Following Pleistocene glacial maxima, species that adapted to temperate climates in low-latitude refugia had to modify their ranges as climate changed, expanding either latitudinally towards the poles, or altitudinally to higher elevations in mountainous regions. Within just a few thousand years, populations taking alternative routes during interglacials became isolated from each other and subjected to different selection pressures, often leading to lineage divergence and speciation. The pine siskin Spinus pinus is a common and widespread songbird showing relative phenotypic uniformity across the North American continent. One exception is the subspecies found in the highlands of northern Central America (S. p. perplexus), which shows marked differentiation in plumage color and shares some traits with the endemic and partly sympatric black-capped siskin S. atriceps, suggesting potential introgression or even a hybrid origin of perplexus. Relationships and species limits among pinus, perplexus and atriceps have been controversial for decades. We provide new molecular evidence to help resolve the evolutionary history of the group. Phylogenetic analysis of mitochondrial DNA and nuclear intron sequences revealed three distinct lineages within the complex, corresponding to: 1) S. pinus individuals from Canada through central Mexico (S. p. pinus and S. p. macropterus), 2) individuals from the highlands of Guatemala and Chiapas (S. p. perplexus), and 3) S. atriceps. Pine siskins across North America show evidence of a recent postglacial population expansion and extremely low levels of diversity and structure. In contrast, S. p. perplexus shows evidence of demographic stasis, reflecting long-term isolation and restricted dispersal. Marked and diagnostic genetic differences among the three lineages in mtDNA and at least one intron, suggest that a hybrid origin of S. p. perplexus is unlikely, yet some degree of introgression between S. p. perplexus and S. atriceps cannot be ruled out in localities where they occur in sympatry.

ND2 haplotype alignemt of the pine siskin and black-capped siskin complexSpinus pinus, Spinus atriceps and Spinus notatus haplotype alignment; NADH dehydrogenase subunit 2 (ND2) gene, partial cds; mitochondrialpisi nd2 haps.fasATP haplotype alignemt of the pine siskin and black-capped siskin complexSpinus pinus, Spinus atriceps and Spinus notatus haplotype alignment; cytochrome c oxidase subunit II gene, partial cds; tRNA-Lys gene, complete sequence; ATP synthase 8 gene, complete cds; and ATP synthase 6 gene, partial cds; mitochondrialpisi atp haps.fasGAPDH haplotype alignemt of the pine siskin and black-capped siskin complexSpinus pinus, Spinus atriceps and Spinus notatus haplotype alignment; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, partial intron 11pisi gapdh ph haps.fasBRM15 haplotype alignemt of the pine siskin and black-capped siskin complexSpinus pinus, Spinus atriceps and Spinus notatus haplotype alignment; brahma protein (BRM) gene, intron 15pisi brm ph haps.fasALDOB haplotype alignemt of the pine siskin and black-capped siskin complexSpinus pinus, Spinus atriceps and Spinus notatus haplotype alignment;aldolase B fructose-bisphosphate (ALDOB) gene, exons 5 and 6, partial cds.pisi aldo ph haps.fas