Molecular characterization of the freshwater snail Lymnaea natalensis (Gastropoda : Lymnaeidae) on Madagascar with an observation of an unusual polymorphism in ribosomal small subunit genes

Recent characterization of nuclear ribosomal small subunit (SSU) genes has shown that variant nucleotides within this region could be useful for speci es and species group identification within the genus Lymnaea (Gastropoda: L ymnaeidae). This study aimed to characterize a range of populations of Lymn aea natalensis Krauss, 1848 on Madagascar, and addressed two related questi ons. First, is there any evidence of intraspecific variation of the SSU and , if so, what might be its significance? Secondly, might this variation jeo pardize the use of SSU for lymnaeid taxonomy and phylogeny? Lymnaea natalen sis (n = 212) was collected from 17 sampling localities, spanning the north ern and southern ends of the island. Variation within a selected region of the SSU known to vary between species, the V1 and V2, was assayed by polyme rase chain reaction (PCR) linked restriction fragment length polymorphism ( RFLP) and denaturing gradient gel electrophoresis (DGGE) analysis. The PCR- RFLP profiles indicated a striking dimorphism across populations at two res triction site loci (CfoI & MspI) within the E10-1 helix of the V2 region. T he observed RFLP variation was confirmed by direct sequencing and by genomi c digestion with subsequent hybridization. Putative heterozygotes were also encountered and in these individuals the SSU arrays composed of two distin ct types approximately 1% divergent. A severe departure from Hardy-Weinberg equilibrium with a highly statistically significant (P < 10(-5)) heterozyg ote deficiency was found and genetic variation among populations was highly structured (F-st = 0.53). The geographic distribution of the variants was mapped, revealing that one variant was restricted to higher, predominately colder environments and was thought to be an adaptation. The molecular basi s of the SSU variation was caused by single nucleotide polymorphisms (SNPs) . To test for the possibility of cryptic taxa, an analysis of individuals r epresentative of the SSU variant types with isoenzyme analysis (ISA), rando mly amplified polymorphic DNA (RAPDs) and PCR-RFLP analysis of the ribosoma l Internal Transcribed Spacer (TTS) was performed. Little variation was rev ealed and none that correlated to the groups suggested by SSU, confirming t hat the SSU variation was intraspecific. The levels of intraspecific diverg ence of the V1 and V2 within Lymnaea were not appreciably different (1%) fr om interspecific and would therefore question the validity of these data fo r lymnaeid taxonomy and phylogeny.