C S A S S C C S

C S A S S C C S
Fisheries and Oceans
Science
Pêches et Océans
Sciences
CSAS
SCCS
Canadian Science Advisory Secretariat
Secrétariat canadien de consultation scientifique
Research Document 2001/097
Document de recherche 2001/097
Not to be cited without
permission of the authors *
Ne pas citer sans
autorisation des auteurs *
High Levels of Genetic Variation in Northern Abalone
Haliotis kamtschatkana of British Columbia
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R.E. Withler , A. Campbell , S. Li , K.M. Miller , D. Brouwer , B.G. Lucas
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Fisheries and Oceans Canada
Aquaculture Division, Science Branch
Pacific Biological Station
Nanaimo, B.C.
V9R 5K6
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Fisheries and Oceans Canada
Stock Assessment Division, Science Branch
Pacific Biological Station
Nanaimo, B.C.
V9R 5K6
* This series documents the scientific basis for
the evaluation of fisheries resources in
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documents it contains are not intended as
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* La présente série documente les bases
scientifiques des évaluations des ressources
halieutiques du Canada.
Elle traite des
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Les documents qu’elle contient ne
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ISSN 1480-4883
Ottawa, 2001
1.0
ABSTRACT
Northern abalone (Haliotis kamtschatkana), from 18 sites in British Columbia and one site in
southeastern Alaska, were surveyed for variation at 12 polymorphic microsatellite loci. In all
samples, levels of observed heterozygosity were high (Ho = 0.64-0.74) but lower than values
expected (He = 0.88-0.90) under conditions of Hardy Weinberg equilibrium (HWE), due to
heterozygote deficiencies at all 12 loci. Levels of excess homozygosity varied more among loci
(fis = 0.02-0.55) than among samples (fis = 0.16-0.28), indicating that inbreeding alone did not
account for the large homozygote excess observed at some loci. Based on the six loci at which
genotypic frequencies were closest to HWE expectations, the estimated level of inbreeding in
northern abalone aggregations was 0.06. The high level of He characterizing all samples resulted
in a large estimated effective population size for northern abalone (420,000), consistent with a
high estimate for the historical average number of migrants entering abalone aggregations each
generation (~20). Hierarchical analysis of gene diversity revealed that 99.6% of genetic
variation was contained within abalone samples and only 0.4% partitioned among samples.
Approximately 0.2% of variation was accounted for by differentiation between abalone of the
Queen Charlotte Islands and Alaska and those found in central and southern British Columbia,
and the remaining 0.2% was due to differences among samples within each of those two regions.
The results indicate that, historically, northern abalone aggregations did not represent isolated
breeding units and any disruption of gene flow that may have been caused by recent low
abundance levels cannot yet be detected in non-size-structured samples of adult abalone.
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1.1
RESUMÉ
On a étudié les variations de 12 loci de microsatellites polymorphes chez des ormeaux nordiques
(Haliotis kamtschatkana) provenant de 18 endroits de la Colombie-Britannique et d’un site dans
le sud-est de l’Alaska. Tous les échantillons présentaient des niveaux d’hétézygotie élevés (Ho =
0,64-0,74), mais inférieurs aux valeurs prévues en conditions d’équilibre Hardy-Weinberg
(EHW), soit He = 0,88-0,90, en raison de déficits d’hétérozygotie aux 12 loci. Les niveaux
d’homozygotie excédentaire variaient davantage entre les loci (coefficient de consanguinité =
0,02-0,55) qu’entre les échantillons (coefficient de consanguinité = 0,16-0,28), ce qui indique
que la consanguinité ne peut expliquer à elle seule les grands excès d’homozygotie observés pour
certains loci. En se fondant sur les six loci dont les fréquences génotypiques s’approchaient le
plus des valeurs à l’EHW, on estime à 0,06 le niveau de consanguinité des concentrations
d’ormeaux nordiques. En raison de la valeur élevée de He pour tous les échantillons, la taille
effective estimée de la population était élevée (420 000), ce qui correspond à l’estimation élevée
du nombre moyen des immigrants qui se joignaient par le passé à des concentrations d’ormeaux
chaque génération (~20). L’analyse hiérarchique de la diversité génétique a montré que 99,6 %
de la variation génétique se trouvait à l’intérieur des échantillons, contre seulement 0,4 % d’un
échantillon à l’autre. Les différences entre les ormeaux des îles de la Reine-Charlotte et de
l’Alaska, d’une part, et du centre et du sud de la Colombie-Britannique, d’autre part,
représentaient environ 0,2 % de la variation inter-échantillons, tandis que le 0,2 % restant était
attribuable aux différences entre les échantillons dans chacune de ces deux régions. Les résultats
indiquent que les concentrations d’ormeaux nordiques ne constituaient pas des unités de
reproduction isolées par le passé et qu’on ne peut pas encore détecter, dans des échantillons
d’ormeaux adultes non classés par taille, de perturbation des flux géniques qui aurait été causée
par les faibles abondances récentes de ce mollusque.
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2.0
INTRODUCTION
Northern or pinto abalone (Haliotis kamtschatkana) are confined to the northeastern
Pacific Ocean, inhabiting shallow (low intertidal to 30 m depth) coastal waters from southern
California to Alaska. This abalone species occurs in patchy distribution on exposed and semiexposed rocky coasts in British Columbia. The biology and ecology of H. kamtschatkana was
reviewed by Sloan and Breen (1988). The abundance of northern abalone declined by 75-80%
during 1978-90 and, for over a decade, remained low in spite of a complete harvest closure since
1990 in British Columbia (Campbell 2000). On 23 April, 1999, northern abalone was listed as a
“threatened” species (i.e., one likely to become in imminent danger of extinction or extirpation if
limiting factors are not reversed) by the Committee on the Status of Endangered Wildlife in
Canada and has continued with this status to date (COSEWIC 2000). An examination of
population structure in northern abalone has been undertaken as an initial step in the process of
developing a comprehensive management plan for the species in British Columbia.
Species at low abundance partitioned into isolated small populations are at risk of
extirpation and extinction from stochastic demographic, environmental and genetic factors. The
biology of northern abalone indicates that this species may be especially vulnerable to processes
in all three of these categories. The current low abundance and low densities of mature abalone
(Campbell 2000) may reflect not only the commercial harvesting that occurred between 1970
and 1990, but also adverse environmental conditions that may have hindered successful
recruitment over the period 1975-1983 (Breen 1986) and that may persist. In turn, the low
abalone abundance hinders successful spawning because external fertilization requires highdensity aggregations of mature individuals (Babcock and Keesing 1999). Finally, reduced
spawning success may lead to the loss of genetic variation within local populations due to
inbreeding and genetic drift, and the disruption of larval-mediated gene flow among local
populations that might normally counteract the erosion of diversity within local populations.
The level and distance of larval dispersal are central to both demographic and genetic
processes in sedentary marine organisms. Typically, levels of dispersal for abalone species are
not known but are apparently sufficiently low to ensure that demographic processes occur on a
local scale (i.e., recruitment is primarily local, ranging from a few m or km) and sufficiently high
enough to prevent strong genetic differentiation over large geographic ranges (Brown 1991;
Hamm and Burton 2000; Huang et al. 2000). Nevertheless, along the coasts of both southern
Australia and California, genetic studies have provided evidence of different scales of population
structure in sympatric abalone species, indicating that factors such as habitat utilization,
spawning season, and larval duration may result in very different population structures among
species. Alternately, Kyle and Boulding (2000) suggested that historical demographic events
rather than differences in current levels of gene flow might underlie different degrees of
population structure in two sympatric species of littorinid snails with planktonic larvae.
For blacklip abalone, H. rubra, sampled along the southern coastline of Australia, genetic
data from allozyme, RAPD, minisatellite and microsatellite loci all indicated that there was
‘isolation by distance’, but that even the most geographically distant (>1000 km) populations
were genetically similar. The FST value for this species estimated from allozymes was 0.022
(Brown 1991) and from microsatellites was 0.077 (Huang et al. 2000). Greater microspatial
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genetic heterogeneity was observed in an Australian greenlip abalone, H. laevigata, a species
with a more patchy distribution than H. rubra, at both allozyme and RAPD loci. However,
whereas the allozyme study indicated some isolation-by-distance for H. laevigata the estimated
FST value (0.014) was not greater than that of the blacklip abalone (Brown and Murray 1992;
Shepherd and Brown 1993). Microspatial variability was also evident in the sympatric Roe’s
abalone, H. roei, a species demonstrating even greater genetic homogeneity over large distances.
For this species, the FST value estimated from allozyme loci for samples collected over an almost
3000 km stretch of coastline was 0.009 (Hancock 2000).
Differences in population structure have also been observed in two sympatric abalone
species along the coast of California. Samples of red abalone, H. rufescens, from northern and
southern California were little differentiated at allozyme loci, in mitochondrial DNA sequence,
or at a single microsatellite locus (Gaffney et al. 1996; Kirby et al. 1998; Burton and Tegner
2000). The FST value of 0.012 estimated among three samples from allozyme data was not
significantly different from zero (Burton and Tegner 2000). In contrast, allozyme data for black
abalone, H. cracherodii, indicated significant genetic differentiation among samples collected
along the central Californian coast (FST = 0.039), a relatively high level of population subdivision
that was attributed to a restricted spawning season that limits larval dispersal (Hamm and Burton
2000). These results led the authors to conclude that immigration from distant sources was
unlikely to be sufficiently great to accelerate recovery in the depleted black abalone populations
of southern California, estimated to have declined in abundance by as much as 97% (Altstatt et
al. 1996).
The relatively low levels of intraspecific differentiation observed in abalone species has
led to the suggestion that the partitioning of genetic diversity within abalone species may be
most amenable to examination with highly polymorphic, rapidly evolving microsatellite loci
(Huang et al. 2000; Withler 2000). In the present study, we survey variation at twelve
polymorphic microsatellite loci in northern abalone collected from 18 sites in British Columbia
and one site in southeast Alaska. We analyze the observed allelic and genotypic frequencies in
the abalone samples to determine the levels of genetic variation within and among aggregations
of abalone in British Columbia, and to estimate effective population sizes and inbreeding levels
for the species. We examine allele frequency distributions for evidence of recent bottlenecks in
population abundance that might have reduced genetic variation within, or increased variation
among, extant abalone populations. We incorporate the genetic data into recommendations for
conservation efforts likely to benefit the northern abalone of British Columbia.
3.0
MATERIALS AND METHODS
Epipodial tissue samples from adult abalone were collected from 18 sites within British
Columbia and one site in southeast Alaska during 1998, 1999 and 2000 (Table 1, Fig. 1).
SCUBA dive teams searched for emergent or exposed (visible on rocks) individuals because
most are easily found, whereas immature abalone tend to be cryptic (Campbell 1996). Samples
from abalone within 10-200m were used to represent each collection area. The small epidodial
tissue sample removal from each abalone was considered non-destructive, causing no mortality
to the abalone (A. Campbell unpublished data on a laboratory experiment). In addition to the 95
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tissue samples collected from Denman/Chrome Island in 2000, tissue samples were taken from
45 illegally harvested abalone putatively collected near Chrome Island in the Strait of Georgia in
1999 (Fig. 1). Samples were stored in 95% ethanol prior to DNA extraction using DNeasy kits
(Qiagen, Valencia, CA).
Variation at 12 microsatellite loci isolated from northern abalone was surveyed using the
primers and protocols outlined by Miller et al. (in press). For each abalone sampled, alleles were
amplified for each locus using the polymerase chain reaction (PCR) and sized using standard
electrophoretic techniques on an ABI 377 automated DNA sequencer. The 12 microsatellite loci
consisted of simple and compound di-, tri- and tetra-nucleotide repeat sequences. Alleles at each
locus were generally differentiated by the number of basepairs (bp) of the predominant repeat
unit, but alleles differentiated by a single bp were observed at some of the complex loci (Table
2).
Analysis of the allelic and genotypic frequency data was carried out using the Genetic
Data Analysis (GDA) program of Lewis and Zaykin (2000) and GENEPOP version 3.1d
(Raymond and Rousset 1995). Genotypic frequencies at each locus in each sample were tested
for conformance to Hardy Weinberg equilibrium (HWE) distributions in GENEPOP. FST and
Nei’s (1972) genetic distance values were computed using GDA among all samples. The
significance of multilocus FST values was determined by bootstrapping over loci and of singlelocus FST values by jackknifing over samples. In GDA, FST was calculated for multiple alleles
and loci according to Weir and Cockerham (1984). Non-zero estimates of FST values for a group
of samples indicate that the individuals of each sample are more closely related to each other (i.e.
have a more recent common ancestor) than they are to individuals of the other samples. Nei’s
(1972) genetic distance is a standard distance measurement based on differences in allele
frequencies between samples. Cavalli-Sforza and Edward’s (1967) chord distance among
samples was computed using PHYLIP (Felsenstein 1993). GENEPOP was used to perform
Mantel’s (1967) regression of the pairwise linearized FST values [(1-FST)/FST] on the natural
logarithm of geographic distance to test for ‘isolation by distance’ among abalone samples.
The genetic distance calculated from the six loci with the smallest heterozygote
deficiencies relative to HWE expectations and the chord distance values calculated from all
twelve loci were independently clustered with the neighbor-joining algorithm to provide
dendrograms of the genetic relationships among abalone samples. The pairwise average number
of migrants (Nm) between samples was estimated by the private alleles method of Barton and
Slatkin (1986) using GENEPOP and with the expression FST = 1/(4Nm + 1), a relationship based
on the assumption of island model of population structure (Whitlock and McCauley 1999). The
effective population size (Ne) for northern abalone was calculated from expected heterozygosity
(He) values for the 12 microsatellite loci using the relationship Ne = (1/[1-He]2-1)/8µ, where µ is
the mutation rate for the microsatellite loci (Lehmann et al. 1998). Little is known of the
mutation rate of microsatellite loci in invertebrate organisms except Drosophila, in which the
observed rate (~10-6) is much lower than in mammals (~10-4). Ne for northern abalone was
estimated in this study using the conservative assumption that µ = 10-4, with recognition that Ne
values are 100 times greater if the true value is 10-6.
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Hierarchical analyses of allele frequency variation were carried out with nested ANOVA
(random effects model) as described by Weir (1996). The proportions of the observed variation
attributable to regions (the Queen Charlotte Islands [QCI], central coast of British Columbia,
west coast of Vancouver Island and Georgia Strait), to sample sites within regions, and to genetic
variation within sample sites were determined. Geographic variation was examined with two
models. The four-region model examined variation among the four regions listed above
(samples shown by region in Table 1). The two-region model examined differentiation between
the QCI and the Alaskan sample, which were distinctive in the dendrograms, and all other
samples. Both models were used with the data from all 12 microsatellite loci and with the data
from six loci at which genotypes mostly closely approximated HWE frequencies.
Heterogeneity among cohorts (age classes) within samples was investigated in the samples from
five sites. Abalone were divided into four size classes based on shell length (SL): ≤50 mm
(immature), 51-69 mm (transition of immature to mature), 70-99 mm (mature) and >99 mm
(fishery), defined by size at maturity estimates by Campbell et al. (1992). Each of these size
groups contained a range of ages whose growth rates could have been influenced by local
environmental conditions: < 2 to < 4 years (≤50 mm SL), between 2 and 7 years (51-69 mm SL),
between 3 and 14 years (70-99 mm SL) and >6 or >14 years (>99 mm SL) estimated from Sloan
and Breen (1988 see Fig. 8). The maximum age of H. kamtschatkana is not known, but
individuals reach ages of 30 years and older (Breen 1980). Thus, the potential number of cohorts
contained within each size class increases with size class. Allele frequencies in the two or three
size classes containing the most abalone at each site were analyzed by ANOVA to examine the
possibility that small numbers of adults contribute to recruitment in individual cohorts of
northern abalone, leading to low genetic variability within cohorts and significant variation
among cohorts within abalone aggregations.
4.0
RESULTS
4.1
Genetic variation within populations
All microsatellite loci examined were highly polymorphic, exhibiting high numbers of
alleles and high values of both observed (Ho) and expected (He) (under conditions of HWE)
heterozygosities (Table 2). Genotypes at all twelve loci showed a significant excess of
homozygotes in comparison to those expected under HWE, but the level of heterozygote
deficiency varied greatly among loci (Table 2). Estimates of fis (the level of inbreeding if the
excess of homozygotes was due entirely to assortative mating among relatives) ranged from 0.02
at Hka43 to 0.55 at Hka85.
All 20 samples of northern abalone displayed high levels of genetic variability. Allelic
diversity (mean numbers of alleles observed over all loci) was high and relatively constant
among samples (Table 3). Average Ho by sample ranged from 0.64 – 0.74 (mean of 0.71), but in
all cases was less than the He which was essentially 0.89 for all samples (Table 3). Thus, the
estimated fis value varied much less among samples (from 0.16 to 0.28) than among loci. The
great range of fis values among loci and the consistency of the fis values for a given locus among
samples indicate that inbreeding is not the sole explanation for the observed heterozygote
deficits.
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Using the mammalian microsatellite mutation rate (10-4) and He values estimated for the
abalone microsatellite loci of this study, we obtained locus-specific estimates of effective
population size (Ne) ranging from 10,000 to 1,390,000, and a mean value of 420,000 (Table 2).
Use of the possibly more realistic mutation rate of 10-6 provides estimates 100 times larger.
4.2
Genetic variation among samples
The FST value calculated from all 12 loci among all samples was low but significantly
greater than zero (0.003; SE 0.0007). Calculated from the six loci closest to HWE that could be
consistently scored in over 80% of abalone among samples (Hka12, Hka37, Hka40, Hka43,
Hka56 and Hka65), the FST value was reduced but remained significantly greater than zero
(0.002; SE 0.0006). Examined on a single locus basis, FST values ranged from 0.0 to 0.010, and
were significantly greater than 0 for 8 of the 12 loci examined (Table 2).
There was not a strong geographically-based grouping of samples apparent in either the
dendrogram based on CSE chord distance calculated from 12 loci (Fig. 2) or the dendrogram
based on Nei’s genetic distance values calculated from 6 loci (Fig. 3). In both dendrograms, the
six QCI and single Alaskan sample clustered together, but the central coast, Georgia Strait and
west coast Vancouver Island samples did not cluster geographically. In both dendrograms the
two samples from the west coast of Vancouver Island (Elbow Island and Vargas Island) not only
failed to cluster together, but one (Elbow Island) clustered with the QCI samples whereas the
other (Vargas Island) clustered with the non-QCI samples. In contrast, the two samples from
Chrome/Denman Island (1999 and 2000) did cluster together, albeit not strongly (as indicated by
the relatively long branch distances joining them). However, because the 1999 sample consisted
of illegally harvested abalone and was attributed to the Chrome Island site by hearsay, the
significance of this relationship cannot be evaluated. The central coast samples did not form a
strong group based on either the 6-locus or the 12-locus analysis. None of the nodes in the CSE
dendrogram was supported by a bootstrap value of greater than 50%, indicating weak support for
the depicted sample structure.
The hierarchical analyses of gene diversity indicated that 99.6% of the observed genetic
variation occurred within samples and only 0.4% was attributable to differentiation among
samples (Table 4). Of the differentiation among samples, approximately half (0.2%) was due to
differences among regions and the other half to differences among samples within regions. This
result did not vary among the various models used to test regional and sample differentiation
(Table 4). Thus, the use of the six loci listed above at which genotypes within samples were
closest to HWE expectations gave the same result as the use of all 12 loci. Similarly, including
the single Alaskan sample from Sitka Sound in model as a member of the QCI region did not
change the proportion of variation accounted for by region. Finally, region accounted for 0.2%
of the total variation in both the four-region and two-region models, but the effect of region was
not significant in either the four-region (F3,16 = 1.75, P > 0.10) or the two-region model (F1,18 =
2.53, P > 0.10). Thus although it was primarily the differentiation between QCI and non-QCI
samples that accounted for the regional variance component, the distinction in allele frequencies
between the two regions was small. Similarly, regardless of the model or number of loci used,
the variation among samples within region was not significant (all P > 0.05).
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The correlation of the linearized FST values with geographic distance approached
significance (P=0.06), but geographic distance accounted for very little of the observed variation
in FST values (r2 = 0.05)(Fig. 4). Again, pairwise FST values tended to be less in comparisons of
QCI samples with each other (0.003), non-QCI samples with each other (0.003), than between
QCI and non-QCI samples (0.004). The distinctiveness of the QCI and Alaskan samples and
their relatively great geographic distance from many of the remaining samples accounts for the
weak relationship between geographic and genetic differentiation.
The average number of migrants per generation into the abalone aggregations represented
by each sample was estimated by the private alleles method as 19.9, a number consistent with the
observed lack of genetic differentiation among samples. This value changed little when only
QCI/Alaskan samples (18.4) or only non-QCI samples (20.3) were considered. Calculating the
average number of migrants using the standard expectation for the relationship between FST and
Nm provided an estimated 83 migrants entering abalone aggregations each generation.
4.3
Genetic variation between size (age) groups within samples
FST values calculated over two or three size (age) samples of abalone from five sites
averaged 0.001, and ranged from 0 to 0.027. Allele frequencies did not differ significantly in
pairwise comparisons between size classes within each site. The numbers of alleles observed in
the smaller (younger) abalone at each site tended to be less than in the older abalone (Table 5).
This was likely due to the smaller sample sizes of younger abalone rather than a real reduction in
allelic diversity because the expected heterozygosity values were the same among size classes
(Table 5). The data provided an indication that levels of inbreeding within the smaller size
classes, composed of fewer cohorts, was greater than in larger size classes. Only in the sample
from Hankin Point, was the inbreeding level higher in the larger abalone (Table 5). Thus, the
inbreeding observed in northern abalone likely results from the settlement and recruitment of
abalone in locations very close to their parents. However, the fact that the smaller size classes
retained high levels of allelic diversity and that age groups were not strongly differentiated in
allele frequencies indicated that the number of abalone participating in individual spawning
events was not extremely low.
5.0
DISCUSSION
This study provides the first analysis of genetic structure in H. kamtschatkana and the
first comprehensive examination of population structure in any abalone species based on
microsatellite variation. The study provided evidence for very high levels of genetic variation
within northern abalone aggregations and very low levels of differentiation among samples
collected from throughout British Columbia, including a single sample from southeast Alaska.
Fully 99.6% of the observed variation was contained within samples, with only 0.2% attributable
to two regional groupings of abalone (QCI/Alaska vs non-QCI) and 0.2% attributable to
variation among the samples within those two regions. The lack of strong differentiation among
samples and weak evidence for ‘isolation by distance’ suggests that gene flow among abalone
breeding aggregations throughout British Columbia is, or has been, extensive. If recent low
levels of abundance have disrupted historical patterns of gene flow, it is not yet evident among
mature abalone of the age groups encompassed in this study.
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The abalone of the QCI were slightly differentiated from abalone in other areas of British
Columbia, perhaps as the result of restricted larval exchange in oceanographic currents, or
perhaps because of historical isolation. During the last Cordilleran glaciation of North America,
which ended approximately 12,000 years ago, the QCI and Alaskan coastal regions may have
provided refugial habitat for terrestrial and marine organisms (Warner et al. 1982). Northern
abalone throughout much of coastal British Columbia and those of the QCI (and perhaps
northern BC and southeast Alaska) may be descendants of different refugial populations. Two
distinctive clades in mitochondrial DNA sequences of the littorinid snail Littorina subrotundata
throughout British Columbia and Washington have been attributed to dispersal from separate
glacial refugia (Kyle and Boulding 1998, 2000). An examination of mtDNA variability in
northern abalone might enable determination if the small degree of differentiation at
microsatellite loci is due to historical isolation in separate refugia or more recent restrictions of
gene flow between coastal and QCI habitats. Even if extant abalone are descendants of different
refugial populations, the high level of intraspecific variability and low level of intersample
differentiation indicate that refugial population sizes were large and limited genetic divergence
occurred during isolation, or that gene flow has occurred since the glacial period.
All of the microsatellite loci examined in this study exhibited an excess of homozygosity
relative to genotypic distributions expected under Hardy Weinberg equilibrium conditions.
Homozygote excess has been observed in other population surveys of abalone at microsatellite
and allozyme loci and was generally attributed to inbreeding (Huang et al. 2000; Brown 1991;
Hara and Kikuchi 1992). In this study, variation among loci in the level of heterozygote
deficiency observed makes it likely that locus-specific factors are also involved. As in the study
of Huang et al. (2000), we tested multiple sets of primers for each locus in an attempt to
eliminate the problem of non-amplifying alleles that result from mutations in flanking sequences
used for primer design. For some of the loci in this study, judicious choice of primers reduced or
eliminated non-amplifying alleles, whereas for other loci, the various primer sets tested all gave
equivalent levels of excess homozygosity. Levels of excess homozygosity differed little among
samples, with the fis values (ranging from 0.16-0.28) merely reflecting the average of the fis
values among loci (0.21). Thus, some level of inbreeding may occur in northern abalone, as in
other abalone species, the level of which is best estimated by those loci showing the least
evidence of non-amplifying alleles (i.e. those loci with genotypic frequencies closest to HWE).
At two loci, Hka3 and Hka85, only approximately half of the individuals surveyed were
heterozygous although the high levels of polymorphism indicated that virtually all individuals
should be heterozygous under HWE conditions. One or both of these loci may be located on
only one member of a dimorphic pair of sex chromosomes, so that the gender which carries
heteromorphic sex chromosomes is hemizygous for the locus (i.e. carries an allele on only one
sex chromosome) but was scored as homozygous in the present study. Although both Hka3 and
Hka85 may be located on a sex chromosomes they were in linkage equilibrium (i.e. they are not
linked to each other)
Six of 12 loci could be consistently scored in all samples and exhibited fis values of 0.10
or less, with an average value of 0.06. This may represent the typical level of inbreeding in
northern abalone populations. High levels of local larval recruitment and/or asynchronous
spawning on a small geographic scale may contribute to inbreeding in H. kamtschatkana, as
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suggested for blacklip abalone (Huang et al. 2000). The analysis of inbreeding among size
classes in this study indicated that inbreeding tended to be less in larger size classes (potentially
containing individuals from more cohorts) than in smaller size classes (potentially containing
individuals from fewer cohorts). Thus, relatedness may be greater among the recruits within a
site from individual spawning events than among those from different years. Recruitment may
be very local with progeny settling near their parents and/or the number of individuals that
spawn successfully at any one time may be small. To some extent, these results are consistent
with a model of “sweepstakes-style” recruitment success (Hedgecock 1994). According to this
model, only a small fraction of mature adults effectively contribute to reproduction in each
generation because of a limited window of oceanographic conditions compatible with successful
spawning and/or recruitment. The resultant spatial and temporal variability in recruitment
success may lead to detectable genetic drift among cohorts and to ‘chaotic genetic patchiness’, in
which samples in very close proximity are as genetically differentiated as ones very far apart
(Larson and Julian 1999). Although proximal samples of northern abalone in this study were
sometimes as different as distal ones, all samples were highly polymorphic and little
differentiated. Moreover, FST values were on average less (0.001) among age classes within a
site than among sites (0.002), and in no case was the FST value among age classes within a site
significantly greater than zero. Thus, there was little evidence that the successful spawners at
any given time were sufficiently small in number or closely related to result in genetic drift.
Moberg and Burton (2000) found significant spatial and temporal genetic variability
among and within three size classes of red sea urchin (Strongylocentrotus franciscanus) along
the coast of California. Adult, sub-adult and recruit urchins collected from the same site
frequently possessed significantly different allozyme frequencies, a finding largely attributed to
inter-family variation in reproductive success. For northern abalone, the pooling of multiple
ages (recruitment events) in each of the size classes examined may have obscured variation in
individual spawning seasons, but the low levels of differentiation observed among sites over the
1200 km range examined in this study supports the indication that stochastic variation in
reproduction/recruitment may be less in northern abalone than in sea urchin species.
All samples of northern abalone in this study were characterized by a high abundance of
rare alleles (>80% of alleles were present at frequencies < 0.1), thus producing L-shaped
distributions of allele frequencies. This indicates that populations have existed at long-term
stable sizes (i.e., not suffered recent bottlenecks) (Luikart et al. 1998). The high values of
heterozygosity (He) observed for northern abalone in this study led to high estimates of effective
population size, indicating that the small local aggregations of mature abalone observed in
census studies (Campbell 2000; Wallace 1999) did not represent genetically isolated breeding
units. ‘Cryptic’ abalone, not recently included in census counts, possibly also contribute to
reproduction in northern abalone. However, it is evident that local northern abalone
aggregations are, or have been, connected by gene flow as the result of larval dispersal.
Spawning is seasonal, usually restricted to summer months (i.e. May-August), and the pelagic
larval stage is of short duration, although it varies (4 to 8 days) with local factors such as
temperature (14 to 10oC) (Sloan and Breen 1988).
The finding that larval dispersal has been sufficient to prevent strong genetic
differentiation throughout much of coastal British Columbia in an abalone species in which
- 11 -
spawning is seasonal is contrary to the results for another seasonal spawner, the black abalone, in
California (Hamm and Burton 2000). In that species, FST values ten times higher than those
estimated in this study were obtained from variation at three allozyme loci and the level of
population differentiation was attributed at least in part to the limited spawning season and
strong seasonal differences in oceanographic patterns in the coastal waters of California. The
lack of genetic structure in northern abalone is more similar to the low level of genetic
differentiation observed in the red abalone of California, which spawns throughout the year
(Burton and Tegner 2000) and in three sympatric abalone species inhabiting the waters of
southern Australia. The Australian blacklip, greenlip and Roe’s abalone all show low levels of
genetic differentiation over spatial scales as large or larger than those encompassed in the present
study (Brown 1991, Brown and Murray 1992, Hancock 2000). For H. roei, the FST value
estimated from eight allozyme loci among samples collected over almost 3000 km was 0.009.
As for northern abalone, FST values between proximate samples of Roe’s abalone could be as
great as between distal samples, especially over distances of 1200 km or less. However, the
greater differentiation of samples separated by more than 1500 km in that species gave a clear
indication of isolation by distance over large distances. Hancock (2000) suggested that the
small-scale heterogeneity in allele frequencies in Roe’s abalone was due to predominantly local
recruitment, with the high gene flow resulting more from large effective population sizes than
from large migration rates. Moreover, he suggested that rare cases of successful long-distance
dispersal might play a role in maintaining the observed large-scale genetic homogeneity.
For northern abalone, in which the mean FST value (0.002) was even lower than in Roe’s
abalone, large effective population sizes likely have also contributed to the observed genetic
homogeneity. However, estimates of the average number of successful migrants among the
samples of this study were also relatively large. Whether successful larval dispersal in the
northern abalone occurs on a regular basis or is predominantly the result of rare, but highly
effective, long distance dispersal events is not known. In either case, the lack of genetic
differentiation at neutral genetic markers such as microsatellite loci does not preclude the
possibility of adaptive genetic differentiation over the range. Although small breeding
aggregates of northern abalone clearly do not represent strongly isolated subpopulations,
adaptation to environmental conditions may be assumed to occur on a broad regional basis.
Long-lived species may maintain genetic variation even in the face of fluctuating
environments and recruitment because of the “storage capacity’ that results from the large cohort
of adults produced from each strong recruitment (Warner and Chesson 1985; Ellner and Hairston
1994; Ellner 1996; Gaggiotti and Vetter 1999). These species effectively ‘store’ a large number
of genotypes within the reproductive population over many reproductive periods that are capable
of contributing to both population size and genetic diversity when favourable spawning and
recruitment conditions return. However, extended periods of low reproductive/recruitment
success may be masked in genetic surveys heavily influenced by the genetic variability being
stored in, but not transmitted from, the older age groups. Gaggiotti and Vetter (1999) suggest
that even when marine fisheries collapse, exploited species may be close to extinction as the
result of demographic or environmental stochasticity before a marked reduction in genetic
variation occurs. For a species such as northern abalone, in which historical effective population
sizes have been large and the reservoir of neutral genetic variation in the older size classes is
correspondingly great, genetic surveys conducted on a non-size-structured basis may fail to
- 12 -
detect population fragmentation and increased genetic drift due to low abundance levels if and
when they occur. Early detection of genetic changes would be facilitated by size-structured
analysis of samples on the finest scale achievable.
The analysis of genetic variation in different size (age) classes of abalone at several sites
in this study provided no indication that younger age classes were less diverse than older ones,
but the sampling of the younger ages was restricted and did not include newly recruited ‘cryptic’
individuals. In the black abalone of southern California, recruitment failure was observed after
abalone abundance dropped by approximately 50% (Richards and Davis 1993). Because of the
‘storage capacity’ in the older individuals of abalone populations, it is essential that recruitment
be measured to determine current levels of reproductive success. Longterm genetic monitoring
of newly recruited abalone would reveal the loss of genetic diversity and population
fragmentation that might follow a disruption of gene flow at low abundances, but only some
years after the fact.
As for other overfished marine organisms, efforts to rebuild depleted abalone populations
by out-planting of hatchery-reared larvae or juveniles have been largely unsuccessful. In
addition to considerable technical problems with producing healthy seed, out-planted abalone
tend to experience high mortality due to natural or human predation (Tegner 2000; Shepherd et
al. 2000). Nevertheless, given good quality seed, favourable environmental conditions and
sufficient protection after out-planting, there may be circumstances under which enhancement
can contribute to stock rebuilding efforts (Shepherd et al. 2000). Genetic concerns associated
with enhancement include the possibility of disrupting natural populations by out-planting
abalone from a distant source that are adapted to different environmental conditions. Such
abalone may not survive to reproduce but, if they do, threaten the genetic integrity of natural
abalone populations in the transplant area (Tringali and Bert 1998, Shaklee and Bentzen 1998;
Utter 1998). For northern abalone, transplantation should be confined within major coastal
regions (e.g. Strait of Georgia, west coast of Vancouver Island, central coast, Queen Charlotte
Islands) in order to avoid the introgression of maladaptive genes into natural populations. Two
other concerns associated with the out-plant of hatchery produced organisms are the random loss
of genetic diversity due to a limited number of spawners and, if the broodstock is maintained in
the hatchery over generations, the development of a strain that is not well adapted to survival and
reproduction in the wild. Hatchery strains that are intended for reseeding into natural
populations should be carefully monitored to ensure that high levels of genetic variation are
maintained, and should be open populations that incorporate naturally produced individuals on a
regular basis. Genetic monitoring may also contribute to evaluation of the success of
enhancement efforts (Burton and Tegner 2000).
The northern abalone of British Columbia likely constitute a single evolutionarily
significant unit (ESU) (Moritz 1994), although additional genetic investigation may establish the
abalone of the QCI and northern British Columbia as an independent ESU. The microsatellite
data of this study indicate that an extremely high level of genetic variation exists in adult
aggregates throughout the province. This reflects historical and/or current gene flow that has
been of sufficient magnitude to prevent population subdivision even in the face of highly
localized larval recruitment and resultant inbreeding levels of ≥ 0.06. Nevertheless, stock
assessment data for areas in close proximity experiencing different environmental conditions and
- 13 -
levels of harvest restriction indicate that the demographic characteristics of abalone aggregates
can be independent on small geographic scales (for literature reviews see Sloan and Breen 1988;
Wallace 1999; Campbell 2000). The migration rate estimated from the microsatellite data of this
study is not sufficiently high to preclude the possibility of local adaptation (genetic
differentiation at loci under natural selection) among abalone inhabiting different environments
within British Columbia. The current picture of northern abalone, as a collection of spawning
aggregates connected by gene flow, indicates that population abundance levels have not been
low for sufficiently long time periods to result in the loss of genetic diversity within aggregates
or to cause random genetic differentiation among aggregates. Nevertheless, whereas the data
indicate that extant spawning aggregates large enough to have been sampled in this study contain
high levels of genetic variation, they can not be used to determine if current levels of
reproductive success and gene flow are sufficient to maintain the diversity. Prudent management
activities would include the identification, protection and monitoring of spawning aggregates
(and recruits) on a regional basis to examine both demographic and genetic parameters for signs
of population recovery or decline.
6.0
RECOMMENDATIONS
1. This study has shown that northern abalone aggregates are not genetically depauperate or
heavily inbred, and have the genetic capacity for population expansion when favourable
environmental conditions prevail. Since adult northern abalone are sedentary, and the stock
recruitment relationships and larval dispersal mechanisms are unknown and difficult to
assess, rehabilitation management should focus on preserving existing brood stock in situ,
perhaps establishing a number of reserve areas where increased protection of local
populations is possible.
2. Attempts to re-establish or enhance abalone aggregates through out-planting have generally
been found to be unsuccessful when evaluated rigorously. The most promising method to
increase local population abundance may be to ensure successful fertilization/reproduction in
as many areas as possible. Techniques such as artificially aggregating adult abalone in areas
of low density or the out-planting of hatchery-reared juveniles may be useful but would
require careful monitoring for evaluation.
3. The number of abalone broodstock used to produce larvae or juveniles for out-planting to the
wild should be at least 50 and preferably 100, with equal numbers of males and females, in
order to maintain genetic diversity in the enhanced population. Controlled single pair mating
and/or genetic monitoring of the progeny produced in the hatchery should be undertaken to
maximize the number of parents contributing to juvenile production.
4. Out-planting and/or transplanting of northern abalone within British Columbia should be
carried out within existing management regions (west coast Vancouver Island, east coast
Vancouver Island, central coast and the Queen Charlotte Islands) to avoid the introgression
of deleterious genes into natural populations, as well as reduce the possibility of disease
transfer.
- 14 -
5. Research to determine the effect of low abundance on genetic structure in northern abalone
and evaluation of enhancement efforts will both require the ability to measure and sample
recruitment into abalone aggregates. Establishing study sites in which larval dispersal,
recruitment and microspatial and temporal genetic variability could be monitored would
enable greater understanding of the current population genetics and demographics of
northern abalone.
7.0
ACKNOWLEDGEMENTS
We thank S. Carignan, B. DeFrietas, J. Disbrow, R. Gurr, J. Harding, M. McNab, T.
Norgard, D. Miller, and D. Woodby for help with sample collections, and Dr. R.S. Burton for a
helpful review of this paper.
8.0
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- 18 -
Table 1. Locations and years in which adult H. kamtschatkana were sampled for microsatellite
analysis.
Site
Latitude
Longitude
Year
West coast Vancouver Island
Elbow Island
Vargas Island
48 54.060
49 09.429
125 16.556
125 57.729
2000
2000
Georgia Strait
Chrome/Denman Island
49 28.883
124 41.209
1999, 2000
BC central coast
Cranstown Point
Nalau Passage
Simonds Group
Iroquois Island
Stryker Island
Nowish Islands
Higgins Pass
Lotbiniere Bay
Hankin Point
51 22.500
51 47.000
51 57.800
52 02.895
52 05.990
52 31.000
52 28.500
53 00.000
54 42.400
127 46.500
128 06.500
128 16.700
128 19.445
123 23.207
128 26.000
128 45.500
129 33.000
130 24.000
1999
1999
1999
1999
1999
1999
1999
2000
2000
Queen Charlotte Islands
Louscoone Inlet
Montserrat Bay
Skincuttle inlet
Faraday Island
Virago Sound
Bruin Bay
52 07.692
52 06.227
52 20.780
52 36.770
54 04.000
54 10.017
131 14.127
130 59.170
131 14.260
131 27.800
132 31.000
132 58.752
1999
1998
1998
1998
1998
1999
Alaska
Sitka Sound
57 03.100
135 20.500
1999
- 19 -
Table 2. Microsatellite loci surveyed in H. kamtschatkana from 19 sites in British Columbia and Alaska. The nature of the
microsatellite repeat is shown as di (dinucleotide), tri (trinucleotide), tetra (tetranucleotide) or compound. The number of alleles (A)
and size range (in basepairs) for each locus are also shown. The mean levels of observed (Ho) and expected (He) heterozygosity,
estimated levels of inbreeding (fis) and variation among samples (FST) are shown for each locus. The effective population size (Ne) of
northern abalone estimated from the He values is shown.
Locus
Hka3
Hka48
Hka56
Hka65
Hka80
Hka85
Repeat
Compound
tri-tetra
Compound
tri-tetra
di
di
Compound
tri-tetra
di
tetra
di
di
di
di
tri
Mean
-
Hka6
Hka12
Hka28
Hka37
Hka40
Hka43
A
74
Size Range
200-350
He
0.97
Ho
0.45
FST
0.003 *
fis
0.53
Ne/1000
1390
32
107-206
0.75
0.47
0.010 *
0.38
20
81
38
29
171-377
183-271
236-330
0.93
0.95
0.70
0.89
0.57
0.66
0.0002
0.002 *
0.004 *
0.04
0.40
0.06
250
500
10
36
24
73
32
59
25
45
112-210
163-263
93-250
93-160
115-250
89-150
200-390
0.92
0.88
0.97
0.92
0.95
0.93
0.92
0.86
0.87
0.50
0.84
0.85
0.88
0.42
0.002 *
0.006 *
0.003 *
0.0
0.001
0.005 *
0.002
0.07
0.02
0.22
0.08
0.10
0.04
0.55
190
90
1390
190
500
250
200
45.7
-
0.90
0.71
0.003 *
0.21
420
* Significantly greater than 0 at α=0.05
- 20 -
Table 3. Genetic variation at 12 microsatellite loci in 20 samples of H. kamtschatkana
from British Columbia and Alaska. The sample size (N), mean number of alleles
observed per locus (A), observed (Ho) and expected (He) levels of heterozygosity, and
estimated level of within sample inbreeding (fis) are shown.
Site
N
A
He
Ho
Fis
WC Vancouver Isl
Elbow Island
Vargas Island
45
70
20.0
24.4
0.88
0.89
0.64
0.72
0.28
0.20
Georgia Strait
Chrome Island
Chrome Island*
70
45
22.9
20.7
0.88
0.89
0.74
0.70
0.16
0.21
BC central coast
Cranstown Point
Nalau Passage
Simonds Group
Iroquois Island
Stryker Island
Nowish Islands
Higgins Pass
Lotbiniere Bay
Hankin Point
110
115
125
110
90
112
90
28
130
27.9
28.8
26.3
29.3
24.8
27.7
25.4
16.0
28.7
0.90
0.89
0.89
0.90
0.88
0.89
0.88
0.88
0.89
0.73
0.70
0.70
0.71
0.70
0.71
0.71
0.65
0.73
0.19
0.21
0.21
0.21
0.21
0.20
0.20
0.27
0.18
Queen Charlotte Isl
Louscoone Inlet
Montserrat Bay
Skincuttle Inlet
Faraday Island
Virago Sound
Bruin Bay
130
70
73
72
70
90
30.4
25.7
25.8
25.2
24.4
28.5
0.89
0.89
0.89
0.90
0.90
0.90
0.69
0.71
0.70
0.70
0.73
0.73
0.22
0.20
0.21
0.21
0.18
0.19
Alaska
Sitka Sound
95
27.8
0.90
0.70
0.23
1740
25.5
0.89
0.71
0.21
Total/Mean
* sample of confiscated abalone attributed to Chrome Island site
- 21 -
Table 4. Hierarchial analyses of genetic diversity for 20 samples of H. kamtschatkana from British Columbia and Alaska.
Regions consisted of the Queen Charlotte Islands [QCI], central coast of British Columbia, west coast of Vancouver Island and
Georgia Strait in the four-region model, with the sample from southeast Alaska included in the QCI region or excluded from the
analysis (BC only). Regions consisted of the QCI and the remainder of British Columbia in the two-region model, with the Alaskan
sample included in the QCI region or excluded from the analysis (BC only).
Model
Absolute diversity
Total
Relative diversity
Within
Within
Among samples
Among
samples
samples
within regions
regions
Four-region - all loci
10.757
10.717
0.996
0.002
0.002
Four-region - all loci, BC only
10.750
10.712
0.996
0.002
0.002
Four-region - six loci
5.277
5.261
0.997
0.002
0.001
Two-region - all loci
10.759
10.717
0.996
0.002
0.002
Two-region - all loci, BC only
10.75
10.712
0.996
0.002
0.002
Two-region – six loci
5.278
5.261
0.996
0.002
0.002
- 22 -
Table 5. Genetic variation in immature (≤50 mm SL), transition (51-69 mm SL), mature
(70-99 mm SL) and fishery (>99 mm SL) northern abalone from five collection sites in
British Columbia. The sample size (N), the mean number of alleles observed over all loci
(A), the expected (He) and observed (Ho) heterozygosity levels estimated over all loci and
the inbreeding coefficient (fis) estimated from six loci at which genotypes were closest to
HWE distributions are given for each size group.
Size
N
A
He
Ho
6 locus fis
Louscoone
immature
transition
mature
20
66
52
15.1
24.8
21.8
0.89
0.89
0.89
0.66
0.69
0.71
0.06
0.08
0.02
Denman
mature
fishery
36
36
17.7
18.5
0.88
0.88
0.71
0.76
0.01
-0.02
Stryker
transition
mature
18
70
13.8
22.6
0.88
0.88
0.69
0.70
0.06
0.04
West coast VI
mature
fishery
46
70
20.0
24.4
0.89
0.89
0.64
0.72
0.19
0.07
Hankin Point
mature
fishery
49
80
21.8
24.7
0.89
0.89
0.73
0.74
0.04
0.06
- 23 -
N
N
Sitka
Sound
#
AL
AS
KA
BRITISH COLUMBIA
Bruin
Bay
Virago
Sound
#
Hankin Pt.
#
Lotbiniere Bay
#
QCI
Higgins Pass
Faraday
Island
Skincuttle
Inlet
Montserrat
Bay
Nowish Islands
Stryker Island
Iroquois Island
#
#
#
#
Louscoone
Inlet
#
#
#
Simonds Group
#
Nalau Pass
#
#
#
Cranstown Pt.
#
Chrome
Island
VA
N
CO
U
VE
R
IS
LA
N
Vargas
Island Elbow
#
D
#
#
Island
0
60
120
180
240 Kilometers
U.S.A.
Figure 1. Sample locations of Haliotis kamtschatkana in British Columbia and Alaska.
- 24 -
Figure 2. Neighbor joining dendrogram of Haliotis kamtschatkana samples based on
Cavalli-Sforza and Edwards (1967) chord distance calculated from all 12 microsatellite
loci.
- 25 -
Figure 3. Neighbor joining dendrogram of Haliotis kamtschatkana samples based on Nei’s (1972) genetic distance calculated from
the six microsatellite loci for which genotypic frequencies were closest to HWE expectations.
- 26 -
Fst / 1-Fst
0.012
0.008
0.004
0
2
3
4
5
6
7
8
Ln Dista nce (km )
Figure 4. Relationship between FST/(1-FST) and geographic distance for samples of Haliotis kamtschatkana from British Columbia
and southeast Alaska.
- 27 -
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