Molecular evidence for long-distance dispersal in the New Zealand pteridophyte flora

Aim To examine the relative importance of long‐distance dispersal in shaping the New Zealand pteridophyte (ferns and lycophytes) flora and its relationships with other floras, with the null hypothesis that the extant New Zealand pteridophyte flora has been isolated since New Zealand’s separation from Gondwana. Location New Zealand. Methods rbcL DNA sequences were assembled for 31 New Zealand pteridophyte genera, with each genus represented by one New Zealand species and the most closely related non‐New Zealand species for which data were available. Maximum‐likelihood, maximum‐parsimony, and Bayesian analysis phylograms were constructed and used as input for r 8s molecular dating, along with 23 fossil calibrations. Divergence estimates less than conservatively recent ages for New Zealand’s geological isolation, namely H_o > 30 Ma for pairs involving New Caledonian and Norfolk Island species and H_o > 55 Ma for all others, were taken as rejection of the null hypothesis. Results The null hypothesis was rejected for all pairs except, under some parameter conditions, for those involving the New Zealand species Cardiomanes reniforme, Lindsaea trichomanoides, Loxsoma cunninghamii, Lygodium articulatum, Marattia salicina, and Pteris comans. However, the Lindsaea and Pteris results probably reflect the absence in the analyses of closely related non‐New Zealand samples, while the Marattia divergence was highly contingent on which fossil calibrations were used. Main conclusions Rejection of the null hypothesis for the majority of pairs implies that the extant New Zealand lineage has undergone long‐distance dispersal either into or out of New Zealand. The notion of a long isolation since geological separation can, therefore, be dismissed for much of New Zealand’s pteridophyte flora. The analyses do not identify the direction of the long‐distance dispersal, and these New Zealand lineages could have had vicariant origins with subsequent long‐distance emigration. However, the alternative that many extant New Zealand pteridophyte lineages only arrived by long‐distance immigration after geological isolation seems likely.     

Vaccine entrapment in liposomes.

The use of liposomes as carriers of peptide, protein, and DNA vaccines requires simple, easy-to-scale-up technology capable of high-yield vaccine entrapment. Work from this laboratory has led to the development of techniques that can generate liposomes of various sizes, containing soluble antigens such as proteins and particulate antigens (e.g., killed or attenuated bacteria or viruses), as well as antigen-encoding DNA vaccines. Entrapment of vaccines is carried out by the dehydration-rehydration procedure which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free vaccines. On rehydration, the large multilamellar vesicles formed incorporate up to 90% or more of the vaccine used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped vaccine still associated with the vesicles. A similar technique applied for the entrapment of particulate antigens (e.g., Bacillus subtilis spores) consists of freeze-drying giant vesicles (4-5 microm in diameter) in the presence of spores. On rehydration and sucrose gradient fractionation of the suspension, up to 30% or more of the spores used are associated with generated giant liposomes of similar mean size.     

Morphological and molecular concordance for the recognition of two species in the New ZealandPolystichum richardii (Hook.) J. Smith complex

The New Zealand fernPolystichum richardii is shown to comprise at least two evolutionary lineages. A narrow-scaled morphological form and a wide-scaled morphological form are recognised. Principal Component Analysis of a set of frond, soral and spore characters supports this morphological separation. Amplified Fragment Length Polymorphism (AFLP) DNA fingerprinting shows that the two morphological forms are also genetically distinct. They are sympatric across a broad area of central New Zealand, and often grow together without apparent ecological separation at the local level. Both are known to form sterile hybrids withP. vestitum, suggesting that both morphological forms ofP. richardii are at least partially outcrossing. The morphological and genetic distinctiveness of these two morphological forms, combined with their sympatric distributions and their (at least partially) outcrossing mating systems, merits their recognition as separate species.     

Molecular phylogenetic and molecular dating of the New Zealand Gleicheniaceae

The Gleicheniaceae are an ancient family of ferns, with three of the six extant genera occurring in New Zealand:Dicranopteris, Gleichenia, andSticherus. The biogeographic origins of this family in New Zeland are unknown, and the taxonomy ofGleichenia in particular is still unclear. To address aspects of these two issues, DNA sequences from thetrnL-trnF locus and therbcL gene were produced for all of the common Gleicheniaceae species in New Zealand, as well as forGleichenia alpina from Tasmania andSticherus cryptocarpus from Chile. SeveraltrnL-trnF haplotypes were found amongst New ZealandG. dicarpa. One of these haplotypes was also observed in TasmanianG. alpina, while the other New ZealandG. dicarpa trnL-trnF haplotypes were more similar to those ofG. microphylla. These results suggest the taxonomy of New ZealandGleichenia may be more complex than presently recognized. Molecular dating of therbcL sequences with the program r8s rejected vicariant explanations for the disjunct distributions between New Zealand and elsewhere for each ofDicranopteris, Gleichenia, andSticherus. However, the direction of the inferred long-distance dispersal was not resolved.     

α,α′-trehalose 6,6′-dibehenate in non-phospholipid-based liposomes enables direct interaction with trehalose, offering stability during freeze-drying

Trehalose is a well known protector of biostructures like liposomes and proteins during freeze-drying, but still today there is a big debate regarding its mechanism of action. In previous experiments we have shown that trehalose is able to protect a non-phospholipid-based liposomal adjuvant (designated CAF01) composed of the cationic dimethyldioctadecylammonium (DDA) and trehalose 6,6′-dibehenate (TDB) during freeze-drying [D. Christensen, C. Foged, I. Rosenkrands, H.M. Nielsen, P. Andersen, E.M. Agger, Trehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying, Biochim. Biophys. Acta, Biomembr. 1768 (2007) 2120-2129]. Furthermore it was seen that TDB is required for the stabilizing effect of trehalose. Herein, we show using the Langmuir-Blodgett technique that a high concentration of TDB present at the water-lipid interface results in a surface pressure around 67 mN/m as compared to that of pure DDA which is approximately 47 mN/m in the compressed state. This indicates that the attractive forces between the trehalose head group of TDB and water are greater than those between the quaternary ammonium head group of DDA and water. Furthermore, addition of trehalose to a DDA monolayer containing small amounts of TDB also increases the surface pressure, which is not observed in the absence of TDB. This suggests that even small amounts of trehalose groups on TDB present at the water-lipid interface associate free trehalose to the liposome surface, presumably by hydrogen bonding between the trehalose head groups of TDB and the free trehalose molecules. Hence, for CAF01 the TDB component not only stabilizes the cationic liposomes and enhances the immune response but also facilitates the cryo-/lyoprotection by trehalose through direct interaction with the head group of TDB. Furthermore the results indicate that direct interaction with liposome surfaces is necessary for trehalose to enable protection during freeze-drying.     

Conflict amongst chloroplast DNA sequences obscures the phylogeny of a group of Asplenium ferns

A previous study of the relationships amongst three subgroups of the Austral Asplenium ferns found conflicting signal between the two chloroplast loci investigated. Because organelle genomes like those of chloroplasts and mitochondria are thought to be non-recombining, with a single evolutionary history, we sequenced four additional chloroplast loci with the expectation that this would resolve these relationships. Instead, the conflict was only magnified. Although tree-building analyses favoured one of the three possible trees, one of the alternative trees actually had one more supporting site (six versus five) and received greater support in spectral and neighbor-net analyses. Simulations suggested that chance alone was unlikely to produce strong support for two of the possible trees and none for the third. Likelihood permutation tests indicated that the concatenated chloroplast sequence data appeared to have experienced recombination. However, recombination between the chloroplast genomes of different species would be highly atypical, and corollary supporting observations, like chloroplast heteroplasmy, are lacking. Wider taxon sampling clarified the composition of the Austral group, but the conflicting signal meant analyses (e.g., morphological evolution, biogeographic) conditional on a well-supported phylogeny could not be performed.     

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.