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Phylogeographic population structure of great reed warblers: an analysis of mtDNA control region sequences
Biological Journal of the Linnean Society (1999), 66: 171–185. With 5 figures Article ID: bijl.1998.0261, available online at http://www.idealibrary.com on
Phylogeographic population structure of great reed warblers: an analysis of mtDNA control region sequences STAFFAN BENSCH∗ AND DENNIS HASSELQUIST Department of Animal Ecology, Ecology Building, Lund University, S-223 62 Lund, Sweden Received 2 February 1998; accepted for publication 20 June 1998
A portion of the mitochondrial control region (494 bp) was sequenced in 106 great reed warblers sampled in six breeding populations in Europe and one wintering population in Africa. In total, 33 different haplotypes were found. There was little evidence of divergence between populations in northern and western Europe whereas the sample from Greece differed significantly from the other European breeding populations. The lowest haplotype diversity was found near the distribution range limit in Sweden and in The Netherlands suggesting recent effects of bottlenecks/founder events in these areas. A neighbour-joining analysis of the different haplotypes placed the haplotypes into two distinctive clades, A and B. The divergence of the two clades was on average 1.29%. Accounting for the within clade variation suggested a divergence time between these lines approximately 70 000 years BP. The frequency of the two clades changed longitudinally across Europe with the A haplotype in the west and the B haplotype in the east. All birds from Kenya carried the B haplotype suggesting an origin of these birds east of Latvia/Greece. The long-term female effective population size was estimated to be 20 000 individuals, which is approximately 2% of current population size. 1999 The Linnean Society of London
ADDITIONAL KEY WORDS—mtDNA – geographic variation – Acrocephalus – effective population size – Afrotropical migrant. CONTENTS
Introduction . . . . . . . . . . . Material and methods . . . . . . . Population samples and DNA isolation The control region . . . . . . . Amplification and sequencing . . . Descriptive statistics and analyses . . Results . . . . . . . . . . . . Sequence variation . . . . . . . Relationships between haplotypes . . Discussion . . . . . . . . . . . Variation between populations . . .
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∗ Corresponding author Email: [email protected] 0024–4066/99/020171+15 $30.00/0
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Comparing samples from breeding and winter quarters . Relationships between haplotypes and population history Acknowledgements . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .
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INTRODUCTION
The population-genetic structure of a species depends on both historical events and current processes (Avise, 1994; Templeton et al., 1995). Variation in effective population size between populations, and dynamics of range extension or contraction, may contribute to create geographic differences in allele frequencies through the act of genetic drift and founder events. The genetic divergence of populations may, or may not, be magnified by selection for local adaptations or retarded by gene flow. The level of gene flow is determined by the degree of geographic isolation between populations and behaviours of individuals, e.g. whether they are philopatric or show mating preferences related to geographical origin of potential partners. Overall, species with limited dispersal potentials will show stronger phylogeographic structuring than species with high degree of dispersal (e.g. Janson, 1987). Because of a relatively high mutation rate and haploid maternal inheritance, mitochondrial DNA (mtDNA) has been extensively used in studies of phylogeographic population structure within animal species (Avise, 1994). In general, terrestrial vertebrates show substantial mtDNA phylogeographic structuring, although the degree of structuring may vary considerably between species (Avise, 1994). Several species expected to show substantial phylogeographic structuring due to extensive morphological variation across ranges show limited mtDNA structuring (e.g. Ball et al., 1988; Seutin et al., 1995; Zink & Dittmann, 1993). Moreover, unexpected longdistance gene flow has been detected in species believed to show limited dispersal, e.g. in co-operatively breeding birds (Edwards, 1993). On the other hand, a substantial phylogeographic structuring of mitochondrial DNA lineages has been reported in species with high intrinsic dispersal capabilities, e.g. migratory birds (Wenink et al., 1996) and cetaceans (Baker et al., 1994; Brown Gladden et al., 1997; Walton, 1997). Hence, at present, mtDNA phylogeographic structure cannot be conclusively predicted from observed morphological variation across regions and capacity of dispersal. The great reed warbler Acrocephalus arundinaceus is a polygynous long-distance migratory passerine bird that breeds in marshy habitats in most of the Palaearctic between latitudes 35° and 60°N. Within Europe, available data do not reveal any morphological differentiation (Cramp, 1992). The species has shown range expansion in northern Europe over the past 100 years (Hagemeijer & Blair, 1997). At present, numbers are stable or still increasing in the Baltic countries (Sweden, Estonia, Latvia, Lithuania) whereas populations in western Europe (Denmark, The Netherlands, Belgium and France) have declined by more than 50% over the past thirty years. The recent and large changes of the breeding range in NW Europe and the high dispersal capability of this long-distance migratory species may have led to limited phylogeographic structuring among great reed warbler populations in Europe. On the other hand, we have observed two behaviours in great reed warblers that may act to maintain or restore population-genetic structuring: (1) both males and females show high fidelity to breeding sites (Bensch & Hasselquist, 1991), and (2) philopatric
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T 1. Sampling localities, co-ordinates and years Country
Site
Coordinates
Sampling year
Spain The Netherlands Sweden Germany Latvia Greece Kenya
Hondo Natural Park, Alicante Zwarte Meer/Weerribben Kvismaren, Na¨rke Mu¨ggelsee, Berlin Engure/Kanieris Limni Mikri Prespa, Florina Ngulia, Tsavo National Park
38°12′N, 0°42′W 52°37′N, 5°55′E 59°10′N, 15°25′E 52°26′N, 13°39′E 57°07′N, 23°20′E 40°50′N, 21°05′E 3°00′S, 38°08′E
1996 1995 1987–1990 1992,1993 1992 1990 1990,1991
individuals have higher fitness than immigrants (Bensch et al. 1998). Hence, at present, we cannot make unequivocal predictions about phylogeographic structuring of great reed warblers. However, because range expansions most likely involve repeated bottlenecks with associated loss of genetic material as new areas are colonized (Merila¨ et al., 1997), we expect to find less genetic variability within the newly colonized northern great reed warbler populations compared to southern populations. Moreover, as winter populations likely consist of a mix of birds from different breeding populations, a sample from African wintering areas should contain more genetic diversity than samples from any breeding population. In the present study we analyse mtDNA sequence variation of great reed warblers from six populations of breeding birds in Europe and one population of wintering birds in East Africa to address the following questions: (1) whether there is any evidence of population-genetic structuring among European breeding populations; (2) whether the degree of genetic variation declines towards north; (3) whether the population of wintering birds shows more genetic variation than any of the breeding populations, and (4) what the relationships and frequencies of different haplotypes in Europe tell us about historical population size and distribution.
MATERIAL AND METHODS
Population samples and DNA isolation Samples of great reed warblers were obtained from six European breeding populations and from one locality in African winter quarters (Table 1). For all individuals, except the samples from The Netherlands, total DNA was extracted from whole blood (5–50 lL) stored in SET-buffer (500 lL) by a phenol-chloroform protocol (Hasselquist et al., 1995b). The samples from The Netherlands consisted of dried contour feathers plucked from nestlings (different nests) and total DNA was extracted by using 5% Chelex 100 (Biorad) following the method of Mundy et al. (1996).
The control region The mitochondrial control region II in great reed warblers was first amplified and sequenced by primers BCML1 (5′-CCCAACTTGCTCTTTTGCGC-3′) and
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Figure 1. The mitochondrial control region II in great reed warblers with flanking genes and locations of primers.
FTPH2 (5′-CCATCTTGACATCTTCAGTGCCATGC-3′) designed for loggerhead shrikes Lanius ludovicianus (Mundy et al., 1996). Because these original primers did not produce a single product, two new primers, BCML4 (5′-TTCACAGATACAAATGCTTGGG-3′) and FTPH3 (5′-AAGGCTGGGAGAGTTGTTGA-3′) were designed (Fig. 1), using the aligned great reed warbler and loggerhead shrike sequences. These two primers, which give a product of 577 bp, were then used to amplify the control region II in all individuals included in this study. In order to confirm that this fragment was from the control region, the primer BCML4 was used together with 12SH1 (5′-AATGTTTACTACTGCTGAGAACCCG-3′) (Mundy et al., 1996) (Fig. 1) and their product sequenced. The 81 bases flanking the control region (3′) of great reed warblers were aligned with sequences of the tRNAPhe gene of chicken and logger-head shrikes (Fig. 2). As expected, the sequences diverge quickly as one goes into the control region (downstream of the tRNAPhe gene). From this observation alone we cannot exclude the possibility that the obtained sequences origin from a numt, i.e. a mtDNA copy incorporated into the nuclear genome (Quinn, 1997). However, from the samples at our Swedish study site we have extensive family data of which parentage has been confirmed by DNA fingerprinting (Hasselquist et al., 1995). This enabled us to confirm that the haplotypes are maternally inherited (n=5 families of which parents carried different haplotypes). Hence, because the 3′ flanking sequence aligns with the tRNAPhe gene in other species, and that haplotypes are maternally inherited, it is safe to conclude that our results are based on the control region of the mitochondria. Amplification and sequencing Polymerase chain reaction (PCR) was performed in volumes of 25 lL and included 10–50 ng of total genomic DNA, 0.125 mM of each nucleotide, 1.5 mM MgCl2, 0.6 lM of each primer and 0.5 units of Taq DNA polymerase. The PCR’s were run using the following conditions: 30s at 94°C, 30s at 50°C, 30s at 72°C (35 cycles). Before the cyclic reactions the samples were incubated at 94°C for 3 min, and after completion at 72°C for 5 min. We used 2.5 lL of the final reaction to run on a 2% agarose gel in 0.5X TBE buffer to check the success of the reaction. The remainder of the PCR product was precipitated by adding 10 lL of 8M NH4Ac and 32 lL of ethanol. Following centrifugation the air-dried DNA pellet was dissolved in 20 lL of water; 2–4 lL was then used for sequencing. The double stranded PCR products were either sequenced using the AmpliCycle Sequencing Kit (Perkin Elmer) with c-32P-ATP end-labelled primers according to the manufacturer’s recommendation and separation of fragments in 8% denaturing polyacrylamide gel (60% of the samples), or with dye terminator cyclic sequencing
Figure 2. mtDNA sequence alignments (light strand shown) of the 3′ end of control region and flanking genes. arund: Acrocephalus arundinaceus (bases 476–596, Genbank AF111791), orient: A. orientalis, stent: A. stentoreus, Lanius: Lanius ludovicianus (bases 324–447 Genbank S82910) and Chick: Chicken (bases 1186–1308 from Desjardins & Morais, 1990).
MITOCHONDRIAL POPULATION STRUCTURE OF GREAT REED WARBLERS 175
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on an ABI PRISM 310 (Perkin Elmer). Ten samples previously analysed by the first method were rerun on the new system and confirmed to produce consistent results. Descriptive statistics and analyses Nucleotide diversity (p) (Nei, 1987) and D statistics (Tajima, 1989) were calculated using the program DnaSP 2.52 (Rozas & Rozas, 1997). Estimates of haplotype diversity (h), was calculated as h=(n/(n−1))(1−Rfi2), where fi is the frequency of the ith haplotype in a sample of n individuals (Nei, 1987). We estimated long term effective female population size using the equation (Wilson et al., 1985): Nf=106∗(p/s)/g where p is the mean pair-wise sequence divergence, s the rate of sequence divergence (proportion substitutions/genome/Myr) and g is mean generation time (years). We assumed s=0.148 (Wenink et al., 1996) and g=2.0. Because the result is sensitive to errors in assumptions of s and g, estimates of Nf will provide information of order of magnitude only. The degree of geographical structuring of populations was tested using the program AMOVA (Excoffier et al., 1992) that calculates φST statistics (a measure analogous to FST) using Jukes and Cantor’s distance (Kumar et al., 1993). Significance of variance components between populations was tested with a randomization procedure provided in AMOVA (1000 permutations). Analyses of isolation by distance of φST statistics were tested by a Mantel’s permutation test provided in the program GENEPOP 3.1 (Raymond & Rousset, 1995). Evolutionary relationships between haplotypes were assessed by the neighbour-joining method with MEGA (Kumar et al., 1993). We used Tamura and Nei’s (1993) distance measure and a gamma correction parameter alpha of 0.04 to account for among-site variation in evolutionary rate (Yang, 1996), as estimated from the present data set with PUZZLE (Strimmer & von Haeseler, 1997). Sequences obtained from the oriental reed warbler Acrocephalus orientalis and clamorous reed warbler A. stentoreus (primers BCML4 and 12SH2 (5′-AGCAACAACCAACGGTAAG-3′) (Fig. 1) were used as outgroups, as these are the two most closely related taxa to the great reed warbler (Leisler et al., 1997). RESULTS
Sequence variation We sequenced 494 bp of the control region II in 106 samples from seven populations. This region contained 29 variable sites which defined 33 different haplotypes between 0.2 and 2.2% (Table 2). None of the individuals appeared to be heteroplasmic, i.e. carrying two different haplotypes (cf. Mundy et al., 1996), and the variable sites never showed shadow bands of the alternative nucleotide. All substitutions except two were transitions (A↔G=11, C↔T=17). The two transversions were noted at positions 386 and 431 (Table 2). The most common haplotype (#20) was detected in 37 (35%) of the samples and was also the most widespread and recorded at all sites except in Kenya.
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T 2. Variable sites in the control region II segment of great reed warblers. Origin of specimens indicated by GE (Germany), GR (Greece), KE (Kenya), LA (Latvia), NL (The Netherlands), SP (Spain) and SW (Sweden) Haplotype (base number) SP 1112222 2333333444 444444444 1152395569 9114688122 233333489 6960152410 2250267905 801678932 TCAGCAGTGT CCTTCATATT ACGATAGTC
Code
.......... .......... ......... C......... .......... ......... C......... .......... G........ C......... .......G.. ......... .......... .........C ......... C......... .......... .....G... C......... .......... G....G... C......... .......... ...G..... C.....AC.. .......... .....G... C......... .......... .......C. .T......A. .......... ......ACT .T......A. .T........ ......ACT .T........ .T........ ......ACT C.......A. .T........ ......ACT C...T...A. .T........ ......ACT C....G..A. .T........ ......ACT C.......A. TT........ ......ACT C.......A. .T........ G.....ACT C.......A. .T........ ....C.ACT CT......A. .T........ ......ACT CTG.....A. .T........ ......ACT CT.A....A. .T........ ......ACT CT......A. .T......C. G.....ACT CT......A. .T........ .T....ACT CT......A. .T....C... ......ACT CT......AC .T........ ......ACT CT......A. .T........ ..A...ACT CT......A. .T...T.... ......ACT CT........ .T........ G.....ACT CT........ .T..T..... ......ACT CT........ .T........ ......ACT CT........ .TCC...... ..T...ACT CT...G.... .T........ ......ACT
#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 #14 #15 #16 #17 #18 #19 #20 #21 #22 #23 #24 #25 #26 #27 #28 #29 #30 #31 #32 #33 Total
NL
SW
Population GE LA
1
1
2
1 1
2
GR
1 1 1 1 2
3 1
2 2 1 1
6
7
1 1 1 3
1 13
5 1
4 1 1 4
2
1 1 1 1 1 1 1 1
1 2 1
2 1 1 11
10
22
20
20
3 1 2 1 1 5
1 1
KE Total
10
1 6 1 3 1 4 2 1 1 10 1 1 2 8 1 1 1 4 1 37 1 1 1 1 1 1 1 1 1 2 3 1 1
13 106
Nucleotide diversity and heterozygosity was lowest in the western European samples and highest in Greece (Table 3). It is notable that the sample from the wintering grounds in Kenya, which supposedly consists of birds from different breeding localities, showed slightly lower levels of heterozygosity and nucleotide diversity. The estimates of Tajima’s (1989) D statistic were negative in six out of the seven sampled great reed warbler populations (Table 3). Under the neutral mutation hypothesis, the probability that D is negative is approximately 0.52 for samples sizes between 10 and 20 (Table 1 in Tajima, 1989). Under this assumption, the observed bias of negative D tends to be more extreme than expected by chance (binomial test, P=0.077). The overall φST for the European breeding populations was 0.115 suggesting that 11.5% of total variation is due to between population differences (P<0.001). However,
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T 3. Sample size (n), number of haplotypes (nH), haplotype diversity (H), nucleotide diversity (p), standard deviation of p (sd), and Tajimas (1989) D statistics for seven populations of great reed warblers nH
H
p
SD
D1
11 10 22 20 20 10 13
5 4 7 13 11 8 6
0.709 0.533 0.649 0.926 0.916 0.955 0.821
0.00201 0.00204 0.00478 0.00646 0.00653 0.00932 0.00342
0.00055 0.00085 0.00131 0.00112 0.00092 0.00122 0.00077
−1.03 −1.74∗ −0.50 −1.27 −0.21 1.28 −0.99
106
33
0.860
0.00706
0.00048
−1.21
n
Population Spain The Netherlands Sweden Germany Latvia Greece Kenya Pooled
Significance levels obtained from Table 2 in Tajima (1989), ∗ P<0.05
1
T 4. Comparison of nucleotide differences between populations expressed as φST (below diagonal). Values above the diagonals are estimates of the probability that the observed differences between samples would occur by chance (1000 permutations by the AMOVA program) Spain
The Netherlands
Spain The Netherlands −0.038 Sweden 0.010 Germany 0.042 Latvia 0.096 Greece 0.426 Kenya 0.776
0.911
Sweden 0.407 0.258
0.024 0.031 0.108 0.428 0.783
−0.006 0.005 0.293 0.625
Germany
Latvia
Greece
Kenya
0.171 0.259 0.380
0.033 <0.001 0.435 0.398
<0.001 <0.001 <0.001 <0.001 <0.001
<0.001 <0.001 <0.001 <0.001 <0.001 0.038
−0.014 0.223 0.563
0.163 0.492
0.158
0.45
PhiST
0.35 0.25 0.15 0.05 –0.05
0
500
1000
2000 1500 Distance (km)
2500
3000
Figure. 3. Plot between φST and geographical distance between European breeding populations of great reed warblers. rs=0.45 (P=0.15, Mantel’s test).
only the samples from Greece differed significantly and consistently from the other European breeding populations (Table 4). The Kenyan sample was significantly different from all the European samples, with the largest difference to the Dutch/ Spanish and least to the Greek sample (Table 4). To examine whether differences in mtDNA variation increased with distance between the sampled populations, we plotted the between population φST values against great circle distances (Fig. 3).
MITOCHONDRIAL POPULATION STRUCTURE OF GREAT REED WARBLERS 22 25 21 26 28 24 27
20
12 88 31
11 13
A
30 32
33 18
10
2
3
8 4
23 29
17 15 16 19
14
179
B
1 6
5 7
9
Figure 4. Phylogenetic relationships among great reed warbler mtDNA haplotypes based on 494 bases of the control region II. Values at the nodes represent bootstrap replication scores (% of 500 resamplings). Haplotype codes as in Table 2.
There was no significant association between genetic differentiation and distance (rs= 0.45, P=0.15, Mantel’s test); however, because of low sample size (six populations) the power of this test is low. The mean pair-wise sequence divergence (p) between all the individuals at the European breeding grounds was 0.60% which translates into a long-term female effective population size of 20 000 individuals.
Relationships between haplotypes A neighbour-joining analysis of the different haplotypes in Table 2 inferred a gene-tree with one major branching event which was supported by a 88% bootstrap value (Fig. 4). Trees in which either or both the oriental and clamorous reed warblers were used as outgroups were similar and supported the placement of haplotypes in two well defined clades A (#11–33) and B (#1–10). When the frequencies of the two clades are plotted on a map, it appears that A haplotypes represent a western clade and B haplotypes an eastern clade (Fig. 5). All birds sampled in Kenya carried mtDNA belonging to the B clade. The mean sequence divergence between the two clades A and B (counting each haplotype once) was 1.29% (range 0.82–2.23%). The mean sequence divergences between the oriental reed warbler and the two clades A and B were 8.52% and 8.88%, respectively. The corresponding values between the two clades and the clamorous reed warbler were 8.34% and 9.09%. The sequence divergence between the oriental and the clamorous reed warbler was 5.71%. Hence, both of the two outgroups were slightly more similar to clade A than to clade B. The analysis of among-site variation in evolutionary rate revealed large heterogeneity as the parameter alpha was small (0.04, SE=0.05). Accounting for this gamma distributed rate heterogeneity in the distance model (Tamura & Nei, 1993),
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Figure 5. Map and frequency of the A (grey) and B (black) mtDNA haplotypes at the six sampled great reed warbler breeding sites in Europe. All individuals sampled from winter ground in Kenya carried B haplotypes.
the mean sequence divergence between the two clades A and B was 2.51% (range 1.05–4.54%), i.e. larger than when comparing the percentage of sequence divergence.
DISCUSSION
Variation between populations Haplotype diversity in the breeding populations of great reed warblers was intermediate to high compared to other species (Seutin et al., 1995; Baker & Marshall, 1997; Mundy et al., 1997). As predicted, most of the variation in control region sequences was found between individuals within populations. This was particularly so when the Greek samples were excluded from the analysis. Hence, there is little evidence of mitochondrial DNA differentiation between North European great reed
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warbler populations. Consistent with this genetic pattern, the species shows no strong morphological variation across Europe (Cramp, 1992), although slight differences might exist if one analyses sex and age groups separately. We found the lowest values of heterozygozity in The Netherlands and Sweden. Both these populations are situated close the species range limit; the Dutch population has decreased drastically over the last decades whereas the Swedish population was founded recently and has increased from approximately 50 to 500 pairs during the last 30 years (Holmbring, 1979; Tyrberg, 1996). Loss of genetic variability at nuclear loci has been suggested by DNA fingerprinting studies of Swedish great reed warblers which showed relatively high similarity between unrelated individuals compared to other species (Bensch et al., 1994). The results suggest that edge populations exhibit lower genetic variation than populations from the interior of the geographical range, which presumably have existed for a longer time. Similarly, declining levels of genetic diversity towards north have been observed in a number of species which expanded their ranges during Pleistocene (e.g. Merila¨ et al., 1997; Takahata, 1995).
Comparing samples from breeding and winter quarters An important finding of our study is that the level of haplotype diversity was not higher in Kenya than in the samples from the breeding populations in Europe. This is surprising since samples collected from one wintering area are likely to contain individuals from several breeding localities (e.g. Wenink et al., 1996); in the present case, this is even more likely because the samples from Kenya are from a stopover site where birds are in mid-winter migration across East Africa (Backhurst & Pearson, 1984). The low variation observed in the Kenyan sample may be due to breeding populations showing more specific wintering areas than previously assumed, though more data from wintering populations are needed before this hypothesis can be evaluated. However, it should be noted that great reed warblers seem to be faithful to their winter grounds in successive years (De Roo & Deheegher, 1969; Bensch et al., 1991). The great reed warbler is a commonly found winter visitor to most of tropical Africa as far as 25°S (Cramp, 1992). Birds ringed on western and Central European breeding grounds have been recovered in tropical West Africa (Schlenker, 1986; Dowsett et al., 1988). The samples from Kenya were significantly different from all the six sampled European breeding populations. In Europe, the frequency of the B haplotypes increased eastwards to reach a maximum of 60% in Greece. That all 13 birds from Kenya carried B-haplotypes suggests that these originated from populations east of Latvia/Greece. At present, there are not enough ringing recoveries to support this conclusion.
Relationships between haplotypes and population history The total European population has recently been estimated at one million pairs (Hagemeijer & Blair, 1997) which is approximately 50-fold larger than our estimate of long term effective population size (Nf ). Long-term effective population sizes estimated from mtDNA genetic distances are commonly smaller by 1–3 orders of magnitude than present-day census sizes (Avise, 1992). The discrepancy between
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population census estimates of Nf and long term estimates of Nf might be caused by population bottlenecks or selective sweeps (Avise, 1992). However, a large variance in reproductive success between females can also cause the long-term female reproductive success to be lower than census estimates. In female great reed warblers, the variance of lifetime reproductive success is as large as in males, despite high levels of polygyny in this species (Hasselquist, 1995). Hence, the observed discrepancy between present day and long-term population size might partly be explained by a large variance in female reproductive success. The relatively few sampling localities may also contribute to the discrepancy. The coexistence of two divergent mtDNA clades within the same population indicates recent secondary contact between allopatrically evolved populations (Avise et al., 1987). Assuming an evolutionary rate of 14.8% per Myr for the control region (Wenink et al., 1996), the average sequence divergence of the two haplotype clades A and B at 1.29% would correspond to a separation of the haplotype clades for 90 000 years. The frequency of the two clades changes with longitude in Europe, suggesting that the coexistence of distinct haplotypes is due to recent admixture of previously isolated populations. Accounting for the within clade diversity (Edwards, 1997) we can calculate the interpopulation sequence divergence (d) to be 0.98%. This suggests that the two populations housing the two clades separated approximately 70 000 years before present. The inferred time since divergence (70 000 years) and the geographical distribution of the two haplotypes suggest that the mtDNA clades evolved in two refugia during the last glaciation, e.g. one in western Europe and one in eastern Europe/Asia. It should be noted that the above calculations of dates of divergence is, among other things, sensitive to rate variation among sites which, in the present data set, was shown to be large. At temperate and higher latitudes, many species which at present have continuous ranges may during the Pleistocene Ice Age have been confined to two or more allopatric populations. Remaining evidence of latitudinal range expansions in temperate regions from refugia further south are the many existing contact zones, as characterized by e.g. sharp morphological clines (Hewitt, 1993) or migratory divides (e.g. Helbig, 1992). When the first examples of large within species divergence of mtDNA sequences were detected, this was considered to be exceptional, although examples of within species divergence at levels above 1% are accumulating (Avise et al., 1990; Quinn, 1992; Taberlet et al., 1992; Wenink et al., 1993; Friesen et al., 1996). In all these cases the most likely explanation seems to be the intermingling of populations which previously have diverged allopatrically. In the case of the great reed warbler, the cline of mtDNA does not coincide with a similar cline in morphology or migratory direction (Cramp, 1992). Similarly, in the northern flicker Colaptes auratus, a pronounced geographical cline of mtDNA haplotype groups does not coincide with the cline in morphology (Moore et al., 1991). Moreover, different populations of the knot Calidris canutus have evolved highly specialized migratory patterns but are still almost identical at their mitochondrial DNA (Baker et al., 1994). A challenge for coming studies will henceforth be to understand the seemingly complex and not always congruent pattern of genetic and morphological divergence. As selective sweeps and population bottlenecks may have considerably hampered information available on the mitochondria, successful studies are likely to include analyses of nuclear markers (Avise, 1994). ACKNOWLEDGEMENTS
We thank N.I. Mundy for advice and primers used to isolate the control region in great reed warblers. Samples were kindly provided by R.T. Pinheiro (Spain), S.
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Fischer (Germany), I. Nishiumi (A. orientalis), A. Helbig (A. stentoreus) and J. Graveland ˚ . Bensch assisted with data collection in (The Netherlands). M. Svensson and A Greece and Latvia. We are indebted to M. Nordborg for discussions about methods to analyse sequence variation. The manuscript benefited from comments by A.J. Baker, S.V. Edwards, B. Hansson and N.I. Mundy. The study was financially supported by the Royal Swedish Academy of Science (Ahlo¨fska), O. Engkvist foundation, Magn. Bergvalls foundation, Swedish Natural Science Research Council (NFR) and Swedish Forestry and Agricultural Research Council (SJFR).
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Phylogeographic population structure of great reed warblers: an analysis of mtDNA control region sequences STAFFAN BENSCH∗ AND DENNIS HASSELQUIST Department of Animal Ecology, Ecology Building, Lund University, S-223 62 Lund, Sweden Received 2 February 1998; accepted for publication 20 June 1998
A portion of the mitochondrial control region (494 bp) was sequenced in 106 great reed warblers sampled in six breeding populations in Europe and one wintering population in Africa. In total, 33 different haplotypes were found. There was little evidence of divergence between populations in northern and western Europe whereas the sample from Greece differed significantly from the other European breeding populations. The lowest haplotype diversity was found near the distribution range limit in Sweden and in The Netherlands suggesting recent effects of bottlenecks/founder events in these areas. A neighbour-joining analysis of the different haplotypes placed the haplotypes into two distinctive clades, A and B. The divergence of the two clades was on average 1.29%. Accounting for the within clade variation suggested a divergence time between these lines approximately 70 000 years BP. The frequency of the two clades changed longitudinally across Europe with the A haplotype in the west and the B haplotype in the east. All birds from Kenya carried the B haplotype suggesting an origin of these birds east of Latvia/Greece. The long-term female effective population size was estimated to be 20 000 individuals, which is approximately 2% of current population size. 1999 The Linnean Society of London
ADDITIONAL KEY WORDS—mtDNA – geographic variation – Acrocephalus – effective population size – Afrotropical migrant. CONTENTS
Introduction . . . . . . . . . . . Material and methods . . . . . . . Population samples and DNA isolation The control region . . . . . . . Amplification and sequencing . . . Descriptive statistics and analyses . . Results . . . . . . . . . . . . Sequence variation . . . . . . . Relationships between haplotypes . . Discussion . . . . . . . . . . . Variation between populations . . .
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∗ Corresponding author Email: [email protected] 0024–4066/99/020171+15 $30.00/0
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Comparing samples from breeding and winter quarters . Relationships between haplotypes and population history Acknowledgements . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .
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INTRODUCTION
The population-genetic structure of a species depends on both historical events and current processes (Avise, 1994; Templeton et al., 1995). Variation in effective population size between populations, and dynamics of range extension or contraction, may contribute to create geographic differences in allele frequencies through the act of genetic drift and founder events. The genetic divergence of populations may, or may not, be magnified by selection for local adaptations or retarded by gene flow. The level of gene flow is determined by the degree of geographic isolation between populations and behaviours of individuals, e.g. whether they are philopatric or show mating preferences related to geographical origin of potential partners. Overall, species with limited dispersal potentials will show stronger phylogeographic structuring than species with high degree of dispersal (e.g. Janson, 1987). Because of a relatively high mutation rate and haploid maternal inheritance, mitochondrial DNA (mtDNA) has been extensively used in studies of phylogeographic population structure within animal species (Avise, 1994). In general, terrestrial vertebrates show substantial mtDNA phylogeographic structuring, although the degree of structuring may vary considerably between species (Avise, 1994). Several species expected to show substantial phylogeographic structuring due to extensive morphological variation across ranges show limited mtDNA structuring (e.g. Ball et al., 1988; Seutin et al., 1995; Zink & Dittmann, 1993). Moreover, unexpected longdistance gene flow has been detected in species believed to show limited dispersal, e.g. in co-operatively breeding birds (Edwards, 1993). On the other hand, a substantial phylogeographic structuring of mitochondrial DNA lineages has been reported in species with high intrinsic dispersal capabilities, e.g. migratory birds (Wenink et al., 1996) and cetaceans (Baker et al., 1994; Brown Gladden et al., 1997; Walton, 1997). Hence, at present, mtDNA phylogeographic structure cannot be conclusively predicted from observed morphological variation across regions and capacity of dispersal. The great reed warbler Acrocephalus arundinaceus is a polygynous long-distance migratory passerine bird that breeds in marshy habitats in most of the Palaearctic between latitudes 35° and 60°N. Within Europe, available data do not reveal any morphological differentiation (Cramp, 1992). The species has shown range expansion in northern Europe over the past 100 years (Hagemeijer & Blair, 1997). At present, numbers are stable or still increasing in the Baltic countries (Sweden, Estonia, Latvia, Lithuania) whereas populations in western Europe (Denmark, The Netherlands, Belgium and France) have declined by more than 50% over the past thirty years. The recent and large changes of the breeding range in NW Europe and the high dispersal capability of this long-distance migratory species may have led to limited phylogeographic structuring among great reed warbler populations in Europe. On the other hand, we have observed two behaviours in great reed warblers that may act to maintain or restore population-genetic structuring: (1) both males and females show high fidelity to breeding sites (Bensch & Hasselquist, 1991), and (2) philopatric
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T 1. Sampling localities, co-ordinates and years Country
Site
Coordinates
Sampling year
Spain The Netherlands Sweden Germany Latvia Greece Kenya
Hondo Natural Park, Alicante Zwarte Meer/Weerribben Kvismaren, Na¨rke Mu¨ggelsee, Berlin Engure/Kanieris Limni Mikri Prespa, Florina Ngulia, Tsavo National Park
38°12′N, 0°42′W 52°37′N, 5°55′E 59°10′N, 15°25′E 52°26′N, 13°39′E 57°07′N, 23°20′E 40°50′N, 21°05′E 3°00′S, 38°08′E
1996 1995 1987–1990 1992,1993 1992 1990 1990,1991
individuals have higher fitness than immigrants (Bensch et al. 1998). Hence, at present, we cannot make unequivocal predictions about phylogeographic structuring of great reed warblers. However, because range expansions most likely involve repeated bottlenecks with associated loss of genetic material as new areas are colonized (Merila¨ et al., 1997), we expect to find less genetic variability within the newly colonized northern great reed warbler populations compared to southern populations. Moreover, as winter populations likely consist of a mix of birds from different breeding populations, a sample from African wintering areas should contain more genetic diversity than samples from any breeding population. In the present study we analyse mtDNA sequence variation of great reed warblers from six populations of breeding birds in Europe and one population of wintering birds in East Africa to address the following questions: (1) whether there is any evidence of population-genetic structuring among European breeding populations; (2) whether the degree of genetic variation declines towards north; (3) whether the population of wintering birds shows more genetic variation than any of the breeding populations, and (4) what the relationships and frequencies of different haplotypes in Europe tell us about historical population size and distribution.
MATERIAL AND METHODS
Population samples and DNA isolation Samples of great reed warblers were obtained from six European breeding populations and from one locality in African winter quarters (Table 1). For all individuals, except the samples from The Netherlands, total DNA was extracted from whole blood (5–50 lL) stored in SET-buffer (500 lL) by a phenol-chloroform protocol (Hasselquist et al., 1995b). The samples from The Netherlands consisted of dried contour feathers plucked from nestlings (different nests) and total DNA was extracted by using 5% Chelex 100 (Biorad) following the method of Mundy et al. (1996).
The control region The mitochondrial control region II in great reed warblers was first amplified and sequenced by primers BCML1 (5′-CCCAACTTGCTCTTTTGCGC-3′) and
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Figure 1. The mitochondrial control region II in great reed warblers with flanking genes and locations of primers.
FTPH2 (5′-CCATCTTGACATCTTCAGTGCCATGC-3′) designed for loggerhead shrikes Lanius ludovicianus (Mundy et al., 1996). Because these original primers did not produce a single product, two new primers, BCML4 (5′-TTCACAGATACAAATGCTTGGG-3′) and FTPH3 (5′-AAGGCTGGGAGAGTTGTTGA-3′) were designed (Fig. 1), using the aligned great reed warbler and loggerhead shrike sequences. These two primers, which give a product of 577 bp, were then used to amplify the control region II in all individuals included in this study. In order to confirm that this fragment was from the control region, the primer BCML4 was used together with 12SH1 (5′-AATGTTTACTACTGCTGAGAACCCG-3′) (Mundy et al., 1996) (Fig. 1) and their product sequenced. The 81 bases flanking the control region (3′) of great reed warblers were aligned with sequences of the tRNAPhe gene of chicken and logger-head shrikes (Fig. 2). As expected, the sequences diverge quickly as one goes into the control region (downstream of the tRNAPhe gene). From this observation alone we cannot exclude the possibility that the obtained sequences origin from a numt, i.e. a mtDNA copy incorporated into the nuclear genome (Quinn, 1997). However, from the samples at our Swedish study site we have extensive family data of which parentage has been confirmed by DNA fingerprinting (Hasselquist et al., 1995). This enabled us to confirm that the haplotypes are maternally inherited (n=5 families of which parents carried different haplotypes). Hence, because the 3′ flanking sequence aligns with the tRNAPhe gene in other species, and that haplotypes are maternally inherited, it is safe to conclude that our results are based on the control region of the mitochondria. Amplification and sequencing Polymerase chain reaction (PCR) was performed in volumes of 25 lL and included 10–50 ng of total genomic DNA, 0.125 mM of each nucleotide, 1.5 mM MgCl2, 0.6 lM of each primer and 0.5 units of Taq DNA polymerase. The PCR’s were run using the following conditions: 30s at 94°C, 30s at 50°C, 30s at 72°C (35 cycles). Before the cyclic reactions the samples were incubated at 94°C for 3 min, and after completion at 72°C for 5 min. We used 2.5 lL of the final reaction to run on a 2% agarose gel in 0.5X TBE buffer to check the success of the reaction. The remainder of the PCR product was precipitated by adding 10 lL of 8M NH4Ac and 32 lL of ethanol. Following centrifugation the air-dried DNA pellet was dissolved in 20 lL of water; 2–4 lL was then used for sequencing. The double stranded PCR products were either sequenced using the AmpliCycle Sequencing Kit (Perkin Elmer) with c-32P-ATP end-labelled primers according to the manufacturer’s recommendation and separation of fragments in 8% denaturing polyacrylamide gel (60% of the samples), or with dye terminator cyclic sequencing
Figure 2. mtDNA sequence alignments (light strand shown) of the 3′ end of control region and flanking genes. arund: Acrocephalus arundinaceus (bases 476–596, Genbank AF111791), orient: A. orientalis, stent: A. stentoreus, Lanius: Lanius ludovicianus (bases 324–447 Genbank S82910) and Chick: Chicken (bases 1186–1308 from Desjardins & Morais, 1990).
MITOCHONDRIAL POPULATION STRUCTURE OF GREAT REED WARBLERS 175
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on an ABI PRISM 310 (Perkin Elmer). Ten samples previously analysed by the first method were rerun on the new system and confirmed to produce consistent results. Descriptive statistics and analyses Nucleotide diversity (p) (Nei, 1987) and D statistics (Tajima, 1989) were calculated using the program DnaSP 2.52 (Rozas & Rozas, 1997). Estimates of haplotype diversity (h), was calculated as h=(n/(n−1))(1−Rfi2), where fi is the frequency of the ith haplotype in a sample of n individuals (Nei, 1987). We estimated long term effective female population size using the equation (Wilson et al., 1985): Nf=106∗(p/s)/g where p is the mean pair-wise sequence divergence, s the rate of sequence divergence (proportion substitutions/genome/Myr) and g is mean generation time (years). We assumed s=0.148 (Wenink et al., 1996) and g=2.0. Because the result is sensitive to errors in assumptions of s and g, estimates of Nf will provide information of order of magnitude only. The degree of geographical structuring of populations was tested using the program AMOVA (Excoffier et al., 1992) that calculates φST statistics (a measure analogous to FST) using Jukes and Cantor’s distance (Kumar et al., 1993). Significance of variance components between populations was tested with a randomization procedure provided in AMOVA (1000 permutations). Analyses of isolation by distance of φST statistics were tested by a Mantel’s permutation test provided in the program GENEPOP 3.1 (Raymond & Rousset, 1995). Evolutionary relationships between haplotypes were assessed by the neighbour-joining method with MEGA (Kumar et al., 1993). We used Tamura and Nei’s (1993) distance measure and a gamma correction parameter alpha of 0.04 to account for among-site variation in evolutionary rate (Yang, 1996), as estimated from the present data set with PUZZLE (Strimmer & von Haeseler, 1997). Sequences obtained from the oriental reed warbler Acrocephalus orientalis and clamorous reed warbler A. stentoreus (primers BCML4 and 12SH2 (5′-AGCAACAACCAACGGTAAG-3′) (Fig. 1) were used as outgroups, as these are the two most closely related taxa to the great reed warbler (Leisler et al., 1997). RESULTS
Sequence variation We sequenced 494 bp of the control region II in 106 samples from seven populations. This region contained 29 variable sites which defined 33 different haplotypes between 0.2 and 2.2% (Table 2). None of the individuals appeared to be heteroplasmic, i.e. carrying two different haplotypes (cf. Mundy et al., 1996), and the variable sites never showed shadow bands of the alternative nucleotide. All substitutions except two were transitions (A↔G=11, C↔T=17). The two transversions were noted at positions 386 and 431 (Table 2). The most common haplotype (#20) was detected in 37 (35%) of the samples and was also the most widespread and recorded at all sites except in Kenya.
MITOCHONDRIAL POPULATION STRUCTURE OF GREAT REED WARBLERS
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T 2. Variable sites in the control region II segment of great reed warblers. Origin of specimens indicated by GE (Germany), GR (Greece), KE (Kenya), LA (Latvia), NL (The Netherlands), SP (Spain) and SW (Sweden) Haplotype (base number) SP 1112222 2333333444 444444444 1152395569 9114688122 233333489 6960152410 2250267905 801678932 TCAGCAGTGT CCTTCATATT ACGATAGTC
Code
.......... .......... ......... C......... .......... ......... C......... .......... G........ C......... .......G.. ......... .......... .........C ......... C......... .......... .....G... C......... .......... G....G... C......... .......... ...G..... C.....AC.. .......... .....G... C......... .......... .......C. .T......A. .......... ......ACT .T......A. .T........ ......ACT .T........ .T........ ......ACT C.......A. .T........ ......ACT C...T...A. .T........ ......ACT C....G..A. .T........ ......ACT C.......A. TT........ ......ACT C.......A. .T........ G.....ACT C.......A. .T........ ....C.ACT CT......A. .T........ ......ACT CTG.....A. .T........ ......ACT CT.A....A. .T........ ......ACT CT......A. .T......C. G.....ACT CT......A. .T........ .T....ACT CT......A. .T....C... ......ACT CT......AC .T........ ......ACT CT......A. .T........ ..A...ACT CT......A. .T...T.... ......ACT CT........ .T........ G.....ACT CT........ .T..T..... ......ACT CT........ .T........ ......ACT CT........ .TCC...... ..T...ACT CT...G.... .T........ ......ACT
#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 #14 #15 #16 #17 #18 #19 #20 #21 #22 #23 #24 #25 #26 #27 #28 #29 #30 #31 #32 #33 Total
NL
SW
Population GE LA
1
1
2
1 1
2
GR
1 1 1 1 2
3 1
2 2 1 1
6
7
1 1 1 3
1 13
5 1
4 1 1 4
2
1 1 1 1 1 1 1 1
1 2 1
2 1 1 11
10
22
20
20
3 1 2 1 1 5
1 1
KE Total
10
1 6 1 3 1 4 2 1 1 10 1 1 2 8 1 1 1 4 1 37 1 1 1 1 1 1 1 1 1 2 3 1 1
13 106
Nucleotide diversity and heterozygosity was lowest in the western European samples and highest in Greece (Table 3). It is notable that the sample from the wintering grounds in Kenya, which supposedly consists of birds from different breeding localities, showed slightly lower levels of heterozygosity and nucleotide diversity. The estimates of Tajima’s (1989) D statistic were negative in six out of the seven sampled great reed warbler populations (Table 3). Under the neutral mutation hypothesis, the probability that D is negative is approximately 0.52 for samples sizes between 10 and 20 (Table 1 in Tajima, 1989). Under this assumption, the observed bias of negative D tends to be more extreme than expected by chance (binomial test, P=0.077). The overall φST for the European breeding populations was 0.115 suggesting that 11.5% of total variation is due to between population differences (P<0.001). However,
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T 3. Sample size (n), number of haplotypes (nH), haplotype diversity (H), nucleotide diversity (p), standard deviation of p (sd), and Tajimas (1989) D statistics for seven populations of great reed warblers nH
H
p
SD
D1
11 10 22 20 20 10 13
5 4 7 13 11 8 6
0.709 0.533 0.649 0.926 0.916 0.955 0.821
0.00201 0.00204 0.00478 0.00646 0.00653 0.00932 0.00342
0.00055 0.00085 0.00131 0.00112 0.00092 0.00122 0.00077
−1.03 −1.74∗ −0.50 −1.27 −0.21 1.28 −0.99
106
33
0.860
0.00706
0.00048
−1.21
n
Population Spain The Netherlands Sweden Germany Latvia Greece Kenya Pooled
Significance levels obtained from Table 2 in Tajima (1989), ∗ P<0.05
1
T 4. Comparison of nucleotide differences between populations expressed as φST (below diagonal). Values above the diagonals are estimates of the probability that the observed differences between samples would occur by chance (1000 permutations by the AMOVA program) Spain
The Netherlands
Spain The Netherlands −0.038 Sweden 0.010 Germany 0.042 Latvia 0.096 Greece 0.426 Kenya 0.776
0.911
Sweden 0.407 0.258
0.024 0.031 0.108 0.428 0.783
−0.006 0.005 0.293 0.625
Germany
Latvia
Greece
Kenya
0.171 0.259 0.380
0.033 <0.001 0.435 0.398
<0.001 <0.001 <0.001 <0.001 <0.001
<0.001 <0.001 <0.001 <0.001 <0.001 0.038
−0.014 0.223 0.563
0.163 0.492
0.158
0.45
PhiST
0.35 0.25 0.15 0.05 –0.05
0
500
1000
2000 1500 Distance (km)
2500
3000
Figure. 3. Plot between φST and geographical distance between European breeding populations of great reed warblers. rs=0.45 (P=0.15, Mantel’s test).
only the samples from Greece differed significantly and consistently from the other European breeding populations (Table 4). The Kenyan sample was significantly different from all the European samples, with the largest difference to the Dutch/ Spanish and least to the Greek sample (Table 4). To examine whether differences in mtDNA variation increased with distance between the sampled populations, we plotted the between population φST values against great circle distances (Fig. 3).
MITOCHONDRIAL POPULATION STRUCTURE OF GREAT REED WARBLERS 22 25 21 26 28 24 27
20
12 88 31
11 13
A
30 32
33 18
10
2
3
8 4
23 29
17 15 16 19
14
179
B
1 6
5 7
9
Figure 4. Phylogenetic relationships among great reed warbler mtDNA haplotypes based on 494 bases of the control region II. Values at the nodes represent bootstrap replication scores (% of 500 resamplings). Haplotype codes as in Table 2.
There was no significant association between genetic differentiation and distance (rs= 0.45, P=0.15, Mantel’s test); however, because of low sample size (six populations) the power of this test is low. The mean pair-wise sequence divergence (p) between all the individuals at the European breeding grounds was 0.60% which translates into a long-term female effective population size of 20 000 individuals.
Relationships between haplotypes A neighbour-joining analysis of the different haplotypes in Table 2 inferred a gene-tree with one major branching event which was supported by a 88% bootstrap value (Fig. 4). Trees in which either or both the oriental and clamorous reed warblers were used as outgroups were similar and supported the placement of haplotypes in two well defined clades A (#11–33) and B (#1–10). When the frequencies of the two clades are plotted on a map, it appears that A haplotypes represent a western clade and B haplotypes an eastern clade (Fig. 5). All birds sampled in Kenya carried mtDNA belonging to the B clade. The mean sequence divergence between the two clades A and B (counting each haplotype once) was 1.29% (range 0.82–2.23%). The mean sequence divergences between the oriental reed warbler and the two clades A and B were 8.52% and 8.88%, respectively. The corresponding values between the two clades and the clamorous reed warbler were 8.34% and 9.09%. The sequence divergence between the oriental and the clamorous reed warbler was 5.71%. Hence, both of the two outgroups were slightly more similar to clade A than to clade B. The analysis of among-site variation in evolutionary rate revealed large heterogeneity as the parameter alpha was small (0.04, SE=0.05). Accounting for this gamma distributed rate heterogeneity in the distance model (Tamura & Nei, 1993),
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Figure 5. Map and frequency of the A (grey) and B (black) mtDNA haplotypes at the six sampled great reed warbler breeding sites in Europe. All individuals sampled from winter ground in Kenya carried B haplotypes.
the mean sequence divergence between the two clades A and B was 2.51% (range 1.05–4.54%), i.e. larger than when comparing the percentage of sequence divergence.
DISCUSSION
Variation between populations Haplotype diversity in the breeding populations of great reed warblers was intermediate to high compared to other species (Seutin et al., 1995; Baker & Marshall, 1997; Mundy et al., 1997). As predicted, most of the variation in control region sequences was found between individuals within populations. This was particularly so when the Greek samples were excluded from the analysis. Hence, there is little evidence of mitochondrial DNA differentiation between North European great reed
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warbler populations. Consistent with this genetic pattern, the species shows no strong morphological variation across Europe (Cramp, 1992), although slight differences might exist if one analyses sex and age groups separately. We found the lowest values of heterozygozity in The Netherlands and Sweden. Both these populations are situated close the species range limit; the Dutch population has decreased drastically over the last decades whereas the Swedish population was founded recently and has increased from approximately 50 to 500 pairs during the last 30 years (Holmbring, 1979; Tyrberg, 1996). Loss of genetic variability at nuclear loci has been suggested by DNA fingerprinting studies of Swedish great reed warblers which showed relatively high similarity between unrelated individuals compared to other species (Bensch et al., 1994). The results suggest that edge populations exhibit lower genetic variation than populations from the interior of the geographical range, which presumably have existed for a longer time. Similarly, declining levels of genetic diversity towards north have been observed in a number of species which expanded their ranges during Pleistocene (e.g. Merila¨ et al., 1997; Takahata, 1995).
Comparing samples from breeding and winter quarters An important finding of our study is that the level of haplotype diversity was not higher in Kenya than in the samples from the breeding populations in Europe. This is surprising since samples collected from one wintering area are likely to contain individuals from several breeding localities (e.g. Wenink et al., 1996); in the present case, this is even more likely because the samples from Kenya are from a stopover site where birds are in mid-winter migration across East Africa (Backhurst & Pearson, 1984). The low variation observed in the Kenyan sample may be due to breeding populations showing more specific wintering areas than previously assumed, though more data from wintering populations are needed before this hypothesis can be evaluated. However, it should be noted that great reed warblers seem to be faithful to their winter grounds in successive years (De Roo & Deheegher, 1969; Bensch et al., 1991). The great reed warbler is a commonly found winter visitor to most of tropical Africa as far as 25°S (Cramp, 1992). Birds ringed on western and Central European breeding grounds have been recovered in tropical West Africa (Schlenker, 1986; Dowsett et al., 1988). The samples from Kenya were significantly different from all the six sampled European breeding populations. In Europe, the frequency of the B haplotypes increased eastwards to reach a maximum of 60% in Greece. That all 13 birds from Kenya carried B-haplotypes suggests that these originated from populations east of Latvia/Greece. At present, there are not enough ringing recoveries to support this conclusion.
Relationships between haplotypes and population history The total European population has recently been estimated at one million pairs (Hagemeijer & Blair, 1997) which is approximately 50-fold larger than our estimate of long term effective population size (Nf ). Long-term effective population sizes estimated from mtDNA genetic distances are commonly smaller by 1–3 orders of magnitude than present-day census sizes (Avise, 1992). The discrepancy between
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population census estimates of Nf and long term estimates of Nf might be caused by population bottlenecks or selective sweeps (Avise, 1992). However, a large variance in reproductive success between females can also cause the long-term female reproductive success to be lower than census estimates. In female great reed warblers, the variance of lifetime reproductive success is as large as in males, despite high levels of polygyny in this species (Hasselquist, 1995). Hence, the observed discrepancy between present day and long-term population size might partly be explained by a large variance in female reproductive success. The relatively few sampling localities may also contribute to the discrepancy. The coexistence of two divergent mtDNA clades within the same population indicates recent secondary contact between allopatrically evolved populations (Avise et al., 1987). Assuming an evolutionary rate of 14.8% per Myr for the control region (Wenink et al., 1996), the average sequence divergence of the two haplotype clades A and B at 1.29% would correspond to a separation of the haplotype clades for 90 000 years. The frequency of the two clades changes with longitude in Europe, suggesting that the coexistence of distinct haplotypes is due to recent admixture of previously isolated populations. Accounting for the within clade diversity (Edwards, 1997) we can calculate the interpopulation sequence divergence (d) to be 0.98%. This suggests that the two populations housing the two clades separated approximately 70 000 years before present. The inferred time since divergence (70 000 years) and the geographical distribution of the two haplotypes suggest that the mtDNA clades evolved in two refugia during the last glaciation, e.g. one in western Europe and one in eastern Europe/Asia. It should be noted that the above calculations of dates of divergence is, among other things, sensitive to rate variation among sites which, in the present data set, was shown to be large. At temperate and higher latitudes, many species which at present have continuous ranges may during the Pleistocene Ice Age have been confined to two or more allopatric populations. Remaining evidence of latitudinal range expansions in temperate regions from refugia further south are the many existing contact zones, as characterized by e.g. sharp morphological clines (Hewitt, 1993) or migratory divides (e.g. Helbig, 1992). When the first examples of large within species divergence of mtDNA sequences were detected, this was considered to be exceptional, although examples of within species divergence at levels above 1% are accumulating (Avise et al., 1990; Quinn, 1992; Taberlet et al., 1992; Wenink et al., 1993; Friesen et al., 1996). In all these cases the most likely explanation seems to be the intermingling of populations which previously have diverged allopatrically. In the case of the great reed warbler, the cline of mtDNA does not coincide with a similar cline in morphology or migratory direction (Cramp, 1992). Similarly, in the northern flicker Colaptes auratus, a pronounced geographical cline of mtDNA haplotype groups does not coincide with the cline in morphology (Moore et al., 1991). Moreover, different populations of the knot Calidris canutus have evolved highly specialized migratory patterns but are still almost identical at their mitochondrial DNA (Baker et al., 1994). A challenge for coming studies will henceforth be to understand the seemingly complex and not always congruent pattern of genetic and morphological divergence. As selective sweeps and population bottlenecks may have considerably hampered information available on the mitochondria, successful studies are likely to include analyses of nuclear markers (Avise, 1994). ACKNOWLEDGEMENTS
We thank N.I. Mundy for advice and primers used to isolate the control region in great reed warblers. Samples were kindly provided by R.T. Pinheiro (Spain), S.
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Fischer (Germany), I. Nishiumi (A. orientalis), A. Helbig (A. stentoreus) and J. Graveland ˚ . Bensch assisted with data collection in (The Netherlands). M. Svensson and A Greece and Latvia. We are indebted to M. Nordborg for discussions about methods to analyse sequence variation. The manuscript benefited from comments by A.J. Baker, S.V. Edwards, B. Hansson and N.I. Mundy. The study was financially supported by the Royal Swedish Academy of Science (Ahlo¨fska), O. Engkvist foundation, Magn. Bergvalls foundation, Swedish Natural Science Research Council (NFR) and Swedish Forestry and Agricultural Research Council (SJFR).
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