Comparative analysis of sequence data and AFLP data resulted in generally congruent delimitation of species, and supported the inferences of distinct lineages and hybridisation made in Dyer et al. (2012). However, evidence for additional genetic diversity within the
A.monanthes lineages, supported in both the AFLP data and the sequence data, also indicated
that genetic segregation or unequal meiosis within apomictic taxa could not be ruled out. Moreover, species delimitation was not applicable for the nuclear pgiC tree, indicating that inferences of hybridisation from this data need to be tested against lineage sorting and genetic segregation.
The inference of sexual progenitors species spec.nov.1 and spec.nov.2 was not supported in the AFLP data. However, this may indicate shortcomings in the use of AFLPs for the detection of parentage in the presence of variation in ploidy level.
Inference of a species tree was not possible from phylogenetic analysis of AFLP data alone. However, the AFLP data did support the species relationships observed in the nuclear phylogeny. In order to move towards a species tree, a greater understanding of reticulate relationships is required. This could be achieved through intraspecific analysis of AFLP data and/or a greater sampling of nuclear genes, and the application of a number of recent methods that are able to detect hybridisation and even distinguish between reticulation and incomplete lineage sorting (see Blanco-Pastor et al., 2012).
CHAPTER 4
Genome size expansion and the evolutionary
relationship between nuclear DNA content and
spore size in the Asplenium monanthes fern
complex (Aspleniaceae)
Prepared for submission to New Phytologist under the co-authorship of Jaume Pellicer, Ilia J. Leitch, Vincent Savolainen, & Harald Schneider.
4.1 Summary
Genome size measurements are an under exploited tool in homosporous ferns. The application of this tool shows great potential to provide an overview of the mechanisms that define genome evolution in these ferns, and test evolutionary hypotheses based on the correlated evolution between genome size and a range of traits, such as spore size. The purpose of this study is to investigate the relationship between genome size and spore size and explore the evolution of genome size within the Asplenium monanthes fern complex and related lineages. We use flow cytometry to measure DNA amount of specimens, and comparative methods to test for correlation between genome size and spore size across the phylogeny. Our findings show that spore size and genome size are not correlated and thus they challenge the widely held assumption that spore size can be used to infer ploidy levels within apomictic fern complexes. The data also provide evidence for marked genome size variation between lineages of the A.monanthes complex and its relatives. We argue that the observed genome size variation is likely to have arisen via both polyploidy and chromosome size expansion. That latter is consistent with retrotransposon-driven chromosome/genome size expansion which to date, has not been considered to be an important process of genome evolution within homosporous ferns. We infer that genome evolution, at least in some homosporous fern lineages, is a more dynamic process than existing studies would suggest. However, it is uncertain to what degree our findings are associated with the prevalence of apomixis in this lineage.
4.2 Introduction
Homosporous ferns are renowned for their high chromosome numbers, with Ophioglossum
reticulatum having the highest chromosome number (2n=ca.1400) so far reported for any
eukaryote (Ghatak, 1977). Moreover, the mean chromosome number for homosporous ferns (n=57.05) is significantly higher than for any other plant group (including heterosporous ferns, n=13.6; and angiosperms, n=15.99) (Klekowski and Baker 1966). Nevertheless, the reasons for such disparity between these different plant groups still remain enigmatic, and are a major focus of ongoing research in this field (e.g. Nakazato et al., 2008; Barker & Wolf, 2010; Barker, 2013).
The application of novel genome-wide analytical methods, including genome size analysis, is providing significant insight into the processes that shape homosporous fern genomes (Nakazato et al., 2008; Barker & Wolf, 2010; Bainard et al., 2011a). Although our knowledge of genome sizes in ferns is limited (< 1% of species have been analysed), available data suggest that patterns of genome size evolution, which include (i) polyploidisation; (ii) changes in chromosome size; and, (iii) paleopolyploidsation, are not operating uniformly across all fern lineages (Bennett & Leitch, 2001, 2010; Obermayer et al., 2002; Bainard et al., 2011a; Leitch & Leitch, 2013). In contrast to the extreme diversity of genome sizes encountered in angiosperms, which range c. 2,400-fold (Pellicer et al., 2010), genome sizes in ferns (monilophytes) are less variable, ranging just c. 94-fold (Bennett & Leitch, 2010; Leitch & Leitch, 2013).
In fact, if we focus within homosporous ferns, this variation in nuclear DNA contents only spans c. 25-fold, from 1C=2.95pg in Athyrium filix-femina (Grime et al., 1988) to 1C=72.68 pg in Psilotum nudum var. rubra (Obermayer et al., 2002). The 1C-value is the DNA content of the un-replicated reduced chromosome compliment (see Greilhuber et al., 2005). While some of this diversity arises from polyploidy (e.g. Ophioglossum petiolatum, 2n=32x=c. 960, and 1C=65.55pg, see Obermayer et al., 2002), genome size changes can also arise within the same ploidy level in some genera. For example, the 1.5-fold range of genome sizes encountered in Davallia have taken place at the diploid level (2n = 80) with the different genome sizes between species reflected in contrasting chromosome sizes (Obermayer et al., 2002). Such differences may arise through different balances between transposable element
activity (especially retrotransposons) leading to genome and chromosome size increases, and DNA elimination, as frequently observed in angiosperms (Grover & Wendel, 2010; Leitch & Leitch, 2012). Nevertheless, available cytogenetic data indicates that despite a few examples (Britton, 1953) most homosporous ferns are characterized by possessing small and rather conserved chromosome sizes with little evidence of retrotransposon activity (Wagner & Wagner, 1980; Brandes et al., 1997; Nakazato et al., 2008; Bainard et al., 2011a). This observation is supported by the relatively small variation in the monoploid genome size reported for homosporous ferns (1Cx=2.95pg - 21.02pg; 1Cx-value is the DNA content of one unreplicated chromosome set sensu Greilhuber et al., 2005), and the absence of any apparent relationship between 1Cx-value and chromosome numbers (Bainard et al., 2011a).
In ferns, studies on closely related species in Dryopteris (Ekrt et al., 2009, 2010) and
Polypodium (Bures et al., 2003) have shown that genome size can be a powerful marker for
taxonomic delimitations. However, as far as we are aware, no study to date has investigated the evolution of genome size and whether it is correlated with breeding system or any morphological traits, in a closely related group of fern species. This is in contrast to studies in angiosperms which have demonstrated that genome size is correlated with several ecological and morphological traits, such as seed mass and stomatal density (e.g. Beaulieu et al., 2007, 2008; Zedek et al., 2010; Greilhuber & Leitch, 2013). Such studies would be highly informative in ferns, as traits such as spore size and stomatal cell size are often used to infer changes in ploidy levels among closely related species (Moran, 1982; Barrington et al., 1986; Beck et al., 2010). This is based on the implicit assumption that species with higher ploidy levels (and hence larger genomes) will have larger spores, although this has never been systematically tested for genome size within a phylogenetically well defined group.
In this study we investigate the evolution of genome size and spore size within the Asplenium
monanthes complex and related lineages (Fig. 4.1), a group of closely related species whose
phylogenetic relationships have been recovered (Dyer et al., 2012). These studies have uncovered evidence of reticulate evolution, and multiple apomictic lineages in this complex of black-stemmed rock spleenworts. In addition, polyploidy is known to occur, based on previously reported chromosome counts in some taxa (see Material and Methods in Dyer et
Our work had two aims. First we wanted to test the widely held assumption that DNA amount and spore size are correlated. To do this we compared DNA content and spore length for multiple taxa within the A.monanthes complex. Our second aim was to investigate genome size evolution within this group of ferns to determine to what extent genome size variation reflects changes in chromosome size or ploidy levels. To do this we used genome size estimations made for taxa with known ploidy level (i.e. karyologically determined) to infer DNA ploidies for those species without available chromosome counts. The data were then analysed within the phylogenetic framework of Dyer et al. (2012) to provide insights into genome size dynamics.