Characterizing the mechanisms of reproductive isolation between lineages is key to determining how new species are formed and maintained. In flowering plants, interactions between the reproductive organs of the flower-the pollen and the pistil-serve as the last barrier to reproduction before fertilization. As such, these pollen-pistil interactions are both complex and important for determining a suitable mate. Here, we test whether differences in style length (a part of the pistil) generate a postmating prezygotic mechanical barrier between five species of perennial Phlox wildflowers with geographically overlapping distributions. We perform controlled pairwise reciprocal crosses between three species with long styles and two species with short styles to assess crossing success (seed set). We find that the heterospecific seed set is broadly reduced compared to conspecific cross success and reveal a striking asymmetry in heterospecific crosses between species with different style lengths. To determine the mechanism underlying this asymmetric reproductive isolating barrier, we assess pollen tube growth in vivo and in vitro. We demonstrate that pollen tubes of short-styled species do not grow long enough to reach the ovaries of long-styled species. We find that short-styled species also have smaller pollen and that both within- and between-species pollen diameter is highly correlated with pollen tube length. Our results support the hypothesis that the small pollen of short-styled species lacks resources to grow pollen tubes long enough to access the ovaries of the long-styled species, resulting in an asymmetrical, mechanical barrier to reproduction. Such reproductive isolating mechanisms, combined with additional pollen-pistil incompatibilities, may be particularly important for closely related species in geographic proximity that share pollinators.

Premise of research. Reinforcement is the process through which prezygotic reproductive barriers evolve in sympatry owing to selection against hybridization between co-occurring, closely related species. The role of self-fertilization in reinforcement and reproductive isolation is uncertain, in part because its efficiency as a barrier against heterospecific mating can depend on the timing of autonomous selfing. Methodology. To investigate whether increased autonomous selfing has evolved as a mechanism for reinforcement, we compared Phlox cuspidata populations across their native Texas range using both estimates of genetic diversity and experimental manipulation with morphological measurements. Specifically, we investigated patterns of variation in floral traits and timing of selfing between individuals from allopatric populations of P. cuspidata and from populations sympatric with the closely related species Phlox drummondii. Pivotal results. We infer intermediate rates of selfing across field-collected individuals with no significant difference between allopatric and sympatric populations. Among greenhouse-grown plants, we find no differences in timing of selfing or other floral traits, including anther dehiscence timing, anther-stigma distance, autonomous selfing rate, and self-seed count, between allopatric and sympatric populations. However, our statistical analyses indicate that P. cuspidata individuals sympatric with P. drummondii seem to have generally larger flowers compared with allopatric individuals. Conclusions. Despite strong evidence of costly hybridization with P. drummondii, we find no evidence of trait divergence due to reinforcement in P. cuspidata. Although we document nearly complete autonomous self-seed set in the greenhouse, estimates of selfing rates from genetic data imply that realized selfing is much lower in nature, suggesting an opportunity for reinforcing selection to act on this trait.

Genetic mechanisms to recognize and reject self-pollen (termed “self-incompatibility”) have evolved independently many times throughout flowering plant evolution. Of the four known mechanisms, each achieves self-incompatibility through distinct molecular pathways with a unique genetic basis, and most self-recognition systems remain uncharacterized. We combine classical genetic crosses with genomic tools to identify the genetic basis of a novel self-recognition mechanism in the wildflower, *Phlox drummondii*. We find no expression of known self-recognition genes (so called “S-loci”) in pistil transcriptomes, consistent with a currently uncharacterized self-recognition mechanism having evolved in the lineage containing *Phlox*. We use a bulk segregant analysis to map self-recognition to a single genomic region in two independent populations. Analyses of expression and polymorphism for genes within this region narrow our search to a single gene candidate. Allelic variation at this gene predicts cross-compatibility in a full diallel cross, providing further confirmation of its role in self-recognition. Our work identifies the genetic basis of a novel self-recognition system in flowering plants, adding to our understanding of the diverse mechanisms through which flowering plants achieve this evolutionarily significant phenotype.

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.

Non-neuronal cells are key to the complex cellular interplay that follows central nervous system insult. To understand this interplay, we generated a single-cell atlas of immune, glial and retinal pigment epithelial cells from adult mouse retina before and at multiple time points after axonal transection. We identified rare subsets in naive retina, including interferon (IFN)-response glia and border-associated macrophages, and delineated injury-induced changes in cell composition, expression programs and interactions. Computational analysis charted a three-phase multicellular inflammatory cascade after injury. In the early phase, retinal macroglia and microglia were reactivated, providing chemotactic signals concurrent with infiltration of CCR2+ monocytes from the circulation. These cells differentiated into macrophages in the intermediate phase, while an IFN-response program, likely driven by microglia-derived type I IFN, was activated across resident glia. The late phase indicated inflammatory resolution. Our findings provide a framework to decipher cellular circuitry, spatial relationships and molecular interactions following tissue injury. Benhar and colleagues provide an atlas of non-neuronal cells in the adult mouse retina at steady state and after optic nerve injury and identify key cellular and molecular events along the path of neuronal degeneration after injury.

Plant–pollinator interactions are major drivers of flowering plant evolution. There are numerous examples of associations between floral traits and diversification rate and of parallel evolution of floral characters in unrelated groups that use similar pollinator types (Kay and Sargent, 2009). These macroevolutionary patterns highlight pollinators as selective agents driving floral trait variation, reproductive isolation, and diversification. However, the role of pollinators is even more fundamental to the plant life cycle than this adaptative framework suggests—pollinators are key dispersal agents. As dispersal agents, pollinators may also drive the distribution of neutral genetic variation and population connectivity within plant species. Population genetic studies of both plants and their pollinators can offer novel insight into how pollinators move across the environment, clarifying how pollinators affect plant evolution at the population scale. Many plants rely on external vectors for pollen and seed dispersal. Data suggest that dispersal via seed is often limited, and the movement of pollen via pollinators represents a significant mechanism through which alleles move across the landscape (Ennos, 1994; Sork and Smouse, 2006; Browne and Karubian, 2018). Whether the distribution of genetic variation within a plant species reflects its pollinator's foraging characteristics has been of interest for decades (Loveless and Hamrick, 1984). Recent studies leverage sequence data to demonstrate that pollinator identity and mobility correlate with outcrossing rates, gene flow, and measures of genetic distance across plant lineages (Gamba and Muchhala, 2020; Wessinger, 2021). The notion that highly mobile pollinators carry pollen across long distances, while less-mobile pollinators move pollen locally offers a mechanism to explain these correlations (Schmidt-Lebuhn et al., 2019). Known as the pollen dispersal-dependent speciation hypothesis, this framework posits that differences in pollinator mobility may scale up to affect the likelihood of speciation and extinction (Harvey et al., 2019; Wessinger, 2021). While evidence supporting this hypothesis is growing, the mechanisms that link pollinator identity to patterns of plant diversity are unclear. If variation in pollinator mobility explains the correlation between pollinator identity and plant diversification, then the genetic structure within a plant species will reflect the movement pattern of its pollinator(s) across space. Testing this hypothesis will bring deeper insight into the microevolutionary processes underlying recognized patterns of pollinator-mediated plant diversification. Because pollinators are an important factor controlling gene dispersal in plants, an intuitive prediction is that plant population genetic structure will reflect pollinator movement patterns (Figure 1). Population genetic tools are useful to test this prediction because they can infer how pollinators are distributed across the landscape. The distribution of pollinators across a plant species’ range is broadly important for pollen dispersal and subsequent gene flow. By pairing population genetic studies of plants and their pollinators, we can explore how plant population structure reflects (1) variation in pollinator visitation frequency, (2) the pattern of pollinator movement among populations, and (3) pollinator responses to the environment. Differences in pollinator visitation frequency to each plant population will affect how genetic variation is distributed within and between populations. Enhanced pollinator attraction will increase the likelihood of pollen movement between a wider number of individuals within and between populations (Harder and Barrett, 1996). For individuals in populations that receive relatively few pollinator visits (particularly from distant populations), the opportunity for diverse mating events is limited. As a result, inbreeding is more likely. Over time, inbreeding reduces the effective population size and can promote population differentiation (Charlesworth, 2003). Simultaneously, individuals in populations for which pollination events are rare will have infrequent opportunities for pollen receipt and/or export between individuals in other populations. Particularly when pollination occurs across short distances, these populations will tend to be more genetically isolated. Additionally, the pattern of pollinator trips among populations will affect plant genetic structure. Because plants are sessile, we might assume that genetic structure reflects geographic location, therefore following a pattern of isolation by distance (Loveless and Hamrick, 1984; Cruzan and Hendrickson, 2020). However, when pollinators repeatedly move between spatially distant populations, these populations will be more genetically similar than predicted by geographic distance (Sork and Smouse, 2006). For example, land-use has fragmented populations of the prairie endemic, Oenothera harringtonii, and yet, measures of genetic distance suggest that hawkmoth pollinators maintain population connectivity (Skogen et al., 2019). In this system, pollinator movement defines dispersal and contributes to plant genetic structure beyond isolation by distance. Finally, patterns of pollinator response to the environment will affect pollen dispersal and plant population structure. Pollinators are distributed heterogeneously due to a variety of factors including territoriality and ecological adaptation. Landscape genetic distance/resistance approaches infer how environmental variables affect pollinator gene flow and can also identify landscape factors influencing pollen dispersal (Cruzan and Hendrickson, 2020; Emel et al., 2021). Whether landscape genetic factors affecting pollinator genetic structure in turn affect the structure of plant species remains unexplored. Although these mechanisms intuitively link pollinator movement patterns to plant population structure, there are many reasons why plant structure may not reflect pollinator movement (Figure 1). Selection during the post-pollination and establishment phase may dominate in determining which alleles persist in a population and thus could obscure patterns of pollen flow. In some plant species, seed/propagule dispersal may contribute more than pollen dispersal to population connectivity (Nazareno et al., 2021). For plant species using multiple pollinators (generalists), the individual impact of each pollinator may be swamped by a collective effect not attributable to any given pollinator. Alternatively, the effects of each pollinator type may be observable at different spatial scales or geographic regions. Pollinator behaviors including grooming or territoriality can impact pollen dispersal probabilities, breaking the connection between pollinator movement and realized pollen flow. Therefore, exploring the connection between plant and pollinator genetic structure remains an exciting future direction. To determine whether and how plant population structure responds to pollinator movement in any specific biological system, two patterns must be described: (1) patterns of pollen movement by pollinators and (2) the distribution of genetic variation within the plant species. Pairing population genetic data of plants and their pollinators can achieve both goals. Historical approaches for characterizing pollinator-mediated pollen dispersal include direct observations (e.g., Levin and Kerster, 1974), pollinator tagging, or the use of pollen grain analogues (e.g., fluorescing quantum dots), which have the additional advantage of incorporating variation in pollen pickup/deposition (e.g., Schmidt-Lebuhn et al., 2019). Looking to the future, we advocate for studies using population genetics of pollinators to reveal their geographic distribution and infer movement patterns. While habitat and/or mating preferences likely also contribute to genetic structure, genetic measures of pollinator population connectivity infer how frequently individuals move between populations and across geography (Lowe and Allendorf, 2010). When these movements correlate with foraging, pollinator genetic structure will reveal potential patterns of pollen flow. Given the growing accessibility of genomic sequencing and advances in analytic methods, population genetic tools are increasingly tractable for exploring pollinator movement across the environment. However, population genetic studies remain rare for many pollinator types, with a notable dearth of data on insect pollinators. Particularly when paired with behavioral observations, population genetic data can be a powerful tool to connect pollinator movement, realized pollen flow, and plant population structure. To explore the effect of pollinator movement on plant population structure, researchers can apply a variety of approaches using genetic data. Plant parentage analyses and assignment tests can reveal pollen movement within a reproductive season (Sork and Smouse, 2006; Bode et al., 2018). Estimates of genetic distance between plant populations describe the cumulative impact of pollen dispersal and gene flow across generations (Cruzan and Hendrickson, 2020). Using pollinator movement patterns as an explanatory variable for plant genetic structure, researchers can then quantitatively test the prediction that pollinators affect the distribution of genetic variation within a plant species beyond isolation by distance (as is done with other environmental variables; e.g., Bradburd et al., 2013). Quantifying the impact of pollinator movement on plant genetic structure will expand our understanding of the ecological and evolutionary consequences of plant–pollinator interactions and generate predictions that can inform species management. If pollinators provide key dispersal services, the predicted impacts of pollinator loss will affect not only plant reproductive output but also population connectivity. Environmental disturbances affecting pollinators will also affect plant population structure. Alternatively, if habitat disturbance fragments plant populations, mobile pollinators may maintain gene flow. Emphasizing pollinators as dispersal vectors links patterns of pollinator driven diversification at the macroevolutionary scale to within species demographic processes (Harvey et al., 2019). When plant genetic structure reflects pollinator movement, pollinator-mediated gene flow may contribute to the likelihood of population isolation and species extinction, persistence, or divergence. Although the relationship between dispersal dynamics, metapopulation structure, and diversification is complex, growing evidence supports an association across diverse taxonomic groups (Harvey et al., 2019; Wessinger et al., 2019; Wessinger, 2021). While pollinator-mediated selection on adaptive floral traits is certainly important to flowering plant evolution, investigating pollinators as drivers of plant population structure can offer an additional path towards reconciling macroevolutionary patterns of floral diversity with microevolutionary processes. G.B. and R.H. contributed equally to conceptualization, drafting, and writing. We thank all those who reviewed drafts of this essay including Austin Garner, Andrea Berardi, and Jacob Suissa. In particular, we thank Pamela Diggle for the opportunity to write this essay and for her helpful comments. The feedback of two anonymous reviewers greatly improved this essay and we thank them for their time and consideration. Work for this essay was supported by NSF DEB-1844906 to R.H.

Genome-wide association studies (GWAS) have revealed risk alleles for ulcerative colitis (UC). To understand their cell type specificities and pathways of action, we generate an atlas of 366,650 cells from the colon mucosa of 18 UC patients and 12 healthy individuals, revealing 51 epithelial, stromal, and immune cell subsets, including BEST4+ enterocytes, microfold-like cells, and IL13RA2+IL11+ inflammatory fibroblasts, which we associate with resistance to anti-TNF treatment. Inflammatory fibroblasts, inflammatory monocytes, microfold-like cells, and T cells that co-express CD8 and IL-17 expand with disease, forming intercellular interaction hubs. Many UC risk genes are cell type specific and co-regulated within relatively few gene modules, suggesting convergence onto limited sets of cell types and pathways. Using this observation, we nominate and infer functions for specific risk genes across GWAS loci. Our work provides a framework for interrogating complex human diseases and mapping risk variants to cell types and pathways.

Christopher S. Smillie1,19, Moshe Biton1,2,19, José Ordovas-Montañes1,3,4,5,6,7,19, Keri M. Sullivan8, Grace Burgin1, Daniel B. Graham2,8,9,10,11, Rebecca H. Herbst1,12, Noga Rogel1, Michal Slyper1, Julia Waldman1, Malika Sud1, Elizabeth Andrews8, Gabriella Velonias8, Adam L. Haber1, Karthik Jagadeesh1, Sanja Vickovic1, Junmei Yao14, Christine Stevens9, Danielle Dionne1, Lan T. Nguyen1, Alexandra-Chloé Villani1,13, Matan Hofree1, Elizabeth A. Creasey14, Hailiang Huang15,16, Orit Rozenblatt-Rosen1, John J. Garber8, Hamed Khalili8, A. Nicole Desch9,14, Mark J. Daly15,16,17, Ashwin N. Ananthakrishnan8,*, Alex K. Shalek1,3,4,5,6,*, Ramnik J. Xavier2,8,9,10,11,14,*, Aviv Regev1,18,20,*

This is a pretreatment for RNAscope optimized for fixed frozen retina sections (emphasizes adhesion of tissue to the slides)

To isolate immune and epithelial cells from human colon biopsies

In the small intestine, a cellular niche of diverse accessory cell types supports the rapid generation of mature epithelial cell types through self-renewal, proliferation, and differentiation of intestinal stem cells (ISCs). However, not much is known about interactions between immune cells and ISCs, and it is unclear if and how immune cell dynamics affect eventual ISC fate or the balance between self-renewal and differentiation. Here, we used single-cell RNA-seq (scRNA-Seq) of intestinal epithelial cells (IECs) to identify new mechanisms for ISC–immune cell interactions. Surprisingly, MHC class II (MHCII) is enriched in two distinct subsets of Lgr5+ crypt base columnar ISCs, which are also distinguished by higher proliferation rates. Using co-culture of T cells with intestinal organoids, cytokine stimulations, and in vivo mouse models, we confirm that CD4+ T helper (Th) cells communicate with ISCs and affect their differentiation, in a manner specific to the Th subtypes and their signature cytokines and dependent on MHCII expression by ISCs. Specific inducible knockout of MHCII in intestinal epithelial cells in mice in vivo results in expansion of the ISC pool. Mice lacking T cells have expanded ISC pools, whereas specific depletion of Treg cells in vivo results in substantial reduction of ISC numbers. Our findings show that interactions between Th cells and ISCs mediated via MHCII expressed in intestinal epithelial stem cells help orchestrate tissue-wide responses to external signals.

To isolate epithelial cells for sorting from the mouse small intestine

To isolate immune cells from a mouse spleen before sorting

The airways of the lung are the primary sites of disease in asthma and cystic fibrosis. Here we study the cellular composition and hierarchy of the mouse tracheal epithelium by single-cell RNA-sequencing (scRNA-seq) and in vivo lineage tracing. We identify a rare cell type, the Foxi1 + pulmonary ionocyte; functional variations in club cells based on their location; a distinct cell type in high turnover squamous epithelial structures that we term ‘hillocks’; and disease-relevant subsets of tuft and goblet cells. We developed ‘pulse-seq’, combining scRNA-seq and lineage tracing, to show that tuft, neuroendocrine and ionocyte cells are continually and directly replenished by basal progenitor cells. Ionocytes are the major source of transcripts of the cystic fibrosis transmembrane conductance regulator in both mouse ( Cftr ) and human ( CFTR ). Knockout of Foxi1 in mouse ionocytes causes loss of Cftr expression and disrupts airway fluid and mucus physiology, phenotypes that are characteristic of cystic fibrosis. By associating cell-type-specific expression programs with key disease genes, we establish a new cellular narrative for airways disease.

Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens.