Kappe, Anique Lamberta

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   University of GroningenDetecting genetic variationKappe, Anique LambertaIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1998Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Kappe, A. L. (1998). Detecting genetic variation: Application of molecular techniques in conservationbiology. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 20-06-2022CHAPTER 7IxrnorucrroNAs emphasized in the introduction and tkoughout the different chapters in this thesis, therelative importance of genetic, demographic and environmental factors in the extinctionchances ofnatural populations has not unequivocally been established. Scrupulousinvestigation of many different populations, taking into account all three components, isessential to gain more insight into the complex, and ever changing interplay ofthesefactors. Assessing the genetic component has been impaired by the fact that classicaltechniques did not possess enough resolving power to detect very low levels ofgeneticvariation, that sometimes characterize endangered populations.The development ofmolecular techniques made it possible to reveal these low levels of genetic variation. Inthis thesis, the applications ofmolecular techniques for the evaluation ofthe geneticstruature of populations are described.Gnxnrrc vARTATIoN REvf,ALED By rHE RArD TECHNIeUEThe Random Amplified Polymorphic DNA technique (RAPD) (Welsh & McClelland,1990, Williams et a|.,1990) has been used in many studies involving mapping strategiesand genomic fingerprinting (Welsh et al., 1991 Wilde et al., 1992). The resolving powerofthis technique to assess genetic variation in Phoca vitulinq (the harbour seal) wasexamined in chapter 2. Two populations of the harbour seal (Phoca vitulina), known tohave low levels of genetic variation, were analysed with the RAPD technique. Thistechnique showed a very low number of polymorpic bands (only 2 out of 50 testedprimers generated polymorphic bands). Genetic variation in the harbour seal could not beassessed by this method, the resolving power was not suffcient to measure geneticdifferentiation. Results found with the RAPD method were in agreement with resultsfound with allozyme electrophoresis, whereby total absence of polymorphism was foundfor 24 loci (Swart et al., 1996). Thus, although in other population and evolutionarystudies genetic variation could be assessed, the amount ofvariation in the harbour sealwas too low to be detected by the RAPD technique (Martin et a1.,7991;Welsh et al.,1991; Wilde et al.,1992).Gnxnnc vARIATToNxFINGERPRINTING TECIThe harbour sealThe multilocus DNA fingerPallozyme electroPhoresis antmultilocus DNA fi ngerPrintivariation in the harbour sealprobes 33.6 and 33 15 (JeffiapproximatelY 0'80, withinWadden Sea PoPulation an<indicates a low level ofovesurveys in geograPhicallY isfingerPrints in other animalGr lion (Gitbert et al'' 199al.,l99l) and the Northenpopulations and sPecies wigenetic variabilitY were ge(JeffreYs et a\.,7985 a,b'I1e93).The frngerPrint dapoPulations in the Dutch'within and between,PoPulpoPulations are not comppoPulations cannot be exrbased uPon these similariThe data on the PoPulatitalleles leading to higher tlarger PoPulation size ofThe low geneticNorth Sea PoPulations ocharacteristic for the sPevitulinq comPlex (P' v' t,rs in this thesis, the; in the extinctionScrupulousae components, isrplay oftheset that classicallevels ofgeneticrelopment ofretic variation. Inofthe geneticIUE: McClelland,pping strategiesresolving powerrr seal) wasrna), known toque. This50 tested:al could not bee geneticvith resultssm was foundllutionarytarbour seallelsh et al.,SUMMARY AND EVALaATIONGnrurrrc vARrATroN REvEALED By rHE MULTILoCUS DNAFINGERPRINTING TECHNIQUEThe harbour sealThe multilocus DNA fingerprinting technique is a much more sensitive method thanallozyme electrophoresis and the RAPD technique. In chapter 2 is described how themultilocus DNA fingerprinting technique was used to determine the amount of geneticvariation in the harbour seal. The DNA fingerprint data showed for the two Jeffreysprobes 33.6 and 33.15 (Jeffreys et q|.,1985a,b) a high degree ofband-sharing,approximately 0.80, within and between two populations of the harbour seal: theWadden Sea population and a Scottish population. This high-degree of bandsharingindicates a low level of overall genetic variation in both populations. Comparativesurveys in geographically isolated and small populations showed also less variable DNAfingerprints in other animal species, like the Channel Island fox (Gilbert et al., 1990), theGir lion (Glbert et al., 1991), mysticete whales (Amos & Hoelzel, 1990; Van Pijlen etal., 1991) and the Northern Elephant seal (Hoelzel et al.,1993). However, inpopulations and species with relatively large large population sizes, high levels ofgenetic variability were generally reported by DNA fingerprinting with the same probes(Jeffreys et al., 1985 a,b, Burke & Bruford, 1987; Georges et al., 1988,Baker et al.,re93).The fingerprint data can be used to calculate similarity between the harbour sealpopulations in the Dutch Wadden Sea and Scotland. No significant differences, eitherwithin and between,populations were found. These results indicate that the twopopulations are not completely isolated, and that some geneflow between thesepopulations cannot be excluded. Nevertheless, when heterozygosities were estimatedbased upon these similarity values, a slight difference between populations was detected.The data on the population from Scotland showed a more frequent occurrence ofrarealleles leading to higher values for estimated heterozygosities, which may reflect thelarger population size ofthe Scottish population.The low genetic variation found by multilocus DNA fingerprinting, in the twoNorth Sea populations ofthe harbour seal could indicate that such low levels arecharacteristic for the species Phoca vitulina. In chapter 3 three subspecies of the Phocqvitulina complex (P. v. vitulina (East Atlantic), P. v. concolor (West Atlantic) and P. v.93CHAPTERTrichordsi (East Pacific)) were analysed with respect to the level of genetic diversity bymultilocus DNA fingerprinting. Bandsharing similarities between subspecies weresignificantly lower than within subspecies. This indicates that gene flow betweensubspecies is limited, which could be expected from the geographical position of thediverse subspecies. Especially, the subspecies P. v. richardsl is clearly separated from theother subspecies. These data indicate that low levels ofgenetic variation are notcharacteristic for all Phoca vitulina subspecies. Moreover, the grey seal (HalichoerusWpus), a marine carnivore living in the same habitat, has significantly higher levels ofgenetic variation, revealed by DNA fingerprinting. Data on cytochrome 6 sequences haveindicated that the grey seal should be included inthe Phocq genus (Arnason et al, 1995),which implies furthermore that the low level of overall genetic variation is notcharacteristic for the genus.The pauperization of genetic variation found in the subspecies in the North Seaarea shown by the heterozygosities estimated from DNA fingerprinting data, could alsobe due to historical events, and more specifically by the last Northern Hemisphereglaciation. The latter event caused a dramatic reduction of size and fragmentation ofpopulations in many large mammal species (Marshall et al., 1982; Menotti-Raymond &O'Brien, 1993). The subspecies P. v. richardsi and P. v. concolor probably didexperience significantly less severe bottlenecks during the last glaciation than thesubspecies P. v. vitulina, because both the West Atlantic and East Pacific regions wereless covered with ice (Peltier, 1994). The date of the bottleneck, causing loss of almostall minisatellite variation in P. v. vitulina, was estimated in chapter 3. The present levelof genetic variation in the subspecies P. v. vitulinq does agree with the assumption of abottleneck some 10.000 years ago.The Seychelles warblerIn chapter 4 the use of multilocus DNA fingerpriting to reveal the amount of geneticvariation in the Seychelles warbler is described. The world population of the Seychelleswarbler population consisted of 26-29 birds, between 1959 and 1968, and thepopulation was confined to Cousin Island. The warbler population started to recoverfrom 1968 with numbers rising to 320 in 1982, since then the population fluctuatedaround that number. The genetic variation was estimated to determine the ef[ects of a tenyear bottleneck in the Cousin Island population and the possible effects ofthe94translocation to Aride islato establish a new PoPulalhigh (about 0.50), when cspecies (Burke & Bruforcbandsharing similaritY betfingerprinting no effect o1In addition, estimifingerprint and demograPpopulation sizebY 67 -78Bruford, 1987; Wetton e,data suggest hat the bottreduced the level of heterSeychelles warblers havefeature, the PoPulation dof founding a new, raPidother avian sPecies. A Pt0.76, this poPulation statpopulation size of arounLow levels of gedepression, which can cifound in the SeYchelles'reported, though, of cotofgenetic load bY earlieoccuned. However, as Ibe reduced and thereforAnd although no culrerchanges of such a PoPuGrNnrrc vARIATThe microsatellite metlvariation. Advantage cto score codominant gfollow genetic Procest)netic diversity byspecies were)w betweeni position of they separated from the;ion are not,eal (Halichoerusly higher levels ofme 6 sequences haveirnason et al, l99S),ion is nots in the North Seang data, could alsorHemisphereagmentation ofnotti-Raymond &rbably didon than the:ific regions wereng loss of almostIhe present level)assumption of amt of genetic,fthe Seychellesmd the9d to recovsrr fluctuatedre effects ofa tenrf theSUMMARY AND EVALUATIONtranslocation to Aride island. In 1988 29 birds were translocated to Aride Island, in orderto establish a new population. Mean bandsharing similarity within both populations ishigh (about 0.50), when compared to values, around 0.25, obtained from outbred avianspecies (Burke & Bruford, 1987; Wetton et al., 1987 Westneat, 1990). No ef;lects onbandsharing similarity between populations on both islands are observed, thus with DNAfingerprinting no effect ofthe translocation could be assigned.In addition, estimates of effective population size were made based on DNAfingerprint and demographic data. The estimates indicate a reduction of effectivepopulation size by 6'7-78%, compared to the situation in outbred avian species (Burke &Bruford, 1987; Wetton et a|,1987; Westneat, 1990). The multilocus DNA fingerprintdata suggest hat the bottleneck of29 animals during at least ten years has considerablyreduced the level ofheterozygosity ofthe Seychelles warbler population. Concluding theSeychelles warblers have probably undergone considerable genetic erosion. Despite thisfeature, the population did recover after protective measures on Cousin and was capableof founding a new, rapidly growing population on Aride. Similar results were found forother avian species A population of the Canada geese showed band similarity values of0.76, this population stafied with 5 individuals and was rapidly increasing to a currentpopulation size ofaround 30 - 50,000 individuals (TegelstrOm & Sjdberg, 1995).Low levels ofgenetic variation are associated with the occurence ofinbreedingdepression, which can cause impaired fitness. Despite the low levels of genetic variationfound in the Seychelles warbler populations, no flagrant deleterious effects have beenreported, though, ofcourse, no outbred population is available for comparison. Purgingofgenetic load by earlier inbreeding periods, including the 1O-year bottleneck may haveoccurred. However, as stated by Hedrick (1995), the mean fitness of the population maybe reduced and therefore the risk ofextinction in the future may have been increased.And although no current effects are visible, the adaptive potential to environmentalchanges ofsuch a population may have been sevedy affected.GnNnrrc vARTATIoN REvEALED By MrcRosATELLrrE ANALySISThe microsatellite method has a high resolving power to reveal low levels of geneticvariation. Advantage of this method, compared to DNA fingerprint analysis, is the abilityto score codominant genotypes with exact allele sizes. This provides the opportunity tofollow genetic processes, like gene flow. Microsatellite analysis have been applied to the95CHAP|ER 7analysis of genetic variation in the harbour seal (Chapter 6). Five specific microsatellitemarkers were developed for the harbour seal and were used to study the geneticstructure of harbour seal populations in the North Sea, the West Atlantic and the EastPacific Ocean.The degree ofheterozygosity ofthe five microsatellite loci proved to be very low in boththe North Sea and the West Atlantic populations of the harbour seal, and the highestheterozygosity was found in the East Pacific population, which is in agreement with themultilocus fingerprinting data (Chapter 3). No specific alleles were found for the NorthSea populations, the populations from the West Atlantic and the East Pacific showed 2and 7 population specific alleles, respectively. A distance tree was constructed based onNei's genetic distance, and provisionally it can be concluded that all populations aregenetically separated, with exception ofthe Scottish populations.The DNA fingerprinting method provides many loci, which are assumed to beindepent, but specific alleles cannot be assigned and therefore geneflow and migrationcannot be determined. With microsatellite analysis specific alles can be assigned and withthe data an estimate of population divergence and migration can be made based on \,measures (Slatkin, 1995). This measure is based on the estimated variances in allelesizes (see chapter 6). The R* values found for the North Sea and the West Atlanticpopulations are relatively high, indicating a distinct population substructure. For theNorth Sea populations a substructure could be detected within the North Sea. Based on\, values the Scottish populations seemed to be one panmictic population and themigration between the Dutch Wadden Sea population and the Scottish populations isestimated to be low. Therefore it is not to be expected that the low levels of geneticvariation can be replenished by exchange with neighbouring gene pools. Although theDutch Wadden Sea population is recovering and increasing in populationsize, the geneticpauperized situation has not improved. Protective measures, installed for the DutchWadden Sea area, however, may have improved the environment, and therefore this areamay become more attractive for seals to breed. Thus, a part of the increase in numbers ofthe harbour seal population in the Dutch Wadden Sea could be due to immigration fromanimals from e.g. Denmark, Germany or Britain, therefore the isolation ofsubpopulations in the North Sea can be relieved.96Coxcr,uonlc REMARIWith molecular techiques th(harbour seal and the SeYchelexamined. Several techniquestructure. Our results indicatopportunities to detect the astructure of PoPulations. Remeasure the level of geneticpopulation structure a methrfingerprinting , was used Mpolymorphism and the amorpopulations ofthe harbour tand 5). To investigate the gvariation, like the harbour sbecause no sPecific loci canscored, and therefore, geneThe data assessed bY the vacomparison of the data is srinterpretation ofthe data c<organisms with various moAlthough, the moleand on the genetic structurthe obtained data. The signwith resPect o the risk of Ithe impact of loss of variatinteraction between genetiin the future.CunentlY, in conseimportance of genetic Prorpopulation extinction. Lorof fitness, which means depotential. The latter trait i(ChaPter 3 and 4). Thus' ior species PresentlY seem

Detecting genetic variation:Application of molecular techniques in conservation biology

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Detecting genetic variation:Application of molecular techniques in conservation biology

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