Genetics Show Current Decline and Pleistocene 

Expansion in Northern Spotted Owls

Open-File Report 2008–1239

U.S. Department of the Interior

U.S. Geological Survey

Genetics Show Current Decline and Pleistocene 

Expansion in Northern Spotted Owls

By W. Chris Funk, U.S. Geological Survey, Eric D. Forsman, U.S. Forest Service, and Thomas 

D. Mullins,

 and Susan M. Haig, U.S. Geological Survey

Open-File Report 2008-1239

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

DIRK KEMPTHORNE, Secretary

U.S. Geological Survey

Mark D. Myers, Director

U.S. Geological Survey, Reston, Virginia: 2008

For product and ordering information:

World Wide Web: http://www.usgs.gov/pubprod

Telephone: 1-888-ASK-USGS

For more information on the USGS--the Federal source for science about the Earth, its natural and living resources, 

natural hazards, and the environment:

World Wide Web: http://www.usgs.gov

Telephone: 1-888-ASK-USGS

Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the 

U.S. Government.

Although this report is in the public domain, permission must be secured from the individual copyright owners to 

reproduce any copyrighted materials contained within this report.

Suggested citation:

Funk, C.W., Forsman, E.D., Mullins,T.D., and Haig, S.M., 2008, Genetics show current decline and Pleistocene 

expansion in northern spotted owls: U.S. Geological Survey Open-File Report 2008-1239, 12 p.
iii

Contents

Abstract...........................................................................................................................................................1

Introduction.....................................................................................................................................................1

Materials and Methods.................................................................................................................................2

Sampling.................................................................................................................................................2

Microsatellite Data ..............................................................................................................................2

Cornuet and Luikart Method ...............................................................................................................3

Bayesian Methods................................................................................................................................4

Results .............................................................................................................................................................5

Recent Bottlenecks ..............................................................................................................................5

Pleistocene Expansion.........................................................................................................................5

Discussion.......................................................................................................................................................5

Acknowledgments.........................................................................................................................................6

References......................................................................................................................................................6

Appendix 1.......................................................................................................................................................9

Figures

Figure 1. Map showing recent population bottlenecks in northern spotted owls............... 2

Figure 2. Graphs showing Pleistocene expansion in northern spotted owls .................. 4

Tables

Table 1. Mean heterozygosity excess and P-values for northern spotted owls ............... 3
iv

This page left intentionally blank
Genetics Show Current Decline and Pleistocene 

Expansion in Northern Spotted Owls

W. Chris Funk1, Eric D. Forsman2, Thomas D. Mullins1 and Susan M. Haig1

Abstract

The northern spotted owl (Strix occidentalis caurina) is 

one of the most controversial threatened subspecies ever listed 

under the U.S. Endangered Species Act. Because of concern 

for persistence of the subspecies, logging on Federal lands in 

the U.S. Pacific Northwest was dramatically reduced under 

the Northwest Forest Plan in 1994. Despite protection of its 

remaining forest habitat, recent field studies show continued 

demographic declines of northern spotted owls. One potential 

threat to northern spotted owls that has not yet been shown 

is loss of genetic variation from population bottlenecks that 

can increase inbreeding depression and decrease adaptive 

potential. Here, we show recent genetic bottlenecks in northern 

spotted owls using a large genetic dataset (352 individuals 

from across the subspecies’ range and 11 microsatellite loci). 

The signature of bottlenecks was strongest in Washington 

State, in agreement with field data. Interestingly, we also 

found a genetic signature of Pleistocene expansion in the same 

study areas where recent bottlenecks were shown. Our results 

provide independent evidence that northern spotted owls have 

recently declined, and suggest that loss of genetic variation 

is an emerging threat to the subspecies’ persistence. Reduced 

effective population size (Ne

), shown here in addition to field 

evidence for demographic decline, highlights the increasing 

vulnerability of this bird to extinction.

Introduction

The debate over northern spotted owl conservation and 

logging is one of the most famous chapters in conservation 

history. Declines stemming from harvest of the northern 

spotted owl’s old-forest habitat in the U.S. Pacific Northwest 

led to listing of the subspecies as threatened under the U.S. 

Endangered Species Act in 1990 (U.S. Fish and Wildlife 

Service, 1990). In addition, the percentage of federal forest 

1

U.S. Geological Survey, Forest and Rangeland Ecosystem Science Center, 

3200 SW Jefferson Way, Corvallis, Oregon 97331, USA 

2

USDA Forest Service, Pacific Northwest Research Station, 3200 SW 

Jefferson Way, Corvallis, Oregon 97331, USA

land in the range of spotted owls allocated to reserves was 

increased to 77 percent under the Northwest Forest Plan in 

1994, dramatically reducing timber harvest (Stokstad, 2005; 

Noon and Blakesley, 2006). Nonetheless, recent field studies 

indicate that northern spotted owls have continued to decline 

at an average rate of 3.7 percent per year, and that declines 

are most severe in Washington State (Anthony and others, 

2006). Anecdotal evidence also suggests severe declines in 

British Columbia. One possible cause for continued decline is 

competition and hybridization with invasive barred owls (Strix 

varia), which have rapidly expanded into the range of northern 

spotted owls from their historic range in eastern North 

America (Kelly and others, 2003; Haig and others, 2004; Kelly 

and Forsman, 2004; Olson and others, 2005; Anthony and 

others, 2006; Funk and others, 2007). An additional potential 

threat that has not yet been shown for northern spotted owls is 

loss of genetic variation when effective population size (Ne

) 

decreases, known as a genetic bottleneck. Loss of genetic 

variation is expected to increase inbreeding depression 

(Crow and Kimura, 1970), which can reduce survival and 

reproductive rates, in turn decreasing population growth rates 

and increasing extinction probabilities (Saccheri and others, 

1998). Decreased genetic variation also reduces adaptive 

potential (Bürger and Lynch, 1995). Thus, determining 

whether Ne of northern spotted owls are decreasing is essential 

for fully understanding the threats to the subspecies and 

possible reasons for its continued decline.

New genetic methods allow detection of population 

bottlenecks as well as population growth or expansion. 

Although these methods have tremendous potential for 

detecting changes in Ne

, their power and limitations in natural 

populations are still being explored. One potential limitation is 

that the genetic signal of historic expansions may swamp the 

signal of recent declines, or vice versa, since expansions and 

declines have opposite effects on patterns of genetic variation. 

This problem may be particularly acute in temperate species 

such as northern spotted owls that are currently declining, but 

that likely expanded during Pleistocene interglacial cycles as 

has been shown for many temperate species (Milá and others, 

2000; Lessa and others, 2003). A possible solution to this 

problem is to use different methods for detecting changes in Ne

that perform best over different time scales. Another potential 

limitation of genetic methods for detecting changes in Ne is 
2 Genetics Show Current Decline and Pleistocene Expansion in Northern Spotted Owls

that they may not have the power to detect relatively slow, 

steady rates of population decline, as seen in northern spotted 

owls. There are several examples of the utility of genetic 

bottleneck tests for detecting catastrophic population crashes 

(Cornuet and Luikart, 1996; Goossens and others, 2006), but 

their capacity to detect slower rates of decline is less clear. 

Nonetheless, detection of slower declines will be important 

scientific information to obtain before populations decrease to 

the point where recovery is unlikely.

We genotyped 352 northern spotted owls across the 

subspecies’ range at 11 variable microsatellite loci to test 

for recent bottlenecks and Pleistocene expansion. Our three 

primary questions were: (1) is there a genetic signature of 

population decline in northern spotted owls despite relatively 

slow, steady declines; (2) if declines are detected, does the 

geographic pattern of decline identified with genetic data 

match the pattern observed in the field; and (3) is the genetic 

signature of Pleistocene expansion preserved in study areas in 

which recent declines are detected?

Materials and Methods

Sampling

We collected blood samples from 352 northern spotted 

owls from 16 study areas across the subspecies’ range from 

1990 to 2006 following the American Ornithologists’ Union 

protocol (Gaunt and Oring, 1997; fig. 1). Ninety-four percent 

of these were collected from 1994 to 2006. Study areas were 

bounded by landscape features such as mountain ridges, rivers, 

and non-forested habitat. No known close relatives (parentoffspring or siblings) were included.

Microsatellite Data 

DNA extraction, PCR, and fragment analysis were 

performed as described previously (Funk and others, 2007). 

All owls were genotyped at 11 variable microsatellite loci 

(table S1 in appendix 1) developed for Mexican spotted owls 

(S. o. lucida, Thode and others, 2002), Lanyu scops owls 

(Otus elegans botelensis, Hsu and others, 2003, 2006), and 

ferruginous pygmy-owls (Glaucidium brasilianum, Proudfoot 

and others, 2005). One of these loci (Oe128) and an additional 

microsatellite marker (Bb126) are diagnostic of spotted versus 

barred owls (Funk and others, 2007) and were genotyped to 

assure that no barred owls or spotted owl-barred owl hybrids 

were included. One barred owl was detected and removed 

from subsequent analyses.

Figure 1. Recent population bottlenecks in northern spotted owls. 

Points represent the 352 individual owls included in the analysis 

which are grouped into 16 study areas. The thickness of study 

area boundaries is proportional to heterozygosity excess 

which reflects the magnitude of bottlenecks. Study areas with 

statistically significant bottlenecks are shown in red (p =0.01 

critical level) and yellow (p =0.05) under a 10% multi-step mutation 

model. Bottlenecks are most severe in the Washington Cascades 

and southern Oregon Coast Range. Blue shading shows the range 

of northern spotted owls and green the range of California spotted 

owls (S. o. occidentalis).

Standard population genetic analyses were performed 

using Genepop 3.4 (Raymond and Rousset, 1995). There 

were no significant departures from Hardy-Weinberg (HW) 

proportions within any study area, suggesting little or no 

genetic structure within study areas. Out of 869 tests for 

linkage disequilibrium (LD), fewer were significant (41 tests) 

than expected by chance (43.5) and there was no evidence 

for consistent LD between any two loci, indicating that loci 

were independent. FST across all study areas was 0.023 and 

pairwise FSTs ranged from 0.0002 to 0.062.
Materials and Methods 3

Cornuet and Luikart Method

We tested for recent population bottlenecks in all 16 

study areas using an analysis developed by Cornuet and 

Luikart (1996). This method is based on the loss of rare 

alleles predicted in recently bottlenecked populations. It uses 

a single population sample to test whether there has been a 

recent reduction in allelic variation. Simulations (Cornuet and 

Luikart, 1996; Williamson-Natesan, 2005), theory (Garza and 

Williamson, 2001), and case studies (Cornuet and Luikart, 

1996; Goossens and others, 2006; Beebee and Rowe, 2001; 

Spear and others, 2006) all show that this is the best method 

available for detecting recent, low-magnitude declines in Ne. 

Simulation studies (Cornuet and Luikart, 1996;, WilliamsonNatesan, 2005) also demonstrate that this method has low 

type I error rates (that is, falsely detecting a bottleneck when 

there is not one). We used program Bottleneck 1.2.02 (Piry 

and others, 1999) to implement this analysis. The two-phase 

mutation model (TPM) was used to generate null distributions 

under mutation-drift equilibrium, as the TPM is considered to 

be most appropriate for microsatellites (Garza and Williamson, 

2001; Di Rienzo and others, 1994). A wide range of values 

was used for the percent of multi-step mutations (5, 10, 20, 

and 30 percent, table 1). The Wilcoxon signed rank test was 

used to determine significance of heterozygosity excess.

Bottleneck tests assume random mating and population 

closure (no gene flow). Non-random mating can produce 

genealogies that resemble bottlenecks, whereas gene flow may 

resemble recent expansion by introducing rare alleles (Cornuet 

and Luikart, 1996; Goossens and others, 2006). Agreement 

with HW proportions above supported random mating within 

study areas (that is, lack of genetic structure), but low FST 

values suggested gene flow among areas. Yet despite gene 

flow that can mimic recent expansion, we found a consistent 

signature of bottlenecks, providing even stronger evidence for 

recent reductions in Ne. Bottlenecks in northern spotted owls 

may therefore actually be more severe than they appear in our 

analysis.

At one locus, 1C6, extreme heterozygosity deficiency 

was observed while at the other 10 loci, heterozygosity 

excess was observed, a pattern suggesting strong positive 

selection at 1C6 or a linked locus (Maynard Smith and Haigh, 

1974; Watterson, 1978) or a different mutation model. For 

example, mean heterozygosity excess (He, Hardy-Weinberg 

heterozygosity, minus H

eq, equilibrium heterozygosity) across 

all study areas was 0.036 at the other 10 loci, but was -0.269 

at 1C6 under the 10 percent multi-step mutation model. 

He was also significantly lower than expected under mutationdrift equilibrium at 1C6 in 13 out of 16 study areas under this 

model. 1C6 was the only trinucleotide repeat microsatellite 

analyzed (see table S1) which may explain the divergent 

pattern in heterozygosity observed at this locus. For example, 

this locus could be an expanded repeat of an amino acid (TAT 

which encodes tyrosine) that is under selection.

Table 1. Mean heterozygosity excess and P-values for northern spotted owls. 

[Het exc, mean heterozygosity excess across loci; P, probability of observed heterozygosity excess under 

mutation-drift equilibrium and the given two-phase mutation model. Significant P-values (p = 0.05) are 

shown in bold]

Study

area

Percent multi-stepmutations (under two-phase mutation model)

n

5 10 20 30

Het

exc

P Het

exc

P Het

exc

P Het

exc

P

1 13 0.015 0.216 0.024 0.188 0.032 0.138 0.039 0.065

2 9 0.022 0.053 0.026 0.042 0.035 0.042 0.042 0.042

3 13 0.055 0.003 0.063 0.001 0.074 0.001 0.081 0.001

4 51 0.033 0.016 0.047 0.002 0.067 0.0005 0.082 0.0005

5 18 0.048 0.042 0.057 0.005 0.070 0.002 0.082 0.002

6 12 0.020 0.216 0.026 0.188 0.041 0.161 0.046 0.138

7 47 0.009 0.348 0.023 0.188 0.042 0.080 0.056 0.012

8 31 0.044 0.012 0.058 0.007 0.074 0.003 0.085 0.001

9 15 0.007 0.246 0.014 0.188 0.025 0.053 0.032 0.042

10 14 0.014 0.080 0.021 0.053 0.033 0.042 0.041 0.042

11 28 0.022 0.313 0.035 0.080 0.049 0.053 0.061 0.009

12 17 0.008 0.313 0.017 0.246 0.027 0.246 0.038 0.216

13 10 0.027 0.313 0.032 0.216 0.041 0.097 0.047 0.080

14 32 0.042 0.065 0.052 0.012 0.068 0.003 0.079 0.002

15 14 0.027 0.097 0.034 0.042 0.046 0.016 0.053 0.009

16 28 0.028 0.138 0.040 0.080 0.054 0.065 0.068 0.009
4 Genetics Show Current Decline and Pleistocene Expansion in Northern Spotted Owls

Since the pattern of genetic variation at 1C6 appeared 

to reflect selection rather than demographic history, it was 

excluded from further analysis. Nonetheless, inclusion of 

1C6 did not qualitatively affect evidence for population 

bottlenecks. For example, even with 1C6, mean heterozygosity 

excess was still greater than zero in study areas 3–5, 8, and 

14–16 regardless of mutation model and was significant for 

study area 3 under the 10 percent multi-step model and study 

areas 3–5 and 15 under the 20 percent multi-step model. 

Detection of bottlenecks despite inclusion of a locus with 

a strong signal of positive selection reinforces evidence for 

declines.

Mean heterozygosity excess was not correlated with 

mean or median year of sample collection across study areas 

(p=0.320 and p=0.410, respectively). Thus, there was no 

evidence for a sampling-year effect.

Bayesian Methods

We next used two Bayesian coalescent-based methods 

to test whether the signature of Pleistocene expansion was 

preserved in three study areas in which recent declines were 

detected, study areas 4 (n=51), 8 (n=31), and 14 (n=32). 

Only three study areas were chosen for the Bayesian analyses 

because these analyses are extremely computationally 

intensive and time consuming. These methods use the full 

allelic distribution to infer past changes in Ne. The Beaumont 

(1999) method estimates the posterior distribution of r, the 

ratio of current population size (N0) to ancestral population 

size (N1), while the Storz and Beaumont (2002) method 

estimates the posterior distributions of N0, N1, and xa, the time 

in years since population growth or decline. Although these 

methods are capable of detecting recent declines (Goossens 

and others, 2006), they are predicted to be best at detecting 

long-term, gradual changes in Ne (Williamson-Natesan, 2005). 

The Beaumont and Storz and Beaumont methods were 

implemented using the msvar programs (Storz and Beaumont, 

2002). The Beaumont method assumes rectangular prior 

distributions. Wide bounds were used for priors (table S2 in 

appendix 1) so that posterior distributions would be minimally 

affected. For each study area, at least four independent runs 

were performed using different starting values and thinning 

intervals (table S2). All runs had at least 109

 iterations and 

thinning intervals of 105–106. Posterior distributions of log(r) 

were very similar among different runs (fig. S1). The last 

half of each run was combined to generate a final posterior 

distribution for each study area (shown in fig. 2).

The Storz and Beaumont method assumes log-normal 

priors, and broad priors were used once again (table S3). Three 

independent runs were performed for each study area using 

different prior distributions of log(xa

). All runs had at least 109 

iterations and thinning intervals of 105. A mean generation 

time of 8.8 years was calculated from parents of known age 

in 2003 (n=43 parents), 2004 (n=102), and 2005 (n=67). 

Changing generation time to lower or higher values (for 

Figure 2. Pleistocene expansion in northern spotted owls. 

(a) Posterior probability distributions of r, the ratio of current (N0) to 

ancestral (N1

) population size, in study areas 4 (dashed line), 8 (thin 

line), and 14 (thick line). Support was much stronger for population 

growth than decline, particularly for study area 4. The dotted 

line shows the rectangular prior distribution for r. (b) Posterior 

distributions of x

a

, the time in years since population growth for the 

same three study areas. Support was greatest for expansion during 

the Pleistocene, especially in study area 4. Dotted curves show 

prior distributions for x

a

.

example, 5 or 12 years) did not alter the conclusions. Posterior 

distributions were similar among runs for log(N0), log(N1), and 

log(xa) (figs. S2 and S3).
Discussion 5

Results

Recent Bottlenecks

We found a significant signature of population decline 

using the Cornuet and Luikart method for several study areas, 

regardless of the mutation model assumed (fig. 1, table 1). 

The signature of declines was strongest for study areas 3–5 in 

the Washington Cascades and area 8 in the southern Oregon 

Coast Range. Declines were also significant when study areas 

were lumped into regions in the Washington Cascades (study 

areas 3–5; p=0.002 under a 10 percent multi-step model), the 

Oregon Coast Range (areas 6–8; p=0.016), and the Klamath 

Mountains (areas 12–15; p=0.005), demonstrating that 

detection of declines was not sensitive to grouping method. In 

addition, mean heterozygosity excess across loci was greater 

than zero in all 16 study areas regardless of mutation model 

(table 1), significantly more areas than the 50 percent expected 

to show heterozygosity excess by chance (exact binomial 

probability; p=0.00003). This suggests that declines have 

occurred throughout the subspecies’ range. Thus, the Cornuet 

and Luikart method detected recent bottlenecks despite 

relatively slow, steady declines. The geographic pattern of 

decline detected with the genetic data (strongest signal of 

declines in Washington) also matches the large-scale pattern 

observed in the field (Anthony and others, 2006). In addition, 

bottleneck tests were able to detect genetic declines in study 

areas 8 and 14 not detected with field data.

Pleistocene Expansion

Bayesian analysis revealed a genetic signature of 

population growth during the Pleistocene in all three 

study areas examined (fig. 2, see fig. S4 in appendix 1). 

Evidence for Pleistocene expansion was greatest for the 

most northerly study area (study area 4, see fig. 1) which is 

closest to Pleistocene ice sheets (Mann and Hamilton, 1995) 

and, therefore was likely most directly affected by glacial 

cycles. The posterior odds for a growing versus declining 

population were 10.6, 3.2, and 2.7 for study areas 4, 8, and 

14, respectively. Posterior odds for a Pleistocene versus nonPleistocene timing of growth were 4.1, 1.7, and 1.8 for these 

same three study areas. Median values of xa were 3.49 × 105, 

3.82 × 105, and 4.75 × 105 years, respectively, all in the midPleistocene. Thus, a genetic signature of Pleistocene expansion 

was preserved in study areas in which recent declines were 

also detected.

Discussion

These results provide strong evidence for recent declines 

and Pleistocene expansion in northern spotted owls. Although 

the Cornuet and Luikart method does not provide an estimate 

of the timing of decline as does the Storz and Beaumont 

method, two findings strongly suggest that the genetic signal 

of decline reflects recent decreases in Ne. First, the geographic 

pattern of decline detected with the Cornuet and Luikart 

method matches the overall pattern of demographic declines 

observed in a field study from 1985 to 2003 (Anthony and 

others, 2006). Second, the Bayesian analysis provides little 

support for older, historic declines. Additionally, as mentioned 

above, previous work (Cornuet and Luikart, 1996; Beebee 

and Rowe, 2001; Garza and Williamson, 2001; WilliamsonNatesan, 2005; Goossens and others, 2006; Spear and others, 

2006) demonstrates that the Cornuet and Luikart method is 

most effective at detecting recent changes in Ne. Previous 

mitochondrial DNA (mtDNA) studies of northern spotted owls 

did not find a statistically significant signature of bottlenecks 

(Barrowclough and others, 1999, 2005; Haig and others, 

2004), likely due to lower power resulting from much smaller 

sample sizes and use of mtDNA which is inherited as a single 

locus (compared to 11 microsatellite loci used here).

Importantly, evidence for genetic declines detected here 

with bottleneck tests indicates reduction in Ne and genetic 

variation rather than demographic population size. This means 

that in addition to habitat loss and invasive barred owls, 

northern spotted owls may become increasingly threatened 

by genetic factors such as inbreeding depression and loss 

of adaptive genetic variation. Inbreeding depression may 

reduce stage-specific survival and reproductive rates, causing 

an increase in the rate of decline in a process termed an 

extinction vortex (Soulé and Mills, 1998). A large-scale field 

study of northern spotted owls (Anthony and others, 2006) 

found evidence for declines in survival and fecundity in some 

study areas, which could be caused by the loss of genetic 

variation detected here. Thus, it is possible that northern 

spotted owls are already caught in an extinction vortex. At 

this point, however, it is not possible to determine whether 

loss of genetic variation is causing vital rate reductions or vice 

versa. Regardless, future efforts to conserve northern spotted 

owl populations will require greater consideration of genetic 

threats to persistence.
6 Genetics Show Current Decline and Pleistocene Expansion in Northern Spotted Owls

Our results demonstrate the potential of genetic methods 

for monitoring changes in Ne of threatened species (Schwartz 

and others, 2007). First, it was possible to detect current 

declines in northern spotted owls despite historic expansion 

by using a combination of methods with different temporal 

resolution. This demonstrates that genetic methods have 

the capacity to detect reductions in Ne even in populations 

with complex demographic and biogeographic histories. 

Additionally, declines were detected in spite of the relatively 

slow, steady rate of decline in northern spotted owls. For 

example, it is not surprising that the Cornuet and Luikart 

method was able to detect declines in orangutans (Pongo 

pygmaeus) which have experienced declines of up to 33 

percent in a single year (Goossens and others, 2006). In 

contrast, the average rate of decline in northern spotted owls 

from 1985 to 2003 across its range was only 3.7 percent, with 

a maximum average rate of 10.4 percent for a single study 

area (Anthony and others, 2006). This agrees with simulation 

studies showing that the Cornuet and Luikart method is able 

to detect declines of relatively low magnitude (WilliamsonNatesan, 2005). Thus, genetic methods should become 

increasingly useful for population monitoring, especially as it 

becomes easier and less expensive to acquire large numbers of 

molecular markers, which will provide greater power.

Acknowledgments

We are indebted to the many people who helped with 

sample collection and logistics in the field, particularly 

P. Loschl, S. Ackers, R. Anthony, R. Claremont, D. Herter, 

S. Hopkins, J. Reid, and S. Sovern. We also thank F. Allendorf 

and D. Tallmon for comments on the manuscript. Funding 

was provided by the USGS Forest and Rangeland Ecosystem 

Science Center and the USDA Forest Service Pacific 

Northwest Research Station. Samples were collected under 

U.S. Fish and Wildlife Service threatened species permit 

number TE026280-11 and Oregon State University Animal 

Care and Use permit number 3091.

References

Anthony, R.G., Forsman, E.D., Franklin, A.B., Anderson, 

D.R., Burnham, K.P., White, G.C., Schwarz, C.J., Nichols, 

J.D., Hines, J.E., Olson, G.S., Ackers, S.H., Andrews, L.S., 

Biswell, B.L., Carlson, P.C., Diller, L.V., Dugger, K.M., 

Fehring, K.E., Fleming, T.L., Gerhardt, R.P., Gremel, S.A., 

Gutiérrez, R.J., Happe, P.J., Herter, D.R., Higley, J.M., 

Horn, R.B., Irwin, L.L., Loschl, P.J., Reid, J.A. and Sovern, 

S.G. 2006, Status and trends in demography of northern 

spotted owls, 1985–2003: Wildlife Monographs 163, 

p. 1–47. 

Barrowclough, G.F., Gutiérrez, R.J., and Groth, J.G., 1999, 

Phylogeography of spotted owl (Strix occidentalis) 

populations based on mitochondrial DNA sequences: gene 

flow, genetics structure, and a novel biogeographic pattern: 

Evolution 53, p. 919–931.

Barrowclough, G.F., Groth, J.G., Mertz, L.A., and Gutiérrez, 

R.J., 2005, Genetic structure, introgression, and a narrow 

hybrid zone between northern and California spotted owls 

(Strix occidentalis): Molecular Ecology 14, p. 1109–1120. 

Beaumont, M.A., 1999, Detecting population expansion and 

decline using microsatellites: Genetics 153, p. 2013–2029.

Beebee, T., and Rowe, G., 2001, Application of genetic 

bottleneck testing to the investigation of amphibian 

declines: a case study with natterjack toads: Conservation 

Biology15, p. 266–270. 

Bürger, R., and Lynch, M., 1995, Evolution and extinction in 

a changing environment: a quantitative-genetic analysis: 

Evolution 49, p. 151–163.

Cornuet, J.M., and Luikart, G., 1996, Description and power 

analysis of two tests for detecting recent population 

bottlenecks from allele frequency data: Genetics 144, 

p. 2001–2014.

Crow, J.F., and Kimura, M., 1970, An Introduction to 

Population Genetics Theory: Minneapolis, Minnesota, 

Burgess Publishing.

Di Rienzo, A., Peterson, A.C., Garza, J.C., Valdes, A.M., 

Slatkin, M., and Freimer, N.B., 1994, Mutational processes 

of simple-sequence repeat loci in human populations: 

Proceedings of the National Academy of Science USA 91, 

p. 3166–3170.

Funk, W.C., Mullins, T.D., Forsman, E.D. and Haig, S.M., 

2007, Microsatellite loci for distinguishing spotted owls 

(Strix occidentalis), barred owls (Strix varia), and their 

hybrids: Molecular Ecology Notes 7, p. 284–286. 

Garza, J.C., and Williamson, E.G., 2001, Detection of 

reduction in population size using data from microsatellite 

loci: Molecular Ecology 10, p. 305–318. 

Gaunt, A.S., and Oring, L.W., 1997, Guidelines to the 

Use of Wild Birds in Research: Washington, DC, The 

Ornithological Council.

Goossens, B., Chikhi, L., Ancrenaz, M., Lackman-Ancrenaz, 

I., Andau, P., and Bruford, M.W., 2006, Genetic signature 

of anthropogenic population collapse in orang-utans: PLoS 

Biology 4, p. 285–291. 

Haig, S.M., Mullins, T.D., and Forsman, E.D., 2004 

Subspecific relationships and genetic structure in the spotted 

owl: Conservation Genetics 5, p. 683–705. 
References 7

Haig, S.M., Mullins, T.D., Forsman, E.D., Trail, P.W., and 

Wennerberg, L., 2004, Genetic identification of spotted 

owls, barred owls, and their hybrids: legal implications of 

hybrid identity: Conservation Biology18, p. 1347–1357. 

Hsu, Y.-C., Severinghaus, L.L., Lin, Y.-S., and Li, S.-H., 

2003, Isolation and characterization of microsatellite 

DNA markers from the Lanyu scops owl (Otus elegans 

botelensis): Molecular Ecology Notes 3, p. 595–597. 

Hsu, Y.-C., Li, S.-H., Lin, Y.-S., and Severinghaus, L.L., 2006, 

Microsatellite loci from Lanyu scops owl (Otus elegans 

botelensis) and their cross-species application in four 

species of Strigidae: Conservation Genetics 7, p. 161–165. 

Kelly, E.G., and Forsman, E.D., 2004, Recent records of 

hybridization between barred owls (Strix varia) and 

northern spotted owls (S. occidentalis caurina): Auk 121, 

p. 806–810. 

Kelly, E.G., Forsman, E.D., and Anthony, R.G., 2003, Are 

barred owls displacing spotted owls? Condor 105, p. 45–53. 

Lessa, E.P., Cook, J.A,. and Patton, J.L., 2003, Genetic 

footprints of demographic expansion in North America, but 

not Amazonia, during the Late Quaternary: Proceedings of 

the National Academy of Sciences, USA 100, 

p. 10331–10334. 

Mann, D.H., and Hamilton, T.D., 1995, Late Pleistocene and 

Holocene paleoenvironments of the North Pacific Coast: 

Quaternary Science, rev. 14, p. 449–471. 

Maynard Smith, J., and Haigh, J., 1974, The hitch-hiking 

effect of a favourable gene: Genetical Research 23, p. 

23–35.

Milá, B., Girman, D.J., Kimura, M., and Smith T.B., 

2000 Genetic evidence for the effect of a postglacial 

population expansion on the phylogeography of a North 

American songbird: Proc. R. Soc. Lond. B Biol. Sci. 267, 

p. 1033–1040. 

Noon, B.R., and Blakesley, J.A., 2006, Conservation of the 

northern spotted owl under the Northwest Forest Plan: 

Conservation Biology 20, p. 288–296. 

Olson, G.S., Anthony, R.G., Forsman, E.D., Ackers, S.H., 

Loschl, P.J., Reid, J.A., Dugger, K.M., Glenn, E.M., and 

Ripple, W.J., 2005, Modeling of site occupancy dynamics 

for northern spotted owls, with emphasis on the effects 

of barred owls: Journal of Wildlife Management 69, 

p. 918–932. 

Piry, S., Luikart, G., and Cornuet, J.-M., 1999, 

BOTTLENECK: A computer program for detecting recent 

reductions in the effective population size using allele 

frequency data: Journal of Heredity 90, p. 502–503. 

Proudfoot, G., Honeycutt, R., and Slack, R.D., 2005 

Development and characterization of microsatellite 

DNA primers for ferruginous pygmy-owls (Glaucidium 

brasilianum): Molecular Ecology Notes 5, p. 90–92. 

Raymond, M., and Rousset, F., 1995, GENEPOP (version 

1.2): Population genetics software for exact tests and 

ecumenicism: Journal of Heredity 86, p. 248–249.

Saccheri, I., Kuussaari, M., Kankare, M., Vikman, P., 

Fortelius, W., and Hanski, I., 1998, Inbreeding and 

extinction in a butterfly metapopulation: Nature 392, 

p. 491–494. 

Schwartz, M.K., Luikart, G., and Waples, R.S., 2007, Genetic 

monitoring as a promising tool for conservation and 

management: Trends in Ecology and Evolution 22, 

p. 25–33. 

Soulé, M.E., and Mills, L.S., 1998, No need to isolate 

genetics: Science 282, p. 1658–1659. 

Spear, S.F., Peterson, C.R., Matocq, M.D., and Storfer, 

A., 2006, Molecular evidence for historical and recent 

population size reductions of tiger salamanders (Ambystoma 

tigrinum) in Yellowstone National Park: Conservation 

Genetics 7, p. 605–611. 

Stokstad, E., 2005, Learning to adapt. Science 309, 

p. 688–690. 

Storz, J.F., and Beaumont, M.A., 2002, Testing for genetic 

evidence of population expansion and contraction: an 

empirical analysis of microsatellite DNA variation using a 

hierarchical Bayesian model: Evolution 56, p. 154–166. 

Thode, A.B., Maltbie, M., Hansen, L.A., Green, L.D., and 

Longmire, L.L., 2002, Microsatellite markers for the 

Mexican spotted owl (Strix occidentalis lucida): Molecular 

Ecology Notes 2, p. 446–448. 

U.S. Fish and Wildlife Service, 1990, Endangered and 

threatened wildlife and plants: determination of threatened 

status of the northern spotted owl: Federal Register 55, 

p. 26114–26194.

Watterson, G.A., 1978 The homozygosity test of neutrality: 

Genetics 88, p. 405–417.

Williamson-Natesan, E.G., 2005, Comparison of methods for 

detecting bottlenecks from microsatellite loci: Conservation 

Genetics 6, p. 551–562. 
8 Genetics Show Current Decline and Pleistocene Expansion in Northern Spotted Owls

This page left intentionally blank
Appendix 1 9

Appendix 1

Table S1. Microsatellite loci used to test for population decline 

and growth in northern spotted owls. 

[He, Hardy-Weinberg heterozygosity. Primer sequences and annealing 

temperatures can be found in original references. 1C6 was excluded from 

bottleneck tests because of evidence for strong positive selection. See Methods 

and Materials section for details]

Locus Repeat Size range 

(bp)

No. 

alleles

He Reference 

no.

6H8 GATA 86–114 8 0.793 23

15A6 GATA 142–166 7 0.656 23

1C6 ATT 105–138 11 0.392 23

13D8 GATA 167–195 8 0.709 23

4E10.2 ATTTT 196–246 11 0.827 23

Oe3-7 GATA 118–138 6 0.763 24

Oe053 GATA 200–224 7 0.722 24

Oe128 GATA 315–327 4 0.639 24

Oe129 GATA 253–285 9 0.726 25

Oe149 GATA 236–276 10 0.736 25

FEPO5 AGAT 258–286 8 0.795 26

Table S2. Parameters used for Beaumont method runs. 

[Exp, exponential; lin, linear; q = 2N0 m (the product of 2, current population size, and mutation rate); r = N0/

N1 (current population size divided by ancestral population size); t

f = Ta /N0 (number of generations since 

population growth or decline divided by current population size). Starting values are shown in columns 3–5 

and the range of rectangular prior distributions in columns 6–8 (see Beaumont 1999 for details). Thinning is 

the number of update steps between successive lines of output. Parameters which were changed from run 1 are 

underlined. Runs 2 and 3 were only used for study area 8, but all other runs were used for all three study areas 

analyzed (4, 8, and 14)]

Run Model q r t

f log(q) range log(r) range log(tf

) range Thinning

1 Exp 0.1 1 10 -5–3 -3–3 -4–6 105

2 Lin 0.1 1 10 -5–3 -3–3 -4–6 105

3 Exp 0.1 1 10 -5–3 -3–3 -4–6 106

4 Exp 0.1 100 10 -5–3 -3–3 -4–6 105

5 Exp 0.1 0.01 10 -5–3 -3–3 -4–6 105

6 Exp 10 1 1000 -5–3 -3–3 -4–6 105
10 Genetics Show Current Decline and Pleistocene Expansion in Northern Spotted Owls

Figure S1. Posterior distributions of population growth (r = N0 /N1) for independent runs. Posterior distributions 

are shown for study areas 4 (a), 8 (b), and 14 (c). Independent runs gave similar posterior distributions despite 

different starting values and thinning intervals.
Appendix 1 11

Figure S2. Posterior distributions of current (N0) and ancestral (N1) population sizes for independent runs. 

Posterior distributions are shown for study areas 4 (a), 8 (b), and 14 (c). Independent runs gave similar 

posterior distributions.

Table S3. Parameters used for Storz and Beaumont method runs. 

[N0, current population size; N1, ancestral population size; m , mutation rate; x

a

, time in years since population growth or 

decline. In each starting value column, the figures are the starting mean and variance of the corresponding parameters (see 

Storz and Beaumont 2002 for details). In each hyperprior column, the first two figures are the mean and variance of a normal 

distribution from which the mean was drawn for the prior log-normal distribution. The second two figures are the mean 

and variance of a truncated normal distribution from which the variance was drawn for the prior log-normal distribution. 

Parameters which were changed from run 1 are underlined. The three parameter combinations were implemented in all three 

study areas analyzed (4, 8, and 14)]

 

Run

Starting values Hyperpriors

log(N0

) log(N1) log(m) log(xa) log(N0) log(N1) log(m) log(xa)

1 2.5 0.5 2.5 0.5 -4 0.5 4 0.5 2.5 2 0 0.5 2.5 2 0 0.5 -4 0.5 0 0.5 4 3 0 0.5

2 2.5 0.5 2.5 0.5 -4 0.5 4 0.5 2.5 2 0 0.5 2.5 2 0 0.5 -4 0.5 0 0.5 5 3 0 0.5

3 2.5 0.5 2.5 0.5 -4 0.5 4 0.5 2.5 2 0 0.5 2.5 2 0 0.5 -4 0.5 0 0.5 3 3 0 0.5
12 Genetics Show Current Decline and Pleistocene Expansion in Northern Spotted Owls

Figure S4. Current and ancestral population sizes in 

northern spotted owls. Posterior probability distributions 

of current (N0

) and ancestral (N1) population sizes in 

study areas 4 (dashed line), 8 (thin line), and 14 (thick line) 

estimated using the Storz and Beaumont method. Current 

population sizes were larger than ancestral population sizes 

in all study areas, indicating population growth (as found 

with the Beaumont method). The dotted curve shows the 

prior distribution used for N0

 and N1.

Figure S3. Posterior distributions of years since 

population expansion (x

a

) for independent runs. Posterior 

distributions are shown for study areas 4 (a), 8 (b), and 14 

(c). Independent runs gave similar posterior distributions 

in spite of different prior distributions for x

a

.
For more information concerning the research in this report, contact the 

Director, Forest and Rangeland Ecosystem Science Center 

U.S. Geological Survey, 777 NW 9th Street, Suite 400

Corvalis, OR 97330

http://fresc.usgs.gov/
Funk and others— Genetics Show Current Decline and Pleistocene Expansion in Northern Spotted Owls—OFR 2008–1239