RNase-based self-incompatibility in cacti.

Approximately one-half of all flowering plants express genetically based physiological mechanisms that prevent self-fertilisation. One such mechanism, termed RNase-based self-incompatibility, employs ribonucleases as the pistil component. Although it is widespread, it has only been characterised in a handful of distantly related families, partly due to the difficulties presented by life history traits of many plants, which complicate genetic research. Many species in the cactus family are known to express self-incompatibility but the underlying mechanisms remain unknown. We demonstrate the utility of a candidate-based RNA-seq approach, combined with some unusual features of self-incompatibility-causing genes, which we use to uncover the genetic basis of the underlying mechanisms. Specifically, we assembled transcriptomes from Schlumbergera truncata (crab cactus or false Christmas cactus), and interrogated them for tissue-specific expression of candidate genes, structural characteristics, correlation with expressed phenotype(s), and phylogenetic placement. The results were consistent with operation of the RNase-based self-incompatibility mechanism in Cactaceae. The finding yields additional evidence that the ancestor of nearly all eudicots possessed RNase-based self-incompatibility, as well as a clear path to better conservation practices for one of the most charismatic plant families.

Biosystematic studies on the status of Solanum chilense.

PREMISE: Common taxonomic practices, which condition species' descriptions on diagnostic morphological traits, may systematically lump outcrossing species and unduly split selfing species. Specifically, higher effective population sizes and genetic diversity of obligate outcrossers are expected to result less reliable phenotypic diagnoses. Wild tomatoes, members of Solanum sect. Lycopersicum, are commonly used as a source of exotic germplasm for improvement of the cultivated tomato, and are increasingly employed in basic research. Although the section experienced significant early work, which continues presently, the taxonomic status of many wild species has undergone a number of significant revisions and remains uncertain. Species in this section vary in their breeding systems, notably the expression of self-incompatibility, which determines individual propensity for outcrossing METHODS: Here, we examine the taxonomic status of obligately outcrossing Chilean wild tomato (Solanum chilense) using reduced-representation sequencing (RAD-seq), a range of phylogenetic and population genetic analyses, as well as analyses of crossing and morphological data. RESULTS: Overall, each of our analyses provides a considerable weight of evidence that the Pacific coastal populations and Andean inland populations of the currently described Solanum chilense represent separately evolving populations, and conceal at least one undescribed cryptic species. CONCLUSIONS: Despite its vast economic importance, Solanum sect. Lycopersicon still exhibits considerable taxonomic instability. A pattern of under-recognition of outcrossing species may be common, not only in tomatoes, but across flowering plants. We discuss the possible causes and implications of this observation, with a focus on macroevolutionary inference.

Interaction among ploidy, breeding system and lineage diversification.

If particular traits consistently affect rates of speciation and extinction, broad macroevolutionary patterns can be interpreted as consequences of selection at high levels of the biological hierarchy. Identifying traits associated with diversification rates is difficult because of the wide variety of characters under consideration and the statistical challenges of testing for associations from comparative phylogenetic data. Ploidy (diploid vs polyploid states) and breeding system (self-incompatible vs self-compatible states) are both thought to be drivers of differential diversification in angiosperms. We fit 29 diversification models to extensive trait and phylogenetic data in Solanaceae and investigate how speciation and extinction rate differences are associated with ploidy, breeding system, and the interaction between these traits. We show that diversification patterns in Solanaceae are better explained by breeding system and an additional unobserved factor, rather than by ploidy. We also find that the most common evolutionary pathway to polyploidy in Solanaceae occurs via direct breakdown of self-incompatibility by whole genome duplication, rather than indirectly via breakdown followed by polyploidization. Comparing multiple stochastic diversification models that include complex trait interactions alongside hidden states enhances our understanding of the macroevolutionary patterns in plant phylogenies.

Tip-dating and the origin of Telluraves.

Despite a relatively vast accumulation of molecular data, the timing of diversification of modern bird lineages remains elusive. Accurate dating of the origination of Telluraves-a clade of birds defined by their arboreality-is of particular interest, as it contains the most species-rich avian group, the passerines. Historically, neontological studies have estimated a Cretaceous origin for the group, but more recent studies have recovered Cenozoic dates, closer to the oldest known fossils for the group. We employ total-evidence dating to estimate divergence times that are expected to be both less sensitive to prior assumptions and more accurate. Specifically, we use a large collection of morphological character data from arboreal bird fossils, along with combined molecular sequence and morphological character data from extant taxa. Our analyses recover a Late Cretaceous origin for crown Telluraves, with a few lineages crossing the K-Pg boundary. Following the K-Pg boundary, our results show the group underwent rapid diversification, likely benefiting from increased ecological opportunities in the aftermath of the extinction event. We find very little confidence for the precise topological placement of many extinct taxa, possibly due to rapid diversification, paucity of character data, and rapid morphological differentiation during the early history of the group.

Self-compatibility is over-represented on islands.

Because establishing a new population often depends critically on finding mates, individuals capable of uniparental reproduction may have a colonization advantage. Accordingly, there should be an over-representation of colonizing species in which individuals can reproduce without a mate, particularly in isolated locales such as oceanic islands. Despite the intuitive appeal of this colonization filter hypothesis (known as Baker's law), more than six decades of analyses have yielded mixed findings. We assembled a dataset of island and mainland plant breeding systems, focusing on the presence or absence of self-incompatibility. Because this trait enforces outcrossing and is unlikely to re-evolve on short timescales if it is lost, breeding system is especially likely to reflect the colonization filter. We found significantly more self-compatible species on islands than mainlands across a sample of > 1500 species from three widely distributed flowering plant families (Asteraceae, Brassicaceae and Solanaceae). Overall, 66% of island species were self-compatible, compared with 41% of mainland species. Our results demonstrate that the presence or absence of self-incompatibility has strong explanatory power for plant geographical patterns. Island floras around the world thus reflect the role of a key reproductive trait in filtering potential colonizing species in these three plant families.

The scope of Baker's law.

Baker's law refers to the tendency for species that establish on islands by long-distance dispersal to show an increased capacity for self-fertilization because of the advantage of self-compatibility when colonizing new habitat. Despite its intuitive appeal and broad empirical support, it has received substantial criticism over the years since it was proclaimed in the 1950s, not least because it seemed to be contradicted by the high frequency of dioecy on islands. Recent theoretical work has again questioned the generality and scope of Baker's law. Here, we attempt to discern where the idea is useful to apply and where it is not. We conclude that several of the perceived problems with Baker's law fall away when a narrower perspective is adopted on how it should be circumscribed. We emphasize that Baker's law should be read in terms of an enrichment of a capacity for uniparental reproduction in colonizing situations, rather than of high selfing rates. We suggest that Baker's law might be tested in four different contexts, which set the breadth of its scope: the colonization of oceanic islands, metapopulation dynamics with recurrent colonization, range expansions with recurrent colonization, and colonization through species invasions.

Is self-fertilization an evolutionary dead end?

A compound hypothesis positing that self-fertilization is an evolutionary dead end conflates two distinct claims: the transition from outcrossing to selfing is unidirectional; and the diversification rate, or the balance of the speciation and extinction rate, is negative for selfing species. Both claims have enjoyed widespread informal support for decades, but have recently come under suspicion. Sources of data that apparently contradict strongly asymmetric mating system transitions often rely on statistical phylogenetic tests plagued by profound flaws. Although recently developed models mend preceding approaches, they have been employed sparingly, and many problems remain. Theoretical investigations, genetic data and applications of new phylogenetic methods provide indirect support for an association of selfing with negative diversification rates. We lack direct tests of reversals from selfing to outcrossing, and require data concerning the genetic basis and complexity of independently evolved outcrossing adaptations. The identification of the mechanisms that limit the longevity of selfing lineages has been difficult. Limitations may include brief and variable durations of selfing lineages, as well as ongoing difficulties in relating additive genetic and nucleotide variation. Furthermore, a common line of evidence for the stability of mixed mating - based simply on its frequent occurrence - is misleading. We make specific suggestions for research programs that aim to provide a richer understanding of mating system evolution and seriously challenge Stebbins' venerable hypothesis.

kakapo: easy extraction and annotation of genes from raw RNA-seq reads.

kakapo (kakapo) is a Python-based pipeline that allows users to extract and assemble one or more specified genes or gene families. It flexibly uses original RNA-seq read or GenBank SRA accession inputs without performing global assembly of entire transcriptomes or metatranscriptomes. The pipeline identifies open reading frames in the assembled gene transcripts and annotates them. It optionally filters raw reads for ribosomal, plastid, and mitochondrial reads, or reads belonging to non-target organisms (e.g., viral, bacterial, human). kakapo can be employed for targeted assembly, to extract arbitrary loci, such as those commonly used for phylogenetic inference in systematics or candidate genes and gene families in phylogenomic and metagenomic studies. We provide example applications and discuss how its use can offset the declining value of GenBank's single-gene databases and help assemble datasets for a variety of phylogenetic analyses.

Changes at a Critical Branchpoint in the Anthocyanin Biosynthetic Pathway Underlie the Blue to Orange Flower Color Transition in Lysimachia arvensis.

Anthocyanins are the primary pigments contributing to the variety of flower colors among angiosperms and are considered essential for survival and reproduction. Anthocyanins are members of the flavonoids, a broader class of secondary metabolites, of which there are numerous structural genes and regulators thereof. In western European populations of Lysimachia arvensis, there are blue- and orange-petaled individuals. The proportion of blue-flowered plants increases with temperature and daylength yet decreases with precipitation. Here, we performed a transcriptome analysis to characterize the coding sequences of a large group of flavonoid biosynthetic genes, examine their expression and compare our results to flavonoid biochemical analysis for blue and orange petals. Among a set of 140 structural and regulatory genes broadly representing the flavonoid biosynthetic pathway, we found 39 genes with significant differential expression including some that have previously been reported to be involved in similar flower color transitions. In particular, F3'5'H and DFR, two genes at a critical branchpoint in the ABP for determining flower color, showed differential expression. The expression results were complemented by careful examination of the SNPs that differentiate the two color types for these two critical genes. The decreased expression of F3'5'H in orange petals and differential expression of two distinct copies of DFR, which also exhibit amino acid changes in the color-determining substrate specificity region, strongly correlate with the blue to orange transition. Our biochemical analysis was consistent with the transcriptome data indicating that the shift from blue to orange petals is caused by a change from primarily malvidin to largely pelargonidin forms of anthocyanins. Overall, we have identified several flavonoid biosynthetic pathway loci likely involved in the shift in flower color in L. arvensis and even more loci that may represent the complex network of genetic and physiological consequences of this flower color polymorphism.

A 15-Myr-old genetic bottleneck.

Balancing selection preserves variation at the self-incompatibility locus (S-locus) of flowering plants for tens of millions of years, making it possible to detect demographic events that occurred prior to the origin of extant species. In contrast to other Solanaceae examined, SI species in the sister genera Physalis and Witheringia share restricted variation at the S-locus. This restriction is indicative of an ancient bottleneck that occurred in a common ancestor. We sequenced 14 S-alleles from the subtribe Iochrominae, a group that is sister to the clade containing Physalis and Witheringia. At least 6 ancient S-allele lineages are represented among these alleles, demonstrating that the Iochrominae taxa do not share the restriction in S-locus diversity. Therefore, the bottleneck occurred after the divergence of the Iochrominae from the lineage leading to the most recent common ancestor of Physalis and Witheringia. Using cpDNA sequences, 3 fossil dates, and a Bayesian-relaxed molecular clock approach, the crown group of Solanaceae was estimated to be 51 Myr old and the restriction of variation at the S-locus occurred 14.0-18.4 Myr before present. These results confirm the great age of polymorphism at the S-locus and the utility of loci under balancing selection for deep historical inference.

On phylogenetic tests of irreversible evolution.

"Dollo's law" states that, following loss, a complex trait cannot reevolve in an identical manner. Although the law has previously fallen into disrepute, it has only recently been challenged with statistical phylogenetic methods. We employ simulation studies of an irreversible binary character to show that rejections of Dollo's law based on likelihood-ratio tests of transition rate constraints or on reconstructions of ancestral states are frequently incorrect. We identify two major causes of errors: incorrect assignment of root state frequencies, and neglect of the effect of the character state on rates of speciation and extinction. Our findings do not necessarily overturn the conclusions of phylogenetic studies claiming reversals, but we demonstrate devastating flaws in the methods that are the foundation of all such studies. Furthermore, we show that false rejections of Dollo's law can be reduced by the use of appropriate existing models and model selection procedures. More powerful tests of irreversibility require data beyond phylogenies and character states of extant taxa, and we highlight empirical work that incorporates additional information.

The evolutionary history of plant T2/S-type ribonucleases.

A growing number of T2/S-RNases are being discovered in plant genomes. Members of this protein family have a variety of known functions, but the vast majority are still uncharacterized. We present data and analyses of phylogenetic relationships among T2/S-RNases, and pay special attention to the group that contains the female component of the most widespread system of self-incompatibility in flowering plants. The returned emphasis on the initially identified component of this mechanism yields important conjectures about its evolutionary context. First, we find that the clade involved in self-rejection (class III) is found exclusively in core eudicots, while the remaining clades contain members from other vascular plants. Second, certain features, such as intron patterns, isoelectric point, and conserved amino acid regions, help differentiate S-RNases, which are necessary for expression of self-incompatibility, from other T2/S-RNase family members. Third, we devise and present a set of approaches to clarify new S-RNase candidates from existing genome assemblies. We use genomic features to identify putative functional and relictual S-loci in genomes of plants with unknown mechanisms of self-incompatibility. The widespread occurrence of possible relicts suggests that the loss of functional self-incompatibility may leave traces long after the fact, and that this manner of molecular fossil-like data could be an important source of information about the history and distribution of both RNase-based and other mechanisms of self-incompatibility. Finally, we release a public resource intended to aid the search for S-locus RNases, and help provide increasingly detailed information about their taxonomic distribution.

The distribution of plant mating systems: study bias against obligately outcrossing species.

Early models of plant mating-system evolution argued that predominant outcrossing and selfing are alternative stable states. At least for animal-pollinated species, recent summaries of empirical studies have suggested the opposite-that outcrossing rates do not show the expected bimodal distribution. However, it is generally accepted that several potential biases can affect conclusions from surveys of published outcrossing rates. Here, we examine one potential bias and find that published studies of outcrossing rates contain far fewer obligate outcrossers than expected. We approximate the magnitude of this study bias and present the distribution of outcrossing rates after compensating for it. Because this study examines only one potential bias, and finds it to be large, conclusions regarding either the frequency of mixed mating or the shape of the distribution of outcrossing rates in nature are premature.

Association of ploidy and sexual system in Lycium californicum (Solanaceae).

In North American Lycium (Solanaceae), the evolution of gender dimorphism has been proposed as a means of restoring outcrossing after polyploidization causes the loss of self-incompatibility. Previous studies of this process in Lycium focused on comparisons between species that differ in ploidy. We examined intraspecific variation in floral morphology and DNA content in populations of L. californicum to determine correlations between sexual system and cytotype. We also used nuclear ITS and GBSSI sequence data to determine whether diploid and polyploid forms represent the same phylogenetic species, and the phylogeographic relationships among populations and ploidy levels. Within populations, no variation in ploidy was found, although among populations there was a perfect correspondence between sexual system and cytotype. Diploid populations were all hermaphroditic, whereas tetraploid populations were all gender dimorphic. There was no clear geographic pattern to the occurrence of diploid and tetraploid forms. Phylogenetic analysis confirms that L. californicum, regardless of ploidy, forms a monophyletic group within the genus Lycium. Sequences from diploid and polyploid individuals did not form reciprocally monophyletic clades, indicating either multiple gains of polyploidy, ongoing gene flow between cytotypes, or lack of lineage sorting since the evolution of polyploidy. The correspondence between ploidy and sex expression is consistent with the hypothesis that polyploidization triggers the evolution of gender dimorphism in this and other Lycium species.

Recurrent modification of floral morphology in heterantherous Solanum reveals a parallel shift in reproductive strategy.

Floral morphology determines the pattern of pollen transfer within and between individuals. In hermaphroditic species, the spatial arrangement of sexual organs influences the rate of self-pollination as well as the placement of pollen in different areas of the pollinator's body. Studying the evolutionary modification of floral morphology in closely related species offers an opportunity to investigate the causes and consequences of floral variation. Here, we investigate the recurrent modification of flower morphology in three closely related pairs of taxa in Solanum section Androceras (Solanaceae), a group characterized by the presence of two morphologically distinct types of anthers in the same flower (heteranthery). We use morphometric analyses of plants grown in a common garden to characterize and compare the changes in floral morphology observed in parallel evolutionary transitions from relatively larger to smaller flowers. Our results indicate that the transition to smaller flowers is associated with a reduction in the spatial separation of anthers and stigma, changes in the allometric relationships among floral traits, shifts in pollen allocation to the two anther morphs and reduced pollen : ovule ratios. We suggest that floral modification in this group reflects parallel evolution towards increased self-fertilization and discuss potential selective scenarios that may favour this recurrent shift in floral morphology and function.

Tempo and mode in plant breeding system evolution.

Classic questions about trait evolution-including the directionality of character change and its interactions with lineage diversification-intersect in the study of plant breeding systems. Transitions from self-incompatibility to self-compatibility are frequent, and they may proceed within a species ("anagenetic" mode of breeding system change) or in conjunction with speciation events ("cladogenetic" mode of change). We apply a recently developed phylogenetic model to the nightshade family Solanaceae, quantifying the relative contributions of these two modes of evolution along with the tempo of breeding system change, speciation, and extinction. We find that self-incompatibility, a genetic mechanism that prevents self-fertilization, is lost largely by the cladogenetic mode. Self-compatible species are thus more likely to arise from the isolation of a newly self-compatible population than from species-wide fixation of self-compatible mutants. Shared polymorphism at the locus that governs self-incompatibility shows it to be ancestral and not regained within this family. We demonstrate that failing to account for cladogenetic character change misleads phylogenetic tests of evolutionary irreversibility, both for breeding system in Solanaceae and on simulated trees.

The expression of self-incompatibility in angiosperms is bimodal.

Self-incompatibility is expressed by nearly one-half of all angiosperms. A large proportion of the remaining species are self-compatible, and they either outcross using various contrivances or self-fertilize to some extent. Because of the common occurrence of populations and individuals with intermediate levels of self-incompatibility, categorization of the expression of self-incompatibility as an approximately binary trait has become controversial. We collect a widely reported index (index of self-incompatibility [ISI]) used to asses the strength and variation of self-incompatibility from over 1200 angiosperm taxa. Its distribution is bimodal and positively associated with outcrossing rate, albeit with a weak relationship within self-compatible taxa. A substantial fraction of species has intermediate mean values of ISI. Their occurrence can be caused by segregating ephemeral self-compatible mutations, averaging artifacts, and experimental biases, in addition to the often invoked stabilizing selection acting on the expression of self-incompatibility. Selection may also generally favor taxa with high ISI values through increased lineage birth and death rates, and it may counter lower level selection advantages within taxa expressing intermediate and low values of ISI. Such a null hypothesis is nearly universally overlooked, despite the fact that it could adequately explain the observed distribution of mating and breeding systems.

Comparative evidence for the correlated evolution of polyploidy and self-compatibility in Solanaceae.

Breakdown of self-incompatibility occurs repeatedly in flowering plants with important evolutionary consequences. In plant families in which self-incompatibility is mediated by S-RNases, previous evidence suggests that polyploidy may often directly cause self-compatibility through the formation of diploid pollen grains. We use three approaches to examine relationships between self-incompatibility and ploidy. First, we test whether evolution of self-compatibility and polyploidy is correlated in the nightshade family (Solanaceae), and find the expected close association between polyploidy and self-compatibility. Second, we compare the rate of breakdown of self-incompatibility in the absence of polyploidy against the rate of breakdown that arises as a byproduct of polyploidization, and we find the former to be greater. Third, we apply a novel extension to these methods to show that the relative magnitudes of the macroevolutionary pathways leading to self-compatible polyploids are time dependent. Over small time intervals, the direct pathway from self-incompatible diploids is dominant, whereas the pathway through self-compatible diploids prevails over longer time scales. This pathway analysis is broadly applicable to models of character evolution in which sequential combinations of rates are compared. Finally, given the strong evidence for both irreversibility of the loss of self-incompatibility in the family and the significant association between self-compatibility and polyploidy, we argue that ancient polyploidy is highly unlikely to have occurred within the Solanaceae, contrary to previous claims based on genomic analyses.

Species selection maintains self-incompatibility.

Identifying traits that affect rates of speciation and extinction and, hence, explain differences in species diversity among clades is a major goal of evolutionary biology. Detecting such traits is especially difficult when they undergo frequent transitions between states. Self-incompatibility, the ability of hermaphrodites to enforce outcrossing, is frequently lost in flowering plants, enabling self-fertilization. We show, however, that in the nightshade plant family (Solanaceae), species with functional self-incompatibility diversify at a significantly higher rate than those without it. The apparent short-term advantages of potentially self-fertilizing individuals are therefore offset by strong species selection, which favors obligate outcrossing.