Cristina P. Vieira

An S-RNase-based gametophytic self-incompatibility system evolved only once in eudicots.

It has been argued that the common ancestor of about 75% of all dicots possessed an S-RNase-based gametophytic self-incompatibility (GSI) system. S-RNase genes should thus be found in most plant families showing GSI. The S-RNase gene (or a duplicate) may also acquire a new function and thus genes belonging to the S-RNase lineage may also persist in plant families without GSI. Nevertheless, sequences that belong to the S-RNase lineage have been found in the Solanaceae, Scrophulariaceae, Rosaceae, Cucurbitaceae, and Fabaceae plant families only. Here we search for new sequences that may belong to the S-RNase lineage, using both a phylogenetic and a much faster and simpler amino acid pattern-based approach. We show that the two methods have an apparently similar false-negative rate of discovery (~10%). The amino acid pattern-based approach produces about 15% false positives. Genes belonging to the S-RNase lineage are found in three new plant families, namely, the Rubiaceae, Euphorbiaceae, and Malvaceae. Acquisition of a new function by genes belonging to the S-RNase lineage is shown to be a frequent event. A putative S-RNase sequence is identified in Lotus, a plant genus for which molecular studies on GSI are lacking. The hypothesis of a single origin for S-RNase-based GSI (before the split of the Asteridae and Rosidae) is further supported by the finding of genes belonging to the S-RNase lineage in some of the oldest lineages of the Asteridae and Rosidae, and by Baysean constrained tree analyses.

Different Positively Selected Sites at the Gametophytic Self-Incompatibility Pistil S-RNase Gene in the Solanaceae and Rosaceae (Prunus, Pyrus, and Malus)

In this work we perform a comparative study on the location of positively selected sites (those likely responsible for defining specificity differences) at the S-RNase gene, the pistil component of the gametophytic self-incompatibility system. For Plantaginaceae and Rosaceae (Prunus and Pyrus/Malus) this is the first study of this kind. A clear sign of positive selection was observed for 13, 17, and 27 amino acid sites in Solanaceae, Prunus, and Pyrus/Malus, respectively, using two different methodologies. In Plantaginaceae no clear positively selected sites were identified. Possible reasons for this result are discussed. Indirect experimental evidence suggests that the identified positively selected amino acid sites play a role in specificity determination. The percentage of positively selected sites is similar in Solanaceae and Rosaceae but the location of those sites is different.

Genetic and molecular characterization of three novel S-haplotypes in sour cherry (Prunus cerasus L.)

Tetraploid sour cherry (Prunus cerasus L.) exhibits gametophytic self-incompatibility (GSI) whereby the specificity of self-pollen rejection is controlled by alleles of the stylar and pollen specificity genes, S-RNase and SFB (S haplotype-specific F-box protein gene), respectively. As sour cherry selections can be either self-compatible (SC) or self-incompatible (SI), polyploidy per se does not result in SC. Instead the genotype-dependent loss of SI in sour cherry is due to the accumulation of non-functional S-haplotypes. The presence of two or more non-functional S-haplotypes within sour cherry 2x pollen renders that pollen SC. Two new S-haplotypes from sour cherry, S33 and S34, that are presumed to be contributed by the P. fruticosa species parent, the complete S-RNase and SFB sequences of a third S-haplotype, S35, plus the presence of two previously identified sweet cherry S-haplotypes, S14 and S16 are described here. Genetic segregation data demonstrated that the S16-, S33-, S34-, and S35-haplotypes present in sour cherry are fully functional. This result is consistent with our previous finding that ‘hetero-allelic’ pollen is incompatible in sour cherry. Phylogenetic analyses of the SFB and S-RNase sequences from available Prunus species reveal that the relationships among S-haplotypes show no correspondence to known organismal relationships at any taxonomic level within Prunus, indicating that polymorphisms at the S-locus have been maintained throughout the evolution of the genus. Furthermore, the phylogenetic relationships among SFB sequences are generally incongruent with those among S-RNase sequences for the same S-haplotypes. Hypotheses compatible with these results are discussed.

Evolutionary patterns at the RNase based gametophytic self - incompatibility system in two divergent Rosaceae groups (Maloideae and Prunus)

BackgroundWithin Rosaceae, the RNase based gametophytic self-incompatibility (GSI) system has been studied at the molecular level in Maloideae and Prunus species that have been diverging for, at least, 32 million years. In order to understand RNase based GSI evolution within this family, comparative studies must be performed, using similar methodologies.ResultIt is here shown that many features are shared between the two species groups such as levels of recombination at the S-RNase (the S-pistil component) gene, and the rate at which new specificities arise. Nevertheless, important differences are found regarding the number of ancestral lineages and the degree of specificity sharing between closely related species. In Maloideae, about 17% of the amino acid positions at the S-RNase protein are found to be positively selected, and they occupy about 30% of the exposed protein surface. Positively selected amino acid sites are shown to be located on either side of the active site cleft, an observation that is compatible with current models of specificity determination. At positively selected amino acid sites, non-conservative changes are almost as frequent as conservative changes. There is no evidence that at these sites the most drastic amino acid changes may be more strongly selected.ConclusionsMany similarities are found between the GSI system of Prunus and Maloideae that are compatible with the single origin hypothesis for RNase based GSI. The presence of common features such as the location of positively selected amino acid sites and lysine residues that may be important for ubiquitylation, raise a number of issues that, in principle, can be experimentally addressed in Maloideae. Nevertheless, there are also many important differences between the two Rosaceae GSI systems. How such features changed during evolution remains a puzzling issue.

Inferring the evolutionary history of Drosophila americana and Drosophila novamexicana using a multilocus approach and the influence of chromosomal rearrangements in single gene analyses

The evolutionary history of closely related organisms can prove sometimes difficult to infer. Hybridization and incomplete lineage sorting are the main concerns; however, genome rearrangements can also influence the outcome of analyses based on nuclear sequences. In the present study, DNA sequences from 12 nuclear genes, for which the approximate chromosomal locations are known, have been used to estimate the evolutionary history of two forms of Drosophila americana (Drosophila americana americana and Drosophila americana texana) and Drosophila novamexicana (virilis group of species). The phylogenetic analysis of the combined data set resulted in a phylogeny showing reciprocal monophyly for D. novamexicana and D. americana. Single gene analyses, however, resulted in incongruent phylogenies influenced by chromosomal rearrangements. Genetic differentiation estimates indicated a significant differentiation between the two species for all genes. Within D. americana, however, there is no evidence for differentiation between the chromosomal forms except at genes located near the X/4 fusion and Xc inversion breakpoint. Thus, the specific status of D. americana and D. novamexicana is confirmed, but there is no overall evidence for genetic differentiation between D. a. americana and D. a. texana, not supporting a subspecific status. Based on levels of allele and nucleotide diversity found in the strains used, it is proposed that D. americana has had a stable, large population during the recent past while D. novamexicana has speciated from a peripheral southwestern population having had an ancestral small effective population size. The influence of chromosomal rearrangements in single gene analyses is also examined.

Resolving the phylogenetic relationships and evolutionary history of the Drosophila virilis group using multilocus data

The Drosophila virilis group is one of the major lineages of Drosophila previously recognised and it has been used as a model for different types of studies. It comprises 13 species whose phylogenetic relationships are not well resolved. In the present study, six nuclear genes (Adh, fused, Gpdh, NonA, CG9631 and CG7219) and the mitochondrial ribosomal RNA genes (12S-16S) have been used to estimate the evolutionary tree of the group using different methods of phylogenetic reconstruction. Different competing evolutionary hypotheses have also been compared using the Approximately Unbiased test to further evaluate the robustness of the inferred trees. Results are, in general, consistent with previous studies in recovering the four major lineages of the group (D. virilis phylad, Drosophila montana subphylad, Drosophila kanekoi subphylad and Drosophila littoralis subphylad), although D. kanekoi, D. littoralis and Drosophila ezoana are here inferred to be more closely related to the D. virilis phylad than to the D. montana subphylad. The age of the crown group, estimated with a Bayesian method that assumes a relaxed molecular clock, is placed in the late Miocene (∼ 10 Mya). The oldest lineages also appeared during this period (∼ 7.5 to ∼ 8.9 Mya), while the ages of the basal nodes of the montana subphylad and the virilis phylad are located in the early Pliocene (∼ 4.9 and ∼ 4.1 Mya). Major cladogenesis events correlate to geological and palaeoclimatic occurrences that most likely affected the freshwater and deciduous forests where these species are found. The inferred biogeographical history of the group, based on the statistical dispersal-vicariance analysis, indicates that the last common ancestor of the group had a Holarctic distribution from which the North American and the Eurasian lineages evolved as a result of a vicariant event.

RNase -Based Gametophytic Self-Incompatibility Evolution: Questioning the Hypothesis of Multiple Independent Recruitments of the S -Pollen Gene

Multiple independent recruitments of the S-pollen component (always an F-box gene) during RNase-based gametophytic self-incompatibility evolution have recently been suggested. Therefore, different mechanisms could be used to achieve the rejection of incompatible pollen in different plant families. This hypothesis is, however, mainly based on the interpretation of phylogenetic analyses, using a small number of divergent nucleotide sequences. In this work we show, based on a large collection of F-box S-like sequences, that the inferred relationship of F-box S-pollen and F-box S-like sequences is dependent on the sequence alignment software and phylogenetic method used. Thus, at present, it is not possible to address the phylogenetic relationship of F-box S-pollen and S-like sequences from different plant families. In Petunia and Malus/Pyrus the putative S-pollen gene(s) show(s) variability patterns different than expected for an S-pollen gene, raising the question of false identification. Here we show that in Petunia, the unexpected features of the putative S-pollen gene are not incompatible with this gene’s being the S-pollen gene. On the other hand, it is very unlikely that the Pyrus SFBB-gamma gene is involved in specificity determination.

On the location of the gene(s) harbouring the advantageous variant that maintains the X/4 fusion of Drosophila americana

Weak selection is maintaining the Drosophila americana X/4 fusion chromosomal frequency cline. The gene(s) harbouring the advantageous variant(s) that is responsible for the establishment and maintenance of this chromosomal frequency gradient must be located in a region of the X and/or 4th chromosome that is genetically isolated between the X/4 fusion and non-fusion forms. The limits of these regions must thus be determined before an attempt is made to identify these genes. For this purpose, the correspondence between the D. virilis X and 4th chromosome genome scaffolds sequence and the D. americana gene order was established. Polymorphism levels and patterns at seven genes located at the base of the D. americana X chromosome, as well as three genes located at the base of the 4th chromosome, were analysed. The data suggest that the D. americana X/4 fusion is no more than 29,000 years old. At the base of the X chromosome, there is suppression of recombination within X/4 fusion and non-fusion chromosomes, and little recombination between the two chromosomal forms. Apparent fixed silent and replacement differences are found in three of seven genes analysed located at the base of the X chromosome. There is no evidence for suppression of recombination between fusion and non-fusion chromosomes at the base of the 4th chromosome. The advantageous variant responsible for the establishment in frequency and maintenance of the X/4 fusion is thus inferred to be in the D. americana X centromere-inversion Xc basal breakpoint region.

Convergent Evolution at the Gametophytic Self-Incompatibility System in Malus and Prunus

S-RNase-based gametophytic self-incompatibility (GSI) has evolved once before the split of the Asteridae and Rosidae. This conclusion is based on the phylogenetic history of the S-RNase that determines pistil specificity. In Rosaceae, molecular characterizations of Prunus species, and species from the tribe Pyreae (i.e., Malus, Pyrus, Sorbus) revealed different numbers of genes determining S-pollen specificity. In Prunus only one pistil and pollen gene determine GSI, while in Pyreae there is one pistil but multiple pollen genes, implying different specificity recognition mechanisms. It is thus conceivable that within Rosaceae the genes involved in GSI in the two lineages are not orthologous but possibly paralogous. To address this hypothesis we characterised the S-RNase lineage and S-pollen lineage genes present in the genomes of five Rosaceae species from three genera: M. × domestica (apple, self-incompatible (SI); tribe Pyreae), P. persica (peach, self-compatible (SC); Amygdaleae), P. mume (mei, SI; Amygdaleae), Fragaria vesca (strawberry, SC; Potentilleae), and F. nipponica (mori-ichigo, SI; Potentilleae). Phylogenetic analyses revealed that the Malus and Prunus S-RNase and S-pollen genes belong to distinct gene lineages, and that only Prunus S-RNase and SFB-lineage genes are present in Fragaria. Thus, S-RNase based GSI system of Malus evolved independently from the ancestral system of Rosaceae. Using expression patterns based on RNA-seq data, the ancestral S-RNase lineage gene is inferred to be expressed in pistils only, while the ancestral S-pollen lineage gene is inferred to be expressed in tissues other than pollen.

A comparative study of the short term cold resistance response in distantly related Drosophila species: the role of regucalcin and frost.

The molecular basis of short term cold resistance (indexed as chill-coma recovery time) has been mostly addressed in D. melanogaster, where candidate genes (Dca (also known as smp-30) and Frost (Fst)) have been identified. Nevertheless, in Drosophila, the ability to tolerate short term exposure to low temperatures evolved several times independently. Therefore, it is unclear whether variation in the same candidate genes is also responsible for short term cold resistance in distantly related Drosophila species. It should be noted that Dca is a candidate gene for cold resistance in the Sophophora subgenus only, since there is no orthologous gene copy in the Drosophila subgenus. Here we show that, in D. americana (Drosophila subgenus), there is a north-south gradient for a variant at the 5′ non-coding region of regucalcin (a Dca-like gene; in D. melanogaster the proteins encoded by the two genes share 71.9% amino acid identities) but in our D. americana F2 association experiment there is no association between this polymorphism and chill-coma recovery times. Moreover, we found no convincing evidence that this gene is up-regulated after cold shock in both D. americana and D. melanogaster. Size variation in the Fst PEST domain (putatively involved in rapid protein degradation) is observed when comparing distantly related Drosophila species, and is associated with short term cold resistance differences in D. americana. Nevertheless, this effect is likely through body size variation. Moreover, we show that, even at two hours after cold shock, when up-regulation of this gene is maximal in D. melanogaster (about 48 fold expression change), in D. americana this gene is only moderately up-regulated (about 3 fold expression change). Our work thus shows that there are important differences regarding the molecular basis of cold resistance in distantly related Drosophila species.