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Estimates of gene flow between populations of the swallowtail butterfly, Papilio machaon in Broadland, UK and implications for conservation
Biological Conservation 89 (1999) 293±299
Estimates of gene ¯ow between populations of the swallowtail butter¯y, Papilio machaon in Broadland, UK and implications for conservation J.C. Hoole a, D.A. Joyce b, A.S. Pullin b,* b
a Biological Sciences Department, Keele University, Keele, Staordshire ST5 5BG, UK School of Biological Sciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Received 27 May 1998; received in revised form 26 October 1998; accepted 4 November 1998
Abstract The swallowtail butter¯y, Papilio machaon has a recognised subspecies britannicus which is con®ned to the Broadland area of Norfolk, UK. The subspecies is threatened by habitat change and fragmentation. Gene ¯ow was studied between four sites in this area using allozyme and RAPD-DNA analysis to see if there is any evidence of isolation of populations or inbreeding that could pose a threat to the species persistence. The allozyme studies suggest high levels of gene ¯ow and a very low value of Nei's genetic distance (D) between sites. RAPD analysis also suggested extensive gene ¯ow between sites. The results indicate that there is little or no isolation of populations and that the species is capable of colonising and exploiting suitable habitats within the Broadland area. Consequently a landscape scale rather than site-based conservation strategy is recommended. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Allozymes; RAPD-DNA; Gene ¯ow; Genetic distance; Conservation genetics
1. Introduction The swallowtail butter¯y Papilio machaon was once widespread in wetland areas of southern and eastern England, but is now restricted to the Broadland area of Norfolk and part of Suolk (Emmet and Heath, 1990), where it is recognised by some authors as a distinct subspecies, P. m. britannicus endemic to the UK (Dempster, 1995; Emmet and Heath, 1990). This species is listed in the British Red Data Book as vulnerable and is protected under the UK Wildlife and Countryside Act 1981. It is a strong ¯yer, and one of the most spectacular and beautiful of the British butter¯ies. Its decline is probably due to increased drainage of fen areas which resulted in the loss of its larval foodplant milk parsley Peucedanum palustre. Continental subspecies of P. machaon have a greater range of larval foodplants and are less sedentary than the specialised British subspecies (Dempster et al., 1976; Dempster, 1995). The tendency of the British subspecies to have
* Corresponding author. Tel.: +44-121-414-7147; fax: +44-121414-5925; e-mail: [email protected]
restricted breeding areas where the larval foodplant occurs creates diculties in determining whether the colonies in Broadland should be treated as one population for conservation purposes, or as a number of isolated to semi-isolated populations. The aim of the work reported here was to estimate the level of gene ¯ow occurring between breeding areas. This will help identify the type of conservation strategy required for this subspecies, and will also indicate the likelihood of (re)colonization of suitable breeding areas created by appropriate management or vacated following local extinction. 2. Methods and materials 2.1. Sample collection Over a period of 3 years from 1993 to 1995 samples were collected from four sites in Broadland: Hickling Broad, on the River Thurne, Woodbastwick Fen, on the Bure marshes, Cat®eld Fen in the Ant valley and Strumpshaw Fen, on the River Yare (Fig. 1). These are as geographically separated as possible to fully test for isolation by distance.
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J.C. Hoole et al. / Biological Conservation 89 (1999) 293±299
It was intended to collect larvae from all four sites but, because of poor seasons, it was considered inadvisable to take larvae from Strumpshaw, and haemolymph samples only were able to be taken from this site, while larvae and haemolymph samples were removed from the other sites. As far as possible, larvae were reared through to adults before being used for analysis in order to prevent the accidental inclusion of plant DNA from larval gut contents. All larval tissue was ¯ash frozen in liquid nitrogen and stored at ÿ70 C until required. Haemolymph samples were frozen on dry ice in the ®eld, transported back to the laboratory in the same medium and stored at ÿ70 C. 2.2. Choice of techniques A combination of RAPD-PCR (random ampli®ed polymorphic DNA-polymerase chain reaction) and allozyme markers was chosen to complement one another. RAPD-PCR (a dominant PCR-based marker) analysis is a fast and powerful treatment that can provide direct evidence of gene-¯ow between populations (e.g. Gibbs et al., 1994). Since this method is based on random DNA ampli®cation, precautions must be taken to avoid artefactual results. Indeed some workers have criticised the technique on the grounds that repeatability is low since the use of such short primers allows spurious bands to be produced. Black (1993) suggests a system to overcome this based on replication of reactions and distribution among gels to avoid dierences based on run-to-run variation being interpreted as real population dierences. In contrast, allozymes are a co-dominant, proteinbased marker system which provides added information from heterozygosity levels.
In using an approach which relies on model-based estimates of gene ¯ow and in which markers may be of low resolving power, more than one technique is preferred (Bossart and Powell, 1998). In combination, the two techniques can be used for the estimation of heterozygosity, gene ¯ow, genetic distance and variation between populations, all of which are of great importance in planning the conservation of populations of endangered species whose degree of isolation from one another may be in doubt. 2.3. Allozyme analysis Standard methods of starch gel electrophoresis were adapted for allozyme analysis. A single buer TBE buer (0.5 M Tris, 0.65 M boric acid and 0.02 M EDTA) was used for all experiments. Starch gels consisted of an 11% (w/v) solution of potato starch in 195 ml of a 1:9 dilution of TVB buer (Hillis and Moritz, 1991). Gels were run for 4 h at 60 mA in a cooled tank. Eight dierent enzymes were examined using stains adapted from several sources as follows: glutamate-oxaloacetate transaminase (GOT) (E.C. 2.6.1.1) (McKechnie et al., 1975); acid phosphatase (ACP); (E.C. 3.1.3.2), alkaline phosphatase (AKP) (E.C. 3.1.3.1), malic enzyme (ME) (E.C. 1.1.1.40), (Ayala et al., 1972); phosphoglucose isomerase (GPI) (E.C. 5.3.1.9), phosphoglucomutase (PGM) (E.C. 2.7.5.1), adenylate kinase (AK) (E.C. 2.7.4.3), hexokinase (HEX) (E.C. 2.7.1.1), (Shaw and Prasad, 1970) Where agar overlays were required (Krafsur and Black, 1992) (ME, PGM, GPI, HEX, AK) 10 ml 2% agar replaced 10 ml of stain. 2.4. RAPD-PCR analysis
Fig. 1. Sites from which Papilio machaon were collected in the Broadland area of Norfolk, UK. (1) Hickling Broad, (2) Woodbastwick Fen, (3) Cat®eld Fen, (4) Strumpshaw Fen.
Template DNA was prepared from butter¯y thoracic tissue by Proteinase K/phenol chloroform extraction (Hillis and Moritz, 1991) and from larval haemolymph and dried tissue by the use of a silicon dioxide/guanidine thiocyanate extraction, (Cameron, 1996). RAPD-PCR was carried out using standard methods (Apostol et al., 1993; Black et al., 1992). The ampli®cation programme used consisted of: 1 cycle of 94 C for 5 min (denaturing); 35 cycles of 94 C for 30 s (denaturing), 36 C for 1 min (annealing), 72 C for 1 min (extension); 1 cycle of 72 C for 5 min (extension). For each individual sample ampli®ed with each primer, six replicates were performed over three successive runs on the thermal cycle, in order to ensure reproducibility of RAPD pro®les (Black, 1993). PCR products were separated on a 1515 cm 1.4% agarose gel which was run for 16 h at 40V, in 0.5 M TBE buer. Ethidium bromide was added to the running buer. Discrete fragments of DNA were resolved into bands which were
J.C. Hoole et al. / Biological Conservation 89 (1999) 293±299
visualised under UV light. A permanent record was made by photographing the gel and recording it on disc using a UVP gel imaging system. The same system was later used in the analysis of the gels. A set of 20 primers was screened using Pieris brassicae DNA, of which ®ve were eventually used because they showed individual variation, reproducibility and consistent priming. The sequences of the primers used are: (1) TGCGCCCTTC; (2) TTCCCCCGCT; (3) TCCGCTCTGG; (4) CCACAGCAGT; (5) ACCCCCGAAG. The bands to be scored were chosen as a result of their intensity; this was determined by eye. Band size for each individual was assessed using the gel imaging system described previously. Consistently ampli®ed bands were selected by comparison between sample replicates and each individual scored for presence (1) or absence (0) of bands found in the species sample. This information for all primers was combined into a data matrix for analysis. 2.5. Statistical analysis 2.5.1. Allozymes GDA version 1.11 (Lewis and Zaykin, 1998) was used to compute Nei's (1978) genetic distance which corrects for a small sample size. This information was used with the geographic distance (km) to perform a Mantel test using NTSYSpc version 2.01e (Rohlf, 1992). Arlequin version 1.1 (Schneider et al., 1997) was used to obtain a matrix of population pair-wise FST values and their Pvalues after 100 permutations. The P-value of the test is the proportion of permutations leading to an FST value larger or equal to the observed one. FST values can be used as a measure of short term genetic distance between populations with the application of a slight transformation to linearise the distance with population divergence time (Schneider et al., 1997; Reynolds et al., 1983; Slatkin, 1995). The estimator used follows Weir and Cockerham (1984). Population level neighbour joining trees were created using PHYLIP (Felsenstein, 1989) from both Nei's 1978 genetic distance and the population pair-wise FST values as a comparison. Other
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F-statistics were calculated in PopGene version 1.21 (Popgene, 1997) according to Nei (1987). 2.5.2. RAPDs The RAPD-DNA pro®les produced were analysed using NTSYSpc version1.70 (Rohlf, 1992). A measure of genetic identity was produced using two dierent methods; the simple matching method produces matches between all possible pairs of individuals using both presence and shared absences of bands, whereas the Jaccard coecient is calculated by including only presence of bands, ignoring shared absences (FranciscoOrtega et al., 1993). 3. Results 3.1. Allozymes Of eight loci, one was monomorphic (ME) and the remaining seven were characterised by a total of 19 alleles. Nei's (1978) D for P. machaon was very small for all three pairs of populations, but it is slightly greater between those from Cat®eld Fen and Woodbastwick (0.104) and between Cat®eld and Hickling (0.136) than between Hickling and Woodbastick (0.017) (Table 1). Population pair-wise FST values show a slightly dierent pattern, with the largest value (0.122) between Cat®eld Fen and Woodbastwick. However, these values are also very low. Population level neighbour joining trees pictorially represent these discrepancies (Fig. 2), which are probably due to the low sample size (n=5) from Cat®eld Fen. The F-statistics across populations for each locus (Table 2) suggest that there is an excess of heterozygotes at ®ve out of eight loci mostly due to the results from Woodbastwick. The mean FIS is close to zero, implying that there is neither an excess or de®cit of heterozygotes.
Table 1 Geographic distance between sampled sites in km (upper triangular matrix), population pair-wise FST values (where * indicates a signi®cant P value at the 0.05 level) and Nei's (1978) genetic distance between Papilio machaon populations (lower triangular matrix) Woodbastwick Woodbastwick Hickling Cat®eld
(km) FST D FST D
0.042 0.017 0.122* 0.104
Hickling
Cat®eld
10
6 6
0.000 0.136
Fig. 2. Population level neighbour joining phylograms showing the relationship between Papilio machaon populations according to (a) FST values and (b) Nei's (1978) genetic distance.
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Table 2 F-statistics (calculated using Popgene version1.21) for each Papilio machaon population and each enzyme locus, where (n) is the sample size Locus
FIS values (n) Woodbastwick
Hickling
Cat®eld
All populations
FIT values(n) All populations
HEX PGM GPI GOT AK ME ACP AKP
0.0588(24) ÿ0.3589(30) ÿ0.2000(30) ÿ0.0714(30) ÿ0.2857(18) n/a(24) 0.5556(12) 0.0667(14)
ÿ0.0338(40) 0.0170(40) ÿ0.2353(42) 0.3939(44) 0.1667(30) n/a(34) 0.2727(18) 0.2086(22)
ÿ0.1111(10) 0.0909(10) n/a(10) ÿ0.4286(10) n/a(2) n/a(8) 0.6774(10) 0.1304(10)
ÿ0.0294(74) ÿ0.0805(80) ÿ0.2185(82) ÿ0.0525(84) ÿ0.0227(50) n/a(66) 0.4950(40) 0.1340(46)
0.0376(74) ÿ0.0615(80) ÿ0.1351(82) 0.0928(84) 0.1439(50) n/a(66) 0.5607(40) 0.2841(46)
Mean
ÿ0.0422(23)
0.1186(34)
0.0617(65)
0.1560(65)
0.1111(9)
The amount of genetic variance displayed within samples from the three reserves is very small, (FST=0.107) indicating that any one colony will contain 89.3% of the gene variability present in the whole population and that there has been little population subdivision. The value of FIT is 0.15; if the population were wholly panmictic the expected value would be zero. The average heterozygosities, (Table 3) are between 0.364 and 0.476. Estimated allele frequencies for each population are shown in Table 4. A Mantel test to assess whether or not the small amount of structuring observed was due to the distance between colonies sampled, showed no relation between the two. Comparing Nei's (1978) genetic distance and the geographic distance in km between sites showed that the two were not positively related (r=ÿ0.96).
Table 3 Sample number (N), Average heterozygosity (H) and variance (V) for three Papilio machaon populations in Broadland, UK Populations Hickling Broad
Woodbastwick Fen
Cat®eld Fen
N H V
14 0.433 0.078
5 0.364 0.099
22 0.476 0.080
3.2. RAPD-PCR A total of 109 reproducible bands was produced using ®ve primers. Clustering analysis using the simple matching coecient with UPGMA showed clustering at a high level of identity between all individuals (Fig. 3). The deepest level of division, at between 0.5 and 0.55 gave three clusters, one consisting of four butter¯ies from Hickling Broad and one from Strumpshaw Fen, another containing the three individuals from Cat®eld Fen and one from Woodbastwick, and the third containing members from all sites except Cat®eld. Using Jaccard similarity with UPGMA clustering showed divisions at a lower level of identity, but the clusters segregated at the deepest levels contained members of all populations, except for one small cluster consisting of three individuals from Hickling, and the Cat®eld Fen butter¯ies which appeared in one cluster with individuals from all other populations (Fig. 4). The Jaccard matching with the Neighbour-Joining method showed a similar level of identity between most individuals, but the three major clusters divided at a lower level of identity, between 0.05 and 0.1, and each included representatives of all populations except that from Cat®eld Fen (Fig. 5). Once again all three individuals from this site appeared in one cluster, with representatives from all other populations.
Table 4 Allozyme allele frequencies for three Papilio machaon populations in Broadland, UK Population
Allele
Locus: HEX
PGM
GPI
GOT
AK
ME
Hickling Broad
1 2 3
0.167 0.381 0.452
0.300 0.425 0.275
0.786 0.214 Ð
0.727 0.250 0.023
0.625 0.375 Ð
1.000 Ð Ð
Woodbastwick Fen
1 2 3
0.125 0.250 0.625
0.333 0.433 0.233
0.833 0.167 Ð
0.467 0.533 Ð
0.778 0.222 Ð
1.000 Ð Ð
Cat®eld Fen
1 2 3
Ð 0.700 0.300
0.500 0.300 0.200
1.000 Ð Ð
0.500 0.500 Ð
1.000 Ð Ð
1.000 Ð Ð
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4. Discussion
297
to this study because of the small number of individuals sampled from the Cat®eld population. The allele frequencies in each population reveal that two of the loci
The very low values of genetic distance obtained for the three populations of P. machaon and the lack of correlation between genetic and geographical distance are consistent with the hypothesis that there is considerable gene ¯ow between the colonies in Broadland. Neighbour-joining trees based on allozyme data show discrepancies according to whether FST or D was used. This is likely to be due to the small sample size from Cat®eld Fen. The very low value of FIT over all loci also suggests high levels of gene ¯ow between the three populations. The fact that none of the loci shows an average heterozygosity signi®cantly dierent from Hardy±Weinburg expectations, and that FIS is 0.062, indicates that the population as a whole is in Hardy±Weinburg equilibrium and that inbreeding has had very little eect on the populations. The average heterozygosities of the three samples of P. machaon are high for a species which eectively has been reduced to a few colonies within a small geographical area for many generations, especially when compared with the values of 0.16 for P. mnemosyne in the Alps (Napolitano et al., 1988). However, the reservations about the validity of H as a measure of the genetic viability of a population expressed by a number of workers (Nei and Roychoudury, 1974; Singh and Rhomberg, 1987; Munsterman, 1994) apply even more strongly
Fig. 4. Clustering of individual Papilio machaon from Broadland populations by UPGMA using Jaccard coecient. Individuals 1H± 13H are from Hickling Broad, 14W±23W are from Woodbastwick Fen, 24S±27S are from Strumpshaw Fen, 28C±30C are from Cat®eld Fen.
Fig. 3. Clustering of individual Papilio machaon from Broadland populations by UPGMA using simple matching. Individuals 1H±13H are from Hickling Broad, 14W±23W are from Woodbastwick Fen, 24S±27S are from Strumpshaw Fen, 28C±30C are from Cat®eld Fen.
Fig. 5. Clustering of individual Papilio machaon from Broadlands populations by neighbour-joining using the Jaccard coecient. Individuals 1H±13H are from Hickling Broad, 14W±23W are from Woodbastwick Fen, 24S±27S are from Strumpshaw Fen, 28C±30C are from Cat®eld Fen.
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have only two alleles and one is monomorphic, while each of the other ®ve loci has only three alleles. However, the number of alleles which constitutes a `safe' level of genetic variability is largely a matter of interpretation. The results obtained using RAPD analysis also suggest that a considerable amount of gene ¯ow occurs between populations since the individuals analysed could not be separated into their populations by any of the methods of analysis used. Some individuals from each population consistently occurred in the same group, for example numbers 6, 7 and 8, from Hickling, the three from Cat®eld Fen, and numbers 19 and 20 from Woodbastwick Fen, which have the highest genetic identity in all the analyses. This may suggest that these groups of individuals are closely related, possibly from eggs laid by the same female. Since larvae were collected at random from foodplants, and larger instars were taken wherever possible, it is not impossible that members of the same brood should be collected. Both RAPD-PCR and allozyme electrophoresis suggest that the colonies of P. machaon in Broadland have a high rate of migration between them. This is consistent with the predictions of Dempster (1995) and has a number of implications for the species conservation in the UK. Firstly it suggests that any action plan for the species should concentrate on larger scale factors, such as landscape features and availability of habitat patches within Broadland, rather than being concerned with population ¯uctuations in individual sites. It also suggests that sites will be colonised as habitat becomes suitable without the need for costly arti®cial re-establishment. This is in agreement with the distribution map produced by Hall (1991) that shows the species occurring in all tetrads (2 km2) where suitable habitat is known to exist in Broadland, and with Anon (1995) who report that the species is well distributed throughout Broadland and has responded well to appropriate habitat management. Although our results do not provide evidence that the long term persistence of the species in Broadland is dependent on frequent movement between patches, it is possible that a patchy population or metapopulation structure is important and this may have implications for attempts to re-establish the species at isolated sites outside Broadland. From another point of view it may be more dicult to defend expensive or politically unpopular conservation measures taken to protect one colony if it is perceived as being a small part of one large population than if it can be characterised as being unique. However, our results do not devalue individual sites since there is no way of currently estimating how important individual sites (or the genetic diversity in the population at that site) are to the persistence of the species as a whole. Therefore, using the precautionary principle, all sites should be protected.
Acknowledgements The authors thank English Nature, Butter¯y Conservation, the RSPB and the Norfolk Wildlife Trust for their assistance in obtaining ®eld samples, Christina James for assistance with data analysis and three anonymous referees for their comments on an earlier version of this paper. This work was supported by a BBSRC CASE studentship in partnership with English Nature. References Anon., 1995. The Swallowtail butter¯y, Papilio machaon: a Species Action Plan. Butter¯y Conservation, Wareham Apostol, B.L., Black, W.C., Miller, B.R., Reiter, Beaty, B.J., 1993. Estimation of the number of full sibling families at an oviposition site using RAPD-PCR markers: applications to the mosquito Aedes aegypti. Theoretical and Applied Genetics 86, 991±1000. Ayala, F.J., Powell, J.R., Tracey, M.L., Mouras, C.A., Perez-Salas, S., 1972. Enzyme variability in the Drosophila willistoni group. IV. Genetic variation in natural populations of D. willistoni. Genetics 70, 113±139. Black, W.C. IV, 1993. PCR with arbitrary primers: approach with care. Insect Molecular Biology 2 (1), 1±6. Black, W.C., DuTeau, N. M., Puterka, G. J., Necholo, J. R., Pettorini, J. M., 1992. Use of the random ampli®ed polymorphic DNA polymerase chain reaction (RAPD-PCR) to detect DNA polymorphisms in aphids. Bulletin of Entomological Research 82, 151±159. Bossart, J.L., Powell, D.P., 1998. Genetic estimates of population structure and gene ¯ow: limitations, lessons and new directions. Trends in Ecology and Evolution 13, 202±206. Cameron, J., 1996. An assessment of the use of molecular techniques in insect conservation. Ph.D. Thesis, Keele University. Dempster, J.P., 1995. The ecology and conservation of P. machaon in Britain. In: Pullin, A.S (Ed.), Ecology and Conservation of Butter¯ies. Chapman and Hall, London. Dempster, J.P., King, M.L., Lakhani, K.H., 1976. The status of the Swallowtail Butter¯y in Britain. Ecological Entomology 1, 71±84. Emmett, A.M., Heath, J. (Eds.), 1990. The Butter¯ies of Great Britain and Ireland, Vol. 7, Pt 1. Harley Books, Colchester. Felsenstein, J., 1989. PHYLIP; phylogeny inference package (version 3.2). Cladistics 5, 164±166. Francisco-Ortega, J., Newbury, H.J., Ford-Lloyd, B.V., 1993. Numerical analyses of RAPD data highlight the origin of cultivated tagasaste (Chamaecytisus proliferus ssp. palmensis) in the Canary Islands. Theoretical and Applied Genetics 87, 64±270. Gibbs, H.L., Prior, K.A., Weatherhead, P.J., 1994. Genetic analysis of populations of threatened snake species using RAPD markers. Molecular Ecology 3, 329±327. Hall, M.R., 1991. An Atlas of Norfolk Butter¯ies 1984±1988. British Butter¯y Conservation Society, Norfolk. Hillis, D.M., Moritz, C., 1991. Molecular Systematics. Sinauer Assoc. Inc., Sunderland, MA. Krafsur, E.S., Black, W.S. VI, 1992. Analysis of isozyme loci in the face ¯y Musca autumnalis De Geer. Biochemical Genetics 30, 625±634. Lewis, P.O., Zaykin, D., 1998. GDA(v1. 0 d11): software for the analysis of discrete genetic data. Available at http://chee. unm.edu/gda/. McKechnie, S.W., Ehrlich, P.R., White, R.R., 1975. Population genetics of Euphydryas butter¯ies. I. Genetic variation and the neutrality hypothesis. Genetics 81, 571±594. Munstermann, L.E., 1994. Unexpected genetic consequenses of colonization and inbreeding: allozyme tracking in Culicidae (Diptera). Annals of the Entomological Society of America 87 (2), 157±164.
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Estimates of gene ¯ow between populations of the swallowtail butter¯y, Papilio machaon in Broadland, UK and implications for conservation J.C. Hoole a, D.A. Joyce b, A.S. Pullin b,* b
a Biological Sciences Department, Keele University, Keele, Staordshire ST5 5BG, UK School of Biological Sciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Received 27 May 1998; received in revised form 26 October 1998; accepted 4 November 1998
Abstract The swallowtail butter¯y, Papilio machaon has a recognised subspecies britannicus which is con®ned to the Broadland area of Norfolk, UK. The subspecies is threatened by habitat change and fragmentation. Gene ¯ow was studied between four sites in this area using allozyme and RAPD-DNA analysis to see if there is any evidence of isolation of populations or inbreeding that could pose a threat to the species persistence. The allozyme studies suggest high levels of gene ¯ow and a very low value of Nei's genetic distance (D) between sites. RAPD analysis also suggested extensive gene ¯ow between sites. The results indicate that there is little or no isolation of populations and that the species is capable of colonising and exploiting suitable habitats within the Broadland area. Consequently a landscape scale rather than site-based conservation strategy is recommended. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Allozymes; RAPD-DNA; Gene ¯ow; Genetic distance; Conservation genetics
1. Introduction The swallowtail butter¯y Papilio machaon was once widespread in wetland areas of southern and eastern England, but is now restricted to the Broadland area of Norfolk and part of Suolk (Emmet and Heath, 1990), where it is recognised by some authors as a distinct subspecies, P. m. britannicus endemic to the UK (Dempster, 1995; Emmet and Heath, 1990). This species is listed in the British Red Data Book as vulnerable and is protected under the UK Wildlife and Countryside Act 1981. It is a strong ¯yer, and one of the most spectacular and beautiful of the British butter¯ies. Its decline is probably due to increased drainage of fen areas which resulted in the loss of its larval foodplant milk parsley Peucedanum palustre. Continental subspecies of P. machaon have a greater range of larval foodplants and are less sedentary than the specialised British subspecies (Dempster et al., 1976; Dempster, 1995). The tendency of the British subspecies to have
* Corresponding author. Tel.: +44-121-414-7147; fax: +44-121414-5925; e-mail: [email protected]
restricted breeding areas where the larval foodplant occurs creates diculties in determining whether the colonies in Broadland should be treated as one population for conservation purposes, or as a number of isolated to semi-isolated populations. The aim of the work reported here was to estimate the level of gene ¯ow occurring between breeding areas. This will help identify the type of conservation strategy required for this subspecies, and will also indicate the likelihood of (re)colonization of suitable breeding areas created by appropriate management or vacated following local extinction. 2. Methods and materials 2.1. Sample collection Over a period of 3 years from 1993 to 1995 samples were collected from four sites in Broadland: Hickling Broad, on the River Thurne, Woodbastwick Fen, on the Bure marshes, Cat®eld Fen in the Ant valley and Strumpshaw Fen, on the River Yare (Fig. 1). These are as geographically separated as possible to fully test for isolation by distance.
0006-3207/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0006-3207(99)00003-8
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It was intended to collect larvae from all four sites but, because of poor seasons, it was considered inadvisable to take larvae from Strumpshaw, and haemolymph samples only were able to be taken from this site, while larvae and haemolymph samples were removed from the other sites. As far as possible, larvae were reared through to adults before being used for analysis in order to prevent the accidental inclusion of plant DNA from larval gut contents. All larval tissue was ¯ash frozen in liquid nitrogen and stored at ÿ70 C until required. Haemolymph samples were frozen on dry ice in the ®eld, transported back to the laboratory in the same medium and stored at ÿ70 C. 2.2. Choice of techniques A combination of RAPD-PCR (random ampli®ed polymorphic DNA-polymerase chain reaction) and allozyme markers was chosen to complement one another. RAPD-PCR (a dominant PCR-based marker) analysis is a fast and powerful treatment that can provide direct evidence of gene-¯ow between populations (e.g. Gibbs et al., 1994). Since this method is based on random DNA ampli®cation, precautions must be taken to avoid artefactual results. Indeed some workers have criticised the technique on the grounds that repeatability is low since the use of such short primers allows spurious bands to be produced. Black (1993) suggests a system to overcome this based on replication of reactions and distribution among gels to avoid dierences based on run-to-run variation being interpreted as real population dierences. In contrast, allozymes are a co-dominant, proteinbased marker system which provides added information from heterozygosity levels.
In using an approach which relies on model-based estimates of gene ¯ow and in which markers may be of low resolving power, more than one technique is preferred (Bossart and Powell, 1998). In combination, the two techniques can be used for the estimation of heterozygosity, gene ¯ow, genetic distance and variation between populations, all of which are of great importance in planning the conservation of populations of endangered species whose degree of isolation from one another may be in doubt. 2.3. Allozyme analysis Standard methods of starch gel electrophoresis were adapted for allozyme analysis. A single buer TBE buer (0.5 M Tris, 0.65 M boric acid and 0.02 M EDTA) was used for all experiments. Starch gels consisted of an 11% (w/v) solution of potato starch in 195 ml of a 1:9 dilution of TVB buer (Hillis and Moritz, 1991). Gels were run for 4 h at 60 mA in a cooled tank. Eight dierent enzymes were examined using stains adapted from several sources as follows: glutamate-oxaloacetate transaminase (GOT) (E.C. 2.6.1.1) (McKechnie et al., 1975); acid phosphatase (ACP); (E.C. 3.1.3.2), alkaline phosphatase (AKP) (E.C. 3.1.3.1), malic enzyme (ME) (E.C. 1.1.1.40), (Ayala et al., 1972); phosphoglucose isomerase (GPI) (E.C. 5.3.1.9), phosphoglucomutase (PGM) (E.C. 2.7.5.1), adenylate kinase (AK) (E.C. 2.7.4.3), hexokinase (HEX) (E.C. 2.7.1.1), (Shaw and Prasad, 1970) Where agar overlays were required (Krafsur and Black, 1992) (ME, PGM, GPI, HEX, AK) 10 ml 2% agar replaced 10 ml of stain. 2.4. RAPD-PCR analysis
Fig. 1. Sites from which Papilio machaon were collected in the Broadland area of Norfolk, UK. (1) Hickling Broad, (2) Woodbastwick Fen, (3) Cat®eld Fen, (4) Strumpshaw Fen.
Template DNA was prepared from butter¯y thoracic tissue by Proteinase K/phenol chloroform extraction (Hillis and Moritz, 1991) and from larval haemolymph and dried tissue by the use of a silicon dioxide/guanidine thiocyanate extraction, (Cameron, 1996). RAPD-PCR was carried out using standard methods (Apostol et al., 1993; Black et al., 1992). The ampli®cation programme used consisted of: 1 cycle of 94 C for 5 min (denaturing); 35 cycles of 94 C for 30 s (denaturing), 36 C for 1 min (annealing), 72 C for 1 min (extension); 1 cycle of 72 C for 5 min (extension). For each individual sample ampli®ed with each primer, six replicates were performed over three successive runs on the thermal cycle, in order to ensure reproducibility of RAPD pro®les (Black, 1993). PCR products were separated on a 1515 cm 1.4% agarose gel which was run for 16 h at 40V, in 0.5 M TBE buer. Ethidium bromide was added to the running buer. Discrete fragments of DNA were resolved into bands which were
J.C. Hoole et al. / Biological Conservation 89 (1999) 293±299
visualised under UV light. A permanent record was made by photographing the gel and recording it on disc using a UVP gel imaging system. The same system was later used in the analysis of the gels. A set of 20 primers was screened using Pieris brassicae DNA, of which ®ve were eventually used because they showed individual variation, reproducibility and consistent priming. The sequences of the primers used are: (1) TGCGCCCTTC; (2) TTCCCCCGCT; (3) TCCGCTCTGG; (4) CCACAGCAGT; (5) ACCCCCGAAG. The bands to be scored were chosen as a result of their intensity; this was determined by eye. Band size for each individual was assessed using the gel imaging system described previously. Consistently ampli®ed bands were selected by comparison between sample replicates and each individual scored for presence (1) or absence (0) of bands found in the species sample. This information for all primers was combined into a data matrix for analysis. 2.5. Statistical analysis 2.5.1. Allozymes GDA version 1.11 (Lewis and Zaykin, 1998) was used to compute Nei's (1978) genetic distance which corrects for a small sample size. This information was used with the geographic distance (km) to perform a Mantel test using NTSYSpc version 2.01e (Rohlf, 1992). Arlequin version 1.1 (Schneider et al., 1997) was used to obtain a matrix of population pair-wise FST values and their Pvalues after 100 permutations. The P-value of the test is the proportion of permutations leading to an FST value larger or equal to the observed one. FST values can be used as a measure of short term genetic distance between populations with the application of a slight transformation to linearise the distance with population divergence time (Schneider et al., 1997; Reynolds et al., 1983; Slatkin, 1995). The estimator used follows Weir and Cockerham (1984). Population level neighbour joining trees were created using PHYLIP (Felsenstein, 1989) from both Nei's 1978 genetic distance and the population pair-wise FST values as a comparison. Other
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F-statistics were calculated in PopGene version 1.21 (Popgene, 1997) according to Nei (1987). 2.5.2. RAPDs The RAPD-DNA pro®les produced were analysed using NTSYSpc version1.70 (Rohlf, 1992). A measure of genetic identity was produced using two dierent methods; the simple matching method produces matches between all possible pairs of individuals using both presence and shared absences of bands, whereas the Jaccard coecient is calculated by including only presence of bands, ignoring shared absences (FranciscoOrtega et al., 1993). 3. Results 3.1. Allozymes Of eight loci, one was monomorphic (ME) and the remaining seven were characterised by a total of 19 alleles. Nei's (1978) D for P. machaon was very small for all three pairs of populations, but it is slightly greater between those from Cat®eld Fen and Woodbastwick (0.104) and between Cat®eld and Hickling (0.136) than between Hickling and Woodbastick (0.017) (Table 1). Population pair-wise FST values show a slightly dierent pattern, with the largest value (0.122) between Cat®eld Fen and Woodbastwick. However, these values are also very low. Population level neighbour joining trees pictorially represent these discrepancies (Fig. 2), which are probably due to the low sample size (n=5) from Cat®eld Fen. The F-statistics across populations for each locus (Table 2) suggest that there is an excess of heterozygotes at ®ve out of eight loci mostly due to the results from Woodbastwick. The mean FIS is close to zero, implying that there is neither an excess or de®cit of heterozygotes.
Table 1 Geographic distance between sampled sites in km (upper triangular matrix), population pair-wise FST values (where * indicates a signi®cant P value at the 0.05 level) and Nei's (1978) genetic distance between Papilio machaon populations (lower triangular matrix) Woodbastwick Woodbastwick Hickling Cat®eld
(km) FST D FST D
0.042 0.017 0.122* 0.104
Hickling
Cat®eld
10
6 6
0.000 0.136
Fig. 2. Population level neighbour joining phylograms showing the relationship between Papilio machaon populations according to (a) FST values and (b) Nei's (1978) genetic distance.
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Table 2 F-statistics (calculated using Popgene version1.21) for each Papilio machaon population and each enzyme locus, where (n) is the sample size Locus
FIS values (n) Woodbastwick
Hickling
Cat®eld
All populations
FIT values(n) All populations
HEX PGM GPI GOT AK ME ACP AKP
0.0588(24) ÿ0.3589(30) ÿ0.2000(30) ÿ0.0714(30) ÿ0.2857(18) n/a(24) 0.5556(12) 0.0667(14)
ÿ0.0338(40) 0.0170(40) ÿ0.2353(42) 0.3939(44) 0.1667(30) n/a(34) 0.2727(18) 0.2086(22)
ÿ0.1111(10) 0.0909(10) n/a(10) ÿ0.4286(10) n/a(2) n/a(8) 0.6774(10) 0.1304(10)
ÿ0.0294(74) ÿ0.0805(80) ÿ0.2185(82) ÿ0.0525(84) ÿ0.0227(50) n/a(66) 0.4950(40) 0.1340(46)
0.0376(74) ÿ0.0615(80) ÿ0.1351(82) 0.0928(84) 0.1439(50) n/a(66) 0.5607(40) 0.2841(46)
Mean
ÿ0.0422(23)
0.1186(34)
0.0617(65)
0.1560(65)
0.1111(9)
The amount of genetic variance displayed within samples from the three reserves is very small, (FST=0.107) indicating that any one colony will contain 89.3% of the gene variability present in the whole population and that there has been little population subdivision. The value of FIT is 0.15; if the population were wholly panmictic the expected value would be zero. The average heterozygosities, (Table 3) are between 0.364 and 0.476. Estimated allele frequencies for each population are shown in Table 4. A Mantel test to assess whether or not the small amount of structuring observed was due to the distance between colonies sampled, showed no relation between the two. Comparing Nei's (1978) genetic distance and the geographic distance in km between sites showed that the two were not positively related (r=ÿ0.96).
Table 3 Sample number (N), Average heterozygosity (H) and variance (V) for three Papilio machaon populations in Broadland, UK Populations Hickling Broad
Woodbastwick Fen
Cat®eld Fen
N H V
14 0.433 0.078
5 0.364 0.099
22 0.476 0.080
3.2. RAPD-PCR A total of 109 reproducible bands was produced using ®ve primers. Clustering analysis using the simple matching coecient with UPGMA showed clustering at a high level of identity between all individuals (Fig. 3). The deepest level of division, at between 0.5 and 0.55 gave three clusters, one consisting of four butter¯ies from Hickling Broad and one from Strumpshaw Fen, another containing the three individuals from Cat®eld Fen and one from Woodbastwick, and the third containing members from all sites except Cat®eld. Using Jaccard similarity with UPGMA clustering showed divisions at a lower level of identity, but the clusters segregated at the deepest levels contained members of all populations, except for one small cluster consisting of three individuals from Hickling, and the Cat®eld Fen butter¯ies which appeared in one cluster with individuals from all other populations (Fig. 4). The Jaccard matching with the Neighbour-Joining method showed a similar level of identity between most individuals, but the three major clusters divided at a lower level of identity, between 0.05 and 0.1, and each included representatives of all populations except that from Cat®eld Fen (Fig. 5). Once again all three individuals from this site appeared in one cluster, with representatives from all other populations.
Table 4 Allozyme allele frequencies for three Papilio machaon populations in Broadland, UK Population
Allele
Locus: HEX
PGM
GPI
GOT
AK
ME
Hickling Broad
1 2 3
0.167 0.381 0.452
0.300 0.425 0.275
0.786 0.214 Ð
0.727 0.250 0.023
0.625 0.375 Ð
1.000 Ð Ð
Woodbastwick Fen
1 2 3
0.125 0.250 0.625
0.333 0.433 0.233
0.833 0.167 Ð
0.467 0.533 Ð
0.778 0.222 Ð
1.000 Ð Ð
Cat®eld Fen
1 2 3
Ð 0.700 0.300
0.500 0.300 0.200
1.000 Ð Ð
0.500 0.500 Ð
1.000 Ð Ð
1.000 Ð Ð
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4. Discussion
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to this study because of the small number of individuals sampled from the Cat®eld population. The allele frequencies in each population reveal that two of the loci
The very low values of genetic distance obtained for the three populations of P. machaon and the lack of correlation between genetic and geographical distance are consistent with the hypothesis that there is considerable gene ¯ow between the colonies in Broadland. Neighbour-joining trees based on allozyme data show discrepancies according to whether FST or D was used. This is likely to be due to the small sample size from Cat®eld Fen. The very low value of FIT over all loci also suggests high levels of gene ¯ow between the three populations. The fact that none of the loci shows an average heterozygosity signi®cantly dierent from Hardy±Weinburg expectations, and that FIS is 0.062, indicates that the population as a whole is in Hardy±Weinburg equilibrium and that inbreeding has had very little eect on the populations. The average heterozygosities of the three samples of P. machaon are high for a species which eectively has been reduced to a few colonies within a small geographical area for many generations, especially when compared with the values of 0.16 for P. mnemosyne in the Alps (Napolitano et al., 1988). However, the reservations about the validity of H as a measure of the genetic viability of a population expressed by a number of workers (Nei and Roychoudury, 1974; Singh and Rhomberg, 1987; Munsterman, 1994) apply even more strongly
Fig. 4. Clustering of individual Papilio machaon from Broadland populations by UPGMA using Jaccard coecient. Individuals 1H± 13H are from Hickling Broad, 14W±23W are from Woodbastwick Fen, 24S±27S are from Strumpshaw Fen, 28C±30C are from Cat®eld Fen.
Fig. 3. Clustering of individual Papilio machaon from Broadland populations by UPGMA using simple matching. Individuals 1H±13H are from Hickling Broad, 14W±23W are from Woodbastwick Fen, 24S±27S are from Strumpshaw Fen, 28C±30C are from Cat®eld Fen.
Fig. 5. Clustering of individual Papilio machaon from Broadlands populations by neighbour-joining using the Jaccard coecient. Individuals 1H±13H are from Hickling Broad, 14W±23W are from Woodbastwick Fen, 24S±27S are from Strumpshaw Fen, 28C±30C are from Cat®eld Fen.
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have only two alleles and one is monomorphic, while each of the other ®ve loci has only three alleles. However, the number of alleles which constitutes a `safe' level of genetic variability is largely a matter of interpretation. The results obtained using RAPD analysis also suggest that a considerable amount of gene ¯ow occurs between populations since the individuals analysed could not be separated into their populations by any of the methods of analysis used. Some individuals from each population consistently occurred in the same group, for example numbers 6, 7 and 8, from Hickling, the three from Cat®eld Fen, and numbers 19 and 20 from Woodbastwick Fen, which have the highest genetic identity in all the analyses. This may suggest that these groups of individuals are closely related, possibly from eggs laid by the same female. Since larvae were collected at random from foodplants, and larger instars were taken wherever possible, it is not impossible that members of the same brood should be collected. Both RAPD-PCR and allozyme electrophoresis suggest that the colonies of P. machaon in Broadland have a high rate of migration between them. This is consistent with the predictions of Dempster (1995) and has a number of implications for the species conservation in the UK. Firstly it suggests that any action plan for the species should concentrate on larger scale factors, such as landscape features and availability of habitat patches within Broadland, rather than being concerned with population ¯uctuations in individual sites. It also suggests that sites will be colonised as habitat becomes suitable without the need for costly arti®cial re-establishment. This is in agreement with the distribution map produced by Hall (1991) that shows the species occurring in all tetrads (2 km2) where suitable habitat is known to exist in Broadland, and with Anon (1995) who report that the species is well distributed throughout Broadland and has responded well to appropriate habitat management. Although our results do not provide evidence that the long term persistence of the species in Broadland is dependent on frequent movement between patches, it is possible that a patchy population or metapopulation structure is important and this may have implications for attempts to re-establish the species at isolated sites outside Broadland. From another point of view it may be more dicult to defend expensive or politically unpopular conservation measures taken to protect one colony if it is perceived as being a small part of one large population than if it can be characterised as being unique. However, our results do not devalue individual sites since there is no way of currently estimating how important individual sites (or the genetic diversity in the population at that site) are to the persistence of the species as a whole. Therefore, using the precautionary principle, all sites should be protected.
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