An evaluation of the population biology, genetics and future viability of the breeding Wood Duck (Aix
sponsa) population at Arrowwood National Wildlife Refuge
by James Bruce Neill
A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in
Biological Sciences
Montana State University
© Copyright by James Bruce Neill (1995)
Abstract:
A breeding population of Wood Ducks (Aix sponsa), introduced to eastern-central North Dakota in
1968 was evaluated. This population occupies habitat outside the native range of Wood Ducks;
numbers of breeding females have greatly declined over the past ten years. Two potential factors for
this decline in population numbers were evaluated. These are competition from Hooded Mergansers
(Lophodytes cucullatus), and reduced genetic variability of the Wood Duck population from the use of
captive individuals to seed the population. Analysis of historical nesting data yielded no indication that
Hooded Merganser’s nesting activities have had any impact on the reproductive success of Wood
Ducks. An analysis of 17 polymorphic allozymes was made for the Arrowwood population and a
captive population similar to the one originally used to create the Arrowwood population. Using these
data, mean heterozygosity, mean number of alleles/locus, and percent polymorphic loci were
calculated. All of these indices indicate that the Arrowwood Wood Duck population is more genetically
diverse than the captive population, and has levels of genetic variability similar to those reported for
other native avian populations. The differences between the two populations were found not to be
statistically different. Minisatellite DNA fingerprint analysis was carried out for the Arrowwood
population, the captive population and a population from western-central Oregon. These analyses
indicate that the Arrowwood population has significantly more variation than the captive population,
and both of these exhibit more DNA polymorphism variability than the population in Oregon. It is
suggested that the number of nesting females is being underestimated at Arrowwood National Wildlife
Refuge because hens are nesting in natural nesting cavities and in areas outside the refuge boundaries.
It is suggested that the Wood Duck population in North Dakota has high levels of genetic variability
because of a constant influx of novel drakes. Umecorded nesting and high genetic variability suggests
the population of Wood Ducks in eastern-central North Dakota is in no danger of immediate
extirpation. It appears that Wood Ducks in the Pacific Flyway have experienced long population
bottle-necks causing a paucity of genetic variability; further analysis of Pacific Wood Duck populations
is suggested. AN EVALUATION OF THE POPULATION BIOLOGY, GENETICS AND FUTURE
VIABILITY OF THE BREEDING WOOD DUCK (A IX SPONSA) POPULATION AT
ARROWWOOD NATIONAL WILDLIFE REFUGE.
by
James Bruce Neill
A thesis submitted in partial fulfillment
o f the requirements for the degree o f
Doctor o f Philosophy
in
Biological Sciences
MONTANA STATE UNIVERSITY
Bozeman, Montana
April 1995
J>31g
V3n
APPROVAL
o f a thesis submitted by
James Bruce Neill
This thesis has been read by each member o f the thesis committee and has been
found to be satisfactory regarding content, Enghsh usage, format, citations, bibliographic
style, and consistency, and is ready for submission to the College o f Graduate Studies.
2
/
A~pri I
Date
I t??^
C
uate (Committee
Chairperson, Graduate
Approved for the Major Department
21 Apri \ 1995
Date
Head, Major Department
Approved for the CoUege o f Graduate Studies
Date
Graduate Dean
iii
STATEMENT OF PERMISSION TO USE
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ACKNOWLEDGMENTS
There are a great many people I would like to thank for helping me on this long,
twisted road I have been traveling on. First and foremost, I must thank BC & Tim for
their total support; this would never have been possible without their patience and
understanding. Leigh Ellis gave me tremendous support throughout this endeavor, her
understanding and patience are not taken lightly. Jessica Bear NeiJl is the best, and gave
me more inspiration and drive than I would have ever imagined; she is a great person and I
am glad to have her as a daughter. Evelyn Monroe provided enormous support and
motivation during the final phases o f this work; without her I doubt I would have ever
finished. Lance Craighead and Hugh Britten gave me advise, computer programs and the
motivation to keep going while on a desert island. Emie Vyse provided me with
wonderful guidance and leadership and provided a model by which I could complete this
process and not lose sight o f the important things in life. Peter Brassard gave me
inspiration and a vision o f how to examine the world as a population biologist, and how to
better understand species. David Cameron taught me how to profess and to continue to
ask questions and seek answers. Dan Goodman opened my eyes to the wonderful world
o f hypersapce in matrices; this vision has taken me on a long journey. Jack Homer has
forever turned me on to the wily pursuit o f evolutionary biology and given me the ability
to keep on going when I thought I couldn’t make it. He also provided funding which
helped to make it happen; for both o f these I am extremely thankful. In this dissertation, I
see a great many people and many great people’s thoughts, inspiration, patience and
kindness, love and energy. I thank you all (and the nameless others), with all my soul.
vi
TABLE OF CONTENTS
Page
1. OVERVIEW OF DISSERTATION PROJECT.............................................................. I
2. POPULATION ANALYSIS OF NESTING WOOD DUCKS AND HOODED
MERGANSERS AT ARROWWOOD NATIONAL WILDLIFE REFUGE..............4
Introduction...............................................................
4
Materials and Methods......... .................................................................................... 11
Results........................................!................................................................................12
Discussion............................................
16
3. AN INVESTIGATION OF ALLOZYME VARIABILITY IN A CAPTIVE AND
AN INTRODUCED POPULATION OF WOOD DUCKS..............................
„23
Introduction..........................................................................................
23
Materials and Methods.............................................................................................. 27
Results...,............................................................................... ....................,..........!....31
Discussion.................................................................................................
35
4. A COMPARISON OF GENETIC DIVERSITY OF THREE WOOD DUCK
POPULATIONS USING DNA FINGERPRINTING.... .............................................43
Introduction........................................................
Materials and Methods............................
Results..................................................................................................................
Discussion................................
5. SUMMARY OF THE DlSSERTATIONRESEARCH.......................
LITERATURE CITED...............................................................
43
45
51
63
!...70
76
APPENDICES........................................... !.................................................... !............... 87
Appendix A—Raw Data from DNA Fingerprints................................................. 88
Appendix B-- Raw Genotypes from AUozyme Analyses...................................100
vii
LIST OF TABLES
Table
Page
1. Number o f Eggs Laid, Followed by the Number o f Eggs Hatched
for both Wood Ducks and Hooded Mergansers Between
the Years 1979 and 1989.......................
15
2. Symbols, Names, Enzyme Commission Numbers Used for AUozyme
Analysis o f Azx sponsa ..................................................................................29
3. Buffer Systems and Types o f Tissue Samples Used to resolve the Loci
Under Investigation........................................................................................30
4. Allele Frequencies for all Loci from the Arrowwood National Wildlife
Refiige and a Captive Population (Hancock) o f W ood Ducks.................32
5. Mean Heterozygosity (H), Mean Numbers o f Allele/Locus, and
Proportion o f Polymorphic Loci (P) for the two Populations
(ANWR and Hancock), Under Investigation..................................
35
6. Genetic Diversity Values for JTaein/p V47 DNA Fingoiprints............................53
7. Percentage o f Fragments by Fragment Size-Class................................................ 58
8. Frequency o f Fragments and Their Occurrence in the Hancock
and ANWR Populations...........................................................................
9. Results o f a T-Test for Differences in Intrapopulation Similarity
Coefficients (S), Between the Three Populations....................................... 61
10. A Matrix o f Interpopulation (Sy) Coefficients for the Three Populations........61
60
viii
LIST OF FIGURES
Figure
Page
1. The numbers o f nesting females o f Hooded Mergansers and
W ood Ducks at Arrowwood National Wildlife Refuge........................... 13
2. Egg Hatching Success as a Function o f Time Since the Introduction
o f Hooded Mergansers................................................................................. 14
3. A Typical Autoradiogram Produced with HaeJHhpV 47 Fingerprints...............52
4. Frequency (Expressed in Percentages) o f DNA Fragments According
to Fragment Size from the Hancock Population........................................54
5. Frequency (Expressed in Percentages) o f DNA Fragments According
to Fragment Size from the ANWR Population..................
55
6. Frequency (Expressed in Percentages) o f DNA Fragments According
to Fragment Size from the Finley Population............................................56
7. Cumulative Percent Frequency o f DNA Fragments as Expressed as
Fragment Size for all Populations................................
57
8. Percentage o f all DNA Fragments as a Function o f the Frequency o f
Occurrence in the Population for the ANWR and Hancock
Populations..........................................................
59
9. Dendrogram Depicting the Relationship Between the Three
Populations o f Wood Ducks......................................................................... 62
ix
ABSTRACT
A breeding population o f Wood Ducks (Aix sponsa), introduced to eastern-central
North Dakota in 1968 was evaluated. This population occupies habitat outside the native
range o f W ood Ducks; numbers o f breeding females have greatly declined over the past
ten years. Two potential factors for this decline in population numbers were evaluated.
These are competition from Hooded Mergansers {Lophodytes cucullatus), and reduced
genetic variability o f the W ood Duck population from the use o f captive individuals to
seed the population. Analysis o f historical nesting data yielded no indication that Hooded
Merganser’s nesting activities have had any impact on the reproductive success o f Wood
Ducks. An analysis o f 17 polymorphic allozymes was made for the Arrowwood
population and a captive population similar to the one originally used to create the
Arrowwood population. Using these data, mean heterozygosity, mean number of
alleles/locus, and percent polymorphic loci were calculated. All o f these indices'indicate
that the Arrowwood W ood Duck population is more genetically diverse than the captive
population, and has levels o f genetic variability similar to those reported for other native
avian populations. The differences between the tw o populations were found not to be
statistically different. Minisatellite DNA fingerprint analysis was carried out for the
Arrowwood population, the captive population and a population from western-central
Oregon These analyses indicate that the Arrowwood,population has significantly more
variation than the captive population, and both o f these exhibit more DNA polymoiphism
variability than the population in Oregon. It is suggested that the number o f nesting
females is being underestimated at Arrowwood National Wildlife Refuge because hens are
nesting in natural nesting cavities and in areas outside the refuge boundaries. It is
suggested that the W ood Duck population in North Dakota has high levels o f genetic
variability because o f a constant influx o f novel drakes. Umecorded nesting and high
genetic variability suggests the population o f W ood Ducks in eastern-central North
Dakota is in no danger o f immediate extirpation. It appears that W ood Ducks in the
Pacific Flyway have experienced long population bottle-necks causing a paucity o f
genetic variability; further analysis o f Pacific W ood DuCk populations is suggested.
I
C h ap ter I
O V ER V IEW O F D ISSERTA TIO N P R O JE C T
This thesis presents a study o f breeding W ood Ducks (Aix sponsd), on
Arrowwood National Wildlife Refuge, in east-central North Dakota. The principal focal
points o f this study are in the areas o f conservation biology and wildlife management. It is
a multi-disciplinary approach to a single problem o f conservation and wildlife
management. Information on the breeding biology, behavior, and genetics o f this species
is compiled to address the viability o f an introduced population. In this manner, this study
is representative o f how successful conservation endeavors must utilize a spectrum of
information in order to answer a single question concerning the relative health o f
populations.
W ood ducks have not traditionally occurred in this region o f the United States,
and they were purposely introduced to Arrowwood to determine whether or not a
breeding population could survive there. W ood Ducks from captive populations were
introduced to Arrowwood in 1968. The population flourished for several years and then
appeared to decline rather sharply. It was unclear as to why the breeding population o f
wood ducks was declining at Arrowwood. This study attempts to understand better the
apparent decline in breeding W ood Ducks at Arrowwood National Wildlife Refuge and to
analyze the genetics o f that population to determine whether the decline could be due to
genetic factors in the population arising from its origin as a captive population.
In this investigation, three hypotheses are addressed which could potentially
explain the decrease in population o f Wood Ducks at Arrowwood. The three hypotheses
are : I) Competition for nesting spaces from Hooded Mergansers is adversely affecting
the reproduction o f W ood Ducks, 2) Wood Ducks are exposed to environmental toxins at
Arrowwood, and the success o f W ood Duck reproduction is being adversely affected by
exposure to these contaminants, and 3) There is low genetic variability among the Wood
Ducks at Arrowwood and this reduced genetic variability is causing a decrease in
reproductive success among the W ood Ducks at Arrowwood. By testing these different
hypotheses, the overall health and viability o f W ood Ducks at Arrowwood National
Wildlife Refuge is evaluated.
The thesis is arranged into distinct chapters which cover different aspects o f this
investigation. The first chapter is an analysis o f the population trends o f breeding Wood
Ducks at Arrowwood; in it an additional evaluation is made to determine whether another
waterfowl species, the Hooded Merganser {Lophodytes cucullatus), appears to be a
contributory factor to the apparent decline o f nesting wood ducks. In this chapter,
historical nesting data is used to infer whether environmental toxicants appear to be
affecting the reproductive success o f Wood Ducks. The second chapter is an analysis o f
the genetic composition o f the Arrowwood population and a comparison o f that
population to a captive population similar to the population from which the Arrowwood
population first originated. This genetic analysis uses protein (allozyme), variability to
infer levels o f genetic variation within the Arrowwood and captive populations. The third
chapter is a genetic investigation based on DNA fingerprinting analyses which measures
amounts o f genetic variability directly from DNA rather than indirect measurements o f
proteins. In this chapter, a third population is added to the analysis. This population is a
naturally occurring, small population in Oregon. It was analyzed and compared to two
populations analyzed in previous chapters.
Through these combined approaches, the three hypotheses proposed are tested and
the health and viability o f the Arrowwood breeding population o f W ood Duck is assessed.
This combined approach is indicative o f how conservation studies and management
practices must draw on a wide variety o f techniques to ascertain the viability o f natural
populations and o f management techniques used to alter and sustain populations of wild
animals.
4
C h ap ter 2
PO PU LA TIO N ANALYSIS O F N ESTIN G W O O D DUCKS AND HOODED
M ERG AN SERS A T A R RO W W O O D N A TIO N A L W IL D L IFE REFU G E.
Introduction
Arrowwood National Wildlife Refuge (ANWR) in eastern-central North Dakota
was created in 1935 and contains 15,900 acres o f prairie grassland along a 16-mile length
o f the James River. On the refuge there are four large impoundments o f the river
producing extensive shallow lakes and marshes; there is very little natural river channel
remaining on the refuge. In these extensive limnetic zones there are communities o f many
aquatic and sub-aquatic plants which provide large areas suitable for waterfowl use.
This area is outside the traditional breeding range o f the W ood Duck (Aix sponsa).
Wood Ducks were introduced to (ANWR) in 1968 (Doty & Kruse 1972). The initial
introduction was accompanied by the placement o f approximately 300 nest boxes within
the refuge boundaries. These combined activities were parts o f an experiment designed to
evaluate the effectiveness o f establishing Wood Duck populations in novel geographic
locations (Doty & Kruse 1972). Since 1969, nest-box utilization has been monitored and
recorded on an annual basis by the staff o f ANWR as a part o f the waterfowl management
program, and these data have been used to monitor the status o f the breeding Wood Duck
population on the refuge.
5
W ood Ducks faced near extinction in the early parts o f this century; it is estimated
that in 1915 there were more W ood Ducks in captive flocks in Europe than in the wild in
North America (Ripley 1973). This species was saved from extinction by strict legislation
imposing a moratorium on hunting (Bellrose & Heister 1987, Baldassarre & Bolen 1994)
and considerable re-introduction efforts from European captive populations (Ripley 1973).
Since that time, Wood Ducks have made a very successful recovery and are now fairly
common throughout their original range even though this species experienced population
bottlenecks and perhaps extensive inbreeding for a period o f 10-20 generations.
The traditional range o f W ood Ducks occupied the Atlantic, Pacific and
Mississippi waterfowl Flyways o f North America. Within these flyways, W ood Ducks are
most abundant in southern regions during both the breeding and winter seasons. Since the
1970's, W ood Ehicks have experienced a range expansion and they are now known to
breed and winter in the Central Flyway (Ladd 1990); this expansion is attributed to
anthropogenic introductions and a natural colonization o f the southeastern portions o f that
area.
Population densities o f W ood Ducks are extremely difficult to obtain, and accurate
population densities are not available for much o f their range. The principal method by
which population sizes are estimated is from harvest statistics gathered from hunters. This
method does not provide accurate information on actual population numbers, but does
provide insight with regard to general population trends. It is perhaps useful to compare
numbers between different flyways, although changing harvest regulations and the
6
dynamic nature o f hunting efforts can be confounding factors. Nonetheless, population
densities o f W ood Ducks appear generally much higher in the Atlantic and Mississippi
Flyways than in the Central or Pacific Flyways (Belhose 1980, Baldassarre & Bolen
1994). An estimate o f the number o f breeding W ood Ducks for the Central Flyway
(Belhose 1980), was around 50,000 individuals, although this estimate is probably very
conservative and could be off by up to 30-50% (May 1986; Ladd 1990). An estimate o f
the numbers o f individuals in the Pacific Flyway is 60,000 (Bartonek et a/.1990).
Although estimates o f absolute numbers o f individuals are not available for Atlantic and
Mississippi Flyways, harvests in those fiyways combined is in excess o f 1.2 million
individuals per year (Baldassarre & Bolen 1994). From these data, it appears that
populations in the Pacific Flyway are the lowest in North America and could be up to two
orders o f magnitude lower than those o f the eastern United States. The origin o f Wood
Ducks in the Pacific Flyway is unclear, but there has been little or no population mixing
between the Pacific Flyway and the flyways east o f the Rocky Mountains; this isolation
has caused the Pacific populations to remain genetically isolated with respect to other
populations.
During the last 20-30 years, Wood Ducks have been successfully introduced to
regions outside their native breeding ranges (Doty & Kruse 1973, Baldassarre & Bolen,
1994). M ost o f the introductions to novel or peripheral environments have been
accomplished by using individuals from captive breeding stocks to propagate new
breeding populations. One notable introduction was accomplished in 1968, when a
7
breeding population o f Wood Ducks was established in eastern-central North Dakota on
Arrowwood National Wildlife Refuge using stocks from captive populations in North
Dakota (Doty & Kruse 1972).
W ood Ducks often inhabit densely forested aquatic habitats. These aquatic
habitats are extremely difficult ones in which to conduct direct population surveys o f this
species (Hein 1966, BeUrose 1980, Parr & Scott 1978, Brakhage 1990, Moser & Graber
1990, Robb & Bookhout 1990). Many methods have been employed to make population
estimates o f W ood Duck abundance, but no feasible method exists to date. Wood Ducks
are also very secretive nesters, most commonly nesting in naturally occurring cavities in
trees. Such arboreal nesting locations are often difficult to find, and if found they are
commonly placed such that access to nests and their contents is impossible to obtain.
W ood Duck hens will readily nest in artificial nesting structures, and when nesting in these
structures their nesting behaviors are much easier to monitor. Much o f the information
regarding nesting densities, clutch size, reproductive success, and other reproductive
components o f W ood Duck biology has been obtained from analysis o f nesting activities in
artificial nesting structures. Such nest-box monitoring has proved valid to monitor cavity­
nesting breeding waterfowl populations, and it is nearly the only method useful for studies
o f W ood Duck nesting activities (Zicus & Hennes 1987, Ladd 1990, Robb & Bookhout
1988).
The breeding biology o f the ANWR W ood Duck population has been evaluated
through nest-box data analysis and was presented by Doty et al. (1984). During the 13
8
years following their introduction, the number o f nesting W ood Duckfemales at ANWR
has fluctuated widely and appeared to be declining in 1982 (Doty et al. 1984). The
nesting population experienced a marked increase during the initial years, but in the early
1980's it appeared to be steadily declining, and extirpation was imminent in the near
future.
The initial release o f Wood Ducks at ANWR consisted o f 280 ducklings that had
been incubated and reared in the Northern Prairie Wildlife Research Center (Doty & Kruse
1972). The sources for eggs from which these individuals hatched were from captive
flocks at the Center and a captive population in Minnesota. After hatching, the ducklings
were maintained in the Center for 9 to 16 days and then transported to ah open-topped
release pen on ANWR; when the ducklings were between 19 and 26 days o f age they were
sexed, banded and released (Doty & Kruse 1972). O f the 280 initial ducklings released,
approximately 253 (132 females and 121 males), survived to flight stages, and
approximately 193 survived until all Wood Ducks departed in the fall o f 1968 (Doty &
Kruse 1972). Band recovery from the first post-release winter indicated that the ducks
migrated along the western Central flyway to the western portion o f normal Wood Duck
winter range (Doty & Kruse 1972). During the following breeding season, 12 o f the
banded Wood Duck hens from the original release returned to ANWR and nested in nestbox structures on the refuge (Doty et al. 1984). For the following six years the number o f
nesting females continued to increase.
9
Since the introduction o f Wood Ducks to ANWR, Hooded Mergansers
(Lophodytes cucullatus), have naturally expanded their breeding range to include the
aquatic habitats there; in 1973 Hooded Mergansers were first observed to nest at the
refuge (Doty et al. 1984). Hooded Mergansers are cavity nesters, and it is hypothesized
that the two species might compete for nesting cavities and influence one-another's nesting
success. Intra- and interspecific cases o f nest parasitism are known for each o f the species
(Morse & White 1969, Clawson et al. 1979, Doty et al. 1984, Haramis & Thompson
1985, Sherman & Semel 1989). Hooded Mergansers have been reported to initiate
nesting activities earlier in the year than Wood Ducks (Fitzner & Fitzner, 1973). This
temporal difference in nest-initiation times could allow Hooded Mergansers to exclude
W ood Duck hens from nesting structures through a mechanism o f exploitative
competition. The potential negative influence o f Hooded Mergansers to the decline o f
nesting W ood Ducks at ANWR has been hypothesized by scientists (Doty et al. 1984) and
by refuge personnel.
When populations are maintained in captivity, there is often a loss o f genetic
variability due to inbreeding, genetic drift due to low NeZN ratios and other factors which
tend to degrade genetic diversity (Hedrick et al. 1986, Briscoe et al. 1992). The
maintenance o f genetic variability is widely believed important because the long-term
survival and viability o f populations is likely related to levels o f genetic variation among
members o f a population (Soule 1980, Frankel & Soule 1981, Barrett & Vyse 1982,
Beardmore 1983, Lande 1993). Although the exact mechanisms o f this relationship have
10
been recently questioned (Caro & Lanrenson 1994), it is nonetheless widely accepted that
populations having small amounts o f genetic variability are more extinction-prone than
populations with higher levels o f genetic variability. In captive breeding programs
designed to release individuals into the wild, efforts need to be made to insure that genetic
variability is not degraded during captivity so that the individuals released will represent a
significant portion o f the genetic variability naturally present in the species under
management (Hedeiick et al. 1986, Soule 1987). Many different strategies exist in
breeding programs that attempt to minimize degradation o f genetic variance in captive
populations. This crucial need to manage captive populations for genetic diversity is
widely accepted among breeders and game managers now, but it was not recognized at
the time o f the introduction o f W ood Ducks to ANWR in 1968. Consequently, no
attention was paid to the genetic composition o f the W ood Ducks released at ANW R
The long-term viability o f the W ood Duck population at ANWR is uncertain
because: I) ANWR is outside the traditional nesting range o f W ood Ducks, 2) The nesting
population is in decline, 3) a strong potential for interspecific nest competition exists, and
it originated from captive breeding stocks whose genetic variability is unknown but likely
to be below that o f natural populations.
In this analysis, nesting data are presented and analyzed to evaluate further
population trends o f nesting W ood Ducks at A N W R Nesting success data for both
Wood Ducks and Hooded Mergansers are examined to investigate the role o f interspecific
competition that might adversely affect Wood Duck nesting success.
M aterials an d M ethods
Nesting-box structures on ANWR are monitored every fall (after all nesting
activity has ceased) by refuge personnel as part o f the waterfowl management program.
During this monitoring, each nest-box is located, its condition noted, and the contents o f
the box recorded. The presence o f eggs, egg shells, and/or egg membranes is recorded,
quantified, and identified as to the species o f origin. The contents o f each nest box is then
removed, and any repairs and/or modifications needed to the structure or nesting materials
are made in preparation for the next nesting season. Hooded Merganser and Wood Duck
eggs and egg shells can be readily distinguished from one-another (SouUiere 1985),
making it possible to determine which o f these two waterfowl species used a particular
nest during the past nesting season. From this information, the number o f active nests,
number o f successful nests, and hatching success o f eggs is obtained for species.
The original waterfowl nesting data for ANWR was obtained for the years 19681988 from the nest-box survey records maintained by the refuge personnel. In 1989,1
collected the data for 320 nest box structures at tw o different times during the year. The
first data collection period was during late May when nesting activity was ongoing and
Wood Duck hens could be captured on the nests. The Second monitoring was made
during September after the cessation o f nesting activities. The methods I employed were
identical to those used by refuge personnel during the previous years.
12
Results
The numbers o f nesting females o f both species are presented in Figure I. There
was an increase in the W ood Duck population for the first six years followed by a sharp
decline over the next seven years. Since 1982, there has been some variation in the
number o f W ood Duck nests, but the population is essentially stationary at approximately
20-30 nests per year (Mean = 29 ± 9). The numbers o f Hooded Merganser and Wood
Duck nests indicate a very general trend o f W ood Duck nests decreasing and Hooded
Merganser nests increasing (Figure I). The number o f Wood Duck nests does not rapidly
decline with the advent o f Hooded Mergansers at the refuge, and there is no apparent
pattern indicating the size o f the breeding population o f one species closely correlates with
the number o f nests o f the other species. In 1982, the number o f H ooded Merganser nests
surpassed the number o f W ood Duck nests; and the number o f nesting Hooded
Mergansers has always been greater then the number o f nesting W ood Ducks since 1984.
In 1987 both species showed an increased number o f nests over the past 3 years.
The number o f successful egg-hatches per species per nesting season was used as
an index o f reproductive success (Figure 2). A regression o f egg hatching success over
time as expressed as the number, o f years since the initial nesting o f Hooded Mergansers at
ANWR revealed that neither species has experienced a significant change in reproductive
success over the period o f 1979-1989. For W ood Ducks, r2= 0.03, F = 0.44, p(F) = 0.52;
for Hooded Mergansers i 2= 0.02, F - 1.13, p(F) = 0.29.
1988
Number of Nest
Figure I. The Numbers of Nesting Females of Hooded Mergansers and Wood
Ducks Using Nestboxes at Arrowwood National Wildlife Refuge.
Wood Ducks
Hooded Mergansers
Figure 2. Egg Hatching Success as a Function of Time Since the Introduction of
Hooded Mergansers.
90
T03) 80
70
t
x
60
S 50
0 40
0)
1 30
§ 20
tS 10
0
I
2
3
4
5
Years Since Introduction of Hooded Mergansers
Wood Ducks
♦
Hooded Mergansers
6
15
A second analysis was carried out by regressing hatching success o f one species on
the number o f nesting females o f the other species for the period o f 1979-1989. For
Wood Duck success as a function o f Hooded Merganser nesting population size, r2= 0.2,
F = 3.64 , p(F) = 0.08; for Hooded Merganser success as a function o f Wood Duck
nesting population size, r2= -0.08, F = 0.21, p(F) = 0.21. A significant relationship does
not exist between the nesting success o f either species and the number o f nesting females
o f the other species.
Table I. Number o f eggs laid, followed by the number o f eggs hatched for both Wood
Duck and Hooded Mergansers between the years o f 1979 and 1989. The percentages in
parentheses are the total percentage o f eggs hatched per year. Values o f G that are not
significant at the a = 0.05 level are marked with an asterisk.
Year
1979
1980
1981
1982
1983
1984
1985
1986
1987
1988
1989
W ood Duck Eggs-Hatched
3 6 9 -2 1 5 (58.3%)
262 - 147 (56.1%)
265 - 208 (78.5%)
245 - 174 (71.1%)
638 - 324 (50.8%)
253 - 344 (73.5%)
185 - 288 (64.0%)
182 - 235 (77.4%)
332 - 398 (83.4%)
114 - 202 (56.4%)
3 1 9 - 381 (83.7%)
Hooded Merganser Eggs-Hatched
171 - 103 (60.2%)
163 - 115 (70.6%)
238 - 158 (66.4%)
150- 111 (74.0% )
238 - 453 (52.5%)
355 - 531 (66.8%)
350 - 483 (72.5%)
489 - 714 (68.5%)
388 - 689 (56.3%)
232 - 658 (35.3%)
640 - 959 (66.7%)
Gadj
0.187*
8.980
9.244
0.411*
0.326*
4.445
5.677
7.079
88.292
28.3026
41.503
To compare reproductive success between the two species, G-tests o f
independence (Sokal & Rolhf 1981), were carried out for hatching success data between
1979-1989. The number o f eggs hatched for each species in each year was used to
calculate G and Williams' adjustment was made to obtain a better approximation to the %2
16
distribution (Sokal & Rolhf 1981). The results are that hatching success differed between
the two species in seven o f the ten years (Table I). In six o f those years. Wood Ducks
exhibited higher nesting success than Hooded Mergansers (Figure 2), and only in 1985 did
Hooded Mergansers have higher nesting success than W ood Ducks. Hatching success,
when viewed as an indicator o f overall reproductive success, indicates that Wood Ducks
have higher reproductive success per individual than Hooded Mergansers but the number
o f nesting W ood Ducks is below that o f Hooded Mergansers.
To evaluate the possibility that intraspecific competition between nesting females
has adversely affected nesting success o f either species, a Pearson product-moment
correlation coefficient was calculated for the number o f eggs laid hr a season and the
percentage o f eggs hatched for each Species during that season. In both species, the
correlation between number o f eggs and hatching success was not significant; for Wood
Ducks, r = -0.27, P(r) = 0.414 and for Hooded Mergansers, r = 0.14, P(r) = 0.68. This
indicates that there was not a significant intraspecific interaction negatively affecting
nesting success for either species during the time o f this investigation.
Discussion
The number o f W ood Ducks using nest boxes on ANWR has apparently declined
in recent years. A possible explanation for this decline is that more W ood Duck hens are
using natural cavities for nesting and thus go unrecorded as nesting on the refuge. ANWR
has many mature cottonwood trees (Populus deltoides) surrounding the James River
impoundments on the refuge. These trees provide a large number o f natural cavities, and
17
observations have been made o f hen Wood Duck activity in and around such cavities by
refuge personnel. Studies in other areas have indicated that only 10% o f nesting females
in an area may utilize nest boxes when natural cavities are available (Soulliere 1985).
Further investigation (Soulliere 1990), reveals that in the Mississippi Flyway there is a
latitudinal trend o f nest-box utilization among Wood Ducks; in northern latitudes. Wood
Duck hens use nest-box structures less commonly than in southern latitudes. One
hypothesis proposed to explain this phenomenon is that it is density-dependent and that at
lower population densities nest boxes are used less frequently.
W ood Ducks are very
secretive nesters, and it is difficult to survey and quantify nests in natural nesting cavities
(Brakhage 1990, Cottrell & Prince 1990, Sauer & Droege 1990). However, the presence
o f hens in and around natural nesting cavities suggests that unmonitored nesting is
occurring on the refuge.
Since the time o f W ood Duck introductions to ANWR, several nest-box programs
have been instigated on private lands near the refuge. The refuge may no longer contain
the entire breeding population o f Wood Ducks in eastern-central North Dakota, and thus
the decline in nest box use within the refuge boundaries may reflect this. The existence o f
non-refuge nesting is suggested by information gathered during late summer wood-duck
trapping programs on the refuge. Cannon nets are deployed over artificial baits in the
early fall; the netted birds are banded, and their sex and age determined. Specific hatching
areas o f the young-of-the-year are unknown, but capture numbers are often too high to be
accounted for by the number o f Wood Duck nests on the refuge. These surplus young-of-
18
the-year are probably produced in nests in natural cavities and/or nest boxes outside the
refuge boundaries but in the vicinity o f the refuge:
Analysis o f egg-hatching data for the two species at ANWR reveals that Wood
Ducks commonly have a higher egg-hatching success rate than Hooded Mergansers, and
that hatching success has not significantly changed from 1979-1989. The constancy o f
egg-hatching success indicates that the reproductive success per female has not changed
during the decline o f Wood Duck nesting at A N W R The values o f egg-hatching success
at ANWR falls within the boundaries reported elsewhere for native W ood Duck
populations (Clawson et al. 1979, Haramis & Thompson 1985, Semel et al. 1988).
Additional analysis reveals that hatching success o f one species at ANWR is not
significantly related to the nesting density o f the other species. Although the presence o f a
weak negative relationship between W ood Duck nesting success and the number o f
Hooded Mergansers nesting in nest boxes (r2= -0.2, F = 3.64, p(F) = 0.08) may be
indicative o f some interaction between the two species that negatively impacts Wood
Duck nesting success the weak nature o f this relationship suggests that this interaction is
likely not the driving factor behind Wood Duck nesting declines at A N W R A similar
pattern o f increasing Hooded Merganser nesting and concomitant decreasing Wood Duck
nesting has been reported from Maine by Allen et al. (1990); but they found no evidence
that would suggest the Hooded Merganser increase was a contributory factor to the
decline o f nesting success o f W ood Ducks.
19
W ood Ducks have a higher reproductive success than Hooded Mergansers, but
numbers o f nesting Wood Ducks are declining at A N W R These differences may be a
function o f high winter mortality in Wood Ducks. W ood Ducks are highly sought after by
hunters and often represent the third most commonly harvested duck species in the U.S.,
comprising up to 10% o f the total U.S. duck harvest by hunters (Bellrose & Heister 1987,
Baldassarre & Bolen 1994). Annual hunting harvests o f W ood Ducks steadily increased
from 1966 through 1985, and 1.23 million individuals were harvested per year during the
period o f 1981-1986 (Baldassarre & Bolen 1994); approximately 98% o f those harvests
are from the Atlantic and Mississippi -Flyways. Harvest rates have declined since 1986
(Baldassarre & Bolen 1994); such harvest rates probably reflect decreases in total Wood
Duck populations in the U. S. Hooded Mergansers are commonly not a prime target
species and as such have a much reduced hunting-induced mortality (Baldassarre & Bolen
1994). This disparity in winter hunting harvests may account for declining number o f
nesting W ood Ducks and a concomitant increase in Hooded Merganser nests.
Another factor possibly accounting for declining Wood Duck populations is post­
hatching duckling survival. Wood Ducks and Hooded Mergansers utilize different brood­
rearing habitats (Bellrose 1980, Kirby 1990, Baldassarre & Bolen 1994); Wood Ducks
utilize marshy, lotic regions along shorelines whereas Hooded Mergansers utilize limnetic
zones. Such habitat utilization differences could contribute to variable survival rates o f the
two species during the nesting season. However, no evidence suggests that hatchling
20
habitat used by Wood Ducks has been altered or degraded at ANWR since the time o f
initial introductions on the refuge.
Two additional factors that could contribute to a decline in W ood Duck numbers
at ANWR are low genetic diversity resulting from founder effects o f the initial release o f
pen-raised individuals or the presence o f environmental contaminants on the refuge.
Others sections o f this investigation report on levels o f genetic diversity in this populations
and compare it to captive populations and published reports for other waterfowl species.
These results indicate that genetic diversity is not low among ANWR nesting Wood
Ducks as compared to other avian species.
W ood Ducks are known to experience reproductive impairment when
contaminated by dioxins commonly associated with agri-chemicals (White & Seginak
1994). The areas surrounding ANWR are predominantly agricultural lands, and the
decreased flow o f the James River through the impoundments on the refuge would allow
for an accumulation o f agricultural toxins in these waters. When W ood Duck are exposed
to dioxins, egg-hatching success is decreased (White & Seginak 1994). The relatively
high egg-hatching success o f W ood Ducks at ANWR and the constancy o f this success
over time indicates that these birds are probably not heavily influenced by environmental
toxins on the refuge. Wood Duck nesting success decreases at high nesting densities due
to antagonistic intraspecific behavioral interactions between nesting females and nestparasitism (Fellman 1993, Semel et al. 1988). Hooded Mergansers could easily be subject
to such reduced reproductive efficiency at elevated nesting densities as they are known to
21
be intraspecific nest parasites. Hooded Merganser nests are parasitized by both intra- and
interspecific (W ood Duck) females at A N W R However, the average egg-hatching
success found for ANWR mergansers from 1979-1989 is within the range reported
elsewhere for this species (Allen et al. 1990, Zicus 1990). The high breeding success o f
Hooded Mergansers at ANWR may be ephemeral if the population continues to grow;
density dependent factors such as interference competition may limit Hooded Merganser
breeding population numbers in the future.
Both species appear to have experienced rapid population increases immediately
following colonization o f the breeding habitat at A N W R The population fluctuations
suggest the presence o f a founder-flush pattern o f population change following a novel
introduction. During the early stages o f population establishment, high nesting densities
are found for both species. The philopatric nature o f both species, along with their high
fecundity could account for rapid initial population increase and accompanying increases
in competitive interactions that would influence nesting activities. A possible result of
such interactions could be females utilizing alternative nesting sites, such as natural
cavities. The Wood Duck nesting population data (Figure I), indicate that they are
beyond the effects o f a founder-flush population growth phase and are becoming more
stationary in population size. The Hooded Merganser is still likely to be under the
influence o f a founder-flush population change and will perhaps become more stationary in
the future.
22
Although the number o f nesting Wood Duck females has declined in recent years
at ANWR, there does not appear to be evidence that Wood Ducks are in danger o f
extirpation in eastern-central North Dakota. While the numbers o f W ood Ducks nesting
in nest-box structures within the refuge boundaries has declined, evidence indicates that a
viable population o f Wood Ducks now occurs in this region. The refuge has probably
acted as a population center from which dispersal o f nesting females has occurred. The
original release o f Wood Ducks in this geographic region has facilitated a range expansion.
23
C h ap ter 3
AN IN V ESTIG A TIO N O F A LLO ZY M E V A RIA BILITY IN A CA PTIV E AND AN
IN TRO D U CED PO PU LA TIO N O F W O O D DUCKS.
Introduction
Heritable genetic diversity is an important component o f a species’ ability to persist
over time. The presence o f phenotypic variance provides the raw material requisite for
natural selection and evolution to occur. The importance o f phenotypic variability to the
process o f evolution has been understood since the time o f Darwin, and genetic diversity is
widely thought at least to reflect and probably provide much o f the phenotypic variability
observed among individuals. Genetic variance provides the raw material upon which the
mechanism o f evolution (natural selection and differential reproduction) acts, thus
allowing species to persist in the face o f dynamic environmental conditions over
substantial periods o f time.
Studies o f population biology and genetics have provided a. rich knowledge o f the
amounts o f genetic variation in populations and how various factors affect the amount o f
genetic variability among individuals o f populations. From these studies, several factors
emerge as significant in reducing levels o f genetic variation; these include small effective
population sizes, founder effects, and prolonged (more than 4 to 5 generations),
population bottlenecks. These factors tend to erode levels o f genetic diversity and thus
Al
II
24
decrease the likelihood o f a population in adaptation and survival over significantly long
(evolutionary) periods o f time.
Since the early 1970's, allozyme Variation has been a standard technique to
determine levels o f genetic variability in almost all taxa, and allozyme research revealed
patterns and trends in the distribution o f genetic variation among widely differing taxa.
One trend is that vertebrates exhibit much lower amounts o f allozyme variability than do
invertebrates (Powell 1975, Selander 1976). In many cases vertebrates have about half the .
allozyme variability o f invertebrates (Nevo et al. 1984, Evans 1987). Among vertebrates,
larger-bodied species tend to exhibit lower amounts o f genetic variability than do smaller
species (Nevo et al. 1984). An exception to this general pattern among vertebrates is
found within the birds; birds exhibit reduced levels o f allozyme variability in comparison to
most other homeothermic vertebrates regardless o f body size (Nevo 1978, Nevo et al.
1984, Cooke & Buckley 1987). In fact, many large-bodied avian species exhibit more
genetic variation than do smaller-sized birds (Evans 1987). This reduced level of
variability among birds is thought to reflect historical events such as inbreeding and or
repeated and persistent population bottlenecks that counteract social and behavioral
actions that would have increased or at least maintained levels o f genetic variance (Evans
1987).
Recently, there has been an increased interest in breeding programs that attempt to
maintain individuals o f rare or endangered taxa in captive situations. Such breeding
programs are now widely considered to be an essential component o f management and
I
25
preservation o f local and global biotic diversity (Foose 1983). Captive propagation is an
effective method for producing and maintaining large numbers o f individuals to re-stock
populations that are either rare or have become extirpated. In fact, for very rare species,
captive propagation may be the only method o f restoring populations to the wild; this has
been the case in Wood Ducks (Ripley 1973, Baldassarre Sc Bolen 1994), the Black-footed
Ferret (Thome et al. 1988, Thome & Oakleaf 1991) and several other species that have
e>q)erienced population comebacks through concerted conservation efforts.
In many captive breeding programs, the taxa being bred and maintained are often
large vertebrates, birds, or other taxa with low amounts o f genetic variability. One o f the
chief concerns o f maintaining captive populations is the retention o f genetic diversity over
the period o f captivity (Soule & Wilcox 1980, Foose 1983, Ralls Sc Ballou 1986, Hederick
et al. 1986, Soule 1987). The maintenance o f such diversity in captive populations is
often difficult as they are usually small, having experienced severe population bottlenecks,
and may already be highly inbred. AU o f these factors are known to contribute to losses o f
genetic diversity in captive populations (Chapco et al. 1973, Sing et al. 1973, RumbaU
1974, Mina et al. 1991). Even in large captive populations, genetic variance may decrease
over time due to low N eZN ratios and reproductive demographics differing from those in
wUd situations (Foose 1983, Lande 1993, Briscoe et al. 1992).
W ood Ducks were introduced to Arrowwood National WUdlife Refuge in 1968 as
an experiment to evaluate the feasibihty o f creating populations o f this species outside its
native breeding range. During the first breeding season, this population was composed o f
26
twelve hens. Those individuals had been released the previous year from captive Wood
Duck flocks (Doty & Kruse 1972). The W ood Duck population at ANWR flourished for
a period o f time and then began to decline at a constant rate, and it appeared that
extirpation was likely (Doty et al. 1984). At the time o f the introduction, the importance
o f genetic variability, and the likelihood that captive populations have reduced amount o f
genetic variability was not widely recognized and was not considered. One o f the factors
thought to be influential in the decline o f the breeding Wood Duck population at ANWR
is reduced genetic variability due to founder effects associated with the small number o f
individuals introduced from captive stocks.
In this chapter, allozyme variability was investigated using tissue samples collected
from W ood Ducks in a non-destructive fashion from two populations. These populations
include the population at ANWR, and a captive population (Hancock), which serves as a
representative o f the original captive stock used to create the ANWR population. The
captive population used as a source o f colonists for ANWR is no longer in existence, but
the Hancock population is a close approximation o f that original population in size and
longevity o f captivity. The Hancock population is composed o f about 12 breeding hens
and anywhere from 5 to 25 drakes; it is maintained in eastern Montana and has been in
captivity for approximately 12 years (B. Hancock, pers. comm.).
27
M aterials and M ethods
Proteins were extracted from samples o f blood and/or tissue pulp. Emerging
feather quills (blood quills), and blood were collected in the field and immediately stored
under cryogenic conditions until transported to the laboratory for preparation and analysis.
AU blood quiU samples were stored at -80° degrees C until protein extraction and
electrophoretic analysis. Feather-quiU tissues were coUected, stored and prepared
essentiaUy foUowing the methods o f Marsden & May (1984). Feather tissues were
homogenized by crushing them with a micro-pestle in a microfrige tube in 500 jil o f cold
extraction buffer (0.05 M Tris-HCl, 0.05 M Tris-Base, pH 7 .1) on ice. Homogenates
were centrifuged at 12,000 rpm for a period o f 5 minutes and either immediately
electrophoresed or stored at -80° C until electrophoresis was carried out. Blood samples
were coUected by venous puncture o f the brachial vein using a 21 gauge needle on a I ml
hypodermic syringe; 0.5 - 1.0 ml o f blood was coUected. The blood was placed in a
microfrige tube containing 1.0 ml 0.9% saline and 0.1 % sodium citrate to act as an anti­
coagulant (Cooke & Buckley 1987). Buffered blood samples were kept at approximately
4° C until prepared for freezing and enzyme extraction. Blood sample preparation was
always carried out within 16-30 hours of coUection. Whole blood samples were
centrifuged at 2000 rpm for 3-5 minutes; the supernatant was discarded, and the peUeted
red blood ceUs were washed 3x with 0.9 % saline and then lysed with an equal volume o f
distiUed water. Blood samples were then either directly applied to electrophoretic gels or
stored at -80 °C until electrophoresis was carried out.
28
A total o f 34 loci were preliminary tested for resolution during early phases o f
electrophoretic analysis. O f the 34 screened, 28 presumptive allozyme loci were found to
be o f sufficient resolution to be assayed for allelic variation using horizontal starch-gel
electrophoresis (Table 2). Electrophoresis and enzyme staining were carried out following
the methods o f May et al. (1979), with modifications described in Britten & Brassard
(1992). The enzymes analyzed and the specific buffer systems used to resolve them are
fisted in Table 3. "R" buffer is from Ridgeway et al. (1971), "4" buffer is from Selander et
al. (1971), and "9C" buffer is from Cooke & Buckley (1987). Protein samples were
applied to starch gels using filter-paper wicks saturated with homogenate supernatant;
wicks were left in place for a period o f 20-30 minutes after the start o f electrophoresis and
removed after the samples had adsorbed into the gels. Electrophoresis was carried out for
a period o f four to six hours, depending on specific buffer systems; gels were kept cold
during electrophoretic runs. Genotype determination was inferred by direct visual analysis
o f the gels after specific enzyme staining. Any genotypes that were not readily scorable
were tentatively assigned a genotype and then re-run in subsequent electrophoretic runs
for clarity o f genotype determination. Known samples were included as controls in all
electrophoretic runs so that genotype determination could be made with a high degree o f
certainty.
29
Table 2. Symbols, names, Enzyme Commission Number used for allozyme analysis o f Aix
sponsa.
Symbol
Enzyme Name
AAT
ACP
ALB
ALD
E ST -1,2
GAPDH
GPI
H b-1,2
HBDH
ID H -1,2
LDH-1,2
M DH-1,2
MPI
ODH-1,2
PEP-LA-1,2
PEP-GL
PEP-LGG
PEP-LLL
PDG
X D H -1,2
Aspartate aminotransferase
Acid Phosphatase
Albumin
Aldolase
Esterase
Glucose-6-phosphate Dehydrogenase
Glucose Phosphate Isomerase
Hemoglobin
Hydroxybutyric Dehydrogenase
Isocitrate Dehydrogenase
Lactate Dehydrogenase
Malate Dehydrogenase
Mannose Phosphate Isomerase
Octonal Dehydrogenase
Peptidase-C (Leucyl-alanine)
Peptidase-glycyl-leucine
Peptidase-B (Leucyl-glycyl-glycine)
Peptidase-leucyl-leucyl,leucine
6-Phosphogluconate Dehydrogenase
Xanthine Dehydrogenase
E.C. Number
2.6.1.I
3.1.3.2
3 .1.1.1
1.1.1.49
5.3.1.9
1.1.1.42
1.1.1.27
1.1.1.37
5.3.1.8
1.1.1.73
3.4.11/13
3.4.11/13
1.1.1.44
1.2.1.37
The FORTRAN program BIOSYS-1 ( SwofFord & Selander 1981), was used to
analyze the genotypic frequencies obtained from the gels. The following indices of
genotypic variation were analyzed: mean heterozygosity per locus (H), calculated as the
proportion o f individuals that are actually heterozygous (the "direct-count method" o f
SwofiFord & Selander 1981). Estimates o f mean heterozygosity per locus were also
calculated based on Hardy-Weinberg equilibrium predictions; methods that include sample
30
size biases and unbiased estimates were calculated (Selander & Swofiford 1981). The
average number o f alleles/locus was calculated as the total number o f alleles over all loci.
The proportion o f polymorphic loci (P), was calculated using the criterion o f considering a
locus polymorphic when the frequency o f the most common allele is < 0.99. Deviations
from Hardy-Weinberg equilibrium predictions were examined by calculating exact
significance probabilities to overcome the difficulties o f small sample sizes associated with
the Chi-squared distribution (Sokal & Rolhf 1981). The Fixation Index (Fis), was
calculated to analyze patterns o f deviations from Hardy-Weinberg equilibrium conditions
(Wright 1965, 1978, Nei 1977). Statistical comparisons o f genetic indices were made
utilizing the computer package SIGMA-STAT.
Table 3. Buffer systems and types o f tissue samples used to resolve the loci under
investigation. Buffer identification are given in text. For tissues, Q refers to feather quill
tissue and R denotes red blood cell samples.
Enzyme
AAT
ACP
ALB
ALD
E ST -1,2
GAPDH
GPI
H b-1,2
HBDH
ID H -1,2
LD H -1,2
M DH -1,2
Buffer System
C
C
4
4
9C
C
4
9C
9C
C
C
4
Tissue
Q
Q
R
R/Q
Q
R
Q
R
Q
R
R
Q
31
Table 3 (Continued). Buffer systems and types o f tissue samples used to resolve
the loci under investigation. Buffer identification are given in text. For tissues, Q refers to
feather quill tissue and R denotes red blood cell samples.
Enzyme
M PI
ODH-1,2
PEP-LA-1,2
PEP-GL
PEP-LGG
PEP-LLL
PDG
X D H -1,2
Buffer System
9C
C
C
9C
C
9C
9C
C
Tissue
R
Q
Q
Q
Q
Q
Q
Q
Results
O fthe 28 loci resolved for Aix sponsa, 17 were found to be polymorphic for both
populations (Table 4). The invariant loci in both populations were LD H -1,2, H b-1,2,
GAPDH, ID H -1, AAT-2, A C P-1, ODH-1,2, HBDH, X DH-1,2, and ALD. Four other
loci were found to be invariant in one population, but polymorphic in the other. The loci
with private alleles in the ANWR population are Est-2, GPI, M DH -1, while IDH-2
exhibited an allele in the Hancock population that was not sampled in the ANWR
population.
Mean heterozygosity per locus (H), values are given in Table 5. Using a directcount criterion, H was 0.032 (SE 0.015) in the Hancock population and 0.045 (SE 0.012)
in the ANWR population. A t-test was carried out to ascertain whether H values were
significantly different; this analysis revealed no significant differences between the ANWR
32
and Hancock direct-count values o f H (t = 0.908, d f = 53, P = 0.368). The direct-count
method o f calculating H was compared to estimates based on Hardy-Weinberg equilibrium
predictions (Swoflford & Selander 1981) using a Mann-Whitney Rank Sum test; none o f
the values o f H differed Atom one-another significantly in either population (Table 5).
The mean number o f alleles per locus was 1.32 (SE 0.12) for the Hancock
population, while the ANWR population had a mean o f 1.68 (SE 0.15) alleles per locus
(Table 5). A t-test indicated that there is not a significant difference between the mean
number o f alleles/locus o f these two populations (t = -1.84, d f = 54, P = 0.07).
Table 4. Allefic frequencies for all loci from Arrowwood National Wildlife Refuge and
from a captive population (Hancock) o f wood ducks.________________________________
)C U S
ATT
ACP
ALB
ALD
EST-I
EST-2
GAPDH
GPI
Hb-I
Hb-2
HBDH
ID H -I
IDH-2
LDH-I
LDH-2
Allele
A
C
C
B
C
C
D
C
B
D
E
C
C
B
C
C
C
C
C
B
C
C
Hancock
1,0 (33)
1.0 (33)
0.987 (38)
0.013
1.0 (33)
0.811 (37)
0.189
1.0 (37)
1.0 (37)
1.0(33)
1.0 (37)
1.0 (37)
1.0(33)
1.0 (33)
0.985 (33)
1.0 (37)
1.0(37)
ANWR
1.0 (15)
1.0 (27)
0.934 (38)
0.066
1.0 (37)
0.962 (39)
0.038
0.976 (42)
0.024
0.073
0.024
1.0 (41)
0.976 (42)
0.024
1.0 (42)
1.0(42)
1.0 (40)
1.0 (39)
1.0 (39)
0.015
1.0 (42)
1.0 (42)
33
Table 4 (Continued). Allelic frequencies for all loci from Aurrowwood National Wildlife
Locus
M DH-I
MDH-2
MPI
ODH-I
ODH-2
PEP-LLL
PGD
XDH-I
XDH-2
PEP-G Ll
PEP-LAl
PEP-LA2
PEP-LGG
Allele
C
B
C
D
C
D
B
C
C
C
D
B
C
D
B
C
C
C
D
B
C
D
C
B
D
C
B
Hancock
1.0(33)
1.0 (33)
0.987 (38)
0.013
1.0(33)
1.0 (33)
1.0 (33)
1.0 (33)
1.0 (33)
1.0 (33)
0.939 (33)
0.015
0.015
1.0 (33)
0.712 (33)
0.106
0.182
0.955 (33)
0.045
ANWR
0.988(42)
0.012
0.988 (42)
0.012
0.976(42)
0.012
0.012
1.0 (40)
1.0 (40)
0.913 (23)
0.043
0.043
0.551 (39)
0.192
0.256
1.0 (43)
LO (43)
. 0.902 (33)
0,073
0.025
0.936 (33)
0.013
0.863 (40)
0.038
0.100
0.936 (39)
0.064
The proportions o f polymorphic loci (P) for the two populations are given in Table
5. Values are given using three different criteria for designating a locus as polymorphic.
There is substantial difference in each population between the 0.95 and the 0.99 criteria.
A G-test for independence (Sokal & Rolhf 1981), was carried out to test for significant
34
differences between the two populations using a criterion o f the most common allele
having a frequency o f < 0.99. The original locus frequencies o f monomorphic and
polymorphic loci were used to calculate the G statistic. This analysis reveals that the
difference between the two populations is not significant (G = 2.75, d f = I, 0.05 > cc <
0 . 1).
Analyses o f deviations from Hardy-Weinberg expectations revealed that all but 5
loci were within Hardy-Weinberg predictions. O f the five, only PEP-LA-2 was found to
be outside o f equilibrium conditions in both populations. Two loci were out o f HardyWeinberg equihbrium for the Hancock population (Pep-LA-2 and E st-1), and four loci
deviated from Hardy-Weinberg expectations in the ANWR population (Pep-LLL, PepLA-2, PEP-LGG, and GPI). The Fixation Index (Fis), values for the two loci out o f
Hardy-Weinberg equilibrium in the Hancock population were 0.119 and 0.257 for Est-I
and Pep-LA-2 respectively, indicating a heterozygote deficiency for each o f these loci.
The Fis values for the ANWR loci deviating from Hardy-Weinberg were 0.198 (Pep-LLL),
0.183 (Pep-LA-2), 0.359 (Pep-LGG), and 0.784 (GPI). As in the Hancock population,
these values indicate a paucity o f heterozygotes sampled with respect to the predicted
values.
35
Table 5. Mean heterozygosity (H), Mean numbers o f alleles/locus, and percent o f
polymorphic loci (P) for the two populations (ANWR and Hancock), under investigation.
The values in parenthesis are the standard error o f the mean._______
Genetic Index
Population
ANWR
Hancock
H
Biased Estimate1
Unbiased Estimate1
Direct-Count Method
0.064 (0.023)
0.065 (0.024)
0.045 (0.012)
0.037 (0.019)
0.038 (0.020)
0,032 (0.015)
Mean Alleles/Locus
1.680 (0.150)
1.320 (0.120)
P
95 % Criterion
99 % Criterion
No Criterion
25.0
46.4
46.4
10.7
25.0
25.0
1- See SwofFord & Selander (1981), for a further description o f biased and unbiased
estimates o f H.
Discussion
Mean heterozygosity values obtained for Wood Ducks in both populations fall
within the range reported for other avian species (Barrowclough 1983, Cooke & Buckley
1987, Gravin et al. 1991). In a survey o f avian allozyme analyses Cooke & Buckley
(1987), report an overall mean heterozygosity value o f 0.044 for 86 avian species (H
values for individual species ranged from 0.002-0.147). These values are slightly lower
than mean heterozygosities (H=0.049), reported for non-avian vertebrates (Evans 1987).
Few allozyme investigations exist for waterfowl species to use as comparisons for these
results; o f the allozyme information available, most studies on waterfowl have been on
Mallards {Anas platyrhyncos) and closely'related sibling species. Mean heterozygosity
values for Mallards range from 0.015 (Browne et al. 1993), to 0.076 (Ankney et al.
1986); an average o f reported values for mallards is 0.046 (Ankney et al. 1986, Browne
et al. 1993). Other mean heterozygosities range from 0.014 for the Laysan Duck {A.
laysanensis), 0.035 for the Hawaiian Duck (A. wyvtlliana), (Browne et al. 1993), and
0.053 for the American Black Duck {A. rubripes), (Ankney et al. 1986). Average
heterozygosities for Wood Ducks fall in the middle o f reported values for other waterfowl
species, but ANWR values are more similar to those for Mallards, whereas Hancock
populations are more similar to those o f the Hawaiian Duck. Mallards have much higher
population sizes, and have been exposed to fewer population bottlenecks than the
Hawaiian Duck. One would expect an open population such as ANWR to exhibit higher
levels o f heterozygosity than the Hancock population which is smaller, has a greater
likelihood o f being inbred and has reduced levels o f gene flow. Barrett and Vyse (1982),
report mean heterozygosity values for Trumpeter Swans at 0.009; these values are much
lower than those found for Wood Ducks in this investigation. Trumpeter Swans are
known to have experienced long periods o f population bottlenecks and exhibit reduced
levels o f heterozygosity.
The percent o f polymorphic loci was found to be 46.4% for ANWR and 25.0% for
Hancock. Reported values range between 26.4% to 22.4% for Black Ducks and Mallards,
respectively (Ankney et al. 1986); while 5.0% and 17.5% are reported for Laysan and
Hawaiian Ducks (Browne et al. 1993). Both o f these are similar to the findings o f Arise
37
et al. (1990a), for wintering populations o f Mallards. The value for the Hancock
population is very close to that o f Black Ducks and Mallards, while the ANWR
population is much higher than any reported values for duck species. In fact, the P values
in the ANWR population are among the highest reported for avian species (Cooke &
Buckley 1987). Nevertheless, a number o f bird studies have reported still higher levels o f
loci polymorphic (Baker & Manwell 1975, Smith & Zimmerman 1976, Yang & Patton
1981), however, making the ANWR estimates within reason. A loss o f polymorphisms is
commonly attributed to a low N e common in many captive populations, (Chesser 1983).
The Hancock population would be expected to have a much lower Ne than the ANWR
population due to decreased numbers o f immigrant hens. This low N 6 and isolation from
migrants would prevent the influx o f new alleles into the population, thus decreasing the
levels o f polymorphism through the loss o f rare alleles through genetic drift.
Statistical comparisons between Hancock and ANWR genetic indices yield a
consistent pattern o f non-significant differences between the two populations, yet for all
indices, the Hancock population consistently exhibited less genetic variabtiity than did the
ANWR population (Table 4). The most likely explanation for this lack o f significance
comes from the low degrees o f freedom associated with comparisons between two
populations; it is often very difficult to attain significant differences when comparing two
population estimates. It is probably safe to assume that the consistent pattern o f lower
genetic variance in the Hancock population is a meaningful trend while not being
statistically significant. It is not a common practice to make statistical comparisons of
38
genetic indices for different populations; this may result from the difficulty o f obtaining
statistical significance in such comparisons.
Assuming the Hancock population is representative o f the original captive
population used to propagate the ANWR population, the proportion o f polymorphic loci
exhibited in the ANWR population indicates that this population has experienced an
increase in genetic diversity since the time o f its initial introduction. This is perhaps not
what would be predicted in a closed population introduced to the peripheral regions o f a
species range. Studies on the Common Myna (Acridotheres tristis), in Hawaii have shown
that although mean heterozygosity did not change after introduction, the proportion of
polymorphic loci appeared to decrease (Fleischer et al. 1991). This loss o f polymorphisms
without a significant loss o f heterozygosity is predicted by theoretical considerations and is
supported in other evaluations o f genetic variability (Nei 1977, Falconer 1981, Hedrick
1983, Evans 1987). Examination o f the mate selection behaviors o f A. sponsa provides a
mechanism through which an isolated, peripheral population could gain genetic diversity
through outbreeding, making it an open rather than a closed population. In Wood Ducks,
mate selection occurs by hens selecting drakes on the winter range. During the winter
months, there is a concentration o f Wood Ducks in the southern United States; all o f the
Wood Ducks in the Central flyway aggregate in the near-coastal waters o f the Mississippi
River drainage o f Louisiana, Texas, and Mississippi. This provides the opportunity for
females to recruit new males into the population, and the entire Central Flyway may act as
one large, effectively panmictic, population. Similar panmbds is known to occur for other
39
vertebrates that disperse widely to reproduce and after mate selection and fertilization
occurs in large aggregations (Avise et al. 1990b). Effective panmixia is also reported for
the sessile invertebrate Tridacna gigas in apparently isolated populations on the Great
Barrier R eef off Australia; the long-range dispersal mechanism is thought to facilitate gene
flow among these isolates. The presence o f panmictic mating in W ood Ducks in the
Central Flyway would provide a mechanism for the maintenance o f high levels of
polymorphism in small breeding units such as ANWR.
Observed deviations from Hardy-Weinberg equilibrium can result from a variety o f
factors affecting populations (Hedrick 1983). One such factor that has been highly
debated is whether the characters examined are under the influence o f natural selection. A
,
recent review o f allozyme studies (Watt 1994), provides evidence.that some loci used in
allozyme studies do exhibit evidence o f being under strong selective forces, but when
considered in large numbers, it is probably safe to discount natural selection as being the
driving force in maintaining non-equilibrium in these populations. A second source o f
non-equilibrium genotypic frequencies is that o f inaccurate genotype scoring o f gels,
rather than some natural phenomenon acting on populations. Because only one locus
(Pep-LA-2), was found to be outside equilibrium in both populations scoring errors do not
appear to be solely responsible for the observed deviations from Hardy-Weinberg
equilibrium, or if they are the scoring errors occurred equally for both populations.
Browne et al. (1993), report Pep-LA to be out o f Hardy-Weinberg equiftbriumfor
Mallards, and Hawaiian Ducks; other Peptidase loci have been found to be outside o f
40
Hardy-Weinberg equilibrium in non-waterfowl avian species (Rasmussen 1994). This
constancy o f non-equilibrium could indicate that selective pressures could be acting on this
particular locus to maintain it outside o f Hardy-Weinberg equilibrium in a variety o f avian
species, or could be indicative o f the presence o f null alleles in many avian species. A
third factor affecting Hardy-Weinberg equilibrium is inbreeding (Falconer 1981).
Inbreeding is common among captive populations (Chesser 1983, Soule 1987) and it
could result in heterozygote deficiencies. However, an unexpected pattern o f equilibrium
deviation was found; the natural population Was less frequently in Hardy-Weinberg
equilibrium than the captive population. Sampling o f genetic neighborhoods and assuming
they are a large, single population is yet another factor that will cause observed genotypic
frequencies to deviate from expected equilibrium frequencies, this phenomenon is known
as the Wahlund effect (Falconer 1981, Hederick 1983). More extensive geographical
sampling throughout the summer range Ofyli spoma or comparisons with samples
collected in the wintering range o f this species could test this hypothesis.
The results o f this investigation have managerial implications for migratory avian
species that utilize concentrated habitats during the non-breeding season. The genetic
diversity o f such spiatiotemporal sub-divided populations is probably not an important
factor during the breeding season. The genetic integrity o f this species is probably much
more vulnerable to demographic factors caused by the destruction or degradation o f
winter habitat. A loss or fragmentation o f wintering grounds could be o f vital importance
to the genetic structuring o f such species as fragmentation would destroy the potential for
11 r
41
panmictic mate selection to occur. It would appear that to conserve diversity in A.
sponsa, efforts must be concentrated on the wintering range o f this species; if managed
properly there will be minimal consequences o f minor habitat degradation and breeding
range fragmentation in the summer ranges.
W ood Ducks are highly philopatric (Nichols & Johnson 1990, Baldassarre &
Bolen 1994), and this behavior lends itself well to initiate breeding populations outside the
normal breeding range o f this species. When populations in peripheral areas are
originated, individuals migrate along main Hyways (Doty & Kruse 1972, 1984), and join
individuals from other geographic regions in the winter range. During this migratory and
wintering period, mate selection occurs in the presence o f individuals from distant
breeding regions. This type o f mate selection is a mechanism through which high amounts
o f gene flow can be maintained in peripheral and isolated populations. The behaviors o f
mate selection are an obviously important component o f this managerial strategy; those
species in which mate selection does not occur during the winter would be much more
prone to the effects o f isolation. Hence the reliance o f outbreeding through mate selection
must be made on a species-specific basis.
The relatively high levels o f allozyme variability found in the Hancock captive
population are an indicator that the efforts this breeder makes to avoid inbreeding are
working fairly well. Attempts are made to regulate inbreeding by replacing hens in the
captive flock with hens from other captive flocks in other parts o f the country on an
annual or semi-annual basis (B. Hancock, pers. com m ). Although the Hancock
I I
42
population is below that o f the ANWR population in all indices, it does not exhibit signs o f
a highly inbred population. Many captive breeding programs attempt such manipulative
gene flow, and it appears to be a reasonable management strategy for W ood Ducks to help
maintain adequate levels o f genetic diversity.
The ANWR breeding W ood Duck population appears to be declining; whether or
not this is actually the case is in question (see other sections o f this study). The breeding
Wood Ducks at ANWR exhibit high levels o f genetic variability in comparison to other
waterfowl populations; therefore i t is unlikely that reduced genetic variability resulting
from founder effects, inbreeding, or population bottlenecks is a major factor responsible
for the apparent decline.
L
43
C h ap ter 4
A C O M PA R ISO N O F G EN ETIC D IV ER SITY O F T H R E E W O O D DUCK
PO PU LA TIO N S USING DNA FIN G ERPR IN TS.
Introduction
DNA fingerprinting is a powerful molecular technique for analyzing individuals
based on unique genetic characteristics (Jeffreys et al. 1985); because o f this accuracy,
DNA fingerprinting is useful for identifying individuals within populations and for
paternity analyses. This technique utilizes endonuclease recognition sequences in
hypervariable minisatellite DNA to create unique (or nearly unique) patterns o f banding
after exposure to restriction endonucleases, electrophoretic separation, Southern blotting
and hybridization with probes o f known DNA sequences. Since its inception (Jeffreys et
al. 1985), DNA-fingerprinting using probes derived from human DNA has been
successfully used for forensic purposes (Cohen 1990, Devlin et al. 1991), paternity
analyses (Quinn et al. 1987, Longmire et al. 1992), ascertaining the degree o f relatedness
among groups o f individuals (Lynch 1988, Reeve et al. 1992), and sex- determinations in
sexually monomorphic species (Longmire et al. 1993)
Probes derived from minisatellite human genomic sequences have been found
useful in analyzing DNA from nearly all taxa; bacteria (Huey & Hall 1989, Ryskov et al.
1988), plants (Dallas 1988, Nybom & Schaal 1990, Milgroom et al. 1992, Alberte et al.
44
1994), insects (Blanchetot 1991), mammals (Gilbert et al. 1990, Reeve et al. 1990), and
birds (Burke & Bruford 1987, W etton et al. 1987, Kuhnlein et al. 1990, Meng et al. 1990,
Westneat 1990, Piper & Rabenold 1992, Tiiggs et al. 1992). The majority o f these
studies have dealt with relatedness o f individuals within familial lineages or determinations
o f breeding systems. DNA fingerprinting is not as commonly applied to studies concerned
with measurements o f genetic variability on a population level (Flint et al. 1989, Kuhlein
et al. 1990, Gilbert etal. 1990, Rave et al. 1994).
DNA fingerprinting reveals large amounts o f individual genetic variability and is
potentially a good method for measuring genetic variation in taxa with low levels of
allozyme diversity. These genetically depauperate taxa include many rare species or small
populations and/or captive populations which may have reduced genetic variance due to
inbreeding, bottle-necks or low N eZN ratios.
Many studies have been conducted using DNA fingerprinting on birds. Birds have
nucleated red blood cells, therefore they are an attractive subject for DNA studies; the
collection and preparation o f nuclear DNA from red blood cells is rather simple and can be
obtained in a non-destructive fashion (Longmire et al. 1988). Birds also exhibit low levels
o f allozyme variability making traditional genetic approaches difficult; in comparison to
other taxa there is little information on allozyme variability among many avian species. In
feet, for many bird species there is more information regarding DNA than allozyme
variability.
45
Li this chapter, I investigate levels o f genetic variance as measured with DNA
fingerprinting in three populations o f Wood Ducks. These populations analyzed include
the Arrowwood National Wildlife Refuge population, a Captive population, and a natural
population from the Pacific Flyway.
M aterials an d M ethods
The following populations were sampled: Arrowwood National Wildlife Refuge
(ANWR), in eastern-central North Dakota, a captive population maintained by a private
aviaculturist in Montana (herein, Hancock), and a native population from western Oregon.
This Hancock population was chosen because it is similar to the captive flock used to
create the ANWR population; the flock originally used to found the ANWR population is
no longer in existence, but the Hancock population is approximately the same size and has
been in captivity for a similar length o f time. A wild population at the Western Oregon
National Wildhfe Refuge Complex (herein, Finley), in western-central Oregon, also
selected because it is known to have been a small, natural population for a number o f years
and has had no known genetic inputs from releases o f individuals from captive
populations.
W ood Ducks were sampled in the field through either the deployment o f cannonnets or trapping individuals in nest-box structures. Blood samples were collected through
venous puncture o f the brachial vein. The volume o f blood obtained from each bird
ranged between 250-1000 p i Blood samples were placed in a microfuge tube containing
46
400 g l o f SET buffer (0.15 M NaCl, 0.05 M Tris-HCl, 0.1 mM EDTA, pH 8.0), and kept
cool until the DNA was extracted in the laboratory.
DNA was extracted from whole blood samples using a phenol-chloroform
extraction method similar to that described in Maniatas et al. (1982). The specific
extraction procedures were as follows. Cells were lysed with the addition o f 20 p i o f 10%
SDS; 25 p i o f Proteinase-K was added, and the samples were incubated for 12-20 hours at
55°C. This was followed by extractions against 500 p i o f water-saturated phenol and two
subsequent protein digestions with 25 p i o f Proteinase-K, incubated at 37°C for 8-10
hours followed with phenolic extractions. Samples were centrifuged at 12,000 rpm for
10-15 minutes following each phenolic extraction. If samples were still not clear, phenolic
extractions were carried out until the supernatant was clear and appeared free o f
contaminants; final extractions were centrifuged at 12,000 rpm for 30 minutes and the
aqueous phase removed. The aqueous phase was then extracted against 500 p i o f a
phenol: chloroform (1:1), mixture, followed by extraction against an equal volume of
ChCL3:IAA. The aqueous phase was removed, and the DNA was precipitated with 0. IX
volume o f 3 M NaOAC and 2X volumes o f 95% EtOH was added. The samples w ere.
gently agitated to dissolve the DNA and frozen at -20°C overnight. The following day the
DNA was rinsed twice in 70% EtOH and allowed to air dry. The DNA was then hydrated
with 300-750 p i o f distilled H2O. This process usually yielded between 3-75 ng o f DNA.
47
Purified DNA samples were stored at -SO0C until digested with restriction endonuclease
enzymes.
Enzyme digests were carried out according to manufacturer's directions; most
digests were carried out in a total volume o f 20 p i and incubated at 37 °C for 12-17 hours.
Digestion products were then electrophoresed in a 0.8% agarose gel.
;
Electrophoresis was carried out in submerged horizontal electrophoretic chambers
at 25 roA for 12-20 hours. Gels were made with TBE buffer. DNA standards were
placed between every five samples in each gel to insure co-migration o f equal size
fragments across the gel. Phage-Lambda DNA digested with Hind IH was used as DNA
standards in all gels. W ood Duck DNA from a common individual was run as a standard
on most gels to insure alignment o f fragment patterns between separate gels. Following
electrophoresis, gels were stained with Ethidium Bromide (EtBr) and photographed under
ultraviolet (UV) illumination; gels were trimmed and the DNA transferred by Southern
blotting.
Southern blots were made using Zetabind™ nylon membrane screens. Southern
blot transfers were accomplished with a protocol adapted from Maniatis et al. (1982), as
described in Westneat et al. (1988). DNA was denatured by washing twice for 15 minutes
in 1.5 M NaCL, 1.5 M NaOH under constant agitation. Gels were then washed twice for
15 minutes in 0.04 M NaOH, IM NH4Ac. The DNA was transferred to screens following
the methods outlined in Maniatis et al. (1982) for a period o f 16-18 hours at room
48
temperature. Southern blots were dried at 80°C under a pressure o f 20-27 lbs for 2 hours
and then hydrated and washed with 2X SSC for 15 minutes with agitation, followed by a
wash for I hour at 60°C in 1.0 M Tris (pH 7.5), 0. IX SSC, 0.5 % SDS. Excess SSC was
blotted off the screen, and screens were either immediately hybridized or stored at -20°C
until used for hybridization,
Hybridization reactions were carried out with the following procedures. The
human minisatellite probe, pV47 (Longmire et al. 1990), was labeled with the radio­
nuclide [32P]dCTP using nick-translation (Rigby et al. 1977). The radio-isotope was from
New England Nuclear™, io mCi/ml. Nick translation was accomplished using a BRL™
Nick-translation Kit following the directions supplied in the kit. Southern blots were pre­
hybridized for 5-20 hours at 60°C in 7% SDS, 0.01% BSA, 0.5 M EDTA, 0.5 M .
NazHPC^. Radio-labeled probe and hybridization buffer were added and hybridization
was carried out at 60°C for 18-24 hours. Hybridized filters were washed (2X), in 2X
SSC, 0.1% SDS for fifteen minutes at room temperature, followed by a 5 minute wash at
65°C in 0. IX SSC, 0.1% SDS. Autoradiographs were then made using Kodak X-OMAT
safety films with exposures at -80°C for times ranging from 12 to 96 hours depending on
the specific activity o f the filters. Exposures were made until autoradiograms were
produced that could be scored.
Autoradiograms were scored by visual examination after close examination of
multiple autoradiograms to ensure fragment alignment between different autoradiograms.
49
Common fragments were identified and used to align samples from different
autoradiograms. Several autoradiograms were scored multiple (2-4), times to determine
the accuracy o f scoring. Fragments were scored as either present or absent, and the
resulting matrices o f presence-absence data were used to analyze indices o f band-sharing.
The mean and variance o f the number o f fragments for all individuals per
population was calculated. Differences in the mean number o f fragments per individual
between the three populations were evaluated using both a Mann-Whitney U and t-test.
Because the original values used to calculate mean number o f bands per individual are
nominal, the non-parametric Mann-Whitney statistic is probably the most valid test to use
(Sokal & Rolhf^ 1981). The t-test is most commonly used in the literature, and I use it to
achieve consistency with common practices. In cases where there is no discrepancy
between the two tests in their levels o f significance, t-values are reported. To compare the
distribution o f different-sized DNA fragments in the three populations, the occurrence o f
bands was expressed ,as percent frequencies. Fragment frequencies were expressed as
percentages to compensate for unequal sample sizes in the three populations. The
frequency o f bands was analyzed across the three populations to evaluate patterns o f
fragment commonness and rarity between the three populations.
The Similarity coefficient (S), o f Lynch (1990) was calculated between all pairs of
individuals within a population using the following equation:
S = 2N ab/(N a+N b),
50
where N ab is the number o f bands shared between two individuals, and N a and N b are the
total number o f fragments present for individuals A and B. The variance o f S was also
calculated for each population, and this variance was used to compare variability. The
mean number o f fragments per individual per population and its associated variance was
also calculated. Significance testing o f mean S values among the three populations was
carried out using a Student's t-test (Sokal & R olhf 1981).
The expected population homozygosity (EH) was estimated for each population
(Lynch 1990) using the following equation:
EH = (Ziu p2ti )/ L,
where pti is the frequency o f the z'th allele (fragment), at the Ath locus (fragment size), and
L is the total number o f loci (different size fragments) (Lynch 1990). This EH is
equivalent to the parameter that Lynch (1990) defines as H; herein I use the abbreviation
EH so that it is not confused with the heterozygosity value (H) commonly calculated for
allozyme analyses. Under random mating, EH is equivalent to the gametic identity-in-state
(I), and in the absence o f random mating the two parameters are closely correlated,
making EH a reasonable index Of the amounts o f homozygosity within a population
(Lynch 1990).
The interpopulation mean similarity coefficient (S,y), o f Lynch (1990) was
calculated to assess the degree o f differentiation between the populations using the
following equation:
. 51
S y = l + S V -(S /+ S y)/2,
where S,- is the mean similarity o f individuals within population /, S7 is the mean similarity
o f individuals within population j , and S',7 is the mean similarity between pairs o f
individuals in population i and j. The matrix o f S,7 values was used to create a phenogram
o f the relationship o f the populations under investigation.
Results
An endonuclease-probe screen was conducted to ascertain which enzyme-probe
combinations would yield scorable autoradiograms. The enzymes used in this initial
screening were: the five-base recognition sequence enzyme H in f I, four-base recognition
sequence enzymes Hae IQ, Sau IQA, Alu I, and six-base recognition sequence enzymes
Hind m , Bam HI, Apa I, Pst I, Bgl I, Bgl Q, EcoK I, P vm Q. The enzymes yielding
scorable results using the p V47 probe were Hae IQ, H inf I and EcoK I. An example o f a
typical autoradiogram produced from Hae IQ digests and probed with pV47 can be found
in Figure 3.
For the ipV41lHae QI combination, a total o f 58 bands was analyzed for 82
individuals from the three populations; the sizes o f these bands ranged from 1.6 Kb to 20
Kb. Over all populations, there were 10.7 (± 1.2) bands per individual (Table 6). For the
separate populations, Hancock had the highest mean number o f fragments per individual
(12.0), followed by ANWR (10.31) and Finley (9.7), respectively. The standard error o f
these means is 0.72 for Hancock, 1.36 for ANWR and 2.79 for Finley. Statistical
52
Figure 3. A Typical autoradiogram produced with HaeIIH^VAl fingerprints. The lanes
are: I and 8 - Lambda DNA digested with HincRJl. Lanes 2 - 4 , 6 , and 7 are Wood
Ducks from Arrowwood National Wildlife Refuge. Lane 5 is a Wood Duck from the
Hancock Population.
1
2
3
4
5
6
7
8
23.1 Kb
9.4 Kb
6.6 Kb
4.4 Kb
2.3 Kb
53
comparisons reveal that only Hancock and ANWR differ from one-another significantly in
mean number o f bands per individual (t = 3.127, d f = 76, a = 0.002).
Table 6. Genetic diversity values for Hae myp V47 DNA Fingerprints. S is the
intrapopulation similarity coefficient, EH is the estimate o f homozygosity, MF is the mean
number o f fragments per individual per population. SE stands for standard error o f the
mean.
Population
S
SE(S)
n
EH
MF
SE(MF)
ANWR
0.267
0.004
42
0.1341
10.2
1.36
Hancock
0.479
0.014
26
0.2961
12.00
0.72
Finley
0.662
0.005
4
2.937
9.7
2.79
Frequencies o f fragments o f different size classes for individual populations are
given in Figures 4, 5, & 6. The ANWR and Finley populations have more small fragments
than does the Hancock population. Because o f the low fragment variability and small
sample size o f the Finley population, it is not included in further analyses o f band-size
distributions. The cumulative percentages o f occurrence o f fragments per fragment size
class for Hancock and ANWR are given in Table 7 and Figure 7. Although both
populations possess fragments throughout the range o f fragment size classes, 81% o f the
fragments in the ANWR population are smaller than 7.0 kB whereas only 58% o f the
Hancock fragments are less than 7.0 kB in size. The general pattern is that ANWR has
more smaller fragments and fewer larger fragments than the Hancock population. The
Hancock population has a somewhat even distribution o f fragments across all fragment
O O O)
CO
O)
o
CO
m
in
O ' f m n c o ' t o c o
o
Fragment Size (Kbp)
o
CO
o
CM
16.94
equency (Percent!
Figure 4. Frequency (expressed in percentages) of individuals with particular
DNA fragments according to fragment size from the Hancock population.
Figure 5. Frequency (expressed in percentages) of individuals with particular
DNA fragments according to fragment size from the ANWR population.
80 j
70 ^
60 -
C
CD
S 50 -
CL
Ln
>. 40 u
Ul
c o t o m c s i c N r ' - o o o c n ’- c o o c o i n i ^ O i - ' - c o m m m c o c N c o i c o o o ^ - ' d - c D m c N C D c o t D c o i n o m i ^ c M O ' f m m c O ' t o o o n o c N
»- ^
< N c v i c N c > i c Tj c o r n c o r j ; «a; L r ) L f j i r > c b r > > is' r ^ o 6 o d c j o o c N ' t
if>
Fragment Size (Kbp)
16.94
C
CO
CO
CO
c:
m
oo
CN
CO
' C O O O O T O d t N ' t
Fragment Size (Kbp)
18.17
requency (Percent
Figure 6. Frequency (expressed in percentages) of individuals with particular
DNA fragments according to fragment size from the Finley population.
Figure 7. Cumulative percent frequency of DNA fragments as expressed as
fragment size for all populations.
Cumulative Per
"*
Hancock
* ----- ANWR
"♦----- Finley
6
6
6
6
6
o
o
ci
6
6
Fragment Size (Kbp)
Ul
-u
58
size-classes. The Finley population has the highest proportion o f small bands o f the three
populations, and Hancock has the lowest proportion o f small-sized fragments.
Table 7. Percentage o f fragments by fragment size-class. % Frag., is the percentage o f all
fragments in that size class. Cum. % is the cumulative percentage o f fragments in that size
class and smaller size classes.
H ancock
Finley
AN W R
Size (kBP)
% Frag.
Cum. %
% Frag. C um % %Frag. Cum %:
1.0-2.0
10.7
6.5
10.7
6.5
0.0
0.0
2.0-3.0
9.9
20.6
29.3
59.0
59.0
35.9
3.0-4;0
16.3
36.8
20.3
56.2 ‘
15,4
74.4
4.0-5.0
48.0
10.2
66.4
12.8
87.2
11.1
6.0
5.0-6.0
54.0
9.7
76.1
7.7
94.9
6.0-7.0
4.1
58.0
5.2
81.3
0.0
94.9
10.1
68.1
7.0.-8.0
4.5
85.8
0.0
94.9
6.0
74.1
2.0
8:0-9.0
87.8
5,1
100.0
0.0
74.1
9.0-10.0
LI
88.9
0.0
100.0
2.6
10.0-11.0
76.7
2.7
91.6
0.0
100.0
4.3
11.0-12.0
80.9
LI
0.0
100.0
92.8
3.9
84.8
0.0
12.0-13.0
92.8
0.0
100.0
0.0
0.0
13.0-14.0
84.8
92.8
0.0
100.0
4.3
89.1
0.0
100.0
14.0-15.0
95.9
. 3.2
0.0
89.1
1.4
97.3
0.0
100.0
15.0-16.0
16.0-17.0
0.9
89.9
.9.8.4
0.0 . 100.0
LI
95.1
0.0
100.0
17.0-18.0
5.1 '
LI
99.5
0.0
95.1
0.0
99.5
0.0
100.0 .
18.0-19.0
4.9
100.0
0.5
0.0
100.0
19.0-20.0
100.0
The results o f an analysis o f frequency o f all fragments (independent o f size), is
given in Table 8. The ANWR population has more rare bands than the Hancock
population. The Hancock population is somewhat bi-modal (Figure 8) in that many bands
Occur at a frequency o f <20%, and a Significant proportion o f the fragments (40%), occurs
in frequencies o f 50-70% in the samples analyzed. In the ANWR population, 79.6% o f the
Figure 8. Percentage of all DNA fragments as a function of the frequency of
occurence in the population for the ANWR and Hancock populations.
35
30
1
25
20
m
15
vo
10
5
0
< 10
10 -20
20-30
30-40
40-50
Frequency of Occurrence
0)
o>
2
E
Ul
<
'o
0O))
5C
0O)
0)
CL
HHancock
^AN W R
50-60
60-70
70-80
60
fragments occur in a frequency o f <30%, whereas in the Hancock population, only 48.6%
o f the fragments occur in a frequency <30%. The general pattern is that most ANWR
fragments occur in low frequencies (<50%), whereas Hancock has both low- and highfrequency fragment groups. The Finley population is excluded due to a low sample size
and the adverse effects o f low sample size on common and rare fingerprint fragments.
Taible 8. Frequency o f fragments and their occurrence in the Hancock and ANWR
populations. Frequency refers to how often a particular band appears in a population on a
percentage basis.
Frequency
1-10
10-20
20-30
30-40
40-50
50-60
60-70
70-80
Hancock
Cumulative %
No. o f Bands
4
11.4
11
42.9
2
48.6
51.4
I
2
57.1
9
82.9
5
97.1
I
100
ANWR
Cumulative %
No. ofB ands
• 29.6
16
17 '
61.1
10
79,6
5
88.9
4
96.3
I
98.1
0
98.1
I
100
Intrapopulation similarity coefficients (S) and their variances for the three
populations are given in Table 6. The ANWR population exhibited the lowest genetic
similarity, followed by the Hancock and Finley populations. AU o f these values differ
significantly from one-another at the a = 0.01 level (Table 9). The standard error o f S
values is highest for the Hancock population (0.014), foUowed by ANW R (0.004), and
Finley has the lowest standard error o f S (0.005).
Table 9. Results o f a t-test for differences in intrapopulation similarity coefficients (S),
between the three populations.
Population Pair
Hancock-ANWR
Hancock- Finley
ANWR - Finley
t-Value
3.667
2.539
8.842
df
36
19
5
Significance Value
0.00008
0.003
0.0003
Population homozygosity (H), values (Table 6), indicate large differences among
the three populations with respect to inbreeding. The Finley population is much more
homozygous than either o f the other two populations. The H-values for the Hancock and
ANWR populations differ by a factor o f two, while the H-values for the Finley population
is an order o f magnitude higher than it is in these populations.
Interpopulation similarity (Sy), values (Table 10), indicate that the Finley and
Hancock populations are the most similar and that the Finley and ANWR populations are
the least similar. When a dendrogram is created (Figure 9) using these similarity indices, it
is apparent that the Finley and Hancock populations are more similar to one-another and
that the ANWR population is the least similar to the two.
Table 10. A matrix o f interpopulation (Sy), similarity coefficients for the three
populations.
_______________________________________
Hancock
ANWR Finley
Hancock
1.0
ANWR
0.337
1.0
Finley
0.534_____0.158
1.0
6 2
Figure 9. Dendrogram depicting the relationship between the three populations o f Wood
Ducks. This realtionship is based on the interpopulation similarity coefficients (Sy)
calculated from HaeIII/pV47 DNA fingerprints listed in Table 5.
Finley
Hancock
ANWR
63
Discussion
Differences in mean number o f fragments per individual among the three
populations are non-significant with the exception o f the Hancock and Arrowwood
populations. Hancock has a mean o f 12 fragments per individual (SE = 0.72), and ANWR
has a mean o f 10.2 (SE = 1.36) fragments per individual. The increased number o f
fragments per individual in the Hancock population is probably due to a mixing of
individuals representing very different genetic strains in that captive frock. Aanong
aviaculturists, it is a common practice to trade individuals from different captive
populations (B. Hancock, pers. comm.). Such a practice could produce a flock having
many different fragments in a population where they occur at low and moderate
frequencies. This situation o f high fragment diversity at intermediate and moderate
frequencies could be maintained despite inbreeding with a constant addition o f novel
genotypes into the population. It is suggested that captive breeding programs should
attempt to maintain a representation o f total genetic diversity present in native populations
(Hedrick et al. 1986). It appears the methods currently used by avaiculturists are
accomplishing this goal in the Hancock population; there is a high fragment diversity
although the frequencies o f these bands differ from those in natural situations. The
number o f fragments per individual at ANWR has a comparatively high variance around
that mean. It is probably more representative o f a natural population in which there is low
fragment diversity and most fragments occur in lower frequencies producing lower
64
amounts o f variance about that mean value. This population banding pattern can be
explained by fairly high gene flow from genetically similar immigrants. This would be the
case when Wood Ducks partially mix within a given fryway during the winter but rarely
migrate among flyways. The pattern in the captive Hancock population would perhaps
mimic a situation in which interflyway migration and gene flow were more common.
The Hancock and ANWR populations differ with respect to the distribution o f
different size fragments produced with the Hae HI/pV47 combination. The genetic
significance o f such size fragment differences is unclear. These size differences result from
either insertions o f endonuclease recognition sites or the amplification o f regions between
existing recognition sites. Regardless o f its genetic function and significance or lack
thereof this analysis provides a useful marker with which to differentiate the populations.
It is highly unlikely that there are profound functional differences in recognition sequence
insertions or amplifications between adjacent recognition sequences. Under that
assumption, such analyses o f fragment sizes might prove useful in differentiating
populations in other taxa, and such variability might prove useful as a selectively neutral
marker to differentiate closely related groups. Further evaluation o f the functional
significance o f size differences would need to be made to validate this assumption.
Because o f smaller sample size, the Finley population is not considered at length in this
analysis, but that population also appears to have its own pattern o f size fragment
distribution
65
Analysis o f the frequency o f shared fingerprint fragments in each population
(Figure 8), indicated that the Hancock population had many rare and many fairly common
fragments; the common fragments were shared between 50% - 70% o f the individuals
sampled. The ANWR population was found to contain many rare fragments and a few
common fragments. The presence o f the second peak in the bimodal distribution o f the
Hancock population o f fragments probably reflects a larger amount o f inbreeding in that
population in comparison to the ANWR population. This distribution reflects the overall
similarity values which indicate that the Hancock population has a greater degree o f
intrapopulation similarity than does the ANWR population.
Similarity coefficients in all o f the populations investigated fall within the range o f
values reported for other avian species. The ANWR population (S = 0.267), exhibits
similarity values that have been reported for other outbred waterfowl populations (Burke
& Bruford 1987; Westneat et al. 1988; Meng et al. 1990; Triggs et al. 1992; Rave et al.
1994). The Hancock population exhibits levels o f similarity (S = 0.441) that have been
reported for populations believed to be under the influence o f moderate levels o f
inbreeding (Triggs et al. 1992; Rave et al. 1994). The genetic similarity o f the Hancock
population is similar to that reported for the unrelated pairs o f the Hawaiian Goose, or
Nene {Branta sandvicemis) in captive populations, but it is less than similarities reported
for captive Nene flocks known to have originated from very small numbers o f individuals
(Rave et al. 1994). Hancock values fall within the range reported for laboratory and wild
66
populations that are known to be inbred (Burke & Bruford 1987) or for rare waterfowl
species (Triggs et al. 1992). The similarity values for Finley (S = 0.662), are comparable
to those o f populations that are known to have experienced significant levels o f inbreeding
or extensive bottlenecks. The high genetic similarity o f this natural population is likely a
result o f multiple generations o f inbreeding and perhaps long periods o f population
bottlenecks.
Homozygosity values (Table 6) indicate that the Hancock population is twice as
inbred as the ANWR population; a small degree o f inbreeding would be expected in a
captive population regardless o f efforts to maintain outbreeding. Inbreeding is avoided in
the Hancock population by replacing hens in the captive flock on a periodic basis with
individuals from other captive populations (B. Hancock pers. comm.). Within this
strategy o f forced gene flow hens in the flock are allowed to mate without direct
intervention. The relatively low value o f H probably indicates that this method o f
outbreeding is working sufficiently well to avoid drastic losses o f genetic variation. It
might be possible to reduce inbreeding further by manual pairing o f hens with single
drakes to insure that paternal variance is maximized, or attempt to create equalized family
sizes among hens to attempt to minimize the effects o f unequal numbers o f offspring
among breeding females. A pitfall with this method o f hen swapping and a lack o f mating
intervention is that avaiculturists must maintain a sufficiently large support network within
which to exchange females. I f the pool of'migrants' is limited, inbreeding could eventually
67
occur over extended periods o f time despite the swapping o f females between captive
populations; careful records must be kept in order to avoid re-swapping hens between
captive flocks. The Finley population exhibits a very large homozygosity value, and there
are several possible explanations for this high level o f inbreeding. Founder effects could
have played an important role. Wood Duck populations in the Pacific Flyway could have
been founded by a small number o f closely-related colonists; this is very likely if they were
originally founded by human settlers transporting Wood Ducks to the Pacific coast.
Alternatively, this high level o f inbreeding could reflect prolonged or repeated population
bottlenecks which reduced genetic variability, and the low resultant variance has not been
increased by migrants from eastern populations. A third plausible explanation is that
within the Pacific Flyway, Wood Ducks do not migrate to the extent that they do in o th e r.
flyways, and as such they would be more likely to select mates more closely related to
one-another than if they all migrated to a common location before mate selection
occurred. No data exist on the migratory patterns o f W ood Ducks in the Pacific Flyway,
but some band return information does indicate that there is some southerly migration
during the winter months. The mild winters o f the areas west o f the Coast Range in
Oregon and Washington could delay migration until selection o f mates has occurred. I
have observed W ood Ducks engaging in mate selection behavior in Oregon in late
September and October, long before Wood Duck population numbers have been reduced
by migration to more southerly portions o f the flyway. A final possible explanation for
68
this high homozygosity value is that the Finley individuals are from a small family group
and that the high homozygosity is a result o f this sampling error. This is not highly likely
since the individuals sampled were collected from nests on very distant sections o f the
refuge. This assumption o f non-relatedness is confirmed by the high standard error o f the
mean number o f fragments per individual in the Hancock population. If the individuals
sampled were closely related, they should share many fragments and have roughly the
same number o f fragments. Although they do not have as many fragments per individual
as do the other two populations, they exhibit a large amount o f variance in the numbers o f
fragments per individual. This suggests that they are not from a closely related familial
group. Further investigations should be carried out to ascertain the levels o f variability in
different locations in the Pacific Flyway.
These results have managerial implications for populations introduced into
northern regions o f the range o f Wood Ducks in the eastern fryways. The ANWR
population has probably increased its level o f genetic variability since the time o f its
initiation. This increased genetic diversity is most likely a result o f immigrants into the
population. The highly philopatric nature o f wood ducks would tend to make their
populations prone to inbreeding; however the mechanisms o f mate choice probably
counteract this tendency. Hens select drakes on the winter range, and the pair return to
the hen's place o f hatching to nest in the following year. In more northerly regions o f their
range W ood Ducks migrate before mate selection occurs. This facilitates hens to select
69
mates that are not from their summer range arid not likely closely related to them In
northern regions, and particularly in eastern flyways, inbreeding is not likely to be a
problem for populations established from captive individuals.
The breeding population o f Wood Ducks at ANWR appears to be declining (Doty
et al. 1984; previous chapters). A possible explanation for this decline is a reduced
reproductive success due to inbreeding or low survival associated with low amounts o f
genetic variability. The results from DNA fingerprinting do not support the hypothesis
that this breeding population has a reduced amount o f genetic variability. Alternative
hypotheses for this decline have been proposed (previous chapters), which appear to be
more tenable than that o f reduced genetic variability.
The patterns o f genetic variability described with ^VAHHae Hi DNA fingerprints
are corroborated by allozyme analyses (see previous chapters). Although ANWR was
consistently more diverse in all genetic indices analyzed for allozymes, none o f these
differences were statistically significant. Almost all o f the indices calculated from DNA
fingerprinting are statistically significant making DNA fingerprinting a more powerful
technique with which to compare relative amounts o f genetic diversity in populations
exhibiting low amounts o f protein diversity.
Jl
70
C h ap ter 5
SUM M ARY O F TH E D ISSER TA TIO N R ESE A R C H
In order to understand better the current status o f nesting W ood Ducks at ANWR1
a series o f hypotheses relating to the reproduction Ofthis species were tested. By testing
the three hypotheses posed in this investigation, a better understanding o f the population
viability o f W ood Ducks at ANWR has been obtained. In addition to testing these
hypotheses, a number o f generalizations about the W ood Ducks at ANWR and about the
management o f W ood Duck populations have become more clear. In this chapter, I
discuss the hypotheses tested, state some o f the general information gained and make
suggestions based on the findings o f this investigation.
The first hypothesis proposed was that competition for nesting spaces from
Hooded Mergansers is adversely affecting the reproduction o f Wood Ducks at ANWK
The analyses conducted indicate that the data on the history o f W ood Duck nesting at
ANWR do not support this hypothesis. It appears that there is little if any competitive
interaction between these two species that is responsible for a reduction in the
reproductive success on the refuge. Thenumbers o f nesting Hooded Mergansers have
increased while the numbers o f nesting W ood Ducks have decreased, but there is no
indication that the presence o f one species has adversely affected the reproductive success
o f the other.
IL
71
The second hypothesis was that the W ood Ducks at ANWR were exposed to
environmental toxins from ostensibly originating from agricultural practices in the drainage
o f the James river in areas surrounding the refuge, and that the reproduction o f Wood
Ducks was being adversely affected by the accumulation o f environmental toxins by
nesting females. The data on nesting success fail to support this hypothesis. When avian
species are exposed to toxins common in agricultural practices, they typically experience a
marked decrease in reproductive success because o f low hatching rates. The Wood Ducks
at ANWR show no historical changes in hatching success, nor do they differ significantly
in their hatching success from other populations o f this species in other regions.
The third hypothesis was that the W ood Ducks at ANWR have low amounts o f
genetic variability and that paucity o f genetic variability is impacting the reproduction or
survival o f the individuals breeding at AN W R The data on genetic variability do not
support this hypothesis o f low genetic diversity. In fact, the Wood Ducks at ANWR have
fairly high levels o f genetic variability as compared to other avian species and there does
not appear to be any genetic reason to suspect that this breeding population should have
any deleterious traits associated with low levels o f genetic variability that might affect the
reproductive success or survivorship o f individuals in this population. Two independent
types o f genetic markers were analyzed and both o f these yielded concurrent results
rejecting the hypothesis o f low genetic variability.
Aside from the testing o f these three hypotheses, the results o f this investigation
yield some general conclusions regarding the current nesting behavior, population status
72
and viability o f nesting Wood Ducks at A N W R Analysis o f historical nesting data has led
to a number o f conclusions. It appears that the number o f breeding hens at ANWR is
probably being underestimated by the current census methods o f nest-box monitoring. It is
possible that hens are nesting in natural cavities and this nesting is not being included in
the annual nesting census, it is also likely that there is nesting outside the refuge
boundaries that is significant to this population. Although such nesting is unmonitored, it
is probably an important component to the breeding population o f W ood Ducks in this
geographic region. The hatching success has not significantly changed since the initial
introduction o f W ood Ducks to A N W R This indicates that Wood Ducks are not under
the influence o f environmental contaminants to an extent that it interferes with nesting
success. This also indicates that the reproductive success o f W ood Ducks is not being
heavily influenced by the occurrence o f potential nesting competitors, Hooded
Margansers. The appearance o f Hooded Mergansers coincides with the decline o f Wood
Ducks at ANWR, but there is likely no causative interaction between the population
dynamics o f the two species. When all o f these factors are viewed in a cumulative nature,
they indicate that the basic breeding parameters o f the Wood Duck population has not
changed significantly since the initial introduction and that the most parsimonious
explanation for the decline is that the nesting population size is probably being
underestimated by current census methods.
The genetic analyses also yielded information o f general utility beyond that of
merely testing the hypothesis o f low genetic variability. The first point is that DNA
11 ;;
73
fingerprinting is a useful tool to analyze levels o f genetic variability in organisms that have
relatively little amounts o f variability. The differences discovered between the ANWR
population and a captive population using allozymes were not significant; the same general
pattern was elucidated using DNA fingerprint analyses, but these differences were found
to be statistically significant ones.
Another interesting point is that the Wood Duck population has probably
experienced an increase in genetic diversity since being introduced from captivity. This
increase has probably come about through the immigration o f new drakes selected by hens
for breeding on an annual basis. Hens are highly philopatric, but select mates during the
winter months in areas containing males from many disparate regions o f North America;
this ability to select mates from non-natal regions promotes outbreeding. This effective
outbreeding has probably served to increase genetic diversity over time.
It was also discovered that the captive population analyzed (Hancock) has fairly
high levels o f genetic diversity; these levels o f diversity are within the range o f genetic
diversities found in natural avian populations. The managers o f this captive population
actively attempt to minimize inbreeding by exchanging females between captive
populations. The high level o f diversity in this population indicates that this management
method attempting to avoid inbreeding is working fairly well and should continue to be
carried out.
It was discovered that a small, natural population in the Pacific Flyway has very
little genetic diversity. The number o f individuals sampled from this population was very
74
small, and these findings may be a result o f this reduced sample. Further sampling would
be necessary to validate the findings o f this analysis, but assuming that these findings are
representative o f genetic variability present in those populations several causal factors can
be discussed. W ood Duck populations in the Pacific Flyway are much smaller than those
east o f the Rocky Mountains, and have had very little if any recent gene flow with
populations to the east. It is possible that these populations were introduced from captive
stocks originating from the eastern United States, and could have experienced extensive
population bottlenecks. It is also possible that these populations are o f natural origin and
have experienced significant population bottlenecks nonetheless. The levels o f genetic
diversity need to be investigated further for Pacific Flyway populations, but if the low
levels discovered in this investigation are indicative o f these populations it might be
advisable to increase genetic diversity through management practices that would increase
gene flow between this population and those having high levels o f genetic diversity in the
eastern United States.
In general, the results o f this investigation indicate that the nesting population of
Wood Ducks at ANWR is a viable one and is in no immediate threat o f extirpation in the
foreseeable future. Further, this investigation indicates that Wood Duck populations can
probably be introduced to novel environments without fears o f inbreeding if they are in
regions where gene flow can occur through the natural process o f winter-time mate
selection. This management strategy can probably be extended to any philopatric species
that migrates or has access to novel mates through mate selection behaviors. Such an
Jl
ill
75 •
interaction o f management and natural ecological parameters is an example o f how
conservation strategies and wildlife management practices can be used to. heavily influence
populations without adverse affects on the viability o f such populations.
I,
v
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APPENDICES
APPENDIX A
RAW DATA FROM DNA FINGERPRINTS.
Those samples marked with the letter TF are from the Hancock population.
Those marked with the letter ‘A ’ or ‘H A ’ are from the Arrowwood population, and those
marked with the letter ‘F ’ are from the Finley population. In the data matrix, the presence
o f a fragments is denoted by a value o f I, the absence o f a fragment is denoted by the
value o f 0.
89
Size (Kb)
IIA40
1,658
1,740
1,853
1,948
2,131
2,303
2,422
2,495
2 ,6 2 3
2 ,7 7 2
2 ,9 7 0
3,182
3,291
3,465
3 ,6 0 0
3,726
3 ,8 0 4
3,865
3,991
4 ,1 2 4
4 ,3 1 4
4 ,4 7 8
4 ,5 8 0
4,771
4 ,9 9 7
5 ,2 1 7
5 ,3 3 0
5,621
5 ,7 5 0
6,029
6,269
6 ,6 8 3
7 ,0 0 2
7,259
7,409
7,578
7,906
7,967
8 ,3 8 3
8,671
8.847
9,213
9,489
9,681
10,029
10 ,4 0 0
10,882
11,284
12,317
13,055
14,059
14,449
15,213
15,644
16 ,9 4 0
17,189
18,165
19,931
0 .
0
P
0
0
I
I
I
I
I
I
0
I
0
0
0
0
0
0
I
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
0
0
0
0
o •
0
0
I
. 0
0
0
I
0
0
0
0
0
0
0
0
0
0
I
,
IIA42
IIA23
IIA41
IIA22
IIAI
Al
0
0
0
0
0
0
I
I
I
0
I
0
0
0
0
0
I
0
I
I
0
I
0
I
0
0
0
0
0
0
0
0
I
0
I
0
I
0
0
I
0
0
0
I
0
0
0
0
0
I
I
I
I
I
I
0
I
0
I
I
0
I
0
I
0
0
0
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
I
I
0
0
I
I
0
0
I
I
0
0
0
0
0
0
0
I
0
0
0
: i ■
I
I
0
0
I
0
I
■ 0
I
0
0
0
0
0
I .
0
0
I
0
0
0
I
0
P
P
0
0
0
0
0
I
0
0
0
0
0
0.
0
0
0
0
0
0
0
0
0
I
I
0
0
P
0
0
I
P
0
I
0
0
0
0
0
I
0
I
,0 .
0
0
0
0
0
0
0
0
0
0
0
0
0
0
P
0
0
I
0
0
0
I
0
0
0
I
.
P
P
0
0
0
0
0
P
0
0
0
0
0
0
0
' 0
0
0
0
0
P
0
0
0
0
0
0
’
P
0
0
I
I
I
P
P
0
0
0
0
0
• I .
0
I .
0
0
0
’
P
I
0
0
I
0
0
0
I
0
0
P
0
0
P
0
0
0
0
0
0
0
0
0
0
0
0
0
.0
I
I
0
0
I
I
0
I
0
I
0
I
0
I
.
P
P
0
0
0
0
■I
.0
0
0
0
0
0
0
P
P
0
0
I
0
0
0
0
0
0
0
P
P
.
0
0
0
0
0
0
0
0
0
IIA11
0
0
I
I
I
I
0
.I
0
I
I
I
0
I
0
I
0
I
0
0
0
0
- 0
0
0
0
0
0
0
0
P
0
0
0
I
P
0
0
0
0
0
I
0
0
0
0
0
I
0
0
0
■0
0
0
0
0
0
0
0
P
P
0
0
0
P
.
'
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
90
Size (Kb)
HI 7
H52
H72
H 118
H51
H81
HIOI
HI I O
1,658
1,740
1,853
1,948
2,131
2 ,3 0 3
2 ,4 2 2
2,495
2 ,6 2 3
2 ,7 7 2
2 ,9 7 0
3,182
3,291
3,465
3 ,6 0 0
3,726
3 ,8 0 4
3,865
3,991
4 ,1 2 4
4 ,3 1 4
4 ,4 7 8
4 ,5 8 0
4,771
4 ,9 9 7
5 ,2 1 7
5 ,3 3 0
5,621
5 ,7 5 0
6,029
6 ,2 6 9
6 ,6 8 3
7 ,0 0 2
7 ,2 5 9
7 ,4 0 9
7 ,5 7 8
7 ,9 0 6
7 ,9 6 7
8 ,3 8 3
8,671
8 ,8 4 7
9 ,2 1 3
9 ,4 8 9
9,681
10,029
1 0 ,4 0 0
1 0 ,8 8 2
1 1 .2 8 4
12 ,3 1 7
13,055
14,059
14,449
15,213
1 5 ,6 4 4
16,940
17,189
18,165
19,931
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
I
0
I
0
I
0
0
0
0
I
0
0
I
0
0
0
0
0
I
0
0
0
I
0
0
0
0
I
0
0.
0
0
0
0
0
0
0
0
I
0
I
0
0
0
0
0
0
0
0
0
0
I
0
I
0
I
0
0
I
0
0
0
I
0
0
0
0
0
0
0
I
0
0
I
0
0
0
0
0
0
I
0
0
0
0
0
0
0
I
I
0
I
0
0
0
0
I
0
I
0
0
0
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91
Size (Kb)
Hill
HI 3
HI 52
HI 07
HI 23
HI 33
H119
H112
1.658
1,740
1.853
1,948
2,131
2,303
2,422
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2,772
2,970
3,182
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4,314
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4,771
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5,217
5,330
5,621
5,750
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6.269
6,683
7,002
7,259
7,409
7,578
7,906
7,967
8,383
8,671
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9,213
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10,400
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11,284
12,317
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92
Size (Kb)
1,658
1,740
1,853
1,948
2,131
2,303
2,422
2,495
2,623
2,772
2,970
3,182
3,291
3.465
3,600
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4,314
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4,580
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HI 3 4
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0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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0
0
0
0
0
0
0
0
0
0
0
0
95
Size (Kb)
Al
IIA29
1.658
1,740
1,853
1,948
2,131
2 .3 0 3
2 ,4 2 2
2,495
2 ,6 2 3
2 ,7 7 2
2 ,9 7 0
3 ,1 8 2
3,291
3,465
3 ,6 0 0
3 ,7 2 6
3 ,8 0 4
3,865
3,991
4 .1 2 4
4 ,3 1 4
4 ,4 7 8
4 ,5 8 0
4,771
4 ,9 9 7
5 ,2 1 7
5 ,3 3 0
5,621
5 ,7 5 0
6,029
6 ,2 6 9
6 ,6 8 3
7 ,0 0 2
7 ,2 5 9
7 ,4 0 9
7 ,5 7 8
7 ,9 0 6
7 ,9 6 7
8 ,3 8 3
8,671
8 ,8 4 7
9 ,2 1 3
9 ,4 8 9
9,681
1 0 ,0 2 9
1 0 ,4 0 0
1 0 ,8 8 2
1 1 ,2 8 4
1 2 ,3 1 7
13,055
14,059
14,449
15,213
15 ,6 4 4
16 ,9 4 0
17,189
18,165
19,931
0
0
0
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0
0
0
0
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0
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0
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0
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0
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0
0
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0
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0
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0
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0
0
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0
0
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0
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0
0
0
0
0
0
0
0
0
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0
0
IIA34
0
0
0
0
0
0
0
0
I
0
I
I
0
I
0
P
0
0
0
0
0
0
0
0
0
0
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0
0
0
P
0
I
0
P
0
0
0
0
0
0
0
0
0
P
0
0
0
0
0
0
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0
0
0
0
0
0
IIA-6
0
0
0
0
0
0
0
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0
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0
0
0
0
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0
0
0
0
0
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0
0
0
0
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0
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0
0
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0
0
0
0
0
0
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0
0
0
0
0
0
P
0
0
0
0
0
0
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0
0
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0
0
0
0
IIA4
0
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0
0
0
0
0
0
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I
I
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0
0
0
0
0
0
0
0
0
I
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0
0
0
0
I
0
0
0
0
I
0
0
0
P
0
0
0
0
0
0
0
0
0
0
P
0
P
I
0
0
0
0
0
0
0
IIA-13
0
0
0
0
0
0
0
0
I
0
I
0
0
0
0
0
0
0
0
0
0
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0
0
0
0
0
0
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0
0
0
0
0
0
0
0
0
P
0
0
0
0
0 .
0
0
0
0
0
0
P
0
0
0
0
0
0
96
Size (Kb)
1,658
1,740
1,853
1,948
2,131
2,303
2,422
2,495
2,623
2,772
2,970
3,182
3,291
3,465
3,600
3,726
3,804
3.865
3,991
4,124
4,314
4,478
4,580
4,771
4,997
5,217
5,330
5,621
5,750
6,029
6,269
6,683
7,002
7,259
7,409
7,578
7,906
7,967
8.383
8.671
8,847
9,213
9,489
9,681
10,029
10,400
10,882
11,284
12,317
13,055
14,059
14,449
15,213
15,644
16,940
17.189
18,165
19,931
IIA25
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0
I
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
0
I
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0 .
0
0
0
0
0
0
I
0
0
0
IIA37
IIA49
0
0
0
0
0
0
0
0
0
0
I
0
0
I
0
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
I
0
0
0
I
0
0
0
0
0
0
0
0
0
0
0
0
0
I
I
0
0
0
I
0
0
0
0
0
I
I
0
0
0
0
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
0
0
I
0
0
0
0
0
0
0
IIA21
0
0
0
0 .
0
0
0
0
I
0
I
I
0
0
0
I
0
0
0
0
0
0
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0
I
0
0
0
0
I
0
0
I
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
I
0
0
0
I
0
0
0
IIA38
!!Al 8
IIA36
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
I
0
0
0
I
0
0
I
0
0
0
0
I
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
0
I
0
0
I
0
0
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
I
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
I
0
0
I
0
0
0
I
0
0
I
0
0
0
0
I
0
0
I
0
0
I
0
0
0
I
0
0
0
0
0
0
0
0
0
0
0
0
I
I
I
0
I
0
0
0
0
0
I
0
I
I
0
0
0
I
I
0
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
0
IIA33
0
0
0
0
0
0
0
0
0
0
0
0
0
I
0
I
0
0
0
I
0
0
I
0
0
0
I
0
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
■0
0
0
0
0
0
I
0
0
0
I
0
0
0
97
Size (Kb)
1,658
1,740
1,853
1,948
2,131
2,303
2,422
2,495
2,623
2,772
2,970
3,182
3,291
3,465
, 3,600
3,726
3,804
3,865
3,991
4,124
4,314
4,478
4,580
4,771
4,997
5,217
5,330
5,621
5,750
6,029
6,269
6,683
7,002
7,259
7,409
7,578
7,906
7,967
8,383
8,671
8,847
9,213
9,489
9,681
10,029
10,400
10,882
11,284
12,317
13,055
14,059
14,449
15,213
15,644
16,940
17,189
18,165
19,931
IIA27
IIA44
IIA46
IIA74
IIA24
IIA44
IIA46
IIA74
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
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0
0
0
0
0
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0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
I
0
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0
I
0
0
0
I
0
I
0
0
I
0
0
0
0
0
0
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0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
0
0
0
0
I
I
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0
I
0
I
0
I
0
I
0
0
I
0
0
0
0
0
0
I
0
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I. .
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I
0
I
0
I
0
0
0
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0
0
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0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
0
0
I
0
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
0
I
0
I
I
0
0
0
0
I
0
0
0
0
0
I
0
I
0
0
0
I
0
0
0
0
0
I
0
0
0
0
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
0
I
I
0
I
0
0
I
I
0
0
I
0
I
0
I
0
0
I
0
I
0
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
I
I
I
I
0
0
0
I
0
0
0
I
0
0
0
I
0
0
I
0
I
0
0
0
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
I
0
0
I
0
0
0
0
0
0
0
0
0
I
0
0
0
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I
0
0
I
0
I
0
0
0
0
I
0
0
0
0
0
0
0
0
0
0
0
I
0
0
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
0
0
98
S iz e (Kb)
IIA 2 4
IIA 3 0
IIA 3 5
IIA 3
IIA I9
IIA 3 2
IIA 2 6
IIA 3 0
0
1 ,6 5 8
I
0
0
0
0
0
0
1 .7 4 0
0
0
0
0
0
0
0
0
1 ,8 5 3
0
0
I
0
0
0
I
0
0
1 ,9 4 8
0
I
I
I
0
0
I
2 ,1 3 1
0 ■
0
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0
I
I
I
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2 ,3 0 3
0
0
I
0
0
I
0
0
2 ,4 2 2
2 ,4 9 5
0
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0
0
I
0
I
0
I
I
0
0
I
0
0
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2 ,6 2 3
I
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0
I
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0
0
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2 .7 7 2
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0
I
0
0
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I
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0
0
0
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3 ,1 8 2
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0
0
0
0
0
0
0
3 ,2 9 1
0
0
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0
0
0
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3 ,4 6 5
0
0
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0
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0
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0
3 ,6 0 0
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0
0
0
0
0
0
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3 ,7 2 6
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0
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0
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0
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3 ,8 0 4
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0
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3 ,9 9 1
0
0
0
0
0
0
0
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4 ,1 2 4
0
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0
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0
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0
4 ,3 1 4
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0
0
0
0
0
0
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4 ,4 7 8
0
0
0
0
0
0
0
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4 ,5 8 0
I
0
I
I
0
0
0
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4 ,7 7 1
0
0
0
0
0
0
0
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4 ,9 9 7
0
0
0
0
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0
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0
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5 ,2 1 7
0
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5 ,3 3 0
0
0
0
0
0
0
0
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5 ,6 2 1
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0
0
0
0
0
0
0
5 ,7 5 0
0
0
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0
0
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0
6 ,0 2 9
0
0
0
0
0
0
0
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6 ,2 6 9
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I
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0
0
0
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6 ,6 8 3
0
0
0
0
0
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7 ,0 0 2
0
0
0
0
0
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7 ,2 5 9
0
0
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0
0
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7 ,4 0 9
0
0
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7 ,5 7 8
0
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0
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7 ,9 0 6
0
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0
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8 ,3 8 3
0
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0
0
0
0
0
0
0
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8 ,8 4 7
I
0
0
0
0
0
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0
0
0
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0
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0
0
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0
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0
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0
0
0
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0
0
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0
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0
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0
0
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0
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0
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0
0
0
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0
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0
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0
0
0
0
0
0
0
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0
0
0
0
0
0
0
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0
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0
0
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0
0
0
0
0
0
0
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0
0
0
0
0
0
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0
0
0
0
0
0
0
0
1 9 ,9 3 1
0
0
0
0
0
0
0
0
.
0
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I
99
Size (Kb)
IIAI2
IIA23
IIA41
IIA41
1,658
1,740
1,853
1,948
2,131
2,303
2,422
2,495
2,623
2,772
2,970
3,182
3,291
3,465
3,600
3,726
3,804
3,865
3,991
4,124
4,314
4,478
4.580
4,771
4,997
5,217
5,330
5,621
5,750
6,029
6,269
6,683
7,002
7,259
7,409
7.578
7.906
7,967
8,383
8,671
8,847
9,213
9.489
9,681
10,029
10,400
10,882
11,284
12,317
13,055
14,059
14,449
15,213
15,644
16,940
17,189
18,165
19,931
0
0
0
0
0
0
0
0
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I
I
I
I
I
I
I
I
I
I
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0
0
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I
I
I
I
I
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0
0
0
0
0
0
0
I
I
0
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0
0
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0
I
I
0
0
0
0
0
0
0
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0
0
0
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0
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I
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0
0
0
I
I
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0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
I
0
0
0
0
I
I
0
0
I
I
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
0
0
0
I
0
0
0
0
0
0
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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0
0
0
0
0
0
0
0
0
0
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0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
. 0
P
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
I
0
0
0
0
0
0
0
0
0
0
0
0
100
APPENDIX B
RAW GENOTYPES FROM AELOZYME ANALYSES.
Those samples labeled with the prefBx TE are from the Hancock population. AU
otUers are from the Arrowwood population. The numbers preffixed by eQ 5 refer to quiU
sample numbers, whUe the other numbers refer to a blood sample number. The
abbreviation ‘N S’ denotes a genotype that was non-scorable.
H23Q52
CC
CC
CC
CC
CC
CD
CC
CC
CC
CC
CC
BC
CC
DC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
H25Q50
CC
CC
CC
CC
CC
CD
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
H1Q25
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
H27Q49
DD
CC
CC
CC
BC
CC
CC
CC
CC
CC
CC
BB
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
H16Q29
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CD
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
H20
CC
CC
CD
CC
CC
CD
CC
CC
CC
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS .
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
HI 9
CC
CC
CC
CC
CC
DD
CC
CC
CC
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
H55
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
H49
CC
CC
CC
CC
CC
DD
CC
CC
CC
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
H41
CC
CC
CC
CC
CC
CD
CC
CC
CC
NS
NS
NS
NS
NS
NS
NS
NS
NS
■ NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
101
LDH-I
LDH-2
MPI
HB-I
ALB
EST-I
EST-2
GAPDH
HB-2
PEP-LLL
PGD
PEP-LAI
PEP-LA2
PEP-GLI
PEP-GL2
IDH-I
IDH-2
AAT
ACP
GPI
MDH-I
MDH-2
LAP
G6PDH
ODH-I
ODH-2
HBDH
XDH-I
XDH-2
PEP-LGG
21 Ql 7
52048
CC
CC
CC
CC 1
CC
CC
CC
CC
NS
NS
NS
NS
CC
CO
CC
CC
CC
CC
CC
CC
DC
DD
CC
CC
DD
CC
CC
CC
DD
NS
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
CC
Ce
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
27024
CC
CC
CC
CC
NS
NS
. CC
NS
CC
CC
CC
NS
DC
DC
DC
CC
CC
NS
CC
CG
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
802
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
47023
CC
CC
CC
CC
BC
CC
BC
CC
CC
CC
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
5044
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
Ce
CC
CC
CC
CC
CC
54053
NS
NS
NS
NS
NS
NS
NS
NS
NS
CD
CD
CD
■CC
CD
NS
CC
CC
NS
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CB
49052
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
51015
CC
CC
CC
CC
CB
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
NS
CC
CC
CC
CC
CC
NS
CC
Ce
CC
CC
CC
BB
37029
CC
CC
CC
CC
CC
DC
CC
CC
CC
NS
BB
CC
CC
CC
NS
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
43025
CC
CC
CC
CC
CC
DC
CC
CC
CC
BC
CC
BC
CD
BC
NS
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC '
102
LDH-I
LDH-2
MPI
HB-I
ALB
EST-I
ESI-2
GAPDH
HB-2
PEP-LLL
PGD
PEP-LAI
PEP-LA2
PEP-GLI
PEP-GL2
IDH-I
IDH-2
AAT
ACP
GPI
MDH-I
MDH-2
LAP
G6PDH
ODH-I
0DH-2
HBDH
XDH-I
XDH-2
PEP-LGG
55022
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
DC
NS
NS
NS
NS
NS
CC
CC
CC
NS
NS
NS
NS
NS
CC
CC
CC
38041
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
NS
CC
CC
CC.
NS
NS
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
53047
NS
NS
NS
NS
NS
NS
NS
NS
NS
CC
NS
CC
CC
CC
NS
CC
CC
NS
NS
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
36012
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
• NS
NS
' NS
NS
NS
41056
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
CD
CC
CD
CD
NS
CC
CC
NS
NS
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
39013
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CD
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
• NS
44026
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
45028
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
BB
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
Ce
CC
CC
CC
4208
CC
CC
Ce
CC
CC
CC
CC
CC
CC
NS
DD
CB
CC
CD
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
23045
CC
CC
CD
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
103
LDH-I
LDH-2
MPI
HB-I
ALB
EST-I
ESI-2
GAPDH
HB-2
PEP-LLL
PGD
PEP-LAI
PEP-LA2
PEP-GLI
PEP-GL2
IDH-I
IDH-2
AAT
ACP
GPI
MDH-I
MDH-2
LAP
G6PDH
ODH-I
ODH-2
HBDH
XDH-I
XDH-2
PEP-LGG
4607
CC
CC
BC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CB
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
40038
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
6032
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
BB
CC
CC
CC
NS
CC
CC
CC
NS
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
56QN
CC
CC
CC
CC
NS
CC
CC
CC
CC
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
I 2040
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
BB
BC
CD
CD
NS
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
3106
NS
NS
NS
NS
NS
NS
NS
NS
NS
CC
BB
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
NS
CC
AA
CC
CC
CC
CC
CC
CC
58000
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
48019
CO
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
. CC
NS
CC
CC
NS
NS
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
33021
CC
CC
CC
CC
NS
NS
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
NS
NS _
CC
CC
CC
CC
NS
CC
CC
CC
CC
CO
BC
29016
NS
NS
NS
NS
NS
NS
NS
NS
NS
BD
BB
BC
CC
BC
CC
NS
CC
CC
NS
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
60020
CC
CC
CC
CC
BC
CC
CC
CC
CC
CC
CC
CC
BC
CC
NS
CC
CC
NS
NS
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
35058
CC
NS
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
NS
NS
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
104
LDH-I
LDH-2
MPI
HB-I
ALB
EST-I
EST-2
GAPDH
HB-2
PEP-LLL
PGD
PEP-LAI
PEP-LA2
PEP-GLI
PEP-GL2
IDH-I
IDH-2
AAT
ACP
GPI
MDH-I
MDH-2
LAP
G6PDH
ODH-I
ODH-2
HBDH
XDH-I
XDH-2
PEP-LGG
28057
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
■CC
CC
3405
cc :
CC
CC
CC
CC
DC
CC
CC
CC
CC
■ BB
CC
CD
CC
NS
. CC
CC
NS
NS
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
59033
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
NS
NS
CC
CC
CC
CC
NS
CC
CC
Ce
CC
CC
CC
204
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
DD
CC
NS
NS .
CC
CC
CD
CC
CC
CC
CC
CC
CC
CC
CC
2209
CC
CC
CC
CC
BC
CC
CC
CC
CC
NS
BB
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
BC
CC
CC
CC
CC
CC
CC
CC
26010
CC
CC
CC
CC
BC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
Cc
CC
CC
CC
2504
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
• CC
CC
CC
CC
NS
CC
CC
NS
NS
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
11014
CC
CC
CC
CC
CC
CC
CC
CC
CC
Ce
NS
Ce
NS
NS
NS
CC
CC
NS
NS
NS
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
10.35
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
NS
NS
CC
NS
NS
NS
NS
NS
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
50QN1
CC
CC
NS
CC
CC
CC
CC
CC
CC
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
' NS
NS
NS
30040
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
SB
CC
CC
DD
NS
CC
CC
CC
CC
BC
CC
CC
BB
CC
CC
CC
CC
CC
CC
CC
105
LDH-I
LDH-2
MPI
HB-I
ALB
EST-I
EST-2
GAPDH
HB-2
PEP-LLL
PGD
PEP-LAI
PEP-LA2
PEP-GLI
PEP-GL2
IDH-I
IDH-2
AAT
ACP
GPI
MDH-I
MDH-2
LAP
G6PDH
ODH-I
0DH-2
HBDH
XDH-I
XDH-2
PEP-LGG
24027
CC
CC
CC
CC
CC
CC
BC
CC
CC
CC
BB
CC
CC
CC
CC
CC
CC
NS
NS
BC
CC
CC
CC
CC
CC
CC
CC
CC
CC
BC
20039
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
NLI 048
32011
NS
CC
NS
CC
NS
CC
NS
CC
CC
NS
NS
CC
NS
CC
CC
NS
CC
NS
NS CD
NS
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
NS
NS
NS
CC
NS
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
CC ■ CC
CC
CC
CC
CC
NS
CC
NL2Q49
NS
NS
NS
NS
NS
NS
NS
NS
CC
DD
CC
DB
CC
NS
CC
CC
NS
NS
CC
CC
CC
CC
NS
ce
CC
CC
CC
CC
CC
NS
H39Q16
CC
CC
CC
CC
CC
CD
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
H54Q47
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
BB
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CB
H21Q14
CC
CC
CC
CC
CC
CD
CC
CC
CC
CC
CC
CC
ce
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
H44Q35
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CD
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
. CC
CC
CC
CC
CC
CC
CC
HI 5010
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CD
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
■ CC
CC
CC
CC
CC
CC
H9Q13
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
106
LDH-I
LDH-2
MPI
HB-I
■ ALB
EST-I
EST-2
GAPDH
HB-2
PEP-LLL
PGD
PEP-LAI
PEP-LA2
PEP-GLI
PEP-GL2
IDH-I
IDH-2
AAT
ACP
GPI
MDH-I
MDH-2
LAP
G6PDH
ODH-I
0DH-2
HBDH
XDH-I
XDH-2
PEP-LGG
H52Q22
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC ,
H18Q45
CC
CC
CC
CC
CC
CD
CC
CC
CC
CC
CC
CD
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
H22Q21
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CO
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CB
H11Ql5
CC
GC
CC
CC
CC
CD
CC
CC
CC
CC
CC
DD
CC .
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
. CC
H53Q56
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
DD
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
H10Q4
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CD
CC
CC
NS
CC
CC
CC
. CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CB
H56Q1
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
,CC
CC
CC
CC
CC
CC
H13Q32
CC
CC
CC
CC
CC
CD
CC
CC
CC
CC
CC
CD
CC
CC
NS
CC
CC
CC
CC
CC
CC
CO
NS
CC
CC
CC
CC
CC
CC
CC
H8Q18
NS
NS
NS
NS
NS
NS
NS
NS
NS
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
H37Q9
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC ,
CC
CC
CC
CC
CC
107
LDH-I
LDH-2
MPI
HB-I
ALB
EST-I
EST-2
GAPDH
HB-2
PEP-LLL
PGD
PEP-LAI
PEP-LA2
PEP-GLI
PEP-GL2
IDH-I
IDH-2
AAT
ACP
GPI
MDH-I
MDH-2
LAP
G6PDH
ODH-I
ODH-2
HBDH
XDH-I
XDH-2
PEP-LGG
H48Q1I
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CD
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
NS
CC
CC
CC
CC
CC
CC
CC
H 46Q 40
H 2Q 30
H 29Q 27
H 4Q 2
H 14017
H 3Q 41
H 42Q 39
H 26Q 34
HI 2 0 2 4
H 17Q 33
L D H -I
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
L D H -2
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
H7 0 6
M PI
CC
CC
CC
CC
CC
CC
CC
CC
H B -I
ALB
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
E S T -I
CC
CC
CD
CC
CC
cc;
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
.
•
CC
CC
GAPD H
CC
CC
CC
CC
CC
CC
CC
cc
CC
CC
H B -2
CC
CC
CC
CC
CC
CC
CC
Cd
CC
CC
CC
PEP-LLL
CC
CC
CC
CC
CC
CC
CC
dd
CC
CC
CC _
CC
CC
CC
CC
CC
CC
CC
dd
CC
CC
CC
P E P -L A I
CC
CC
CD
CD
CC
BC
CC
dC
BC
CC .
CC .
P E P -L A 2
CC
CC
CC
CC
CC
CC
CC
dc
CC
CC
CC
P E P -G L I
CC
CC
CC
CC
CC
CC
BC
dd
CC
CC
DC
P E P -G L 2
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
ID H -I
CC
CC
CC
CC
CC
CC
cc
dd
CC
CC
CC
ID H -2
BC
CC
CC
CC
CC
CC
CC
dd
CC
CC
CC
AAT
CC
CC
CC
CC
Ce
Ce
CC
dd
CC
CC
CC
ACP
CC
CC
CC
CC
CC
CC
CC
cc
CC
CC
CC
GPI
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
M D H -2
CC
CC
Ce
CC
CC
cc
CC
CC
CC
LAP
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
CC
CC
CC
CC
GC
CC
CC
CC
CC
CC
CC
CC
CC
Cd
CC
CC
CC
CC-
CC
dC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
H BD H
CC
CC
CC
CC
CC
CC
CC
O 'D H -2
CC
CC
CC
CC
CC
CC
CC
dC
CC
CC
CC
CC
CC
CC
CC
Ce
CC
CC
PG D
M D H -I
G 6PD H
O D H -I
X D H -1
X D H -2
P E P-L G G
•
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
.
CC
. CC
CC
CC
CC
CC
c c . .
CC
CC
CC
cc
108
E S T -2
CC