Shifts in genome size associated with the preservation of plant material

In contrast, samples processed using 2 year old silica material showed that 2C-values were on average, 18.6% higher (ranging from 7.83–30.09%), compared with estimates made from fresh material (Table 1). Furthermore, the quality of the measurements made on 2 year old silica samples was notably lower compared with both fresh samples and samples 6 months in age, based on the mean CV% of the peaks in the flow histograms (i.e. fresh samples = 3.37; silica samples 6 month old = 3.25; Silica samples 2 years old = 5.65). Data from 2 year old material were therefore excluded from further analysis.

Variation in genome size and spore size

Within the A.formosum and A.resiliens clades, 2C-values were highly conserved (13.19-13.93 pg and 13.70-14.50 pg respectively), as were the spore lengths (27.85-34.56 μm and 42.26-45.07 μm respectively) (Figure 1). All accessions of A.resiliens formed a well supported monophyletic lineage (clade A, Figure 1), with accessions of A.aff.heterochroum occupying a sister position (clade B, Figure 1). Of the two specimens investigated of A.aff.heterochroum, one (RD75, 2C = 14.38 pg) had a similar 2C DNA content to A.resiliens (mean 2C of four individuals = 13.99 pg), while the other was distinctly higher (RD9a, 2C = 18.67 pg). In contrast to this genome size variability, both specimens showed very similar spore lengths (37.43-38.62 μm), which were smaller compared with the mean value for the four A.resiliens specimens analysed (43.29 μm).

Asplenium fibrillosum, A.soleirolioides, and A.polyphyllum, which together comprise the A.castaneum clade, were observed to have the highest 2C values in the complex (37.78 pg for A.fibrillosum, 29.95 pg for A.polyphyllum, and 50.29 pg for A.soleirolioides) (Table 2 and Table 1), but since these estimates were made from material that had been stored on silica for 2 years their reliability is questioned (as noted above). Interestingly, the mean spore length of the A.fibrillosum specimen was also very large (47.90 μm), although those for A.soleirolioides and A.polyphyllum were distinctly smaller (39.37 μm and 35.42 μm respectively).

Correlation between DNA amount and spore size

Regression analysis of the raw data showed a strong positive correlation (p = 4.57×10^-6, r ²  = 0.542, slope = 10.986, 95% CI’s = 8.665-13.929) between DNA amount and spore length (Figure 2A). However, phylogenetically independent contrasts analysis indicated no significant relationship between contrasts (Grafen branch transformation, p = 0.101) (Figure 2B and Table 3). This indicated that divergences in DNA amount are not associated with divergences in spore length, and therefore there is no correlated evolution between DNA amount and spore length.

Branch transformation method	Phylogenetic signal (κ)				PIC standardisation
	GS		SL		GS		SL
	κ	P	κ	P	r 2	P	r 2	P
Log	2.500	0.001	0.773	0.001	0.314	0.024	0.207	0.076
Pagel	2.814	0.001	0.800	0.001	0.196	0.008	0.021	0.408
Nee	2.317	0.001	0.988	0.001	0.176	0.014	0.019	0.434
Grafen	1.131	0.001	0.273	0.003	0.035	0.289	0.080	0.090

DNA amount values were log transformed prior to analysis. GS = genome size, and SL = spore length. 1C or 2C DNA values were used depending on the reproductive mode: 2C-values are used for apomictic taxa, and 1C-values are used for sexual taxa. Results are given for four branch length transformation methods, as discussed in the Materials and Methods. Significant P values are given in bold.

Genome size and ploidy level

Monoploid genome size (1Cx-values) and ploidy levels were not inferred for A.fibrillosum, A.soleirolioides and A.polyphyllum, due to the shifts observed in the 2C DNA contents of these species, as a consequence of their storage for 2 years in silica (see above). Ploidy levels and 1Cx-values were inferred for all other subclades analysed (Table 4 and Figure 3).

Species	Mean 2C-value (pg)	Mean CV%	Inferred Ploidy (x)	Holoploid 1C-value (pg)	1C-value SE	Monoploid 1C x-value (pg)	1C x-value SE	Mean spore length
A.formosum	13.61 ± 0.10	2.97	2	6.81	0.09	6.81	0.09	32.07
A.resiliens clade
A.resiliens	13.99 ± 0.08	2.87	3	7.00	0.09	4.66	0.06	43.29
A.aff.heterochroum RD9a	18.67 ± 0.08	3.11	4	9.34	-	4.67	-	38.62
A.aff.heterochroum RD75	14.38 ± 0.05	2.39	3	7.19	-	4.79	-	37.43
A.monanthes clade
A.hallbergii	26.11 ± 0.11	3.46	3	13.06	0.03	8.70	0.02	45.24
MO1 I	30.17 ± 0.41	4.09	3	15.09	0.15	10.06	0.10	40.11
MO1 II	28.00 ± 0.13	3.06	3	14.00	0.02	9.33	0.01	44.47
MO1 III	26.86 ± 0.21	3.88	3	13.43	0.05	8.95	0.03	43.72
MO2 I	27.06 ± 0.14	4.28	3	13.53	0.16	9.02	0.11	44.51
MO2 II	18.58 ± 0.09	3.00	2	9.29	-	9.29	-	42.11
Spec.nov.1	17.67 ± 0.19	4.26	2	8.84	-	8.84	-	30.87
Spec.nov.2	22.20 ± 0.28	3.54	2	11.10	-	11.10	-	29.71

Mean 2C DNA content (pg) and mean spore length (μm) were only calculated for sub-clades distinguished by low genome size variation (Figure 1). This resulted in separate inferences of genome size and ploidy for the two A.aff.heterochroum specimens (RD75 and RD9a), and A.monanthes subclades: MO1 (I); MO1 (II); MO1 (III); MO2 (I), and MO2 (II) (Figure 1). Published chromosome counts (A.formosum, 2n = 2x = 72; A.monanthes, 2n = 3x = 108; A.resiliens, 2n = 3x = 108) were used to estimate ploidy and hence calculate monoploid genome size (i.e. 2C-value divided by ploidy level). For lineages with unknown ploidy levels, monoploid genome size and ploidy level were estimated according related species. Estimates of ploidy underlined in bold are based on the spore number and spore measurements [29].

Within the A.resiliens clade, different ploidy levels were inferred for the two A.aff.heterochroum specimens. One specimen of A.aff.heterochroum (RD75; 2C = 14.38 pg) had a similar 2C-value to A.resiliens (mean 2C = 13.99 pg), a species reported to be triploid, suggesting that RD75 could also be a triploid. In contrast, the other A.aff.heterochroum specimen analysed (RD9a) had a significantly higher 2C-value (18.67 pg) and a 1Cx-value approximately four times that inferred for RD75, which lead to the inference that this specimen was a tetraploid.

The suggestion that most A.monanthes specimens are triploid is based on the observation that, with the exception of one report of a tetraploid count, all counts for this species have been 2n = 3x = 108. Under this assumption, the mean 1Cx-values for MO1 sub-clades I, II and III are 10.06 pg, 9.33 g, and 8.95 pg, respectively (Table 4). A triploid level is also assumed for MO2 (I), based on the 2C-values, resulting in a mean 1Cx-value of 9.02 pg. The A.monanthes MO2 (II) sub-clade, which comprised a single specimen (RD96), had a significantly lower 2C-value of 18.58 pg, and we infer that this specimen is likely to be a diploid cytotype. This would result in a 1Cx value of 9.29 pg, which is in broad agreement with the monoploid genome sizes calculated for the remaining A.monanthes sub-clades (Table 4). Nevertheless, it is noted that the mean spore size of RD96 (42.11 μm) conflicts with this finding, suggesting that this specimen is a polyploid.

In A.hallbergii, the similarity of its mean 2C-value (26.11 pg) and mean spore size (45.24 μm) to that of the triploid A.monanthes lineages (MO1 and MO2.I) suggests this species is also a triploid. As for the two new species, spec.nov.1 and spec.nov.2, they differ in 2C-values (17.67 pg and 22.20 pg respectively) but have similarly small spore sizes. They are currently inferred to be diploid although further data are needed to confirm or refute this.

Overall, the results show that monoploid genome size varies 2.4-fold (4.66-11.10 pg), with little variation in 1Cx-values within clades, but considerable variation between some of them (Table 4 and Figure 3). For example, compared with the small 1Cx-values in the A.resiliens clade (4.66-4.79 pg), the mean 1Cx-value for diploid A.formosum (6.81 pg) was markedly higher. A larger monoploid genome size was also noted in the A.monanthes clade compared with the A.resiliens clade. Indeed, the largest monoploid genome sizes were encountered within the A.monanthes clade (8.70-11.10 pg), with spec.nov.2 having the highest 1Cx-value (11.10 pg) of all, which is more than double the mean monoploid genome size of the A.resiliens clade.

Considerations for the use of silica dried material for genome size estimation

The question as to whether it is possible to use silica preserved material to measure genome size in absolute units has recently become the focus of intense debate [30–32]. In order to avoid ambiguous interpretations, some authors only advocate the use of fresh material to estimate genome size [33], and restrict the use of silica-dried material to estimating DNA ploidy levels (e.g. [34]). However, recent studies have started to explore the use of silica dried material for genome size measurements (see [30]). This would help overcome some of the constraints imposed by the need for fresh material, especially when the quality of measurements is not compromised (i.e. <5% of variation between fresh and silica-dried tissues).

In the current analysis some of the accessions were only available as silica dried material (either 6 months or 2 years old), so the reliability of the genome size estimates obtained from such material was investigated. We were able to compare fresh and silica-dried tissues belonging to the same accession, for three separate species (Table 1). We found a significant increase in fluorescence intensity in all silica-dried samples of 2 years in age, relative to the fresh material, which resulted in genome size estimates up to c. 30% larger compared with those estimated on fresh material (Table 1). In addition, as previously reported, %CV was also seen to increase, and in most cases these exceeded acceptable values (i.e. >5%). We therefore conclude that for Asplenium, the use of 2 year old silica dried material is unsuitable for obtaining accurate genome size estimations.

In contrast, samples of two accessions of A.formosum stored for 6 months in silica gave very similar results to those obtained from fresh material (RD28 and RD33; Table 1). In addition, there was no evidence of any reduction in the quality of the flow histogram with the CVs for both fresh and silica samples being similar. Nevertheless, while these results suggest that the short term storage of Asplenium samples in silica is suitable for genome size estimations, the extent to which these findings can be extrapolated to other plant genera, including other ferns remains unknown.

Relationship between DNA amount and spore size

Numerous studies in angiosperms have shown a positive correlation between genome size and a number of morphological traits, including seed mass, cell size and stomatal density (e.g. [23, 24, 35, 36]). Beyond the angiosperms, such studies are relatively rare (e.g. [19, 21]), and while spore size is often used as a proxy for inferring ploidy level (e.g. [26–28]), there have been no empirical studies to date that test these inferences using genome size. In this study we investigated the relationship between genome size and spore size to determine the extent to which such an assumption is valid.

We found a significant and positive correlation between nuclear DNA amount and spore length using the raw data. However, when evolutionary relationships were considered, using phylogenetically independent contrasts (PICs), no significant correlation was found. The discrepancy between analyses is interesting, and although the significance of the raw data should not be discounted it does highlight the importance of using PICs to determine the evolutionary association of statistically non-independent traits [37].

The insignificant relationship observed between DNA amount and spore size calls into question the use of spore size for inferences of ploidy level within homosporous ferns. Previous authors have pointed out that this inference should be restricted to very close relatives, and the distinction between diploids and their autoploid offspring [26, 27]. Our findings are consistent with these suggestions. In addition, our study sample is mainly comprised of apomictic accessions, and due to a low sample number of sexually reproducing species we are unable to test for the effect of reproductive mode on the relationship between traits.

Considerable variation was found in spore length, both within and between specimens of the apomictic lineages. In part this variation may be a consequence of the non-globulose shape of the monolete spores found in this complex, making precise measurements difficult. However, the variation may also be due to the variable nature of Döpp Manton sporogenesis in triploid apomicts of homosporous ferns [38, 39]. The variation in spore size within and between these taxa is reflected by the inability to determine ploidy level (beyond the difference between diploid and polyploids) from spore size measurements in this complex [29].

Overall, our findings challenge the utility of spore size for inferring ploidy level within ferns. Nevertheless, further investigations are needed to test our findings for spore size in a greater number of lineages, and also determine the effect, if any, of reproductive mode on the relationship between DNA amount and spore size.

The evolution of genome size in the Asplenium monanthescomplex

The high chromosome numbers, and conserved chromosome sizes reported for many homosporous ferns has contributed to the hypothesis that the evolution of fern genomes is less dynamic than the evolution of angiosperm genomes [3, 4, 9]. This has been suggested to be due to a higher retention rate of chromosomes and the possible suppression of transposable elements (TEs) in homosporous ferns [5, 6]. The inferred constancy of chromosome size is based on physical measurements [16], the low number of reported retrotransposons [17], and the reported correlation between chromosome number and genome size [6]. Bainard et al. [6] suggest that these observations show that genome size expansion is mainly driven by polyploidy, and that stepwise increases observed in 1Cx-values across monilophytes might be indicative of paleopolyploidy.

Indeed, in the Asplenium monanthes complex studied here some of the 1C-value variation within clades clearly arises from polyploidisation, with ploidy levels ranging from 2x to 4x (Table 4), as inferred by comparing the genome size data with previously published chromosome counts (available for some of the species). This is consistent with the hypothesis that genomes size variation in homosporous ferns is driven by polyploidisation. However, contrary to this hypothesis, we also report variation in the monoploid genome size (1Cx-value), indicating chromosome size variation between species (Figure 3). Our study provides evidence that both processes of genome evolution are occurring in this complex, both are discussed below.

Polyploidy is known to occur in several species of this complex based on previously reported chromosome counts (see Methods). The genome size data obtained in this study proved useful in identifying putative cryptic species (e.g. A.aff.heterochroum, and MO2,II) and in inferring ploidy levels for taxa without reported chromosome numbers. Inferences of ploidy were carried out with caution as even closely related taxa with different ploidy levels can display similar genome sizes, potentially leading to incorrect ploidy determinations [40]. A.formosum and the apomict A.resiliens clearly illustrated this situation as both taxa have very similar 2C-values (mean 2C = 13.61 pg and 13.99 pg respectively) (Figures 1 and 3). Without additional information it might be tempting to assume they had the same ploidy level. Yet complementary information from published chromosome counts strongly supported the presence of different ploidy levels, with A.formosum being a sexual diploid and A.resiliens a triploid apomict. Different ploidy levels were also identified among A.aff.heterochroum specimens (RD75 = 3x and RD9a = 4x), when compared to the genome size of the sister species A.resiliens.

In the A.monanthes complex moderate variation in 1C-values was noted between the accessions identified as A.monanthes (Figure 1). However, in all but one case (i.e. RD96), these specimens were inferred to be triploid. It is suggested that such variation has most likely arisen from differences in the DNA contents of the progenitor species that gave rise to the triploids, a claim supported by the different 1C-values reported for the putative diploid progenitor species, spec.nov.1 and spec.nov.2 [29]. The exception noted in specimen RD96, which belonged to the MO2.II lineage of A.monanthes, was inferred to be a diploid apomict, due to a significantly smaller 2C-value (18.58 pg) than the mean 2C of the remaining triploid taxa (27.64 pg). This suggestion is consistent with the occurrence of diploid apomicts in other ferns, including taxa of the Dryopteris affinis complex [19, 41] and the Pteris cretica complex [42].

Chromosome size expansion/reduction is indicated in this complex by the observed differences in the monoploid genome size of the study species. This finding would suggest that chromosome size may not be as conserved as widely reported in homosporous ferns (e.g. [3]). In our study, the 1Cx-values of specimens within the A.resiliens clade (1Cx = 4.66-4.79 pg) are similar to more distantly related Asplenium species including A.trichomanes ssp. quadrivalens (1Cx = 4.53 pg; a species within A.trichomanes complex) [6, 8]. This indicates that A.formosum and species within the A.monanthes clade have undergone up to a two-fold expansion in monoploid genome size.

An expansion of the chromosome/ monoploid genome size could arise via retrotransposon-driven changes, as suggested for Equisetum and Pstilotum[9, 38, 43, 44]. Retrotransposon proliferation and elimination is linked with genomic and environmental factors such as effective population size, environmental stress, hybridisation and polyploidy [13, 45–49]. Given that the apomictic A.monanthes clade and related lineages show strong patterns of reticulate evolution, it is possible that these processes may be acting as triggers for retrotransposon activity leading to the range of 1Cx-values observed. Indeed, given the extent of hybridization and reticulate evolution reported in homosporous ferns in general [50], it seems likely that retrotransposon driven changes in genome size are probably more widespread across ferns but may have been largely overlooked due to the low level of sampling.

Taxa studied

The Asplenium species included in the present study are listed in Table 2. They were selected based on the phylogenetic investigation of the complex by Dyer et al.[29] and whether the species could be successfully cultivated (see Table 2). Spec.nov1 and spec.nov.2, which belong to the A.monanthes complex, are reported by Dyer et al. [29] to be sexual diploids (based on spore size and nuclear DNA sequence analysis) and considered to be putative progenitor species to the two distinct apomictic lineages of A.monanthes, referred to as MO1 and MO2 (see Figure 1).

Chromosome counts and derived ploidy levels are reported in the literature for: A.formosum, 2n = 2x = 72 [1, 51–54]; A.monanthes, mainly 2n = 3x = 108, although there is a single count of 2n = 4x = 144 [38, 55–60]; A.resiliens, 2n = 3x = 108 and A.heterochroum, 2n = 4x = 144/2n = 6x = 216, 2n = 5x = 180 [52, 57, 61–67].

Fresh material was unavailable for A.fibrillosum, A.polyphyllum, A.soleirolioides, spec.nov.1, spec.nov.2, and some accessions of A.formosum (see Table 2), so silica dried samples were used instead. In total, the study sample comprised 31 fresh samples cultivated from specimen spores, and seven silica-dried samples.

Flow cytometry: genome size and DNA ploidy

The nuclear DNA content of 38 specimens was measured following the one-step procedure described by Dolezel et al.[33]. Individual sporophytes were prepared as follows: several pinnae (after removing the rachis) were co-chopped using new razor blades, together with the appropriate calibration standard (Pisum sativum ‘Ctirad’, 2C = 9.09 pg) [68], in a Petri dish containing 2 mL of ‘General purpose buffer’ (GPB) (Loureiro et al., [69]) supplemented with of 3% PVP-40 . The suspension of nuclei was then filtered through a 30 μm nylon mesh, stained with 100 μl of propidium iodide (Sigma; 1 mg · mL^-1), and treated with 34 μl of 3 mg · mL^-1 ribonuclease A (RNase A; Sigma). Both propidium iodide and RNase A were adjusted to a final concentration of 50 μg/mL. Samples were kept on ice for 30 min and 5,000 particles recorded using a Partec Cyflow SL3 flow cytometer (Partec GmbH) fitted with a 100 mW green solid state laser (532 nm, Cobolt Samba). Flow histograms were analysed with the FlowMax software (v. 2.4, Partec GmbH). Three sporophytes were measured separately for each specimen, and three replicates of each were processed. Measurements obtained from fresh and silica dried materials of the same specimens were compared to determine the extent to which the preservation method influenced the relative fluorescence (i.e. nuclear DNA content) estimate.

We compared 1Cx-values within species (for species with karyologically determined ploidy levels) to check for cryptic ploidy levels. In order to infer DNA ploidy levels (i.e. ploidy levels inferred only from DNA amount cf. [40]) for the species without chromosome counts, we compared their holoploid genome size and mean spore length (see section below), with those of taxa within the same clades whose ploidy level had been karyologically determined. Given the comprehensive variety (quantity and geographic spread) of the previously reported karyological data, we consider our inferences of 1Cx-values for the remaining taxa (by means of the DNA content) to be legitimate.

Spore measurements

To analyse the relationship between spore size and genome size we combined the spore size data from Dyer et al. [29], with additional spore measurements made for the remaining specimens sampled in this study. Spores from individual specimens were mounted onto slides using glycerine jelly. Each spore length was measured using AxioVision on a calibrated light-microscope (v4.8.2, Zeiss). An average of 25 spores were measured per specimen, and a mean spore length was calculated (Table 2). Special care was taken to identify putative abortive spores that were then excluded from the analyses. Individual box plots were compiled to show spore size variance within each specimen, as well as the interquartile range and the median.

The relationship between DNA amount and spore length

We investigated the relationship between DNA amount and spore length by comparing the results obtained from analysing the raw data with those obtained using phylogenetic independent contrasts (PIC),. A standard regression analysis assumes that the data points are independent, which is not the case in related species data. PIC’s take phylogenetic relationships into account by transforming the species data into statistically independent data. Different DNA amount values were used for specimens based on their mode of reproduction: apomictic ferns produce unreduced spores, and so 2C-values were used for analysis; sexually reproducing ferns produce reduced spores, and hence 1C-values were used for analysis. The raw data were not normally distributed; therefore, in order to linearize the data for PIC analysis [70], the mean measurements for DNA amount were log transformed.