Jean-François Manen

Phylogeny and classification of the subfamily Rubioideae (Rubiaceae)

We performed phylogenetic analyses of the subfamily Rubioideae (Rubiaceae) based on three different pieces of chloroplast DNA, the protein codingrbcL gene, the spacer sequence betweenatpB andrbcL (atpB-rbcL), and the recently published (Andersson and Rova 1999)rps16 intron data. NewrbcL sequences have been produced for 41 taxa and there are 52 newatpB-rbcL spacer sequences. All analyses gave similar results concerning the phylogeny, but they differ slightly in resolution and support for the various branches. The minor tribes Ophiorrhizeae, Urophylleae, Lasiantheae, and Coussareeae form a grade to the rest of the subfamily, which consists of two well-supported branches, the Psychotrieae alliance and the Spermacoceae alliance, including a majority of all genera and species. Based on the resulting phylogenies we present a revised classification of the Rubioideae. We accept 16 tribes of which 12 more or less correspond to earlier tribal circumscriptions: Anthospermeae, Argostemmateae, Craterispermeae, Gaertnereae, Morindeae, Paederieae, Psychotrieae, Schradereae, Spermacoceae, Rubieae, Theligoneae, and Urophylleae; two tribes have received new and very different circumscriptions: Ophiorrhizeae and Coussareeae; and two are new to science: Lasiantheae and Danaideae.

Phylogeny ofRubiaceae-Rubieae inferred from the sequence of a cpDNA intergene region

A phylogenetic analysis of 25 species, representing eight genera of theRubieae tribe (Rubiaceae), has been made using the DNA sequence of the chloroplastatp B-rbc L intergene region. Six tropical genera from other tribes ofRubiaceae have been used as outgroups. Whatever the method of analysis (distance, parsimony or maximum likelihood), five groups are clearly separated and described as informal clades. Their relative relationships are not clearly resolved by the parsimony analysis, resulting in eight equally parsimonious trees, 327 steps long, with a consistency index (CI) of 0.749 (excluding uninformative sites). TheRubieae tribe appears monophyletic from the data available. Some new and partly unexpected phylogenetic relationships are suggested. The genusRubia forms a separate clade and appears to be the relatively advanced sister group of the remaining taxa. TheSherardia clade also includes the generaCrucianella andPhuopsis. Galium sect.Aparinoides appears closely attached to theAsperula sect.Glabella clade. The remaining taxa ofGalium are paraphyletic:Galium sect.Platygalium (in theCruciata clade) is linked to the advanced generaCruciata andValantia; the more apomorphic groups ofGalium form theGalium sect.Galium clade, including the perennial sectionsGalium, Leiogalium, andLeptogalium as well as the annual (and possibly polyphyletic) sect.Kolgyda.

The use of herbarium specimens in DNA phylogenetics: Evaluation and improvement

During the last few years we have been confronted with the need to use herbarium specimens in the molecular phylogeny studies, since it is generally difficult to obtain living material of some rare species. Ancient DNA has been sequenced, and there are also reports on successful DNA amplification from herbarium specimens. However, it is not easy to obtain amplified DNA from the first herbarium sample tested. In this paper, experiments are described about trials of DNA amplification from two to 151-year-old herbarium specimens of plant species we needed for our projects. Of the 17 herbarium samples tested only two allowed DNA amplification under standard DNA isolation conditions. Different types of PCR inhibiting activities were demonstrated in DNA extracts. In some of the extracts there was extremely low concentration of template with satisfactory quality. In some instances, PCR inhibiting activities were successfully removed by treating them either with insoluble polyvinylpyrrolidone or by adding bovine serum albumin (BSA) to the amplification mixture. However, some PCR-inhibiting activities were resistant to the treatments described above. When the concentration of template was very low, a second PCR amplification with internal primers was necessary to increase the amount of DNA for sequencing. Nevertheless, contamination of either DNA extract or amplification mixture were sometimes observed, and consequently precautions were taken to minimize them. Finally, successful amplification was obtained in eight samples out of the 17 examined.

Comparison of the evolution of ribulose-1, 5-biphosphate carboxylase (rbcL) and atpB-rbcL noncoding spacer sequences in a recent plant group, the tribe Rubieae (Rubiaceae)

Plastid sequences of the atpB-rbcL spacer and rbcL gene itself were used to evaluate their respective potential in reconstructing the phylogeny of 15 taxa from the tribe Rubieae (Rubiaceae). From our previous analyses using the atpB-rbcL spacer, the 15 selected taxa represent most of the variability of the tribe. Since this group is considered to be relatively recent (Upper Tertiary), it should allow the study of early dynamics of nucleotide substitutions in recent divergences. The results show that the spacer and rbcL inferred phylogenies are not totally congruent; the spacer trees are more similar to interpretations of morphological data. A comparative analysis of the pattern of nucleotide substitution of these two sequences in the Rubieae shows that (1) the overall rate of substitution is similar in the spacer and in rbcL, and the rate of synonymous substitution in rbcL is much higher; (2) the level of homoplasy is higher in rbcL than in the spacer matrix which shows a higher phylogenetic structure; and (3) the pattern of transition and transversion substitutions is different in the two sequences, and is not linear in rbcL. As a result of these observations, we suggest that (1) the spacer is evolving relatively slowly because of unsuspected, and phylogenetically important; selective constraints on its sequence; and (2) in the rbcL sequence, many sites, free of constraint, are changing at high rate, and some of these sites seem to have undergone multiple substitutions even in this recent tribe. This could explain the high level of homoplasy found in Rubieae rbcL sequences.

A fully automatable enzymatic method for DNA extraction from plant tissues

BackgroundDNA 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.ResultsUsing 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.ConclusionIn 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.

The atpB and rbcL promoters in plastid DNAs of a wide dicot range.

The plastid atp B-rbcL intergene has been analyzed within a wide range of plants covering the major dicot lineages. New sequences from 13 plant species were determined and aligned with three already-known sequences. The promoters of the rbcL and the atpB genes were localized and analyzed according to published observations in spinach and tobacco. The evolutionary conservation of two atpB promoters, separated by 113–262 nucleotides, is strong support that both are functionally active, and it also allows a discrimination between the previously reported atpB transcripts. Moreover, the radically distinct sequences of the two atpB promoters suggest that they interact with two distinct initiation complexes. The alignment also confirms the much higher conservation of the leader sequence in the rbcL mRNA than in the atpB mRNA among dicots, presuming a function at the post-transcriptional level.

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.

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.

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

BackgroundIntra-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.ResultsThe 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.ConclusionsData 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.

Chloroplast DNA variation and parentage analysis in 55 apples

The chloroplastic atpB-rbcL spacer and the first 53 codons of the rbcL coding sequence was sequenced for 40 apple cultivars and 15 wild species. This chloroplast DNA region is 904 base pairs long, and only five mutations sites were found among the tested samples. Although the cpDNA variation was low, some parentages are proposed based on the maternal inheritance of plastid DNA: the male and female parents are specified, or else suggested, for Worcester, Discovery, Starking, Starkrimson, Kidds Orange Red, Priscilla, and Gloster, as well as for the putative wild origin for Malus x domestica.