New insights into the evolution of the fern family Dennstaedtiaceae from an expanded molecular phylogeny and morphological analysis.

Dennstaedtiaceae has 270 species, a worldwide distribution, and an edge-colonizing habit that is unusual among ferns. Aneuploidy, polyploidy, and hybrids are common in the family. Combining morphology, anatomy, chromosome number, and geographical distributions with our newly generated molecular phylogeny, we provide new insights into the evolution of the family. We paid special attention to Hypolepis. Our molecular dataset of five cpDNA markers is the most comprehensive to date, comprising 72 species (and a total of 98 taxa), of which 33 are Hypolepis (45 taxa). We also generated divergence-time estimates through BEAST with four fossil calibrations. We recovered three sub-families in Dennstaedtiaceae: Monachosoroideae (monogeneric), Dennstaedtioideae, and Hypolepidoideae. Monachosoroideae has a chromosome base number of x = 28; Hypolepidoideae of x = 26; while in Dennstaedtioideae this is still obscure, with different numbers ranging from 30 to 47. Dennstaedtioideae genera require re-circumscriptions because Dennstaedtia is polyphyletic. In Hypolepidoideae, the six genera are monophyletic. Within Hypolepis, seven geographically distinct clades were recovered; but we found no strong morphological characters to define them. Within the family, the long-creeping rhizome evolved with a change in habit: from shade-tolerant to edge-colonizers, to thicket-formers. Short or extremely large leaves are derived conditions. Sorus shape and position, glandular hairs, and prickles are homoplastic. Hybridization/allotetraploidy in Hypolepis can be suggested by the combined data. In our phylogenetic hypothesis, Dennstaedtiaceae originated around 135 Ma, with the split of Monachosoroideae around 94 Ma, and the split between Dennstaedtioideae/Hypolepidoideae around 78 Ma. All extant genera are inferred to be relatively young. Hypolepis started to diversify around 10 Ma, and it probably originated in east Asia and/or Oceania. Hypolepis reached the Neotropics twice: through elements of the Hypolepis rugosula clade (which originated at 7 Ma), and through the ancestor of the Neotropical clade, which originated at 3.1 Ma and was prickly.

Parallel polyploid speciation: distinct sympatric gene-pools of recurrently derived allo-octoploid Asplenium ferns.

Although polyploidy is widespread, its significance to the generation of biodiversity remains unclear. Many polyploids have been derived recurrently. For a particular polyploid, gene-flow between the products of independent origin is typical where they come into contact. Here, we use AFLP DNA-fingerprinting and chloroplast DNA sequences to demonstrate parallel polyploid speciation within both of the ferns Asplenium cimmeriorum and A. gracillimum. Both of these taxa comprise at least two allopolyploids, recurrently derived from the same progenitor pair. Each of these allopolyploids remain genetically distinguishable even with extensive sympatry, and could therefore be considered distinct species. To our knowledge, parallel speciation on this scale amongst recurrent polyploids has not been previously reported. With their parallel origins, these 'evolutionary replicates' provide an unrivalled opportunity to investigate how the reproductive barriers and ecological differentiation necessary for speciation arise following polyploidy.

Genetic and morphological identification of a recurrent Dicksonia tree fern hybrid in New Zealand.

Hybridization is common in many ferns and has been a significant factor in fern evolution and speciation. However, hybrids are rare between the approximately 30 species of Dicksonia tree ferns world-wide, and none are well documented. In this study we examine the relationship of a newly-discovered Dicksonia tree fern from Whirinaki, New Zealand, which does not fit the current taxonomy of the three species currently recognized in New Zealand. Our microsatellite genotyping and ddRAD-seq data indicate these plants are F1 hybrids that have formed multiple times between D. fibrosa and D. lanata subsp. lanata. The Whirinaki plants have intermediate morphology between D. fibrosa and D. lanata subsp. lanata and their malformed spores are consistent with a hybrid origin. The Whirinaki plants-Dicksonia fibrosa × D. lanata subsp. lanata-are an example of hybridization between distantly related fern lineages, with the two parent species estimated to have diverged 55-25 mya. Our chloroplast sequencing indicates asymmetric chloroplast inheritance in the Whirinaki morphology with D. lanata subsp. lanata always contributing the chloroplast genome.

Low-copy nuclear DNA sequences reveal a predominance of allopolyploids in a New Zealand Asplenium fern complex.

Recent generalisations about polyploidy in plants have been largely based on studies of angiosperms. A compelling group to compare with angiosperms is ferns, because of their high polyploidy. The bi-parental inheritance of nuclear DNA sequence markers makes them advantageous for investigating polyploid complexes, but few such markers have been available for ferns. We have used DNA sequences from the low-copy nuclear LFY locus to study an Asplenium polyploid complex. The New Zealand species of this Austral group comprise seven tetraploids and eight octoploids. LFY sequences indicate that allopolyploidy is much more predominant than previously thought, being implicated in the origins of seven of the octoploids. One of the tetraploids has had a central role, being a progenitor for five of the octoploids. All of the octoploids appear to have relatively recent origins, with the dynamic environmental conditions of the Pleistocene possibly playing a role in their formation and/or establishment.

Fire and ice: volcanic and glacial impacts on the phylogeography of the New Zealand forest fern Asplenium hookerianum.

In the Southern Hemisphere there has been little phylogeographical investigation of forest refugia sites during the last glacial. Hooker's spleenwort, Asplenium hookerianum, is a fern that is found throughout New Zealand. It is strongly associated with forest and is a proxy for the survival of woody vegetation during the last glacial maximum. DNA sequence data from the chloroplast trnL-trnF locus were obtained from 242 samples, including c. 10 individuals from each of 21 focal populations. Most populations contained multiple, and in many cases unique, haplotypes, including those neighbouring formerly glaciated areas, while the predominant inference from nested clade analysis was restricted gene flow with isolation by distance. These results suggest that A. hookerianum survived the last glacial maximum in widespread populations of sufficient size to retain the observed phylogeography, and therefore that the sheltering woody vegetation must have been similarly abundant. This is consistent with palynological interpretations for the survival in New Zealand of thermophilous forest species at considerably smaller distances from the ice sheets than recorded for the Northern Hemisphere. Eastern and central North Island populations of A. hookerianum were characterized by a different subset of haplotypes to populations from the remainder of the country. A similar east-west phylogeographical pattern has been detected in a diverse array of taxa, and has previously been attributed to recurrent vulcanism in the central North Island.

Genetic variation is not concordant with morphological variation in the fern Asplenium hookerianum sensu lato (Aspleniaceae).

Species status cannot be adequately determined when partitions are based on only a single morphological character. For instance, the sympatry of plants with broad and narrow pinnules in the fern Asplenium hookerianum sensu lato from New Zealand creates the impression that two entities are present. The narrow-pinnuled plants are sometimes segregated as a distinct species, A. colensoi. However, this variation in pinnule morphology could equally be infraspecific, and only additional data can resolve this uncertainty. Analyses using AFLP DNA-fingerprinting and DNA sequencing of the chloroplast trnL-trnF region indicate that genetic variation in A. hookerianum sensu lato is not concordant with pinnule morphology. Consequently, the recognition of A. colensoi is not supported.