Greater Phenotypic Plasticity Research Articles

Rapid loss of phenotypic plasticity in the introduced range of the beach daisy, Arctotheca populifolia

Abstract Phenotypic plasticity is thought to be beneficial for species when introduced to a new range, increasing their proficiency in colonising and expanding into new environments. It has been hypothesised that an increase in phenotypic plasticity can be selected for when introduced to a new range. We set out to determine whether there was greater phenotypic plasticity in introduced populations of Arctotheca populifolia, compared with their native source population. We compared levels of phenotypic plasticity in four introduced Australian populations with the one identified source population from South Africa, across two separate glasshouse experiments that varied availability of (i) nutrients and (ii) water. Phenotypic plasticity was assessed at the population level for 13 individual traits, and across a suite of traits using a meta‐analysis to determine whether there was an overall difference in plasticity between ranges. The Australian populations had a significantly lower composite plasticity in response to nutrient availability (−0.56 [95% CI: −0.99; −0.13]), as well as lower plasticity in response to water availability, though this was not significant (−0.36 [95% CI: −0.80; 0.07]). Synthesis. Contrary to our expectations, A. populifolia has lower phenotypic plasticity in its introduced range than in its native range. This may have resulted from founder effects, or from rapid changes in plasticity due to selective pressures differing between the native and introduced ranges.

Alpine plant species converge towards adopting elevation-specific resource-acquisition strategy in response to experimental early snow-melting

Snow-melt is one of the important factors limiting growth and survival of alpine plants. Changes in snow-melt timing have profound effects on eco-physiological characteristics of alpine plant species through alterations in growing season length. Here, we conducted a field experiment and studied species response to experimentally induced early snow-melting (ES) (natural vs. early) at an alpine site (Rohtang) in the western Himalaya region. Eco-physiological response of eight snow-bed restricted alpine plant species from different elevations (lower: 3850 m and upper: 4150 m amsl) and belonging to contrasting resource acquisition strategies (conservative and acquisitive) were studied after 2-years (2019 & 2020) of initiating ES field experiment. We estimated the functional traits related to leaf economic spectrum and physiological performance and assessed their pattern of phenotypic plasticity. Analysis by linear mixed effect model showed that both the ‘conservative’ and ‘acquisitive’ species had responded to ES with significant effects on species specific leaf area, leaf dry matter content, leaf thickness, leaf water content and sugar content. Our results also revealed that ES treatment induced significant increase in leaf C/N ratio (10.57 % to 13.65 %) and protein content (15.85 % to 20.76 %) at both the elevations, irrespective of species groups. The phenotypic plasticity was found to be low and was essentially species-specific. However, for leaf protein content, the upper elevation species exhibited a higher phenotypic plasticity (0.43 ± 0.18) than the lower elevation species (0.31 ± 0.21). Interestingly, we found that irrespective of species unique functional strategy, species adapt to perform more conservative at lower elevation and more acquisitive at upper elevation, in response to ES. We conclude that plants occurring at contrasting elevations respond differentially to ES. However, species showed capacity for short-term acclimation to future environmental conditions, but may be vulnerable, if their niche is occupied by new species with greater phenotypic plasticity and a superior competitive ability.

Growth and Physiological Performance in Growth Phenotypes of the Carpet Shell Clam (Ruditapes decussatus) Fed Diets of Variable Lipid/Carbohydrate Ratios

Mixed suspensions of phytoplankton and yeast cells in different proportions were designed to achieve diets with a range of variation (0.6–2.2) in the ratio of lipid to carbohydrate while maintaining protein content constant. Juvenile specimens of the carpet shell clam (Ruditapes decussatus) from two segregated growth phenotypes (fast and slow growers) were food conditioned and the physiological components of the energy balance were determined with these different diet compositions in order to assess the combined effects of endogenous and nutritional factors on growth performance. Conditioning to lipid-rich diets increased growth rate relative to conditioning to carbohydrate-rich diets and resulted in higher scope for growth values in both growth groups. These dietary effects are mainly driven by differences in the absorption efficiency (AE) found between clams fed different food compositions, although the present results do not allow to ascertain whether the reduced AE recorded with the carbohydrate-rich diets results from reduced digestibility of yeasts cells due to structural restrictions or either reflects the digestive imbalance of lipids associated to higher production of metabolic fecal losses. Greater phenotypic plasticity was seen to enable the fast-growing clams fed a carbohydrate-rich diet to overcome the above digestive limitations through an overfeeding response; however, the resource to such kind of physiological mechanism appeared limited by the nutritional conditions (energetic status) prevailing during the conditioning phase.

Metabolic flexibility during a trophic transition reveals the phenotypic plasticity of greater duckweed (Spirodela polyrhiza 7498).

The greater duckweed (Spirodela polyrhiza 7498) exhibits trophic diversity (photoautotrophic, heterotrophic, photoheterotrophic, and mixotrophic growth) depending on the availability of exogenous organic carbon sources and light. Here, we show that the ability to transition between various trophic growth conditions is an advantageous trait, providing great phenotypic plasticity and metabolic flexibility in S. polyrhiza 7498. By comparing S. polyrhiza 7498 growth characteristics, metabolic acclimation, and cellular ultrastructure across these trophic modes, we show that mixotrophy decreases photosynthetic performance and relieves the CO2 limitation of photosynthesis by enhancing the CO2 supply through the active respiration pathway. Proteomic and metabolomic analyses corroborated that S. polyrhiza 7498 increases its intracellular CO2 and decreases reactive oxygen species under mixotrophic and heterotrophic conditions, which substantially suppressed the wasteful photorespiration and oxidative-damage pathways. As a consequence, mixotrophy resulted in a higher biomass yield than the sum of photoautotrophy and heterotrophy. Our work provides a basis for using trophic transitions in S. polyrhiza 7498 for the enhanced accumulation of value-added products.

Differences in Phenotypic Plasticity between Invasive and Native Plants Responding to Three Environmental Factors.

The phenotypic plasticity hypothesis suggests that exotic plants may have greater phenotypic plasticity than native plants. However, whether phenotypic changes vary according to different environmental factors has not been well studied. We conducted a multi-species greenhouse experiment to study the responses of six different phenotypic traits, namely height, leaf number, specific leaf area, total biomass, root mass fraction, and leaf mass fraction, of native and invasive species to nutrients, water, and light. Each treatment was divided into two levels: high and low. In the nutrient addition experiment, only the leaf mass fraction and root mass fraction of the plants supported the phenotypic plasticity hypothesis. Then, none of the six traits supported the phenotypic plasticity hypothesis in the water or light treatment experiments. The results show that, for different environmental factors and phenotypes, the phenotypic plasticity hypothesis of plant invasion is inconsistent. When using the phenotypic plasticity hypothesis to explain plant invasion, variations in environmental factors and phenotypes should be considered.

Changes in cellular Ca2+ and Na+ regulation during the progression towards heart failure.

In adapting to disease and loss of tissue, the heart shows great phenotypic plasticity that involves changes to its structure, composition and electrophysiology. Together with parallel whole body cardiovascular adaptations, the initial decline in cardiac function resulting from the insult is compensated. However, in the long term, the heart muscle begins to fail and patients with this condition have a very poor prognosis, with many dying from disturbances of rhythm. The surviving myocytes of these hearts gain Na+ , which is positively inotropic because of alterations to Ca2+ fluxes mediated by the Na+ /Ca2+ exchange, but compromises Ca2+ -dependent energy metabolism in mitochondria. Uptake of Ca2+ into the sarcoplasmic reticulum (SR) is reduced because of diminished function of SR Ca2+ ATPases. The result of increased Ca2+ influx and reduced SR Ca2+ uptake is an increase in the diastolic cytosolic Ca2+ concentration, which promotes spontaneous SR Ca2+ release and induces delayed afterdepolarisations. Action potential duration prolongs because of increased late Na+ current and changes in expression and function of other ion channels and transporters increasing the probability of the formation of early afterdepolarisations. There is a reduction in T-tubule density and so the normal spatial arrangements required for efficient excitation-contraction coupling are compromised and lead to temporal delays in Ca2+ release from the SR. Therefore, the structural and electrophysiological responses that occur to provide compensation do so at the expense of (1) increasing the likelihood of arrhythmogenesis; (2) activating hypertrophic, apoptotic and Ca2+ signalling pathways; and (3) decreasing the efficiency of SR Ca2+ release.

Abstract 962: A hybrid epithelial-mesenchymal phenotype identifies lung cancer cells with high metastatic potential due to increased stemness properties and escape from NK immunosurveillance

Abstract Introduction: Epithelial to mesenchymal transition (EMT) is essential for cancer dissemination but its role in overt metastasis establishment remains an open issue. Multiple EMT states and great phenotypic plasticity have also been described, indicating the need for in-depth characterization of biological properties associated with different EMT phenotypes. The so-called hybrid phenotype, characterized by presence of both mesenchymal and epithelial markers, might in fact identify cancer cells with enhanced metastatic propensity. Materials and Methods: To assess the metastatic potential of different EMT phenotypes, we exploited two lung cancer cell lines with different properties (A549/epithelial and LT73/hybrid) and generated their respective sublines through four sequential rounds of in vitro migration assay (named M4). TGFβ treatment was also used to induce EMT. With a previously validated score based on the ratio between expression of epithelial (CDH1) or mesenchymal genes (SNAI2), cells were classified as: epithelial (A549)>hybrid (A549-M4, LT73)>mesenchymal (LT73-M4, A549-TGFβ). The disseminating/metastatic properties of the different lines were tested in vivo by subcutaneous and tail-vein injections in SCID mice. Interaction with NK cells (NKs), known to specifically target EMT cancer cells, was also assessed. Results and Discussion: Compared to parental cells, M4 cells showed up-regulation of EMT-associated genes, expansion of the subset of CD133+ lung cancer stem cells (CSC) and displayed in vivo higher ability to disseminate from primary tumors to murine lungs (2-fold change, p=0.004). However, hybrid cells (LT73 and A549-M4) showed an increased ability to generate overt lung metastases in experimental metastasis assays compared to either epithelial (A549) or mesenchymal cells (LT73-M4 and A549-TGFβ). The higher metastatic ability of hybrid cells was correlated with CSCs expansion and reduced NKs infiltration to the metastatic lungs. Expression of IL-8, a cytokine involved in NKs recruitment, was decreased in hybrid cells and they were less prone to chemoattract NKs in vitro compared to both epithelial and mesenchymal cells. Finally, in vivo prevention of NKs recruitment to the lungs using a neutralizing antibody, massively increased metastasis formation and expansion of CSCs in mice injected i.v. with mesenchymal A549-TGFβ (p=0.0006), but had limited effects in groups injected with both epithelial A549 and hybrid A549-M4. Conclusions: Our data indicate that the hybrid phenotype correlates with the highest metastatic potential related to efficient cancer cell dissemination, increased induction of stemness features and ability to avoid NKs recruitment. Conversely, fully mesenchymal cells show limited metastatic potential, possibly due the control exerted by NKs on their CSC subsets. Citation Format: Giulia Bertolini, Monica Parodi, Massimo Milione, Fabio Murianni, Giovanni Centonze, Giulia Taiè, Gabriella Sozzi, Massimo Vitale, Luca Roz. A hybrid epithelial-mesenchymal phenotype identifies lung cancer cells with high metastatic potential due to increased stemness properties and escape from NK immunosurveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 962.

Phenotypic plasticity, genetic structure, and systematic position of Neoechinorhynchus emyditoides Fisher, 1960 (Acanthocephala: Neoechinorhynchidae) a parasite of emydid turtles from the Nearctic and Neotropical regions.

AbstractThe taxonomy of the 10 recognizedNeoechinorhynchusspecies associated with emydid turtles is complex due to the morphological conservatism. In the present study, specimens ofN. emyditoidesfrom northern and southeastern Mexico exhibit great phenotypic plasticity on its diagnostic characteristics. We sequenced three molecular markers: the internal transcribed spacers ITS1, ITS2 and 5.8S gene, the D2 + D3 domains of the large subunit from nuclear DNA and cytochromecoxidase subunit I (cox1) from mitochondrial DNA. Sequences of the nuclear molecular markers were aligned and compared with other congeneric species associated with emydids available in GenBank. Phylogenetic analyses supported the polyphyly ofNeoechinorhynchus. The species from emydids formed a clade, which was subdivided into five subclades that correspond with each species analysed (N. pseudemydis,N. chrysemydis,N. emydis,N. schmidtiandN. emyditoides). To understand better the genetic structure ofN. emyditoidesa haplotype network was inferred with 29cox1sequences, revealing the presence of 13 haplotypes, two of which were shared and 11 were unique. The high values of fixation index,Fst(0.4227–0.8925) detected between the two populations from southeastern and the two from northern Mexico indicated low genetic flow among the populations. Our data suggest that theNeoechinorhynchusspecies associated with emydid turtles diversified in the eastern USA and that ofN. emyditoidesexpanded its distribution range reached southeastern Mexico.

Resident birds are more behaviourally plastic than migrants

Species subjected to more variable environments should have greater phenotypic plasticity than those that are more restricted to specific habitat types leading to the expectation that migratory birds should be relatively more plastic than resident birds. We tested this comparatively by studying variation in flight initiation distance (FID), a well-studied antipredator behaviour. We predicted that variation in FID would be greater for migratory species because they encountered a variety of locations during their lives and therefore had less predictable assessments of risk compared to more sedentary species. Contrary to our prediction, we found that non-migratory species (sedentary) had greater variation in FID than migratory ones. Migratory and partially migratory birds had greater average FIDs than sedentary birds, suggesting that they were generally more wary. These results suggest that the predictability associated with not migrating permits more nuanced risk assessment which was seen in the greater variation in FID of sedentary bird species.

Phenotypic plasticity and genetic diversity elucidate rarity and vulnerability of an endangered riparian plant

AbstractEnvironmental change, accelerated by anthropogenic activities, threatens many species and can be especially challenging for rare species given their potentially limited capacity for migration and adaptation relative to more common species. The ability to acclimate via phenotypic plasticity could provide an important path to species persistence in the face of such change. We investigated the responses of an endangered plant species endemic to a highly dynamic riparian habitat in southeastern Tennessee, USA, and its most widespread congener to environmental change to elucidate their current statuses and future vulnerability. Specifically, we compared the population‐ and species‐level plasticity of rarePityopsis ruthiiand commonP. graminifoliato contrasting light, temperature, and water conditions in a growth chamber experiment to evaluate their potential to acclimate to environmental change. Contrary to our expectations,P. ruthiihad greater phenotypic plasticity than its common congener in response to both altered light and water availability. But this plasticity was not associated with increased fitness, suggesting that it was not adaptive. Concurrently, we genotyped these individuals at nine putatively neutral microsatellite loci to contrast genetic diversity across the range of each species. As expected,P. ruthiiexhibited reduced genetic diversity relative to its more common congener. Overall, our findings accord with the narrow range and current habitat specificity ofP. ruthii, especially its tolerance of highly variable water, and highlight its potential vulnerability to future environmental change.

Temperature Extremes and Sex-Related Physiology, Not Environmental Variability, Are Key in Explaining Thermal Sensitivity of Bimodal-Breathing Intertidal Crabs

Global temperature increases are predicted to have pronounced negative effects on the metabolic performance of both terrestrial and aquatic organisms. These metabolic effects may be even more pronounced in intertidal organisms that are subject to multiple, abruptly changing abiotic stressors in the land-sea transition zone. Of the available studies targeting the intertidal environment, emphasis has largely been on water-breathing model organisms and this selective focus resulted in limited reliable forecasts on the impact of global warming on primarily air-breathing intertidal species. We investigated the thermal sensitivity of six phylogenetically related fiddler crab species that occupy different microhabitats on intertidal shores from south America and east Asia to test how bimodal-breathing intertidal ectotherms cope with thermal stress. We examined the metabolic physiology and thermal limits of the crabs by measuring their cardiac function and oxygen consumption along a thermal gradient. Their specific thermal microhabitat was also appraised. We found that subtropical fiddler crab species inhabiting vegetated microhabitats have lower upper lethal temperatures and therefore greater thermal sensitivity in comparison to their tropical counterparts. Additionally, females exhibited higher oxygen consumption and lower lethal temperatures in comparison to males. Our results contradict previous predictions that species from higher latitudes that experience greater temperature variability have broader latitudinal distributions, greater phenotypic plasticity and lower thermal sensitivity. Furthermore, the higher thermal sensitivity demonstrated by female fiddler crabs with respect to males strongly suggests a role of both gametogenesis and physiological dimorphism on the thermal performance of tropical and subtropical intertidal organisms. These observations ultimately, advocates for further studies on sex-biased and development-biased thermal sensitivity before drawing any generalizations based on a single sex or life stage.

Carbon, nitrogen and phosphorus stoichiometry of two submerged macrophytes are more affected by plant species and organs than by light

Context Submerged macrophytes develop great phenotypic plasticity responding to light stress. However, the light effects on carbon (C), nitrogen (N) and phosphorus (P) stoichiometry of submerged macrophytes are unknown. Aims To determine the stoichiometric response of Myriophyllum spicatum and Vallisneria natans to a light gradient. Methods We determined the C, N and P stoichiometry of two aquatic plants grown in pots subjected to an incident light gradient ranging from 100 to 17% of incident light. Key results The C, N and P stoichiometry of V. natans (except C:N of root) were not significantly different among the light treatments, whereas those of M. spicatum (except leaf element concentrations and root element ratios) showed significant differences across the light gradient. The analysis of variance results implied that the species, plant organs and their interactions explained more than 75% of total variance of all measured indices (except C). Conclusions The C, N and P stoichiometry of the two studied submerged macrophytes are more affected by plant organs and species than by light irradiance. Implication Although C, N and P stoichiometry of submerged macrophytes was little affected by light irradiance, whether the stoichiometry of submerged macrophytes is stable under other environmental factors should be focused on in future investigation.

Invasion of alien slugs in disturbed habitats: role of behavioural phenotype, plasticity and interspecific competition

Habitat disturbance can promote the invasion success of alien species, but the underlying mechanisms remain unclear, especially in animal species. Alien species may have trait values that facilitate invasion (ideal weed hypothesis) or greater phenotypic plasticity than native species (plasticity hypothesis), thus promoting their invasion success in disturbed habitats. Invasion success and ultimately alien species dominance in disturbed habitats can also be a direct consequence of competitive exclusion of native species. We performed experiments in controlled conditions to determine which of these mechanisms could explain the invasion success of Arion subfuscus s.l. (hereafter Arion ), an alien slug species complex that lives in various disturbed ecosystems of eastern Canada such as managed forests. To do so, we compared the behavioural response of Arion and native sympatric philomycid slugs (hereafter Philomycus ) to a decrease in soil moisture content, which often occurs after forest harvesting (i.e. habitat disturbance). We also investigated whether the presence of Arion alters the behaviour of Philomycus , which would indicate that Arion has a greater ability to compete by interference than these native species. Our findings show that Arion did not have higher boldness or locomotor activity (i.e. behaviours that promote invasion success) or greater plasticity than Philomycus , and that the presence of Arion did not affect the behaviour of Philomycus . However, Arion may be able to exploit resources more efficiently than native species since it mainly used the closest refuge from the food source in the present study. These findings are in line with the ideal weed hypothesis but do not support the phenotypic plasticity hypothesis or any role of interference competition in Arion invasion success in disturbed habitats such as harvested forests. Our results also suggest that the direction, but not the magnitude, of behavioural plasticity might be a key aspect in alien species invasion success in disturbed habitats. • Alien slugs were not bolder or more active than native species. • Plasticity of these behaviours was not higher in alien slugs than in native slugs. • Interference competition does not seem to be involved in alien slug invasion success. • Alien slugs may be more efficient in exploiting resources than native species.

Experimental Evolution of West Nile Virus at Higher Temperatures Facilitates Broad Adaptation and Increased Genetic Diversity.

West Nile virus (WNV, Flaviviridae, Flavivirus) is a mosquito-borne flavivirus introduced to North America in 1999. Since 1999, the Earth’s average temperature has increased by 0.6 °C. Mosquitoes are ectothermic organisms, reliant on environmental heat sources. Temperature impacts vector–virus interactions which directly influence arbovirus transmission. RNA viral replication is highly error-prone and increasing temperature could further increase replication rates, mutation frequencies, and evolutionary rates. The impact of temperature on arbovirus evolutionary trajectories and fitness landscapes has yet to be sufficiently studied. To investigate how temperature impacts the rate and extent of WNV evolution in mosquito cells, WNV was experimentally passaged 12 times in Culex tarsalis cells, at 25 °C and 30 °C. Full-genome deep sequencing was used to compare genetic signatures during passage, and replicative fitness was evaluated before and after passage at each temperature. Our results suggest adaptive potential at both temperatures, with unique temperature-dependent and lineage-specific genetic signatures. Further, higher temperature passage was associated with significantly increased replicative fitness at both temperatures and increases in nonsynonymous mutations. Together, these data indicate that if similar selective pressures exist in natural systems, increases in temperature could accelerate emergence of high-fitness strains with greater phenotypic plasticity.

Macrophages in Chronic Liver Failure: Diversity, Plasticity and Therapeutic Targeting.

Chronic liver injury results in immune-driven progressive fibrosis, with risk of cirrhosis development and impact on morbidity and mortality. Persistent liver cell damage and death causes immune cell activation and inflammation. Patients with advanced cirrhosis additionally experience pathological bacterial translocation, exposure to microbial products and chronic engagement of the immune system. Bacterial infections have a high incidence in cirrhosis, with spontaneous bacterial peritonitis being the most common, while the subsequent systemic inflammation, organ failure and immune dysregulation increase the mortality risk. Tissue-resident and recruited macrophages play a central part in the development of inflammation and fibrosis progression. In the liver, adipose tissue, peritoneum and intestines, diverse macrophage populations exhibit great phenotypic and functional plasticity determined by their ontogeny, epigenetic programming and local microenvironment. These changes can, at different times, promote or ameliorate disease states and therefore represent potential targets for macrophage-directed therapies. In this review, we discuss the evidence for macrophage phenotypic and functional alterations in tissue compartments during the development and progression of chronic liver failure in different aetiologies and highlight the potential of macrophage modulation as a therapeutic strategy for liver disease.

Morphological plasticity in Chlamydomonas reinhardtii and acclimation to micropollutant stress

Phytoplankton are characterized by a great phenotypic plasticity and amazing morphological variability, both playing a primary role in the acclimation to changing environments. However, there is a knowledge gap concerning the role of algal morphological plasticity in stress responses and acclimation to micropollutants. The present study aims at examining palmelloid colony formation of the green alga Chlamydomonas reinhardtii upon micropollutants exposure. Cells were exposed to four micropollutants (MPs, copper, cadmium, PFOS and paraquat) with different modes of action for a duration of 72 h. Effects of MPs on palmelloid formation, growth and physiological traits (chlorophyll fluorescence, membrane integrity and oxidative stress) were monitored by flow cytometry and fluorescence microscopy. Palmelloid formation was observed upon treatment with the four micropollutants. Number of palmelloid colonies and their size were dependent on MP concentration and exposure duration. Cells reverted to their unicellular lifestyle when colonies were harvested and inoculated in fresh medium indicating that palmelloid formation is a plastic response to micropollutants. No physiological effects of these compounds were observed in cells forming palmelloids. Palmelloid colonies accumulated lower Cd concentration than unicellular C. reinhardtii suggesting that colony formation protects the cells from MPs stress. The results show that colony formation in Chlamydomonas reinhardtii is a stress response strategy activated to face sub-lethal micropollutant concentrations.

Speciation in sympatric species of land snails from the genus Trochulus (Gastropoda, Hygromiidae)

AbstractThe identification and designation of land snail species in the genus Trochulus on the basis of shell characteristics are problematic because of their great phenotypic plasticity. Some genetic analyses have proved inconclusive, with much variation within populations and apparent gene flow among them. We examined this issue by morphometric and molecular approaches on the morphologically similar species T. coelomphala, T. hispidus and T. striolatus, co‐occurring in the Alpenvorland of Germany. While these species differed in shell and reproductive system morphology, there were forms that turned out intermediate in shell characters between T. coelomphala and T. hispidus but had genital morphology similar to T. coelomphala. Phylogenetic analysis, however, showed that these forms clustered neither with T. coelomphala nor T. hispidus but are sister to T. striolatus from the same region, which suggests that they evolved by way of sympatric speciation. Further, these analyses suggest that T. coelomphala diverged within T. hispidus; a crossing experiment indicated that they were interfertile. Expanding the study to include all available Trochulus sequences enabled us to infer evolutionary relationships between them and showed that T. hispidus is polyphyletic. Some Trochulus samples of one nominal species were grouped within others. The combination of phenotypic plasticity and possible mitochondrial DNA introgression illustrates the complex nature of evolutionary processes and the need for caution in the application of traditional taxonomic practice.

Forest stratification shapes allometry and flight morphology of tropical butterflies

Studies of altitudinal and latitudinal gradients have identified links between the evolution of insect flight morphology, landscape structure and microclimate. Although lowland tropical rainforests offer steeper shifts in conditions between the canopy and the understorey, this vertical gradient has received far less attention. Butterflies, because of their great phenotypic plasticity, are excellent models to study selection pressures that mould flight morphology. We examined data collected over 5 years on 64 Nymphalidae butterflies in the Ecuadorian Chocó rainforest. We used phylogenetic methods to control for similarity resulting from common ancestry, and explore the relationships between species stratification and flight morphology. We hypothesized that species should show morphological adaptations related to differing micro-environments, associated with canopy and understorey. We found that butterfly species living in each stratum presented significantly different allometric slopes. Furthermore, a preference for the canopy was significantly associated with low wing area to thoracic volume ratios and high wing aspect ratios, but not with the relative distance to the wing centroid, consistent with extended use of fast flapping flight for canopy butterflies and slow gliding for the understorey. Our results suggest that microclimate differences in vertical gradients are a key factor in generating morphological diversity in flying insects.

Stem Cell Pluripotency Genes Klf4 and Oct4 Regulate Complex SMC Phenotypic Changes Critical in Late-Stage Atherosclerotic Lesion Pathogenesis.

Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis. We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11-, Lgals3+ population with a chondrocyte-like gene signature that was markedly reduced with SMC-Klf4 knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene-positive, extracellular matrix-remodeling, "pioneer" cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3+ osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.

MicroRNAs as Key Players in Melanoma Cell Resistance to MAPK and Immune Checkpoint Inhibitors.

Advances in the use of targeted and immune therapies have revolutionized the clinical management of melanoma patients, prolonging significantly their overall and progression-free survival. However, both targeted and immune therapies suffer limitations due to genetic mutations and epigenetic modifications, which determine a great heterogeneity and phenotypic plasticity of melanoma cells. Acquired resistance of melanoma patients to inhibitors of BRAF (BRAFi) and MEK (MEKi), which block the mitogen-activated protein kinase (MAPK) pathway, limits their prolonged use. On the other hand, immune checkpoint inhibitors improve the outcomes of patients in only a subset of them and the molecular mechanisms underlying lack of responses are under investigation. There is growing evidence that altered expression levels of microRNAs (miRNA)s induce drug-resistance in tumor cells and that restoring normal expression of dysregulated miRNAs may re-establish drug sensitivity. However, the relationship between specific miRNA signatures and acquired resistance of melanoma to MAPK and immune checkpoint inhibitors is still limited and not fully elucidated. In this review, we provide an updated overview of how miRNAs induce resistance or restore melanoma cell sensitivity to mitogen-activated protein kinase inhibitors (MAPKi) as well as on the relationship existing between miRNAs and immune evasion by melanoma cell resistant to MAPKi.