Genetic Interrelationship

Genetic Interrelationship

Populations of Populations of Gila cypha 

Colorado River Ecosys

Colorado River Ecosys

Marlis R. Douglas

Michael E. Douglas

Dept. Fish, Wildlife & Conservat

Colorado State Universi
Humpback Chub ( Humpback Chub (Gila cypha)

Relationships among Popu

Relationships among Popu

Humpback Chub (H

Humpback Chub (H
HBC in

Grand Cn

9 locations

=

aggregates
• Do aggregates represent discrete 

Do aggregates represent discrete 

• migration among aggregates? migration among aggregates?• local reproduction? local reproduction?• local recruitment? local recruitment?

Gila cypha in Grand Ca

in Grand Ca
HBC in

Grand Cn

locations locations

3

Middle 

Granite Granite 

Gorge

Western Western 

Grand 

Canyon
• Relationships among 9 Relationships among 9 “aggregate

aggregate

• Mainstem Mainstem / LCR continuum ? / LCR continuum ?

• 30-Mile vs LCR?

• LCR vs Middle Granite Gorge Middle Granite Gorge• LCR vs Western Grand Canyon Western Grand Canyon

Gila cypha in Grand Ca

in Grand Ca
HBC in

Grand Cn

LCR vs 30M

LCR vs MGG

LCR vs WGC

aggregates
• Examine population structure of Examine population structure of 

• genetic genetic interrelationships am

interrelationships am

aggregates in Grand Canyon aggregates in Grand Canyon

• Is there a Is there a baseline baseline?

• how much differentiation to 

how much differentiation to 

• which methods to use? which methods to use?

Gila cypha in Grand Can

in Grand Can
Gila cypha in 

Colorado Colorado 

River Basin River Basin

Upper Basin Upper Basin

Grand Canyon Grand Canyon
Colorado R Colorado R

Gila Humpback Chub Humpback Chub

Roundtail Roundtail Chub

Bonytail Bonytail 

John N. John N. Rinne
Colorado R Colorado R

Gila Humpback Chub Humpback Chub

Roundtail Roundtail Chub

Bonytail Bonytail 

John N. John N. Rinne
Colorado R Colorado R

Gila Humpback Chub Humpback Chub

Roundtail Roundtail Chub

Bonytail Bonytail 

HBC

BTC

RTC

John N. John N. Rinne
Entire Basin Entire Basin

Upper Basin Upper Basin• HBC• RTC

Grand Canyon Grand Canyon• HBC
• Examine population structure amo

Examine population structure amo

Colorado River Ecosystem Colorado River Ecosystem

• genetic interrelationships genetic interrelationships of popu

of popu

• spatial scales spatial scales (= population str

(= population str

• temporal scales temporal scales (= evolutionary 

(= evolutionary 

Study Objectives Study Objectives
Approach: Molecular Geneti

Approach: Molecular Geneti

Tissue collection

DNA

-Extraction Extraction

DNA Amplificat

DNA Amplificat

(PCR) 
Molecular Genetic Met

Molecular Genetic Met

• mitochondrial DNA mitochondrial DNA sequence anal

sequence anal

>> evolutionary history >> evolutionary history

>> long >> long

-term conservation g

term conservation g

• microsatellite microsatellite DNA analysis analysis

>> population structure >> population structure

>> management units ( >> management units (MUs

)
Msat Loci & Sample S

Loci & Sample S

• high variability high variability

+ population population-level analyses level analyses

- # alleles >> # sample sizes # alleles >> # sample sizes

• codominant codominant inheritance inheritance

+ species ecology (e.g., introgre

species ecology (e.g., introgre

- larger sample size to encapsul

larger sample size to encapsul

pop. variation pop. variation
• 9 aggregates / 234 HBC 9 aggregates / 234 HBC

- 30-Mile Springs Mile Springs 

- Little Colorado River Little Colorado River - Lava to Lava to Hance - Shinumo Shinumo Ck - Stephen Stephen’s Aisle s Aisle 

- Randy’s Rock - Kanab Ck Kanab Ck - Havasu Ck Havasu Ck - Western Grand Western Grand Cn 

Sampling: Grand Cany

Sampling: Grand Cany

Table 5 Table 5
• 5 locations / 186 HBC + 208 R

5 locations / 186 HBC + 208 R

HBC 

- Yampa R Yampa R 7 

- Desolation Desolation Cn 51 - Black Rocks Black Rocks Cn 51 - West Water West Water Cn 77 

- 15-mile Reach mile Reach - 

Sampling: Upper Bas

Sampling: Upper Bas

Table 5 Table 5
Sampling Sampling

Upper Basin Upper Basin

208 RTC 208 RTC186 HBC 186 HBC

Grand Canyon Grand Canyon

234 HBC 234 HBC

Broodstock Broodstock

15 BTC
• 8 HBC/RTC with HBC/RTC with BTC haploty

haploty

- HBC Desolation HBC Desolation Cn 

- HBC 30-Miles - HBC LCR HBC LCR - HBC Randy HBC Randy’s Rock - RTC Desolation RTC Desolation Cn 

Results: Results: mtDNA Analy

Analy

Table 2 Table 2
Parsimony Parsimonyhaplotype haplotype

network network

mtDNA sequence datap-distance: 1-8Jukes-Cantor: 0.05-0.4% 
Comparison Comparison % Sequence 

% Sequence 

HBCUB / HBCGC no dif

no dif

HBC / RTCUB ~ 0.1% 

BTC / HBC+ RTCUB ~ 5%

RTCUB / RTCLB ~ 4%

Ptychocheilus Ptychocheilus / Gila ~ 6%

Mitochondrial DNA An

Mitochondrial DNA An

Table 3 Table 3
Results: Results: mtDNA Analy

Analy

• many haplotypes haplotypes

but low divergence among but low divergence among h

h

• historic population bottleneck historic population bottleneck

thus low effective populatio

thus low effective populatio

• population expansion since bott

population expansion since bott

re-colonization of upper bas

colonization of upper bas

Table 2 + 4 Table 2 + 4
mtDNA + Colorado Rive

+ Colorado Rive

• Douglas, Brunner, Douglas (2003

Douglas, Brunner, Douglas (2003

Freshwater Biology Freshwater Biology 

• Drought in Drought in “recent” geologic tim

geologic tim

severe impacts severe impacts on Colorado 

on Colorado 
10

9

8

6

Climatic Optima Climatic Optima for End for End

-of

-

Pleistocene Pleistocene Hypsithermal Hypsithermal
• Historic Population Bottleneck Historic Population Bottleneck

• Low effective population size Low effective population size • Shorter evolutionary time sca

Shorter evolutionary time sc

• Different molecular approach

Different molecular approach

Historic Demography & Ma

Historic Demography & Ma
Results: Results: Msat Analysis Analysis - D

D

• High allelic diversity High allelic diversity within popula

• Moderate to high levels of hetero

hetero

• Unique alleles Unique alleles for populations, bas

Table 7 + 8, Appendix 5 + 6 Table 7 + 8, Appendix 5 + 6 
Results: Results: Msat Analysis Analysis - D

D

• Allele frequencies Allele frequencies between popula

show differences (Bonferroni correct

...... but 

• sample size effects!

Table 9 Table 9
• Data support recognition of six Data support recognition of six M

(1) HBCGC : Marble/Grand : Marble/Grand Cn

(2) HBCDES + RTCDES : Desolation : Desolation 

(3) HBCUB : upper basin : upper basin

(4) RTCUB : upper basin (except 

: upper basin (except 

(5) RTCYR : Yampa R : Yampa R(6) BTC : Bonytail Bonytail

Conclusion (1) Conclusion (1)
HBC

DES

msat – STRUCTURE: 6 Clu

STRUCTURE: 6 Clu

HH

A

V

H

B

K

R

H

D

E

S

HWW

C

H

Y

A

M

H

30

M

H

L

C

R

H

S

HN

H

L

A

H

H

S

AA

H

R

A

N

HW

G

C

H

K

A

N

R

B

K

R

HBC-UB

HBC-GC
• Populations: Populations: 

- Yampa River Yampa River

- Desolation Desolation Cn- Black Rocks Black Rocks Cn- Westwater Westwater Cn- Cataract Cataract Cn

Gila cypha – Upper Ba

Upper Ba

no samples no samples
 HBC in Marble/Grand HBC in Marble/Grand Cn genetic

genetic

• aggregates connected by aggregates connected by gene

gene

pairwise pairwise tests non tests non-significant significant

GP5 = genepool genepool identified by TFPGA entified by TFPGA 

>> downstream drift of larvae + j

>> downstream drift of larvae + j

>> LCR likely primary source >> LCR likely primary source>> occasional local reproduction >> occasional local reproduction

Conclusion (2) Conclusion (2)
H

H

A

V

UB-H

DC/BT

UB-H

GC-H

UB-

H

B

K

R

H

D

E

S

HWW

C

H

Y

A

M

H

30

M

H

L

C

R

H

S

HN

H

L

A

H

H

S

AA

H

R

A

N

HW

G

C

H

K

A

N

R

B

K

R

msat – STRUCTURE: 5 Clus

STRUCTURE: 5 Clu
HBC-UB HBC-GC

HBC-Deso R

Four-Group Clustering (Progra

msat - Bayesian Bayesian Assignme

Assignm
 30-Mile population in Marble Mile population in Marble Cn

• admixture with admixture with BTC (mtDNA)

• association with association with HBCUB

• distinct distinct genepool genepool>> occasional local reproduction >> occasional local reproductionBUT – small sample size small sample size, low pop. 

, low pop

Conclusion (3) Conclusion (3)
HBC

DES

msat – STRUCTURE: 6 Clu

STRUCTURE: 6 Clu

HH

A

V

H

B

K

R

H

D

E

S

HWW

C

H

Y

A

M

H

30

M

H

L

C

R

H

S

HN

H

L

A

H

H

S

AA

H

R

A

N

HW

G

C

H

K

A

N

R

B

K

R

HBC-UB

HBC-GC
msat – STRUCTURE: 6 Clu

STRUCTURE: 6 Clu

HBC 

HBC-UB HBC-G
 Upper Basin HBC / RTC Upper Basin HBC / RTC

• RTC in Yampa River forms dis

RTC in Yampa River forms dis

>> different from other Upper B

>> different from other Upper B

BUT – small sample size small sample size for HBC 

for HBC 

Conclusion (4) Conclusion (4)
HBC

DES

msat – STRUCTURE: 6 Clu

STRUCTURE: 6 Clu

HH

A

V

H

B

K

R

H

D

E

S

HWW

C

H

Y

A

M

H

30

M

H

L

C

R

H

S

HN

H

L

A

H

H

S

AA

H

R

A

N

HW

G

C

H

K

A

N

R

B

K

R

HBC-UB

HBC-GC
 Bonytail Bonytail

• admixture with HBC admixture with HBC>> historic signal of introgression >> historic signal of introgression>> stronger signal Desolation Can

>> stronger signal Desolation Can

Conclusion (5) Conclusion (5)
H

H

A

V

UB-H

DC/BT

UB-H

GC-H

UB-

H

B

K

R

H

D

E

S

HWW

C

H

Y

A

M

H

30

M

H

L

C

R

H

S

HN

H

L

A

H

H

S

AA

H

R

A

N

HW

G

C

H

K

A

N

R

B

K

R

msat – STRUCTURE: 5 Clus

STRUCTURE: 5 Clu
Msat Analysis Analysis - Admix

Admix

• Bonytail Bonytail

• characteristic allele size ranges 

some BTC in HBC some BTC in HBC some HBC/RTC in BTC some HBC/RTC in BTC
Conclusion (5) Conclusion (5)

Allele Frequency for G99

0.0000.0200.0400.0600.0800.1000.1200.140

337

341

345

349

353

357

361

365

369

373

377

381

385

389

393

397

401

405

409

413

417

421

425

429

433

437

445

449

453

461

465

473

477

485

489

G99

Frequency 
• mtDNA reflects evolutionary h

reflects evolutionary h

– admixture admixture HBC / RTC HBC / RTC

• msat also reflect morphology also reflect morphology

- distinct distinct morphs

– locality locality effect

Species Species still described morph

described morph

Compendium Compendium
• Morphometric Morphometric Characters Characters (museum 

(museum 

• Discriminant Discriminant Function Analysis Function Analysis• Adults: 95% allocated to species Adults: 95% allocated to species

• Juveniles: 97% allocated to RTC or 

Juveniles: 97% allocated to RTC or 

Morphology & Morphology & Gila

Dou

Dou
Relative Warp 1

RW2

GCN

WWC

BRK

DES

CCC

YAM

DES

DEB

RIF

CCC

BKR WWC

YAM

0

0

-0.015

0.015

-0.002 

Shape Variation: Shape Variation: Relative Wa

Relative Wa

D
Thank You! Thank You!