Divergent dynamics of sexual and habitat isolation at the transition between stick insect populations and species.

Speciation is often viewed as a continuum along which populations diverge until they become reproductively-isolated species. However, such divergence may be heterogeneous, proceeding in fits and bursts, rather than being uniform and gradual. We show in Timema stick insects that one component of reproductive isolation evolves non-uniformly across this continuum, whereas another does not. Specifically, we use thousands of host-preference and mating trials to study habitat and sexual isolation among 42 pairs of taxa spanning a range of genomic differentiation and divergence time. We find that habitat isolation is uncoupled from genomic differentiation within species, but accumulates linearly with it between species. In contrast, sexual isolation accumulates linearly across the speciation continuum, and thus exhibits similar dynamics to morphological traits not implicated in reproductive isolation. The results show different evolutionary dynamics for different components of reproductive isolation and highlight a special relevance for species status in the process of speciation.

Online cognitive bias modification for interpretation to reduce anxious thinking during the COVID-19 pandemic.

Anxiety disorders are highly prevalent, and rates increased during the COVID-19 pandemic. However, most individuals with elevated anxiety do not access treatment due to barriers such as stigma, cost, and availability. Digital mental health programs, such as cognitive bias modification for interpretation (CBM-I), hold promise in increasing access to care. Before widely disseminating CBM-I, we must rigorously test its effectiveness and determine whom it is best positioned to benefit. The present study (which is a substudy of a parent trial) compared CBM-I against psychoeducation offered through the public website MindTrails, and also tested whether baseline anxiety tied to COVID-19 influenced the rate of change in anxiety and interpretation bias during and after each intervention. Adults with moderate-to-severe anxiety symptoms were randomly assigned to complete five sessions of either CBM-I or psychoeducation as part of a larger trial, and 608 enrolled in this substudy after Session 1. As predicted (https://osf.io/2dyzr), CBM-I was superior to psychoeducation at reducing anxiety symptoms (on the OASIS but not the DASS-21-AS: d = -0.31), reducing negative interpretation bias (d range = -0.34 to -0.43), and increasing positive interpretation bias (d = 0.79) by the end of treatment. Results also indicated that individuals higher (vs. lower) in baseline COVID-19 anxiety had stronger decreases in anxiety symptoms while receiving CBM-I but weaker decreases in anxiety symptoms (on the DASS-21-AS) while receiving psychoeducation. These findings suggest that CBM-I may be a useful anxiety-reduction tool for individuals experiencing higher anxiety tied to uncertain events such as the COVID-19 pandemic.

Minimal Effect of Messaging on Engagement in a Digital Anxiety Intervention.

This study evaluated the effectiveness of different recruitment messages for encouraging enrollment in a digital mental health intervention (DMHI) for anxiety among 1,600 anxious patients in a large healthcare system. Patients were randomly assigned to receive a standard message, or one of five messages designed to encourage enrollment: Three messages offered varying financial incentives, one message offered coaching, and one message provided consumer testimonials. Patients could then click a link in the message to visit the DMHI website, enroll, and start the first session. We examined the effects of message features and message length (short vs. long) on rates of site clicks, enrollment, and starting the first session. We also tested whether demographic and clinical factors derived from patients' electronic health records were associated with rates of enrollment and starting the first session to understand the characteristics of patients most likely to use DMHIs in this setting. Across messages, 19.4% of patients clicked a link to visit the DMHI website, but none of the messages were significantly associated with rates of site clicks, enrollment, or starting the first session. Females (vs. males) had a greater probability of enrollment. No other demographic or clinical variables were significantly associated with enrollment or starting the first session. Findings provide guidance for resource allocation decisions in larger scale DMHI implementations in healthcare settings.

Shifting Episodic Prediction With Online Cognitive Bias Modification: A Randomized Controlled Trial.

Negative future thinking pervades emotional disorders. This hybrid efficacy-effectiveness trial tested a four-session, scalable online cognitive bias modification program for training more positive episodic prediction. 958 adults (73.3% female, 86.5% White, 83.4% from United States) were randomized to positive conditions with ambiguous future scenarios that ended positively, 50/50 conditions that ended positively or negatively, or a control condition with neutral scenarios. As hypothesized (preregistration: https://osf.io/jrst6), positive training participants improved more than control participants in negative expectancy bias (d = -0.58), positive expectancy bias (d = 0.80), and self-efficacy (d = 0.29). Positive training was also superior to 50/50 training for expectancy bias and optimism (d = 0.31). Training gains attenuated yet remained by 1-month follow-up. Unexpectedly, participants across conditions improved comparably in anxiety and depression symptoms and growth mindset. Targeting a transdiagnostic process with a scalable program may improve bias and outlook; however, further validation of outcome measures is required.

Autolysis Affects the Iron Cargo of Ferritins in Neurons and Glial Cells at Different Rates in the Human Brain.

Iron is known to accumulate in neurological disorders, so a careful balance of the iron concentration is essential for healthy brain functioning. An imbalance in iron homeostasis could arise due to the dysfunction of proteins involved in iron homeostasis. Here, we focus on ferritin-the primary iron storage protein of the brain. In this study, we aimed to improve a method to measure ferritin-bound iron in the human post-mortem brain, and to discern its distribution in particular cell types and brain regions. Though it is known that glial cells and neurons differ in their ferritin concentration, the change in the number and distribution of iron-filled ferritin cores between different cell types during autolysis has not been revealed yet. Here, we show the cellular and region-wide distribution of ferritin in the human brain using state-of-the-art analytical electron microscopy. We validated the concentration of iron-filled ferritin cores to the absolute iron concentration measured by quantitative MRI and inductively coupled plasma mass spectrometry. We show that ferritins lose iron from their cores with the progression of autolysis whereas the overall iron concentrations were unaffected. Although the highest concentration of ferritin was found in glial cells, as the total ferritin concentration increased in a patient, ferritin accumulated more in neurons than in glial cells. Summed up, our findings point out the unique behaviour of neurons in storing iron during autolysis and explain the differences between the absolute iron concentrations and iron-filled ferritin in a cell-type-dependent manner in the human brain. The rate of loss of the iron-filled ferritin cores during autolysis is higher in neurons than in glial cells.

Polymethyl methacrylate cure time in simulated in vivo total knee arthroplasty versus in vitro conditions.

BACKGROUND: The present means of confirming the cure of intra-operative polymethyl methacrylate (PMMA) cement are to wait for the remainder cement to harden. To our knowledge, there is no available technique to determine the precise moment of cure for in-vivo cement beneath the tibial tray. This study uses a novel means to determine cement curing time in two environments. One environment represents the operating theater, and the other environment attempts to model cement conditions under the tibial tray during surgery. MATERIALS AND METHODS: We determined the temperature-versus-time plot of cement curing using the following two temperature sensors: one in a simulated implanted tibial tray and another in the remainder cement. We performed 55 tests using dental methyl methacrylate cement mixed in the same ratio as the orthopedic cement. To simulate in vivo conditions, a simulated stainless-steel tibial tray was implanted on a cancellous bone substitute (Sawbones, Vashon Island, WA, USA) using standard cement technique and subsequently placed in a 90°F (32.2 °C) circulating water bath. We positioned a temperature sensor in the cement mantel and positioned a second sensor in a portion of the remaining cement. The temperature from both sensors was measured simultaneously, beginning at 5 min after mixing and continuing for 20 min. The first derivative of the temperature provided the precise curing time for each condition. We analyzed the results of 55 repeated experiments with an independent samples t-test. RESULTS: With the described technique, we were able to accurately determine the moment of cure of the cement beneath the simulated tray. There was a mean difference between cure time of 5 min and 26 s (p value < 0.001) between the two conditions. CONCLUSIONS: We validated that our technique was successful in determining the precise time to cure in two different environments. LEVEL OF EVIDENCE: This was not a clinical trial and did not involve patients as such the level of evidence was Grade A: Consistent 1 and 2.

Multi-session online interpretation bias training for anxiety in a community sample.

The present study assessed target engagement, preliminary efficacy, and feasibility as primary outcomes of a free multi-session online cognitive bias modification of interpretation (CBM-I) intervention for anxiety in a large community sample. High trait anxious participants (N = 807) were randomly assigned to a CBM-I condition: 1) Positive training (90% positive-10% negative); 2) 50% positive-50% negative training; or 3) no-training control. Further, half of each CBM-I condition was randomized to either an anxious imagery prime or a neutral imagery prime. Due to attrition, results from six out of eight sessions were analyzed using structural equation modeling of latent growth curves. Results for the intent-to-treat sample indicate that for target engagement, consistent with predictions, decreases in negative interpretations over time were significantly greater among those receiving positive CBM-I training compared to no-training or 50-50 training, and vice-versa for increases in positive interpretations. For intervention efficacy, the decrease in anxiety symptoms over time was significantly greater among those receiving positive CBM-I training compared to no-training. Interaction effects with imagery prime were more variable with a general pattern of stronger results for those completing the anxious imagery prime. Findings indicate that online CBM-I positive training is feasible and shows some promising results, although attrition rates were very high for later training sessions.

Older Adults' Physical Activity and Healthcare Costs, 2003-2014.

INTRODUCTION: Research has documented the health benefits of physical activity among older adults, but the relationship between physical activity and healthcare costs remains unexplored at the population level. Using data from 50 U.S. states and the District of Columbia, this study investigates the extent to which physical activity prevalence is associated with healthcare costs among older adults. METHODS: Twelve-year state-level data (2003-2014) were obtained from 5 secondary sources (n=611). Healthcare costs were captured by Medicare Parts A and B spending. Fixed-effect models were estimated in 2019 to assess the relationship between the state-level physical activity prevalence and Medicare costs. The potential lagged associations were captured by lagged variables of physical activity prevalence (i.e., t-1, t-2, and t-3). RESULTS: Physical activity prevalence was not associated with Medicare costs occurring in the concurrent and subsequent year (p>0.05); however, the 2-year lagged variable (p=0.03) and the 3-year lagged variable (p=0.01) for physical activity prevalence were negatively associated with Medicare costs, indicating a time-lagged relationship. It was estimated that a 10 percentage point increase in physical activity prevalence in each state is associated with reduced Medicare Parts A and B costs of 0.4% after 2 years and 1.0% after 3 years. CONCLUSIONS: Results revealed a time lag effect highlighted by a delayed inverse relationship between state-level physical activity prevalence and healthcare costs among older adults. This evidence offers governments and communities new insights to guide policymaking on long-term public investment in physical activity intervention programs.

Enhancing Older Adults' Sense of Belonging and Subjective Well-Being Through Sport Game Attendance, Team Identification, and Emotional Support.

Objective: We investigate how (a) attendance at sport games and (b) identification with a sport team as fans (i.e., team identification) influence older adults' perceptions of emotional support, belonging, and subjective well-being (SWB). Method: An experimental pilot study was conducted with 50 older adults, followed by a main survey study administered to 534 older adults from various communities across the United States. Results: Pilot study results indicated that game attendance and team identification had a positive and significant influence on older adults' perceptions of emotional support from fellow team fans. These results were replicated in the main study, which also showed that older adults' perceived emotional support from fellow fans was positively associated with their sense of belonging which predicted their SWB. Discussion: The findings provide insights into how older adults may be engaged in meaningful forms of social life to help them maintain and enhance mental health.

Integrating health education and physical activity programming for cardiovascular health promotion among female inmates: A proof of concept study.

Female inmate populations in the United States tend to be overweight, physically inactive, experience high stress, and have a history of nicotine and other drug dependence. Thus, they bear an elevated risk of cardiovascular (CV) disease than the general population. However, few evidence-based health interventions exist for this population. This study will test proof of concept, feasibility, and potential efficacy of a multiple health behavior change intervention that integrates CV-health promotion education delivered during a physical activity (PA) program (indoor cycling) tailored to this population. This study uses a quasi-experimental 2-group design with two measurement time-points: baseline and 8-week end of treatment. N=120 incarcerated women (18-59years of age) who are medically cleared for participation in PA will be enrolled. Indoor cycling instructors will be trained to deliver five health education topics over an 8-week period during twice-weekly cycling classes. Topics match the American Heart Association recommendations for CV health: (a) nutrition, (b) PA promotion, (c) weight management, (d) stress management, and (e) smoking cessation and relapse prevention. Modes of intervention include instructor advice, written materials and audio/video clips reviewed during class. CV-related and mental health measures will be assessed at both time-points. Results will guide a full scale efficacy study. Future research in this area has potential to impact the health of female inmates, a high-risk population. Moreover, this multiple health behavior change intervention model represents a community approach to health promotion that could generalize to other underserved populations who may benefit most from similar intervention efforts.

Isolation by adaptation in Neochlamisus leaf beetles: host-related selection promotes neutral genomic divergence.

This study tests how divergent natural selection promotes genomic differentiation during ecological speciation. Specifically, we use adaptive ecological divergence (here, population divergence in host plant use and preference) as a proxy for selection strength and evaluate the correlation between levels of adaptive and genetic differentiation across pairwise population comparisons. Positive correlations would reveal the pattern predicted by our hypothesis, that of 'isolation by adaptation' (IBA). Notably, IBA is predicted not only for selected loci but also for neutral loci. This may reflect the effects of divergent selection on neutral loci that are 'loosely linked' to divergently selected loci or on geneflow restriction that facilitates genetic drift at all loci, including neutral loci that are completely unlinked to those evolving under divergent selection. Here, we evaluate IBA in maple- and willow-associated populations of Neochlamisus bebbianae leaf beetles. To do so, we collected host preference data to construct adaptive divergence indices and used AFLPs (amplified fragment length polymorphisms) and mitochondrial sequences to quantify genetic differentiation. Partial Mantel tests showed significant IBA in 'pooled' analyses of putatively neutral and of putatively selected ('outlier') AFLP loci. This pattern was also recovered in 12% of 'locus-specific' analyses that separately evaluated genetic differentiation at individual neutral loci. These results provided evidence for widespread effects of selection on neutral genomic divergence. Our collective findings indicate that host-related selection may play important roles in the population genomic differentiation of both neutral and selected gene regions in herbivorous insects.

Inference is bliss: using evolutionary relationship to guide categorical inferences.

Three experiments, adopting an evolutionary biology perspective, investigated subjects' inferences about living things. Subjects were told that different enzymes help regulate cell function in two taxa and asked which enzyme a third taxon most likely uses. Experiment 1 and its follow-up, with college students, used triads involving amphibians, reptiles, and mammals (reptiles and mammals are most closely related evolutionarily) and plants, fungi, and animals (fungi are more closely related to animals than to plants). Experiment 2, with 10th graders, also included triads involving mammals, birds, and snakes/crocodilians (birds and snakes/crocodilians are most closely related). Some subjects received cladograms (hierarchical diagrams) depicting the evolutionary relationships among the taxa. The effect of providing cladograms depended on students' background in biology. The results illuminate students' misconceptions concerning common taxa and constraints on their willingness to override faulty knowledge when given appropriate evolutionary evidence. Implications for introducing tree thinking into biology curricula are discussed.

Effect of resistance training and aerobic conditioning on muscular strength and submaximal fitness for individuals with chronic heart failure: influence of age and gender.

The hallmark characteristic of chronic heart failure (CHF) is exercise intolerance. This study evaluated a combined exercise program that would be consistent with current cardiac rehabilitation guidelines. The program consisted of progressive resistance training (intensity progressing from 50 to 80% maximum) and aerobic conditioning (70% of heart rate reserve). Outcomes included muscular strength measured as 8-repetition maximum and submaximal aerobic fitness indicators (measured at 50% of predicted maximal oxygen consumption) including heart rate, blood pressure, and rate pressure product. Subjects were taken retrospectively from a database of CHF patients who underwent cardiac rehabilitation between 1998 and 2004 with the Cardiac Rehabilitation Center of River Cities Cardiology in Jeffersonville, IN. The same data set was used to evaluate 2 research questions: to compare training outcomes between gender (men and women) and to compare outcomes between young (65 years) subjects. Forty-two subjects (15 women and 27 men; 19 were 65 years of age) met inclusion and exclusion criteria and underwent 36 sessions of cardiac rehabilitation over a 14-week time period. Repeated-measures analysis of variance was used to test whether strength and submaximal responses differed across gender or age. Analysis indicated that regardless of gender or age, subjects significantly increased their strength on each of 6 strength tests. Heart rate, systolic blood pressure, and rate pressure product were lower after training indicating increased aerobic efficiency. Progressive resistance training and aerobic conditioning prescribed in a manner consistent with cardiac rehabilitation guidelines resulted in similar improvements regardless of gender or age for individuals with CHF suggesting that no training program adjustments are necessary to accommodate age or gender.

Lateral phage transfer in obligate intracellular bacteria (wolbachia): verification from natural populations.

Lateral transfer of mobile DNA is a hallmark of bacteria with a free-living replicative stage; however, its significance in obligate intracellular bacteria and other heritable endosymbionts remains controversial. Comparative sequence analyses from laboratory stocks infected with Wolbachia pipientis provide some of the most compelling evidence that bacteriophage WO-B transfers laterally between infections of the same insect host. Lateral transfer between coinfections, however, has been evaluated neither in natural populations nor between closely related Wolbachia strains. Here, we analyze bacterial and phage genes from two pairs of natural sympatric field isolates, of Gryllus pennsylvanicus field crickets and of Neochlamisus bebbianae leaf beetles, to demonstrate WO-B transfers between supergroup B Wolbachia. N. bebbianae revealed the highest number of phage haplotypes yet recorded, hinting that lab lines could underestimate phage haplotype variation and lateral transfer. Finally, using the approximate age of insect host species as the maximum available time for phage transfer between host-associated bacteria, we very conservatively estimate phage WO-B transfer to occur at least once every 0-5.4 My within a host species. Increasing discoveries of mobile elements, intragenic recombination, and bacterial coinfections in host-switching obligate intracellular bacteria specify that mobile element transfer is common in these species.

Ecologically dependent postmating isolation between sympatric host forms of Neochlamisus bebbianae leaf beetles.

Ecological speciation is the promotion of reproductive isolation via the divergent adaptation of populations to alternative environments. A prediction peculiar to ecological speciation is that hybrids between such populations should be adapted poorly to parental environments, yielding reduced fitness and postmating isolation. However, F(1) analyses alone cannot demonstrate that ecological ("extrinsic") factors contribute to such isolation. Rather, this requires documenting a "switch" in the relative fitnesses of reciprocal backcrosses between environments. Specifically, each backcross should exhibit higher fitness in the environment of its pure parent, with which it shares the most genes, including environment-specific ones. In contrast, because genetic proportions are expected to be similar for all backcrosses ( approximately (3/4) from one parental type and approximately (1/4) from the other), the more general genetic incompatibilities responsible for "intrinsic" isolation predict no such environment-specific fitness switches. Thus, although intrinsic isolation may contribute to the fitness reduction and variation underlying such patterns, it offers an insufficient explanation for them. Here, we present a quantitative genetic "backcross" analysis of sympatric Neochlamisus bebbianae leaf beetle populations adapted to maple versus willow host plants. Results statistically supported ecological speciation predictions, notably the switch in relative fitness for backcross types, the expected rank order of cross type fitnesses, and appreciable extrinsic isolation. We additionally documented genetic variation in host-associated fitness, ruled out nongenetic maternal effects, and discuss the maintenance of ecological differentiation in sympatry. In summary, our study provides a rare and strongly supported demonstration of genetically based, ecologically dependent postmating isolation during ecological speciation.

Divergent selection and heterogeneous genomic divergence.

Levels of genetic differentiation between populations can be highly variable across the genome, with divergent selection contributing to such heterogeneous genomic divergence. For example, loci under divergent selection and those tightly physically linked to them may exhibit stronger differentiation than neutral regions with weak or no linkage to such loci. Divergent selection can also increase genome-wide neutral differentiation by reducing gene flow (e.g. by causing ecological speciation), thus promoting divergence via the stochastic effects of genetic drift. These consequences of divergent selection are being reported in recently accumulating studies that identify: (i) 'outlier loci' with higher levels of divergence than expected under neutrality, and (ii) a positive association between the degree of adaptive phenotypic divergence and levels of molecular genetic differentiation across population pairs ['isolation by adaptation' (IBA)]. The latter pattern arises because as adaptive divergence increases, gene flow is reduced (thereby promoting drift) and genetic hitchhiking increased. Here, we review and integrate these previously disconnected concepts and literatures. We find that studies generally report 5-10% of loci to be outliers. These selected regions were often dispersed across the genome, commonly exhibited replicated divergence across different population pairs, and could sometimes be associated with specific ecological variables. IBA was not infrequently observed, even at neutral loci putatively unlinked to those under divergent selection. Overall, we conclude that divergent selection makes diverse contributions to heterogeneous genomic divergence. Nonetheless, the number, size, and distribution of genomic regions affected by selection varied substantially among studies, leading us to discuss the potential role of divergent selection in the growth of regions of differentiation (i.e. genomic islands of divergence), a topic in need of future investigation.

Selection and genomic differentiation during ecological speciation: isolating the contributions of host association via a comparative genome scan of Neochlamisus bebbianae leaf beetles.

This study uses a comparative genome scan to evaluate the contributions of host plant related divergent selection to genetic differentiation and ecological speciation in maple- and willow-associated populations of Neochlamisus bebbianae leaf beetles. For each of 15 pairwise population comparisons, we identified "outlier loci" whose strong differentiation putatively reflects divergent selection. Of 447 AFLP loci, 15% were outliers across multiple population comparisons, and low linkage disequilibrium indicated that these outliers derived from multiple regions of the genome. Outliers were further classified as "host-specific" if repeatedly observed in "different-host" population comparisons but never in "same-host" comparisons. Outliers exhibiting the opposite pattern were analogously classified as "host-independent." Host-specific outliers represented 5% of all loci and were more frequent than host-independent outliers, thus revealing a large role for host-adaptation in population genomic differentiation. Evidence that host-related selection can promote divergence despite gene flow was provided by population trees. These were structured by host-association when datasets included host-specific outliers, but not when based on neutral loci, which united sympatric populations. Lastly, three host-specific outliers were highly differentiated in all nine different-host comparisons. Because host-adaptation promotes reproductive isolation in these beetles, these loci provide promising candidate gene regions for future molecular studies of ecological speciation.

Heterogeneous genomic differentiation between walking-stick ecotypes: "isolation by adaptation" and multiple roles for divergent selection.

Genetic differentiation can be highly variable across the genome. For example, loci under divergent selection and those tightly linked to them may exhibit elevated differentiation compared to neutral regions. These represent "outlier loci" whose differentiation exceeds neutral expectations. Adaptive divergence can also increase genome-wide differentiation by promoting general barriers to neutral gene flow, thereby facilitating genomic divergence via genetic drift. This latter process can yield a positive correlation between adaptive phenotypic divergence and neutral genetic differentiation (described here as "isolation-by-adaptation"). Here, we examine both these processes by combining an AFLP genome scan of two host plant ecotypes of Timema cristinae walking-sticks with existing data on adaptive phenotypic divergence and ecological speciation in these insects. We found that about 8% of loci are outliers in multiple population comparisons. Replicated comparisons between population-pairs using the same versus different host species revealed that 1-2% of loci are subject to host-related selection specifically. Locus-specific analyses revealed that up to 10% of putatively neutral (nonoutlier) AFLP loci exhibit significant isolation-by-adaptation. Our results suggest that selection may affect differentiation directly, via linkage, or by facilitating genetic drift. They thus illustrate the varied and sometimes nonintuitive contributions of selection to heterogeneous genomic differentiation.

Individual advantages to ecological specialization: insights on cognitive constraints from three conspecific taxa.

The information-processing hypothesis (IPH) posits that specialist herbivores should make host-associated decisions more effectively than generalists and thus enjoy associated fitness advantages that may help explain the evolutionary prevalence of host-specific insects. This is because generalists must evaluate a greater diversity of host plants/cues than specialists and thus face a cognitive challenge that is predicted to constrain the efficiency and accuracy of their choices. Here, we present the first individual-level evaluation of this hypothesis. This involved experimentally quantifying the specificity, efficiency, and accuracy of host selection, as both larvae and adults, for many individuals representing each of three 'host forms' of Neochlamisus bebbianae leaf beetles. These experiments provided several significant findings: host forms differed in larval specificity, with the more specialized host forms more efficiently and accurately selecting optimal hosts as both larvae and adults. Positive correlations between larval specificity and both efficiency and accuracy across test individuals provided the most direct evidence to date for a biological association between these variables. Our results thus provide strong and consistent support for the IPH at the level of both populations and individuals. Because individual N. bebbianae make many host-associated decisions in nature, our results suggest that cognitive constraints may play a major role in the evolutionary dynamics of ongoing ecological specialization and diversification in this species.

The evolution of unisexuality in Calligrapha leaf beetles: molecular and ecological insights on multiple origins via interspecific hybridization.

Interspecific hybridization is a well-established cause of unisexual origins in vertebrates. This mechanism is also suspected in other apomictic taxa, but compelling evidence is rare. Here, we evaluate this mechanism and other hypotheses for the evolutionary origins of unisexuality through an investigation of Calligrapha leaf beetles. This group provides an intriguing subject for studies of unisexual evolution because it presents a rare insect example of multiple apomictic thelytokous species within a primarily bisexual genus. To investigate unisexual evolution, this study conducts the first molecular systematic analysis of Calligrapha. This involved the collection and analysis of about 3000 bp of DNA sequences--representing RNA and protein-coding loci from mitochondrial and nuclear genomes--from 54 specimens of 25 Calligrapha species, including four unisexual tetraploid taxa. Phylogenetic and molecular clock analyses indicated independent and single evolutionary origins of each of these unisexual species during the Pleistocene. Significant phylogenetic incongruence was detected between mitochondrial and nuclear datasets and found to be especially associated with the asexual taxa. This pattern is expected when unisexual lineages arise via interspecific hybridization and thus represent genetic mosaics that possess certain nuclear alleles from the paternal species lineage and mitochondrial DNA (mtDNA) alleles from the maternal parent. Analyzing the mtDNA and nuclear relatedness of unisexuals with corresponding haplotypes of bisexual Calligrapha species allowed the putative identification of these maternal and paternal species lineages for each unisexual species. Strong phenotypic similarities between unisexual taxa and their paternal parent species supported a model that involves both backcrosses of interspecific hybrids with a paternal parent and unreduced gametes. This model accounts for the origins of apomixis, polyploidy, and an overrepresentation of paternal nuclear alleles (and associated phenotypes) in unisexuals. This model is also consistent with the tetraploid karyotypes of unisexual Calligrapha, in which three sets of chromosomes (of presumed paternal ancestry) are quite morphologically homogeneous compared to the fourth. Especially intriguing was a consistent association of unisexual species with the host plant of the paternal parent but never with the maternal host. The statistical implausibility of these patterns occurring by chance further supports our inference of parental species. Moreover, it points to a potentially critical role for host-association in the formation and preservation of unisexual lineages. These findings suggest that ecological factors are critical for the diversification of unisexual as well as bisexual taxa and thus point out new research directions in the area of ecological speciation.