Phenotypic Mosaicism Research Articles

Decoding the phenotypic mosaic of aging.

Decoding the phenotypic mosaic of aging.

Neutrophil Functionalities in Patients with Paroxysmal Nocturnal Haemoglobinuria Are Not Affected By Complement C5- Inhibition

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired haematological stem cell disorder characterized by haemolysis, thrombophilia, and cytopenia. PNH is caused by a somatic mutation in the phosphatidylinositol glycan class A (PIG-A) gene at the level of pluripotent, hematopoietic stem cells leading to a lack of GPI anchored cell surface proteins, such as complement-inactivating proteins, (e.g., CD55, CD59) on stem cells, polymorphonuclear neutrophil granulocytes (PMN), erythrocytes, and platelets. Subsequently, uncontrolled complement overactivity leads to the systemic complications. Phenotypic mosaicism is a distinct characteristic of PNH. For diagnostic purposes, the absence of the GPI-anchor on granulocytes and monocytes in the flow cytometry is considered the gold standard. However, the exact consequences of GPI deficiency in PMNs and monocytes have not been studied sufficiently yet. Since the implementation of complement inhibitors, the primary cause of morbidity and mortality in PNH has been significantly reduced. Despite this, the tendency towards thrombosis, as well as the consequences of the deficiency of GPI-linked proteins on PMNs and monocytes, remain largely unclear. Therefore, the main objective of this project is to gain a deeper understanding of the impact of PNH-PMNs and monocytes on thrombo-inflammation in PNH. Specifically, the impact of complement inhibitors on these factors has been compared. Blood from PNH patients (n=14) with a median clone size of 74 % were compared with an age- and sex-matched control group (n =20). Various cellular markers indicating a state of activation (CD11b, TREM1, CD64) or showing platelet adherence (CD42), were measured using flow cytometry. Unmutated PMNs and monocytes from PNH patients (PMN) served as intrinsic control, GPI positive PMNs (WT-PMN (PNH)) and monocytes from a control group served as extrinsic control. We compared unstimulated PNH and WT cells for their basal phenotype, and then functional changes upon stimulation (phorbol 12-myristate 13-acetate (PMA)). Our data present a contrary perspective to our initial assumptions, indicating that PNH-PMNs have a direct impact on unmutated PMNs, resulting in the generation of a thrombogenic state and increased levels of activation. The unmutated GPI-positive PMNs and monocytes exhibit a significantly stronger direct aggregation with platelets compared to GPI-deficient clones. It is hypothesized that the PNH clones influence the activity and aggregation propensity of the remaining unmutated granulocytes through additional cellular mechanisms. In a subgroup analysis, the impact of complement factor 5 inhibitors (C5i) on the aforementioned factors was investigated detecting no significant differences in terms of PMN activation or platelet aggregation. Thus, it can be hypothesized that thrombosis and hyper-inflammation are not solely driven by the PNH clones directly and modulated C5 inhibition. Additional cellular mechanisms and signaling pathways appear to play an important role, and these aspects have not been adequately addressed by the current therapy.

Metabolic heterogeneity of tissue-resident macrophages in homeostasis and during helminth infection

Tissue-resident macrophage populations constitute a mosaic of phenotypes, yet how their metabolic states link to the range of phenotypes and functions in vivo is still poorly defined. Here, using high-dimensional spectral flow cytometry, we observe distinct metabolic profiles between different organs and functionally link acetyl CoA carboxylase activity to efferocytotic capacity. Additionally, differences in metabolism are evident within populations from a specific site, corresponding to relative stages of macrophage maturity. Immune perturbation with intestinal helminth infection increases alternative activation and metabolic rewiring of monocyte-derived macrophage populations, while resident TIM4+ intestinal macrophages remain immunologically and metabolically hyporesponsive. Similar metabolic signatures in alternatively-activated macrophages are seen from different tissues using additional helminth models, but to different magnitudes, indicating further tissue-specific contributions to metabolic states. Thus, our high-dimensional, flow-based metabolic analyses indicates complex metabolic heterogeneity and dynamics of tissue-resident macrophage populations at homeostasis and during helminth infection.

Male Ejaculatory Endophenotypes: Revealing Internal Inconsistencies of the Concept in Heterosexual Copulating Rats.

Distinct manifestations of sexual behavior are conceived as separate phenotypes. Each sexual phenotype is assumed to be associated with a characteristic brain. These notions have justified the phenotyping of heterosexual copulator males based upon their ejaculation’s latencies (EL) or frequencies (i.e., cumulative ejaculation number; EN). For instance, men and male rats showing premature, normal or retarded ejaculation are assumed to be distinctive endophenotypes. This concept, nonetheless, contradicts past and recent evidence that supports that sexual behavior is highly variable within each sex, and that the brain sexual functional morphology represents an intricate sexual phenotypic mosaic. Hence, for ejaculatory male endophenotypes to be considered as a valid biological concept, it must show internal consistency at various levels of organization (including genetic architectures), after being challenged by intrinsic and/or extrinsic factors. We then judged the internal consistency of the presumed ejaculatory endophenotypes by assessing whether copulatory behavior and the expression of copulation relevant genes and brain limbic structures are specific to each of the presumed EL- or EN-ejaculatory endophenotypes. To do this, copulating male rats were first phenotyped in groups consistently displaying short, average or long ejaculation latencies or very high, high, average, low or very low EN, based in their copulatory performance. Then, the internal consistency of the presumed EL- or EN-endophenotypes was tested by introducing as covariates of phenotyping other copulatory parameters (e.g., number of intromissions) in addition to EL or EN, or by analyzing the expression levels of genes encoding for estrogen receptor alpha, progesterone receptor, androgen receptor, aromatase, DNA methyl-transferase 3a and DNA methyl-transferase 1 in the amygdala, medial preoptic area, ventromedial hypothalamus and olfactory bulb. We found that even though there were group-level differences in all the variables that were studied, these differences did not add-up to create the presumed EL- or EN-ejaculatory endophenotypes. In fact, the extensive overlapping of copulatory parameters and expression levels of copulation relevant genes in limbic structures across EL- or EN-phenotyped copulating male rats, is not consistent with the hypothesis that distinct ejaculatory endophenotypes exist and that they are associated with specific brain characteristics.

Phenomics-Based Quantification of CRISPR-Induced Mosaicism in Zebrafish.

Genetic mosaicism can manifest as spatially variable phenotypes that vary from site to site within an organism. Here, we use imaging-based phenomics to quantitate phenotypes at many sites within the axial skeleton of CRISPR-edited G0 zebrafish. Through characterization of loss-of-function cell clusters in the developing skeleton, we identify a distinctive size distribution shown to arise from clonal fragmentation and merger events. We quantitate the phenotypic mosaicism produced by somatic mutations of two genes, plod2 and bmp1a, implicated in human osteogenesis imperfecta. Comparison of somatic, CRISPR-generated G0 mutants to homozygous germline mutants reveals phenotypic convergence, suggesting that CRISPR screens of G0 animals can faithfully recapitulate the biology of inbred disease models. We describe statistical frameworks for phenomic analysis of spatial phenotypic variation present in somatic G0 mutants. In sum, this study defines an approach for decoding spatially variable phenotypes generated during CRISPR-based screens.

Morphological characterization of sympatric and allopatric populations of Petunia axillaris and P. exserta (Solanaceae)

AbstractFloral morphological traits are frequently used to identify species, including those that are closely related and show low genetic diversity, and floral shape and colour are known to play an important role in diversification and species isolation. Floral morphology in Petunia (Solanaceae) is considered a driver of diversification because of its association with pollinators. Here, flower morphology was characterized through morphometric analyses and floral pigments. Our main aim was to determine corolla shape in populations of Petunia axillaris and P. exserta and their natural hybrids and how floral display, size and colour are involved in pollinator attraction. In addition, we investigated floral pigments in P. exserta and different hybrid classes. The results from morphometric analyses revealed that each species has a specific floral shape, independent of the collection site. By contrast, in two contact zones, a mosaic of floral phenotypes was observed with some hybrid classes based on corolla colour being placed close to P. exserta. The results suggest that several generations of hybrids or backcrossing could have given rise to this floral diversity in contact zones.

Abstract 3777: Exploring phenotypic mosaicism in a mouse model of glioma towards understanding intratumoral heterogeneity

Abstract The lethality of glioblastoma multiforme (GBM) and the failure of treatment is largely attributable to the heterogeneous properties of this cancer. We aim specifically to characterize the driving event for GBMs to acquire a heterogeneous phenotype during progression. Central to achieving this goal is the ability to interrogate possible sources of heterogeneity that guide cellular states that are similar to those identified in human patients. We therefore employ a mouse model of glioma that recapitulates the pathophysiology and gene expression signatures of human GBM. While initiated with identical oncogenic drivers, this mouse model unexpectedly displays heterogeneous phenotypes between animals. Our preliminary data using time-series single-cell transcriptomics approach shows that transformed cells, even without acquiring additional genetic alterations, switch cellular states at the beginning of tumor formation and one population commit themselves to expand to establish gliomas that are mainly comprised of one cell type. We further observe the strong association of microenvironmental cues with glioma cell populations and their roles in the state transitioning of glioma cells. Through the characterization of mechanisms that drive phenotypic mosaicism, we hope to uncover general principles that govern tumor progression and heterogeneity, and then ultimately provide novel therapeutic strategies to cure GBM. Citation Format: Toshiro Hara. Exploring phenotypic mosaicism in a mouse model of glioma towards understanding intratumoral heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3777.

Parallel plumage colour evolution and introgressive hybridization in wheatears.

Genetic and phenotypic mosaics, in which various phenotypes and different genomic regions show discordant patterns of species or population divergence, offer unique opportunities to study the role of ancestral and introgressed genetic variation in phenotypic evolution. Here, we investigated the evolution of discordant phenotypic and genetic divergence in a monophyletic clade of four songbird taxa-pied wheatear (O.pleschanka), Cyprus wheatear (Oenanthe cypriaca), and western and eastern subspecies of black-eared wheatear (O.h.hispanica and O.h.melanoleuca). Phenotypically, black back and neck sides distinguish pied and Cyprus wheatears from the white-backed/necked black-eared wheatears. Meanwhile, mitochondrial variation only distinguishes western black-eared wheatear. In the absence of nuclear genetic data, and given frequent hybridization among eastern black-eared and pied wheatear, it remains unclear whether introgression is responsible for discordance between mitochondrial divergence patterns and phenotypic similarities, or whether plumage coloration evolved in parallel. Multispecies coalescent analyses of about 20,000 SNPs obtained from RAD data mapped to a draft genome assembly resolve the species tree, provide evidence for the parallel evolution of colour phenotypes and establish western and eastern black-eared wheatears as independent taxa that should be recognized as full species. The presence of the entire admixture spectrum in the Iranian hybrid zone and the detection of footprints of introgression from pied into eastern black-eared wheatear beyond the hybrid zone despite strong geographic structure of ancestry proportions furthermore suggest a potential role for introgression in parallel plumage colour evolution. Our results support the importance of standing heterospecific and/or ancestral variation in phenotypic evolution.

Intraspecific morphological divergence in two Cichlid species from Benin

Selection on morphological traits can vary across the range of species, inducing a mosaic of phenotypes across populations. Intraspecific morphological divergence had been demonstrated for many fish groups inhabiting environments with varying abiotic or biotic selective pressures. Such intraspecific phenotypic variation can have a strong influence on the ecologies of species. In the current study, we examined patterns of intraspecific morphological divergence between two populations of Sarotherodon melanotheron and Coptodon guineensis in Lake Ahémé and Porto-Novo lagoon, Benin. Using multiple morphological traits, we demonstrated intraspecific morphological divergence between Lake Ahémé and Porto-Novo lagoon for these species. However, evidence for parallel divergence was found for these two species, implying a similar response to selective pressures might have been acting on labile traits. In addition, species specific morphological changes observed in the current study might be because of differing responses to similar selective forces or taxon-specific selective forces acting on labile traits. The intraspecific trait divergence demonstrated in the current study suggests several possible selective pressures acting on these populations, yet the cause of this divergence remains unknown and additional studies are required to test these inferences.

Assessing Clinical Outcomes in Colorectal Cancer with Assays for Invasive Circulating Tumor Cells.

Colorectal carcinoma (CRC) is the second leading cause of cancer-related mortality. The goals of this study are to evaluate the association between levels of invasive circulating tumor cells (iCTCs) with CRC outcomes and to explore the molecular characteristics of iCTCs. Peripheral blood from 93 patients with Stage I–IV CRC was obtained and assessed for the detection and characterization of iCTCs using a functional collagen-based adhesion matrix (CAM) invasion assay. Patients were followed and assessed for overall survival. Tumor cells isolated by CAM were characterized using cell culture and microarray analyses. Of 93 patients, 88 (95%) had detectable iCTCs, ranging over 0–470 iCTCs/mL. Patients with Stage I–IV disease exhibited median counts of 0.0 iCTCs/mL (n = 6), 13.0 iCTCs/mL (n = 12), 41.0 iCTCs/mL (n = 12), and 133.0 iCTCs/mL (n = 58), respectively (p < 0.001). Kaplan–Meier curve analysis demonstrated a significant survival benefit in patients with low iCTC counts compared with in patients with high iCTC counts (log-rank p < 0.001). Multivariable Cox model analysis revealed that iCTC count was an independent prognostic factor of overall survival (p = 0.009). Disease stage (p = 0.01, hazard ratio 1.66; 95% confidence interval: 1.12–2.47) and surgical intervention (p = 0.03, HR 0.37; 95% CI: 0.15–0.92) were also independent prognostic factors. Gene expression analysis demonstrated the expression of both endothelial and tumor progenitor cell biomarkers in iCTCs. CAM-based invasion assay shows a high detection sensitivity of iCTCs that inversely correlated with overall survival in CRC patients. Functional and gene expression analyses showed the phenotypic mosaics of iCTCs, mimicking the survival capability of circulating endothelial cells in the blood stream.

Predator Perspective Drives Geographic Variation in Frequency-Dependent Polymorphism.

Color polymorphism in natural populations can manifest as a striking patchwork of phenotypes in space, with neighboring populations characterized by dramatic differences in morph composition. These geographic mosaics can be challenging to explain in the absence of localized selection because they are unlikely to result from simple isolation-by-distance or clinal variation in selective regimes. To identify processes that can lead to the formation of geographic mosaics, we developed a simulation-based model to explore the influence of predator perspective, selection, migration, and genetic linkage of color loci on allele frequencies in polymorphic populations over space and time. Using simulated populations inspired by the biology of Heliconius longwing butterflies, Cepaea land snails, Oophaga poison frogs, and Sonora ground snakes, we found that the relative sizes of predator and prey home ranges can produce large differences in morph composition between neighboring populations under both positive and negative frequency-dependent selection. We also demonstrated the importance of the interaction of predator perspective with the type of frequency dependence and localized directional selection across migration and selection intensities. Our results show that regional-scale predation can promote the formation of phenotypic mosaics in prey species, without the need to invoke spatial variation in selective regimes. We suggest that predator behavior can play an important and underappreciated role in the formation and maintenance of geographic mosaics in polymorphic species.

A role of stochastic phenotype switching in generating mosaic endothelial cell heterogeneity.

Previous studies have shown that biological noise may drive dynamic phenotypic mosaicism in isogenic unicellular organisms. However, there is no evidence for a similar mechanism operating in metazoans. Here we show that the endothelial-restricted gene, von Willebrand factor (VWF), is expressed in a mosaic pattern in the capillaries of many vascular beds and in the aorta. In capillaries, the mosaicism is dynamically regulated, with VWF switching between ON and OFF states during the lifetime of the animal. Clonal analysis of cultured endothelial cells reveals that dynamic mosaic heterogeneity is controlled by a low-barrier, noise-sensitive bistable switch that involves random transitions in the DNA methylation status of the VWF promoter. Finally, the hearts of VWF-null mice demonstrate an abnormal endothelial phenotype as well as cardiac dysfunction. Together, these findings suggest a novel stochastic phenotype switching strategy for adaptive homoeostasis in the adult vasculature.

Transcriptome sequencing of two phenotypic mosaic Eucalyptus trees reveals large scale transcriptome re-modelling.

Phenotypic mosaic trees offer an ideal system for studying differential gene expression. We have investigated two mosaic eucalypt trees from two closely related species (Eucalyptus melliodora and E. sideroxylon), which each support two types of leaves: one part of the canopy is resistant to insect herbivory and the remaining leaves are susceptible. Driving this ecological distinction are differences in plant secondary metabolites. We used these phenotypic mosaics to investigate genome wide patterns of foliar gene expression with the aim of identifying patterns of differential gene expression and the somatic mutation(s) that lead to this phenotypic mosaicism. We sequenced the mRNA pool from leaves of the resistant and susceptible ecotypes from both mosaic eucalypts using the Illumina HiSeq 2000 platform. We found large differences in pathway regulation and gene expression between the ecotypes of each mosaic. The expression of the genes in the MVA and MEP pathways is reflected by variation in leaf chemistry, however this is not the case for the terpene synthases. Apart from the terpene biosynthetic pathway, there are several other metabolic pathways that are differentially regulated between the two ecotypes, suggesting there is much more phenotypic diversity than has been described. Despite the close relationship between the two species, they show large differences in the global patterns of gene and pathway regulation.

Age-associated stresses induce an anti-inflammatory senescent phenotype in endothelial cells

Age is the greatest risk factor for cardiovascular disease. In addition, inflammation and age (senescence) have been linked at both the clinical and molecular levels. In general, senescent cells have been described as pro-inflammatory based on their senescence associated secretory phenotype (SASP). However, we have previously shown that senescence induced by overexpression ofSENEX (or ARHGAP18), in endothelial cells results in an anti-inflammatory phenotype. We have investigated, at the individual cellular level, the senescent phenotype of endothelial cells following three of the chief signals associated with ageing; oxidative stress, disturbed flow and hypoxia. All three stimuli induce senescence and, based on neutrophil adhesion and expression of the adhesion molecules E-selectin and VCAM-1, a population of senescent cells is seen that is resistant to inflammatory stimuli and thus we define as anti-inflammatory. The proportion of anti-inflammatory cells increases with time but remains stable at approximately 50% by eight days after induction of senescence, suggesting that these are stable phenotypes of endothelial cell senescence. Similar to other senescent cell types, p38MAPK blockade inhibits the development of the pro-inflammatory phenotype but unique to EC, there is a corresponding increase in the number of anti-inflammatory senescent cells. Thus stress-induced senescent endothelial cells display a mosaic of inflammatory phenotypes. The anti-inflammatory population suggests that senescent endothelial cells may have an unique protective role, to inhibit uncontrolled proliferation and to limit the local inflammatory response.

Timely expression of the Arabidopsis stoma‐fate master regulator MUTE is required for specification of other epidermal cell types

Epidermal differentiation in Arabidopsis thaliana aerial organs involves stomatal lineage development. Lineages derive from meristemoids, which arise from asymmetric divisions of protodermal cells. Each meristemoid divides repeatedly in an inward spiral before it transits to a guard mother cell (GMC) that produces the stoma, leaving a trail of surrounding stomatal lineage ground cells (SLGCs) that eventually differentiate into endoreplicated pavement cells. MUTE is a bHLH transcription factor that is expressed in late meristemoids and drives their transition to GMCs. Loss-of-function mute mutants are stomata-less dwarf plants with arrested lineages, in which stunted putative SLGCs surround a halted meristemoid. We analysed MUTE functions using a chemically inducible system for mute-3 complementation based on conditional MUTE expression in its normal domain. Continuous induction from germination produced stomata-bearing, normal-sized plants with viable mute-3 seeds. In 2-week-old mute-3 cotyledons, meristemoids appeared to retain their identity and synchronously formed stomata in response to induced MUTE expression. However, arrested SLGCs were not complemented: many produced stomata, leading to stomatal clusters, and others remained unexpanded and diploid. In contrast, non-lineage pavement cells, which are under-endoreplicated in mute-3, expanded and increased their ploidy level upon induction, showing that the lack of response of SLGCs is specific to this arrested cell type. Leaf phenotypic mosaics include wild-type lineages and adjacent mute-3 lineages, whose meristemoids and putative SLGCs remained arrested, indicating that the role of MUTE in SLGC fate is strictly lineage-autonomous. These results show that timely MUTE expression is essential to prevent stomatal fate in SLGCs and to promote their differentiation as pavement cells.

Correction: Differences in gene expression within a striking phenotypic mosaic Eucalyptus tree that varies in susceptibility to herbivory

Correction: Differences in gene expression within a striking phenotypic mosaic Eucalyptus tree that varies in susceptibility to herbivory

Differences in gene expression within a striking phenotypic mosaic Eucalyptus tree that varies in susceptibility to herbivory

BackgroundLong-lived trees can accumulate mutations throughout their lifetimes that may influence biotic and abiotic interactions. For example, some Eucalyptus trees display marked variation in herbivore defence within a single canopy. These “mosaic” trees support foliage with distinct chemotypes which are differentially favoured by insect and vertebrate herbivores, resulting in susceptible and resistant branches within a single canopy. These mosaic trees provide a unique opportunity to explore the biosynthesis and genetic regulation of chemical defences in the foliage. The biosynthesis of the principal defence compounds, terpenoid-dominated essential oils, is well understood. However, the regulation of the genes involved and thus the control of phenotypic variation within a single tree canopy remains a mystery.ResultsWe sequenced the transcriptomes of the leaves of the two different chemotypes of a chemically mosaic Eucalyptus melliodora tree using 454 pyrosequencing technology. We used gene set enrichment analysis to identify differentially expressed transcripts and found the proportion of differentially expressed genes in the resistant and susceptible foliage similar to the transcript difference between functionally distinct tissues of the same organism, for example roots and leaves. We also investigated sequence differences in the form of single nucleotide polymorphisms and found 10 nucleotides that were different between the two branches. These are likely true SNPs and several occur in regulatory genes.ConclusionWe found three lines of evidence that suggest changes to a ‘master switch’ can result in large scale phenotypic changes: 1. We found differential expression of terpene biosynthetic genes between the two chemotypes that could contribute to chemical variation within this plant. 2. We identified many genes that are differentially expressed between the two chemotypes, including some unique genes in each branch. These genes are involved in a variety of processes within the plant and many could contribute to the regulation of secondary metabolism, thus contributing to the chemical variation. 3. We identified 10 SNPs, some of which occur in regulatory genes that could influence secondary metabolism and thus contribute to chemical variation. Whilst this research is inherently limited by sample size, the patterns we describe could be indicative of other plant genetic mosaics.

Introgressive hybridization betweenBrahea dulcisandBrahea nitida(Arecaceae) in Mexico: evidence from morphological and PCR–RAPD patterns

Gene introgression usually results from spontaneous hybridization among closely related species in sympatric populations. This phenomenon has recently become a frequent topic of interest in evolutionary and conservation biology. A palm species complex occurring in Mexico formed by two native species, Brahea dulcis (Kunth) C. Martius and Brahea nitida André, and their putative hybrids, was analyzed with morphological and molecular markers in four hybrid zones and two pure sites for each parental species. Our results showed that the closeness of the hybrids to an allopatric site of either parental species (B. nitida, mesic habitat, and B. dulcis, xeric habitat) is directly related to their morphological and genetic similarity. Also, B. dulcis differed significantly from B. nitida in all the examined characters. Morphological and molecular data were highly coincident and supported the hypothesized hybridization. Hybrid phenotypes were morphologically parent-like for 46.4% of traits, intermediate for 33.2% of traits, and transgressive for 10.0% of traits. Thus, individual hybrids appear to be a mosaic of parent-like, intermediate, and transgressive phenotypes. Our results suggest that B. nitida has been genetically assimilated by B. dulcis in Xochiapala, and in the remaining hybrid zones a unidirectional pattern of gene flow towards B. nitida exists. Therefore, hybridization may threaten the conservation status of species through genetic assimilation and may confound the ability to distinguish among taxa.

The use of digital image‐based morphometrics to study the phenotypic mosaic in taxa with porous genomes

ABSTRACTRapid recent developments in DNA sequencing and genetic marker technologies call for the establishment of cost‐effective, automated phenotyping assays for evolutionary biology and systematics, so that the effects of DNA polymorphisms and epigenetic changes on the phenotype can be evaluated. We discuss the use of digital image‐based morphometrics in evolutionary biology and systematics with special emphasis on studies of taxa with porous genomes, i.e., taxa that do not conform to the traditional view of whole‐genome isolation between species. We outline the phenomenon of the 'phenotypic mosaic' in taxa with porous genomes using well‐documented examples from the literature and describe three important challenges arising for taxonomists: (1) character conflict in phylogenetic studies, (2) biased sampling of traits in morphological studies, and (3) cryptic ecological speciation. We demonstrate the use of geometric morphometrics using a combined molecular and morphometric dataset from an interspecific hybrid zone between two divergent Eurasian species of Populus, P. alba (white poplar) and P. tremula (European aspen). Elliptic Fourier analysis (EFA)‐based morphometric data were collected for 527 leaf specimens from 84 trees with known genomic composition as determined by a set of 30 nuclear DNA microsatellites. In addition, to demonstrate the ease of scoring functionally relevant phenotypes via digital image analysis, quantitative differences in leaf reflectance were examined and their structural basis determined using scanning electron microscopy (SEM). The EFA results indicate a heritable inter‐individual component for symmetric aspects and an important intra‐individual component for asymmetric aspects of variation in leaf outlines. Symmetric traits displayed a striking variety of phenotypes in hybrids compared to their parental species, consistent with the notion of the phenotypic mosaic. Linear discriminant analysis of these morphometric traits revealed (1) clear differentiation between parental species and (2) divergence between recombinant hybrids and their sympatric backcross parent P. alba, mediated primarily by two P. tremula–like and two transgressive traits. Our results demonstrate the usefulness of geometric morphometrics to interrogate multiple independent phenotypic characters and detect individual traits affected by introgression and divergence in taxa with porous genomes. Digital image‐based morphometrics holds great promise for large‐scale studies of relationships between DNA polymorphism and phenotypes in evolutionary biology.

Localized Darier's disease in a Blaschkoid distribution: Two cases of phenotypic mosaicism and a review of mosaic Darier's disease

Darier's disease is a rare autosomal dominant genodermatosis. Characteristic skin and nail changes consist of bilateral crusted red-brown papules and plaques in a seborrheic distribution, and V-shaped nicking or longitudinal ridging of the nails. In limited cases, a segmental distribution of this disease may be present in which localized lesions, found along the lines of Blaschko, are otherwise indistinguishable from those of generalized Darier's disease. Genomic mosaicism in localized lesions of Darier's disease is a new concept that was recently demonstrated in individuals of mosaic phenotypes. Both type 1 and type 2 mosaic phenotypes have been reported. We report two cases of type 1 localized Darier's disease in a Blaschkoid distribution and review the genetic implications of phenotypic mosaicism within the field of Darier's disease.