Recent Loss of Self-Incompatibility by Degradation of the Male Component in Allotetraploid Arabidopsis kamchatica

The evolutionary transition from outcrossing to self-fertilization (selfing) through the loss of self-incompatibility (SI) is one of the most prevalent events in flowering plants, and its genetic basis has been a major focus in evolutionary biology. In the Brassicaceae, the SI system consists of male and female specificity genes at the S-locus and of genes involved in the female downstream signaling pathway. During recent decades, much attention has been paid in particular to clarifying the genes responsible for the loss of SI. Here, we investigated the pattern of polymorphism and functionality of the female specificity gene, the S-locus receptor kinase (SRK), in allotetraploid Arabidopsis kamchatica. While its parental species, A. lyrata and A. halleri, are reported to be diploid and mainly self-incompatible, A. kamchatica is self-compatible. We identified five highly diverged SRK haplogroups, found their disomic inheritance and, for the first time in a wild allotetraploid species, surveyed the geographic distribution of SRK at the two homeologous S-loci across the species range. We found intact full-length SRK sequences in many accessions. Through interspecific crosses with the self-incompatible and diploid congener A. halleri, we found that the female components of the SI system, including SRK and the female downstream signaling pathway, are still functional in these accessions. Given the tight linkage and very rare recombination of the male and female components on the S-locus, this result suggests that the degradation of male components was responsible for the loss of SI in A. kamchatica. Recent extensive studies in multiple Brassicaceae species demonstrate that the loss of SI is often derived from mutations in the male component in wild populations, in contrast to cultivated populations. This is consistent with theoretical predictions that mutations disabling male specificity are expected to be more strongly selected than mutations disabling female specificity, or the female downstream signaling pathway.     

Effective population size and tests of neutrality at cytoplasmic genes in Arabidopsis.

Cytoplasmic genomes typically lack recombination, implying that genetic hitch-hiking could be a predominant force structuring nucleotide polymorphism in the chloroplast and mitochondria. We test this hypothesis by analysing nucleotide polymorphism data at 28 loci across the chloroplast and mitochondria of the outcrossing plant Arabidopsis lyrata, and compare patterns with multiple nuclear loci, and the highly selfing Arabidopsis thaliana. The maximum likelihood estimate of the ratio of effective population size at cytoplasmic relative to nuclear genes in A. lyrata does not depart from the neutral expectation of 0.5. Similarly, the ratio of effective size in A. thaliana is close to unity, the neutral expectation for a highly selfing species. The results are thus consistent with neutral organelle polymorphism in these species or with comparable effects of hitch-hiking in both cytoplasmic and nuclear genes, in contrast to the results of recent studies on gynodioecious taxa. The four-gamete test and composite likelihood estimation provide evidence for very low levels of recombination in the organelles of A. lyrata, although permutation tests do not suggest that adjacent polymorphic sites are more closely linked than more distant sites across the two genomes, suggesting that mutation hotspots or very low rates of gene conversion could explain the data.     

Evolution of the S-locus region in Arabidopsis relatives

The S locus, a single polymorphic locus, is responsible for self-incompatibility (SI) in the Brassicaceae family and many related plant families. Despite its importance, our knowledge of S-locus evolution is largely restricted to the causal genes encoding the S-locus receptor kinase (SRK) receptor and S-locus cysteine-rich protein (SCR) ligand of the SI system. Here, we present high-quality sequences of the genomic region of six S-locus haplotypes: Arabidopsis (Arabidopsis thaliana; one haplotype), Arabidopsis lyrata (four haplotypes), and Capsella rubella (one haplotype). We compared these with reference S-locus haplotypes of the self-compatible Arabidopsis and its SI congener A. lyrata. We subsequently reconstructed the likely genomic organization of the S locus in the most recent common ancestor of Arabidopsis and Capsella. As previously reported, the two SI-determining genes, SCR and SRK, showed a pattern of coevolution. In addition, consistent with previous studies, we found that duplication, gene conversion, and positive selection have been important factors in the evolution of these two genes and appear to contribute to the generation of new recognition specificities. Intriguingly, the inactive pseudo-S-locus haplotype in the self-compatible species C. rubella is likely to be an old S-locus haplotype that only very recently became fixed when C. rubella split off from its SI ancestor, Capsella grandiflora.     

Duplication of centromeric histone H3 (HTR12) gene in Arabidopsis halleri and A. lyrata, plant species with multiple centromeric satellite sequences

Arabidopsis halleri and lyrata have three different major centromeric satellite sequences, a unique finding for a diploid Arabidopsis species. Since centromeric histones coevolve with centromeric satellites, these proteins would be predicted to show signs of selection when new centromere satellites have recently arisen. We isolated centromeric protein genes from A. halleri and lyrata and found that one of them, HTR12 (CENP-A), is duplicated, while CENP-C is not. Phylogenetic analysis indicates that the HTR12 duplication occurred after these species diverged from A. thaliana. Genetic mapping shows that HTR12 copy B has the same genomic location as the A. thaliana gene; the other copy (A, at the other end of the same chromosome) is probably the new copy. To test for selection since the duplication, we surveyed diversity at both HTR12 loci within A. lyrata. Overall, there is no strong evidence for an "evolutionary arms race" causing multiple replacement substitutions. The A. lyrata HTR12B sequences fall into three classes of haplotypes, apparently maintained for a long time, but they all encode the same amino acid sequence. In contrast, HTR12A has low diversity, but many variants are amino acid replacements, possibly due to independent selective sweeps within populations of the species.     

Selection on amino acid substitutions in Arabidopsis

Studies of nucleotide diversity have found an excess of low-frequency amino acid polymorphisms segregating in Arabidopsis thaliana, suggesting a predominance of weak purifying selection acting on amino acid polymorphism in this inbreeding species. Here, we investigate levels of diversity and divergence at synonymous and nonsynonymous sites in 6 circumpolar populations of the outbreeding Arabidopsis lyrata and compare these results with A. thaliana, to test for differences in mutation and selection parameters across genes, populations, and species. We find that A. lyrata shows an excess of low-frequency nonsynonymous polymorphisms both within populations and species wide, consistent with weak purifying selection similar to the patterns observed in A. thaliana. Furthermore, nonsynonymous polymorphisms tend to be more restricted in their population distribution in A. lyrata, consistent with purifying selection preventing their geographic spread. Highly expressed genes show a reduced ratio of amino acid to synonymous change for both polymorphism and fixed differences, suggesting a general pattern of stronger purifying selection on high-expression proteins.     

Genome wide gene expression in artificially synthesized amphidiploids of Arabidopsis

The merging of two different genomes occurs during the formation of amphidiploids, and the merged regulatory networks have the potential to generate a new gene expression pattern. We examined the genome-wide gene expression of two newly synthesized amphidiploids between Arabidopsis thaliana and the related species Arabidopsis lyrata subsp. lyrata and Arabidopsis halleri subsp. gemmifera. 1,137 (4.7%) and 1,316 (5.4%) of probesets showed differential gene expression in A. thaliana-A. halleri and A. thaliana-A. lyrata hybrids respectively, compared to the mid parent value and of these, 489 were in common. Genes that differed in expression between the parental lines tended to have an expression level in both hybrids differing from the mid parent value. In contrast to protein coding genes, there is little differential expression of transposons. Genes in the categories of chloroplast-targeted and response to stress were overrepresented in the non-additively expressed genes in both amphidiploids. As these genes have the potential to contribute directly to the plant phenotype, we suggest that rapid changes of gene expression in amphidiploids might be important for producing greater biomass.     

Reticulate evolution in Thuja inferred from multiple gene sequences: implications for the study of biogeographical disjunction between eastern Asia and North America

The eastern Asia-North America disjunction is one of the most interesting biogeographical patterns, but its formation is still in much debate. Here nucleotide sequences of five cpDNA regions, nrDNA ITS and two low-copy nuclear genes (LEAFY, 4CL) were employed to reconstruct the phylogeny and to explore the historical biogeography of Thuja, a typical eastern Asia-North America disjunct genus. High topological discordance was observed between chloroplast and nuclear gene trees, even between different nuclear gene trees, suggesting that Thuja could have a reticulate evolutionary history due to multiple interspecific hybridization events. The eastern Asian species Thuja koraiensis might have obtained its chloroplast genome from the eastern North American species T. occidentalis by chloroplast capture, while the western North American species T. plicata is very likely to have inherited a recombinant cpDNA. Based on the phylogenetic analysis of multiple genes, DIVA-reconstruction of the distribution history, molecular clock estimation and fossil data, we inferred that Thuja could have originated from the high-latitude areas of North America in the Paleocene or earlier with subsequent expansion into eastern Asia through the Bering Land Bridge. The two eastern Asia species T. standishii and T. sutchuenensis have a sister relationship, and their split could have occurred in the Oligocene or early Miocene. In the present study, the selection of molecular markers in biogeographic studies was also discussed. Since most previous studies on the eastern Asia and North America disjunction are based on uniparentally inherited cpDNA and (or) directly sequenced nrDNA ITS data, the historical reticulate evolution in the studied groups might have been underestimated. Therefore, we suggest that multiple genes from different genomes, especially low-copy nuclear genes, be used in this research area in the future.     

The transition to self-compatibility in Arabidopsis thaliana and evolution within S-haplotypes over 10 Myr

A recent investigation found evidence that the transition of Arabidopsis thaliana from ancestral self-incompatibility (SI) to full self-compatibility occurred very recently and suggested that this occurred through a selective fixation of a nonfunctional allele (PsiSCR1) at the SCR gene, which determines pollen specificity in the incompatibility response. The main evidence is the lack of polymorphism at the SCR locus in A. thaliana. However, the nearby SRK gene, which determines stigma specificity in self-incompatible Brassicaceae species, has extremely high sequence diversity, with 3 very divergent SRK haplotypes, 2 of them present in multiple strains. Such high diversity is extremely unusual in this species, and it suggests the possibility that multiple, different SRK haplotypes may have been preserved from A. thaliana's self-incompatible ancestor. To study the evolution of S-haplotypes in the A. thaliana lineage, we searched the 2 most closely related Arabidopsis species Arabidopsis lyrata and Arabidopsis halleri, in which most populations have retained SI, and found SRK sequences corresponding to all 3 A. thaliana haplogroup sequences. Our molecular evolutionary analyses of these 3 S-haplotypes provide an independent estimate of the timing of the breakdown of SI and again exclude an ancient transition to selfing in A. thaliana. Comparing sequences of each of the 3 haplogroups between species, we find that 2 of the 3 SRK sequences (haplogroups A and B) are similar throughout their length, suggesting that little or no recombination with other SRK alleles has occurred since these species diverged. The diversity difference between the SCR and SRK loci in A. thaliana, however, suggests crossing-over, either within SRK or between the SCR and SRK loci. If the loss of SI involved fixation of the PsiSCR1 sequence, the exchange must have occurred during its fixation. Divergence between the species is much lower at the S-locus, compared with reference loci, and we discuss two contributory possibilities. Introgression may have occurred between A. lyrata and A. halleri and between their ancestral lineage and A. thaliana, at least for some period after their split. In addition, the coalescence times of sequences of individual S-haplogroups are expected to be less than those of alleles at non-S-loci.     

Chromosome arrangement and nuclear architecture but not centromeric sequences are conserved between Arabidopsis thaliana and Arabidopsis lyrata

In contrast to the situation described for mammals and Drosophila, chromosome territory (CT) arrangement and somatic homologous pairing in interphase nuclei of Arabidopsis thaliana (n = 5) are predominantly random except for a more frequent association of the chromosomes bearing a homologous nucleolus organizer region. To find out whether this chromosome arrangement is also characteristic for other species of the genus Arabidopsis, we investigated Arabidopsis lyrata ssp. lyrata (n = 8), one of the closest relatives of A. thaliana. First, we determined the size of each chromosome and chromosome arm, the sequence type of centromeric repeats and their distribution between individual centromeres and the position of the 5S/45S rDNA arrays in A. lyrata. Then we demonstrated that CT arrangement, homologous pairing and sister chromatid alignment of distinct euchromatic and/or heterochromatic regions within A. lyrata interphase nuclei are similar to that in A. thaliana nuclei. Thus, the arrangement of interphase chromosomes appears to be conserved between both taxa that diverged about 5 million years ago. Since the chromosomes of A. lyrata resemble those of the presumed ancestral karyotype, a similar arrangement of interphase chromosomes is also to be expected for other closely related diploid species of the Brassicaceae family.     

Contrasting patterns of nucleotide polymorphism at the alcohol dehydrogenase locus in the outcrossing Arabidopsis lyrata and the selfing Arabidopsis thaliana

Nucleotide variation at the alcohol dehydrogenase locus (Adh) was studied in the outcrossing Arabidopsis lyrata, a close relative of the selfing Arabidopsis thaliana. Overall, estimated nucleotide diversity in the North American ssp. lyrata and two European ssp. petraea populations was 0.0038, lower than the corresponding specieswide estimate for A. thaliana at the same set of nucleotide sites. The distribution of segregating sites across the gene differed between the two species. Estimated sequence diversity within an A. lyrata population with a large sample size (0.0023) was much higher than has previously been observed for A. thaliana. This North American population has an excess of sites at intermediate frequencies compared with neutral expectation (Tajima's D = 2.3, P < 0.005), suggestive of linked balancing selection or a recent population bottleneck. In contrast, an excess of rare polymorphisms has been found in A. thaliana. Polymorphism within A. lyrata and divergence from A. thaliana appear to be correlated across the Adh gene sequence. The geographic distribution of polymorphism was quite different from that of A. thaliana, for which earlier studies of several genes found low within-population nucleotide site polymorphism and no overall continental differentiation of variation despite large differences in site frequencies between local populations. Differences between the outcrossing A. lyrata and the selfing A. thaliana reflect the impact of differences in mating system and the influence of bottlenecks in A. thaliana during rapid colonization on DNA sequence polymorphism. The influence of additional variability-reducing mechanisms, such as background selection or hitchhiking, may not be discernible.     

Sequence diversification of 45S rRNA ITS, trnH-psbA spacer, and matK genic regions in several Allium species

Allium is a very diverse genus with over 600 species distributed worldwide. Haplotype analyses of 45S rRNA ITS, trnH-psbA spacer, and matK gene sequences in 9 Allium species were carried out, subsequent to which phylogenetic relations of the nine species were also analyzed. Of the three genes, the nuclear 45S rRNA ITS sequences showed the highest variation with one haplotype in each species. The other two chloroplast genes revealed that more than one haplotype was present in each species, and each haplotype was present in several of the species. In the matK gene, EcoRI restriction revealed heteroplasmy in which the functional gene retains the EcoRI recognition site while the nonfunctional, pseudogene does not. Phylogenetic patterns were not consistent among the haplotypes of the 45 rRNA ITS, trnH-psbA spacer, and matK genic regions. This phylogenetic incongruency might be due to the presence of multiple haplotypes in each of the chloroplast genes. However, the inconsistency of the phylogenetic relationships, based on the 45S rRNA ITS sequences makes a strong case for further analysis.     

Selection on rapidly evolving proteins in the Arabidopsis genome

Genes that have undergone positive or diversifying selection are likely to be associated with adaptive divergence between species. One indicator of adaptive selection at the molecular level is an excess of amino acid replacement fixed differences per replacement site relative to the number of synonymous fixed differences per synonymous site (omega = K(a)/K(s)). We used an evolutionary expressed sequence tag (EST) approach to estimate the distribution of omega among 304 orthologous loci between Arabidopsis thaliana and A. lyrata to identify genes potentially involved in the adaptive divergence between these two Brassicaceae species. We find that 14 of 304 genes (approximately 5%) have an estimated omega > 1 and are candidates for genes with increased selection intensities. Molecular population genetic analyses of 6 of these rapidly evolving protein loci indicate that, despite their high levels of between-species nonsynonymous divergence, these genes do not have elevated levels of intraspecific replacement polymorphisms compared to previously studied genes. A hierarchical Bayesian analysis of protein-coding region evolution within and between species also indicates that the selection intensities of these genes are elevated compared to previously studied A. thaliana nuclear loci.     

Subdivision and haplotype structure in natural populations of Arabidopsis lyrata

High levels of inbreeding are expected to cause a strong reduction in levels of genetic variability, effective recombination rates and in adaptation compared with related outcrossing populations. We examined patterns of DNA polymorphism at five nuclear loci and one chloroplast locus within and between four populations of the outcrossing plant Arabidopsis lyrata, a close relative of the highly self-fertilizing model species A. thaliana. The observed patterns are compared with species-wide polymorphism at orthologous loci, as well as within- and between-population patterns at other studied loci in A. thaliana. In addition to evidence for much higher average within-population diversity, species-wide levels of silent polymorphism are generally higher in A. lyrata than in A. thaliana, unlike the results from a previous study of the ADH locus. However, polymorphism is also low in the North American A. lyrata subspecies lyrata compared with the European subspecies petraea, suggesting either a population bottleneck in North American populations or recent admixture involving diverged European populations. Differentiation between the two subspecies is strong, although there are few fixed differences, suggesting that their isolation is recent. Estimates of intralocus recombination rates and analysis of haplotype structure in European A. lyrata populations indicate lower recombination than predicted based on the variability together with physical recombination rates estimated from A. thaliana. This may be due to strong population subdivision, or to recent departures from demographic equilibrium such as a bottleneck or population admixture. Alternatively, there may be consistently lower recombination rates in the outcrossing species. In contrast, estimates of recombination rates from species-wide samples of A. thaliana are close to the values expected assuming a high rate of self-fertilization. Complex population histories in both A. thaliana and A. lyrata complicate theoretical predictions and empirical tests of the effects of inbreeding on polymorphism and molecular evolution.     

Patterns of genetic variation suggest a single,ancient origin for the diploid hybrid species Helianthus paradoxus

Abstract.— Experimental and comparative evidence implies that homoploid hybrid speciation is a reproducible process, mediated in part by ecological selection. Here, molecular data from the chloroplast genome and 17 nuclear microsatellite loci were employed to determine whether a well-documented homoploid hybrid species, Helianthus paradoxus , has arisen multiple times. Helianthus paradoxus is ecologically divergent from its parental species, and has a disjunct geographic distribution consistent with multiple origins. The molecular data, however, strongly support a single hybrid origin. First, all sampled populations of H. paradoxus are fixed for a single chloroplast DNA (cpDNA) haplotype, whereas local populations of both parental species, H. annuus and H. petiolaris , have multiple cpDNA haplotypes. Second, H. paradoxus populations form a single, well-supported clade (99.8% bootstrap support) in a neighbor-joining tree based on microsatellite allele frequencies. The microsatellite data also tentatively place the origin of H. paradoxus between 75,000 years and 208,000 years before present, indicating that anthropogenic disturbance likely did not play a role in the formation of this species. Finally, the genetic structure of this species is not consistent with passive riparian dispersal, which has been suggested for other wetland plant species, but may be explained by dispersal mechanisms implicated for H. annuus , such as large migratory mammals.     

Balancing selection and low recombination affect diversity near the self-incompatibility loci of the plant Arabidopsis lyrata

The self-incompatibility (S-) locus region of plants in the Brassica family is a small genome region. In Arabidopsis lyrata, the S-genes, SRK and SCR, encode the functional female and pollen recognition proteins, which must be coadapted to maintain correct associations between the two component genes, and thus self-incompatibility (SI). Recombinants would be self-compatible and thus probably disadvantageous in self-incompatible species. Therefore, tight linkage between the two genes in incompatibility systems is predicted to evolve to avoid producing such recombinant haplotypes. The evolution of low recombination in S-locus regions has not been rigorously tested. To test whether these regions' per-nucleotide recombination rates differ from those elsewhere in the genome, and to investigate whether the A. lyrata S-loci have the predicted effect on diversity in their immediate genome region, we studied diversity in genes that are linked to the S-loci but are not involved in incompatibility and are not under balancing selection. Compared with other A. lyrata loci, genes linked to the S-loci have extraordinarily high polymorphism. Our estimated recombination in this region, from fitting a model of the effects of S-allele polymorphism on linked neutral sites, supports the hypothesis of locally suppressed recombination around the S-locus.     

Multilocus analysis of variation and speciation in the closely related species Arabidopsis halleri and A. lyrata

Nucleotide variation in eight effectively unlinked genes was surveyed in species-wide samples of the closely related outbreeding species Arabidopsis halleri and A. lyrata ssp. petraea and in three of these genes in A. lyrata ssp. lyrata and A. thaliana. Significant genetic differentiation was observed more frequently in A. l. petraea than in A. halleri. Average estimates of nucleotide variation were highest in A. l. petraea and lowest in A. l. lyrata, reflecting differences among species in effective population size. The low level of variation in A. l. lyrata is concordant with a bottleneck effect associated with its origin. The A. halleri/A. l. petraea speciation process was studied, considering the orthologous sequences of an outgroup species (A. thaliana). The high number of ancestral mutations relative to exclusive polymorphisms detected in A. halleri and A. l. petraea, the significant results of the multilocus Fay and Wu H tests, and haplotype sharing between the species indicate introgression subsequent to speciation. Average among-population variation in A. halleri and A. l. petraea was approximately 1.5- and 3-fold higher than that in the inbreeder A. thaliana. The detected reduction of variation in A. thaliana is less than that expected from differences in mating system alone, and therefore from selective processes related to differences in the effective recombination rate, but could be explained by differences in population structure.     

Selection at work in self-incompatible Arabidopsis lyrata: mating patterns in a natural population

Identification and characterization of the self-incompatibility genes in Brassicaceae species now allow typing of self-incompatibility haplotypes in natural populations. In this study we sampled and mapped all 88 individuals in a small population of Arabidopsis lyrata from Iceland. The self-incompatibility haplotypes at the SRK gene were typed for all the plants and some of their progeny and used to investigate the realized mating patterns in the population. The observed frequencies of haplotypes were found to change considerably from the parent generation to the offspring generation around their deterministic equilibria as determined from the known dominance relations among haplotypes. We provide direct evidence that the incompatibility system discriminates against matings among adjacent individuals. Multiple paternity is very common, causing mate availability among progeny of a single mother to be much larger than expected for single paternity.     

Confirmation of a natural hybrid species in Petrocosmea (Gesneriaceae) based on molecular and morphological evidence

A natural hybrid species in Petrocosmea named Longianthera in Yanshan County,Yunnan Province is confirmed for the first time based on molecular and morphological evidence.The character count procedure of the variable characters show that Longianthera populations are characteristic of the intermediate morphological traits between its putative parents Yanshan and Petrocosmea martinii.The nuclear ribosomal internal transcribed spacer region and three chloroplast regions of matK,trnL-F,and trnT-L are sequenced in the putativehybrid and the related species.Both alignment of DNA sequences and the phylogenetic trees could exclude all the other species in Petrocosmea as the parental species except for Yanshan and P martinii.Eight haplotypes in the 31 internal transcribed spacer sequences and six haplotypes in 42 cpDNA sequences were found from 14 individuals of Longianthera populations.The analyses of DNA sequences,haplotypes,and phylogenetic trees indicate that Longianthera is likely a hybrid species between its putative parents Yanshan and P martinii,in which Yanshan might be the most possible maternal parent.Several factors may contribute to the natural hybridization between these two parental species in Petrocosmea,such as the overlapped geographic distribution,habitats,flowering periods,and shared pollinators.Finally,the new species of Yanshan and the natural hybrid species of Longianthera are described.     

Self-incompatibility in the genus Arabidopsis: characterization of the S locus in the outcrossing A. lyrata and its autogamous relative A. thaliana

As a starting point for a phylogenetic study of self-incompatibility (SI) in crucifers and to elucidate the genetic basis of transitions between outcrossing and self-fertilizing mating systems in this family, we investigated the SI system of Arabidopsis lyrata. A. lyrata is an outcrossing close relative of the self-fertile A. thaliana and is thought to have diverged from A. thaliana approximately 5 million years ago and from Brassica spp 15 to 20 million years ago. Analysis of two S (sterility) locus haplotypes demonstrates that the A. lyrata S locus contains tightly linked orthologs of the S locus receptor kinase (SRK) gene and the S locus cysteine-rich protein (SCR) gene, which are the determinants of SI specificity in stigma and pollen, respectively, but lacks an S locus glycoprotein gene. As described previously in Brassica, the S haplotypes of A. lyrata differ by the rearranged order of their genes and by their variable physical sizes. Comparative mapping of the A. lyrata and Brassica S loci indicates that the S locus of crucifers is a dynamic locus that has undergone several duplication events since the Arabidopsis--Brassica split and was translocated as a unit between two distant chromosomal locations during diversification of the two taxa. Furthermore, comparative analysis of the S locus region of A. lyrata and its homeolog in self-fertile A. thaliana identified orthologs of the SRK and SCR genes and demonstrated that self-compatibility in this species is associated with inactivation of SI specificity genes.