The C Value Of Ferns Was Determined By FCM And Its Variation In Phylogeny Was Discussed

As transitional group from bryophytes to seed plants, ferns are the earliest vascular plants with vascular tissue and they reproduce by spores instead of seeds. Over the world, there are almost 11,000 lycophytes and monilophytes, of which 10% are lycophytes. Ferns have made significant contribution to the formation of terrestrial ecosystem and the improvement of land ecological environment, and laid a foundation for the origin and early evolution of seed plants. The amount of DNA in the unreplicated haploid nuclear genome (C-value) provides important information for genome biodiversity. Researchers have used DNA C-values to address questions in cellular, developmental, ecological, evolutionary and molecular biology as well as systematics, physiology and paleontology. However, in the Kew plant DNA C-values database, there are only 120 species’ records of lycophytes and monilophytes by now, greatly less than the records of seed plants. Based on DNA sequences, molecular phylogeny can reconstruct the lycophytes and monilophytes’ phylogenetic relationship, combined with the contrast analysis of C-value data, which make great sense to getting better understanding of the occurrence and system evolution of this group and even the whole vascular plants.Based on the research statue of the C-value estimates in lycophytes and monilophytes, in this study, we sampled nearly 300 species of lycophytes and monilophytes, and finally estimated 4 lycophytes and 163 monilophytes’ C-values. Then we downloaded the rbcL sequences of these species who had C-values from GenBank. We constructed the phylogenetic tree of these 197 lycophytes and monilophytes species with MrBayes 3.2.1. By contrast analysis, we got the change characteristic of C-value of these species in different taxon, which furthermore would help to clarify the evolution trend of C-value in lycophytes and monilophytes phylogeny. The main results were as below:1. Flow cytometry is the standard method used for DNA content measurement, combined two kinds of extraction buffers, we estimated 167 species of ferns. We optimized the FCM estimation methods of genome size of ferns by comparing different conditions, such as different extraction buffers(namely Otto and GPB), different standard species (including Oryza sativa, Pisum sativum and Vicia faba) and different types of material, namely fresh leaves and leaves preserved in silica gel.2. With above optimized methods and the use of Otto and GPB extraction buffers, we estimated C-values of 163 species in monilophytes and 4 species in lycophytes. Among these species, Tectariaceae, Lomariopsidaceae, Blechnaceae, Hypodematiaceae and Rhachidosoraceae are 5 new families being estimated C-values for the first time. Pityrogramme with other 37 genus are also estimated C-values for the first time. Angiopteris badia and other 162 species’C-values are all firstly being estimated. The estimated C-values variation range is from 0.64 pg to 103.72 pg, within the already recorded range of 0.17 pg to 145.35 pg. These data provide the foundation to enable phylogenetic analysis of C-value variation in next station.3. The rbcL gene sequences of 193 species in ferns were downloaded from GenBank. With MrBayes 3.2.1, we performed Bayesian analysis and obtained the phylogenetic tree. Combining the C-values from both C-value database and our extimation above, we investigated the variation of ferns’C-values in different taxaon. The results showed that:our phylogenetic tree’s topology was consistent with the existing lycophytes and monilophytes phylogenetic tree with posterior probability of 95% of the branch node more than 50. SPSS analysis of variance showed the mean C-value of lycophytes was significantly smaller than the mean C-value of monilophytes, which was consistent with the generally accepted view that genome size is increasing in the evolution of the land plants. Nevertheless, among orders, families and genus, there was no significant correlation between C-value and its evolutionary position. It may be connected with the low number of species with C-value or the complicated ploidy in lycophytes and monilophytes.