Mitochondrial DNA Data

We examined 63 pygmy shrews from continental Europe for 342 bp of the mitochondrial control region. Of the 342 nucleotides, 108 were polymorphic (including indels). Fifty one (47.2%) polymorphic sites were found in the 5' unique flanking region. Thirty (27.8%) polymorphic sites were found in the last tandem repeat which was 78-80 bp in size and 27 (25%) polymorphic sites were found in the 3' end and part of the Central Domain. Fifty three unique haplotypes were identified (see Table 1). Only one haplotype was shared between localities: Fra6 between two localities in Northern France. NJ, MP, ML, and Bayesian analysis of the 53 haplotypes revealed concordant topologies resulting in five major lineages (Fig. 1), with poor to relatively good statistical support. The Western lineage included individuals from Andorra, central France, and the French island Belle Ile. The Northern lineage included individuals from Northern France and Norway in the West to Siberia in the East. The Spanish lineage was confined to Spain. The Balkan lineage was present as far North as Austria and Slovakia, and the Italian lineage was present in Italy and the Alps in Switzerland and France. One outlier haplotype, Ita7, was identified from this region (Fig. 1). The Northern lineage is separated from both Western and Italian lineages by two indels, 3 bp and 4 bp in size. The Spanish lineage is separated from the Northern lineage by an 8 bp indel, and the Spanish and Balkan lineages are separated by a 2 bp indel. This corroborates the lineages identified by Mascheretti et al. (2003) using cytochrome b but with certain lineages more widespread because of better sampling coverage. An AMOVA showed that the majority of control region variation (62.8%) was distributed among lineages with a little variation (10.13%) found among populations within lineages.

The nucleotide diversity (p) for lineages ranged from 0.01786 to 0.0331 (Table 2). Net divergence (Da ± SE%) between lineages and estimated divergence in 1,000 s of calendar years before present (ky BP) are given in Table 3. Mismatch distributions for four out of the five lineages significantly conformed to the sudden population expansion model (Table 4). The Spanish lineage was excluded because of the small sample size (n = 3). Fu's FS values and estimates for time since expansion for the four lineages based on Rogers (1995) t are given in Table 4.

- 0.005 substitutions/site

Cze1 O

P- Swe10

1-Pol10

Sib1 Ger2Q

Fin10

- Svk10

Swz3 0 -Nor10

Nor4 O Nor2 O Nor3 O Ita2 O

Fra4 O

Svk3

BI5W,

And^

Ita1 Swz1 Swz2 Ita5 _

- S.volnuchini

76/64/65

64/66/83

Northern

Balkan

Western

Italian

Spanish

Fig. 1 Neighbour-joining tree using the HKY85 + I + G model of substitution of 53 control region haplotypes (342 bp) showing the 5 Lineages found in European S. minutus. See Table 1 for haplo-type designations. Bootstrap values obtained by NJ, MP, ML (> 50%) are shown above nodes and Bayesian probabilities (> 0.50) are shown below nodes for each lineage

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