Relationship between population size and genetic diversity inendangered populations of the European bullhead (Cottus gobio):

implications for conservation

Guy Knaepkensa,*, Lieven Bervoetsb, Erik Verheyenc, Marcel Eensa

aDepartment of Biology, University of Antwerp—UIA, Universiteitsplein 1, 2610 Wilrijk, BelgiumbDepartment of Biology, University of Antwerp—RUCA, Groenenborgerlaan 171, 2610 Wilrijk, Belgium

cRoyal Belgian Institute of Natural Sciences, Vautierstraat 29, 1000 Brussels, Belgium

Received 13 June 2002; received in revised form 18 February 2003; accepted 21 February 2003

Abstract

This study investigated the relationship between the current size of endangered bullhead (Cottus gobio) populations and micro-satellite genetic variability. Additionally, the microsatellite data were used to evaluate whether a genetic test for population bottle-

necks was able to provide evidence of recent severe population declines. Finally, our results were used to develop conservationpriorities and measures. Population size appears to be a crucial parameter in determining the amount of genetic diversity that canbe preserved in bullheads, since a significant positive correlation was observed between both variables. Furthermore, in some

populations we were able to detect genetic signatures of the documented decline in population size. We suggest that the mostimmediate goal for bullhead conservation should be to increase the size and the range of the populations, and in doing so minimiseor even reverse further genetic erosion. Potential management actions like habitat quality improvement, reduction of river frag-

mentation and supplementation programmes (translocation, supportive breeding) are discussed.# 2003 Elsevier Ltd. All rights reserved.

Keywords: Conservation; Cottus gobio; Genetic diversity; Microsatellite; Population size

1. Introduction

Due to destruction and fragmentation of their habitats,many species have been forced into small and isolatedpopulations. These populations generally face appreci-able risk from the effects of environmental variation,demographic stochasticity and reduced genetic diversity(Meffe and Carroll, 1997).Maintaining genetic diversity has become a major

issue in conservation biology because it is generallythought to be important for the overall species viabilityand the potential for evolutionary responses to envir-onmental change (Meffe and Carroll, 1997). Populationsize is considered to be important to the maintenance ofgenetic variation. Population genetic theory predictsthat, as a consequence of genetic drift and/or inbreeding,

small populations should show lower levels of geneticvariability than large populations (Hartl and Clark,1997). Reduced levels of genetic diversity have in factbeen observed in small natural populations of differentspecies (for a review, see Frankham, 1996). Never-theless, there are also examples where the size of popu-lations fails to explain a significant part of the geneticvariability. For example, due to historical populationprocesses such as demographic bottlenecks or founderevents, levels of genetic diversity may be low despiteconsiderable current population sizes (Hoelzel et al.,1993; Hartl and Clark, 1997). Conversely, the loss ofgenetic diversity in small populations may be counter-acted if migration (gene flow) among populationsoccurs (Van Rossum et al., 1997). In general, popul-ation size may not always be a good predictor of geneticvariability because it does not necessarily reflect theeffective size of a population, i.e. the size of an idealpopulation experiencing the same rate of genetic changeas the natural population of interest (Crow and Kimura,

0006-3207/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/S0006-3207(03)00156-3

Biological Conservation 115 (2004) 403–410

www.elsevier.com/locate/biocon

* Corresponding author. Tel.: +32-3-820-22-86; fax: +32-3-820-

22-71.

E-mail address: guy.knaepkens@ua.ac.be (G. Knaepkens).

1970). Thus, the benefit of large population size in termsof increased genetic diversity is not self-evident, and thisshould not be forgotten in conservation planning(Fleishman et al., 2001).The European bullhead (Cottus gobio L.) is a small

bottom-dwelling freshwater fish that is widely distributedin Europe. Nevertheless, in recent decades, this species hassuffered a considerable decline in several areas like Swit-zerland, Germany and Flanders (northern part of Bel-gium; Lelek, 1987; Utzinger et al., 1998; Vandelannooteet al., 1998). In the latter, human activities such as pol-lution, fragmentation and river canalisation have mod-ified the natural riverine habitat of this species andconsequently had a large impact on the size, range andviability of the local bullhead populations (Vande-lannoote et al., 1998; Knaepkens et al., 2002b). Conse-quently, the species is regarded as highly vulnerable andis fully protected by law in Belgium (Vandelannoote etal., 1998).In the present study, we examined the relationship

between the current size of 10 Flemish bullhead popu-lations and the level of genetic diversity using variationat seven microsatellite loci, i.e. DNA sequences thatconsist of a variable number of tandemly repeated (1–6bp) DNA motifs (Goldstein and Schlotterer, 1998). Thisinformation may lead to a better understanding of theimportance of current population size to the main-tenance of genetic diversity in these natural fish popu-lations. More importantly, since the WorldConservation Union has recognised genetic diversity asone of the three levels of diversity requiring conserva-tion (McNeely et al., 1990), the results of our study mayalso contribute to the development of specific managementpriorities and measures to preserve genetic diversity inthe investigated bullhead populations.Additionally, conservation and evolutionary bio-

logists have recently developed methods, especially in theuse of microsatellites, to evaluate whether populations

have experienced reductions in size. A very promisingmethod is based on the premise that reduced popu-lations develop an excess of heterozygosity at selectivelyneutral loci relative to the heterozygosity expected atmutation-drift equilibrium (Cornuet and Luikart, 1996).Using this approach, severe demographic contractionshave successfully been detected in a series of naturalpopulations but not in others (Luikart and Cornuet,1998). An additional aim of our study was therefore touse the bullhead microsatellite data to evaluate whetherthis method was able to detect a genetic signature of thedocumented decline in the Flemish bullhead popu-lations during recent decades.

2. Materials and methods

2.1. Microsatellite analysis

We extracted data on genetic variability at sevenmicrosatellite loci from a recent population geneticstudy of 10 Flemish bullhead populations (Knapen etal., 2003), belonging to the main drainage basins, i.e.Scheldt and Meuse (Table 1; Fig. 1). Because of theendangered status of the bullhead, sample size wassometimes relatively low, ranging between 9 and 48individuals (Table 1). Microsatellite genetic diversitywithin populations was measured as the mean numberof alleles per locus (MNA), mean observed hetero-zygosity (HO) and mean expected heterozygosity (HE)with GENETIX (Belkhir et al., 1996). Microsatellitegenetic diversity was used to calculate estimates of evo-lutionarily effective population size (Ne) given particularassumptions about mutation patterns and rates. Atequilibrium, the heterozygosity under the infinite allelemodel (IAM) is expressed as: HE ¼ 4Ne�= 4Ne�þ 1ð Þ,which leads to Ne ¼ HE=1� HEð Þ=4� (Crow andKimura, 1970), where � is the mutation rate. Under the

Table 1

Drainage basin, sample size (N), mean number of alleles (MNA), observed (HO) and expected (HE) heterozygosity, effective population size (Ne)

under IAM and SMM, and patch size (PS; m) for Flemish bullhead populations

Nr

Population Basin N MNA HO HE Ne(IAM) Ne(SMM) PS

1

Veurs Meuse 37 1.43 0.085 0.116 65 70 150

2

Voer Meuse 41 1.71 0.213 0.208 131 148 4080

3

Nethen Scheldt 14 2.71 0.347 0.445 400 561 –

4

Steenputbeek Scheldt 42 1.43 0.082 0.080 43 45 2449

5

Verrebeek Scheldt 48 1.43 0.012 0.012 6 6 1525

6

Molenbeek Scheldt 9 1.14 0.032 0.067 36 37 2415

7

Laarse Beek Scheldt 43 2.57 0.316 0.370 294 381 5070

8

Zwanebeek Scheldt 24 3.57 0.399 0.504 509 767 3200

9

Tappelbeek Scheldt 21 6.00 0.680 0.684 1084 2258 5630

10

Witte Nete Scheldt 11 2.00 0.299 0.294 209 252 3790

Mean

2.36 0.23 0.26 278 453 3145

S.E.

1.56 0.22 0.23 329 682 1724

– no data available; SE, standard error. Estimates of genetic variability from Knapen et al. (2003).

404 G. Knaepkens et al. / Biological Conservation 115 (2004) 403–410

stepwise mutation model (SMM), the equilibrium het-erozygosity is equal to: HE ¼ 1� 1= 1þ 8Ne�ð Þ

0:5� �

,which gives Ne ¼ 1= 1� HEð Þ

2� �

� 1� �

=8� (Ohta andKimura, 1973). Both mutation models were used in thisstudy since the exact mutation pattern of microsatellitesremains uncertain at this moment (Goldstein andSchlotterer, 1998) Following the two mutation models,we obtain two estimates of Ne which are a direct func-tion of our mutation rate estimates. Studies indicatethat microsatellites exhibit mutation rates usuallybetween 10�3 and 10�5 (Goldstein and Schlotterer,1998). As a conservative estimate, we consider thatbullhead microsatellites do not fall outside this rangeand we use m=5�10�4, a value which is commonlyaccepted for microsatellites across a wide range of ani-mal taxa, including fish (Angers and Bernatchez, 1998).Using this approach, reliable estimates of the long-termNe have been computed for natural populations of dif-ferent species including fish (DeWoody and Avise, 2000;Spong et al., 2000).Populations that have experienced a severe reduction

in size will display a reduction in both allele numbersand gene diversity (heterozygosity), but allelic loss willoccur more rapidly than loss of heterozygosity. Basedon this phenomenon, tests have been developed todetect excess heterozygosity in a population at muta-tion-drift equilibrium as an indication of recent popu-lation decline (Cornuet and Luikart, 1996). Sincebullhead populations have declined dramatically inFlanders in recent decades, we analysed our micro-satellite data with this approach. The programBOTTLENECK is a recently developed method (espe-cially for microsatellites) for the detection of this geneticsignature of population decline (Cornuet and Luikart,1996; Piry et al., 1999). The method assumes mutation-drift equilibrium to compute the distribution of genediversity expected from the observed number of alleles.This expected average heterozygosity (HEQ) is calcu-lated through simulation under various possible muta-tion models: IAM, SMM and two phase mutation

model (TPM). Populations without a recent change insize will be in mutation-drift equilibrium where theexpected heterozygosity (HEQ) will equal the Hardy–Weinberg equilibrium heterozygosity (HE). Given thenumber of loci and sample sizes in our dataset, we fol-lowed the authors’ recommendation in using the Wil-coxon sign-rank test to test the significance ofheterozygosity excess. In addition, the BOTTLENECKprogram also provides a qualitative descriptor of allelefrequency distributions (the mode-shift indicator) thatcan be used to examine additional evidence for recentpopulation declines (Luikart et al., 1998).

2.2. Population size

In the absence of empirical information on currentpopulation size, the geographical range of populationscan be used as a surrogate for population size (Frank-ham, 1996). Hanfling et al. (1998) showed that the spa-tial extent of bullhead populations is an appropriatesurrogate for population size. Since rivers are linearhabitats, Hanfling et al. (1998) estimated the spatialextent as the length of the river system occupied by alocal population and called this measure ‘patch size’.Similarly in our study, the size of Flemish bullheadpopulations was estimated using the spatial extent of apopulation in a river. Between November 1999 andJanuary 2000, we obtained information about the patchsize of the Flemish populations by sampling the differ-ent rivers using electric fishing gear. Since the exactlocation of these populations is often well documented(Fig. 1; Vandelannoote et al., 1998), determining thegeographical range of the investigated populations wasrelatively easy. To test for associations between popu-lation size (patch size) and the different estimates ofgenetic variability (MNA, HO, HE, Ne), we used thePearson product–moment correlation coefficient (STA-TISTICA). The MNA and Ne data were log-trans-formed to meet the requirements for this parametric test(Sokal and Rohlf, 1997).

Fig. 1. Distribution of the investigated bullhead populations in Flanders (northern part of Belgium). Population numbers are listed in Table 1.

G. Knaepkens et al. / Biological Conservation 115 (2004) 403–410 405

3. Results

3.1. Genetic diversity of Flemish bullhead populations

Genetic diversity differed considerably among Flemishbullhead populations (Table 1). Mean number ofmicrosatellite alleles (per locus) (MNA) ranged between1.14 and 6.00 (2.36�1.56), observed heterozygosity(HO) between 0.01 and 0.68 (0.23�0.22), and expectedheterozygosity (HE) between 0.01 and 0.68 (0.26�0.23).Furthermore, genetic variability was relatively low inseveral populations. Five bullhead populations (Veurs,Voer, Steenputbeek, Verrebeek and Molenbeek) hadless than two different alleles per locus and in fourpopulations (Veurs, Steenputbeek, Verrebeek andMolenbeek) the observed heterozygosity was <0.1. Wetested for a possible influence of sample size on the dif-ferent estimates of genetic variability. However, none ofthe correlations was significant (P>0.39 in all cases),indicating that the differences in genetic variability ofpopulations are not heavily biased by variation in sample

size. Estimates of the effective size of the bullheadpopulations, based on the levels of genetic diversity, arealso shown in Table 1. Based on IAM, we found Ne

values ranging from 6 to 1084 (278�329). Under theSMM we found somewhat larger Ne values, rangingfrom 6 to 2258 (453�682).

3.2. Relationship between genetic diversity and populationsize

The spatial extent (patch size) of the Flemish bullheadpopulations ranged from 150 to 5630 m (3145�1724;Table 1). All measures of genetic diversity were sig-nificantly positively correlated with patch size (MNA:Pearson correlation coefficient r=0.72, P=0.029; HO:r=0.79, P=0.011; HE: r=0.76, P=0.018; Fig. 2), indicat-ing that genetic variation was lower in small populationsthan in larger ones. Similarly, estimates of Ne underboth mutation models were significantly correlated withpatch size (under IAM: r=0.73, P=0.024; under SMM:r=0.74, P=0.021; Fig. 2).

Fig. 2. The relationship between (a) mean number of alleles (MNA), (b) observed heterozygosity (HO), (c) expected heterozygosity (HE) and

(d) effective population size (Ne) under IAM versus patch size (population size) for the investigated Flemish bullhead populations. The MNA and Ne

data were log-transformed to meet the requirements for parametric testing.

406 G. Knaepkens et al. / Biological Conservation 115 (2004) 403–410

3.3. Genetic signature of population decline

We employed Cornuet and Luikart’s (1996) test forheterozygosity excess to test if allele distributions withinpopulations had been perturbed by recent changes inpopulation size (Table 2). Under the expectations ofIAM there were three populations (Nethen, Laarse Beekand Tappelbeek) that exhibited a significant hetero-zygosity excess. In addition, Voer, Zwanebeek andWitte Nete showed a marginally significant excess.Under TPM, a significant excess was found in onepopulation (Nethen), while for Voer and Laarse Beekthe P-value was approaching significance at the 0.05level. Under SMM, only one population showed amarginally significant excess (Nethen). Finally, threepopulations (Voer, Nethen and Molenbeek) exhibitedallele frequency shifts indicative for recent populationdeclines. However, this qualitative method can not beused with high confidence with samples of fewer than 30individuals since the type I error rate varies with sam-ples size (Cornuet and Luikart, 1996). Therefore, cau-tion should be applied when interpreting thedistribution shape results of Nethen and Molenbeek(sample size <30) as signs of recent population declines.

4. Discussion

4.1. Genetic diversity and population size

The positive correlation that we observed betweengenetic variability and the size of the degraded bullheadpopulations implies that (1) current population size is acrucial factor in determining the amount of geneticvariability that can be maintained in these populations,(2) population size, estimated as the spatial extent of apopulation in a river, is an appropriate surrogate for theeffective size of bullhead populations and (3) the concerns

about loss of genetic diversity in small populationsindeed are warranted.At this moment, there is no consensus among

researchers what the minimum size of populationsshould be to ensure viability. At first, it was suggestedthat the effective population size must be at least 50 toavoid inbreeding depression and thereby ensure short-term viability, and at least 500 to maintain adaptivegenetic variation and thereby ensure long-term viability(Soule and Wilcox, 1980). Recent experimental work,however, suggests that Ne�50 is inadequate to ensureshort-term viability (Reed and Bryant, 2000). Further-more, theoretical work suggests that Ne�500 is inade-quate to ensure long-term viability. Effective sizes of theorder of 1000–5000 may be necessary to maintain evo-lutionary flexibility and to avoid the accumulation andfixation of mildly deleterious mutations (Lynch andLande, 1998). In our study, estimates of Ne indicate thatall bullhead populations, except the Tappelbeek, aresmall and far below these suggested values (under IAM:Ne between 6 and 509; under SMM: Ne between 6 and767). Of course, the Ne estimates we propose are neces-sarily based on the mutation model and rate, for whichsome uncertainty remains (see Section 2), so probablythe relative rather than absolute Ne estimates should beafforded greater credence (DeWoody and Avise, 2000).Nevertheless, the estimates of the spatial extent of theFlemish populations are congruent with the conclusionthat these degraded populations are dangerously small.Average river length for Flemish populations is 3.145(�1.724) km, while in other European bullhead popu-lations like those in Germany, average patch size isnoticeably higher (41�55 km; Hanfling and Brandl,1998). In these larger populations, the mean levels ofmicrosatellite variability (HE: 0.46, MNA: 4.53; Han-fling et al., 2002) are also much higher than in theFlemish populations (HE=0.26, MNA=2.36).

4.2. Genetic signature of population decline

The genetic test for population bottlenecks providedsome evidence of the documented decline in four bull-head populations. However, in the other populations,this test was not able to detect a significant genetic sig-nature of population decline. Nevertheless, this may bebecause heterozygosity excess observed in populationsthat have declined in size is a transient feature, expectedto last only a few generations (Luikart and Cornuet,1998). In many cases, researchers do not know whetherthey examined the populations within this time period.Similarly, although anthropogenic disturbance ofFlemish rivers has been going on for several decades, itremains unclear at what time these activities have actu-ally resulted in the decline of the bullhead populations(Vandelannoote et al., 1998). Furthermore, limitedsample sizes (range 9–48) in some populations and

Table 2

Results of the BOTTLENECK analysis

Population

IAM TPM SMM Distribution

shape

Veurs

0.250 0.250 0.250 Normal

Voer

0.062 0.094 0.156 Shifted

Nethen

0.008 0.016 0.078 Shifted

Steenputbeek

0.250 0.875 0.875 Normal

Verrebeek

1.000 1.000 1.000 Normal

Molenbeek

0.250 0.250 0.250 Shifted

Laarse Beek

0.039 0.078 0.343 Normal

Zwanebeek

0.055 0.406 0.710 Normal

Tappelbeek

0.039 0.344 0.961 Normal

Witte Nete

0.078 0.312 0.312 Normal

Significance of heterozygote excess according to the Wilcoxon sign-

rank test under the IAM, TPM and SMM for each population. Also

shown is the shape of the allele frequency distributions. Significant P-

values are indicated italic.

G. Knaepkens et al. / Biological Conservation 115 (2004) 403–410 407

number of loci (7) used in our study may also haveaffected the detection of recent population declines.Although the Wilcoxon sign-rank test can be used withas few as four polymorphic loci and any number ofindividuals, to achieve high power, 15–40 individualsand 10–15 polymorphic loci are recommended (Cornuetand Luikart, 1996). A recent genetic study of EuropeanCottus gobio populations (including Flanders) indicatedthat populations in the Witte Nete and Verrebeek alsoshowed signs of a recent population decline using theheterozygosity excess test, while populations in theVeurs and Steenputbeek showed, similar to our study,no signs of a recent decline. Furthermore, four out offive other studied Flemish bullhead populations, notinvestigated in our study, also showed signs of a recentdecline (Hanfling et al., 2002). Sample size (19–40) andnumber of loci (10) examined in these populations were,however, always within the recommended range toachieve high power with this test.

4.3. Implications for conservation and management

In the degraded Flemish bullhead populations, popu-lation size appears to be very important to the main-tenance of genetic variation. Therefore, to minimise oreven reverse further genetic erosion in the bullhead, themost immediate and effective conservation prioritieswould be to (1) maintain the populations at existingsites by protecting these sites and (2) increase the size andthe range of these populations. Essential requirements toachieve these goals are improvement of the habitat qual-ity and reduction of the river fragmentation.Vital habitat components for the bullhead are stones

or other hard substrates. They not only provide shelter,but are also essential for reproduction because they areused as spawning substrate (Smyly, 1957; Lelek, 1987).Nevertheless, in anthropogenically perturbated rivers,natural substrates like iron-sandstone aggregates (typi-cal for many parts of Flanders) have degraded, conse-quently limiting the survival of the local populations(Knaepkens et al., 2002a). Since the natural recovery ofsuch habitat structures is a long-term or even unrealisticapproach due to cultural, demographic and/or eco-nomic reasons, improving the quality and variety ofdegraded fish habitats may also be possible by the con-struction of artificial habitat structures (Cowx andWelcomme, 1998; Collares et al., 2002). Recent studiesindicated that the use of artificial structures such as tilesis a very promising spawning habitat enhancementtechnique for threatened Cottidae like the pygmy bull-head (Cottus paulus) or the European bullhead (John-ston, 2001; Knaepkens et al., unpublished).To ensure free migration of bullheads in these pertur-

bated rivers, all physical obstacles (520 cm; Utzinger etal., 1998) have to be removed or equipped with attrac-tive and efficient fish passes like by-pass channels or fish

ladders. Important requirements for such fish passesare: sufficient capacity, adapted to the swimming capa-cities of the fish, permanently functional and a goodpositioning of entrance (Cowx and Welcomme, 1998).Additionally, since the bullhead is considered to be veryvulnerable to pollution (Lelek, 1987; Vandelannoote etal., 1998), chemical obstacles may also act as a potentialmigration barrier. Therefore, water pollution abatementprogrammes should be executed to improve waterquality and connectivity of populations.If, despite implementation of these management stra-

tegies, bullhead populations continue to decline, addi-tional measures may become necessary. Translocation,i.e. intentional release of wild caught individuals fromlarge, genetically diverse conspecific populations, is acontroversial strategy. It has been successfully used toenhance the genetic variability, fitness and size ofendangered populations of the greater prairie chicken(Tympanuchus cupido pinnatus) and adder (Vipera berus)(Westemeier et al., 1998; Madsen et al., 1999), but hasfailed for other threatened species like the ibex (Capraibex; Templeton, 1997). A major risk to the recipientpopulation is outbreeding depression, i.e. interbreedingbetween genetically distinct populations may result in adecline in fitness due to the the breakdown of co-adap-ted gene complexes and/or local adaptation (Templeton,1997). On the basis of genetic data available for theFlemish populations, we would therefore expect that theworst candidates to supplement reduced populationslike those at Verrebeek and Molenbeek would be indi-viduals from populations belonging to the Meuse basin:the latter are part of a different evolutionary lineage(Knapen et al., 2003). One of the few candidates to actas a possible source population for augmentation is theTappelbeek population since (1) its size and level ofgenetic variability are still relatively high and (2) itbelongs to the same evolutionary lineage as the popula-tions of the Verrebeek and Molenbeek. Nevertheless,there may still be a considerable chance of outbreedingdepression since the source and recipient populationsshow significant divergence of allele frequencies atmicrosatellite loci. At this moment, the questionremains whether the benefits brought by an increase innumbers and reduced inbreeding due to translocationswill outweigh the tangible threats of outbreedingdepression.Alternatively, to augment bullhead populations with-

out introducing exogenous genes, supportive breedingprogrammes may be developed. This implies that poten-tial parents are caught in a wild population and broughtinto captivity to increase reproductive and reduce mor-tality rates (for an overview of the practical aspects ofcaptive breeding of fish, see Philippart, 1995). Followingbreeding, some or all of the offspring are returned to thesame wild population. Nevertheless, although suppor-tive breeding may increase the census population size,

408 G. Knaepkens et al. / Biological Conservation 115 (2004) 403–410

there are several recognised possible problems such asreduced population fitness due to selection in captivity(Ford, 2002) or a reduction of the Ne (Ryman andLaikre, 1991). To limit or even circumvent such pro-blems, parameters such as the number of generations ofsupport, the proportion of individuals in captivity ateach generation and the level of gene flow between cap-tive and wild population, should be taken into account(Wang and Ryman, 2001; Duchesne and Bernatchez,2002; Ford, 2002). Nevertheless, because of the difficul-ties and risks in terms of genetic conservation, suppor-tive breeding should remain a temporary safeguardmeasure, and may only be justified when the hazardsthat caused the population’s decline are treated.

Acknowledgements

We thank T. Dillen, D. Knapen, V. Mees and M.Taylor for assistance in the field and/or in the lab. B.Hanfling and P. Galbusera provided comments anddiscussions which improved the quality of the manu-script. This work was conducted within the frameworkof a nature development programme of the Flemishgovernment (VLINA/9903).

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