Daniela Bártová, Tomáš Pavlíček and Petr Bureš (šéf nakonec?)
Background and aims. Dactylis glomerata and Hordeum spontaneum are two species with previously documented genome size variation. 1. In Dactylis glomerata, a frequently cited study of Reeves et al. (1998) reported a negative association of genome size with site altitude. Our aim was to reinvestigate this report and explain the variation using climatic data or infraspecific taxonomic traits. 2. For in Hordeum spontaneum, environmentally induced proliferation of transposable elements was reported, but without an apparent impact on genome size estimations. Our aim was to study the variation in this species on a sharp climatic gradient and explain it by fitting appropriate climatic variables.
Methods. Genome size was estimated using FCM with DAPI staining. Climatic variables were extracted from the global WORLDCLIM model. In D. glomerata, maximum leaf width was recorded as a proxy of the plant traits.
Results. D. glomerata samples varied by 1.11 fold in genome size and the variation was proven by double-peaks. Genome size and leaf width showed a strong positive correlation together and both also with annual precipitation and the geographical distribution. Genome size in H. spontaneum varied 1.02 fold.
Conclusions. The previously estimated relationship of genome size and altitude in Dactylis glomerata was not confirmed, thus this hypothesis of the abovementioned study of Reeves et al. should be rejected.
Higher genome size was found in Spanish populations, where the distribution ranges of subsp. glomerata and subsp. izcoi overlap. Further study is needed to confirm whether higher genome size is inherent to the subsp. izcoi or results from an environmental adaptation.
In Hordeum spontaneum, a detailed assessment of its genome size variation is still beyond the actual limits of flow cytometric measurements.
The period of doubts about the existence and extent of intraspecific genome size (GS onwards) variation, mostly caused by imprecise GS estimates (reviewed by Greilhuber 2005, Šmarda et Bureš 2010), is already over. Nowadays, evidence for taxa with genuine variation accumulates again (e.g. Šmarda et Bureš 2006, Leong-Škorničková 2007, Achigan-Dako 2008, Balao 2009, Cires 2010). When proper measurement conditions are met (i. e. internal standardization, cytosolic compounds avoidance, more in the review by Loureiro et al. 2010), FCM (FCM onwards) yields high-quality data, in suitable amounts and timespans, hence allowing detailed population screening in a single species. Distinct individuals can be examined in a simultaneous measurement, where, depending on tissue properties, even relatively small differences in GS can be proven.
Proximate causes of GS variation have been largely uncovered (e.g. Bennetzen et al. 2005), as well for the increases and decreases of the GS. However, the ultimate causes of GS variation remain still unknown. That's why observation studies, bringing new hypotheses on the constraints and advantages of specific GSs are still needed. In such studies, environmental conditions are used to as first-choice explanatory variables. and in the background, there are general theories as the nucleotypic theory (Bennett, 1971) or an analogy of the Large genome constraint hypothesis (Knight et al. 2005), but independent of phylogenetic relations.
Because the response of plants to abiotic factors is obviously species-dependent, the most suitable objects of such a study are individuals from a single genus. Intraspecific GS variation is believed not to be a very common feature of plant taxa, nevertheless only few studies so far have examined species in deep at the population level (because not species, but populations evolve), with respect to e. g. demographic processes, species history, or the effects of its distribution range. The observed variation might result from gradualistic as well as punctuacionalistic mechanisms. The question of the limit size of differences in GS, having already observable (significant) consequences on plant fitness is unansewered. Which extent of GS variation has still the form of a neutral, slightly deletrious or advantageous mutation and what extent poses real constraints on the plants' life, remains also unclear. Our aim was to study these peculiarites using two plants with variable GS: orchard grass (Dactylis glomerata) and wild barley (Hordeum spontaneum).
Orchard grass is a perennial herb, cultivated worldwide as forage crops. Its Eurasian wild relatives form a taxonomic complex of diploid, tetraploid and one hexaploid subspecies (reviewed in Jogan, 2002), although taxonomic rankings of some might differ according to different authors. A frequent production of unreduced gametes (Lumaret et Barrientos, 1990) causes gene flow among sympatric subspecies and also the formation of autotetraploids. Autotetraploids grow in sympatry with their parental diploids and are not reproductively isolated from them (e.g. Bretagnolle and Thompson, 2001, Gauthier et al. 1997). Apart from these local diploid-tetraploid complexes, there are two widely distributed tetraploid subspecies in continental western Europe, subsp. glomerata and subsp. hispanica, very variable, and probably due to convergent evolution, they form rather a morphological continuum along the cline from temperate to mediterranean (Borrill, 1961). These are supposed to originate from hybridization of two diploids, but only one parental species is known in both cases (Stebbins et Zohary 1959, Mizianty 1991). Other hypotheses discuss their autopolyploid origin (reviewed in Mizianty 1990).
Interestingly, local ecological specialization, was recorded in transplanting experiments in D. glomerata (Gauthier, Lumaret et Bedecarratas 1996), even regardless the taxonomic rank (Joshi et al. 2001).
GS investigations in Dactylis glomerata
First single estimates of GS from the early era of GS investigations are reviewed by Vilhar et al. (2002). Population-based studies within one ploidy level have been carried out by Creber et al. (1994) and Reeves et al. (1998) using densitometry with Feulgen staining. The study assessed 19 accessions from three altitudinal gradients in SW Europe and the reported 1.33-fold intraspecific variation was one of the highest values published in that period. Moreover, a significant negative correlation of GS and altitude was found, hence, there emerged a question about the puissant ecological constraint, that shapes this relation. However, the cold hydrolysis protocol published in the study of Creber et al. (1994) and utilized as well in the latter (Reeves et al. 1998), was later criticized (Greilhuber et Baranyi, 1999). Nevertheless, Reeves's study has been cited as a proper example of GS variation until recently (e.g. Chen et al. 2010, Xie et al. 2010).
A comparative experiment was done in Slovenia, on an altitudinal transect along the Krvavec mountain. Only a 1.021-fold variation was found using Feulgen-stained image densitometry and no relation of GS to altitude was observed. (Vilhar et al. 2001).
Wild barley has been extensively studied in order to explore genetic variation of breeding resources of this closest wild relative of a major crops – Hordeum vulgare. H. spontaneum and H. vulgare are diploid (n=7). According to the narrowness of a taxonomic concept applied both are distinguished as subspecies of H. vulgare (subsp. vulgare and subsp. spontaneum) or two distinct species.
Genetic variation with respect to environmental conditions in H. spontaneum has been assessed multiple times and the results differ according to the markers used and the sampling scale. Forster (1997) found specific AFLP markers for salt tolerance and SSR markers for drought tolerance. Ivandic et al. (2002) found microsatellite markers with large-scale geographical interpretation, but no specific ecological relations. Huang (2002) conducted micro-scale screening of SSR on the area of one mountain (Mt. Tabor) and found a non-random distribution of genotypes reflecting heat and edaphic stress. More recent papers agree on markers for salt and drought tolerance, used for breeding purposes – RAPD, AFLP, SSR, rDNA, SNP and QTL loci (reviewed in Nevo et Chen, 2010). Local ecological specialization of H. spontaneum populations is thus very probable.
GS investigations in Hordeum spontaneum
One of the first studies of GS variation in populations of H. spontaneum was performed using FCM by Kankanpaa et al. (1996). Nine ecologically distinct populations, each represented by a single accession, showed a 1.13-fold variation but failed to reflect any significant environmental pattern. However, these GS estimates (i) lacked verifications using double peaks and (ii) lacked information about the quality of measurements, expressed by CVs. The sample peak, as it can be seen in the Figure 1 in the concerned paper, is clearly bimodal and asymmetric, which could indicate that conditions for proper measurements of small variation in GS were not met. Finally (iii) the possible variation inside the populations was not considered.
The data of Kankanpaa et al. (1996) was later compared to the results of quantification of transposable elements BARE-1, but did not show any significant relationship to the copy number. Even the relations of GS and the environment were denoted just as trends (Vicient et al. 1999). The connection of BARE1 copy number to the site conditions was later confirmed in a micro-scale experiment in the Evolution Canyon, nonetheless without a relation to the almost invariable GSs of the samples (Kalendar et al. 2000). Minor importance is assigned to GS variation in the works of Eilam et al. (2007) and Jakob et al. (2004); both of these studies are based on FCM on a limited number of accessions (12 and 4, respectively). An other mid-scale FCM study analyzed 97 accessions from 10 Israeli populations and found a 1,05-fold variation in GS. This variation was significantly associated to January temperature (Turpeinen, Kumala et Nevo, 1999). However, the observed variation was not proven by double-peaks.
The putative mechanism behind the shifts in GS in the genus Hordeum is BARE-1 proliferation (Vicient et al. 1999) and their subsequent environmentally induced removal by LTR-LTR recombination (Schulman et Kalendar, 2005), which manifests itself as an excess of solo-LTRs. BARE-1 removal and consequentially solo-LTR abundance is associated with micro-scale gradients of environmental conditions (Kalendar et al. 2000). It is therefore rather paradoxical that the effect of GS changements seems not to be observable on the whole-genome level among micro-scale differentiated sites (Kalendar et al. 2000).
We attempted to re-investigate the same accessions of Dactylis glomerata, as had used Reeves et al. (1998) in order to (I) confirm or reject the GS variation in this species and its relation to altitude. Further, we (II) searched for more precise explanatory variables, since “altitude” is not only an ecological factor per se, but rather a complex of more, correlated and uncorrelated variables - temperature, moisture, exposition, UV irradiation etc. (Korner, 2007). A key question for D. glomerata was also, (III) whether the observed variation is not attributable to taxonomic heterogeneity. In Hordeum spontaneum, we wanted to (IV) to complete the link between the known environmentally induced variation in transposable elements and GS estimates by the means of precise FCM measurements. Similarly to D. glomerata, (V) to propose more precise environmental explanatory variables to this variation.