The Asplenium normale complex contains the widespread A. normale and several geographically restricted species: A. boreale, A. hobdyi, A. kiangsuense, A. oligophlebium and A. shimurae. The taxonomy of this group is unclear with some entities treated infraspecifically or as synonyms. Furthermore, the existence of diploids and tetraploids in this species complex is suggestive of reticulate evolution. In order to formulate a natural classification and to investigate the relationships in this complex, phylogenetic analyses of plastid and nuclear sequence data and inference of ploidy level were used to assess the distinctiveness of putative taxonomic units and their relationships. The Asplenium normale complex was recovered as a monophyletic group comprising six principal chloroplast lineages. The results support the hypothesis that A. normale s.l. contains several species. Based on our inferences, we outline an improved species classification recognizing three diploid and four tetraploid entities. Incongruence of chloroplast and nuclear phylogenies was interpreted to be a result of recurrent reticulation events in the A. normale complex.

Polyploidy is widely considered as a major process in the evolution of plants but the accumulation of polyploid species diversity is still controversial. Some recent studies proposed increased extinction risk in neopolyploids compared with their diploid ancestors. The high proportion of polyploid ferns is expected to be formed mainly by neopolyploids, whereas paleopolyploid species are predicted to be clustered in clades founded by whole genome duplications. Here, we test this prediction by exploring the evolution of polyploidy in the derived fern family Aspleniaceae. The family has a global distribution and shows the highest frequency of polyploid taxa among all ferns. To test the hypothesis, we obtained a comprehensive phylogeny using chloroplast DNA sequences of 883 specimens representing 292 species. All published chromosome counts were mapped onto this phylogenetic framework in order to explore the evolution of polyploids. We recovered evidence for several whole genome duplications in the history of Aspleniaceae. Phylogenetic relationships of polyploids exceeding the tetraploid level suggest that tetraploid Asplenium species may have replaced their diploid ancestors as the main evolutionary players in some clades of this family.

The Asplenium normale D. Don complex comprises several taxa that are either diploid or tetraploid. The tetraploids are assumed to have originated from diploid ancestors by relatively recent autopolyploidization or allopolyploidization. Some of the diploids are readily recognized morphologically but most of the taxa have until now been placed into a single species. However, phylogenetic studies have challenged this treatment and emphasized the notion that the taxonomic treatment of this complex needs to be revised. An integrative taxonomic approach was employed to delimit species in the complex using cytological, morphological, and DNA sequence data. Initially, we employed a diploid first approach to establish a robust taxonomic framework. Special efforts were made to collect and identify the diploid progenitors of each polyploid lineage identified in the plastid DNA based phylogenetic hypothesis. A total of six distinct diploid species were identified. The distinctive nature of the six diploids is strongly supported by sequence differences in plastid DNA and nuclear loci, as well as by the results of morphometric analysis. Diagnostic morphological characters were identified to distinguish the six diploid species, resulting in their revised taxonomy, which includes two novel species, namely, Asplenium normaloides and A. guangdongense. Further studies to strengthen the taxonomic classification of all of the tetraploid taxa are warranted.

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