Club-mosses (Diphasiastrum, Lycopodiaceae) from the Far East - Introgression and possible cryptic speciation.Hybridization occurs often in the genus Diphasiastrum (Lycopodiaceae), which corroborates reports for the two other recognized lycophyte families, Isoëtaceae and Selaginellaceae.  Here we investigate the case of D. alpinum and D. sitchense from the Russian Far East (Kamchatka).  Their hybrid, D. × takedae, was morphologically recognizable in 16 out of 22 accessions showing molecular signatures of hybridization; the remaining accessions displayed the morphology of either D. alpinum (3) or D. sitchense (3).  We sequenced markers for chloroplast microsatellites (cp, 175 accessions from Kamchatka) and for the two nuclear markers RPB and LFY (175 and 152 accessions).  A selection of 42 accessions, including all hybrid accessions, was analysed via genotyping by sequencing (GBS).  We found multiple, but apparently uniparental hybridization, clearly characterized by a deviating group of haplotypes for D. sitchense and all hybrids.  All accessions showing molecular signatures of hybridization in nuclear markers revealed the parental haplotype of D. sitchense, however only the LFY marker differentiated between the parent species.  GBS, including 69,819 quality-filtered single nucleotid polymorphisms, unambiguously identified the hybrids and revealed introgression to occur.  Most of the hybrids were F1, but three turned out to be backcrosses with D. alpinum (one) and with D. sitchense (two).  These observations are in contrast to prior findings on three European species and their intermediates where all three hybrids turned out to be independent F1 crosses without evidence of recent backcrossing.  In this study, backcrossing was detected, which indicates a limited fertility of the hybrid taxon D. × takedae.  A comparison of accessions of Kamchatkian D. alpinum with plants from Europe indicated possible cryptic speciation.  Accessions from the Far East had  (i) a lower DNA content (7.0 vs. 7.5 pg/2C),  (ii) different prevailing cp haplotypes, and  (iv) a clearly different SNP pattern in GBS.  Diphasiastrum sitchense and the similar D. nikoënse, for the latter additional accessions from Japan were investigated, appeared as forms of one diverse species, sharing genotypes in both nuclear markers, although chloroplast haplotypes and DNA content show slight variations.
C