Molnupiravir maintains antiviral activity against SARS-CoV-2 variants and exhibits a high barrier to the development of resistance.

Molnupiravir, an oral prodrug of N-hydroxycytidine (NHC), previously demonstrated broad in vitro antiviral activity against multiple RNA viruses and has shown a high barrier to the development of resistance. Here, we present the antiviral activity of NHC against recent SARS-CoV-2 variants and the results of resistance selection studies to better understand the potential for viral resistance to NHC. NHC activity against SARS-CoV-2 variants omicron (BA.1, BA.1.1, BA.2, BA.4, BA.4.6, BA.5, BQ.1.1, XBB.1, and XBB.1.5), alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), lambda (C.37), and mu (B.1.621) was evaluated in Vero E6 cells using cytopathic effect assays. Resistance selection studies were performed by passaging SARS-CoV-2 (WA1) in the presence of NHC or a 3C-like protease inhibitor (MRK-A) in Vero E6 cells. Supernatants from cultures exhibiting a cytopathic effect score of ≥2 were re-passaged, and IC50 values were estimated. Whole-genome deep sequencing was performed on viral RNA isolated at each passage. NHC demonstrated similar potency against all SARS-CoV-2 variants evaluated. No evidence of SARS-CoV-2 phenotypic or genotypic resistance to NHC was observed following 30 passages. A random pattern of nucleotide changes was observed in NHC cultures, consistent with the drug's mechanism of action. In contrast, resistance was readily selected in all three MRK-A control cultures with the selection of a T21I substitution in the 3C-like protease. In conclusion, molnupiravir maintains antiviral activity across all major SARS-CoV-2 variants. Furthermore, no evidence of viral resistance to NHC was observed, supporting previous reports that NHC has a high barrier to developing resistance.

Short paired-end reads trump long single-end reads for expression analysis.

BACKGROUND: Typical experimental design advice for expression analyses using RNA-seq generally assumes that single-end reads provide robust gene-level expression estimates in a cost-effective manner, and that the additional benefits obtained from paired-end sequencing are not worth the additional cost. However, in many cases (e.g., with Illumina NextSeq and NovaSeq instruments), shorter paired-end reads and longer single-end reads can be generated for the same cost, and it is not obvious which strategy should be preferred. Using publicly available data, we test whether short-paired end reads can achieve more robust expression estimates and differential expression results than single-end reads of approximately the same total number of sequenced bases. RESULTS: At both the transcript and gene levels, 2 × 40 paired-end reads unequivocally provide expression estimates that are more highly correlated with 2 × 125 than 1 × 75 reads; in nearly all cases, those correlations are also greater than for 1 × 125, despite the greater total number of sequenced bases for the latter. Across an array of metrics, differential expression tests based upon 2 × 40 consistently outperform those using 1 × 75. CONCLUSION: Researchers seeking a cost-effective approach for gene-level expression analysis should prefer short paired-end reads over a longer single-end strategy. Short paired-end reads will also give reasonably robust expression estimates and differential expression results at the isoform level.

Electrophysiological evidence that psychopathic personality traits are associated with atypical response to salient distractors.

Event-related potentials (ERPs) were used to assess the neural mechanisms underlying visual-spatial attention abnormalities associated with psychopathic personality traits. Sixty-nine undergraduates (56 women, 13 men) completed the Psychopathic Personality Inventory-Revised (PPI-R; Lilienfeld & Widows, 2005) and performed two cognitive tasks in which search displays containing a lateralized singleton encircled a fixation point that changed luminance from trial-to-trial. When searching for the singleton as a target, PPI-R scores were uncorrelated with ERP measures of its salience (Ppc), goal-directed selection (N2pc), and working memory evaluation (negative amplitude CDA). In contrast, when responding to the changes in luminance at fixation and ignoring the lateral singleton as a salient distractor, PPI-R Self-Centered Impulsivity factor scores were positively correlated with a potential indicator of distractor suppression (a sustained positive amplitude CDA). These findings provide support for a neurophysiological interpretation of the changes in visual-spatial attention associated with psychopathic personality traits: normal selection of target information accompanied by greater elimination of distractor information at a later visual working memory stage.

People with schizophrenia show enhanced cognitive costs of maintaining a single item in working memory.

BACKGROUND: Working memory (WM) deficits are seen as a core deficit in schizophrenia, implicated in the broad cognitive impairment seen in the illness. Here we examine the impact of WM storage of a single item on the operation of other cognitive systems. METHODS: We studied 37 healthy controls (HCS) and 43 people with schizophrenia (PSZ). Each trial consisted of a sequence of two potential target stimuli, T1 and T2. T1 was a letter presented for 100 ms. After delays of 100-800 ms, T2 was presented. T2 was a 1 or a 2 and required a speeded response. In one condition, subjects were instructed to ignore T1 but respond to T2. In another condition, they were required to report T1 after making their speeded response to T2 (i.e. to make a speeded T2 response while holding T1 in WM). RESULTS: PSZ were dramatically slowed at responding to T2 when T1 was held in WM. A repeated measures ANOVA yielded main effects of group, delay, and condition with a group by condition interaction (p's < 0.001). Across delays, the slowing of the T2 response when required to hold T1 in memory, relative to ignoring T1, was nearly 3 times higher in PSZ than HCS (633 v. 219 ms). CONCLUSIONS: Whereas previous studies have focused on reduced storage capacity, the present study found that PSZ are impaired at performing tasks while they are successfully maintaining a single item in WM. This may play a role in the broad cognitive impairment seen in PSZ.

Inability to suppress salient distractors predicts low visual working memory capacity.

According to contemporary accounts of visual working memory (vWM), the ability to efficiently filter relevant from irrelevant information contributes to an individual's overall vWM capacity. Although there is mounting evidence for this hypothesis, very little is known about the precise filtering mechanism responsible for controlling access to vWM and for differentiating low- and high-capacity individuals. Theoretically, the inefficient filtering observed in low-capacity individuals might be specifically linked to problems enhancing relevant items, suppressing irrelevant items, or both. To find out, we recorded neurophysiological activity associated with attentional selection and active suppression during a competitive visual search task. We show that high-capacity individuals actively suppress salient distractors, whereas low-capacity individuals are unable to suppress salient distractors in time to prevent those items from capturing attention. These results demonstrate that individual differences in vWM capacity are associated with the timing of a specific attentional control operation that suppresses processing of salient but irrelevant visual objects and restricts their access to higher stages of visual processing.

NGmerge: merging paired-end reads via novel empirically-derived models of sequencing errors.

BACKGROUND: Advances in Illumina DNA sequencing technology have produced longer paired-end reads that increasingly have sequence overlaps. These reads can be merged into a single read that spans the full length of the original DNA fragment, allowing for error correction and accurate determination of read coverage. Extant merging programs utilize simplistic or unverified models for the selection of bases and quality scores for the overlapping region of merged reads. RESULTS: We first examined the baseline quality score - error rate relationship using sequence reads derived from PhiX. In contrast to numerous published reports, we found that the quality scores produced by Illumina were not substantially inflated above the theoretical values, once the reference genome was corrected for unreported sequence variants. The PhiX reads were then used to create empirical models of sequencing errors in overlapping regions of paired-end reads, and these models were incorporated into a novel merging program, NGmerge. We demonstrate that NGmerge corrects errors and ambiguous bases better than other merging programs, and that it assigns quality scores for merged bases that accurately reflect the error rates. Our results also show that, contrary to published analyses, the sequencing errors of paired-end reads are not independent. CONCLUSIONS: We provide a free and open-source program, NGmerge, that performs better than existing read merging programs. NGmerge is available on GitHub ( https://github.com/harvardinformatics/NGmerge ) under the MIT License; it is written in C and supported on Linux.

DNA methylome and transcriptome alterations and cancer prevention by curcumin in colitis-accelerated colon cancer in mice.

Inflammation is highly associated with colon carcinogenesis. Epigenetic mechanisms could play an important role in the initiation and progression of colon cancer. Curcumin, a dietary phytochemical, shows promising effects in suppressing colitis-associated colon cancer in azoxymethane-dextran sulfate sodium (AOM-DSS) mice. However, the potential epigenetic mechanisms of curcumin in colon cancer remain unknown. In this study, the anticancer effect of curcumin in suppressing colon cancer in an 18-week AOM-DSS colon cancer mouse model was confirmed. We identified lists of differentially expressed and differentially methylated genes in pairwise comparisons and several pathways involved in the potential anticancer effect of curcumin. These pathways include LPS/IL-1-mediated inhibition of RXR function, Nrf2-mediated oxidative stress response, production of NO and ROS in macrophages and IL-6 signaling. Among these genes, Tnf stood out with decreased DNA CpG methylation of Tnf in the AOM-DSS group and reversal of the AOM-DSS induced Tnf demethylation by curcumin. These observations in Tnf methylation correlated with increased and decreased Tnf expression in RNA-seq. The functional role of DNA methylation of Tnf was further confirmed by in vitro luciferase transcriptional activity assay. In addition, the DNA methylation level in a group of inflammatory genes was decreased in the AOM+DSS group but restored by curcumin and was validated by pyrosequencing. This study shows for the first time epigenomic changes in DNA CpG methylation in the inflammatory response from colitis-associated colon cancer and the reversal of their CpG methylation changes by curcumin. Future clinical epigenetic studies with curcumin in inflammation-associated colon cancer would be warranted.

High Level of Trait Anxiety Leads to Salience-Driven Distraction and Compensation.

Individuals with high levels of anxiety are hypothesized to have impaired executive control functions that would otherwise enable efficient filtering of irrelevant information. Pinpointing specific deficits is difficult, however, because anxious individuals may compensate for deficient control functions by allocating greater effort. Here, we used event-related-potential indices of attentional selection (the N2pc) and suppression (the PD) to determine whether high trait anxiety is associated with a deficit in preventing the misallocation of attention to salient, but irrelevant, visual search distractors. Like their low-anxiety counterparts ( n = 19), highly anxious individuals ( n = 19) were able to suppress the distractor, as evidenced by the presence of a PD. Critically, however, the distractor was found to trigger an earlier N2pc in the high-anxiety group but not in the low-anxiety group. These findings indicate that, whereas individuals with low anxiety can prevent distraction in a proactive fashion, anxious individuals deal with distractors only after they have diverted attention.

DMRfinder: efficiently identifying differentially methylated regions from MethylC-seq data.

BACKGROUND: DNA methylation is an epigenetic modification that is studied at a single-base resolution with bisulfite treatment followed by high-throughput sequencing. After alignment of the sequence reads to a reference genome, methylation counts are analyzed to determine genomic regions that are differentially methylated between two or more biological conditions. Even though a variety of software packages is available for different aspects of the bioinformatics analysis, they often produce results that are biased or require excessive computational requirements. RESULTS: DMRfinder is a novel computational pipeline that identifies differentially methylated regions efficiently. Following alignment, DMRfinder extracts methylation counts and performs a modified single-linkage clustering of methylation sites into genomic regions. It then compares methylation levels using beta-binomial hierarchical modeling and Wald tests. Among its innovative attributes are the analyses of novel methylation sites and methylation linkage, as well as the simultaneous statistical analysis of multiple sample groups. To demonstrate its efficiency, DMRfinder is benchmarked against other computational approaches using a large published dataset. Contrasting two replicates of the same sample yielded minimal genomic regions with DMRfinder, whereas two alternative software packages reported a substantial number of false positives. Further analyses of biological samples revealed fundamental differences between DMRfinder and another software package, despite the fact that they utilize the same underlying statistical basis. For each step, DMRfinder completed the analysis in a fraction of the time required by other software. CONCLUSIONS: Among the computational approaches for identifying differentially methylated regions from high-throughput bisulfite sequencing datasets, DMRfinder is the first that integrates all the post-alignment steps in a single package. Compared to other software, DMRfinder is extremely efficient and unbiased in this process. DMRfinder is free and open-source software, available on GitHub ( github.com/jsh58/DMRfinder ); it is written in Python and R, and is supported on Linux.

Suppression of salient objects prevents distraction in visual search.

To find objects of interest in a cluttered and continually changing visual environment, humans must often ignore salient stimuli that are not currently relevant to the task at hand. Recent neuroimaging results indicate that the ability to prevent salience-driven distraction depends on the current level of attentional control activity in frontal cortex, but the specific mechanism by which this control activity prevents salience-driven distraction is still poorly understood. Here, we asked whether salience-driven distraction is prevented by suppressing salient distractors or by preferentially up-weighting the relevant visual dimension. We found that salient distractors were suppressed even when they resided in the same feature dimension as the target (that is, when dimensional weighting was not a viable selection strategy). Our neurophysiological measure of suppression--the PD component of the event-related potential--was associated with variations in the amount of time it took to perform the search task: distractors triggered the PD on fast-response trials, but on slow-response trials they triggered activity associated with working memory representation instead. These results demonstrate that during search salience-driven distraction is mitigated by a suppressive mechanism that reduces the salience of potentially distracting visual objects.

FlowClus: efficiently filtering and denoising pyrosequenced amplicons.

BACKGROUND: Reducing the effects of sequencing errors and PCR artifacts has emerged as an essential component in amplicon-based metagenomic studies. Denoising algorithms have been designed that can reduce error rates in mock community data, but they change the sequence data in a manner that can be inconsistent with the process of removing errors in studies of real communities. In addition, they are limited by the size of the dataset and the sequencing technology used. RESULTS: FlowClus uses a systematic approach to filter and denoise reads efficiently. When denoising real datasets, FlowClus provides feedback about the process that can be used as the basis to adjust the parameters of the algorithm to suit the particular dataset. When used to analyze a mock community dataset, FlowClus produced a lower error rate compared to other denoising algorithms, while retaining significantly more sequence information. Among its other attributes, FlowClus can analyze longer reads being generated from all stages of 454 sequencing technology, as well as from Ion Torrent. It has processed a large dataset of 2.2 million GS-FLX Titanium reads in twelve hours; using its more efficient (but less precise) trie analysis option, this time was further reduced, to seven minutes. CONCLUSIONS: Many of the amplicon-based metagenomics datasets generated over the last several years have been processed through a denoising pipeline that likely caused deleterious effects on the raw data. By using FlowClus, one can avoid such negative outcomes while maintaining control over the filtering and denoising processes. Because of its efficiency, FlowClus can be used to re-analyze multiple large datasets together, thereby leading to more standardized conclusions. FlowClus is freely available on GitHub (jsh58/FlowClus); it is written in C and supported on Linux.

Discrimination of plant-parasitic nematodes from complex soil communities using ecometagenetics.

Many plant pathogens are microscopic, cryptic, and difficult to diagnose. The new approach of ecometagenetics, involving ultrasequencing, bioinformatics, and biostatistics, has the potential to improve diagnoses of plant pathogens such as nematodes from the complex mixtures found in many agricultural and biosecurity situations. We tested this approach on a gradient of complexity ranging from a few individuals from a few species of known nematode pathogens in a relatively defined substrate to a complex and poorly known suite of nematode pathogens in a complex forest soil, including its associated biota of unknown protists, fungi, and other microscopic eukaryotes. We added three known but contrasting species (Pratylenchus neglectus, the closely related P. thornei, and Heterodera avenae) to half the set of substrates, leaving the other half without them. We then tested whether all nematode pathogens-known and unknown, indigenous, and experimentally added-were detected consistently present or absent. We always detected the Pratylenchus spp. correctly and with the number of sequence reads proportional to the numbers added. However, a single cyst of H. avenae was only identified approximately half the time it was present. Other plant-parasitic nematodes and nematodes from other trophic groups were detected well but other eukaryotes were detected less consistently. DNA sampling errors or informatic errors or both were involved in misidentification of H. avenae; however, the proportions of each varied in the different bioinformatic pipelines and with different parameters used. To a large extent, false-positive and false-negative errors were complementary: pipelines and parameters with the highest false-positive rates had the lowest false-negative rates and vice versa. Sources of error identified included assumptions in the bioinformatic pipelines, slight differences in primer regions, the number of sequence reads regarded as the minimum threshold for inclusion in analysis, and inaccessible DNA in resistant life stages. Identification of the sources of error allows us to suggest ways to improve identification using ecometagenetics.

Difficulty suppressing visual distraction while dual tasking.

Human beings must often perform multiple tasks concurrently or in rapid succession. Laboratory research has revealed striking limitations in the ability to dual task by asking participants to identify two target objects that are inserted into a rapid stream of irrelevant items. Under a variety of conditions, identification of the second target (T2) is impaired for a short period of time following presentation of the first target (T1). Several theories have been developed to account for this "attentional blink" (AB), but none makes a specific prediction about how processing of T1 might impact an observer's ability to ignore a salient distractor that accompanies T2. Using event-related potentials (ERPs) to track target and distractor processing, we show that healthy young adults are capable of suppressing a salient visual-search distractor (D2) while dual tasking (as measured by the PD component, which has been associated with suppression) but struggle to do so shortly after the appearance of T1. In fact, the impairment was more severe for distractor processing than it was for target processing (as measured by the N2pc component). Whereas, the T2-elicited N2pc was merely delayed during the AB, the distractor PD was reduced in magnitude and was found to be statistically absent. We conclude that the inhibitory control processes that are typically engaged to prevent distraction are unavailable while an observer is busy processing a target that appeared earlier.

Application of multi-source minimum variance beamformers for reconstruction of correlated neural activity.

Linearly constrained minimum variance beamformers are highly effective for analysis of weakly correlated brain activity, but their performance degrades when correlations become significant. Multiple constrained minimum variance (MCMV) beamformers are insensitive to source correlations but require a priori information about the source locations. Besides the question whether unbiased estimates of source positions and orientations can be obtained remained unanswered. In this work, we derive MCMV-based source localizers that can be applied to both induced and evoked brain activity. They may be regarded as a generalization of scalar minimum-variance beamformers for the case of multiple correlated sources. We show that for arbitrary noise covariance these beamformers provide simultaneous unbiased estimates of multiple source positions and orientations and remain bounded at singular points. We also propose an iterative search algorithm that makes it possible to find sources approximately without a priori assumptions about their locations and orientations. Simulations and analyses of real MEG data demonstrate that presented approach is superior to traditional single-source beamformers in situations where correlations between the sources are significant.

Antisaccade Deficits in Schizophrenia Can Be Driven by Attentional Relevance of the Stimuli.

The antisaccade task is considered a test of cognitive control because it creates a conflict between the strong bottom-up signal produced by the cue and the top-down goal of shifting gaze to the opposite side of the display. Antisaccade deficits in schizophrenia are thought to reflect impaired top-down inhibition of the prepotent bottom-up response to the cue. However, the cue is also a highly task-relevant stimulus that must be covertly attended to determine where to shift gaze. We tested the hypothesis that difficulty in overcoming the attentional relevance of the cue, rather than its bottom-up salience, is key in producing impaired performance in people with schizophrenia (PSZ). We implemented 3 versions of the antisaccade task in which we varied the bottom-up salience of the cue while holding its attentional relevance constant. We found that difficulty in performing a given antisaccade task-relative to a prosaccade version using the same stimuli-was largely independent of the cue's bottom-up salience. The magnitude of impairment in PSZ relative to control subjects was also independent of bottom-up salience. The greatest impairment was observed in a version where the cue lacked bottom-up salience advantage over other locations. These results indicate that the antisaccade deficit in PSZ does not reflect an impairment in overcoming bottom-up salience of the cue, but PSZ are instead impaired at overcoming its attentional relevance. This deficit may still indicate an underlying inhibitory control impairment but could also reflect a hyperfocusing of attentional resources on the cue.

Oculomotor Inhibition of Salient Distractors: Voluntary Inhibition Cannot Override Selection History.

Several studies have demonstrated that salient distractors can be proactively inhibited to prevent attentional capture. Traditional theories frame attentional guidance effects such as this in terms of explicit goals. However, several researchers have recently argued that that unconscious factors-such as the features of attended and ignored items on previous trials (called selection history)-play a stronger role in guiding attention and can overpower explicit goals. The current study assessed whether voluntary inhibition can overpower selection history. We directly compared both forms of top-down control by measuring the control of eye movements, which offer an unambiguous measure of which location has won the competition for attention. We repeatedly found that selection history overpowered any effects of voluntary goals, such that observers were unable to avoid fixating a salient distractor of a known color if the target had been presented in that color on the previous trial. Moreover, a salient distractor of a particular color captured gaze even when the observer had voluntarily chosen this color to be the distractor color just moments before. Taken together, these experiments suggest that the ability to inhibit a salient color singleton is primarily a result of recent experience and not a result of explicit goals.

Histone acetylation maps in aged mice developmentally exposed to lead: epigenetic drift and Alzheimer-related genes.

AIM: Early life exposure to lead (Pb) has been shown to increase late life biomarkers involved in Alzheimer's disease (AD) pathology. Here, we tested the hypothesis that latent over expression of AD-related genes may be regulated through histone activation pathways. METHODS: Chromatin immunoprecipitation sequencing was used to map the histone activation mark (H3K9Ac) to the mouse genome in developmentally Pb exposed mice on postnatal days 20, 270 and 700. RESULTS: Exposure to Pb resulted in a global downregulation of H3K9Ac across the lifespan; except in genes associated with the Alzheimer pathway. DISCUSSION: Early life exposure to Pb results in an epigenetic drift in H3K9Ac consistent with latent global gene repression. Alzheimer-related genes do not follow this trend.

Regulated nuclear accumulation of a histone methyltransferase times the onset of heterochromatin formation in C. elegans embryos.

Heterochromatin formation during early embryogenesis is timed precisely, but how this process is regulated remains elusive. We report the discovery of a histone methyltransferase complex whose nuclear accumulation and activation establish the onset of heterochromatin formation in Caenorhabditis elegans embryos. We find that the inception of heterochromatin generation coincides with the accumulation of the histone H3 lysine 9 (H3K9) methyltransferase MET-2 (SETDB) into nuclear hubs. The absence of MET-2 results in delayed and disturbed heterochromatin formation, whereas accelerated nuclear localization of the methyltransferase leads to precocious H3K9 methylation. We identify two factors that bind to and function with MET-2: LIN-65, which resembles activating transcription factor 7-interacting protein (ATF7IP) and localizes MET-2 into nuclear hubs, and ARLE-14, which is orthologous to adenosine 5'-diphosphate-ribosylation factor-like 14 effector protein (ARL14EP) and promotes stable association of MET-2 with chromatin. These data reveal that nuclear accumulation of MET-2 in conjunction with LIN-65 and ARLE-14 regulates timing of heterochromatin domains during embryogenesis.

Mechanisms of colitis-accelerated colon carcinogenesis and its prevention with the combination of aspirin and curcumin: Transcriptomic analysis using RNA-seq.

Colorectal cancer (CRC) remains the leading cause of cancer-related death in the world. Aspirin (ASA) and curcumin (CUR) are widely investigated chemopreventive candidates for CRC. However, the precise mechanisms of their action and their combinatorial effects have not been evaluated. The purpose of the present study was to determine the effect of ASA, CUR, and their combination in azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colitis-accelerated colorectal cancer (CAC). We also aimed to characterize the differential gene expression profiles in AOM/DSS-induced tumors as well as in tumors modulated by ASA and CUR using RNA-seq. Diets supplemented with 0.02% ASA, 2% CUR or 0.01% ASA+1% CUR were given to mice from 1week prior to the AOM injection until the experiment was terminated 22weeks after AOM initiation. Our results showed that CUR had a superior inhibitory effect in colon tumorigenesis compared to that of ASA. The combination of ASA and CUR at a lower dose exhibited similar efficacy to that of a higher dose of CUR at 2%. RNA isolated from colonic tissue from the control group and from tumor samples from the experimental groups was subjected to RNA-seq. Transcriptomic analysis suggested that the low-dose combination of ASA and CUR modulated larger gene sets than the single treatment. These differentially expressed genes were situated in several canonical pathways important in the inflammatory network and liver metastasis in CAC. We identified a small subset of genes as potential molecular targets involved in the preventive action of the combination of ASA and CUR. Taken together, the current results provide the first evidence in support of the chemopreventive effect of a low-dose combination of ASA and CUR in CAC. Moreover, the transcriptional profile obtained in our study may provide a framework for identifying the mechanisms underlying the carcinogenesis process from normal colonic tissue to tumor development as well as the cancer inhibitory effects and potential molecular targets of ASA and CUR.

Levels of DNA polymorphism vary with mating system in the nematode genus caenorhabditis.

Self-fertilizing species often harbor less genetic variation than cross-fertilizing species, and at least four different models have been proposed to explain this trend. To investigate further the relationship between mating system and genetic variation, levels of DNA sequence polymorphism were compared among three closely related species in the genus Caenorhabditis: two self-fertilizing species, Caenorhabditis elegans and C. briggsae, and one cross-fertilizing species, C. remanei. As expected, estimates of silent site nucleotide diversity were lower in the two self-fertilizing species. For the mitochondrial genome, diversity in the selfing species averaged 42% of diversity in C. remanei. Interestingly, the reduction in genetic variation was much greater for the nuclear than for the mitochondrial genome. For two nuclear genes, diversity in the selfing species averaged 6 and 13% of diversity in C. remanei. We argue that either population bottlenecks or the repeated action of natural selection, coupled with high levels of selfing, are likely to explain the observed reductions in species-wide genetic diversity.