Clonal Richness Research Articles

Patients with juvenile idiopathic arthritis have decreased clonal diversity in the CD8+ T cell repertoire response to influenza vaccination.

Recurrent exposures to a pathogenic antigen remodel the CD8+ T cell compartment and generate a functional memory repertoire that is polyclonal and complex. At the clonotype level, the response to the conserved influenza antigen, M158-66 has been well characterized in healthy individuals, but not in patients receiving immunosuppressive therapy or with aberrant immunity, such as those with juvenile idiopathic arthritis (JIA). Here we show that patients with JIA have a reduced number of M158-66 specific RS/RA clonotypes, indicating decreased clonal richness and, as a result, have lower repertoire diversity. By using a rank-frequency approach to analyze the distribution of the repertoire, we found several characteristics of the JIA T cell repertoire to be akin to repertoires seen in healthy adults, including an amplified RS/RA-specific antigen response, representing greater clonal unevenness. Unlike mature repertoires, however, there is more fluctuation in clonotype distribution, less clonotype stability, and more variable IFNy response of the M158-66 specific RS/RA clonotypes in JIA. This indicates that functional clonal expansion is altered in patients with JIA on immunosuppressive therapies. We propose that the response to the influenza M158-66 epitope described here is a general phenomenon for JIA patients receiving immunosuppressive therapy, and that the changes in clonal richness and unevenness indicate a retarded and uneven generation of a mature immune response.

Sex or Fission? Genetics Highlight Differences in Reproductive Strategies of Two Sympatric Fissiparous Sea Cucumber Species in Reunion Island (Southwestern Indian Ocean)

Holothuria leucospilota and Stichopus chloronotus are among the most widespread tropical sea cucumber species usually harvested for food and medicine in Asian countries, for which natural stocks have collapsed worldwide. Both species can reproduce sexually and asexually, and a better understanding of their reproductive strategy can provide useful information for conservation purposes. To describe the genetic structure and diversity of sympatric populations from these species in space and time, individuals were sampled over different sites and seasons in Reunion Island (Southwestern Indian Ocean). They were genotyped using 24 and 9 specific microsatellite markers for H. leucospilota and S. chloronotus, respectively. Multi-locus genotypes (MLG) and lineages (MLL) were identified, and analyses of population structure were performed among sites and seasons. No repeated MLG nor MLL were found for H. leucospilota, demonstrating the absence of asexual reproduction. Populations of H. leucospilota were not genetically differentiated, acting as a metapopulation, with larval exchanges within the reef. Contrarily, repeated MLGs were found for S. chloronotus and all populations were genetically differentiated. Asexual reproduction seems to reach a high level for this species (mean clonal richness = 0.24). For both species, genetic structure was stable through seasons. Thus, these sympatric fissiparous sea cucumber species use two different strategies of reproduction, which may allow them to reduce interspecific competition.

Abstract 2271: Characteristics of peripheral blood T cell receptor repertoire and correlation with response to chemotherapy in patients with breast cancer

Abstract Purpose: The success of immune checkpoint inhibitors therapy in triple negative breast cancer highlights the potential immunogenic characteristics of breast cancer. Increasing evidence have suggested that conventional chemotherapy can exert anti-cancer activity through various immune-based mechanisms. T cell receptor (TCR) diversity is crucial for immune responses and TCR repertoire has been reported acting as predictive and prognostic markers for cancer outcome. Here, we reported the TCR repertoire profiling in breast cancer and its association with chemotherapy and treatment response in breast cancer patients. Experimental design: This study is part of VGH-TAYLOR study, a comprehensive precision medicine study protocol on the heterogeneity of Taiwanese breast cancer patients (NCT04626440). A total of 856 female patients with luminal A, luminal B1, luminal B2, HER2-enriched, or triple-negative breast cancers were recruited. The enrolled subjects contains patients who receive surgery as the first-line treatment followed by adjuvant therapy and who receive neoadjuvant therapy as the first-line treatment followed by surgery. The blood samples from patients during chemotherapy were collected for TCR sequencing. The Oncomine TCR Beta-LR Assay was applied to determine the dynamic change in TCR repertoire. We examined TCR repertoire in terms of clonality, TCR richness/evenness and convergence. Results: Higher TCR clonality in breast cancer patients was associated with lower clonal richness. TCR clonality was positively correlated with age and stage but not tumor size. Patients with recurrence had higher TCR clonality and lower Shannon diversity. Patients with luminal B2 breast cancer showed lower TCR diversity compared to luminal A and triple-negative breast cancer. However, there were no significant differences in CDR3 length and the usage of variable and joining genes among breast cancer subtypes. Notably, both of adjuvant and neoadjuvant chemotherapies increased convergence and clonality while decreased Shannon diversity of TCR. In neoadjuvant settings, lower post-treatment blood TCR richness was associated with complete pathologic response. Conclusion: These data suggested that TCR repertoire is associated with clinicopathological characteristics including stage and recurrence. TCR clonal richness might provide prognostic value for patients receiving neoadjuvant chemotherapy. Citation Format: Chun-Yu Liu, Chi-Cheng Huang, Ji-Lin Chen, Yi-Fang Tsai, Ta-Chung Chao, Pei-Ju Lien, Yen-Shu Lin, Chin-Jung Feng, Yen-Jen Chen, Jiun-I Lai, Jen-Hwey Chiu, Chih-Yi Hsu, Ling-Ming Tseng. Characteristics of peripheral blood T cell receptor repertoire and correlation with response to chemotherapy in patients with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2271.

Clonal diversity and recruitment strategy of the two dominant seagrass species Cymodocea rotundata and Enhalus acoroides in the southern Philippines

The studied fifteen populations of Cymodocea rotundata and Enhalus acoroides in Mindanao, southern Philippines revealed a remarkable strategy for reproduction and dispersal. E. acoroides populations exhibits high rates of sexual recruitment with high clonal richness ranged from 0.9 to 1.0, while C. rotundata exhibited monoclonal to high clonal richness ranged from 0 to 1.0. Overall, clonal richness was high indicating sexual reproduction is prevalent, but varied for C. rotundata. The largest genet found in the study was at Rizal (RIZ), northern Mindanao wherein throughout the sampling area only one genet was identified, maybe influenced by environmental condition and hydrodynamic of the water currents and tidal fluctuation in this range-edge population. Significant pairwise population genetic differentiation was found among many sites. Isolation by distance (IBD) was detected in C. rotundata (P < 0.05) but not in E. acoroides (P = 0.253). IBD manifested among populations maybe influenced by the ocean currents in Mindanao, but it has to be confirmed yet in the future study. The results have implications for understanding on how the environmental conditions can influenced the dispersal strategy of these species in natural settings. These species manifested high plasticity most likely to adopt to environmental conditions favoring natural selection for long term demographic stability and fitness. The genetic diversity, structure, reproduction and dispersal strategies observed in this study may prove useful for eventual genetic conservation plans of these two foundational seagrass species.

High Prevalence of Clonal Reproduction and Low Genetic Diversity in Scutellaria floridana, a Federally Threatened Florida-Endemic Mint

The threatened mint Florida skullcap (Scutellaria floridana) is endemic to four counties in the Florida panhandle. Because development and habitat modification extirpated several historical occurrences, only 19 remain to date. To inform conservation management and delisting decisions, a comprehensive investigation of the genetic diversity and relatedness, population structure, and clonal diversity was conducted using SNP data generated by ddRAD. Compared with other Lamiaceae, we detected low genetic diversity (HE = 0.125-0.145), low to moderate evidence of inbreeding (FIS = -0.02-0.555), and moderate divergence (FST = 0.05-0.15). We identified eight populations with most of the genetic diversity, which should be protected in situ, and four populations with low genetic diversity and high clonality. Clonal reproduction in our circular plots and in 92% of the sites examined was substantial, with average clonal richness of 0.07 and 0.59, respectively. Scutellaria floridana appears to have experienced a continued decline in the number of extant populations since its listing under the Endangered Species Act; still, the combination of sexual and asexual reproduction may be advantageous for maintaining the viability of extant populations. However, the species will likely require ongoing monitoring, management, and increased public awareness to ensure its survival and effectively conserve its genetic diversity.

Genetic Structure of The Dominant Tropical Seagrasses Cymodocea rotundata and Enhalus acoroides in Southern Philippines for Conservation Management

Large‐scale genetic population study of the two dominant tropical species of seagrass is conducted in Mindanao, southern Philippines. The goal of the study was to understand population genetic status of the dominant tropical seagrass species, Cymodocea rotundata and Enhalus acoroides for appropriate management. Population genetics structure for the 15 sites was performed by using polymorphic microsatellite markers. The results showed that the clonal richness was high in E. acoroides than C. rotundata. The largest genet found in the study was at Rizal, northern Mindanao wherein throughout the sampling area (i.e. 300 x 40 m) only one genet was identified for C. rotundata. The mean FIS (coefficient of local inbreeding) values was positive (heterozygous deficit) with some sites deviated from Hardy-Weinberg Equilibrium. Isolation by distance (IBD) was detected in C. rotundata (P&lt;0.05) but not in E. acoroides (P=0.253), with the Mindanao ocean currents influenced genetic connectivity and structure. Genetic differentiation did not show any relationship between the geographical location and distance exhibiting high FST values for E. acoroides (0.183) and C. rotundata (0.205). The floating, buoyant fruits of E. acoroides may play a role in their long-distance dispersal; however, such dispersal is not frequent. Almost all of the seeds and fruits of C. rotundata are derived from self-recruitment in the natal meadow. This study suggests that C. rotundata and E. acoroides populations possess a weak genetic connectivity, and that the persistence of the meadow is threatened due to the low genetic diversity and high degree of population isolation.

Spatial genetic structure reveals migration directionality in Mediterranean Ruppia spiralis (Western Sicily)

Mediterranean salinas, originally built for salt production, function as alternative wetlands. A variety of accompanying lagoon, ditch, and marsh systems are suitable habitats for salt-tolerant submerged macrophytes and often characterized by monospecific beds of Ruppia. Traditionally, birds are considered the main dispersal vector of submerged macrophytes. However, Ruppia spiralis habitats are under marine influence and therefore interference of coastal currents in their connectivity might be expected. In this study, we aim to infer connectivity and spatial patterns from population genetic structures. Using nuclear microsatellite loci, the nuclear ribosomal cistron and chloroplast sequences, we investigated the genetic diversity, genetic structure, and demographic history of 10 R. spiralis populations along a 25-km coastal stretch of western Sicily encompassing a variety of saline habitats. We tested for local fine-scaled structures, hypotheses of regional isolation by distance, and migration directionality. Our results revealed a high degree of allele and gene diversity that was locally maintained by outcrossing. At the regional level, we detected isolation by distance and identified three genetically differentiated clusters, with a significant structure that matches an overall north-to-south unidirectional migration model. This directionality follows the main sea current, hence indicating the importance of hydrological connectivity in regional conservation management. Significant fine-scale spatial structures only emerged in some populations and were absent in the ‘salina fridda’ habitat that showed the largest clonal richness. The local site-dependent patterns emphasize a need to examine the influence of disturbances on seed recruitment and clonal growth over small distances.

Global Analysis of Hemileia vastatrix Populations Shows Clonal Reproduction for the Coffee Leaf Rust Pathogen Throughout Most of Its Range.

Hemileia vastatrix is the most important fungal pathogen of coffee and the causal agent of recurrent disease epidemics that have invaded nearly every coffee growing region in the world. The development of coffee varieties resistant to H. vastatrix requires fundamental understanding of the biology of the fungus. However, the complete life cycle of H. vastatrix remains unknown, and conflicting studies and interpretations exist as to whether the fungus is undergoing sexual reproduction. Here we used population genetics of H. vastatrix to infer the reproductive mode of the fungus across most of its geographic range, including Central Africa, Southeast Asia, the Caribbean, and South and Central America. The population structure of H. vastatrix was determined via eight simple sequence repeat markers developed for this study. The analyses of the standardized index of association, Hardy-Weinberg equilibrium, and clonal richness all strongly support asexual reproduction of H. vastatrix in all sampled areas. Similarly, a minimum spanning network tree reinforces the interpretation of clonal reproduction in the sampled H. vastatrix populations. These findings may have profound implications for resistance breeding and management programs against H. vastatrix.

Exploring the Pocillopora cryptic diversity: a new genetic lineage in the Western Indian Ocean or remnants from an ancient one?

Cryptic species and lineages have been widely reported during the last decades, particularly in the marine realm. Misidentifications and ignoring species complexes imply many consequences, notably biasing biodiversity and connectivity assessments, which in turn mislead our understanding of ecosystems and impact the effective design and management of conservation plans. Focusing on the Indo-Pacific coral genus Pocillopora, playing key roles in reef ecosystems as one of the main bio-constructors, we report the first Pocillopora PSH16 (ORF53; sensu Gélin et al. 2017) colonies (N = 19) in the Western Indian Ocean (Nosy Tanikely, Madagascar), 6000 km further from its current distribution. Colonies were identified according to their mitochondrial open reading frame (ORF) haplotype and Bayesian assignment tests based on 13-microsatellite genotypes. Additionally, we performed genetic structure and diversity analyses with sympatric colonies from other Pocillopora species and Pocillopora PSH16 colonies from the tropical southwestern Pacific, revealing (1) a weak clonal richness, (2) a weak genetic diversity and (3) a relative isolation for the newly reported PSH16 colonies. These colonies thus represent either a new, distinct and uncommon, genetic lineage or isolated remnants of a wider one. In any case, unless specific management measures are implemented, their long-term maintenance seems compromised due to restricted gene flow within a restricted pool of genes.

Cyanobacterial bloom associated with a complete turnover of a Daphnia population in a warm‐temperate eutrophic lake in Eastern China

Abstract The effect of cyanobacterial blooms on aquatic ecosystems has received wide attention, yet little is known about their impacts on zooplankton genetic structure. For 26 months (April 2012–May 2014), we monitored zooplankton in a warm‐temperate ice‐free lake in Eastern China, with an emphasis on seasonal population dynamics of the cladoceran, Daphnia galeata. There was a seasonal succession in the zooplanktonic community (composed of Cladocera, Copepoda, and Rotifera). Genetic analysis (based on 14 microsatellite loci) of the 21 monthly samples in which D. galeata was present demonstrated that several clones could successfully overwinter and/or persist for many months. However, all clones, including these long‐lived clones, were completely replaced by new ones after October 2013, probably due to cyanobacteria blooms in that summer. A high clonal richness coupled with high turnover rate was observed in the D. galeata population overall, suggesting frequent sexual reproduction. Daphnia galeata experimentally fed with a diet of Microcystis aeruginosa (the most abundant toxic cyanobacterial species in the lake) had a substantially reduced survivorship, consistent with the field observations. Our findings highlight the changes of zooplanktonic population dynamics in terms of both community and clonal structure in a warm‐temperate ice‐free lake, and call for further investigation on ecological responses of zooplankton to cyanobacterial blooms.

Clonal Architecture and Evolutionary Dynamics in Acute Myeloid Leukemias.

Simple SummaryAcute myeloid leukemias (AML) results from the accumulation of genetic and epigenetic alterations, often in the context of an aging hematopoietic environment. The development of high-throughput sequencing—and more recently, of single-cell technologies—has shed light on the intratumoral diversity of leukemic cells. Taking AML as a model disease, we review the multiple sources of heterogeneity of leukemic cells and discuss the definition of a leukemic clone. After introducing the two dimensions contributing to clonal diversity, namely, richness (number of leukemic clones) and evenness (distribution of clone sizes), we discuss the mechanisms at the origin of clonal emergence and the causes of clonal dynamics including neutral drift, and cell-intrinsic and -extrinsic influences on clonal fitness. After reviewing the prognostic role of leukemic diversity on patients’ outcome, we discuss how a better understanding of AML as an evolutionary process could lead to the design of novel therapeutic strategies for this disease. Acute myeloid leukemias (AML) results from the accumulation of genetic and epigenetic alterations, often in the context of an aging hematopoietic environment. The development of high-throughput sequencing—and more recently, of single-cell technologies—has shed light on the intratumoral diversity of leukemic cells. Taking AML as a model disease, we review the multiple sources of genetic, epigenetic, and functional heterogeneity of leukemic cells and discuss the definition of a leukemic clone extending its definition beyond genetics. After introducing the two dimensions contributing to clonal diversity, namely, richness (number of leukemic clones) and evenness (distribution of clone sizes), we discuss the mechanisms at the origin of clonal emergence (mutation rate, number of generations, and effective size of the leukemic population) and the causes of clonal dynamics. We discuss the possible role of neutral drift, but also of cell-intrinsic and -extrinsic influences on clonal fitness. After reviewing available data on the prognostic role of genetic and epigenetic diversity of leukemic cells on patients’ outcome, we discuss how a better understanding of AML as an evolutionary process could lead to the design of novel therapeutic strategies in this disease.

Genetic homogeneity, lack of larvae recruitment, and clonality in absence of females across western Mediterranean populations of the starfish Coscinasterias tenuispina

We here analysed the populations’ genetic structure of Coscinasterias tenuispina, an Atlantic-Mediterranean fissiparous starfish, focusing on the western Mediterranean, to investigate: the distribution and prevalence of genetic variants, the relative importance of asexual reproduction, connectivity across the Atlantic-Mediterranean transition, and the potential recent colonisation of the Mediterranean Sea. Individuals from 11 Atlantic-Mediterranean populations of a previous study added to 172 new samples from five new W Mediterranean sites. Individuals were genotyped at 12 microsatellite loci and their gonads histologically analysed for sex determination. Additionally, four populations were genotyped at two-time points. Results demonstrated genetic homogeneity and low clonal richness within the W Mediterranean, due to the dominance of a superclone, but large genetic divergence with adjacent areas. The lack of new genotypes recruitment over time, and the absence of females, confirmed that W Mediterranean populations were exclusively maintained by fission and reinforced the idea of its recent colonization. The existence of different environmental conditions among basins and/or density-depend processes could explain this lack of recruitment from distant areas. The positive correlation between clonal richness and heterozygote excess suggests that most genetic diversity is retained within individuals in the form of heterozygosity in clonal populations, which might increase their resilience.

Persistent Clones and Local Seed Recruitment Contribute to the Resilience of Enhalus acoroides Populations Under Disturbance

Human-induced land use in coastal areas is one of the main threats for seagrass meadows globally causing eutrophication and sedimentation. These environmental stressors induce sudden ecosystem shifts toward new alternative stable states defined by lower seagrass richness and abundance. Enhalus acoroides, a large-sized tropical seagrass species, appears to be more resistant toward environmental change compared to coexisting seagrass species. We hypothesize that reproductive strategy and the extent of seedling recruitment of E. acoroides are altered under disturbance and contribute to the persistence and resilience of E. acoroides meadows. In this research, we studied eight populations of E. acoroides in four lagoons along the South Central Coast of Vietnam using 11 polymorphic microsatellite loci. We classified land use in 6 classes based on Sentinel-2 L2A images and determined the effect of human-induced land use at different spatial scales on clonal richness and structure, fine-scale genetic structure and genetic diversity. No evidence of population size reductions due to disturbance was found, however, lagoons were strongly differentiated and may act as barriers to gene flow. The proportion and size of clones were significantly higher in populations of surrounding catchments with larger areas of agriculture, urbanization and aquaculture. We postulate that large resistant genets contribute to the resilience of E. acoroides meadows under high levels of disturbance. Although the importance of clonal growth increases with disturbance, sexual reproduction and the subsequent recruitment of seedlings remains an essential strategy for the persistence of populations of E. acoroides and should be prioritized in conservation measures to ensure broad-scale and long-term resilience toward future environmental change.

The discernible and hidden effects of clonality on the genotypic and genetic states of populations: Improving our estimation of clonal rates.

Partial clonality is widespread across the tree of life, but most population genetic models are designed for exclusively clonal or sexual organisms. This gap hampers our understanding of the influence of clonality on evolutionary trajectories and the interpretation of population genetic data. We performed forward simulations of diploid populations at increasing rates of clonality (c), analysed their relationships with genotypic (clonal richness, R, and distribution of clonal sizes, Pareto β) and genetic (FIS and linkage disequilibrium) indices, and tested predictions of c from population genetic data through supervised machine learning. Two complementary behaviours emerged from the probability distributions of genotypic and genetic indices with increasing c. While the impact of c on R and Pareto β was easily described by simple mathematical equations, its effects on genetic indices were noticeable only at the highest levels (c>0.95). Consequently, genotypic indices allowed reliable estimates of c, while genetic descriptors led to poorer performances when c<0.95. These results provide clear baseline expectations for genotypic and genetic diversity and dynamics under partial clonality. Worryingly, however, the use of realistic sample sizes to acquire empirical data systematically led to gross underestimates (often of one to two orders of magnitude) of c, suggesting that many interpretations hitherto proposed in the literature, mostly based on genotypic richness, should be reappraised. We propose future avenues to derive realistic confidence intervals for c and show that, although still approximate, a supervised learning method would greatly improve the estimation of c from population genetic data.

Shifts in coral clonality along a gradient of disturbance: insights on reproduction and dispersal of Pocillopora acuta

Pocillopora acuta, formerly synonymized with P. damicornis, is an ecologically important reef-building coral that exhibits mixed reproductive modes, geographic variation in clonality, and conflicting reports of population genetic structure. Using 16 polymorphic microsatellite loci, this study examined clonality, genetic differentiation, and connectivity of genetically identified P. acuta (n = 428) in the Bolinao–Anda Reef Complex (BARC), Philippines, characterized by varying levels of wave exposure. Estimates of clonal richness indicate that the populations are largely derived from asexual reproduction, more likely via dispersal of ameiotic larvae. Clonal richness, population density, and mean colony size vary with wave exposure, suggesting the potential influence of local-scale disturbance on clonality, reproductive mode, and population structure. Populations in low-energy environments were characterized by greater colony density, larger colonies, and a greater proportion of clones compared to high-energy environments. Despite evidence for realized clonal dispersal of P. acuta extending up to 22 km, significant genetic differentiation among BARC populations reveals restricted gene flow at small spatial scales. Moreover, genetic differentiation is more pronounced when considering the spatial distribution of clones (FST including clones = 0.059; FST excluding clones = 0.028), suggesting that (1) asexually produced propagules are likely retained locally and across-site settlement is not as common; and (2) sexually derived propagules may have broader scales of dispersal. This study reexamines the population genetics of this often-problematic coral and underlines the importance of contextualizing site and species biology in designing or enhancing management towards the maintenance of functional genetic diversity and pathways of connectivity among populations.

Linking Spatial Patterns of Adult and Seed Diversity Across the Depth Gradient in the Seagrass Zostera marina L.

Sexual reproduction is the primary source of genotypic diversity in seagrass meadows, with important implications for both the evolutionary potential and ecological function of these habitats. However, relatively little is known about seagrass pollination dynamics or how genetic diversity in life history stage may influence genetic diversity in another. We used microsatellite DNA markers to assess how genetic diversity of adult shoots and seeds covaried across the depth gradient in two Zostera marina L. (eelgrass) meadows in Massachusetts (USA). Despite strong spatial patterns in the density of reproductive shoots and genetic evidence for limited pollen dispersal, we found modest corresponding variation in outcrossing rates and paternal diversity. Across spatial scales from quadrats to sites, there was a positive association between the density of flowering shoots and clonal richness; however, other measures of diversity (e.g., allelic richness and heterozygosity) showed weak or opposing patterns with reproductive effort, and neither flowering effort nor local genotypic richness of adults were good proxies for seed diversity. Self-pollinated seeds were significantly smaller than their outcrossed half-siblings, and our data suggest that selection against selfed seeds varies spatially within and among meadows, possibly due to high competition with vegetative shoots in the dense shallow margin. Understanding the processes that shape genetic diversity in seagrass populations is key to their conservation and management; our study highlights the challenges of inferring the contribution of sexual reproduction to population structure using a single metric or life history stage.

Abstract 5490: Scales and dynamics of intratumor heterogeneity

Abstract Current dogma stipulates that more heterogenous tumor cell populations results from compounding genetic and epigenetic changes and instability, ultimately driving unfavorable outcomes for these patients. Nonetheless, some cancers, including many pediatric cancers and some leukemias, have limited genomic diversity. As a result, we have a limited number of biomarkers available to stratify patients into high versus low-risk groups by their mutational cellular diversity or heterogeneity. For these cases, we have developed a method to quantify intratumor heterogeneity, for example at the transcriptomic level, using a generalized diversity index (GDI) from ecology. This index allows control of a scaling parameter called the order of diversity, q, to tailor the scale or resolution of the cellular diversity to a given context. However, the question remains, what role does the order of diversity play in the context of relative tumor age or disease stage? We show that the order of diversity parameter allows to either emphasize clonal richness (at low values of q), while high values shift the analysis toward the abundance of potential drivers of the tumor evolution. To gain deeper quantitative insight, we use evolutionary dynamics approaches that model the dynamics of heterogeneous cell populations over time, to understand how a patient's tumor diversity could emerge, persist, and change over time. Applying the replicator-mutator dynamics framework, we explore the effects and magnitude of frequency-dependent (positive or negative) selection on GDI. These analyses suggest that GDI often peaks at intermediate time points and, when run over a sufficiently long timescale, they reduce as the tumor population presumable becomes clonally dominant or otherwise stable before a major transition (e.g. metastasis). We explore GDI changes over time further in vitro in sequential single-cell RNA sequencing samples of fused breast cancer cells, where at earlier passages after fusion, the GDI peaks, and at later passages after fusion the GDI returns to levels similar to that of the parental breast cancer cell lines. Overall, GDI, as a means to quantify intratumor heterogeneity at select time points, can be a powerful tool to stratify high-risk patients. Our analyses show how computational analyses and mathematical modeling tools can be coupled with clinical and experimental data to understand eco-evolutionary dynamics of a patient's tumor. Citation Format: Meghan C. Ferrall-Fairbanks, Gregory J. Kimmel, Philipp M. Altrock. Scales and dynamics of intratumor heterogeneity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5490.

Abstract 173: Profiling T cell clonality in FFPE samples using a targeted sequencing approach: Critical parameters and limitations

Abstract With the rapid development of immuno-therapy approaches for cancer treatment, tools that characterize tumor-infiltrating immune cells fuel new opportunities to better understand the natural defense mechanisms against tumor cells. The AmpliSeqTM for Illumina Immune Repertoire Plus, TCR beta SR Panel is a targeted sequencing assay that assesses T cell clonal diversity by amplifying the CDR3 region of the TCRβ gene in both DNA or RNA. This study describes the performance of this assay on peripheral blood mononuclear cells (PBMC) as high-quality input as well as formalin-fixed paraffin embedded (FFPE) samples, using the MiXCR Immune Repertoire Analyzer app for clonotype analysis. Multiple replicate libraries prepared from RNA (n=6) and DNA (n=12) extracted from the same PBMC donor showed a minimum of 96.4% alignment, with high reproducibility between replicates for clonotypes (&amp;lt;7% CV). Interestingly the RNA input had a 25x higher clonal diversity compared to its DNA counterpart, highlighting a fundamental difference between the two inputs to be taken into consideration for optimal sequencing requirements. Indeed, when we tested various inputs of DNA or RNA at different sequencing depths, we found that the clonotype count reaches saturation at a much lower read depth for DNA samples than for RNA. Looking at the top 50 clones ranked by abundance, we also noticed that the DNA assay only showed 58% common clones between 4 replicates whereas the respective RNA assay showed a much higher replicate reproducibility (96% of common clones). To further investigate the sensitivity of this assay, we modelled the biological representation of immune cells and nucleic acid quality in FFPE tumor samples by titrating heat-treated PBMC RNA (10%, 1% and 0.5%) into a TCRβ-negative RNA background. The Shannon index, a classic TCRβ clonal richness metric, showed that RNA quality was a more critical parameter than the input amount. Evenness (clone frequency), however, was robust to both parameters. Looking at the top 50 clones in four FFPE samples, we noticed a clear split between two distinct trace patterns: a very skewed frequency distribution with a couple of highly represented clones (“activated pattern”) versus a more even distribution, like PBMC controls. Except for the lymph node biopsy, all three skin biopsies showed a direct correlation between the plot pattern and the pathologist inflammation assessment. Taken together, with this limited dataset, this study shows that AmpliSeqTM for Illumina Immune Repertoire Plus, TCR beta SR Panel is a flexible assay that delivers T cell clonal diversity information, even in challenging samples like FFPE. While the data significance still remains to be established, its availability to the immuno-oncology research field opens up promising avenues for future cancer treatment discoveries. Citation Format: Valerie N. Montel, Stephanie B. Greene, Jeff H. Tsai. Profiling T cell clonality in FFPE samples using a targeted sequencing approach: Critical parameters and limitations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 173.

Long‐term seagrass degradation: Integrating landscape, demographic, and genetic responses

Abstract Seagrass meadows are crucial habitats on nearshore areas, which are exposed to human disturbances that frequently cause seagrass loss. Although demographic and mapping data have been widely used in long‐term monitoring programmes, the integration of multi‐locus genotype data through time remains rare. The present work links, for the first time, landscape, demographic, and genetic data for a seagrass, so as to explore long‐term responses to persistent human impacts. The temporal patterns in meadow area, shoot density, and clonal and genetic diversity of three Cymodocea nodosa meadows were compared: in one meadow directly affected by the construction of an industrial port and in two control areas. The hypothesis tested was that seagrass deterioration and subsequent habitat loss at the affected meadow would be associated with a reduction of seagrass clonal and genetic diversity. The results show significant reductions in both meadow area (from approx. 21 to 1.5 ha) and shoot density (from approx. 1,800 to 400 shoots per m2) at the impacted meadow, with concurrently reduced clonal and allelic richness (by approx. 22%), as well as heterozygosity (HLmean, approx. 21%; Hobs, approx. 12%). These descriptors, however, remained stable, or even increased, in the controls. Importantly for seagrass management, multi‐disciplinary analysis made it possible to link slower, large‐scale, landscape and genetic degradation processes with faster demographic deterioration on an intermediate meadow scale. Multi‐locus genotype data were also crucial for revealing long‐term degradation processes at the genetic level, which is undetectable by traditional monitoring techniques. In summary, this holistic approach provides a valuable framework for long‐term seagrass monitoring programmes to detect cumulative signals of seagrass meadow degradation across a range of scales.

Genetic structure in hybrids and progenitors provides insight into processes underlying an invasive cattail (Typha × glauca) hybrid zone.

Traditional models of hybrid zones have assumed relatively low hybrid fitness, and thus focussed more on interspecific gene flow than on hybrid dispersal. Therefore, when hybrids have high fitness and the potential for autonomous dispersal, we have limited understanding of whether hybrid dispersal or repeated local hybrid formation is more important for maintaining hybrid zones. The invasive hybrid cattail Typha × glauca occupies an extensive hybrid zone in northeastern North America where it is sympatric with its progenitors T. latifolia and T. angustifolia. We characterized genetic diversity and genetic structure of the three taxa across a broad spatial scale where the maternal parent is relatively rare, and tested the hypothesis that the hybrid shows stronger evidence of gene flow than its progenitor species, particularly among disturbed sites (ditches) compared with established wetlands. Support for this hypothesis would suggest that dispersal, rather than repeated local formation, is more important for maintaining hybrid zones. Within each taxon, genetic differentiation among ditches was comparable to that among wetlands, although clonal richness was consistently greater in ditches, suggesting more frequent seed establishment. Genetic structure across sites was more pronounced in the hybrid compared with either progenitor species. Overall, our data reflect relatively low gene flow in hybrids, and suggest that hybrids are more likely to be created in situ than to be introduced from other sites. Despite the high fitness of invasive T. × glauca and its potential for autonomy, local processes appear more important than dispersal in maintaining this hybrid zone.