Phylogeography and genetic structuring of European nine-spined sticklebacks (Pungitius pungitius)-mitochondrial DNA evidence

Abstract

As a consequence of colonisation from different glacial refugia, many northern European taxa are split into distinct western and eastern lineages. However, as for the nine-spined stickleback (Pungitius pungitius), the exact location of the contact zone between lineages often remains poorly known. We assessed the genetic differentiation and diversity in the nine-spined stickleback within Europe using 1037 base pairs of cytochrome b sequence for 320 individuals from 57 locations, including pond, lake, river, and coastal habitats. Our main aims were (i) to locate the contact zone between the previously recognized western and eastern lineages, (ii) investigate latitudinal patterns in genetic diversity, (iii) compare genetic diversity among different habitat types, and (iv) date the known split between eastern and western lineages. The data revealed the split between eastern and western to be located across the Danish Straits and roughly following the Norway/Sweden border to the North. Reference sites from Canada form their own clades, and one of the Canadian sites was found to have a haplotype common to the Eastern European lineage, possibly representing an ancestral polymorphism. The split between the two European clades was dated to approximately 1.48 million years ago (Mya), and between Canada and Europe to approximately 1.62 Mya. After controlling for habitat effects, nucleotide (but not haplotype) diversity across populations decreased with increasing latitude. Coastal populations showed significantly higher haplotype diversity (but not nucleotide diversity) than pond populations, but there were no detectable differences in haplotype diversity among different freshwater habitat types (viz. river, lake and pond populations), or between coastal and lake/river populations. Sequences were found to cluster according to their geographic proximity, rather than by habitat type, and all habitat types were found within each major clade, implying that colonisation and adaptation between the coastal and freshwater environments in different regions must have occurred in parallel.

Figure 1. Map showing the sampling locations within Europe.

Different marker shapes represent different habitat types (circle = sea, square = river, triangle = pond, inverted triangle = lake), whilst different colours represent the Eastern clade (black), and the Western clade (white). Markers with two colours show sites where haplotypes from multiple clades were found. In addition, there are two sites in Canada that are not shown – one is a pond site, and one is a lake site. Both sites form their own clade, however one sample from the pond site has haplotype E3, the most common Eastern haplotype. The distribution of the nine-spined stickleback is overlayed in grey (from Paepke [32]), and the major seas are labelled.

Figure 2. Bayesian phylogeny of 102 cytochrome b haplotypes, with main clades marked.

Haplotype numbers are given (E1–106), followed by the site types represented (P = pond, L = lake, R = river, S = sea), and the number of such sites in brackets. Posterior probabilities >0.7 are indicated at the major nodes, and major clades are labelled. The outgroup is labelled by its accession number (GU227782), and is a previously published nine-spined stickleback sequence from Japan.

Figure 3. Box plots showing the distribution of haplotype and nucleotide diversity for each habitat type.

Outliers are shown with open circles, the range (excluding outliers) is marked by the upper and lower horizontal lines, the main boxes denote the inter-quartile range, and the heavy central lines show the medians.