Using stomatal length to indicate ploidy in the Dryopteris affinis (Dryopteridaceae) complex: does sampling location on the frond matter?

The scaly male ferns of the Dryopteris affinis (Lowe) Fraser-Jenk. complex pose problems for taxonomists and botanists because of their hybrid origins, polyploidy, and partly apomictic and partly sexual reproduction. Diploid, triploid, tetraploid, and pentaploid taxa are known, and knowing the ploidy level of an individual is essential for identification. Stomatal length increases with higher ploidy levels and can be used as a proxy for ploidy level. Previous studies have measured stomata from the pinnae (primary frond divisions) in the middle part of the frond. The aim of this study was to determine whether the location of sampling of stomata on the pinnules (pinna segments) and on the pinna locations on the frond is important. Thirty stomata were measured from each of three regions on acroscopic (upper) and basiscopic (lower) pinnules on three pinnae from each of 18 plants representing six taxa. Ploidy level of all plants under study was examined by flow cytometry. The results show that in most cases (89.8%) the correct ploidy level can be inferred from samples from any part of the pinnule, and from three levels in the lower half of the frond (92%). The lower pinnae had the most regions with stomatal length values outside the expected range (5.6%) for the ploidy level. This study supports previous findings that average stomatal length is a useful proxy for ploidy level determination in the Dryopteris affinis complex, but we would recommend using the proximal areas of pinnules from the inner part of pinnae from the middle of the frond.