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6/01 cJCIRC/DataDuepesvaS

DEMOGRAPHIC AND LIFE HISTORY CHARACTERISTICS OF REMNANT LAKE
STURGEON POPULATIONS IN THE UPPER GREAT LAKES BASIN:
INFERENCES BASED ON GENETIC ANALYSES
By

Patrick William DeHaan

A THESIS
Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Fisheries and Wildlife

2003

ABSTRACT
DEMOGRAPHIC AND LIFE HISTORY CHARACTERISTICS OF REMNANT LAKE
STURGEON POPULATIONS IN THE UPPER GREAT LAKES BASIN:
IN FERENCES BASED ON GENETIC ANALYSES
By
Patrick William DeHaan
While once abundant throughout the Great Lakes, lake sturgeon (Acipenser
fulvescens) have experienced dramatic population declines over the past 100 years.
Declines are due mainly to anthropogenic activities including over-harvest, blockage of
spawning habitat by dams, and loss of spawning habitat due to sedimentation and
pollution. While the need for rehabilitation has been recognized, knowledge of many
fundamental aspects of this species life history are currently lacking. The objectives of
this study were to employ molecular genetic markers to 1) assess the degree of genetic
variability and assess levels of population structure present between remnant lake
sturgeon populations in the Upper Great Lakes Basin 2) to determine parentage and
describe the lake sturgeon mating system 3) to examine the genetic consequences of a
supportive breeding program designed to rehabilitate a declining lake sturgeon
population. The results of this study show that a signiﬁcant degree of population structure
exists among remnant lake sturgeon populations. Using genetic determination of
parentage, this study describes the lake sturgeon mating system and documents a high
degree of polygyny and polyandry as well as variance in male and female reproductive
success. Results of this study also indicate that a supportive breeding program for lake

sturgeon has led to an overall increase in relatedness. Results and implications of these

data are discussed in light of declining population numbers.

TABLE OF CONTENTS

LIST OF TABLES .................................................................................... v

LIST OF FIGURES .................................................................................. vi

INTRODUCTION .................................................................................... 1

CHAPTER I

GENETIC POPULATION STRUCTURE OF REMNANT LAKE STURGEON

POPULATIONS IN THE UPPER GREAT LAKES BASIN .................................. 7
Introduction ......................................................................................... 7
Methods .......................................................................................... 11
Results ...... . ..................................................................................... 16
Discussion ....................................................................................... 19
Conclusion ....................................................................................... 25

CHAPTER II

LAKE STURGEON REPRODUCTIVE ECOLOGY AND CONSERVATION STATUS

BASED ON GENETIC DETERMINATION OF PARENTAGE ............................ 26
Introduction ....................................................................................... 26
Methods ........................................................................................... 31
Results ............................................................................................. 35
Discussion ........................................................................................ 38
Conclusion ....................................................................................... 43

CHAPTER III

GENETIC CONSEQUENCES OF SUPPORTIVE BREEDING FOR A DECLINING

POPULATION OF LAKE STURGEON ........................................................ 45
Introduction ...................................................................................... 45
Methods .......................................................................................... 50
Results ............................................................................................ 53
Discussion ....................................................................................... 55
Conclusion ....................................................................................... 59

APPENDIX I

Estimates of Microsatellite Allele Frequencies and Mitochondrial DNA Haplotype

Frequencies from Chapter I ........................................................................ 62

APPENDIX II

Tables and Figures .................................................................................. 66

LITERATURE CITED ............................................................................. 86

iv

LIST OF TABLES

Table 1. Measures of genetic diversity based on 8 microsatellite loci for the 10
populations used in this study ..................................................................... 66

Table 2. Pair-wise estimates of Fst based on 8 microsatellite loci (above the diagonal) and
(1)“ based on mtDNA (below the diagonal) ....................................................... 67

Table 3. Hierarchical partitioning of variance based on microsatellite and mitochondiral
DNA for the 10 populations used in this study .................................................. 68

Table 4. Measures of genetic diversity for the 8 microsatellite loci used in the parentage
study ................................................................................................... 69

Table 5. Allele frequencies based on 8 microsatellite loci for the two groups of sturgeon
used in this study. One group was composed of wild adults that spawned in the spring of
2001 and the other group was composed of individuals likely to be the progeny of
supportive breeding events ........................................................................ 70

Table 6. Measures of genetic diversity calculated for each of the two groups of lake
sturgeon used in this study ......................................................................... 71

LIST OF FIGURES

Figure 1. Map showing the locations of the 10 lake sturgeon populations in Michigan and
Wisconsin that were sampled for this study ..................................................... 72

Figure 2. Graph showing the relative contribution of each of the 10 populations to total
gene diversity (CT). Gene diversity is broken down into two separate components; Cd,
the contribution of each population to the overall diversity based on differentiation from
other populations and Cs, the contribution of each population to the overall diversity
based on its own diversity .......................................................................... 73

Figure 3. Neighbor joining tree based on Cavali-Sforza and Edwards chord distance
showing the genetic structuring of remnant lake sturgeon populations in the Upper Great
Lakes. Values indicate bootstrap support over 2000 replicates ............................... 74

Figure 4. Map showing the Black Lake system including the Lower and Upper Black
Rivers and Black Lake .............................................................................. 75

Figure 5. Graphs showing the number of mates per female (5a) and the number of mates
per male (5b) for the 111 adult lake sturgeon in this study .................................... 76

Figure 6. Graph showing the relationship between total length of males and females that
spawned with one another in Black Lake in 2001. The low correlation observed suggests
that size based assortive mating is not taking place ............................................. 77

Figure 7. Graph showing the numbers of offspring produced for females (7a) and males
(7b) for the 111 lake sturgeon in this study ...................................................... 78

Figure 8a. Graph showing the number of offspring each female produced in each one of
the groups of out-migrating juveniles ............................................................ 79

Figure 8b. Graph showing the number of offspring each male produced in each one of the
three groups of out-migrating juveniles .......................................................... 80

Figure 9. Relationship between total length and the number of offspring produced for
each male and female ............................................................................... 81

Figure 10. Relationship between the number of mates and the number of offspring
produced for male and female lake sturgeon .................................................... 82

Figure 11. Percent of catch during population estimates in Black composed of individuals
of different lengths. The two peaks between 90 and 130 cm in 1997 and 2002 likely
represent individuals produced by supportive breeding efforts. 1997 data adapted from
Baker and Borgeson 1999, 2002 data adapted from MDNR unpublished data 2002 ...... 83

vi

Figure 12. Distribution of the levels of relatedness (rxy) for the two groups of lake
sturgeon used in this study ......................................................................... 84

Figure 13. Conceptual model showing the ways in which different aspects of lake
sturgeon life history and the demographic characteristics of this population will effect the
level of inbreeding in this population. Factors such as population decline, increasing
recruitment of hatchery individuals and the nature of the lake sturgeon mating system
will all cause levels of inbreeding to increase more rapidly and subsequently cause
population ﬁtness to become reduced more rapidly ............................................ 85

vii

Introduction

The lake sturgeon, Acipenserfulvescens, is found in three major drainages
throughout North America; The Great Lakes, Hudson Bay, and the Mississippi River.
This distribution spans 18 states and 5 Canadian provinces (Houston 1987). Lake
sturgeon are members of the order Acipenseriformes, which includes 25 species of
sturgeon and two species of paddleﬁsh (Birstein and DeSalle 1998). Species in the order
Acipenseriformes are often referred to as “living fossils” because they have existed since
the Upper Cretaceous period, over one hundred million years ago. Modern day
Acipenseriformes still posses many primitive characteristics, including a cartilaginous
skeleton, a heterocercal tail, and ﬁve rows of bony scutes along their body (Auer 1999a,
Birstein 1993). Acipenseriformes also posses the unique genetic characteristic of
polyploidy and have chromosome numbers of 4n, 8n, or l6n (Blacklidge and Bidwell,
1993)

Lake sturgeon have a life history strategy that is common among members of the
genus Acipenser. Lake sturgeon are mostly potamodromous ﬁshes and spend most of
their lives in fresh waters between 4 and 9 meters deep (Houston 1987). In the spring,
lake sturgeon migrate up large river systems where they spawn in groups in areas
characterized by fast moving water and rocky substrate (Auer 1999a, Beamesderfer and
Farr 1997). Lake sturgeon do not construct any type of nest for egg deposition. Lake
sturgeon spawn once water temperatures are between 10 and 20° C (Auer 1996,
Kempinger 1988, Houston 1987). After the female releases her eggs and they are

fertilized by the male, the adhesive eggs stick to rocks and gravel in the river, where they

incubate for 5 to 14 days (Kempinger 1988, Houston 1987). Lake sturgeon typically
leave spawning grounds after spawning and make no parental investment in their
offspring. After hatching, larval sturgeon remain in the river for 1 to 3 weeks before
drifting downstream to larger bodies of water (Auer 1999a, Kempinger 1988).

During the ﬁrst year of life, lake sturgeon experience rapid growth. In subsequent
years, however, growth slows considerably (Harkness and Dymond 1961). Although lake
sturgeon have a slow growth rate, they typically grow to sizes between 1 and 2 m and
weights of S to 40 kg. Lake sturgeon reach sexual maturity in ﬁfteen to twenty-ﬁve years;
longer than any other freshwater ﬁsh (Auer 1999a, Houston 1987). Males generally reach
sexual maturity before females (Threader and Brousseau 1986). Once mature, lake
sturgeon do not spawn every year, males may spawn every 1 to 4 years and females may
spawn every 3 to 7 years (Auer 1999b, Bearnish et a1. 1996). Both growth rate and age at
maturity seem to be inversely related to latitude, which implies a direct relation to
temperature (Bearnish et a1. 1996). Lake sturgeon are one of the longest-lived freshwater
ﬁsh, often reaching ages of over 100 years (Auer 1999a, Houston 1987).

Historically lake sturgeon were abundant throughout their native range including
the Great Lakes. In the late 18808 lake sturgeon were among the ﬁve most abundant
commercial ﬁshes in the Great Lakes with catches in the millions of kilograms annually
(Auer 1999a). While their longevity has historically buffered lake sturgeon and other
Acipenseriformes to changes in their environment, in recent years life history
characteristics such as delayed maturity, periodicity of spawning and low survivorship of
offspring have made lake sturgeon extremely vulnerable to increasing anthropogenically-

mediated environmental changes such as increases in pollution and loss of spawning

habitat (Beamesderfer and Farr 1997, Houston 1987). Auer (1999a) identiﬁed the three
greatest threats to sturgeon populations worldwide as: 1) physical impacts on spawning
and nursery habitat, 2) barriers to migration, and 3) effects of ﬁshing. Lake Sturgeon are
vulnerable to predation for only a short period of time during the ﬁrst year of life before
their scutes have fully formed (Houston 1987). During this time juvenile lake sturgeon
may spend much of their time in rivers or near river mouths aﬁer migration. These areas
often contain high levels of pollutants from upstream industrial activities. Other
environmental stresses, such as water level manipulation by hydroelectric facilities, can
also be harmful to juvenile lake sturgeon. In the late 18005 and early 19005 many dams
were built on Great Lakes tributaries that prevented lake sturgeon from accessing historic
spawning grounds. Erosion due to deforestation and other anthropogenic activities has
further degraded many historic lake sturgeon spawning grounds (Auer 1999a, Houston
1987)

Overﬁshing has been the greatest cause for decline not only for lake sturgeon, but
for all species of sturgeon worldwide (Birstein et a1. 1997). Prior to the late 18005, lake
sturgeon were largely viewed as a nuisance species because their sharp scutes would
often damage or destroy ﬁshing nets (Auer 1999a, Hay-Chmielewski and Whelan 1997).
However, around 1860 the value of smoked sturgeon ﬂesh was realized and the annual
harvest in the Great Lakes was over a million kilograms before the turn of the century.
Sturgeon are also valued for their eggs (caviar) and increased demand for caviar has also
contributed signiﬁcantly to their decline (Houston 1987). Due to drastic declines in lake
sturgeon populations throughout their historic range, commercial ﬁshing for lake

sturgeon has been closed in the United States but still exists in some areas in Canada

(Auer 1999a). Some recreational ﬁsheries still exist for lake sturgeon in areas with
relatively large, self-sustaining populations (Bruch 1999, Thuemler 1997).

Today lake sturgeon populations are believed to be at less than 1% of their former
size and very few remnant spawning populations exist in the Great Lakes (Hay-
Chmielewski and Whelan 1997). Currently the lake sturgeon is classiﬁed as either
endangered, threatened, or a species of “special concern” in all of its 18 native states
(Auer 1996) and it is considered a species of regional concern by the United States Fish
and Wildlife Service (USFWS). Because of the threatened status of the lake sturgeon, the
Michigan Department of Natural Resources (MDNR) has developed a lake sturgeon
rehabilitation strategy (Hay-Chmielewski and Whelan 1997) which lists its goal as “To
conserve and rehabilitate self-sustaining populations of lake sturgeon to a level that will
permit delisting as a threatened species under the Michigan Endangered Species Act.”
Several other states including Wisconsin, Ohio, and New York have developed similar
rehabilitation plans for lake sturgeon.

Plans to help rehabilitate lake sturgeon populations in the Great Lakes should be
based on an understanding of the species biology and life history. Due to low population
numbers, delayed maturity, long life span, and infrequent spawning, little information is
known about many aspects of lake sturgeon life history that are critical to the
development of effective rehabilitation plans. Genetic information represents one source
of data that can be used to help elucidate many of the unknown aspects of lake sturgeon
biology and life history. Genetic markers have become increasingly popular for use in
ﬁsheries management. Wirgin and Waldman (1994) cite several examples of the utility of

genetic data in ﬁsheries management including, stock identiﬁcation, taxonomic

investigation, identiﬁcation of hybrids, discrimination of wild and hatchery reared ﬁsh,
restoration of extinct populations, and forensic analysis.

My thesis utilizes genetic markers to assess different demographic and life history
traits that are important to the conservation and management of remnant lake sturgeon
populations in the Great Lakes. The ﬁrst chapter of my thesis focuses on the use of
microsatellite and mitochondrial DNA markers to assess the level of genetic diversity and
population structuring among remnant lake sturgeon populations in the upper Great
Lakes basin and the historical, ecological and anthropogenic forces inﬂuencing levels of
population structure. Based on this information, decisions regarding stocking can be
made which will maintain current levels of population structure and conserve genetic
diversity of these remnant stocks.

The second chapter of my thesis examines lake sturgeon reproductive ecology
based on genetic determination of parentage. Using microsatellite markers, parentage is
determined for a group of juvenile lake sturgeon from the Black River in Michigan.
Using this data, estimates of the number of mates per male and female and male and
female variance in reproductive success can be made. Based on this behavioral data, an
estimate of effective numbers of breeders is calculated as a means to infer the
conservation status of this declining population.

The ﬁnal chapter of my thesis examines the effects of hatchery supplementation
of declining lake sturgeon populations. Using genetic markers 1 assess the levels of
relatedness (rxy: Queller and Goodnight 1989) in two groups of lake sturgeon. The ﬁrst
group was composed of ﬁsh that spawned naturally in Black Lake, MI during the spring

of 2001. The second group was composed of individuals from supportive breeding efforts

in which very few adults were spawned to produce large numbers of offspring. Using
data on levels of relatedness, along with demographic data on the Black Lake lake
sturgeon population and the lake sturgeon mating system, I identify the factors that will
lead to an increase in the amount of inbreeding in this population and the effects that

increased inbreeding will likely have on the long-term viability of this population.

CHAPTER I

Genetic Population Structure of Remnant Lake Sturgeon Populations
In the Upper Great Lakes Basin

Introduction

Prior to the 19005 lake sturgeon were abundant throughout all of the Great Lakes.
Historic numbers of lake sturgeon were estimated to be in the hundreds of thousands in
the Great Lakes prior to the 18805 and tens of thousands more in inland lakes and rivers
in Canada alone (Houston 1987). In the early 18805, increased demand for sturgeon ﬂesh
and caviar led to the development of a commercial ﬁshery for lake sturgeon in the Great
Lakes. Lake sturgeon quickly became one of the ﬁve most abundant commercial species
in the Great Lakes by the late 18805 with harvests totaling in the millions of kilograms
annually (Auer 1999a).

By the early 19005, however, the lake sturgeon ﬁshery rapidly declined as a result
of over-exploitation (Auer 1999a). By 1929 most Great Lakes states had closed their lake
sturgeon ﬁsheries. Today commercial ﬁsheries for lake sturgeon only exist in Canadian
waters (Auer 1999a). Overﬁshing, along with the construction of dams that blocked
access to historic spawning grounds, and the loss of suitable spawning habitat due to
pollution and sedimentation have led to continual and drastic declines in lake sturgeon
population numbers throughout the Great Lakes (Auer 1999a, Houston 1987, Harkness
and Dymond 1961). Today, lake sturgeon numbers in the Great Lakes are thought to be
less than 1% of historic ntunbers (Hay-Chmielewski and Whelan 1997).

Because of this dramatic decline in population numbers, lake sturgeon have

become a species of conservation concern throughout their native range. Many states

have drafted management strategies for lake sturgeon that list stocking of hatchery-reared
individuals as a primary means for rehabilitation (WDNR 2000, Hay-Chmielewski and
Whelan 1997). Sound management plans for remnant populations and plans to repOpulate
extirpated populations should be based on a ﬁmdamental understanding of the population
structure of remnant lake sturgeon populations.

Most organisms exhibit some degree of genetic differentiation between
populations. Genetic structuring can often be partitioned hierarchically across macro and
micro-geographic scales. For example, populations within the Great Lakes may be
structured by lake basin, by river drainage or even by tributaries within river drainages.
Genetic population structure of lake sturgeon in the Great Lakes is likely to be inﬂuenced
by historical, ecological, and anthropogenic factors.

The most signiﬁcant historical process that inﬂuenced the population structure of
native ﬁsh species in the Great Lakes was the Wisconsin Glaciation. During the
Wisconsin ice age, the Great Lakes were covered by ice until approximately 15,000 years
before the present time (Mandrak and Crossman 1992, Underhill 1986). Dming this time,
Great Lakes ﬁsh species either persisted in low numbers in glacial refugia or became
extirpated (Underhill 1986). Species that persisted in glacial refugia were likely to have
reduced population sizes as well as reduced levels of genetic diversity (Bematchez and
Wilson 1998). As glacial ice sheets receded, species that persisted in glacial refugia were
able to recolonize the Great Lakes via a series of proglacial lakes and their associated
drainages (Mandrak and Crossman 1992, Underhill 1986). Several different
recolonization routes were utilized by ﬁsh species, and this in turn helped to shape

present day population genetic structure (Mandrak and Crossman 1992). In a study on

ﬁsh species that re-colonized previously glaciated areas, Bematchez and Wilson (1998)
found that past glacial events have had a signiﬁcant inﬂuence on levels of genetic
diversity and population structuring in various species of Great Lakes ﬁshes.

Population structure of lake sturgeon in the Great Lakes is also likely to be
signiﬁcantly inﬂuenced by ecological and evolutionary forces. Gene ﬂow is one
evolutionary force that can inﬂuence population structure. Lake sturgeon are capable of
migrating large distances (Auer 1999b). Typically, populations with high dispersal rates
show reduced levels of population structure due to an increase in migration of individuals
among populations (Stabile et a]. 1996). Species that exhibit natal philopatry should show
a higher degree of spatial population structure. While tag return data on lake sturgeon are
limited due to infrequency of spawning, data suggest that lake sturgeon do exhibit some
degree of natal philopatry, where the same individuals are repeatedly captured spawning
in the same stream (Auer 1999b, R. Elliott, USFWS, personal communication). Other
species of sturgeon have also been found to exhibit some degree of natal phiolopatry
based on tag return data (Smith et al. 2002, Stabile et al. 1996). Given the broad
geographic range occupied by lake sturgeon (Houston 1987), presumably populations
further apart geographically should show a greater degree of divergence in allele
frequency from one another than populations in close geographic proximity.

Anthropogenic factors are likely to have affected the degree to which remnant
lake sturgeon populations are genetically structured as well. Lake sturgeon population
numbers have declined substantially over the past 100 years (approximately 5 sturgeon
generations) largely due to anthropogenic forces (Auer 1999a, Hay-Chmielewski and

Whelan 1997). Organisms that have experienced drastic reductions in population size are

likely to show a strong degree of population structure due to an increase in genetic drift
(Avise 1994). While current population sizes are unknown for most populations of lake
sturgeon in the Great Lakes, it is believed that the majority of the remnant populations in
the Great Lakes are relatively small (i.e. less than 200 spawning adults annually) (Elliott
2003, Holey et al. 2000).

Previous lake sturgeon genetics studies have primarily utilized mitochondrial
DNA. These studies found levels of genetic variation to be low in lake sturgeon
populations across a broad geographic range (Ferguson and Duckworth 1997, Ferguson et
a1. 1993). These studies concluded that highly variable nuclear DNA markers would be
needed to detect and adequately characterize levels of genetic variation within
populations and to detect structuring among lake sturgeon populations. Recently,
polymorphic microsatellite markers have been developed for lake sturgeon that are
effective tools for assessing population structure (Welsh et a1. 2003, McQuown et a1.
2002, McQuown et a1. 2000, May et a1. 1997). King et al. (2001) found that signiﬁcant
differences in allele frequencies existed among Atlantic sturgeon (Acipenser oxyrinchus
oxyrinchus) populations based on microsatellite DNA data. Other studies have also
demonstrated the efficacy of using mitochondrial DNA markers, primarily direct mtDNA
sequencing, as a means of inferring population structure in shortnose (Acipenser
brevirostrum), Atlantic and Gulf of Mexico sturgeon (Acipenser oxyrinchus desotoi)
(Grunwald et a1. 2002, Waldman et a1. 2002, Stabile et al. 1996). Brown et a1. (1996) and
Ludwig et a1. (2000) have also identiﬁed an 82 base pair repeated region in the lake
sturgeon mtDNA control region that could also be used to assay for population-level

genetic differences.

10

Given the magnitude and duration over which historical, ecological and
anthropogenic forces that have affected lake sturgeon populations, it is likely that lake
sturgeon exhibit some degree of genetic population structure. The objective of this study,
therefore, was to use genetic markers to assess the amount of genetic variability and to
examine the degree of population structure among remnant populations of lake sturgeon
in the Upper Great Lakes basin. Through the use of new and more polymorphic genetic
markers, this study is likely to be able to document genetic variability on a ﬁner scale
than previous studies and to answer previously unknown questions regarding population
structuring of Great Lakes lake sturgeon populations. This information is critical for the
development of sound management plans and effective restoration strategies for

extirpated populations and remnant lake sturgeon populations across the Great Lakes.

Methods

Sample Collection

Lake sturgeon were captured during spring spawning migrations in 10 different
Great Lakes tributaries (Figure 1) during 1999, 2000, 2001 and 2002. Two of the
populations sampled, Black Lake (MI) and the Wolf River (WI), are presently isolated
due to the construction of dams that prevent out-migration to the Great Lakes. Sturgeon
were captured using large dip nets, gill nets, setlines, electroﬁshing and trawls. F in clips
were taken from the caudal or dorsal ﬁn from all lake sturgeon captured. Tissue samples

were preserved in either tissue storage buffer (4M Urea, 0.2M NaCl, 0.1M Tris-HCl, 5%

ll

Sarcosine, IOmM EDTA) at -70° C or dried and placed in scale envelopes and stored at

ambient temperatures. The number of samples per locale ranged from 9 to 111 (Table 1).

Laboratory Analyses

Two classes of genetic markers were utilized, microsatellite and mitochondrial
DNA. Microsatellites are bi-parentally inherited regions of non-coding nuclear DNA that
contain short (2-5 base pairs) repeated motifs (i.e. (GATA),.) and are generally
characterized by a high degree of polymorphism (Scribner et al. 1996). Microsatellites
offer many advantages over other genetic markers including a greater level of variation
and the ability to sample using non-lethal and non-invasive techniques. Further, they can
often be used in different species of similar taxa (Welsh et al. 2003, King et al. 2001,
McQuown et al. 2000, May et al. 1997, Scribner et al. 1996). Microsatellites have been
widely employed in population level studies of several organisms and have shown to be
an effective tool for determining population structure for many species of ﬁshes,
including sturgeons (Nielsen and Sage 2002, King et al. 2001, Scribner et al. 1996).

The other class of genetic markers used was mitochondrial DNA (mtDNA).
Mitochondrial DNA is a closed circular molecule that is inherited exclusively maternally
(Brown 1985). The most variable region of the mtDNA molecule is the control region or
D-Loop. This region serves as the origin of mtDNA replication and variations are usually
comprised of base pair substitutions (Brown 1985). Mitochondrial DNA has also been
shown to be a useful tool for identifying population structure in ﬁsh species including

sturgeons (Grunwald et al. 2002, Stabile et al. 1996).

12

Total DNA was extracted from ﬁn clips using either the Puregene® (Gentra
Systems) or DNeasy® (QIAGEN Inc.) protocols. Final re-suspensions were in SOul TE
buffer for Puregene extractions and 50ul AE buffer for QIAGEN extractions. DNA was
quantiﬁed using ﬂuorometry. For microsatellite analysis, DNA was ampliﬁed at 8 loci,
LS-68 (May et al. 1997), Aﬁ468b (McQuown et al. 2002), Sp1120(McQuown et al. 2000),
on27 (King et al. 2001) Aqu9, Aqu63, Aqu 74 and AquI 12 (Welsh et al. 2003).
Microsatellite Polymerase Chain Reactions (PCR) were conducted in 25111 volumes and
consisted of 100mg template DNA, 10X PCR2 Buffer (1M Tris-HCl, 1M MgClz, 1M
KCl, 10% Gelatin, 10% NP-40, 10% Triton-X), 2mM of each dNTP, 10pmol of
ﬂuorescently labeled forward and reverse primer, and 0.3 pl Taq polymerase. PCR
reactions were conducted using Stratagene Robocycler® 96 (Stratagene Inc.) and Perkin
Elmer 9600 therrnocyclers. PCR conditions were as follows: 94° C for 2 minutes,
followed by 30 cycles of 94° C for 1 minute, 1 minute for primer speciﬁc annealing
temperature, and 72° C for 1 minute, and ﬁnally a 2.5 minute ﬁnal extension at 72° C.
Microsatellite PCR products were run on 6% denaturing polyacrylamide gels and
visualized using the Hitachi F MBIO® II scanner (Hitachi Corp) and associated software.
Allele sizes were determined using a commercially available size standard
(MapMarkerTM, BioVentures Inc.) and using standard samples of known genotype.

Size polymorphisms due to an 82 base pair repeat in the control region as
described in Ludwig et al. (2000) was also assayed for the examination of population
structure. For this assay the, primer tPr0123 (Brown 1996) was used and a new primer,
SturngRl (5’ CAT AGA GAT AAT GGT GTG GAT 3’), was developed that ﬂanked

this repeated region. This primer was developed using lake sturgeon mtDNA control

13

region sequence available from the National Center for Biotechnology Information
website (accession # U32309). PCR reactions for this primer were conducted in 50 ul
volumes and consisted of 100ng template DNA, 10X Kocher buffer (1M Tris, 1M MgClz,
10% Tween 20), IOmM dNTPs, 50pmol tPr0123, 50pmol SturngRl, and 0.5ul Taq
polymerase. PCR conditions were as follows: 94° C for 2 minutes, followed by 40 cycles
of 94° C for 30 seconds, 50° C for 30 seconds and 72° C for 1 minute, and a ﬁnal
extension cycle of 72° C for 5 minutes. PCR reactions were conducted in Robocycler®
96 therrnocyclers (Stratagene Inc.). PCR products were then run on 1% agarose gels with
Sul of ladder. Gels were stained with ethidium bromide and visualized using UV trans-
illumination. The number of repeated units was counted and recorded and each individual

was checked for heteroplasmy (presence of multiple mitochondrial genomes).

Statistical Analyses

Estimates of microsatellite allele frequencies and tests for Hardy-Weinberg
equilibrium were calculated using the program Fstat v. 2.9.3 (Goudet 2001). Tests for
genotypic disequilibrium for each pair of loci were also calculated using F stat. F stat was
also used to calculate Weir and Cockerham’s (1984) F -statistics and pair-wise F S, values.
P-values associated with pair-wise Fst values were adjusted for multiple comparisons
using a Bonferroni correction (Rice 1989). Estimates of genetic variability including
mean number of alleles per locus, and observed and expected heterozygosities were
calculated for each population using the program BIOSYS-l (Swofford and Selander

1981). A neighbor-joining tree based on Cavalli-Sforza and Edwards chord distance over

14

2000 bootstrapping replicates was produced using the program “NJ BP” (Comuet,
unpublished).

Hierarchical F -statistics were calculated using the program GDA (Lewis and
Zaykin 2001) to determine how variation was partitioned. Populations were grouped
according to their basin of origin (Lake Michigan, Lake Superior, or Lake Huron).
Estimates of F is, F it, 05 (variance among populations within basins) and 0p (variance
among basins) were generated for all loci. Conﬁdence intervals for Fig, Fit, 0, and 0', were
generated based on 1000 bootstrapping replicates over the 8 loci used.

The program Contribute (Petit et al. 1998) was used to calculate allelic richness to
adjust estimates of allelic diversity for population differences in sample size. The total
contribution each population made to the overall gene diversity (CT) was also estimated
using Contribute. Each population’s contribution to the overall diversity was partitioned
into two components; C,, the contribution of that population to the overall diversity based
on its own diversity, and Cd, the contribution of a population to the overall diversity
based on differentiation from other populations.

Analysis of molecular variance for mtDNA was performed to calculate (1)-
statistics using the program AMOVA (Excofﬁer et al. 1992). Hierarchical analyses of
molecular variance were performed by grouping populations together based on their
basin of origin (Lake Michigan, Lake Superior, Lake Huron) in order to estimate (1),,
(variance within populations), (D5, (variance among populations within basins) and (Dc,
(variance among basins). Incidences of heteroplasmy (multiple mtDNA genomes) were
documented for each population. In incidences of heteroplasmy, individuals were

assigned to the haplotype that appeared more intense when viewed on agarose gels.

15

Individuals that exhibited equal intensity of more than one haplotype were omitted from

the analysis.

Results

Levels of Genetic Variation Within Populations

Allelic richness for the 10 populations in this study ranged from 2.3 for the
Manistee River to 2.7 for the St. Clair River. Observed heterozygosity across the 10
populations ranged from 0.541 for the Manistee River to 0.681 for the Oconto River
(Table 1). All populations except the Black River and the Manistee River were in Hardy
Weinberg equilibrium. These two populations deviated from Hardy-Weinberg
equilibrium at a single locus (LS-68). Tests for genotypic disequilibrium showed that
none of the 8 loci used in this study were linked.

F our different size polymorphisms were observed in the mtDNA control region as
described by Ludwig et al (2000). Polymorphisms reﬂected variable numbers of an 82
base pair repeated segment. Individuals were assigned to haplotypes based on the number
of repeated units they possessed. The Menominee River population was the only
population that exhibited only 1 haplotype. The Wolf River population had a relatively
high frequency of haplotype 2 (frequency = 0.680). The two populations from Lake
Superior, the Bad and Sturgeon Rivers, exhibited high frequencies of haplotypes l and 3,
however, haplotype 2 was not observed in any individuals from Lake Superior and
haplotype 4 was found at a relatively low frequency in the Bad river (0.129) and was not

found in any individuals in the Sturgeon River (Appendix 1).

16

Heteroplasmy was observed in 11.9% (35 out of 293) of all the individuals
assayed for the mtDNA size polymophism. The frequency of heteroplasmic individuals
was relatively low (<0.10) in all but three of the populations. Two of these populations,
the Bad and the Sturgeon River, are located in Lake Superior. The other population with
a relatively high frequency of heteroplasmy was the isolated Black Lake population.
Heteroplasmy was not observed in any individuals in the Menominee River or the Oconto
River populations.

Not all populations contributed equally to the overall gene diversity (Figure 2).
The populations from lake Michigan contributed less than average to the total diversity
(indicated by negative CT values) and the populations from Lakes Superior and Huron
contributed the most to the total diversity. High relative contributions from populations in
the Lake Superior Basin were based primarily on these populations differentiation from
the other populations. In the populations from Lake Huron high contributions to total

gene diversity are based mostly on the diversity within these populations.

Levels of Genetic Differentiation Among Populations

Mean values for F“, F is, and F5: for all populations over all loci were 0.114, 0.059,
and 0.059 respectively. All values were found to be statistically signiﬁcant (P < 0.05).
The majority of the pair-wise F 5, values were found to be statistically signiﬁcant (Table
2). The exceptions were the pair-wise F5, values between the Oconto River and the other
Green Bay tributaries to Lake Michigan (Menominee, Peshtigo, Fox, and Wolf Rivers)

and the pair-wise F5, between the Fox and Wolf Rivers.

17

Mean (1)5, (variance among populations) was estimated to be 0.201 (P<0.001).

Pairwise estimates of (D5, (Table 2) showed that the Menominee River exhibited a

relatively high degree of variance from all of the other populations in the study. The Wolf
River also showed a high degree of variance from all other populations in the study
except for the Fox River. Similar to microsatellite analyses, a low degree of variance was
found between the Oconto River and the rest of the Green Bay tributaries (Fox, Peshtigo,
Wolf) except for the Menominee River.

The hierarchical analysis of variance for microsatellite DNA showed that more

variance exists among populations within basins (mean 05 = 0.065; P<0.05) than among

basins (mean 0p = 0.021; P<0.05). Hierarchical analysis of mtDNA variance showed
similar results. The majority of the mtDNA variance, 80.45% was partitioned within
populations, with 21.38% of the variance being partitioned among populations (<1)SC =

0.201, P<0.001). Variance partitioned among basins was negligible (Table 3).

The relationships among populations are shown in a neighbor-j oining tree that
shows three distinct population assemblages (Figure 3). One assemblage consists of the
Bad and Sturgeon Rivers from Lake Superior. Strong genetic divergence was observed
between the other two population assemblages (84% bootstrap support; Figure 3). One of
these assemblages consisted of the populations from the Green Bay Basin in western
Lake Michigan (the Menominee, Peshtigo, Oconto, Fox, and Wolf Rivers). The other
population assemblage consisted of populations from eastern Lake Michigan (Manistee

River) and Lake Huron (Black Lake and St. Clair River).

18

Discussion

Lake sturgeon in the Great Lakes have been subject to historical, ecological, and
anthropogenic forces which have inﬂuenced the amount of genetic variation present
within populations as well as the degree of genetic structuring among remnant
populations. Previous population level genetic studies focused exclusively on
mitochondrial DNA and revealed that lake sturgeon exhibited low levels of genetic
diversity (Ferguson and Duckworth 1997, Ferguson et al.1993). By focusing on more
polymorphic regions of the mtDNA genome as well as highly variable microsatellite
markers, this study addresses previously unanswered questions regarding levels of
genetic variation and the degree of genetic population structure. The results of this study
indicate that lake sturgeon exhibit higher levels of genetic variability than previously
detected and that lake sturgeon populations in the Upper Great Lakes Basin are spatially

genetically structured.

Description of Population Structure

Signiﬁcant levels of overall F st (0.059) and (1),, (0.210), as well as signiﬁcant pair-

wise levels of F st and (1),, values (Table 2) provide evidence that remnant lake sturgeon

populations in the upper Great Lakes are genetically structured. The neighbor- joining
tree indicates that populations from the same basin are more similar to one another than
to populations from other basins. This tree shows three distinct population assemblages

corresponding to Lake Superior (Bad and Sturgeon Rivers), tributaries from the Green

19

Bay basin (Fox, Oconto, Peshtigo, Menominee, and Wolf Rivers) and populations from
eastern Lake Michigan (Manistee) and Lake Huron (Black and St.Clair).

The two hierarchical analyses of variance based on microsatellite DNA and
mitochondrial DNA showed similar results as to how genetic variability is partitioned

(Table 3). The microsatellite DNA data showed that there is more variance among
populations within basins than there is among basins (0S = 0.065 vs. 0p = 0.021).
Similarly the mtDNA data shows that there is a higher variance among populations
within basins than there is among basins (<1)SC = 0.210 vs. (Dc, = 0.0). While variance does

exist at multiple levels, both microsatellite and mtDNA analyses show that the majority
of this variance is partitioned among populations within basins. Data indicating
signiﬁcant levels of population structure along with data on partitioning of variance
suggest that remnant lake sturgeon populations in the Great Lakes do not exist as a single
mixed population.

The pair-wise F5, values for the Oconto River and the other tributaries from the
Green Bay Basin (The Fox, Wolf, Peshtigo, and Menominee Rivers) were not found to be
signiﬁcant. This is likely due to the small size of this population and the subsequent small
sample size (n = 9) from this population used in this study. Since the Oconto River
represents the smallest remnant population in this study and one of the smaller
populations in the Green Bay Basin (Elliott 2003, Holey et al. 2000), it is conceivable
that individuals that may have historically spawned in the Oconto River are searching for
more favorable spawning opportunities in other nearby river systems.

The pair-wise F5, between the Fox and Wolf Rivers was also not found to be

signiﬁcant. In addition, the pair-wise (Dst for the Fox and Wolf Rivers was relatively low.

20

Although lake sturgeon were once abundant in the Fox River, until recently their numbers
during spawning periods had been low (Cochran 1995). In past years, however, lake
sturgeon numbers in the Fox River have been increasing with over 50 adults seen in
spawning areas in past years. Approximately 5% of the adult lake sturgeon captured in
the Fox River since 2000 have been tagged previously in the Lake Winnebago/Wolf
River system (R. Elliott, USFWS, personal communication). While it is not likely that
ﬁsh can ascend the Fox River to Lake Winnebago and the Wolf River due to the presence
of multiple dams, ﬁsh from Lake Winnebago and the Wolf River do seem to be traveling

downstream over dams. This downstream movement of ﬁsh is a likely explanation for the
low levels of pair-wise F 5, and (1),, observed between these two populations.

The results of this study are similar to studies on other species of sturgeon that
demonstrate a signiﬁcant amount of population structure. To date, one other study has
employed nuclear microsatellite markers to examine population structure in a sturgeon
species. King et al. (2001) observed a signiﬁcant degree of population structure in
Atlantic sturgeon based upon microsatellite markers. Pair-wise values of F5, for Atlantic
sturgeon were found to range from 0.023 to 0.278 (compared to 0.0 to 0.146 in this
study). Similar to lake sturgeon, this study found regional genetic differences to exist
between populations of Atlantic sturgeon. Several studies using mitochondrial DNA
markers have also shown sturgeon populations to be signiﬁcantly genetically structured
(Waldman et al. 2002, Grunwald et al. 2002, Stabile et al. 1996). While the degree of
genetic population structure ranged from species to species, mtDNA markers documented
a signiﬁcant degree of regional genetic differences in shortnose, Atlantic, and Gulf of

Mexico sturgeon.

21

One interesting result of this study was the observation of heteroplasmy (the
presence of multiple mitochondrial DNA genomes) in the mtDNA control region.
Heteroplasmy was observed in 11.9% of the individuals that were assayed for mtDNA
size polymorphism. This represents a much higher incidence of heteroplasmy than
observed in previous studies, Brown et al. (1996) found no incidence of heteroplasmy in
21 lake sturgeon and Ludwig et al (2000) found heteroplasmy in only 0.064% of the lake
sturgeon they assayed. Heteroplasmy has been observed in other species of sturgeon as
well. Grunwald et al. (2002) observed heteroplasmy in approximately 1/3 of the
shortnose sturgeon assayed and Miracle and Campton (1995) observed heteroplasmy in
18.5% of Gulf of Mexico sturgeon they assayed. The ability to detect heteroplasmy is
dependant on the ability to visually detect bands of low copy numbers of DNA. Because

of this, heteroplasmy may be underestimated in this study.

Factors Inﬂuencing Population Structure

Past Glacial events have been found to inﬂuence population structure in many
species of ﬁshes (Bematchez and Wilson 1998). The northern range of lake sturgeon was
extensively glaciated during the Wisconsin Ice Age and therefore populations in this
study must have originated from glacial refugia (Mandrak and Crossman 1992, Underhill
1986). Previous studies have suggested that lake sturgeon persisted in multiple refugia
with one glacial refugium re-colonizing the Great Lakes and another re-colonizing the
Hudson/James Bay area (F ergeson and Duckworth 1997).

The results of this study seem to support a multiple refuge hypothesis as well.

These results indicate the possibility that multiple refugia contributed to populations

22

within the Great Lakes. A fundamental divide in the neighbor-joining tree suggests that
the populations in Lake Superior are greatly different from the other populations sampled

in the Great Lakes. This differentiation is further supported by pair-wise F 5, values, pair-
wise ¢st values, and the fact that Lake Superior populations exhibit a high degree of gene

diversity based on differentiation from other populations. Populations in Lake Superior
were also found to exhibit a high degree of heteroplasmy compared to other populations
in the Great Lakes and one of the mtDNA haplotypes, haplotype 2, which was found in
other Great Lakes populations was not observed in either of the Lake Superior
populations. These data suggest that populations in Lake superior have been isolated
considerably longer than other Great Lakes populations, possibly the result of these
populations originating from a different glacial refugium than other p0pulations in the
Great Lakes.

Levels of contemporary gene ﬂow are also likely to inﬂuence genetic population
structure. Species that are highly migratory typically have high levels of gene ﬂow
between populations that in turn leads to a reduction in differences between populations
and a reduction in population structure (Stabile et al. 1996, Chakraborty and Leimar
1987). While Lake sturgeon migrate large distances in non-spawning periods (Auer
1999b), adults appear to exhibit a high degree of natal philopatry given the high degree of
population structure observed. This hypothesis is further substantiated by tag return data
that has yet to ﬁnd adults straying between populations during spawning periods (R.
Elliott, USFWS, personal communication). High levels of population structure have been
attributed to a high level of natal philopatry in other sturgeon species as well (Waldman

et al. 2002, Grunwald et al. 2002, King et al. 2001, Stabile et al. 1996).

23

Anthropogenic forces have also signiﬁcantly inﬂuenced the genetic structure of
remnant lake sturgeon populations in the Great Lakes. Populations that have experienced
signiﬁcant declines and a subsequent decrease in effective population size are likely to
experience an increase in genetic drift (Lynch 1996). As genetic drift increases, the
genetic differences between populations will become accentuated and as a result the
degree of population structure will increase (Avise 1994). Lake sturgeon populations in
the Great Lakes have been drastically reduced as a result of anthropogenic forces over the
past 100 years (Hay-Chmielewski and Whelan 1997). Currently the majority of
populations in the Great Lakes, including the majority of the populations in this study,
have annual spawning runs of 200 or fewer adult ﬁsh (Elliott 2003, Holey et al. 2000).

Mitochondrial DNA data provides evidence of genetic drift in lake sturgeon
populations in the Great Lakes. Because of its rapid rate of evolution, mtDNA can often
provide a better view of the degree of genetic differences between populations than
nuclear markers (Ferris and Berg 1987). Because of its maternal mode of inheritance
mitochondrial DNA experiences approximately 4 times higher levels of genetic drift than
would be expected for nuclear DNA (Avise 1994). The Menominee River represents one
population that seems to be experiencing high levels of genetic driﬁ. Only 1 mtDNA
haplotype (haplotype 1) was observed in the Menominee River, whereas 4 haplotypes
were observed in only 9 individuals sampled in the nearby Oconto River. The
Menominee River is one of the few populations that remains subject to harvest. The
exploitation rate in the un-impounded section of the river was found to be 18.7%, which
is higher than any other section of the river that contains sturgeon (Thuemler 1997).

Spawning habitat for sturgeon in the Menominee River returning from Green Bay and

24

Lake Michigan is also signiﬁcantly reduced due to the construction of a dam 3.9km
upstream from the mouth of the Menominee River. It is likely that the combination of
exploitation and habitat loss in this system have accelerated the process of genetic drift
and subsequently reduced the effective population size. This population provides an
example of the relatively short time period required for anthropogenic effects to cause an
increase in levels of genetic drift and subsequently cause lake sturgeon populations to

become highly differentiated.

Conclusion

Remnant lake sturgeon populations in the upper Great Lakes exhibit a high degree
of population structure. This structure appears to be inﬂuenced by historical, ecological,
and anthropogenic factors. Past glacial events and a strong degree of natal philopatry
have led to signiﬁcant genetic differences among populations within basins. The dramatic
reduction in lake sturgeon population size in the Great Lakes over the past 100 years has
further accentuated these differences through the process of genetic drift. These results
have important implications for conservation efforts. Based on the fact that signiﬁcant
genetic differences do exist between remnant lake sturgeon populations in the upper
Great Lakes, lake sturgeon in the Great Lakes should not be managed as a single
population. Furthermore, genetic differences between populations should be considered

when utilizing hatcheries as a means to rehabilitate declining and extirpated populations.

25

CHAPTER II

Lake Sturgeon Reproductive Ecology and Conservation Status
Based on Genetic Determination of Parentage

Introduction

Fishes have evolved many different reproductive strategies. These strategies have
evolved to maximize ﬁtness under a wide variety of conditions including varying
population densities and habitats (Avise et al. 2002). Fishes that reside in high densities,
including many species of sunﬁsh, have evolved reproductive strategies in which larger
bourgeois males build and guard nests from younger cuckolding males who attempt to
sneak mating opportunities (Avise et al. 2002, Mackiewicz et al. 2002, Taborsky 2001,
DeWoody et al. 1998). Certain salmonids exhibit ontogentic shifts in reproductive
strategies in which males may spawn as either freshwater parr or as fully deve10ped
adults returning from marine environments (Avise et a1. 2002, Moran et al. 1996, Gross
1991). Other reproductive strategies that have evolved include group spawning, oral
incubation, sex-role reversals, and live bearing of young (Avise et al. 2002).‘

Direct observation of ﬁsh mating behavior can be difﬁcult given that ﬁshes may
spawn in areas with high turbidity or at depths that make observation difﬁcult. Different
spawning behaviors (i.e. large group spawning, multiple extra-pair matings) may also
make observation of ﬁsh mating systems difﬁcult. Recent advances in genetic techniques
have made it possible to uncover many aspects of ﬁsh mating systems that have
previously been difﬁcult to observe (Avise et al. 2002). To date, most genetic studies of
ﬁsh mating systems involve cases where one parent is known and the other parent is

determined through the process of excluding all but one of the potential unknown parents

26

(Avise et al. 2002). Due to their highly variable nature and their Mendelian mode of
inheritance, microsatellite markers are well suited for examining mating systems in ﬁshes
(Avise et al. 2002, Mackiewicz et al. 2002, DeWoody et al. 2000, DeWoody et al. 1998,
Colboume et al. 1996, Queller et al. 1993). Highly variable microsatellite markers allow
for exclusion of potential parents when the genotypes of potential parents are inconsistent
with offspring genotypes (Dewoody and Avise 2001).

Genetic studies of mating systems in ﬁshes can provide valuable information for
conservation efforts. As populations of many ﬁsh species continue to decline, the 'use of
genetic markers can provide data on numbers of spawning adults, skews in sex ratios,
variance in reproductive success and reductions in effective population sizes (Bekkevold
et al. 2002, Garant et al. 2001, Moran and Garcia-Vazquez 1998). As spawning habitat is
modiﬁed and/or lost, ﬁshes are likely to exhibit changes in their mating behavior
including increased hybridization (Scribner et al. 2001) and shifts in mating strategies
(Gross 1991). These changes in mating behavior may lead to a subsequent loss of genetic
diversity. Species that are unable to adapt their mating behavior to changes and
reductions in spawning habitat, and are experiencing declining population sizes and low
reproductive outputs are likely to be at an increased risk of extinction as spawning habitat
continually declines.

Information on mating systems can be used to assess conservation status
including estimation of effective population size, N. The effective population size of a
population refers to the size of an idealized population that would have the same genetic
characteristics (i.e. rate of genetic drift, increase in inbreeding, decrease in

heterozygosity) as the population under consideration (Kimura and Crow 1963). Multiple

27

methods, parameterized using different demographic and genetic variables, exist for
calculating effective population size (Frankham 1995). Three main factors that
signiﬁcantly inﬂuence effective population size are unequal sex ratios, variance in
reproductive success, and ﬂuctuations in population size (Frankham 1995). Ideally ratios
of NJN should be close to 1, however this is not typically the case. Anthropogenic
factors such as overharvest and loss of habitat can skew sex ratios and increase variance
in reproductive success which in turn decreases Ne. Populations with a low Nc are likely
to lose genetic diversity more rapidly and experience higher rates of extinction (Newman
and Pilson 1997).

Small populations with low effective population sizes are likely to be subject to
an Allee effect. The Allee effect has been deﬁned as a decrease in population growth rate
when population size becomes low (Saether et a1. 1996). Principles of the Allee-effect
can be applied to ﬁsh species and ﬁsh mating systems in multiple ways. Fishes that
exhibit group behavior such as schooling or group spawning are likely to be less
successful at defending themselves from predators when group sizes are low (Stephens
and Sutherland 1999, Saether et al. 1996). When population sizes are low, ﬁshes that
exhibit group spawning behavior may also experience difﬁculties ﬁnding mates or may
be forced to pair with poor mates, resulting in lower reproductive success and ultimately
a reduction in population growth rate (Stephens and Sutherland 1999, Saether et al.
1996)

Observational studies have shown that lake sturgeon have evolved a group
spawning system. Lake sturgeon are broadcast spawners and congregate in large groups

during spawning periods (Bruch and Binkowski 2002, Kempinger 1988). Unlike other

28

species of ﬁshes, sturgeon construct no nest, they do not protect their eggs after
deposition, and they make no parental investment in their offspring (Kempinger 1988).
The unprotected lake sturgeon eggs and juveniles are prone to high rates of predation.
Lake sturgeon have developed compensatory mechanisms to minimize predation levels
on eggs and offspring. Lake sturgeon eggs incubate rapidly (5-14 days) (LaHaye et al.
1992, Kempinger 1988, Houston 1987), which helps to reduce predation on the
unprotected eggs. Lake sturgeon are capable of producing large numbers of offspring, a
single female lake sturgeon can produce over 100,000 eggs in one spawning period
(Houston 1987). The large number of offspring produced also helps to ensure that
adequate numbers of lake sturgeon survive.

Lake sturgeon migrate up their natal river to spawn in the spring. Lake sturgeon
do not spawn annually; males may spawn every 1 to 4 years, and females may spawn
every 3 to 7 years (Auer 1999b, Houston 1987). The tendency for males to spawn more
frequently than females leads to male-biased sex ratios during spawning periods
(Kempinger 1988). Sturgeon spawning grounds are characterized as having rocky
substrate and high ﬂow rates (LaHaye et al. 1992, Harkness and Dymond 1961). Lake
sturgeon spawning is temperature dependent with spawning observed primarily between
10 and 15° C (LaHaye et al. 1992, Kempinger 1988, Houston 1987). Often, a drop in
temperature during spawning periods will cause lake sturgeon to cease spawning and
leave spawning areas until temperatures increase, at which time spawning will resume.
(Bruch and Binkowski 2002, Kempinger 1988, E. Baker, MI DNR, personal

communication).

29

Several male sturgeon have been observed to spawn with one female
simultaneously. Kempinger (1988) observed between 6 and 8 males spawning with one
female, and Bruch and Binkowski (2002) observed similarly skewed spawning sex ratios.
Prior to egg release, female lake sturgeon have been observed to swim upstream,
presumably to attract potential mates. Immediately before the female lake sturgeon
releases her eggs, males compete to gain the closest possible position to females (Bruch
and Binkowski 2002). Lake sturgeon eggs incubate for 5 to 14 days before they hatch.
Soon after hatching the larval lake sturgeon drift downstream to larger bodies of water
(LaHaye et al. 1992, Kempinger 1988, Houston 1987).

One remnant lake sturgeon population that still has relatively large annual
spawning migrations is in Black Lake, MI (Figure 4). Historically sturgeon could migrate
from Lake Huron to the Upper Black River to spawn. Following the construction of the
Alvemo Dam on the Lower Black River in 1903, sturgeon became isolated in Black Lake
and immigration from Lake Huron was cut off (Baker and Borgeson 1999). Sturgeon in
Black Lake still spawn in the Upper Black River, however, construction of the Kleber
Dam on the Upper Black River has reduced the amount of available spawning habitat.
Adult lake sturgeon in Black Lake spend relatively little time in the upper Black River
during spawning periods. Typically ﬁsh will remain in the river for a few days and leave
the river immediately following spawning. Recent population estimates suggest that there
are approximately 1,250 individuals currently in the Black Lake population, with about
550 of those being ﬁsh of reproductive age (Baker and Borgeson 1999). Comparing their

data to previous studies, these authors found evidence for a substantial reduction in

30

population size over approximately 25 years. These authors also found recruitment in this
population to be low (Baker and Borgeson 1999).

The objective of this study, therefore, was to use microsatellite markers to
determine parentage in the remnant population of lake sturgeon in Black Lake. Based on
this information, inferences can be made in regards to many aspects of the lake sturgeon
mating system about which little information is currently available, including the number
of mates each male and female is spawning with, variation in reproductive success, and
the underlying mechanisms responsible for variance in reproductive success. These data
will also be used to estimate the effective number of breeding adults. These data have
important conservation implications for this declining population as well as other

populations across the species range.

Methods

Sample Collection

Field sampling was conducted during the spring and summer of 2001. Adult lake
sturgeon were captured during spring spawning migrations in the Upper Black River in
the 11km section below the Kleber Dam. Three periods of spawning activity were
observed between April 28 and May 26 2001, in the Upper Black River. Many ﬁsh were
observed to return to Black Lake between periods of peak spawning activity. A total of
114 adult lake sturgeon (70 males and 44 females) were captured in the Upper Black

River using large dip nets. All adults were measured, weighed, and sexed based on the

31

presence of eggs or milt. A small tissue sample was taken from the upper lobe of the
caudal ﬁn and dried and stored at ambient temperatures.

Once spawning behavior had been observed, sampling also began for out-
migrating larval sturgeon. D-frame drift nets were used to capture larval lake sturgeon
downstream of spawning grounds in the Upper Black River. Sampling occurred during
the peak drift hours, between 10:00pm and 2:00am. Three peaks were observed in the
numbers of drifting larvae, presumably coinciding with the 3 peaks in adult spawning
activity. All larval lake sturgeon that were captured were transported to the Michigan
DNR Wolf Lake State Fish Hatchery. Juvenile lake sturgeon were divided into three
groups at the hatchery based on the three periods of larval drift.

Lake sturgeon were reared in the hatchery and released the following fall back
into the Upper Black River. All hatchery mortalities (n = 779) were preserved in 95%
non-denatured ethanol for genetic analyses. Prior to their release into the Upper Black
River, ﬁn clips were taken from all juvenile lake sturgeon (n = 912) and preserved in

scale envelopes at ambient temperatures.

Laboratory Analyses

DNA was extracted from 111 adult tissue samples using DNeasy® kits (QIAGEN
Inc.) according to the manufactures protocol. DNA was extracted from 576 juvenile lake
sturgeon also using the DNeasy® protocol. 192 juvenile lake sturgeon were selected from
each of the three out-migrating groups for genetic analyses. Juvenile lake sturgeon were
sampled proportionally from the hatchery mortalities and the ﬁsh that were released back

into the Upper Black River. From the ﬁrst group of juveniles samples from 48 alive and

32

144 dead individuals were used. From the second group samples from 176 alive and 16
dead ﬁsh were used. From the third group samples from 106 alive and 86 dead ﬁsh were
used. DNA was quantiﬁed using a Beckman DU® 7400 spectrophotometer.

DNA was ampliﬁed at 8 microsatellite loci, LS—68 (May et al. 1997), Afu68b
(McQuown et a1. 2002), Sp1120 (McQuown et al. 2000), on27 (King et al. 2001) Aqu9,
Aqu63, AﬁtG74 and AquI 1 2 (Welsh et al. 2003). PCR reactions were conducted in 25
ul volumes containing 100ng DNA, 10X PCR Buffer (1M Tris-HCl, 1M MgClz, 1M
KCl, 10% gelatin, 10% NP—40, 10% Triton-X), 2mM of each dNTP, 10 pmol of forward
and reverse reverse primer and 0.3 ul Taq polymerase. PCR conditions were as follows;
94° C for 2 minutes, followed by 30 cycles of 94° C for 1 minute, 1 minute at primer
speciﬁc annealing temperatures, and 72° C for 1 minute, and a ﬁnal extension at 72° C
for 2.5 minutes. PCR products were run on 6% denaturing polyacrylamide gels and
visualized on a Hitachi F MBIO II scanner. Allele sizes were determined using
commercially available size standards (MapMarkerTM, BioVentures Inc.) and based on

standard samples of known genotype.

Statistical Analyses

Estimates of allele frequencies, mean number of alleles per locus, and observed
and expected heterozygosityies were calculated using the computer program CERVUS
v2.0 (Marshall et al. 1998). Chi-squared goodness of ﬁt tests were performed using
CERVUS for each locus to test for Hardy Weinberg equilibrium. CERVUS was also used

to generate probabilities of excluding unrelated individuals from parentage assignment.

33

All adults that were captured in the Upper Black River prior to spawning were
considered as potential parents of each offspring. Parentage assignments were made using
a maximum likelihood-based approach (Meagher 1986, Thompson 1975) employed by
the program CERVUS. Parental pairs were assigned based on total exclusion of all other
potential parents or when CERVUS assigned parents at either 95% (strict) or 80%
(relaxed) probabilities relative to other parental pairs. Parentage was assessed by ﬁrst
determining maternity then paternity, and then validated by performing the analysis in
reverse order. Conﬁdence levels associated with each parental pair were derived based on
population allele frequencies and representative levels of tolerance of false parentage (see
Marshall et al. 1998 for details). In cases in which only a male parent or female parent
could be identiﬁed for a particular offspring based on total exclusion of all other males or
females, maternity or paternity only was assigned.

Mean and variance for numbers of mates and numbers of offspring produced were
estimated from the sample. To determine if size-related assortive mating was taking
place, the correlation between the total length of males and females that spawned with
one another was estimated using SAS (SAS Institute Inc.). In order to determine the
factors underlying reproductive success, the correlation between male and female total
length and the number of offspring produced was estimated using SAS. The correlation
between the number of mates and the number of offspring produced was also calculated
using SAS to examine the number of mates as another potential factor responsible for
variance in male and female reproductive success. Relationships were considered

signiﬁcant when P<0.05.

34

Because only a subset of the adult lake sturgeon population in Black Lake spawns
each year, it is not possible to estimate a true effective population size based on parentage
data from only 1 spawning season. Instead, an effective number of breeders (N b) was
estimated based on the parentage data from 2001. To account for unequal sex ratio and
variance in reproductive success, the effective number of breeders was calculated as in

Ballou and Foose (1996):

 

 

 

_ 4NefNem

Nb - (Nef-l- Nem) (I)
_ Mk —1

W _ [k—1+(Vk/k)] (2)

Nem N’” ’1 (3)

=[k—1+(Vk/k)

Ne; represents the effective number of females, with mean family size It and variance in
family size V1. and Ne," represents the effective number of males with mean family size k

and variance in family size Vk.

Results

None of the 8 loci were found to signiﬁcantly deviate from Hardy-Weinberg
equilibrium. The mean number of alleles per locus was 6.63 and the observed
heterozygosity ranged from 0.448 for the locus on27 to 0.818 for the locus Aqu9

(Table 4). Overall exclusion probability was 0.967. For 146 offspring a single parental

35

pair was assigned. 103 of these assignments (70.5%) were at the 95% conﬁdence level,
33 of the parentage assignments (22.6%) were at the 80% conﬁdence level and 10 of the
parentage assignments (6.9%) were below the 80% conﬁdence level. All of the cases
where parentage assignments were made below 80% conﬁdence represented situations
where parentage was assigned based on total exclusion. Additionally a single parent only

was assigned for 139 of the offspring in the study based on total exclusion.

Mating System

Multiple incidences of polygyny and polyandry were observed. Estimates of the
number of males spawning with each female ranged from 0 to 11 (mean = 3.10; variance
= 6.19) (Figure 5a). The number of females spawning with each male ranged from 0 to 5
(mean = 1.93; variance = 2.20) (Figure 5b). Three females and 8 males were not assigned
as potential parents to any of the offspring sampled. Low correlation (0.068; P>0.436)
was observed between the total length of males and females that spawned with one
another (Figure 6). Females whose offspring were found in only one of the groups of
drifting larvae spawned with an average of 1.86 males, whereas females whose offspring
were found in more than one group spawned with an average of 4. 16 males. Similarly,
males whose offspring were found in only one of the groups of drifting larvae spawned
with an average of 1.25 females and males whose offspring were found in multiple

groups spawned with an average of 2.74 females.

36

Reproductive Success

Reproductive success varied considerably among males and females. Estimates of
the number of offspring each female produced ranged from 0 to 22 (mean = 5.05;
variance = 21.02) (Figure 7a). The number of offspring produced per male ranged from 0
to 17 (mean = 3.16; variance = 8.02) (Figure 7b).

The numbers of offspring that each adult produced in each of the three out-
migrating groups of juveniles varied considerably (Figure 8a, b). Fourteen females and 24
males produced offspring in a single out-migrating group. The mean number of offspring
produced by adults that produced offspring in only 1 group was 2.21 for females and 1.75
for males. Alternatively, 25 females and 38 males produced offspring in 2 or 3 different
groups. The mean number of offspring produced by adults that produced offspring in
multiple groups was 7.24 for females and 4.71 for males.

Overall, correlation between total length and number of offspring produced was
low (r = 0.064, P>0.520) (Figure 9). For males, correlation between total length and
number of offspring produced was —0.093 (P>0.05) and for females correlation between
total length and number of offspring produced was 0.180 (P>0.05). In contrast, a high
degree of correlation was found between the number of known mates and the number of
offspring produced (r = 0.888, P<0.0001) (Figure 10). For males, correlation between the
number of mates and the number of offspring produced was found to be 0.810
(P<0.0001). For females correlation between the number of mates and the number of

offspring produced was 0.927 (P<0.0001).

37

Effective Number of Breeders
Based on equations (2) and (3) above, the effective number of females and males
was estimated to be 20.71 and 31.72 respectively. Substituting these estimates of Nef and

Nem into equation (1) produced an estimate of 50.12 effective breeders for 2001.

Disscussion

To date, studies of the lake sturgeon mating system have focused exclusively on
observational data (see Bruch and Binkowski 2002, Kempinger 1988). Observational
studies have provided insight into some of the spawning behaviors lake sturgeon exhibit
and have observed polygyny and polyandry, however, many lingering questions
regarding the mating system of lake sturgeon exist. Estimates presented in this study
represent the ﬁrst attempt to examine the mating system of any sturgeon species using
genetic markers. This study also represents one of the few studies in which no prior

information is known about either of the parents of any given offspring.

Mating System

Because lake sturgeon spawn in large groups and are broadcast spawners, it is
difﬁcult to assess visually which males are spawning with which females and vice versa.
The fact that lake sturgeon often leave spawning grounds due to changes in water
temperature and then return later (Bruch and Binkowski 2002, Kempinger 1988), further

complicates observational studies. This study allows a much more accurate method of

38

assessing the lake sturgeon mating system. The results of this study conﬁrm direct
observations of the lake sturgeon mating system but also provide evidence for many
aspects of the lake sturgeon mating system which were previously unknown.

Previous observational studies on the lake sturgeon mating system have observed
likely evidence for polygyny and polyandry. Kempinger (1988) observed 6 to 8 males
spawning with one female and Bruch and Binkowski (2002) observed between 2 and 8
males spawning with a single female and also observed males spawning with multiple
females. The results of the genetic analyses in this study found polygyny and polyandry
to be widespread. Whereas females in the Lake Winnebago system were observed to be
spawning with an average of 5-6 males (Bruch and Binkowski 2002), genetic analyses
showed that female lake sturgeon in the Black River were observed to spawn with an
average of 3.10 males. This discordance is likely to be a result of more heavily skewed
sex ratios during spawning periods in the Winnebago system (1:5.7 females to males
(Bruch and Binkowski 2002) vs. 1:1.6 females to males in this study). It is important to
consider that since the number of mates was based on successful assignment of
parentage, the number of mates per male and female in this study represents a
conservative estimate. The low correlation between the total length of males and females
that spawned with one another in Black Lake indicates that assortive mating based on
size is not occurring place in this population.

Eleven of the adults in this study, 3 females and 8 males, were not assigned as
potential parents to any of the 576 offspring. One possible explanation for this is that the
offspring from these adults were not represented in the sub-sample used in this study.

Another possible explanation is that these individuals were present in the Black River

39

during spawning periods but did not spawn. Given the large number of offspring that
were surveyed, it is likely that these individuals did not successfully spawn during 2001.
This may be attributed to a low number of potential mates while these individuals were in
spawning areas or the fact that these individuals were less aggressive for mating

opportunities.

Reproductive Success

A high level of variation in reproductive success was found among both male and
female lake sturgeon. Multiple studies have utilized genetic markers to examine variance
in male and female reproductive success in other ﬁsh species and have found similar
trends whereby males and females exhibited a high degree of variance in reproductive
success (Bekkevold et al. 2002, Fuimera et al. 2002, Garant et al. 2001, Moran et al.
1996). One potential factor underlying this variation in reproductive success is the size of
the ﬁsh. Since larger sturgeon can hold greater numbers of gametes it is possible that
larger sturgeon may have higher reproductive success. In a study on cod, Bekkevold et al.
(2002) found that on average, larger males did indeed sire higher numbers of offspring.
This does not appear to be the case in lake sturgeon, however. In this study the
correlation between total length and number of offspring produced was very low (0.064).
Garant et al. (2001) similarly found a low correlation between the body size of Atlantic
salmon and the number of offspring produced.

A signiﬁcantly higher correlation was found between the numbers of mates a
male or female had and the number of offspring produced (0.888). Data suggest that for

lake sturgeon, where multiple males mate with a single female simultaneously and a high

40

degree of physical competition exists between males to obtain optimal spawning
positions, distributing gametes among several mates rather than concentrating
reproductive efforts on just one or two mates maximizes reproductive success. Garant et
al. (2001) also observed a high degree of correlation between the number of mates and
the number of offspring produced in Atlantic salmon.

A high degree of variation was also observed in the numbers of offspring that
each male and female produced in each of the three groups of out-migrating juveniles.
These data, coupled with data on the numbers of mates adults had, suggests that two
different mating strategies exist in Black Lake. One group of sturgeon appear to be
migrating up the Black River, releasing all their gametes at once and then returning to
Black Lake. On average these individuals have fewer mates and produce fewer offspring.
Another group of the adult population in Black Lake appear to be releasing a portion of
their gametes at one time, leaving the stream and then returning at a later time and
releasing the remainder of their gametes. These individuals on average had more mates
and greater numbers of offspring. The mating strategy whereby males and females
apportion gametes among multiple spawning events appears to lead to higher
reproductive success. Incidences of lake sturgeon leaving spawning areas and then
returning at a later time have been documented previously and are thought to be a result
of ﬂuctuations in temperature (Bruch and Binkowski 2002, Auer 1999b, Kempinger
1988). Based on this information, the decision to apportion gametes over a single versus
multiple spawning episodes may be random event in response to environmental

conditions rather than a conscious decision.

41

Eﬂective Number of Breeders

The effective number of breeders for the 2001 adult spawning population in Black
Lake was found to be lower than the census number of adults observed in the Upper
Black River (N b/N = 0.45). Frankham (1995) reviewed reported estimates of effective
population size for 102 different species from several taxa and found that the three most
important factors inﬂuencing effective population size were ﬂuctuations in population
size, variance in family size, and sex ratio. Lake sturgeon in Black Lake exhibit male
biased sex ratios during spawning periods as well as a high variance in reproductive
success. The combination of these two factors along with the declining nature of this
population has contributed to a reduction in the effective number of breeders in the Black
Lake population.

The low number of effective breeders raises conservation concerns for this
population. Populations that experience reduced effective population sizes are likely to
experience an increase in the loss of genetic diversity as well as an increase in the rate of
extinction (Newman and Pilson 1997). The size of the lake sturgeon population in Black
Lake was found to have signiﬁcantly declined in a span of approximately 25 years (Baker
and Borgeson 1999). Many other populations of lake sturgeon in the Great Lakes have
population sizes much lower than Black Lake (Holey et al. 2000). Populations that have
experienced more severe declines than Black Lake will likely have much more heavily
skewed sex ratios during spawning periods. Because of this it is reasonable to assume
that many populations around the Great Lakes are currently experiencing effective
population sizes much smaller than in Black Lake, and thus are at a much higher risk of

extinction.

42

The lake sturgeon mating system evolved at a time when lake sturgeon were
much more abundant than they are presently. Mechanisms including rapid incubation of
eggs and the production of large numbers of offspring likely evolved to counter the
effects of predation on eggs and offspring and to help increase reproductive ﬁtness.
Currently lake sturgeon numbers in the Great Lakes are signiﬁcantly reduced. Given the
aggregate nature of their spawning behavior and the fact that juvenile lake sturgeon drift
in large groups after hatching, low population sizes are likely to lead to a decrease in
population growth. Low population numbers will make it difﬁcult for lake sturgeon to

ﬁnd adequate breeding opportunities to ensure long-term population viability.

Conclusion

This study offers the ﬁrst analysis of the lake sturgeon mating system from a
genetic viewpoint. The genetic data presented in this study conﬁrm the results of previous
observational studies and also provide new insight into aspects of lake sturgeon
reproductive ecology about which little information had been known. The lake sturgeon
mating system evolved at a time when populations had much greater numbers of
individuals than they do presently. Currently, lake sturgeon experience high variance in
reproductive success and skewed sex ratios, factors that have been shown to signiﬁcantly
reduce effective population sizes. Other remnant populations of lake sturgeon that have
fewer individuals and more heavily skewed sex ratios than the population in Black Lake
are likely to experience an even greater variance in reproductive success and in turn much

lower effective population sizes. Historically this mating system served as an effective

43

means of preserving population viability, however, given the reduced size of most
populations of lake sturgeon and the factors causing lake sturgeon effective population

sizes to be low, the viability of this mating system is greatly reduced.

44

Chapter III

Genetic Consequences of Supportive Breeding
For a Declining Population of Lake Sturgeon

Introduction

As lake sturgeon populations in the Great Lakes continue to decline, it is likely
that hatchery supplementation will be increasingly used to rehabilitate declining and
extirpated populations (Hay-Chmielewski and Whelan 1997). While hatchery programs
have been a popular management and conservation strategy for many species of ﬁshes,
such programs have also received much critical attention (Ford 2002, Meffe 1992,
Krueger and May 1991). Production goals for lake sturgeon hatchery programs may be
easily met by spawning relatively few females given their high fecundity (Houston 1987).
The development of lake sturgeon hatchery programs that successfully accomplish
conservation goals of maintaining genetic diversity will be difﬁcult, however, due to
small sizes of potential source populations (Holey et al. 2000) and limited opportunities
to capture large numbers of adults in spawning condition due to infrequency of spawning
(Auer 1996) and male-skewed sex ratios during spawning periods (Bruch and Binkowski
2002). In addition to obtaining suitable numbers of adults, hatchery programs for lake
sturgeon will also need to focus on choosing donor stocks that are genetically similar to
those populations that are targeted for rehabilitation in order to preserve the genetic
diversity of wild populations (Meffe 1995).

One remnant lake sturgeon population that has received hatchery supplementation
in the recent past is a landlocked p0pulation in Black Lake, MI. This population

represents one of the few remnant lake sturgeon populations that still has a relatively

45

large number of individuals. Recent population estimates found that there were
approximately 550 ﬁsh of reproductive age in this population (Baker and Borgeson
1999). Comparisons of recent population estimates to estimates from approximately 25
years prior have shown that the size of this population is declining and recruitment is low
(Baker and Borgeson 1999).

Anthropogenic factors are largely responsible for this decline. This population has
been isolated from other populations of lake sturgeon in the Great Lakes for the past 100
years due to the construction of the Alvemo Dam on the Lower Black River (Figure 4).
Additionally the construction of the Kleber Dam on the Upper Black River has caused a
signiﬁcant decline in the amount of available spawning habitat. This population also
supports one of the few sport ﬁsheries for lake sturgeon. Currently the ﬁshery is
restricted to a harvest of ﬁve ﬁsh annually, however, less conservative harvest regulations
historically likely contributed to the decline of this population as well (Baker and
Borgeson 1999).

Hatchery supplementation was utilized in Black Lake as a means to supplement
this declining population and to sustain the sport ﬁshery. In the spring of 1983, 1984 and
1988, male and female lake sturgeon in spawning condition were captured in the Black
River and gametes were taken for supplemental breeding. Each year gamete takes were
performed, eggs were taken from 1 or 2 females, the exact number of females used each
year is not known. The exact number of males used each year for gamete takes is also
unknown, though it is likely to be no more than 2 males per year (D. Borgeson, MI DNR,
personal communication). Each year gametes were taken, eggs were fertilized and

juvenile lake sturgeon were reared in a hatchery prior to their release the following fall

46

into the Black Lake system. During 1983, 1984, and 1988, 1,187, 6,698 and 3,587
juvenile lake sturgeon were released respectively. The probability of survival of these
juveniles was greatly increased given the fact that the time period when these ﬁsh were
most vulnerable to natural mortality and predation was spent in captivity (Houston 1987).
A 1997 population estimate of the Black Lake sturgeon population showed that a large
number of individuals in the population ﬁt a size range that was consistent with length at
age data for cohorts from 1983, 1984 and 1988 (Baker and Borgeson 1999) (Figure 11).
Based on the delayed maturation of lake sturgeon (Houston 1987, Harkness and Dymond
1961), it is expected that these cohorts will shortly be recruiting into the adult spawning
population in Black Lake. Given the large proportion of the total breeding population that
these offspring will shortly represent and the high levels of coancestry present due to the
extremely small number of adults used to produce these individuals, it is likely that some
level of inter-breeding among these cohorts will take place.

The practice of breeding wild caught individuals in a captive environment and
then returning their offspring to their native environment, as was the case in Black Lake,
has commonly been referred to as supportive breeding (Ryman and Laikre 1991).
Supportive breeding programs favor the reproduction rate of a small segment of the
population by increasing the probability of survival of the offspring of captively bred
individuals (Ryman and Laikre 1991). When one segment of the population experiences
much higher reproductive success, there is an overall increase in the variance in family
size for the population, a factor that has been shown to signiﬁcantly reduce effective
population size (Frankham 1995). The use of supportive breeding as a means of

p0pulation restoration often results in a tradeoff between numbers of individuals in the

47

population and the population’s effective population size (N). This phenomenon has
previously been referred to as a the Ryman and Laikre effect and can be represented by
the equation:

_1__x_2+(1-x)2
Ne Nc Nw

 

where N. represents the number of effective parents reproducing in the wild, NC
represents the number of effective parents bred in captivity and x is the relative
contribution of offspring from captive parents and (1 - x) the relative contribution from
wild offspring. This tradeoff is accentuated in species that have high reproductive outputs
such as sturgeons (Ryman and Laikre 1991).

Another potential consequence associated with supportive breeding programs is
an increase in the number of related individuals in a population. Supportive breeding
programs for ﬁshes often rely on small numbers of adults to produce large numbers of
offspring (Wang et a1 2002). Depending on the numbers of parents used for mating and
the number of offspring produced and released back into the wild population, this
practice can result in a disproportionate amount of individuals in a population that are
closely related (i.e., full or half siblings). As the number of related individuals in a
population increases, the likelihood of inbreeding in future generations increases.

The term inbreeding generally refers to the mating between two individuals who
share one or more common ancestors (Templeton and Read 1994). Inbreeding is likely to
occur to some degree in all natural populations. In ﬁsh species the level of inbreeding can
be inﬂuenced by factors including reductions in effective population sizes, variance in

reproductive success, dispersal rates and natal ﬁdelity (Lynch 1996, Bekkevold et al.

48

2002, Gerlach et al. 2001, Wang et al. 2002). Low effective population size in wild
populations will lead to a reduction in genetic diversity and an increase in genetic drift
(Keller and Waller 2002, Wang et al. 2002). Inbreeding may also lead to an increase in
the expression of deleterious alleles in a population (Keller and Waller 2002). Increased
levels of inbreeding in salmonid populations have been shown to have a negative effect
on phenotypic traits including body weight and juvenile survivorship (Wang et al. 2002).
High levels of inbreeding are also likely to increase the probability of extinction,
particularly in small populations (Bijlsma et al. 2000).

Direct negative effects of supportive breeding have been previously documented
in other species of ﬁshes. Tessier et al. (1997) found that that a supportive breeding
program for Atlantic salmon caused a signiﬁcant change in allele ﬁ‘equencies between
wild and F1 generation hatchery ﬁsh. This supportive breeding program also had
negative impacts on effective population sizes of the populations receiving stocked
individuals. Hedrick et al. (2000) similarly found that in certain years supportive breeding
practices for winter run Chinook salmon led to a reduction in effective population size
when non winter run individuals were used as parents.

Reductions in effective population size and increases in levels of inbreeding will
ultimately lead to a reduction in population size. As population size decreases, the effects
of genetic drift become more signiﬁcant (Lynch 1996). As the amount of genetic drift
increases in a population, the rate of accumulation of deleterious mutations will also
increase, causing populations to decline even further. This phenomenon has been referred
to as a mutational meltdown (Lynch 1996). Mutational meltdowns may be further

exacerbated in situations where supportive breeding is used. Mutations that are

49

deleterious in the wild may be rendered neutral in captive environments (such as ﬁsh
hatcheries) that provide more favorable environmental conditions. This can lead to a
much more rapid accumulation of these deleterious mutations which will presumably be
incorporated into the wild population once individuals from captive environments are
released into the wild (Lynch 1996).

Given the potentially harmful effects of the supportive breeding program for lake
sturgeon in Black Lake, the objective of this study was to examine the effect that
hatchery supplementation had on levels of relatedness (er) among lake sturgeon in Black
Lake. Levels of relatedness were assessed for two groups of lake sturgeon in Black Lake.
The ﬁrst group was composed of wild adult ﬁsh that spawned in the spring of 2001. The
second group was composed of individuals that were likely from the 1983, 1984 and
1988. This study examines how aspects of the lake sturgeon mating system along with
low numbers of breeding adults will inﬂuence the likelihood of mating between closely
related individuals. A conceptual model is used to examine the ways in which aspects of
the lake sturgeon mating system as well as the declining nature of the Black Lake lake

sturgeon population will inﬂuence the amount of inbreeding.

Methods

Sample Collection
Two groups of lake sturgeon were used for this study. The ﬁrst group of sturgeon
in this study was composed of 114 adult ﬁsh collected during spring 2001 spawning

migrations (see chapter 2 for collection methods). These individuals represented a sample

50

of the population that was produced by natural matings between wild adults. The second
group of ﬁsh was composed of individuals that, based on length at age data, were likely
to be from the 3 cohorts stocked in 1983, 1984 and 1988. The second group of sturgeon
was collected during the summer of 2002 by personnel from the Michigan DNR using
large mesh gill nets. These individuals ﬁt a size range of 104-1300m. All ﬁsh captured
were measured, weighed, tagged and ﬁn clips were taken from the upper lobe of the

caudal ﬁn for genetic analyses and stored at ambient temperatures in scale envelopes.

Laboratory Methods

DNA was extracted from all sturgeon sampled in the summer of 2002 that ﬁt into
this size range (n= 53). All DNA extractions were performed using the DNeasy®
(QIAGEN Inc.) protocol with ﬁnal re-suspensions in 50pl AE buffer. DNA was
quantiﬁed using a Beckman DU® 7400 spectrophotometer.

DNA was ampliﬁed at 8 microsatellite loci, LS-68 (May et al. 1997), Afu68b
(McQuown et al. 2002), Sp1120 (McQuown et al. 2000), on27 (King et al. 2001) Aqu9,
Aqu63, Aqu 74 and AquI 12 (Welsh et al. 2003). Microsatellite PCR reactions were
conducted in 25 pl volumes containing 100ng DNA, 10X PCR2 Buffer (1M Tris-HCl,
1M MgClz, 1M KCl, 10% Gelatin, 10% NP-40, 10% Triton-X), 2mM of each dNTP, 10
pmol of forward and reverse primer and 0.3pl Taq polymerase. PCR conditions were as
follows; 94° C for 2 minutes, followed by 30 cycles of 94° C for 1 minute, 1 minute at
primer speciﬁc annealing temperatures, and 72° C for 1 minute, and a ﬁnal extension at
72° C for 2 minutes and 30 seconds. All PCR reactions were conducted using Stratagene

Robocycler® 96 (Stratagene Inc.) therrnocyclers. PCR products were run on 6%

51

denaturing polyacrylamide gels and visualized on a Hitachi F MBIO II scanner. Allele
sizes were determined using commercially available size standards (MapMarkerTM,

BioVentures Inc.) and using samples of known genotype.

Statistical Analyses

The program GENEPOP v3.1 (Raymond and Rousset 1995) was used to generate
estimates of allele frequencies and to perform exact tests of Hardy-Weinberg Equilibrium
using a Markov Chain method. A Bonferroni correction (Rice 1989) was performed to
correct nominal alpha levels for multiple comparisons for tests of Hardy Weinberg. The
program BIOSYS-l (Swofford and Selander 1984) was used to estimate measures of
genetic diversity including observed and expected heterozygosities and mean numbers of
alleles per locus for individuals in the two groups. A Chi-squared contingency test was
used to determine if observed heterozygosity and number of alleles per locus were
signiﬁcantly different between the two groups of sturgeon in this study.

Levels of relatedness among individuals were inferred using estimates of
coefﬁcients of relatedness (er) calculated as in Queller and Goodnight (1989) using the

formula:

r =2(Py-P*)
"’ Zest—13*)

 

where P, represents the frequency of a particular allele in individual x, P, represents the
frequency of the same allele in individual y and P* represents the frequency of the allele
in the entire population. Coefﬁcients of relatedness were calculated for both groups of

lake sturgeon using the program Kinship v2.1 (Goodnight and Queller 1999). Proportions
52

of individuals signiﬁcantly related at the half and full sibling level in each of the two
groups of sturgeon were estimated using likelihood methods employed using the program
Kinship. A Wilcoxon test was performed using the program SAS to test for differences in
distributions of rxy values between the two groups.

A conceptual model was used in order to determine some of the demographic and
life history characteristics of the Black Lake lake sturgeon population that would effect
the level of inbreeding in this population and how these different factors would effect
inbreeding. This model accounted for factors including sex ratios, recruitment of hatchery
individuals into the adult population as well as senescence/ mortality of the wild adult

population and declining nature of this population.

Results

Levels of genetic diversity differed for the two groups of sturgeon used in this
study. Estimates of allele frequencies showed that 8 alleles present in the 2001 spawning
population were not present in the group of sturgeon that were likely derived from
supportive breeding (Table 5). Additionally 2 alleles that were not present in the 2001
spawning group were present in the ﬁsh sampled in 2002. In the 2001 spawning group all
but one locus (L568) were in Hardy Weinberg equilibrium. In the group of individuals
that were likely derived from supportive breeding, 4 of the loci deviated from Hardy
Weinberg equilibrium (LS68, on2 7, Aqu9, and Aqu63). The mean number of alleles
per locus was greater in the group that spawned in 2001 (6.0) than the group composed of

suspected stocked individuals (5.3). A chi-squared contingency test showed that the

53

number of alleles per locus was not signiﬁcantly different between the two groups (P =
0.497). Observed and expected values of heterozygosity were slightly lower for the group
of sturgeon believed to be derived from supportive breeding (Table 6). The Chi-squared
test showed that differences in heterozygosity between the two groups of sturgeon were
not signiﬁcantly different (P = 0.99)

Mean rxy values were —0.020 for the 2001 spawning adults and 0.061 for the
group of sturgeon that were likely derived from supportive breeding. While both groups
of sturgeon appear to have r,‘y values that are skewed to the right (Figure 12), the group
of individuals likely representing the stocked cohorts shows a much greater skew. The
Wilcoxon test showed that the distribution of rxy values for the two groups signiﬁcantly
differed (Test statistic = 6372571 .5; P<0.0001).

In the group of adult sturgeon that spawned in spring 2001, estimates showed that
90.4% of the pair-wise comparisons among individuals were not indicative of relatedness
at the level of half or full siblings, 7.5% of comparisons were indicative of individuals
signiﬁcantly related at the level of half siblings (P<0.05), and 2.1% of the comparisons
were indicative of individuals signiﬁcantly related at the level of full siblings (P<0.05). In
the group of sturgeon that were likely the progeny produced from supportive breeding,
estimates showed that 84.5% of the pair-wise comparisons were indicative of individuals
that were not signiﬁcantly related at the level of half or full siblings, 10.2% of the
comparisons were indicative of individuals were signiﬁcantly related at the level of half
siblings (P<0.05) and 5.3% of the comparisons were indicative of individuals that were

signiﬁcantly related at the level of full siblings (P<0.05).

54

Discussion

The remnant lake sturgeon population in Black Lake was targeted for supportive
breeding to counteract population decline and to support a sport ﬁshery. 11,472 lake
sturgeon were stocked into Black Lake and the immediate goal of increasing the
population size was accomplished. As is evidenced by recent population estimates and
size distributions of the Black Lake lake sturgeon population, individuals from these
stocking events currently represent a large portion of the lake sturgeon population in
Black Lake (Baker and Borgeson 1999), and will shortly begin to recruit into the adult
population. While the short-term goals of the supportive breeding efforts in Black Lake
(increasing population size) were met, this action does not represent a viable management
strategy for this population.

One of the primary goals of a hatchery program should be to maintain genetic
characteristics (i.e., levels of heterozygosity) of the wild source population in the
hatchery stock (Allendorf and Ryman 1987). The use of genetically suitable source
populations represents one method in which this goal can be accomplished (Meffe 1995).
While the source population used in the Black Lake supportive breeding program was
identical to the target population, the goal of effectively capturing the genetic
characteristics of the wild population was clearly not met. Due to the fact that only a
small portion of the adult population in Black Lake was used for this supportive breeding
program, several rare alleles that were present in the wild population were lost in the
hatchery individuals and there was a decrease in the mean number of alleles per locus.

Additionally, signiﬁcant departures from Hardy Weinberg equilibrium were also

55

observed in the group of sturgeon that were likely to be the progeny produced by
supportive breeding efforts. These data clearly illustrate that the genetic characteristics of
the progeny of the supportive breeding efforts do not represent those of the wild source
population. Data in this study clearly illustrates the need for captive mating scenarios to
focus on factors besides simply genetic compatibility of source and recipient populations.

It is likely that supportive breeding has also led to a decline in the effective
population size of the Black Lake lake sturgeon population. Ryman and Laikre (1991)
showed that as the relative captive contribution to a population increased, the effective
population size decreased proportionally, primarily the result of an increase in the
variance in family size. Reductions in effective population size due to supportive
breeding programs can occur rapidly as was the case in Atlantic salmon when a
supportive breeding program was predicted to cause signiﬁcant reductions in effective
population size in a single generation (Tessier et al. 1997). Proper use of supportive
breeding in which steps are taken to maximize effective population size and minimize
inbreeding can result in a net effect of maintaining Ne (Hedrick et al. 2000). With the
case of lake sturgeon in Black Lake, however, these factors clearly were not taken into
account. The small number of adults used for this supportive breeding program combined
with the large number of offspring produced will signiﬁcantly inﬂuence the variance in
family size, which in turn will result in further declines in effective population size in this
population which already has a reduced Nc (Chapter 2).

Another consequence of the supportive breeding program was an increase in the
number of closely related individuals in the Black Lake lake sturgeon population. This

hypothesis is directly evidenced by the increase in both estimated levels of relatedness

56

among individuals (rxy) in the group of sturgeon likely to be derived from supportive
breeding, and by the increase in estimates of proportions of half and full siblings in this
group. Even if individuals mate randomly during future spawning events, the increase in
the number of related individuals due to the addition of the hatchery cohorts will lead to
an increase in the level of inbreeding in the population. More of the progeny of the
supportive breeding program will recruit into the adult spawning population coinciding
with senescence of wild spawning adults and leading to further increased levels of
inbreeding.

When considering the levels of inbreeding in this population there are many
aspects of lake sturgeon biology and the nature of the population in Black Lake that are
likely to inﬂuence levels of inbreeding. The fact that this population has been isolated
from all other lake sturgeon populations for approximately 100 years (roughly 5 lake
sturgeon generations) signiﬁcantly increases the likelihood of elevated levels of
inbreeding in this population (Keller and Waller 2002). In small isolated populations
rates of genetic drift and accumulations of deleterious alleles will be accentuated as the
amount of inbreeding increases (Keller and Waller 2002, Lynch 1996). It is likely that
when this population became isolated, a ntunber of related juvenile sturgeon were present
in Black Lake as well as adult ﬁsh that were also related at some level. As time
progresses and the population size is reduced this level of relatedness in the population
will gradually increase.

The fact that this population is declining is also likely to inﬂuence the amount of
inbreeding in this population. As the number of adults in this population decreases, the

number of potential mates for each male or female will also decrease. As a result,

57

spawning adults will have a greater chance of mating with individuals with whom they
are related at some level. As matings of this type become more common, more closely
related offspring will be produced that will in turn recruit into the adult population and
will experience some probability of mating with one another.

The nature of the lake sturgeon mating system is also likely to inﬂuence
inbreeding in this population. Lake sturgeon typically spawn with multiple individuals
each spawning season (Bruch and Binkowski 2002, Kempinger 1988, Chapter 2 this
document). While mating does appear to be random, in a ﬁnite population such as Black
Lake, the more mates an individual has, the greater the chances are that one or more of
those mates will be a close relative. The fact that lake sturgeon spawn multiple times
throughout their life can also affect the amount of inbreeding in this population. While
lake sturgeon may not mate with a close relative one year, the chance of mating with a
close relative still exists in subsequent spawning years. Lake sturgeon may spawn with a
close relative one year and spawn with another close relative in a subsequent year as well.

Lake sturgeon that were produced as a result of supportive breeding efforts in
Black Lake will soon be recruiting into the adult spawning p0pulation. Each year as the
proportion of highly related individuals in this population increases, the chance of mating
among closely related individuals also increases. The chance of inbreeding will be further
increased each generation as some proportion of the adult population senesces and less
opportunities exist for individuals to mate with older individuals with whom they
presumably have little close relation.

The manner in which these factors inﬂuence the amount of inbreeding in this

population is depicted in Figure 13. Adults in Black Lake may spawn with individuals

58

that they are closely related to, or individuals to whom they have little relation. When
matings occur between adults that are not closely related, the increase in inbreeding in the
population will be gradual and depend on the background levels of inbreeding in the
population. When closely related individuals mate, the increase in the amount of
inbreeding in this population will be much more rapid. This rapid increase in inbreeding
will be directly inﬂuenced by the recruitment of hatchery individuals into this population
coupled with the loss of some proportion of the adult spawning population due to
senescence and mortality. Male biased sex ratios will further inﬂuence this decline. As
the number of mates per female increases, females have more mates and the likelihood of
one of those mates being a close relative also increases. Population decline will also
cause inbreeding to increase more rapidly. As the number of potential mates in the
population declines, more individuals will be forced to mate with close relatives. Both the
rapid and gradual increase in the amount of inbreeding in this population will cause a
reduction in population ﬁtness that over time will lead to an increased likelihood of

extinction.

Conclusions

Past supportive breeding efforts for lake sturgeon in Black Lake are likely to have
had serious negative conservation implications for this declining population. Supportive
breeding in this population has failed to capture the genetic characteristics of the wild
population in the hatchery-reared offspring and has also signiﬁcantly increased the

number of closely related individuals in this population. As closely related individuals

59

begin to recruit into the adult spawning population in Black Lake and the current
spawning population senesces and experiences mortality, levels of inbreeding will
increase in this population, effective population size will become further reduced and an
increase in deleterious mutations will also likely occur. Inbreeding is likely to be
inﬂuenced by several of the biological characteristics of lake sturgeon as well as the
declining nature of this population. It is likely that levels of inbreeding in this population
will cause population ﬁtness to be reduced to the point that this population is no longer
self-sustaining. This situation clearly illustrates the need to consider more information
than the source population used for supportive breeding in order to develop biologically

sound management strategies.

60

APPENDIX 1

Estimates of Microsatellite Allele Frequencies and Mitochondrial DNA
Haplotype Frequencies from Chapter I

61

8:8 903

 

 

 

 

000.0 00N0 000.0 NN00 00N0 00F.0 00F.0 000.0 000.0 00F.0 00F
500.0 0 F00 000.0 000.0 0NF.0 vFNO F000 NN00 F000 NF00 v0F
V000 N050 30.0 050.0 000.0 000.0 #050 30.0 000.0 .000 00F

F0 0N 0 0N 00 00 F F F 0v 00 0v 2

566... M33

000.0 0 F00 000.0 000.0 000.0 000.0 000.0 000.0 000.0 000.0 50F
000.0 F500 000.0 0.00 03.0 0NNO 000.0 NF0.0 500.0 NOF.0 00F
0vF .0 000.0 50F.0 000.0 0N00 000.0 000.0 50F.0 50F.0 000.0 00F
500.0 F500 000.0 500.0 00F.0 VF00 000.0 03.0 050.0 F F00 00F
03.0 F500 50F.0 000.0 000.0 0N00 0N00 05F.0 F F00 0vF .0 FOF
00v0 FOF.0 v.30 V5F0 00N0 500.0 00.0 000.0 #000 00N0 55F
00 F .0 NON0 000.0 00F.0 00 F .0 0¢N0 000.0 000.0 500.0 00 F .0 05F
0000 .000 000.0 00F.0 0NNO 0N00 050.0 000.0 NON0 500.0 00F
000.0 000.0 000.0 00F.0 0N0.0 000.0 000.0 VN00 5 F00 0N00 00F
0N00 000.0 000.0 NN00 000.0 000.0 000.0 N50 530 000.0 FOF
0.00 00N0 F F F .0 00 F .0 000.0 00 F .0 000.0 000.0 000.0 500.0 50 F
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62

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V000 500.0 055.0 F050 V000 F F00 0N00 005.0 055.0 FV00 0 FN
05 5N 0 0N 0V 5N 0F F 0V 50 V Z
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000 .0 500.0 50F.0 NOV0 000.0 0000 N000 00V.0 0N00 0 F00 00F
00F.0 000.0 F F F0 0V00 00F.0 000.0 000.0 VVFO 0N00 0V00 00F
VNNO 00F.0 05N0 00N0 00N0 00F.0 00V0 05N0 0N00 00N0 5NF
05 5N 0 NN 00 00 0F F 0V 00 V 2
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NVF .0 0N00 05N0 FV00 0NNO 000.0 05N0 5V0.0 00V0 00N0 VVF
500.0 00V0 VVVO V000 0FV.0 000.0 VON0 000.0 0V00 NF00 0VF
0 F00 000.0 000.0 000.0 000.0 000.0 N000 NF00 000.0 000.0 00F
030 000.0 000.0 0N00 0V00 000.0 N000 000.0 NF00 NF00 NOF
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50 0N 0 NN 0V 00 00F 0V F0 0V 2
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500.0 F500 000.0 000.0 00F.0 VF00 000.0 0 FNO FOF.0 000.0 NON
000.0 0 F00 000.0 000.0 0N00 50F.0 500.0 0 F00 000.0 NF00 05N
00N0 0VF.0 50F.0 050.0 0NF.0 0N00 550.0 00N0 N000 50N.0 V5N
000.0 000.0 NNNO 00N0 000.0 00F.0 00F.0 000.0 N000 50N.0 NON
0V00 VOV0 000.0 00F.0 00F.0 00V0 000.0 03.0 0VF.0 00F.0 00N
000 .0 VFNO 000.0 00V0 05V.0 VFNO 05N0 00V0 000.0 0N00 VON
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63

 

 

 

 

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000.0 000.0 NNNO 000.0 050.0 000.0 F500 00V.0 050.0 0NF .0 N
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05 mm 0 0.. 00 FN 00F 0V N0 3 2
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4

6

APPENDIX 11

Tables and Figures

65

Table 1. Measures of genetic diversity based on 8 microsatellite loci for the 10

populations used in this study.

 

 

Population N A H99; Hm,—
Peshtigo River 44 2.424 0.609 0.618
Manistee River 89 2.264 0.541 0.588
Fox River 46 2.401 0.588 0.600
Black River 111 2.630 0.606 0.679
Bad River 37 2.373 0.646 0.653
St. Clair 50 2.647 0.664 0.690
Menominee River 23 2.300 0.567 0.580
Oconto River 9 2.458 0.681 0.615
Sturgeon River 30 2.384 0.544 0.613
Wolf River 81 2.440 0.591 0.626

 

A = Allelic Richness as in Petit et al. (1998)
Hob, = Observed Heterozygosity
chp = Expected Heterozygosity

66

5:00:00 _:0t0..:om .000 00.0 n 0:20 .05. 0:0 00 00:85:90 0000205 .

 

 

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20>> 500.30 2:000 00:_Eo:0_2 00.0.0 000 3.00.0 x0“. 090:0: 000500

 

.2000me 05 3206 0.298 00 0800 5.6 000 2000006 05 0.603 60— 858080008 0 00 00000 am 00 83:58 803-000 .N 030,—.

67

Table 3. Hierarchical partitioning of variance based on microsatellite and mitochondiral
DNA for the 10 populations used in this study.

 

 

 

 

 

Among
Within Indvivvégaals Populations Among
individuals populations within basins Basins
Microsatellite
DNA Locus F f (Os) (9p)
LS68 0.255 0.195 0.074 0.003
Afu68b 0.103 0.023 0.081 0.009
on27 0.150 0.089 0.068 0.058
Spl120 0.073 0.000 0.091 0.050
AquQ 0.120 0.060 0.064 0.011
Aqu63 0.100 0.051 0.052 0.008
Aqu74 0.122 0.082 0.043 0.027
Aqu112 0.055 0.017 0.039 0.000
Overall 0.120 0.059 0.065 0.021
mtDNA (1)5: (Dsc (Dot
N.A. 0.195 0.210 0.000

 

68

Table 4. Measures of genetic diversity for the
8 microsatellite loci used in the parentage study.

 

 

Number of
Locus Alleles Hobs I—Im
L868 8 0.517 0.754
Afu68b 11 0.735 0.739
Spl120 6 0.749 0.740
on27 4 0.448 0.436
Aqu9 11 0.818 0.812
Aqu63 3 0.585 0.663
Aqu74 3 0.450 0.492
Aqu112 7 0.774 0.781
Mean 6.63 0.630 0.680

 

Hobs = Observed Heterozygosity
chp = Expected Heterozygosity

69

Table 5. Allele frequencies based on 8 microsatellite loci for the two groups of sturgeon
used in this study. One group was composed of wild adults that spawned in the Spring of
2001 and the other group was composed of individuals likely to be the progeny of
supportive breeding events.

 

 

 

Locus Group Locus Group
Supportive Supportive
L868 2001 Adults Breeding AquQ 2001 Adults Breeding

112 0.311 0.387 124 0.035 0.038
116 0.033 0.019 132 0.045 0.009
120 0.108 0.123 136 0.054 0.019
124 0.278 0.255 140 0.252 0.302
128 0.203 0.217 144 0.277 0.189
132 0.009 0.000 148 0.109 0.236
136 0.014 0.000 152 0.168 0.113
140 0.042 0.000 156 0.054 0.000

Afu68b 160 0.005 0.094
133 0.000 0.019 Aqu63
153 0.018 0.085 127 0.408 0.509
157 0.005 0.000 139 0.335 0.274
161 0.000 0.009 143 0.257 0.217
165 0.009 0.000 Aqu74
169 0.078 0.113 218 0.630 0.698
173 0.349 0.274 222 0.088 0.151
177 0.358 0.434 226 0.282 0.151
181 0.023 0.009 Aqu112
185 0.096 0.019 244 0.354 0.198
189 0.060 0.038 248 0.005 0.000
193 0.005 0.000 252 0.161 0.255

Spl120 256 0.254 0.208
254 0.285 0.283 260 0.161 0.075
258 0.370 0.330 264 0.073 0.264
262 0.140 0.094
274 0.080 0.160
278 0.060 0.085
282 0.065 0.047

on27
130 0.735 0.802
134 0.084 0.104
138 0.181 0.094

 

70

Table 6. Measures of Genetic diversity calculated for each of the
two groups of lake sturgeon used in this study.

 

 

Mean #

alleles per Heterozygosity Heterozygosity
Group locus Observed Expected
2001 Spawning
Adults 6.0 0.624 0.679
1983, 1984,
1988 Stocked
Cohort 5.3 0.594 0.656

 

71

K To Po ulations

1. Bad River

2. Sturgeon River

3. Menominee River
4. Peshtigo River
5. Oconto River

6. Wolf River
7. Fox River

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Figure 1. Map showing the locations of the 10 lake sturgeon populations in Michigan and Wisconsin that were sampled for

this study.

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Mean=1.93 02:2.20

 

 

Figure 5. Graphs showing the number of mates per female (5a) and the number of mates
per male (5b) for the 111 adult lake sturgeon in this study.

76

 

 

 

 

190
o o
o o
o o e. I

A o o o o o
E o o 3“. on . o
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To 150-
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LL

14o - o o

r = 0.068; P>0.436
130 l I I l
100 120 140 160 180 200

Male Total Length (cm)

Figure 6. Graph showing the relationship between total length of males and females that
spawned with one another in Black Lake in 2001. The low correlation observed suggests
that size-based assortive mating is not taking place.

77

 

11>

Offspring Produced

 

13 5 7 911131517192123252729313335373941
Female

 

Mean = 5.05 dz = 21.02

 

 

 

Offspring Produced

 

. 159131721252933374145495357616569
l Male

Mean = 3.16 02 = 8.02

 

 

Figure 7. Graph showing the numbers of offspring produced for females (7a) and males
(7b) for the 111 lake sturgeon in this study.

78

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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79

Figure 8a. Graph showing the number of offspring each female produced in each one of the groups of out-migrating juveniles.

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