Clonal Fitness Research Articles

The Impact of PI3-kinase/RAS Pathway Cooperating Mutations in the Evolution of KMT2A-rearranged Leukemia.

Leukemia is an evolutionary disease and evolves by the accrual of mutations within a clone. Those mutations that are systematically found in all the patients affected by a certain leukemia are called "drivers" as they are necessary to drive the development of leukemia. Those ones that accumulate over time but are different from patient to patient and, therefore, are not essential for leukemia development are called "passengers." The first studies highlighting a potential cooperating role of phosphatidylinositol 3-kinase (PI3K)/RAS pathway mutations in the phenotype of KMT2A-rearranged leukemia was published 20 years ago. The recent development in more sensitive sequencing technologies has contributed to clarify the contribution of these mutations to the evolution of KMT2A-rearranged leukemia and suggested that these mutations might confer clonal fitness and enhance the evolvability of KMT2A-leukemic cells. This is of particular interest since this pathway can be targeted offering potential novel therapeutic strategies to KMT2A-leukemic patients. This review summarizes the recent progress on our understanding of the role of PI3K/RAS pathway mutations in initiation, maintenance, and relapse of KMT2A-rearranged leukemia.

Somatic Mutations Increase Hepatic Clonal Fitness and Regeneration in Chronic Liver Disease

Normal tissues accumulate genetic changes with age, but it is unknown if somatic mutations promote clonal expansion of non-malignant cells in the setting of chronic degenerative diseases. Exome sequencing of diseased liver samples from 82 patients revealed a complex mutational landscape in cirrhosis. Additional ultra-deep sequencing identified recurrent mutations in PKD1, PPARGC1B, KMT2D, and ARID1A. The number and size of mutant clones increased as a function of fibrosis stage and tissue damage. To interrogate the functional impact of mutated genes, a pooled invivo CRISPR screening approach was established. In agreement with sequencing results, examination of 147 genes again revealed that loss of Pkd1, Kmt2d, and Arid1a promoted clonal expansion. Conditional heterozygous deletion of these genes in mice was also hepatoprotective in injury assays. Pre-malignant somatic alterations are often viewed through the lens of cancer, but we show that mutations can promote regeneration, likely independent of carcinogenesis.

A software tool for the quantification of metastatic colony growth dynamics and size distributions in vitro and in vivo.

The majority of cancer-related deaths are due to metastasis, hence improved methods to biologically and computationally model metastasis are required. Computational models rely on robust data that is machine-readable. The current methods used to model metastasis in mice involve generating primary tumors by injecting human cells into immune-compromised mice, or by examining genetically engineered mice that are pre-disposed to tumor development and that eventually metastasize. The degree of metastasis can be measured using flow cytometry, bioluminescence imaging, quantitative PCR, and/or by manually counting individual lesions from metastatic tissue sections. The aforementioned methods are time-consuming and do not provide information on size distribution or spatial localization of individual metastatic lesions. In this work, we describe and provide a MATLAB script for an image-processing based method designed to obtain quantitative data from tissue sections comprised of multiple subpopulations of disseminated cells localized at metastatic sites in vivo. We further show that this method can be easily adapted for high throughput imaging of live or fixed cells in vitro under a multitude of conditions in order to assess clonal fitness and evolution. The inherent variation in mouse studies, increasing complexity in experimental design which incorporate fate-mapping of individual cells, result in the need for a large cohort of mice to generate a robust dataset. High-throughput imaging techniques such as the one that we describe will enhance the data that can be used as input for the development of computational models aimed at modeling the metastatic process.

A role for chromatin regulatory dynamics in breast cancer evolution.

Enhancer profiling of breast tumors reveals that chromatin regulatory elements contribute to the clonal fitness landscape, treatment resistance and phenotypic divergence.

Combining Mutational Signatures, Clonal Fitness, and Drug Affinity to Define Drug-Specific Resistance Mutations in Cancer

SummaryThe emergence of mutations that confer resistance to molecularly targeted therapeutics is dependent upon the effect of each mutation on drug affinity for the target protein, the clonal fitness of cells harboring the mutation, and the probability that each variant can be generated by DNA codon base mutation. We present a computational workflow that combines these three factors to identify mutations likely to arise upon drug treatment in a particular tumor type. The Osprey-based workflow is validated using a comprehensive dataset of ERK2 mutations and is applied to small-molecule drugs and/or therapeutic antibodies targeting KIT, EGFR, Abl, and ALK. We identify major clinically observed drug-resistant mutations for drug-target pairs and highlight the potential to prospectively identify probable drug resistance mutations.

Abstract 1178: Genotype-fitness maps guide targeted therapy combination in lung adenocarcinoma

Abstract Targeted EGFR inhibition in lung cancer leads to dramatic responses. Nonetheless, disease evolution to resistance is the rule. As this evolutionary process is fueled by intra-tumoral genetic diversity, a comprehensive mapping of clonal fitness is required to inform strategies to overcome resistance. To model intra-tumoral diversity in vitro, we performed a genome-wide, over-expression genetic perturbation assay in EGFR-driven non-small cell lung cancer (NSCLC) PC9 cells. Our screen covered 17,255 ORFs (open reading frame constructs) representing 12,728 wildtype and mutated genes, and examined the effects of a first-generation EGFR inhibitor (erlotinib), a third-generation EGFR inhibitor (osimertinib) and a MEK inhibitor (binimetinib) on evolutionary selection, alone or in combination. Specifically, to obtain genotype-to-fitness maps for each drug, we measured clonal abundance up to 4 times during the screen and mathematically resolved their growth behavior. Finally, for each drug, we applied multiple doses to obtain dose-fitness relationships, which may impact tumor evolution in patients due to high inter- and intra-tumoral variability in drug delivery. In total, we have performed 78 genome-wide screens to map the evolutionary landscape of PC9 resistance to targeted therapy. Erlotinib and osimertinib both result in an overwhelming reduction of fitness, as expected from their clinical benefit. Known clinical resistance mechanisms involving ERBB2, PIK3CA, AXL, and BRAF confer a pronounced fitness advantage in the presence of both drugs, while EGFR (T790M) improves fitness only with erlotinib. Novel resistance mechanisms include alternative tyrosine kinases (NTRK, PDGFRB, CSF1R, KIT), KRAS, G-coupled protein receptors, transcription factors (SOX15, FOXA1), and cellular transporters (ABCG2). Notably, we observe significant divergence in dose-fitness relationships between different resistance mechanisms. For example, PIK3CA confers a modest but persistent advantage across the dose range, in contrast to BRAF which results in a fitness advantage only when sufficiently high drug levels are added. The fitness landscape for binimetinib resistance appeared to be largely orthogonal to that of the EGFR inhibitors, with decreased fitness noted for many tyrosine kinases as well as KRAS mutations. MEK inhibitor resistance results from RAF family member overexpression (ARAF, BRAF, RAF1) and MAPK activation. The orthogonal resistance landscapes of EGFR inhibition and MEK inhibition translate into highly synergistic effects in drug combination, with effective prediction of combinatorial fitness from the single-agent fitness landscapes using generalized linear models. These results validate this approach as a systematic method to address the combinatorial problem of optimizing drug combinations and doses to directly anticipate and address cancer evolution. Citation Format: Asaf Zviran, Patrick Bolan, Lisa Brenan, Amy Goodale, Denisse Rotem, Viktor Adalsteinsson, Federica Piccioni, Cory Johannessen, Dan Landau. Genotype-fitness maps guide targeted therapy combination in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1178.

Evidence for plasticity, but not local adaptation, in invasive Japanese knotweed (Reynoutria japonica) in North America

While local adaptation and phenotypic plasticity are commonly observed in species occupying heterogeneous environments, these phenomena are less well understood in invasive species. However, plant invasions offer the opportunity to study these dynamics as they occur in species colonizing new habitats. In this study, we examined local adaptation and phenotypic plasticity in an invasive plant, Reynoutria japonica, across a broad latitudinal range within North America. We performed full-factorial reciprocal transplants using plants from three sites and examined fitness responses in both sexual and clonal reproductive measures, as well as vegetative responses in height, basal stem diameter, and biomass. For all vegetative traits, there was a significant effect of source population, indicating genetic differentiation among populations. There was also a significant effect of transplant site, suggesting phenotypic plasticity. However, there was no evidence of local adaptation at the North American meta-population level for either measure of sexual or clonal fitness. All three comparisons for sexual fitness failed to show any differences between source populations, indicating a lack of local adaptation. For clonal fitness, two of the three comparisons showed local maladaptation, and only one showed greater fitness at the home compared to foreign sites, but this population had greater fitness at all sites, indicating greater fitness overall for this population rather than local adaptation. The fact that we did not detect consistent patterns of local adaptation in these populations across a broad geographic range is somewhat surprising given that local adaptation appears common in many species, including invasives, and that the populations have been established for over a century. However, the lack of local adaptation observed in this species may indicate that phenotypic plasticity within the species is sufficient to allow the persistence of R. japonica in a variety of environments across its invaded range.

De novo activating mutations drive clonal evolution and enhance clonal fitness in KMT2A-rearranged leukemia

Activating signaling mutations are common in acute leukemia with KMT2A (previously MLL) rearrangements (KMT2A-R). These mutations are often subclonal and their biological impact remains unclear. Using a retroviral acute myeloid mouse leukemia model, we demonstrate that FLT3ITD, FLT3N676K, and NRASG12D accelerate KMT2A-MLLT3 leukemia onset. Further, also subclonal FLT3N676K mutations accelerate disease, possibly by providing stimulatory factors. Herein, we show that one such factor, MIF, promotes survival of mouse KMT2A-MLLT3 leukemia initiating cells. We identify acquired de novo mutations in Braf, Cbl, Kras, and Ptpn11 in KMT2A-MLLT3 leukemia cells that favored clonal expansion. During clonal evolution, we observe serial genetic changes at the KrasG12D locus, consistent with a strong selective advantage of additional KrasG12D. KMT2A-MLLT3 leukemias with signaling mutations enforce Myc and Myb transcriptional modules. Our results provide new insight into the biology of KMT2A-R leukemia with subclonal signaling mutations and highlight the importance of activated signaling as a contributing driver.

KRAS Allelic Imbalance: Strengths and Weaknesses in Numbers

The identification of therapeutic vulnerabilities in mutant KRAS tumors has proven difficult to achieve. Burgess and colleagues recently reported in Cell that mutant/wild-type Kras allelic dosage determines clonal fitness and MEK inhibitor sensitivity in a leukemia model, demonstrating that KRAS allelic imbalance is likely an important and overlooked variable.

Doubling Down on Mutant RAS Can MEK or Break Leukemia

Targeting of the RAS pathway has long been a critical therapeutic challenge in oncology. Burgess etal. examine how the relative expression of mutant and wild-type KRAS modulates clonal fitness and sensitivity to MEK inhibitors in a model of KrasG12D mutant acute myeloid leukemia and propose its use as a predictive biomarker.

Transition of Chronic Myeloid Leukemia to Chronic Myelomonocytic Leukemia As a Tool to Observe Development of Chronic Myelomonocytic Leukemia

Introduction. Development of abnormal Philadelphia (Ph) negative clones following treatment of chronic myeloid leukemia (CML) patients with imatinib has been observed in 3 to 9% of patients. Here we report on a 77 year old male diagnosed with CML that responded to imatinib treatment and subsequently developed chronic myelomonocytic leukemia (CMML). He achieved major cytogenetic response within 3 months but this response coincided with the emergence of monocytosis diagnosed as CMML. Five months after starting imatinib treatment the patient succumbed to CMML. We analyzed five sequential samples to determine whether a chronological order of mutations defined the emergence of CMML and to characterize the clonal evolution of the CMML population.Materials and Method. Five samples (diagnostic and four follow up samples) were available for analysis. CMML mutations were identified by whole exome sequencing (WES) in CD14+ cells following the onset of CMML, using CD3+ cells as constitutional control. Mutations were validated by Sequenom MassARRAY and Sanger sequencing and quantified by pyrosequencing. Deep WES was performed on the diagnostic sample to determine whether the mutations were present at CML diagnosis. To determine the clonal architecture of the emerging CMML, colony formation assays were performed on the diagnostic and the next two follow-up samples (Samples 1-3). More than 100 colonies per sample were plucked for DNA and RNA isolation. The DNA from these colonies were tested for the presence of the confirmed CMML mutations and the RNA was used for detection of BCR-ABL1 transcript using a Taqman real time assay.Results. Four mutations were identified by Sequenom and WES throughout the patient's time course [KRASG12R, MSLNP462H, NTRK3V443I and EZH2I669M ]. Sequenom did not identify these at diagnosis while deep WES did. Clones derived from colony formation assay revealed three distinct clones present in all samples analysed. Clone 1 had only KRASG12R, clone 2 had KRASG12R, MSLNP462H, and NTRK3V443I, and clone 3 had all four mutations. All clones containing any of these four mutations were BCR/ABL1 negative. Analysis of clonal architecture indicated that KRASG12R was acquired first and EZH2I669M last, while MSLNP462H and NTRK3V443I were acquired in between. These CMML clones increased proportionately as clinical CML metamorphosed into clinical CMML after initiation of imatinib therapy. Consistent with the colony data, pyrosequencing revealed that the ratio between the mutants remained largely stable throughout the follow up period.Conclusion. This case illustrates how targeted therapy impacts clonal competition in a heterogeneous MPN. While the CML clone was dominant in the absence of imatinib, it was quickly outcompeted by the CMML clones upon initiation of imatinib therapy. The clonal architecture analysis, in combination with in vivo kinetics data, suggest that the KRASG12R mutation alone was able to produce a CMML phenotype as clones with just KRASG12R remained at a relatively stable ratio during follow up. Unexpectedly, acquisition of additional mutations, including EZH2I669M as the last mutational event identified in this patient, did not increase clonal competitiveness, at least in the peripheral blood. These data show that clonal evolution may not invariably increase clonal fitness, suggesting that factors other than Darwinian pressures contribute to clonal diversity in myeloproliferative neoplasms. [Display omitted] DisclosuresDeininger:Gilead: Research Funding; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Abstract 3747: Identification of NSCLC biomarkers underlying quantifiable drug-induced clonal fitness

Abstract Lung cancer accounts for nearly 20% of all cancer-related deaths worldwide. Subsets of non-small-cell lung cancer (NSCLC; >80% of all lung cancers) harboring activating epidermal growth factor receptor (EGFR) mutations are highly sensitive to erlotinib, a small-molecule EGFR tyrosine kinase inhibitor. However, clinical responses to erlotinib are variable and limited in duration as all tumors eventually relapse. Clonal heterogeneity is thought to be a major cause of relapse, but it has been difficult to quantify in drug-sensitive cancer cell populations. To overcome this obstacle, we quantify clonal fitness via live-cell imaging as a drug-induced proliferation (DIP) rate that incorporates cell division, death, and quiescence into a single metric. This fitness rate varies across cell types from positive (division prevails) to negative (death prevails) and, unlike traditional IC50 measurements, can effectively discriminate intricate degrees of drug sensitivity. Moreover, in monoclonal sublines isolated naïve-to-drug from NSCLC parental cell lines, erlotinib-treated DIP rates calculated in vitro predict the long-term growth rates (250+ days) of in vivo tumor xenografts. Using global proteomics across a panel of monoclonal sublines, we have characterized networks of biomarkers that act as molecular determinants of phenotypic drug fitness. We have identified unsuspected molecular correlates in the EGFR and AKT pathways and are currently investigating whether they can act as lead targets for small molecule inhibition in combination therapies. Our data indicate that a multi-scale systems approach integrating molecular subtyping with quantifiable clonal fitness can yield insights into clonal heterogeneity and lead to novel strategies to overcome tumor relapse. Note: This abstract was not presented at the meeting. Citation Format: Katherine L. Jameson, Peter L. Frick, Darren R. Tyson, Thomas E. Yankeelov, Vito Quaranta. Identification of NSCLC biomarkers underlying quantifiable drug-induced clonal fitness. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3747. doi:10.1158/1538-7445.AM2015-3747

Dynamics of Tumor Heterogeneity Derived from Clonal Karyotypic Evolution.

Numerical chromosomal instability is a ubiquitous feature of human neoplasms. Due to experimental limitations, fundamental characteristics of karyotypic changes in cancer are poorly understood. Using an experimentally inspired stochastic model, based on the potency and chromosomal distribution of oncogenes and tumor suppressor genes, we show that cancer cells have evolved to exist within a narrow range of chromosome missegregation rates that optimizes phenotypic heterogeneity and clonal survival. Departure from this range reduces clonal fitness and limits subclonal diversity. Mapping of the aneuploid fitness landscape reveals a highly favorable, commonly observed, near-triploid state onto which evolving diploid- and tetraploid-derived populations spontaneously converge, albeit at a much lower fitness cost for the latter. Finally, by analyzing 1,368 chromosomal translocation events in five human cancers, we find that karyotypic evolution also shapes chromosomal translocation patterns by selecting for more oncogenic derivative chromosomes. Thus, chromosomal instability can generate the heterogeneity required for Darwinian tumor evolution.

Quantifying heterogeneity and dynamics of clonal fitness in response to perturbation.

The dynamics of heterogeneous clonal lineages within a cell population, in aggregate, shape both normal and pathological biological processes. Studies of clonality typically relate the fitness of clones to their relative abundance, thus requiring long-term experiments and limiting conclusions about the heterogeneity of clonal fitness in response to perturbation. We present, for the first time, a method that enables a dynamic, global picture of clonal fitness within a mammalian cell population. This novel assay allows facile comparison of the structure of clonal fitness in a cell population across many perturbations. By utilizing high-throughput imaging, our methodology provides ample statistical power to define clonal fitness dynamically and to visualize the structure of perturbation-induced clonal fitness within a cell population. We envision that this technique will be a powerful tool to investigate heterogeneity in biological processes involving cell proliferation, including development and drug response.

Identification of a Gene Expression Signature Characterizing Clonal Fitness and Dominance in Vivo in a Murine Model of MLL-AF9 Leukemia

Rationale:Human malignancies are often composed of multiple, related clones that arise through a process of branching Darwinian evolution. In acute myeloid leukemia (AML), high-throughput DNA sequencing identifies clonal heterogeneity at the mutational level, but the downstream molecular pathways driving clonal fitness, and their impact on response to therapy are still poorly understood. We report on the development of a novel experimental tool that allows prospective tracking of clonal evolution at the functional level. Using a combination of murine models of AML and fluorescent protein labeling, we can measure clonal evolution in real time, serially isolate live clones competing in the same environment for phenotypic characterization, and correlate these findings with mutational, epigenetic and gene expression profiling.Methods:A clonal pre-leukemic cell line, derived from a murine granulocyte-macrophage progenitor infected with a retrovirus enforcing expression of the MLL-AF9 fusion protein, was labeled with a pool of lentiviruses driving the expression of multiple fluorescent proteins (FPs). From this cell line, fluorescently distinguishable sub-clones were selected and expanded. This pool of clones was transplanted in multiple recipients. Disease competition and evolution was tracked prospectively in primary recipients through repeated sampling of blood and bone marrow. Secondary leukemic cell lines were established from individual clones harvested at serial time points during growth in vivo. In order to study the cell-intrinsic characteristics acquired following exposure to the bone marrow microenvironment, these secondary cell lines were transplanted into additional recipients to measure engraftment potential, growth rate, cell cycle and apoptosis, gene expression profile and acquisition of secondary mutations.Results:By transplanting in competition multiple pre-leukemic clones in a cohort of primary recipients, we observed emergence of dominant clones during the development of AML. By sampling “winner” clones at different time points, we could observe the gradual acquisition of a cell-intrinsic growth advantage that was preserved in secondary transplantation, whereas control “loser” clones grew at a significantly lower rate. Functional characterization of the winner clones demonstrated a higher engraftment rate in vivo, whereas the self-renewal potential and growth rate in vitro was similar among winner and loser clones. By comparing the gene expression profile of leukemic stem cells isolated from various clones, we observed that the most aggressive clones share a common signature characterized by activation of multiple metabolic pathways, such as steroid biosynthesis and response to oxidative stress. Since these pathways are not activated in the less aggressive clones, we hypothesize that secondary genetic events that promote growth spontaneously occured in vivo. Functional validation of these pathways is underway and, in parallel, constitutes the basis of ongoing pooled genetic perturbation screens to identify novel therapeutic targets that impede malignant cell growth. DisclosuresNo relevant conflicts of interest to declare.

Mutation, clonal fitness and field change in epithelial carcinogenesis.

Developments in lineage tracing in mouse models have revealed how stem cells maintain normal squamous and glandular epithelia. Here we review recent quantitative studies tracing the fate of individual mutant stem cells which have uncovered how common oncogenic mutations alter cell behaviour, creating clones with a growth advantage that may persist long term. In the intestine this occurs by a mutant clone colonizing an entire crypt, whilst in the squamous oesophagus blocking differentiation creates clones that expand to colonize large areas of epithelium, a phenomenon known as field change. We consider the implications of these findings for early cancer evolution and the cancer stem cell hypothesis, and the prospects of targeted cancer prevention by purging mutant clones from normal-appearing epithelia.

Cooperative interactions in the West Nile virus mutant swarm

BackgroundRNA viruses including arthropod-borne viruses (arboviruses) exist as highly genetically diverse mutant swarms within individual hosts. A more complete understanding of the phenotypic correlates of these diverse swarms is needed in order to equate RNA swarm breadth and composition to specific adaptive and evolutionary outcomes.ResultsHere, we determined clonal fitness landscapes of mosquito cell-adapted West Nile virus (WNV) and assessed how altering the capacity for interactions among variants affects mutant swarm dynamics and swarm fitness. Our results demonstrate that although there is significant mutational robustness in the WNV swarm, genetic diversity also corresponds to substantial phenotypic diversity in terms of relative fitness in vitro. In addition, our data demonstrate that increasing levels of co-infection can lead to widespread strain complementation, which acts to maintain high levels of phenotypic and genetic diversity and potentially slow selection for individual variants. Lastly, we show that cooperative interactions may lead to swarm fitness levels which exceed the relative fitness levels of any individual genotype.ConclusionsThese studies demonstrate the profound effects variant interactions can have on arbovirus evolution and adaptation, and provide a baseline by which to study the impact of this phenomenon in natural systems.

A closer look at the spatial architecture of aphid clones

Nearly 25 years ago, Ellstrand & Roose (1987) reviewed what was known at the time of the genetic structure of clonal plant species. What is the relationship between space and clonal fitness, they asked. What is the best way for a clone to grow within its ecological neighbourhood? The pot had been stirred 10 years previously by Janzen (1977), who pointed out how little we know about the population biology of clonal organisms like dandelions and aphids. He wondered whether, like good curries, outward appearances masked common ingredients. Because in no small part of the advent of molecular ecology, we know more about clonal life histories today, particularly in plants (van Dijk 2003; Vallejo-Marin et al. 2010). Surprisingly, studies of the spatial architecture of aphid clones have been comparably rare. In this issue of Molecular Ecology, Vantaux et al. characterize the finescale distribution of the black bean aphid (Aphis fabae) and in so doing, help to fill that gap. They describe a moderate degree of intermingling between aphid clones over a growing season--A. fabae clones are ‘sticky’, but only a bit. By mixing, clones directly compete with each other as well. The results of Vantaux et al. (2011) will help to integrate evolutionary patterns in aphids with the appropriate ecological scales out of which those patterns emerge.

Feed-backs between genetic structure and perturbation-driven decline in seagrass (Posidonia oceanica) meadows

We explored the relationships between perturbation-driven population decline and genetic/genotypic structure in the clonal seagrass Posidonia oceanica, subject to intensive meadow regression around four Mediterranean fish-farms, using seven specific microsatellites. Two meadows were randomly sampled (40 shoots) within 1,600 m2 at each site: the “impacted” station, 5–200 m from fish cages, and the “control” station, around 1,000 m downstream further away (considered a proxy of the pre-impact genetic structure at the site). Clonal richness (R), Simpson genotypic diversity (D*) and clonal sub-range (CR) were highly variable among sites. Nevertheless, the maximum distance at which clonal dispersal was detected, indicated by CR, was higher at impacted stations than at the respective control station (paired t-test: P < 0.05, N = 4). The mean number of alleles (Â) and the presence of rare alleles ( r) decreased at impacted stations (paired t-test: P < 0.05, and P < 0.02, respectively, N = 4). At a given perturbation level (quantified by the organic and nutrient loads), shoot mortality at the impacted stations significantly decreased with CR at control stations (R 2 = 0.86, P < 0.05). Seagrass mortality also increased with  (R 2 = 0.81, P < 0.10), R (R 2 = 0.96, P < 0.05) and D* (R 2 = 0.99, P < 0.01) at the control stations, probably because of the negative correlation between those parameters and CR. Therefore, the effects of clonal size structure on meadow resistance could play an important role on meadow survival. Large genotypes of P. oceanica meadows thus seem to resist better to fish farm-derived impacts than little ones. Clonal integration, foraging advantage or other size-related fitness traits could account for this effect.

A threefold genetic allee effect: population size affects cross-compatibility, inbreeding depression and drift load in the self-incompatible Ranunculus reptans.

A decline in population size can lead to the loss of allelic variation, increased inbreeding, and the accumulation of genetic load through drift. We estimated the fitness consequences of these processes in offspring of controlled within-population crosses from 13 populations of the self-incompatible, clonal plant Ranunculus reptans. We used allozyme allelic richness as a proxy for long-term population size, which was positively correlated with current population size. Crosses between plants of smaller populations were less likely to be compatible. Inbreeding load, assessed as the slope of the relationship between offspring performance and parental kinship coefficients, was not related to population size, suggesting that deleterious mutations had not been purged from small populations. Offspring from smaller populations were on average more inbred, so inbreeding depression in clonal fitness was higher in small populations. We estimated variation in drift load from the mean fitness of outbred offspring and found enhanced drift load affecting female fertility within small populations. We conclude that self-incompatibility systems do not necessarily prevent small populations from suffering from inbreeding depression and drift load and may exacerbate the challenge of finding suitable mates.