be ideal to
determine the allelic diversity for loci that may confer adaptation to current or future environments, it is generally
problematic to estimate levels of variation in quantitative
traits (Falconer 1981; Young et al. 1996) and nearly impossible to accurately identify traits with adaptive potential. As
a consequence, most investigations of genetic variation have
concentrated on (nearly) neutral genetic markers as a gauge
of contemporary and historical processes affecting the maintenance and distribution of genetic variation. The level of
genetic diversity for neutral markers is not always a perfect
predictor of genetic variation for quantitative traits (Hamrick
et al. 1991; Young et al. 1996); however, variation at marker
loci is much more tractable and can be analyzed with population genetic models that describe a wide range of evolutionary processes (Hedrick 1983; Weir 1996). Neutral markers are particularly useful for species with relatively small
effective population sizes, where the observed patterns of
diversity should be representative of the level and distribution
of variation across a range of loci that are neutral or affected
by weak to moderately strong selection (Hedrick and Miller
1992; Young et al. 1996; Montgomery et al. 2000). Studies
using neutral markers have made important contributions to
our knowledge of the influences of mating systems, popu-
Accepted April 23, 2001.
lation sizes, and gene flow to the distribution and maintenance of genetic variation (reviewed in Hamrick and Godt
1990; Avise 1994; Cruzan 1998).
Of the characteristics potentially affecting levels of genetic
variation, the size and distribution of populations have received the most attention, particularly in the context of species’ responses to anthropogenic disturbances (Barrett and
Kohn 1991; Ellstrand and Elam 1993; Hamrick and Godt
1996; Young et al. 1996). Population size is of primary interest because of the manifold effects it is expected to have
on the maintenance of variation and the effectiveness of selection. A sustained reduction in population size may result
in the loss of diversity because of genetic drift that is a
consequence of sampling effects between generations, inbreeding as a result of higher frequencies of consanguineous
matings, and for many flowering plants, increased selfing due
to reduced pollinator activity (Barrett and Kohn 1991; Young
et al. 1996; Kearns et al. 1998). For species with substantial
amounts of genetic load, increased selfing and biparental inbreeding may be accompanied by fitness losses through the
exposure of deleterious recessive alleles to selection (Barrett
and Kohn 1991; Young et al. 1996). Genetic load may be
purged after several generations of selfing (Barrett and
Charlesworth 1991), or these fitness losses may become permanent through the fixation of deleterious recessive alleles
in cases where genetic load is determined by many loci of
small effect (Willis 1999). The persistence of small populations that are suffering the effects of increased frequencies
of deleterious alleles may be enhanced by gene flow from
1569
q 2001 The Society for the Study of Evolution. All rights reserved.
1570
MITCHELL B. CRUZAN
neighboring populations, which would tend to counteract the
potentially negative effects of genetic drift (Templeton et al.
1990).
Individual populations may be interconnected by dispersal
to form integrated groups or metapopulations (Hanski 1996;
Husband and Barrett 1996). Although it is often assumed that
genetic diversity is maintained on a regional scale by gene
flow, the degree of interdependence among populations has
only rarely been tested (e.g., Young et al. 1993; Prober and
Brown 1994; Hall et al. 1996; Barrett and Husband 1997;
Nason and Hamrick 1997; Vrijenhoek 1997). In particular,
the effects of the geographic distribution of populations and
consequences of different levels of fragmentation for the
preservation of genetic diversity are not well known (Templeton et al. 1990; Husband and Barrett 1996; Young et al.
1996). For example, analyses of the distribution of genetic
variation typically do not assess the contribution of the spatial
arrangement of populations to levels of genetic variation and
patterns of gene flow (Kudoh and Whigham 1997; Vrijenhoek
1997; Bossart and Prowell 1998; Whitlock and McCauley
1999; but see Barrett and Husband 1997; Goodell et al. 1997).
Populations that have been isolated for long periods, either
because of disjunct distributions or metapopulation decay
(i.e., regional extinction of populations), would be expected
to have reduced genetic diversity compared to populations
that were subject to gene flow from neighboring populations
(Templeton et al. 1990; Young et al. 1996). The combination
of geographic dispersion of populations and relative dispersal
ability will influence regional patterns of genetic variation
and may affect long-term evolutionary processes such as the
spread of novel adaptations across a species’ range (Wright
1931; Wade and Goodnight 1998).
Here I use spatially explicit analyses of genetic parameters
to assess the effects of population dispersion on the maintenance of genetic diversity. In particular, I examine the effects of local population extinction on genetic erosion by
investigating the relationship between the degree of population isolation and measures of genetic diversity. Unfortunately, a theoretical framework for the analysis of the effects
of the spatial distribution of populations on the maintenance
of genetic variation is not well developed (Husband and Barrett 1996). However, the lack of such models does not preclude the statistical analysis of spatial patterns. In this study
I obtain an approximate assessment of the effects of spatial
dispersion on patterns of gene diversity and selfing using
stepwise regression analysis of metapopulation circle diameter (i.e., the number of populations present within a circumscribed region around each population) on within-population genetic parameters. My expectation is that the metapopulation diameter that displays the strongest association
with variation in genetic parameters will be indicative of the
geographic scale over which changes in the number of local
populations have the largest impact on genetic processes. For
example, in a system in which dispersal contributes to the
maintenance of genetic variation, the metapopulation density
within circle diameters that are smaller than the effective
dispersal distance would not be expected to be strongly associated with differences in the level of genetic diversity
within individual populations. However, as the circle diameter approaches the maximum effective dispersal distance,
the correlation between the metapopulation density and genetic diversity should increase. The explanatory power of
even larger circle diameters would be lower because the metapopulation would include populations that may be beyond
the effective dispersal distance. Thus, stepwise regression
analyses of the number of populations present within concentric circles of increasing size provides an appraisal of
geographic scale for genetic processes and allows the characterization of fragmentation thresholds for metapopulation
decay.
In this paper I assess the effects of population size and
metapopulation structure on patterns of genetic diversity and
inbreeding in Scutellaria montana Chapman (Lamiaceae), an
herbaceous perennial endemic to southeastern North America. This is an ideal system for analyses of metapopulation
structure because it grows over a restricted range, so populations can be sampled at a relatively high density; extensive
surveys for occurrences have been conducted, so accurate
estimates of metapopulation density can be made; and regional variation in population density is substantial enough
to allow an assessment of the degree of population isolation
on population genetic parameters. I use the results of these
analyses to identify critical levels of fragmentation and population size for the long-term maintenance of genetic diversity.
MATERIALS
AND
METHODS
The large-flowered skullcap (Scutellaria montana) is an
herbaceous perennial found in dry and semimesic deciduous
forests of southeastern Tennessee and northwestern Georgia
(Fig. 1). Plants tend to be sparsely distributed in the forest
understory, with individuals often separated by one to several
meters and densities rarely exceeding a few individuals per
square meter. Individual plants consist of one to several stems
(20–40 cm tall) that emerge from the same location each year
and do not display any tendency for clonal spread (based on
a four-year demographic study of individually marked plants;
unpubl. data). Newly germinated plants typically do not flower for the first few seasons. Larger plants produce between
two and 20 flowers per stem during the month of May. Flowers are light blue to white, with some darker blue markings
near the opening of the floral tube. The long floral tube (3–
4 cm) and a sucrose-hexose ratio near 50% (unpubl. data)
are indicative of an historical association with moths or longtonged bees as the primary pollen vector (Baker and Baker
1979; Southwick 1982; Kearns and Inouye 1993). However,
several hundred hours of observation over four seasons suggest that these pollinators may be rare or lacking (unpubl.
data). Plants are self-compatible, but most individuals produce very few seeds, largely because seed set is limited by
low levels of pollen deposition on stigmas (unpubl. data).
In the spring of 1998, 31 populations were sampled across
the range of S. montana in Tennessee and Georgia. Surveys
of the occurrence of this species by resource managers have
been extensive (Shea and Hogan 1998), so in most cases I
was able to change the intensity of population sampling along
with the density of populations in each region. However, a
number of additional populations were discovered in Georgia
after the genetic sampling was complete, so my sampling
FRAGMENTATION THRESHOLDS IN S. MONTANA
1571
FIG. 1. The distribution of populations of Scutellaria montana in Tennessee and Georgia. Filled circles indicate populations that were
sampled. Populations are divided into two regions: the Tennessee River region northwest of Taylor Ridge and the Oostanaula River
region southeast of Taylor Ridge.
density is relatively sparse in some areas (Fig. 1). Areas with
high concentrations of known sites (e.g., Lookout Mountain
and Signal Mountain in TN and Floyd Co. in GA) were sampled more intensively, and an effort was made to include
many isolated populations (Table 1, Fig. 1).
Populations were sampled by removing a single leaf from
each plant, and sampling was distributed across the population to cover the majority of each area of occurrence (generally between 100 m2 and 500 m2). Leaves were sampled
from up to 40 plants (larger populations) or all of the individuals that could be located (smaller populations) at each
site (Table 1). Three of the larger populations had slightly
fewer than 40 samples because some were lost after collection. Leaf samples were stored on ice until they could be
returned to the laboratory for processing.
The total census population size (Nest) was estimated by
direct counts (for populations less than 100 individuals) or
by subsampling and extrapolating to the total area. To take
into account individuals that were not immediately apparent
during surveys, estimates were rounded up to the nearest digit
evenly divisible by 10 for population sizes that were less than
100, and to the nearest 100 for population sizes that were
greater than 100 but less than 500 (Table 1). Four populations
that were very large were recorded as being greater than 500
individuals. When possible, size estimates were confirmed
with censuses available from databases maintained by the
Tennessee and Georgia Natural Heritage Programs (Shea and
Hogan 1998).
Leaf samples were prepared for allozyme electrophoresis
by placing 0.5-cm2 samples of fresh leaf material in 1.5-ml
microcentrifuge tubes. Samples were pulverized in liquid nitrogen before adding 200 ml of buffer (0.028 M ascorbic acid,
0.01 M MgCl2, 0.1 M KCl, 0.001 M EDTA, 0.2 M sucrose,
0.005 M sodium bisulfate, 2%PVP-40T,0.006 M diethyl dithiocarbamate, and a trace of b-mercaptoethanol in 0.1 M
tris HCl buffer at pH 8.0) followed by 30 sec of additional
maceration. The resulting supernatant was soaked onto filter
paper wicks (Whatman no. 3, Whatman International, Maidstone, England) that were stored at 2708C. Wicks were loaded into 11% hydrolyzed starch gels (Sigma S-4501, Sigma
Co., St. Louis, MO) and proteins separated as described in
Acquaah (1992) for a type I electrophoresis system. All gels
were run for 5 h at 50 mA before they were sliced, stained
for specific enzyme activity using previously described pro-
1572
MITCHELL B. CRUZAN
TABLE 1. Collection site locations, latitudes, number of neighboring populations within radii of 1 km (n2) and 4 km (n8), estimated population
size (Nest), and the number of plants sampled (Nsamp) for populations of Scutellaria montana in Tennessee Georgia. Population size estimates
are based on surveys conducted at the time of leaf sample collection and from databases maintained by conservation organizations.
Population
Elsi A. Holmes
Taylor Ridge
Blacks Bluff
Flower Glenn
Milner Lake 1
Milner Lake 2
Milner Lake 3
Turnip Mt.
Big Ridge
Bowater Creek
Chigger Point
Fairview Slopes
Glen Falls
Grasshopper Creek
Gum Springs
Murphey Hill
Old Hog Lot
Reflection Ridge
Rainbow Lake
Sanders Picnic
Skyuka Trail
Stanley Branch
Truck Trail
Upper Truck
Whiteside Trail
Ellis Spring S
Ellis Spring N
Huff Branch
Mullens Creek
McBrien Ln.
County
State
Latitude (N)
n2
n8
Nest
Nsamp
Catoosa
Chattooga
Floyd
Floyd
Floyd
Floyd
Floyd
Floyd
Hamilton
Hamilton
Hamilton
Hamilton
Hamilton
Hamilton
Hamilton
Hamilton
Hamilton
Hamilton
Hamilton
Hamilton
Hamilton
Hamilton
Hamilton
Hamilton
Hamilton
Marion
Marion
Marion
Marion
Marion
GA
GA
GA
GA
GA
GA
GA
GA
TN
TN
TN
TN
TN
TN
TN
TN
TN
TN
TN
TN
TN
TN
TN
TN
TN
TN
TN
TN
TN
TN
34.872
34.474
34.209
34.243
34.187
34.187
34.187
34.252
35.106
35.236
35.334
35.135
34.988
35.219
35.002
35.301
35.159
35.002
35.126
34.991
35.007
35.153
35.002
35.005
35.003
35.035
35.053
35.026
35.071
35.080
0
1
0
0
5
5
5
1
0
1
0
0
3
0
9
1
5
8
3
3
7
7
8
9
2
0
1
0
0
1
3
1
1
1
8
8
8
4
1
5
0
4
16
0
17
4
19
17
18
17
17
20
17
17
17
7
10
7
6
6
500
500
200
500
50
100
500
200
20
200
40
20
60
20
20
100
100
60
200
300
60
100
100
20
300
100
100
10
100
50
40
40
38
40
34
40
40
40
13
41
29
11
40
16
12
37
40
40
40
40
40
40
40
16
40
39
40
7
40
37
cedures (Acquaah 1992; Wang and Cruzan 1998), and documented with a video image copier. Interpretations of banding patterns were made according to the known quaternary
structure of proteins to identify the separate loci and allelic
associations for each enzyme system. An initial screen of
allozyme variation was conducted to identify resolvable loci
by testing 20 enzymes on four different buffer systems.
Population genetic data were analyzed to assess variation
in the level of inbreeding, the distribution of genetic variation, and to determine whether equilibrium conditions existed
at local or regional scales for levels of gene flow and genetic
drift. I estimate the degree of inbreeding from the level of
heterozygote deficiency as compared to the Hardy Weinberg
expectation (i.e., the inbreeding coefficient or fixation index:
F 5 [He 2 Ho]/He; Hedrick 1983). Although variation in this
parameter will potentially capture the effects of all types of
consanguineous matings, it is primarily determined by the
level of selfing and sibling mating (Hedrick 1983; Ritland
and Ganders 1987). The level of population differentiation
(GST), expected and observed levels of heterozygosity (He
and Ho, respectively), effective number of alleles per locus
(Ae), and proportion of polymorphic loci (P) were estimated
using the PopGene software package (Yeh and Boyle 1997).
The relationship between geographic distance and level of
genetic differentiation was assessed to determine the contributions of gene flow and genetic drift to patterns of genetic
diversity using paired-FST analysis (Hutchison and Templeton 1999). The strength of the association between geographic distance and genetic similarity was estimated using Mantel
tests to compare pairwise genetic and geographic distance
matrices (Smouse et al. 1986). Matrices were constructed for
each separate region (see below) using FSTAT (ver. 2.8;
Goudet 1995) for the paired-FST matrix and by calculating
the straight-line distance between each pair of coordinates
for the geographic distance matrix. Mantel tests of significance for the correlation between the paired-FST and geographic distance matrices were made using 1000 permutations of the data with the MATCOM program (D. J. Perry,
Lakehead Univ., Thunder Bay, Ontario, Canada).
Data were analyzed to assess the effects of population size
and the regional density of populations on the level of genetic
diversity and the degree of selfing and biparental inbreeding.
The number of neighboring populations was estimated from
the geographic coordinates of all known occurrences of S.
montana using ArcView GIS (Environmental Systems Research Institute, Redlands, CA). This analysis was possible
because extensive surveys for occurrences of S. montana have
been conducted across its distributional range (Shea and Hogan 1998). The number of neighboring populations (nd, where
d refers to the circle diameter) was estimated by counting
the number of populations present within concentric circles
around each population. Six different estimates of nd were
made for each population using circle diameters of 0.5, 1, 2,
4, 8, and 16 km to assess the effects of long-distance pollen
dispersal on the genetic parameters measured. Stepwise regression models were used to examine the influence of latitude, the estimated population size (Nest), and the number
of neighboring populations (nd) on the population genetic
1573
FRAGMENTATION THRESHOLDS IN S. MONTANA
parameters described above using the REG procedure of SAS
(SAS Institute 1989). For each model, the latitude and the
estimated population size were retained, whereas the nd variables for each circle diameter was entered one at a time to
identify the metapopulation diameter that maximized R2 for
the model (using the MAXR selection method and limiting
the model size to three independent variables). Separate stepwise regression models were analyzed to assess the amount
of variance explained by different circle diameters for the
observed heterozygosity (Ho), the fixation index (F), the effective number of alleles per locus (Ae), and the proportion
of polymorphic loci (P). All dependent variables, latitude,
and Nest values were log-transformed to improve normality.
All nd values were square-root transformed to avoid giving
excessive weight to larger samples.
RESULTS
Our initial screening of 20 enzymes on four buffer systems
yielded a total of eight resolvable loci (Est-1, Mdh-1, Mdh2, Pgm-2, Pgi-1, Pgi-2, Pgi-3, and Skd-1, where the lowest
numbers designate the locus closest to the origin). All of the
resolved loci were polymorphic with between two and seven
alleles per locus (Table 2). Populations were divided into two
major regions for analysis because it was determined that
populations southeast of Taylor Ridge in Georgia (Oostanaula River region; Fig. 1, Table 2) were genetically distinct
and lacked a number of alleles present in populations northwest of Taylor Ridge (Tennessee River region; Fig. 1, Table
2). This division is supported by analysis of chloroplast DNA
variation, which indicates that populations of S. montana are
divided into two geographically distinct groups of populations that are probably derived from separate Pleistocene refugia (unpubl. data).
Levels of genetic variation within populations were relatively high, with the majority of loci surveyed being polymorphic in nearly all of the populations sampled (Table 3).
Genetic diversity, as measured by the mean expected heterozygosity across populations, was also high. The observed
levels of heterozygosity were substantially lower than expected levels, which led to high fixation indices (F) for the
majority of populations (Table 3).
Statistics describing the distribution of genetic variation
indicate that the majority of variation is contained within the
populations in each geographic region delineated by Taylor
Ridge rather than among populations (Table 4). Levels of
differentiation within the Tennessee and Oostanaula River
drainages were relatively low, suggesting that gene flow
among populations in each region may be relatively high.
However, Mantel tests indicate that the relationship between
geographic and genetic distance in each region was relatively
weak (r 5 0.136, P 5 0.051; r 5 0.346, P 5 0.098 for the
Tennessee and Oostanaula drainages, respectively), indicating a lack of equilibrium between gene flow and drift, which
may render gene flow estimates that are based on levels of
differentiation unreliable (Hutchison and Templeton 1999;
Whitlock and McCauley 1999).
Examination of variation in metapopulation size indicates
that aggregations of populations were more effective at reducing the frequency of inbreeding (measured by heterozy-
TABLE 2. Allele frequencies for populations of Scutellaria montana
in the Tennessee River and Oostanaula River subregions and for all
populations combined.
Tennessee River
Oostanaula River
All populations
Est-1
a
b
Locus
0.952
0.048
0.948
0.052
0.951
0.049
Mdh-1
a
b
0.980
0.020
0.998
0.002
0.984
0.016
Mdh-2
a
b
c
0.003
0.965
0.032
—
1.000
—
0.003
0.973
0.024
Pgm-2
a
b
c
d
e
f
0.054
0.603
0.013
0.000
0.327
0.003
—
0.569
0.017
0.002
0.412
—
0.041
0.595
0.014
0.001
0.347
0.002
Pgi-1
a
b
c
0.438
0.398
0.164
0.123
0.020
0.857
0.365
0.311
0.324
Pgi-2
a
b
c
d
e
f
g
0.001
0.495
0.018
0.474
0.001
0.010
0.001
0.004
0.420
0.024
0.550
—
0.002
—
0.001
0.477
0.019
0.492
0.001
0.009
0.001
Pgi-3
a
b
c
d
e
f
0.006
0.582
0.025
0.325
0.018
0.044
—
0.684
0.082
0.232
0.002
—
0.005
0.606
0.038
0.304
0.014
0.033
Skdh
a
b
c
0.001
0.997
0.002
—
0.998
0.002
0.001
0.997
0.002
gote deficiency as compared to the Hardy-Weinberg expectation) and maintaining genetic diversity than isolated populations. Genetic parameters associated with variation in the
level of selfing and biparental inbreeding (Ho and F) were
most strongly affected by the number of populations within
the immediate vicinity of a population (metapopulation diameters of 1–2 km; Fig. 2A). The increase in the level of
observed heterozygosity (Ho), and the decrease in the severity
of heterozygote deficiency (F) for larger metapopulations was
best predicted using a circle diameters of 1 km and 2 km,
respectively, but the variance explained by metapopulation
size dropped off steeply for larger diameters (Fig. 2A). For
measures of genetic diversity, however, the predictive power
of metapopulation diameters increased from smaller circles
and peaked for diameters between 4 km and 8 km (Fig. 2B).
Both the effective number of alleles (Ae) and the proportion
1574
MITCHELL B. CRUZAN
TABLE 3. Genetic diversity statistics for poulations of Scutellaria montana. Measures of population genetic variation include the effective
number of alleles per locus (Ae), the observed and expected level of heterozygosity (Ho and He, respectively) with standard deviations in
parentheses, the number of polymorphic loci (Pn), the proportion of polymorphic loci (P), and the heterozygote deficiency (F 5 He 2 Ho /He).
Population
Ae
Big Ridge
Glen Falls
McBrien Ln.
Chigger Point
Elsie A. Holmes
Skyuka Trail
Murphey Hill
Fairview Slopes
Grasshopper Creek
Whiteside Trail
Taylor Ridge
Huff Branch
Ellis Spring S
Ellis Spring N
Mullens Creek
Turnip Mt.
Flower Glenn
Milner Lake 1
Milner Lake 2
Milner Lake 3
Blacks Bluff
Reflection Ridge
Rainbow Lake
Upper Truck
Gum Springs
Truck Trail
Bowater Creek
Sanders Picnic
Old Hog Lot
Stanley Branch
Mean
1.50
2.50
2.13
2.50
2.50
2.89
2.00
1.87
2.50
2.38
1.88
2.00
2.00
2.75
2.38
2.13
2.00
2.13
2.00
2.00
1.75
2.00
2.13
2.00
2.25
2.38
2.25
2.63
2.50
2.38
2.21
Ho
0.16
0.19
0.15
0.18
0.25
0.17
0.15
0.18
0.22
0.14
0.13
0.10
0.18
0.19
0.18
0.23
0.17
0.20
0.17
0.20
0.20
0.18
0.26
0.23
0.30
0.17
0.23
0.23
0.22
0.20
0.19
0.21
0.32
0.27
0.30
0.31
0.31
0.26
0.28
0.35
0.30
0.23
0.25
0.29
0.33
0.26
0.25
0.22
0.25
0.17
0.22
0.23
0.29
0.31
0.28
0.35
0.24
0.28
0.33
0.29
0.27
0.28
(0.22)
(0.16)
(0.18)
(0.17)
(0.23)
(0.19)
(0.20)
(0.32)
(0.19)
(0.15)
(0.19)
(0.12)
(0.17)
(0.17)
(0.17)
(0.27)
(0.25)
(0.32)
(0.27)
(0.28)
(0.30)
(0.19)
(0.22)
(0.23)
(0.29)
(0.20)
(0.25)
(0.22)
(0.21)
(0.15)
of polymorphic loci (P) were higher when more populations
were present in the associated metapopulation circles of 4
km and 8 km, respectively (Fig. 2B).
The level of genetic diversity in populations of S. montana
appears to be affected by latitudinal position, the estimated
size of the population (Nest), and the presence of other populations within the immediate region (nd; Fig. 3, Table 5).
Moving from southern to northern populations, there was a
tendency for the effective number of alleles per locus (Ae)
and the proportion of polymorphic loci (P) to increase (Table
5). Once the effect of latitude is held constant, it is apparent
that both population size (Nest) and the number of neighboring
He
Pn
(0.23)
(0.27)
(0.29)
(0.24)
(0.27)
(0.31)
(0.27)
(0.31)
(0.24)
(0.30)
(0.27)
(0.25)
(0.27)
(0.29)
(0.23)
(0.26)
(0.24)
(0.26)
(0.23)
(0.23)
(0.22)
(0.27)
(0.23)
(0.27)
(0.24)
(0.24)
(0.26)
(0.27)
(0.26)
(0.25)
4
7
5
7
8
5
6
4
7
6
4
5
6
7
8
6
5
5
6
5
5
6
7
5
6
6
7
8
7
8
6.03
P
F
0.24
0.41
0.44
0.40
0.19
0.45
0.42
0.36
0.37
0.53
0.43
0.60
0.38
0.42
0.31
0.08
0.23
0.20
0.00
0.09
0.13
0.38
0.16
0.18
0.14
0.29
0.18
0.30
0.24
0.26
0.29
50.0
87.5
62.5
87.5
100.0
62.5
75.0
50.0
87.5
75.0
50.0
62.5
75.0
87.5
100.0
75.0
62.5
62.5
75.0
62.5
62.5
75.0
87.5
62.5
75.0
75.0
87.5
100.0
87.5
100.0
75.42
populations (nd) have an effect on the maintenance of genetic
variation (Fig. 3, Table 5). The effect of population size on
these parameters is apparently nonlinear (Fig. 3), making it
difficult to discern using multiple regression analyses (Table
5). However, the effect of population size on genetic diversity
parameters can be seen by dividing populations into small
(Nest , 100) and large (Nest $ 100) groups. This analysis
indicates that a higher number of the populations that were
less than 100 individuals tended to have lower proportions
of polymorphic loci (t 5 3.81, P 5 0.0021, with 23 df for
the comparison of P between populations ,100 and $100
plants; Fig. 3, Table 5). The effect of small population size
TABLE 4. Genetic statistics for populations of Scutellaria montana in the Tennessee River and Oostanaula River subregions and for all
populations combined. Total genetic variation (HT) is partitioned into its within (HS) and among (GST) population components.
Oostanaula River
Tennessee River
Locus
Est-1
Mdh-1
Mdh-21
Pgm-2
Pgi-1
Pgi-2
Pgi-3
Skdh
Mean
1
All populations
HT
HS
GST
HT
HS
GST
HT
HS
GST
0.125
0.276
0.066
0.108
0.755
0.306
0.355
20.002
0.375
0.087
0.135
0.033
0.054
0.720
0.274
0.309
20.012
0.314
0.042
0.163
0.034
0.057
0.127
0.044
0.067
0.010
0.075
0.032
20.002
—
0.062
0.915
20.440
0.537
20.002
0.154
0.008
20.013
—
0.017
0.909
20.482
0.482
0.012
0.104
0.024
0.010
—
0.046
0.062
0.028
0.107
0.010
0.057
0.106
0.275
0.072
0.102
0.812
0.162
0.392
20.002
0.374
0.071
0.132
0.033
0.047
0.738
0.122
0.338
20.013
0.287
0.038
0.164
0.040
0.058
0.281
0.045
0.081
0.010
0.121
This locus was not analyzed for the Oostanaula River because all populations in this region were fixed for allele b (see Table 2).
1575
FRAGMENTATION THRESHOLDS IN S. MONTANA
FIG. 2. The explanatory power (mean square) of each metapopulation circle diameter (nd) for variation in parameters associated
with the level of selfing or sibling mating (A) and genetic diversity
(B) in Scutellaria montana. Mean squares are from general linear
models (GLM procedure; SAS Institute 1989) for the analysis of
each genetic parameter that included geographic region, latitude,
and estimated population size as independent variables. Parameters
associated with selfing or sibling mating for individual populations
include the observed heterozygosity (Ho) and the fixation index (F).
Population genetic diversity parameters are the effective number of
alleles (Ae) and the proportion of polymorphic loci (P). Asterisks
indicate circle diameters identified by stepwise regression analysis
as producing models with the highest R2.
on the mean level of allelic diversity (Ae) is not as clear (t
5 1.07, P 5 0.149, df 5 23; Table 5), but there was higher
among-population variance in the effective number of alleles
for small than for large populations (F 5 2.93, P , 0.023,
df 5 12, 16; Fig. 3).
FIG. 3. Effects of census population size on the proportion of
polymorphic loci (A) and the effective number of alleles per locus
(B) in Scutellaria montana. In each case the values plotted are
residuals from a general linear analysis (GLM procedure; SAS Institute 1989) that included the river drainage region and latitude as
independent variables. Lines are based on the backtransformed predicted values from linear regression models that used the natural
log of the estimated population size.
DISCUSSION
The maintenance of genetic diversity in S. montana is associated with the size of individual populations and the number of populations within close dispersal distances to each
other. The loss of genetic variation is apparent in smaller
populations, but genetic erosion is offset by gene flow from
neighboring populations producing a rescue effect (Brown
and Kodric-Brown 1977) for the maintenance of genetic di-
TABLE 5. The effects of latitude, the estimated population size (Nest), and the estimated number of neighboring populations (nd) on levels of
allelic diversity (Ae), observed heterozygosity (Ho), expected heterozygosity (He), percent polymorphic loci (P), and the heterozygote deficiency
(F) in Scutellaria montana. Dependent variables used were the residuals from general linear model analysis (GLM procedure; SAS Institute
1989) of the effects of river drainage region on each genetic parameter. Slopes from multiple regression models are given along with their
corresponding probabilities (in parentheses). Significant values (P , 0.05) are shown in bold.
Dependent Variable
Ho
He
P
F
Metapopulation diameter 5 2 km
Latitude
2.440 (0.148)
Nest1
0.115 (0.160)
0.089 (0.073)
n2
Source
29.216 (0.058)
20.132 (0.150)
0.189 (0.034)
20.049 (0.208)
20.003 (0.439)
0.066 (0.420)
11.605 (0.009)
0.195 (0.048)
0.039 (0.185)
219.807 (0.192)
20.176 (0.271)
20.151 (0.040)
Metapopulation diameter 5 8 km
Latitude
1.818 (0.169)
Nest1
0.133 (0.180)
0.095 (0.042)
n8
28.440 (0.117)
20.144 (0.270)
0.078 (0.134)
20.054 (0.197)
20.001 (0.428)
0.044 (0.252)
10.177 (0.006)
0.231 (0.036)
0.111 (0.049)
4.456 (0.359)
20.157 (0.478)
20.049 (0.251)
1
Ae
Slopes for the natural log of Nest 3 1000.
1576
MITCHELL B. CRUZAN
versity. It appears that the combined effect of isolation and
small population size leads to the greatest loss of alleles and
reduction in the proportion of polymorphic loci. The expected
loses in genetic diversity in association with restricted ranges
(Karron 1987, 1991; Hamrick and Godt 1990, 1996) or in
smaller populations (Barrett and Kohn 1991) have often been
investigated, but have not always been detected (Ellstrand
and Elam 1993). The approach that I have taken to study the
distribution of genetic diversity in S. montana provides a
clearer picture of the contributions of gene flow and population size to patterns of genetic erosion. Using information
on the number of neighboring populations within different
geographic ranges has allowed me to infer the distance over
which gene flow potentially contributes to the maintenance
of genetic diversity. The analyses of the number of neighboring populations on patterns of inbreeding suggest that
ecological conditions in locally isolated populations may
have led to increases in the level of selfing or sibling mating.
However, it is not clear that these circumstances have existed
for very long because analyses of genetic diversity at the
same spatial scale do not indicate a substantial loss of genetic
diversity in these populations.
An implicit assumption of this analysis is that populations
of S. montana were historically more abundant and that within
each geographic region they had similar levels of genetic
diversity. Under this scenario, the observed loss of genetic
variation in geographically isolated populations would be due
to the local extinction and the resulting loss of gene flow
from neighboring populations. Alternatively, isolated populations could be the result of recent colonizations, in which
case the observed lower levels of genetic diversity would be
a consequence of population bottlenecks rather than genetic
drift. The latter scenario is unlikely because phylogeographic
evidence indicates that S. montana has recently undergone
range contraction (unpubl. data), isolated populations (i.e.,
n8 , 5) are distributed throughout the range of S. montana
and not just in a region of recent expansion, and populations
of S. montana are not growing rapidly and have low rates of
seed production (unpubl. data), so colonization rates for the
metapopulation are probably low (Hanski 1996). Moreover,
this species has large, gravity-dispersed seeds, so I would
expect that long distance dispersal of seed would be extremely rare. The low rates of seed production, reduced population
growth, and the restricted dispersal ability of S. montana
suggest that the sparse distribution of this species is a consequence of local and regional extinctions.
The variance in genetic diversity estimates among populations of different size provides additional evidence of recent
changes in the abundance of S. montana. Large populations
had consistently high levels of genetic variation, whereas
smaller populations displayed a wider range of levels of genetic diversity. It is important to note that substantial loss of
genetic diversity in small populations is expected only if their
reduced size were sustained for extended periods of time
(Barrett and Kohn 1991; Montgomery et al. 2000). The high
level of genetic variation present in many of the smaller
populations suggests that they may have only recently experienced reductions in size, and the consistently high level
of genetic diversity across larger populations indicates that
their size has remained relatively constant. Furthermore, none
of the larger populations display reduced levels of genetic
diversity, which would be expected if they had recently been
derived from smaller populations (Barrett and Kohn 1991).
Thus, the high variance in genetic diversity among small
populations and low variance for estimates from large populations is consistent with the hypothesis that populations of
S. montana are generally in the process of demographic decline.
The stepwise regression approach used in this study provides an approximate assessment of the scale over which
metapopulation structure affects population genetic processes. Although application of these methods could potentially
provide important information on the effect of population
dispersion on a wide range of ecological and evolutionary
processes, there are several limitations to this procedure that
should be recognized. First, the methods presented produce
a quantitative analysis of spatial patterns, however, the analytical relationship between the patterns detected and actual
biological processes that produce them have not been discerned. Thus, the fragmentation thresholds reported are largely qualitative, and spatially explicit models would have to
be developed before quantitative estimates of the processes
responsible for these patterns could be made. Second, because
populations that are in close proximity are connected by dispersal, they will tend to covary for the parameters being
analyzed, which would tend to reduce the level of independence when more than one focal population is used from each
metapopulation. This problem is difficult to circumvent because using only a single population from each population
cluster would restrict the sample sizes for larger metapopulations. However, as long as samples are taken from multiple
population clusters (as in the current analysis), the fragmentation thresholds detected with this stepwise regression approach should be relatively robust to covariation introduced
by geographic proximity.
Distribution of Genetic Variation
Levels of genetic variation in S. montana were relatively
high compared to other plant species with similar life-history
characteristics. The average level of genetic diversity in this
herbaceous endemic was higher than widespread herbaceous
perennials and other species that are primarily outcrossed and
associated with animal pollinators (Hamrick and Godt 1990).
Genetic variation in this species may be inflated because there
may be relatively high levels of gene flow among populations,
plants in each population may be relatively long-lived, and
populations appear to have persistent seed banks, which may
buffer them from the effects of genetic drift (Templeton and
Levin 1979). The weak among-population genetic differentiation within each river drainage suggests that gene flow
may be relatively high, perhaps because of a historical relationship with pollinators that are capable of long flight distances, such as large moths or bees (Chase et al. 1996). This
hypothesis is consistent with analyses of the floral morphology and the sugar composition of nectar of S. montana,
which also suggest an association with large-bodied pollinators (unpubl. data). A persistent seed bank is likely in this
species because cold treatments failed to break seed dormancy (unpubl. data). This same treatment resulted in rela-
FRAGMENTATION THRESHOLDS IN S. MONTANA
tively high germination frequencies for the closely related S.
pseudoserrata, thus dormancy in the seeds of S. montana is
probably complex, requiring a combination of factors to promote germination (Baskin and Baskin 1998). Both high rates
of gene flow as a result of long-distance pollen dispersal and
large effective population sizes due to persistent seed banks
may have contributed to the maintenance of high levels of
genetic variation in populations of S. montana.
Although gene flow among populations of S. montana is
evident, the dispersal range of this species may be restricted
because the distribution of genetic variation was uneven and
varied with the number of neighboring populations. Stepwise
regression analyses of metapopulation circle diameters indicates that the number of populations present within 8-km
circles had the greatest predictive power for differences in
levels of genetic diversity within populations. These analyses
suggest that dispersal is frequent enough over distances of 4
km or less (the radius of the circle) to effectively maintain
allelic diversity within populations. It is interesting to note,
however, that although dispersal rates over these distances
are substantial enough to maintain genetic variation, the frequency of pollen movement among populations was not frequent enough to render these metapopulations genetically
homogenous. The paired-FST analyses indicate that there is
not a strong relationship between genetic similarity and geographic distance as would be expected under the equilibrium
conditions of an isolation-by-distance model (Hutchison and
Templeton 1999). However, even very low rates of gene flow
among populations may be adequate to counteract the effects
of genetic drift (Lacy 1987; Young et al. 1996). The present
study suggests that low levels of gene flow may be great
enough to infuse genetic variation into populations without
having a strong influence on native allele frequencies.
Patterns and Probable Causes of Inbreeding
Heterozygote deficiency as a consequence of inbreeding is
generally associated with an increase in the frequency of
selfing or with small population size, when matings among
close relatives may be prevalent (Barrett and Kohn 1991;
Prober and Brown 1994). However, an apparent discrepancy
between the observed and expected number of heterozygotes
could be produced in the absence of inbreeding. For example,
sampling across genetically differentiated patches within
populations would tend to inflate the fixation index (i.e., due
to a Wahlund effect; Hedrick 1983). This does not appear to
be the case in the present study because the density of individuals was similar across populations and heterozygote
deficiencies were not greater in large populations, as would
be expected if they were subdivided into distinct patches. It
is more likely that the patterns of heterozygote deficiency
among populations of S. montana are largely do to variation
in the level of inbreeding.
Remote populations of S. montana would be expected to
have lower levels of genetic diversity because of reduced
levels of gene flow, and the loss of variation in these populations could be exacerbated by higher frequencies of selfing
or sibling mating. The observation that isolated populations
have more substantial heterozygote deficiencies is surprising
and would not be predicted from the consideration of prin-
1577
ciples from population genetics alone. The reduced frequencies of heterozygotes are typically due to higher levels of
selfing or sibling mating, which is more common in small
populations (Raijmann et al. 1994; Young et al. 1996), but
it is unclear why there would be more frequent mating among
close relatives in both large and small populations that were
remote from other populations. One possibility is that the
level of inbreeding is equivalent across populations, but high
levels of gene flow among populations that are in close proximity serves to reduce the fixation index (Allendorf 1983).
However, the effectiveness of this mechanism has been questioned (Ellstrand and Elam 1993). Moreover, such high rates
of dispersal would rapidly homogenize allele frequencies
among local populations, which was not the case in S. montana; thus, gene flow is probably not responsible for the
observed lack of heterozygote deficiency in clusters of populations.
Gene flow appears unlikely to account for the observed
patterns of heterozygote deficiency, and there are much more
plausible ecological explanations for the increased levels of
inbreeding in remote populations. In particular, changes in
the pollinator fauna in isolated populations of S. montana
may result in higher levels of inbreeding because of more
frequent selfing. Lack of pollinator service is known to lead
to higher selfing frequencies in self-compatible taxa (Thomson and Stratton 1985; Barrett et al. 1989; Rigney et al. 1993;
Washitani 1996; Kearns et al. 1998), and reduced pollinator
service and depauperate pollinator faunas have been frequently documented in both habitat fragments and isolated
populations (Powell and Powell 1987; Aizen and Feinsinger
1994; Washitani et al. 1994; Didham 1996; Steffan-Dewenter
and Tscharntke 1999). It is possible that the observed variation in inbreeding in populations of S. montana is closely
tied to the availability of its specialist pollinators. Surveys
of pollen loads on stigmas indicate that even in areas where
S. montana is regionally abundant, lack of pollination severely limits seed set (unpubl. data). If the availability of
pollinators has historically been lower in isolated populations, then more frequent autogamous selfing (i.e., self pollination in the absence of a pollen vector) would be expected
to result in higher levels of heterozygote deficiency.
It has been suggested that plant species with more specialized pollinator associations are more vulnerable to variation in the abundance of their floral visitors, but there are
few examples of the detrimental effects of the loss of pollinator service in species with extreme floral morphologies
(e.g., Washitani et al. 1994). In the case of S. montana, the
floral tube is exceptionally long (3.0–3.5 cm) compared to
the other insect-pollinated species of Scutellaria in North
American, which generally have tubes of 2.5 cm or less
(Leonard 1927; Epling 1942). Such an exaggerated floral
morphology is indicative of an association with a specific
subset of the pollinator fauna (Grant and Grant 1965; Faegri
and van der Pijl 1966) because an excessively long toung
would be required to achieve pollination during the process
of foraging. The hypothesized loss of specialized pollinators
in this species is corroborated by field observations of floral
visitors and by the lack of pollen deposition on stigmas (unpubl. data). Over a period of four years and several hundred
hours of observation the only floral visitors that were ob-
1578
MITCHELL B. CRUZAN
served on S. montana were small-bodied bees, which did not
effectively contact the reproductive organs, and large bumblebees (Bombus sp.), which acted as nectar thieves and foraged by chewing holes at the base of the corolla (unpubl.
data). It is probable that the absence or rarity of one particular
class of pollinators has had detrimental effects on the fecundity and possibly on the level of selfing in S. montana populations. Absence of long-tounged pollinators may ultimately
have consequences for rates gene flow and the levels and
distribution of genetic variation among populations of S.
montana.
ditions in contemporary populations of S. montana may be
relatively stable, it is possible that the hypothesized loss of
specialist pollinators for this species would ultimately have
negative consequences for its regional abundance and for the
preservation of genetic diversity. In the absence of effective
pollinators, lack of gene flow, increased selfing, and reductions in fecundity may ultimately precipitate the extinction
of isolated populations. Further studies of gene flow and reproductive patterns in S. montana may help clarify the consequences of variation in the pollinator fauna for the distribution of genetic variation and the levels of selfing in isolated
populations of this endemic species.
Metapopulation Structure
It is apparent from these analyses that metapopulation processes are functioning at two spatial scales in S. montana.
The response of mating system parameters to variation in the
metapopulation circle diameter indicates that local clusters
of populations (i.e., within 2 km of each other) may be more
effective at maintaining a pollinator fauna, or that the same
conditions that are favorable for the persistence of S. montana
in these areas are also favorable for its specialized floral
associates. The latter case could be true if the local flora
contained other species that the putative pollinators of S.
montana could forage on when it was not in bloom. At broader spatial scales (i.e., within a circle diameter of 8 km) the
pollinators of S. montana have apparently been making infrequent movements among populations that have contributed
to the maintenance of allelic diversity across the metapopulation. Because the predictive power of metapopulation size
for variation in genetic diversity drops off at the largest circle
diameter (16 km), it may be indicative of the limits of longdistance pollinator flight movements. This analysis also provides insights into the impact of pollinators on evolutionary
processes in S. montana; the higher frequencies of selfing
and sibling mating inferred in locally isolated populations
may be counteracted by infrequent gene flow from populations within the larger metapopulation. However, populations
that are isolated at distances greater than 8 km may be more
susceptible to genetic erosion because long-distance gene
flow may not occur at a high enough rate to offset the effects
of genetic drift.
Lack of pollinator service for populations of S. montana
that have been historically outcrossed would be expected to
have cascading effects on genetic diversity. The initial response to a reduction in effective pollinator visits could be
an increase in the frequency of selfing, which would result
in the production of highly homozygous progeny. Persistent
selfing would increase the rate of loss of alleles from populations. Initial increases in the level of homozygosity may
also lead to the exposure of deleterious recessive alleles to
selection (Charlesworth and Charlesworth 1987) and a loss
of fecundity for plants in recently inbred populations as purging of genetic load proceeds (Barrett and Charlesworth 1991;
Willis 1999). Thus, loss of pollinators could have a twofold
effect on fecundity of S. montana, with a primary decrease
in seed production due to lack of pollination and a secondary
reduction in seed numbers after fertilization because of a high
frequency of inviable selfed progeny (van Treuren et al. 1993;
Willis 1993; Ouborg and van Treuren 1994). Although con-
ACKNOWLEDGMENTS
I thank G. Baucom, J. Estill, A. Morris, and C. Murren for
comments on earlier versions of this manuscript and S. Case,
J. Estill, N. Laszlo, A. Morris, J. Ferguson, S. Hopkins, and
S. Vege for assistance in the laboratory and the field. This
research would not have been possible without the cooperation and assistance of A. Shea and the Tennessee Natural
Heritage Program; T. Patrick, A. Levy, and T. Govus of the
Georgia Natural Heritage Program; L. Collins and T. Smith
of the Tennessee Valley Authority; J. Brown of the Tennessee
River Gorge Trust; B. Henderson of Elsie A. Holmes Nature
Park; J. Schild of the Reflection Riding Nature Center; and
C. Weaver and C. Spearman of the Lookout Mountain National Military Park. Funding was provided by the U.S. Fish
and Wildlife Service and the Tennessee Department of Environment and Conservation.
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Corresponding Editor: O. Savolainen