Phylogenomic analyses re-examine the evolution of reinforcement and hypothesized hybrid speciation in Phlox wildflowers.

The tree of life is riddled with reticulate evolutionary histories, and some clades, such as the eastern standing Phlox, appear to be hotspots of hybridization. In this group, there are two cases of reinforcement and nine hypothesized hybrid species. Given their historical importance in our understanding of plant speciation, the relationships between these taxa and the role of hybridization in their diversification require genomic validation. Using phylogenomic analyses, we resolve the evolutionary relationships of the eastern standing Phlox and evaluate hypotheses about whether and how hybridization and gene flow played a role in their diversification. Our results provide novel resolution of the phylogenetic relationships in this group, including paraphyly across some taxa. We identify gene flow during one case of reinforcement and find genomic support for a hybrid lineage underlying one of the five hypothesized homoploid hybrid speciation events. Additionally, we estimate the ancestries of four allotetraploid hybrid species. Our results are consistent with hybridization contributing to diverse evolutionary outcomes within this group; although, not as extensively as previously hypothesized. This study demonstrates the importance of phylogenomics in evaluating hypothesized evolutionary histories of non-model systems and adds to the growing support of interspecific genetic exchange in the generation of biodiversity.

Characterizing each step of pollination in Phlox drummondii reveals that a single butterfly species predominates in the pollinator assemblage.

PREMISE: A central goal of pollination biology is to connect plants with the identity of their pollinator(s). While predictions based on floral syndrome traits are extremely useful, direct observation can reveal further details of a species' pollination biology. The wildflower Phlox drummondii has a floral syndrome consistent with pollination by Lepidoptera. We tested this prediction using empirical data. METHODS: We observed each step of pollination in P. drummondii. First, we observed 55.5 h of floral visitation across the species range. We used temporal pollinator exclusion to determine the contribution of diurnal and nocturnal pollination to reproductive output. We then quantified P. drummondii pollen transfer by the dominant floral visitor, Battus philenor. Finally, we tested the effect of B. philenor visitation on P. drummondii reproduction by quantifying fruit set following single pollinator visits. RESULTS: Battus philenor is the primary pollinator of P. drummondii. Pollination is largely diurnal, and we observed a variety of lepidopteran visitors during the diurnal period. However, B. philenor was the most frequent visitor, representing 88.5% of all observed visits. Our results show that B. philenor is an extremely common visitor and also an effective pollinator by demonstrating that individuals transfer pollen between flowers and that a single visit can elicit fruit set. CONCLUSIONS: Our data are consistent with the prediction of lepidopteran pollination and further reveal a single butterfly species, B. philenor, as the primary pollinator. Our study demonstrates the importance of empirical pollinator observations, adds to our understanding of pollination mechanics, and offers a specific case study of butterfly pollination.

Flower orientation influences floral temperature, pollinator visits and plant fitness.

Effective insect pollination requires appropriate responses to internal and external environmental cues in both the plant and the pollinator. Helianthus annuus, a highly outcrossing species, is marked for its uniform eastward orientation of mature pseudanthia, or capitula. Here we investigate how this orientation affects floral microclimate and the consequent effects on plant and pollinator interactions and reproductive fitness. We artificially manipulated sunflower capitulum orientation and temperature in both field and controlled conditions and assessed flower physiology, pollinator visits, seed traits and siring success. East-facing capitula were found to have earlier style elongation, pollen presentation and pollinator visits compared with capitula manipulated to face west. East-facing capitula also sired more offspring than west-facing capitula and under some conditions produced heavier and better-filled seeds. Local ambient temperature change on the capitulum was found to be a key factor regulating the timing of style elongation, pollen emergence and pollinator visits. These results indicate that eastward capitulum orientation helps to control daily rhythms in floral temperature, with direct consequences on the timing of style elongation and pollen emergence, pollinator visitation, and plant fitness.

Genomic analyses overturn two long-standing homoploid hybrid speciation hypotheses.

The importance of hybridization in generating biological diversity has been historically controversial. Previously, inference about hybridization was limited by dependence on morphological data; with the advent of the next-generation sequencing tools for nonmodel organisms, the evolutionary significance of hybridization is more evident. Here, we test classic hypotheses of hybrid origins of two species in the Phlox pilosa complex. Morphological intermediacy motivated the hypotheses that Phlox amoena lighthipei and Phlox pilosa deamii were independent homoploid hybrid lineages derived from P. amoena amoena and P. pilosa pilosa. We use double-digest restriction site-associated DNA sequencing of individuals from throughout the range of these taxa to conduct the most thorough analysis of evolutionary history in this system to date. Surprisingly, we find no support for the hybrid origin of P. pilosa deamii or P. amoena lighthipei. Our data do identify a history of admixture in individuals collected at a contemporary hybrid zone between the putative parent lineages. We show that three very different evolutionary histories, only one of which involves hybrid origin, have produced intermediate or recombinant morphological traits between P. amoena amoena and P. pilosa pilosa. Although morphological data are still an efficient means of generating hypotheses about past gene flow, genomic data are now the standard of evidence for elucidating evolutionary history.

A Linkage-Based Genome Assembly for the Mosquito Aedes albopictus and Identification of Chromosomal Regions Affecting Diapause.

The Asian tiger mosquito, Aedes albopictus, is an invasive vector mosquito of substantial public health concern. The large genome size (~1.19-1.28 Gb by cytofluorometric estimates), comprised of ~68% repetitive DNA sequences, has made it difficult to produce a high-quality genome assembly for this species. We constructed a high-density linkage map for Ae. albopictus based on 111,328 informative SNPs obtained by RNAseq. We then performed a linkage-map anchored reassembly of AalbF2, the genome assembly produced by Palatini et al. (2020). Our reassembled genome sequence, AalbF3, represents several improvements relative to AalbF2. First, the size of the AalbF3 assembly is 1.45 Gb, almost half the size of AalbF2. Furthermore, relative to AalbF2, AalbF3 contains a higher proportion of complete and single-copy BUSCO genes (84.3%) and a higher proportion of aligned RNAseq reads that map concordantly to a single location of the genome (46%). We demonstrate the utility of AalbF3 by using it as a reference for a bulk-segregant-based comparative genomics analysis that identifies chromosomal regions with clusters of candidate SNPs putatively associated with photoperiodic diapause, a crucial ecological adaptation underpinning the rapid range expansion and climatic adaptation of A. albopictus.

Genomic Signatures of Reinforcement.

Reinforcement is the process by which selection against hybridization increases reproductive isolation between taxa. Much research has focused on demonstrating the existence of reinforcement, yet relatively little is known about the genetic basis of reinforcement or the evolutionary conditions under which reinforcement can occur. Inspired by reinforcement's characteristic phenotypic pattern of reproductive trait divergence in sympatry but not in allopatry, we discuss whether reinforcement also leaves a distinct genomic pattern. First, we describe three patterns of genetic variation we expect as a consequence of reinforcement. Then, we discuss a set of alternative processes and complicating factors that may make the identification of reinforcement at the genomic level difficult. Finally, we consider how genomic analyses can be leveraged to inform if and to what extent reinforcement evolved in the face of gene flow between sympatric lineages and between allopatric and sympatric populations of the same lineage. Our major goals are to understand if genome scans for particular patterns of genetic variation could identify reinforcement, isolate the genetic basis of reinforcement, or infer the conditions under which reinforcement evolved.

Circadian regulation of sunflower heliotropism, floral orientation, and pollinator visits.

Young sunflower plants track the Sun from east to west during the day and then reorient during the night to face east in anticipation of dawn. In contrast, mature plants cease movement with their flower heads facing east. We show that circadian regulation of directional growth pathways accounts for both phenomena and leads to increased vegetative biomass and enhanced pollinator visits to flowers. Solar tracking movements are driven by antiphasic patterns of elongation on the east and west sides of the stem. Genes implicated in control of phototropic growth, but not clock genes, are differentially expressed on the opposite sides of solar tracking stems. Thus, interactions between environmental response pathways and the internal circadian oscillator coordinate physiological processes with predictable changes in the environment to influence growth and reproduction.

Genetic loci with parent-of-origin effects cause hybrid seed lethality in crosses between Mimulus species.

In flowering plants, F1 hybrid seed lethality is a common outcome of crosses between closely related diploid species, but the genetic basis of this early-acting and potentially widespread form of postzygotic reproductive isolation is largely unknown. We intercrossed two closely related species of monkeyflower, Mimulus guttatus and Mimulus tilingii, to characterize the mechanisms and strength of postzygotic reproductive isolation. Then, using a reciprocal backcross design, we performed high-resolution genetic mapping to determine the genetic architecture of hybrid seed lethality and directly test for loci with parent-of-origin effects. We found that F1 hybrid seed lethality is an exceptionally strong isolating barrier between Mimulus species, with reciprocal crosses producing < 1% viable seeds. This form of postzygotic reproductive isolation appears to be highly polygenic, indicating that multiple incompatibility loci have accumulated rapidly between these closely related Mimulus species. It is also primarily caused by genetic loci with parent-of-origin effects, suggesting a possible role for imprinted genes in the evolution of Mimulus hybrid seed lethality. Our findings suggest that divergence in loci with parent-of-origin effects, which is probably driven by genomic coevolution within lineages, might be an important source of hybrid incompatibilities between flowering plant species.