title: Genetic diversity in Cypripedium calceolus (Orchidaceae) with a focus on north-western Europe, as revealed by plastid DNA length polymorphisms.
abstract: OBJECTIVE
  Cypripedium calceolus, although widespread in Eurasia, is rare in many countries in which it occurs. Population genetics studies with nuclear DNA markers on this species have been hampered by its large nuclear genome size. Plastid DNA markers are used here to gain an understanding of variation within and between populations and of biogeographical patterns.
  
  
  METHODS
  Thirteen length-variable regions (microsatellites and insertions/deletions) were identified in non-coding plastid DNA. These and a previously identified complex microsatellite in the trnL-trnF intergenic spacer were used to identify plastid DNA haplotypes for European samples, with sampling focused on England, Denmark and Sweden.
  
  
  RESULTS
  The 13 additional length-variable regions identified were two homopolymer (polyA) repeats in the rps16 intron and a homopolymer (polyA) repeat and ten indels in the accD-psa1 intergenic spacer. In accD-psa1, most of these were in an extremely AT-rich region, and it was not possible to design primers in the flanking regions; therefore, the whole intergenic spacer was sequenced. Together, these new regions and the trnL-trnF complex microsatellite allowed 23 haplotypes to be characterized. Many were found in only one or a few samples (probably due to low sampling density), but some commoner haplotypes were widespread. Most of the genetic variation was found within rather than between populations (83 vs. 18%, respectively). Two haplotypes occurred from the Spanish Pyrenees to Sweden.
  
  
  CONCLUSIONS
  Plastid DNA data can be used to gain an understanding of patterns of genetic variation and seed-mediated gene flow in orchids. Although these data are less information-rich than those for nuclear DNA, they present a useful option for studying species with large genomes. Here they support the hypothesis of long-distance seed dispersal often proposed for orchids.
authors.affiliation: Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK. m.fay@kew.org
authors.forename: Michael F
authors.initials: MF
authors.lastname: Fay
authors.forename: Ruth
authors.initials: R
authors.lastname: Bone
authors.forename: Peter
authors.initials: P
authors.lastname: Cook
authors.forename: Imalka
authors.initials: I
authors.lastname: Kahandawala
authors.forename: Jennifer
authors.initials: J
authors.lastname: Greensmith
authors.forename: Stacey
authors.initials: S
authors.lastname: Harris
authors.forename: Henrik A E
authors.initials: HA
authors.lastname: Pedersen
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Ingrouille
authors.forename: Christian
authors.initials: C
authors.lastname: Lexer
mesh_terms: D000483:Alleles; D020029:Base Pairing / Q000235:genetics*; D018742:DNA, Chloroplast / Q000235:genetics*; D005060:Europe; D005828:Genetics, Population; D006239:Haplotypes; D029595:Orchidaceae / Q000235:genetics*; D011110:Polymorphism, Genetic
chemical_list: D018742:DNA, Chloroplast