The genetic structure of steelhead and spring Chinook salmon in the upper

Willamette River, Oregon

Marc A. Johnson1, Thomas A. Friesen1, David J. Teel2, Donald M. Van Doornik2

1Oregon Department of Fish and Wildlife,Corvallis Research Laboratory, 28655 Highway 34, Corvallis, OR 97333

2NOAA Fisheries, Northwest Fisheries Science Center, Manchester

Research Laboratory, PO Box 130, Manchester, WA 98353

Upper Willamette River Chinook and Steelhead

Chinook salmon

•Spring Chinook are native to the basin •Hatchery stocks founded from local, wild stocks

•Integrated hatcheries

•Minimize genetic divergence

•Reduce consequences of HxW interactions

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Steelhead

•Winter steelhead are native to some subbasins •Hatchery summer steelhead are not a native UWR stock

•Segregated hatcheries

•Reduce frequency of HxW interactions

•Minimize natural production and interbreeding

“One of the most serious problems faced by wild and hatchery populations is the permanent

loss of genetic material. Not only can such losses affect the immediate performance of a

stock, but they also limit its flexibility to respond to changing conditions in the future.”

-Waples et al. 1990. Fisheries 15:19-25

Willamette River Spring Chinook

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Objectives

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Describe how genetic diversity is distributed within and

among hatchery and wild spring Chinook populations

Evaluate how alternate wild integration and hatchery

straying (migration) rates could affect genetic diversity

Sampling

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Fin tissue collected from adult hatchery and wild spring Chinook

•MF Willamette (H & W) •McKenzie (H & W) •Calapooia (W) •South Santiam (H & W) •North Santiam (H & W) •Molalla (W) •Clackamas (H & W) •Catherine Cr., Grande Ronde (H)

“Wild” determined by adipose fin and no otolith thermal mark

In the Lab Isolated genomic DNA

Amplified and scored 17 microsatellites • 13 GAPS markers • 4 “immune-relevant” markers*

An electrophoretogram for a single microsatellite locus from a Willamette River Chinook salmon. This individual is a heterozygote, with two major “peaks” representing the two different alleles, or character states, for this marker.

*Tonteri et al. (2008) Molecular Ecology Resources 8:1486-1490

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Data Analyses Describe how genetic diversity is distributed within and among hatchery and wild spring

Chinook populations

Estimated

• Mean heterozygosity1

• Allelic richness2

• Pairwise θ1

Inferred

• Phylogenetic relationships among hatchery and wild populations3

Tested

• Locus-specific signatures of selection with FST outlier4

Evaluate how alternate wild integration and hatchery straying (migration) rates could affect

genetic diversity

Simulated

• Effects of alternate migration rates on heterozygosity, θ, total allele

count5

1 GENETIX – Belkhir et al. 2004. Available at http://kimura.univ-montp2.fr/genetix/ 2 FSTAT – Goudet. 1995.Journal of Heredity 86:485-486 3 CONTML – Felsenstein. 2009. Available at http://evolution.genetics.washington.edu/phylip/doc/ 4 LOSITAN – Antao et al. 2008. Bioinformatics 9: 323 5 NEMO – Guillaume & Rougemont. 2006. Bioinformatics 22:2556-2557

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Samples

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Subbasin n wild n hatchery

Clackamas 51 80

Molalla 8 -

North Santiam 72 95

South Santiam 62 94

Calapooia - -

McKenzie 67 95

Middle Fork Willamette 12 144

Catherine Creek - 33

Total 272 541

Total of 813 samples included in statistical analyses

Heterozygosity & Allelic Richness

• Higher heterozygosities in hatchery populations

• No pattern of difference for allelic richness

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Subbasin Wild Ho Hatchery Ho

Clackamas 0.752 0.815

Molalla 0.823

North Santiam 0.777 0.820

South Santiam 0.746 0.813

McKenzie 0.788 0.805

Middle Fork Willamette 0.620 0.818

Catherine Creek 0.735

Heterozygosity and

Allelic Richness

RANK Population He Ho AR

1 Lewis Hatchery (spring) 0.866 0.87 15.2

2 Cowlitz Hatchery (spring) 0.861 0.853 14.9

3 Klickitat River (spring) 0.864 0.846 15.9

4 Kalama Hatchery (spring) 0.865 0.837 15.1

5 McKenzie Hatchery (spring) 0.817 0.812 12.9

6 North Santiam Hatchery (spring) 0.82 0.812 13.1

7 Winthrop Hatchery, Carson stock (spring) 0.792 0.809 12.5

8 Wenatchee River (spring) 0.795 0.803 13.4

9 Tucannon River (spring)a 0.791 0.803 11.6

10 Battle Creek (spring) 0.841 0.801 15

11 Cle Elum Hatchery (spring) 0.816 0.796 13.2

12 Red River (spring)a 0.795 0.795 13

13 Entiat Hatchery (spring) 0.782 0.793 11.7

14 Imnaha River (spring)a 0.783 0.793 12.7

15 Sawtooth Hatchery (spring)a 0.79 0.793 13

16 Dworshak Hatchery (spring)a 0.793 0.792 13.5

17 Pahsimeroi River (spring)a 0.78 0.79 11.5

18 Lochsa River–Powell Trap (spring)a 0.788 0.789 13.1

19 Methow River (spring) 0.793 0.788 13.4

20 Minam River (spring)a 0.79 0.788 13.5

21 South Fork Clearwater (spring)a 0.785 0.782 12.8

22 Big Creek-b (spring)a 0.76 0.782 11.3

23 West Fork Yankee Fork (spring)a 0.758 0.779 10.3

24 Marsh Creek (spring) 0.782 0.777 12.1

25 Catherine Creek (spring)a 0.775 0.776 12.7

26 Johnson Creek supplementation (spring)a 0.779 0.776 12.2

27 Johnson Creek (spring)a 0.776 0.775 11.9

28 Lolo Creek (spring)a 0.787 0.767 13.6

29 Rapid River Hatchery (spring)a 0.762 0.767 11.3

30 Big Creek-a (spring)a 0.754 0.764 11.7

31 Lostine River (spring)a 0.754 0.763 11

32 Secesh River (spring)a 0.773 0.763 12.1

33 Newsome Creek (spring)a 0.765 0.76 12

34 Shitike Creek (spring) 0.763 0.757 12.2

35 East Fork Salmon River (spring)a 0.769 0.757 12

36 John Day River (spring) 0.78 0.755 13.5

37 Warm Springs Hatchery (spring) 0.725 0.728 10.9

Narum et al. 2010. Transactions of the

American Fisheries Society 139:1465-1477

RANK Population He Ho AR

1 Klickitat River (spring) 0.864 0.846 15.9

2 Lewis Hatchery (spring) 0.866 0.87 15.2

3 Kalama Hatchery (spring) 0.865 0.837 15.1

4 Battle Creek (spring) 0.841 0.801 15

5 Cowlitz Hatchery (spring) 0.861 0.853 14.9

6 Lolo Creek (spring)a 0.787 0.767 13.6

7 Dworshak Hatchery (spring)a 0.793 0.792 13.5

8 Minam River (spring)a 0.79 0.788 13.5

9 John Day River (spring) 0.78 0.755 13.5

10 Wenatchee River (spring) 0.795 0.803 13.4

11 Methow River (spring) 0.793 0.788 13.4

12 Cle Elum Hatchery (spring) 0.816 0.796 13.2

13 North Santiam Hatchery (spring) 0.82 0.812 13.1

14 Lochsa River–Powell Trap (spring)a 0.788 0.789 13.1

15 Red River (spring)a 0.795 0.795 13

16 Sawtooth Hatchery (spring)a 0.79 0.793 13

17 McKenzie Hatchery (spring) 0.817 0.812 12.9

18 South Fork Clearwater (spring)a 0.785 0.782 12.8

19 Imnaha River (spring)a 0.783 0.793 12.7

20 Catherine Creek (spring)a 0.775 0.776 12.7

21 Winthrop Hatchery, Carson stock (spring) 0.792 0.809 12.5

22 Johnson Creek supplementation (spring)a 0.779 0.776 12.2

23 Shitike Creek (spring) 0.763 0.757 12.2

24 Marsh Creek (spring) 0.782 0.777 12.1

25 Secesh River (spring)a 0.773 0.763 12.1

26 Newsome Creek (spring)a 0.765 0.76 12

27 East Fork Salmon River (spring)a 0.769 0.757 12

28 Johnson Creek (spring)a 0.776 0.775 11.9

29 Entiat Hatchery (spring) 0.782 0.793 11.7

30 Big Creek-a (spring)a 0.754 0.764 11.7

31 Tucannon River (spring)a 0.791 0.803 11.6

32 Pahsimeroi River (spring)a 0.78 0.79 11.5

33 Big Creek-b (spring)a 0.76 0.782 11.3

34 Rapid River Hatchery (spring)a 0.762 0.767 11.3

35 Lostine River (spring)a 0.754 0.763 11

36 Warm Springs Hatchery (spring) 0.725 0.728 10.9

37 West Fork Yankee Fork (spring)a 0.758 0.779 10.3

Pairwise θ Values

Small but significant values among subbasins

Pairwise θ values among hatchery (H) and wild (W) origin spring Chinook populations from the Willamette River and

Catherine Creek Hatchery (Grande Ronde River), estimated from genotypic data for 13 GAPS microsatellite loci. Values not significantly different from zero (p > 0.05) are indicated in bold.

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Clackamas

Hatchery

Clackamas

Wild

Willamette

Hatchery

McKenzie

Hatchery

McKenzie

Wild

N.Santiam

Hatchery

N. Santiam

Wild

S. Santiam

Hatchery

S. Santiam

Wild

Catherine Cr. H 0.111 0.106 0.106 0.107 0.102 0.100 0.110 0.099 0.104

Clackamas H 0.007 0.012 0.013 0.013 0.010 0.012 0.010 0.009

Clackamas W 0.004 0.003 0.003 0.004 0.005 0.002 0.001

Willamette H 0.007 0.006 0.008 0.009 0.003 0.004

McKenzie H 0.000 0.003 0.006 0.004 0.005

McKenzie W 0.004 0.006 0.004 0.003

N. Santiam H 0.002 0.005 0.005

N. Santiam W 0.005 0.005

S. Santiam H 0.000

Phylogenetic Relationships Hatchery populations most similar to local wild populations

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Maximum likelihood trees depicting genetic relationships among hatchery (H) and wild origin (W) spring Chinook populations

from the Willamette River and (right) Catherine Creek Hatchery population. Phylogeny inferred from genotypic data for 13

microsatellite loci. Branch lengths represent Cavalli-Sforza chord measures of genetic distances (Cavalli-Sforza and Edwards

1967). Bootstrap values are indicated for nodes with >50% support (left tree only). Branch lengths of the South Santiam H-

South Santiam W-MF Willamette H clade are not significantly different from zero (95% confidence interval).

Evidence of Selection • Among UWR populations: NO • UWR populations & Catherine Cr: YES

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Overall FST values for 17 microsatellite loci (markers) plotted against heterozygosity, as characterized from the

Catherine Creek hatchery and nine Willamette River spring Chinook populations. Gray shaded area defines the

99.5% CI of expected FST values for all possible heterozygosities, constructed from 50,000 data simulations.

Shaded areas indicate regions associated with positive (red) and balancing (yellow) selection.

Simulations of Migration and Diversity

Migration: Represents symmetrical pHOS and pNOB

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Generation

0 5 10 15 20 25 30 35

Hete

rozygosity

0.94

0.95

0.96

0.97

0.98

m = 0.00

m = 0.02

m = 0.05

m = 0.10

Generation

0 5 10 15 20 25 30 35

Theta

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

m = 0.00

m = 0.02

m = 0.05

m = 0.10

Migration of 5% preserves similar level of diversity as 10%

Simulated change in mean heterozygosity and theta for 17 neutral microsatellite loci in hatchery and wild populations of

McKenzie River spring Chinook. Data were simulated under four migration rates (m) across 30 generations.

Simulations of Migration and Diversity

Migration: Represents symmetrical pHOS and pNOB

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Migration of 5% preserves similar level of diversity as 10%

Generation

0 5 10 15 20 25 30 35

Mean N

um

ber

of

Alle

les

29

30

31

32

33

34

35

36

37

m = 0.00

m = 0.02

m = 0.05

m = 0.10

Simulated change in total allele count for 17 neutral microsatellite loci in hatchery and

wild populations of McKenzie River spring Chinook. Data were simulated under four

migration rates (m) across 30 generations.

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Key Points

• Heterozygosity in hatchery populations higher than in wilds

• Genetic structure is weak but present among subbasins

• Hatchery populations are genetically most similar to local wild populations

• No evidence for locus-specific selection among Willamette populations • Selection appears to drive divergence between UWR and Catherine

Creek populations at two of the loci examined

• Symmetrical migration of 5% appeared to preserve most (neutral) genetic diversity – nearly as well as 10%

Willamette River Steelhead

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Objectives

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Describe genetic structure among Oncorhynchus mykiss

populations

Estimate the proportion of summer steelhead among natural

origin O. mykiss smolts sampled at Willamette Falls and other

locations (subbasins) of the upper Willamette River

Estimate the proportion of summer steelhead hybrids among

natural origin O. mykiss smolts sampled at Willamette Falls and

various locations (subbasins) of the upper Willamette River

Sampling and Data Collection

Sample Collections • Samples of known type– baseline and phylogeny • Unmarked juvenile and some adult O. mykiss • Juveniles

• Willamette Falls (2009-2011) • Subbasins (2011; McKenzie 2005 & 2011)

Data Collection • All samples genotyped at 15 GAPS microsatellite loci

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Analyses Describe genetic structure among Oncorhynchus mykiss populations

Estimate the proportion of summer steelhead among natural origin O. mykiss smolts sampled at

Willamette Falls and other locations (subbasins) of the upper Willamette River

Genetic Stock Identification (GSI) • Constructed phylogeny to identify reporting groups1

• Unknown samples assigned with a Bayesian GSI approach2

Estimate the proportion of summer steelhead hybrids among natural origin O. mykiss smolts

sampled at Willamette Falls and various locations (subbasins) of the upper Willamette River

Introgression Analysis • Bayesian clustering method3 • Samples classified to group or hybrid group 1PHYLIP - Felsenstein. 2009. Available at http://evolution.genetics.washington.edu/phylip/doc/ 2ONCOR – Kalinowski. 2007. Available at http://www.montana.edu/kalinowski/Software/ONCOR.htm 3STRUCTURE – Pritchard et al. 2000. Genetics 155: 945-959.

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Introgression Analyses (continued) Introgression Analysis Based on a four-group stock structure, we estimated the proportion (q) of each individual’s genome descended from each group We then used q values to classify individual samples into the following general categories:

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Pure: q > 0.50 for a single population and q < 0.20 for all other populations

Two-way hybrid: 0.20 < q < 0.80 for exactly two populations

Three-way hybrid: 0.20 < q < 0.80 for exactly three populations

S EW RB WW SxEW SxRB

Perc

ent q

0

20

40

60

80

100

SUMMER

EAST-SIDE WINTER

WEST-SIDE WINTER

RAINBOW

Willamette Oncorhynchus mykiss

Van Doornik & Teel 2010

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Neighbor-joining dendrogram of Cavalli-Sforza Edwards genetic distances

among Willamette River steelhead populations. Bootstrap values (%) greater

than 50% are shown. The last two digits of the brood year for the earliest

samples are included in the sample names. Major groupings, which also

correspond to the reporting groups used for GSI analyses, are circled.

GSI of unmarked juvenile Oncorhynchus mykiss: Willamette Falls

VanDoornik & Teel 2010, 2011, 2012

Location Year n EW S WW RB

Willamette Falls 2009 240 88.3% 7.5% 4.2% 0.0%

Willamette Falls 2010 287 78.0% 13.2% 8.7% 0.0%

Willamette Falls 2011 56 89.3% 5.4% 5.4% 0.0%

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GSI of unmarked juvenile Oncorhynchus mykiss: Willamette River subbasins

VanDoornik & Teel 2010, 2011, 2012

Location Year n EW S WW RB

Willamette R., various mainstem 2011 29 58.6% 13.8% 0.0% 27.6%

Santiam R., mouth 2011 11 90.9% 9.1% 0.0% 0.0%

North Santiam R. 2011 36 94.4% 2.8% 0.0% 2.8%

South Santiam R. 2011 27 100.0% 0.0% 0.0% 0.0%

McKenzie R., Leaburg Bypass 2005 72 25.0% 75.0% 0.0% 0.0%

McKenzie R., Leaburg Bypass 2011 91 27.5% 68.1% 0.0% 4.4%

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Genetic introgression

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no

wn

s

Un

kn

ow

ns

East-side

Winter Rainbow Summer

West-side

Winter

Genetic introgression: Juveniles

Year Location n S EW RB WW SxWW SxEW SxRB WWxEW WWxRB EWxRB 3x Hybrid

2009 Willamette Falls 240 19 126 1 34 1 23 1 31 0 1 3

2010 Willamette Falls 287 39 144 1 37 4 29 0 25 0 3 5

2011 Willamette Falls 56 3 29 0 13 1 3 0 5 0 0 2

Percent of Total 10.5 51.3 0.3 14.4 1.0 9.4 0.2 10.5 0.0 0.7 1.7

2005 McKenzie R., Leaburg 72 56 1 0 0 1 11 1 1 0 0 1

2011 McKenzie R., Leaburg 91 63 2 4 0 1 11 6 0 0 2 2

Percent of Total 73.0 1.8 2.5 0.0 1.2 13.5 4.3 0.6 0.0 1.2 1.8

2010 Mainstem Willamette R. 30 3 10 10 0 1 1 0 0 0 5 0

Percent of Total 10.0 33.3 33.3 0.0 3.3 3.3 0.0 0.0 0.0 16.7 0.0

2011 N. Santiam R. 36 0 25 0 1 0 4 0 4 0 1 1

Percent of Total 0.0 69.4 0.0 2.8 0.0 11.1 0.0 11.1 0.0 2.8 2.8

2011 Santiam R., Mouth 11 0 6 2 0 0 1 0 1 0 0 1

Percent of Total 0.0 54.5 18.2 0.0 0.0 9.1 0.0 9.1 0.0 0.0 9.1

2011 S. Santiam R. 27 0 20 0 1 0 4 0 2 0 0 0

Percent of Total 0.0 74.1 0.0 3.7 0.0 14.8 0.0 7.4 0.0 0.0 0.0

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Summer steelhead introgression

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q

0.0 0.2 0.4 0.6 0.8 1.0

Pro

port

ion

0.0

0.2

0.4

0.6

0.8

1.0

Willamette Falls 2009 n = 240

q

0.0 0.2 0.4 0.6 0.8 1.0

Pro

port

ion

0.0

0.2

0.4

0.6

0.8

1.0

Willamette Falls 2011n = 56

Summer steelhead introgression introduction methods results summary questions

q

0.0 0.2 0.4 0.6 0.8 1.0

Pro

port

ion

0.0

0.2

0.4

0.6

0.8

1.0North Santiam 2011n = 36

q

0.0 0.2 0.4 0.6 0.8 1.0

Pro

port

ion

0.0

0.2

0.4

0.6

0.8

1.0

South Santiam 2011n = 27

q

0.0 0.2 0.4 0.6 0.8 1.0

Pro

port

ion

0.0

0.2

0.4

0.6

0.8

1.0McKenzie 2011n = 91

q

0.0 0.2 0.4 0.6 0.8 1.0

Pro

port

ion

0.0

0.2

0.4

0.6

0.8

1.0McKenzie 2005n = 72

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Key Points

• About ~10% of naturally produced juvenile steelhead sampled at Willamette Falls were summer-run type

• Little evidence for natural production of pure summer

steelhead in the Santiam rivers, but most juveniles from the McKenzie River were summer steelhead

• Evidence for some genetic introgression from summer steelhead detected at all locations

Questions?

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Acknowledgments ODFW Rich Carmichael,

Hatchery managers, Field crews et al.

Sample collections

WDFW Jeffrey Grimm Otolith analyses

OSU Michael Banks, Kathleen O’Malley, Amelia Whitcomb, Dave Jacobson et al.

Genetic laboratory services

USACE David Griffith, Rich Piaskowski, David Leonhardt et al.

Funding

Genetic introgression: Adults

Year Location n S EW RB WW SxWW SxEW SxRB WWxEW WWxRB EWxRB 3x Hybrid

2009 S. Santiam R., Foster 50 0 42 0 0 0 5 0 2 0 1 0

Percent of Total 0.0 84.0 0.0 0.0 0.0 10.0 0.0 4.0 0.0 2.0 0.0

2004 N. Santiam R., Bennett 28 2 7 0 0 0 16 0 1 0 1 1

2009 N. Santiam R., Minto 11 0 8 0 0 0 2 0 1 0 0 0

2010 N. Santiam R., Minto 1 0 1 0 0 0 0 0 0 0 0 0

Percent of Total 5.0 40.0 0.0 0.0 0.0 45.0 0.0 5.0 0.0 2.5 2.5

2005 Mainstem Willamette R. 1 0 1 0 0 0 0 0 0 0 0 0

2010 Willamette R., Fall Cr. 19 0 16 0 0 0 0 0 0 0 3 0

2011 Willamette R., Fall Cr. 16 0 16 0 0 0 0 0 0 0 0 0

Percent of Total 0.0 91.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 8.3 0.0

2005 McKenzie R., Mohawk R. 1 0 1 0 0 0 0 0 0 0 0 0

2011 McKenzie R., Leaburg 6 3 0 1 0 0 1 1 0 0 0 0

Percent of Total 42.9 14.3 14.3 0.0 0.0 14.3 14.3 0.0 0.0 0.0 0.0

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