Exceptionally high rates of positive selection on the rbcL gene in the genus Ilex (Aquifoliaceae).

BACKGROUND: The genus Ilex (Aquifoliaceae) has a near-cosmopolitan distribution in mesic habitats from tropical to temperate lowlands and in alpine forests. It has a high rate of hybridization and plastid capture, and comprises four geographically structured plastid groups. A previous study showed that the plastid rbcL gene, coding for the large subunit of Rubisco, has a particularly high rate of non-synonymous substitutions in Ilex, when compared with other plant lineages. This suggests a strong positive selection on rbcL, involved in yet unknown adaptations. We therefore investigated positive selection on rbcL in 240 Ilex sequences from across the global range. RESULTS: The rbcL gene shows a much higher rate of positive selection in Ilex than in any other plant lineage studied so far (> 3000 species) by tests in both PAML and SLR. Most positively selected residues are on the surface of the folded large subunit, suggesting interaction with other subunits and associated chaperones, and coevolution between positively selected residues is prevalent, indicating compensatory mutations to recover molecular stability. Coevolution between positively selected sites to restore global stability is common. CONCLUSIONS: This study has confirmed the predicted high incidence of positively selected residues in rbcL in Ilex, and shown that this is higher than in any other plant lineage studied so far. The causes and consequences of this high incidence are unclear, but it is probably associated with the similarly high incidence of hybridization and introgression in Ilex, even between distantly related lineages, resulting in large cytonuclear discordance in the phylogenies.

Correction: Phylogeny, character evolution and spatiotemporal diversification of the species-rich and world-wide distributed tribe Rubieae (Rubiaceae).

[This corrects the article DOI: 10.1371/journal.pone.0207615.].

Phylogeny, character evolution and spatiotemporal diversification of the species-rich and world-wide distributed tribe Rubieae (Rubiaceae).

The Rubiaceae tribe Rubieae has a world-wide distribution with up to 1,000 species. These collectively exhibit an enormous ecological and morphological diversity, making Rubieae an excellent group for macro- and microevolutionary studies. Previous molecular phylogenetic analyses used only a limited sampling within the tribe or missed lineages crucial for understanding character evolution in this group. Here, we analyze sequences from two plastid spacer regions as well as morphological and biogeographic data from an extensive and evenly distributed sampling to establish a sound phylogenetic framework. This framework serves as a basis for our investigation of the evolution of important morphological characters and the biogeographic history of the Rubieae. The tribe includes three major clades, the Kelloggiinae Clade (Kelloggia), the Rubiinae Clade (Didymaea, Rubia) and the most species-rich Galiinae Clade (Asperula, Callipeltis, Crucianella, Cruciata, Galium, Mericarpaea, Phuopsis, Sherardia, Valantia). Within the Galiinae Clade, the largest genera Galium and Asperula are para- and polyphyletic, respectively. Smaller clades, however, usually correspond to currently recognized taxa (small genera or sections within genera), which may be used as starting points for a refined classification in this clade. Life-form (perennial versus annual), flower shape (long versus short corolla tube) and fruit characters (dry versus fleshy, with or without uncinate hairs) are highly homoplasious and have changed multiple times independently. Inference on the evolution of leaf whorls, a characteristic feature of the tribe, is sensitive to model choice. Multi-parted leaf whorls appear to have originated from opposite leaves with two small interpetiolar stipules that are subsequently enlarged and increased in number. Early diversification of Rubieae probably started during the Miocene in western Eurasia. Disjunctions between the Old and the New World possibly are due to connections via a North Atlantic land bridge. Diversification of the Galiineae Clade started later in the Miocene, probably in the Mediterranean, from where lineages reached, often multiple times, Africa, eastern Asia and further on the Americas and Australia.

The history of extant Ilex species (Aquifoliaceae): evidence of hybridization within a Miocene radiation.

The history and diversification of the genus Ilex (Aquifoliaceae), based on 108 different species (116 specimens), are inferred from the analysis of two nuclear (ITS and nepGS) and three plastid (rbcL, trnL-F and atpB-rbcL) sequences. Nuclear and plastid trees are highly incongruent and the nuclear tree is more compatible with current taxonomic classifications than the plastid one. The most recent common ancestor (MRCA) of extant species is dated from the Miocene, although the Ilex stem lineage can be traced back to the late Cretaceous, according to fossil records. This suggests extensive lineage extinctions between the Cretaceous and Miocene and may also explain the difficulties encountered in defining the relationships between Ilex and its closest relatives. The MRCA ancestral area was identified as being in the North Hemisphere (North America and/or East Asia). Several bidirectional North America/East Asia and North America/South America dispersal events are proposed to explain observed geographic and phylogenetic patterns. Hybridization and introgression events between distantly related lineages are also inferred, indicating weak reproductive barriers between species in Ilex.

Potential DNA transfer from the chloroplast to the nucleus in Eryngium alpinum.

Very divergent psbA-trnH chloroplast sequences were, for some Eryngiun alpinum individuals, repeatedly obtained and could not be attributed to contaminations nor to casual intraspecific variation. The design of external primers allowed the amplification of two different sequences for the same individuals. The divergent sequences were found to be more variable than their counterparts, to have a low GC content and to display a nonsynonymous substitution in psbA C-terminal region, all reasons that led us to hypothesize that they are paraloguous fragments transferred into the nucleus (NuPt). Quantitative polymerase chain reactions confirmed this hypothesis. Such NuPt might have severe implications in plant phylogeography and barcoding.

A plastid gene phylogeny of the non-photosynthetic parasitic Orobanche (Orobanchaceae) and related genera.

The phylogenetic relationships of the non-photosynthetic Orobanche sensu lato (Orobanchaceae), which includes some of the economically most important parasitic weeds, remain insufficiently understood and controversial. This concerns both the phylogenetic relationships within the genus, in particular its monophyly or lack thereof, and the relationships to other holoparasitic genera such as Cistanche or Conopholis. Here we present the first comprehensive phylogenetic study of this group based on a region from the plastid genome (rps2 gene). Although substitution rates appear to be elevated compared to the photosynthetic members of Orobanchaceae, relationships among the major lineages Cistanche, Conopholis plus Epifagus, Boschniakia rossica (Cham. & Schltdl.) B. Fedtsch., B. himalaica Hook. f. & Thomson, B. hookeri Walp. plus B. strobilacea A. Gray, and Orobanche s. l. remain unresolved. Resolution within Orobanche, however, is much better. In agreement with morphological, cytological and other molecular phylogenetic evidence, five lineages, corresponding to the four traditionally recognised sections (Gymnocaulis, Myzorrhiza, Orobanche, Trionychon) and O. latisquama Reut. ex Boiss. (of sect. Orobanche), can be distinguished. A combined analysis of plastid rps2 and nuclear ITS sequences of the holoparasitic genera results in more resolved and better supported trees, although the relationships among Orobanche s. l., Cistanche, and the clade including the remaining genera is unresolved. Therefore, rps2 is a marker from the plastid genome that is well-suited to be used in combination with other already established nuclear markers for resolving generic relationships of Orobanche and related genera.

Horizontal gene transfer of a plastid gene in the non-photosynthetic flowering plants Orobanche and Phelipanche (Orobanchaceae).

Plastid sequences are among the most widely used in phylogenetic and phylogeographic studies in flowering plants, where they are usually assumed to evolve like non-recombining, uniparentally transmitted, single-copy genes. Among others, this assumption can be violated by intracellular gene transfer (IGT) within cells or by the exchange of genes across mating barriers (horizontal gene transfer, HGT). We report on HGT of a plastid region including rps2, trnL-F, and rbcL in a group of non-photosynthetic flowering plants. Species of the parasitic broomrape genus Phelipanche harbor two copies of rps2, a plastid ribosomal gene, one corresponding to the phylogenetic position of the respective species, the other being horizontally acquired from the related broomrape genus Orobanche. While the vertically transmitted copies probably reside within the plastid genome, the localization of the horizontally acquired copies is not known. With both donor and recipient being parasitic plants, a possible pathway for the exchange of genetic material is via a commonly attacked host.

A fully automatable enzymatic method for DNA extraction from plant tissues.

BACKGROUND: DNA extraction from plant tissues, unlike DNA isolation from mammalian tissues, remains difficult due to the presence of a rigid cell wall around the plant cells. Currently used methods inevitably require a laborious mechanical grinding step, necessary to disrupt the cell wall for the release of DNA. RESULTS: Using a cocktail of different carbohydrases, a method was developed that enables a complete digestion of the plant cell walls and subsequent DNA release. Optimized conditions for the digestion reaction minimize DNA shearing and digestion, and maximize DNA release from the plant cell. The method gave good results in 125 of the 156 tested species. CONCLUSION: In combination with conventional DNA isolation techniques, the new enzymatic method allows to obtain high-yield, high-molecular weight DNA, which can be used for many applications, including genome characterization by AFLP, RAPD and SSR. Automation of the protocol (from leaf disks to DNA) is possible with existing workstations.

Are both sympatric species Ilex perado and Ilex canariensis secretly hybridizing? Indication from nuclear markers collected in Tenerife.

BACKGROUND: Intra-specific and intra-individual polymorphism is frequently observed in nuclear markers of Ilex (Aquifoliaceae) and discrepancy between plastid and nuclear phylogenies is the rule in this genus. These observations suggest that inter-specific plastid or/and nuclear introgression played an important role in the process of evolution of Ilex. With the aim of a precise understanding of the evolution of this genus, two distantly related sympatric species collected in Tenerife (Canary Islands), I. perado and I. canariensis, were studied in detail. Introgression between these two species was previously never reported. One plastid marker (the atpB-rbcL spacer) and two nuclear markers, the ribosomal internal transcribed spacer (ITS) and the nuclear encoded plastid glutamine synthetase (nepGS) were analyzed for 13 and 27 individuals of I. perado and I. canariensis, respectively. RESULTS: The plastid marker is intra-specifically constant and correlated with species identity. On the other hand, whereas the nuclear markers are conserved in I. perado, they are highly polymorphic in I. canariensis. The presence of pseudogenes and recombination in ITS sequences of I. canariensis explain this polymorphism. Ancestral sequence polymorphism with incomplete lineage sorting, or past or recent hybridization with an unknown species could explain this polymorphism, not resolved by concerted evolution. However, as already reported for many other plants, past or recent introgression of an alien genotype seem the most probable explanation for such a tremendous polymorphism. CONCLUSIONS: Data do not allow the determination with certitude of the putative species introgressing I. canariensis, but I. perado is suspected. The introgression would be unilateral, with I. perado as the male donor, and the paternal sequences would be rapidly converted in highly divergent and consequently unidentifiable pseudogenes. At least, this study allows the establishment of precautionary measures when nuclear markers are used in phylogenetic studies of genera having experienced introgression such as the genus Ilex.

Phylogeny and intraspecific variability of holoparasitic Orobanche (Orobanchaceae) inferred from plastid rbcL sequences.

The rbcL sequences of 106 specimens representing 28 species of the four recognized sections of Orobanche were analyzed and compared. Most sequences represent pseudogenes with premature stop codons. This study confirms that the American lineage (sects. Gymnocaulis and Myzorrhiza) contains potentially functional rbcL-copies with intact open reading frames and low rates of non-synonymous substitutions. For the first time, this is also shown for a member of the Eurasian lineage, O. coerulescens of sect. Orobanche, while all other investigated species of sects. Orobanche and Trionychon contain pseudogenes with distorted reading frames and significantly higher rates of non-synonymous substitutions. Phylogenetic analyses of the rbcL sequences give equivocal results concerning the monophyly of Orobanche, and the American lineage might be more closely related to Boschniakia and Cistanche than to the other sections of Orobanche. Additionally, species of sect. Trionychon phylogenetically nest in sect. Orobanche. This is in concordance with results from other plastid markers (rps2 and matK), but in disagreement with other molecular (nuclear ITS), morphological, and karyological data. This might indicate that the ancestor of sect. Trionychon has captured the plastid genome, or parts of it, of a member of sect. Orobanche. Apart from the phylogenetically problematic position of sect. Trionychon, the phylogenetic relationships within sect. Orobanche are similar to those inferred from nuclear ITS data and are close to the traditional groupings traditionally recognized based on morphology. The intraspecific variation of rbcL is low and is neither correlated with intraspecific morphological variability nor with host range. Ancestral character reconstruction using parsimony suggests that the ancestor of O. sect. Orobanche had a narrow host range.

The cpcB-cpcA locus as a tool for the genetic characterization of the genus Arthrospira (Cyanobacteria): evidence for horizontal transfer.

To investigate the genetic diversity of the genus Arthrospira and to compare it with other cyanobacteria, sequences of 670 nt from the phycocyanin operon were determined for 23 natural, cultivated or commercial strains of Arthrospira and compared with sequences from 20 other non-Arthrospira cyanobacterial strains. The sequenced DNA fragment comprises the last 255 nt of cpcB, the cpcB-cpcA spacer and the first 304 nt of cpcA. The resulting phylogenetic tree confirms that the genus Arthrospira is not related to Spirulina. So far, cpcB-cpcA data suggest that the closest relative of Arthrospira is Planktothrix. Based on this locus, the genus Arthrospira consists of three genetically clustered lineages. However, the distribution of nucleotide substitutions indicates that these three lineages are not the result of a simple cladogenesis characterized by the accumulation of independent substitutions. Instead, the observed clustering is the result of horizontal transfers of blocks of sequences. Analysis of the distribution of substitutions in the sequenced fragment indicates a point of intragenic recombination close to the stop codon of cpcB. The capacity of exchange of genetic material among strains probably explains why morphology and geographical origin do not correlate with the cpcB-cpcA clusters. Nevertheless, this study shows for the first time that the genus Arthrospira, represented here by cultivated and wild specimens, is clearly monophyletic. Moreover, the cpcB-cpcA DNA fragment, comprising both highly and moderately variable regions, allows (1) a strict differentiation of the taxon Arthrospira from other cyanobacteria (using the coding regions only) and (2) the study of relationships inside Arthrospira (using both the coding and non-coding regions).