Changes in the composition of the RNA virome mark evolutionary transitions in green plants

Background: The known plant viruses mostly infect angiosperm hosts and have RNA or small DNA genomes. The only other lineage of green plants with a relatively well-studied virome, unicellular chlorophyte algae, is mostly infected by viruses with large DNA genomes. Thus RNA viruses and small DNA viruses seem to completely displace large DNA virus genomes in late branching angiosperms. To understand better the expansion of RNA viruses in the taxonomic span between algae and angiosperms, we analyzed the transcriptomes of 66 non-angiosperm plants characterized by the 1000 Plants Genomes Project. Results: We found homologs of virus RNA-dependent RNA polymerases in 28 non-angiosperm plant species, including algae, mosses, liverworts (Marchantiophyta), hornworts (Anthocerotophyta), lycophytes, a horsetail Equisetum, and gymnosperms. Polymerase genes in algae were most closely related to homologs from double-stranded RNA viruses leading latent or persistent lifestyles. Land plants, in addition, contained polymerases close to the homologs from single-stranded RNA viruses of angiosperms, capable of productive infection and systemic spread. For several polymerases, a cognate capsid protein was found in the same library. Another virus hallmark gene family, encoding the 30 K movement proteins, was found in lycophytes and monilophytes but not in mosses or algae. Conclusions: The broadened repertoire of RNA viruses suggests that colonization of land and growth in anatomical complexity in land plants coincided with the acquisition of novel sets of viruses with different strategies of infection and reproduction.We thank the colleagues at the 1000 Plant Genomes Project for helping us to access the transcriptomes used in this study via the iPlant Collaborative. We are grateful to Javier Forment (IBMCP-CSIC), Vincent Lefort (PhyML), and the E-Biothon team (E-Biothon platform is supported by CNRS, IBM, INRIA, l'Institut Francais de Bioinformatique and SysFera) for expert help with high-performance computing; to Yuri Wolf, Jan Kreuze, Eddie Holmes, and Mang Shi for sharing sequence data and alignments; to Sejo Sabanadzovic, Jan Kreuze, and the anonymous reviewers for helpful virtual discussions and critical remarks; and to Natalia Mushegian for technical assistance. SFF was supported by grants BFU2015-65037P from Spain Ministry of Economy and Competitiveness and PROMETEOII/2014/021 from Generalitat Valenciana. 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The natural behavior of Drosera: Sundews do not catch insects on purpose

Volume: 38Start Page: 114End Page: 12

With 7 figures

Four plastid markers, four nuclear markers and 14 morphometric characters were used in this study to investigate the evolution of Dactylorhiza baltica (Orchidaceae) in European Russia. In total, 98, 214 and 775 samples from 85, 112 and 121 populations were involved in the combined and separate molecular and morphometric analyses, respectively. In most cases, morphometric measures were done on exactly the same plants that were used for DNA studies. ADDITIONAL KEYWORDS: allotetraploids -microsatellites -morphometrics -systematics. INTRODUCTION Dactylorhiza Necker ex Nevski (Orchidaceae) is, along with Epipactis and Ophrys , one of the most taxonomically controversial orchid genera in Europe. There is great instability in the accepted number of species and infraspecific taxa. The borders between many species are unclear, and there are considerable difficulties in the determination of single plants (Averyanov, 1990; Reinhard, 1990; Delforge, 1995; Stace, 1997; Bateman, 2001). Many of the most problematic taxa are allotetraploids, most of which are believed to be the result of multiple hybridization events between two broadly defined parental species, D. fuchsii (Druce) Soó and D. incarnata (L.) Soó (Heslop-Harrison, 1968; Hedrén, 2002; Devos et al ., 2003; Y. Pillon, unpubl. data). Multiple lines of evidence indicate that this complex is an '. . . unusually dynamic system of polyploid speciation and extinction in which polyploids evolve continuously from the same set of broadly defined parental lineages' (Hedrén, 2003(Hedrén, : 2678. Furthermore, the limits of the diploid parental taxa are sometimes made less clear by the exchange of genetic material, hypothesized to be via allotetraploids (Hedrén, Fay & Chase, 2001; Hedrén, 2003), in spite of the differences in their ploidy. One good example of such polyploid species and at the same time a less well known member of this complex is D. baltica (Klinge) Orlova, for which the distribution, unlike other named allotetraploids of this complex (which occur principally in western Europe), is restricted to the eastern part of Germany, Poland, the Baltic countries, southern Finland and Russia. The eastern parts of its distribution are less definite; some authors (Nevski, 1935; Smoljaninova, 1976) have argued that it is restricted to the western parts of European Russia (Pskov and Leningrad regions) together with some localities in the northern Urals and southern Siberia, whereas others expand the European portion across all of European Russia (between the Arctic Circle and 50 ∞ N latitude) to the Urals (Soó, 1980; Averyanov, 1990). The most recent evidence is, however, that ' D. baltica ' populations in the southern Urals have been misidentified and should be assigned to D. fuchsii (Kulikov & Filippov, 1999a). Thus, current opinion limits the distribution of D. baltica in European Russia to between 50 and 60 ∞ N latitude (with two exceptions in the northern Urals) and west of 60 ∞ longitude The epithet baltica was first used by Klinge (1895, 1898) for a subspecies of ' Orchis' latifolia L., nom. illeg. [ = Dactylorhiza majalis (Reichb.) P.F.Hunt & Summerhayes], a species with a western European distribution, long believed to be another member of the polyploid complex (Averyanov, 1990). This subspecies was later upgraded to species rank by Nevski (1935) Vermeulen, 1947; Senghas, 1968; Averyanov, 1990 (Hedrén et al. 2001; Hedrén, 2003; Devos et al ., 2003; Y. Pillon, unpubl. data; Shipunov et al ., 2004). (Averyanov, 1990; Kulikov & Filippov, 1999a). Each collection site is labelled with an abbreviated region name (see Appendix 1). However, no molecular analysis has yet been performed on D. baltica , which is unique among other Dactylorhiza allotetraploids due its eastern distribution and relative isolation from other allotetraploids. Moreover, there are few morphometric studies of Russian dactylorchids (Kulikov & Filippov, 1999a, b). Our recent investigation of European Russian Dactylorhiza showed good agreement between morphometric characters and molecular markers such as plastid microsatellites and ITS alleles (Shipunov et al ., 2004). Plastid microsatellites had previously been shown to be useful for revealing geographical patterns, the maternal parentage of hybrids, and even some relationships among populations (Y. Pillon, unpubl. data; Shipunov et al ., 2004 Shipunov et al . (2004) used the same markers to study general patterns of these same species and allotetraploid complexes in Russian Europe. The goal of this study is to explore diversity in detail in one of the Russian allotetraploid taxa, D. baltica , via morphological and molecular markers in the context of its likely origin via hybridization between the D. fuchsii and D. incarnata aggregates (both are treated as broadly defined species for simplicity). To the two sets of markers developed in Shipunov et al . (2004) and Y. Pillon (unpubl. data), we have added a set of two nuclear microsatellite markers, which we hope will be more variable than ITS and thus reveal more structure among populations of the putative parental taxa. We chose to focus on this allotetraploid taxon because it appeared to us that it was likely to be operating locally as a 'bridge' between the diploid taxa and would therefore make an appropriate subject for a more detailed study to determine whether we could detect evidence of this phenomenon though the study of both morphological and molecular markers. MATERIAL AND METHODS Some of the samples were used in a previous study (Shipunov et al ., 2004), but many samples from European Russia (mostly from central and north-western regions) and Britain were newly collected for this investigation (see Appendix). All incoming samples were initially identified and assigned to a priori species by experts in regional floras (G. Konechnaja M OLECULAR MARKERS Samples for DNA extraction were dried in silica gel (Chase & Hills, 1991). DNA was extracted by the 2 ¥ CTAB protocol (Doyle & Doyle, 1987 but without an RNA treatment). PCR was performed with a set of primers designed by Y. Pillon & M. F. Fay (unpubl. data) to amplify four polymorphic plastid loci: Orch1, Msf, Ms1 and Ms2, located in three plastid DNA regions: the trnS-trnG spacer, trnL intron and trnLtrnF spacer. Two pairs of specific primers were also used to amplify length-variable regions of ITS ribosomal DNA that, taken together, indicate which ITS alleles are found in each sample (Shipunov et al ., 2004; Y. Pillon, unpubl. data). To identify other molecular makers that are sufficiently polymorphic to reveal interpopulational structure, we have developed several nuclear microsatellites, two of which proved useful for this study. To develop these markers, we used a strategy proposed by Fisher, Gardner & Richardson (1996), which employs a degenerate primer PCT4 (Brachet et al ., 1999) that contains a (CT) 6 repeat at its 3 ¢ end. The conditions for PCR amplification were those of Fisher et al. (1996). Several PCR products were cloned using the Promega pGEM-T Easy Vector System. These were reamplified from transformed bacterial colonies by touching them with a sterile toothpick and using that sample as the template in a further round of PCR. Primers for this PCR were located on the vector. Amplified DNA fragments were purified using QIAquick PCR mini-columns (QIAGEN, Inc.), following the manufacturer's protocols, and sequenced on a 3100 genetic analyser (Applied Biosystems Inc.), following the manufacturer's protocols (we again used the primers that annealed to sites on the vector). Sequence editing and assembly of the two complementary strands used SequenceNavigator and AutoAssembler (Applied Biosystems Inc.) software. Several pairs of specific primers were subsequently designed to amplify the most promising microsatellite loci. The resulting fragments were checked to determine whether they revealed any polymorphisms, and two loci were then chosen for this investigation M ORPHOLOGY We used the set of 14 morphological characters, slightly modified from previous work (Shipunov et al ., 2004). These characters were measured in nature on either the same plants that were used for DNA extractions or, on a few occasions (e.g. for D. praetermissa and several populations of D. baltica ), we measured neighbouring plants in the same population. We used principal component analysis (PCA) and multidimensional scaling (MDS) of individual and population data. In the latter case, population medians (because these are usually more robust than means; Fowler, Cohen & Jarvis, 1999) were used. The analysis of population data was wider than the analysis of individual data because we included some species and populations for which DNA sampling and morphometric measurements were made on different plants. We have also analysed correlation from individual data (all species included) for all morphological measurements and nuclear DNA markers, and used recursive partitioning analysis, which is the model-based version of discriminant analysis, describing which character values best predict the existing classification (Breiman et al., 1984 (da114, da118, ds130, ds153). For most population samples, multiple alleles were amplified (see Appendix); putative diploids displayed 1-2 alleles, and several of the allotetraploids had up to four alleles. Some samples collected as D. fuchsii (a diploid) have 3-4 alleles, but these plants are in fact 'northern tetraploids' (Shipunov et al., 2004) Although the repeats selected were all in triplets MULTIVARIATE ANALYSES Both PCA and MDS of the morphological data revealed similar patterns. There are three overlapping groups In both cases the most important characters (which have relatively high loadings in the first component, PC1) are for individuals, plant heights, all leaf characters and inflorescence lengths, and for populations, bract lengths, stem diameters and leaf lengths. Simultaneous analysis of morphology, ITS alleles and nuclear microsatellites produced a less ambiguous structure An analysis of individuals combining all characters, including the uniparentally inherited plastid sequences, changed the picture completely To represent better some interpopulation relationships and possible geographical patterns, we constructed a UPGMA tree in PAUP for the nuclear DNA data (presence/absence of molecular markers) for populations of D. baltica and its putative parental species. This analysis showed that most D. baltica populations have clear relationships with their putative parents, either D. fuchsii, D. incarnata or even, in some cases, both. The tree The morphological characters formed three correlation groups: (1) most of the vegetative characters, including bract and inflorescence length but not leaf spots, (2) leaf spots and (3) floral characters in which the largest significant correlation is between lateral lobe and mid-lobe lengths (r = 0.83, P << 0.05). The DNA characters most correlated with species parti- . UPGMA tree derived from nuclear DNA data of investigated populations. All labels contain the species epithet, population number and code for the collection site (see Recursive partitioning revealed that the three characters most important in the analysis of data for D. fuchsii, D. incarnata and D. baltica ( The UPGMA results also demonstrate that D. baltica is related to D. incarnata and D. fuchsii (not D. maculata), but it is clear that in this region the two Exchange of alleles/haplotypes between the parental diploids is consistent with the allotetraploids forming a 'bridge' for gene flow between the two diploids. There is also some correlation between the proportion of D. incarnata ITS allele (determined from the peak height in the PCR of the length-variable ITS fragments) in D. baltica plants and their morphology, which could be explained by backcrossing with D. incarnata. Some D. baltica samples (especially from north-western populations) lack or have only a small proportion of D. fuchsii (da114 or da118) nuclear microsatellite alleles, which again could be evidence of backcrossing. The D. incarnata plants from populations 210 and 215 with the A haplotype fall into the D. fuchsii group in the combined analysi