Antidepressant-Associated Mania in Bipolar Disorder: A Review and Meta-analysis of Potential Clinical and Genetic Risk Factors.

PURPOSES/BACKGROUND: Antidepressants (ADs) play a valuable role in treating the depressive episodes of bipolar disorder. However, 14% of these individuals taking ADs experience AD-associated mania (AAM) within a few weeks of starting treatment. Numerous studies have suggested potential clinical and genetic risk factors. We aimed to conduct a comprehensive systematic review and meta-analysis that integrates the past literature with the recent studies and identifies important predictors for AAM. METHODS/PROCEDURES: The review was limited to experimentally designed studies that contain the relevant search terms in PubMed and PsychInfo. After removing studies that were in discordance with our criteria, the review included 24 reports examining clinical risk factors and 10 investigating genetic risk factors. Our meta-analysis was conducted on 5 clinical risk factors, each of which had at least 4 articles with extractable data. FINDINGS/RESULTS: The only clinical factors in the literature that have been shown to be more indicative of AAM risk are AD monotherapy and tricyclic ADs. Among genetic factors, the serotonin transporter gene polymorphism may play a minor role in AAM. Our meta-analysis provided support for the number of prior depressive episodes. IMPLICATIONS/CONCLUSION: Prevention of AAM may be served by early detection of recurrent depression episodes. Further large-scale longitudinal studies are required to determine the underpinnings of AAM.

Mitochondrial Genetics.

The maternally inherited mitochondrial DNA (mtDNA) is located inside every mitochondrion, in variable number of copies, and it contains 37 crucial genes for cellular bioenergetics. This chapter will discuss the unique features of this circular genome including heteroplasmy, haplogroups, among others, along with the corresponding clinical relevance for each. The discussion also covers the nuclear-encoded mitochondrial genes (N > 1000) and the epistatic interactions between mtDNA and the nuclear genome. Examples of mitochondrial diseases related to specific mtDNA mutation sites of relevance for humans are provided. This chapter aims to provide an overview of mitochondrial genetics as an emerging hot topic for the future of medicine.

Origin and dynamics of admixture in Brazilians and its effect on the pattern of deleterious mutations.

While South Americans are underrepresented in human genomic diversity studies, Brazil has been a classical model for population genetics studies on admixture. We present the results of the EPIGEN Brazil Initiative, the most comprehensive up-to-date genomic analysis of any Latin-American population. A population-based genome-wide analysis of 6,487 individuals was performed in the context of worldwide genomic diversity to elucidate how ancestry, kinship, and inbreeding interact in three populations with different histories from the Northeast (African ancestry: 50%), Southeast, and South (both with European ancestry >70%) of Brazil. We showed that ancestry-positive assortative mating permeated Brazilian history. We traced European ancestry in the Southeast/South to a wider European/Middle Eastern region with respect to the Northeast, where ancestry seems restricted to Iberia. By developing an approximate Bayesian computation framework, we infer more recent European immigration to the Southeast/South than to the Northeast. Also, the observed low Native-American ancestry (6-8%) was mostly introduced in different regions of Brazil soon after the European Conquest. We broadened our understanding of the African diaspora, the major destination of which was Brazil, by revealing that Brazilians display two within-Africa ancestry components: one associated with non-Bantu/western Africans (more evident in the Northeast and African Americans) and one associated with Bantu/eastern Africans (more present in the Southeast/South). Furthermore, the whole-genome analysis of 30 individuals (42-fold deep coverage) shows that continental admixture rather than local post-Columbian history is the main and complex determinant of the individual amount of deleterious genotypes.

Mitochondrial dysfunction in schizophrenia: an evolutionary perspective.

Schizophrenia (SCZ) is a severe psychiatric illness with a lifetime prevalence of 0.4 %. A disturbance of energy metabolism has been suggested as part of the etiopathogenesis of the disorder. Several lines of evidence have proposed a connection between etiopathogenesis of SCZ and human brain evolution, which was characterized by an increase in the energy requirement, demanding a co-evolution of the mitochondrial system. Mitochondria are key players in brain energy homeostasis and multiple lines of evidence suggest that the system is disrupted in SCZ. In this review, we will describe the current knowledge on pathways/system involved in the human brain evolution as well as the main theories regarding the evolutionary origin of SCZ. We will furthermore discuss the role of mitochondria in the context of brain energy metabolism and its role in the etiopathogenesis of SCZ. Understanding SCZ in the context of human brain evolution opens a new perspective to elucidate pathophysiological mechanisms involved in the origin and/or portions of the complex symptomatology of this severe mental disorder.

Association study of the complement component C4 gene and suicide risk in schizophrenia.

Schizophrenia is a severe mental illness and a major risk factor for suicide, with approximately 50% of schizophrenia patients attempting and 10% dying from suicide. Although genetic components play a significant role in schizophrenia risk, the underlying genetic risk factors for suicide are poorly understood. The complement component C4 gene, an immune gene involved in the innate immune system and located in the major histocompatibility complex (MHC) region, has been identified to be strongly associated with schizophrenia risk. In addition, recent findings have also suggested that the MHC region has been associated with suicide risk across disorders, making C4 a potential candidate of interest for studying suicidality in schizophrenia patients. Despite growing interest in investigating the association between the C4 gene and schizophrenia, to our knowledge, no work has been done to examine the potential of C4 variants as suicide risk factors in patients with schizophrenia. In this study, we investigated the association between different C4 copy number variants and predicted C4 brain expression with suicidal outcomes (suicide attempts/suicidal ideation). We directly genotyped 434 schizophrenia patients to determine their C4A and C4B copy number variants. We found the C4AS copy number to be marginally and negatively associated with suicide risk, potentially being protective against suicide attempts (OR = 0.49; p = 0.05) and suicidal ideation (OR = 0.65; p = 0.07). Furthermore, sex-stratified analyses revealed that there are no significant differences between males and females. Our preliminary findings encourage additional studies of C4 and potential immune dysregulation in suicide.

Exosome-associated mitochondrial DNA in late-life depression: Implications for cognitive decline in older adults.

BACKGROUND: Disrupted cellular communication, inflammatory responses and mitochondrial dysfunction are consistently observed in late-life depression (LLD). Exosomes (EXs) mediate cellular communication by transporting molecules, including mitochondrial DNA (EX-mtDNA), playing critical role in immunoregulation alongside tumor necrosis factor (TNF). Changes in EX-mtDNA are indicators of impaired mitochondrial function and might increase vulnerability to adverse health outcomes. Our study examined EX-mtDNA levels and integrity, exploring their associations with levels of TNF receptors I and II (TNFRI and TNFRII), and clinical outcomes in LLD. METHODS: Ninety older adults (50 LLD and 40 controls (HC)) participated in the study. Blood was collected and exosomes were isolated using size-exclusion chromatography. DNA was extracted and EX-mtDNA levels and deletion were assessed using qPCR. Plasma TNFRI and TNFRII levels were quantified by multiplex immunoassay. Correlation analysis explored relationships between EX-mtDNA, clinical outcomes, and inflammatory markers. RESULTS: Although no differences were observed in EX-mtDNA levels between groups, elevated levels correlated with poorer cognitive performance (r = -0.328, p = 0.002) and increased TNFRII levels (r = 0.367, p = 0.004). LLD exhibited higher deletion rates (F(83,1) = 4.402, p = 0.039), with a trend remaining after adjusting for covariates (p = 0.084). Deletion correlated with poorer cognitive performance (r = -0.335, p = 0.002). No other associations were found. LIMITATION: Cross-sectional study with a small number of participants from a specialized geriatric psychiatry treatment center. CONCLUSION: Our findings suggest that EX-mtDNA holds promise as an indicator of cognitive outcomes in LLD. Additional research is needed to further comprehend the role of EX-mtDNA levels/integrity in LLD, paving the way for its clinical application in the future.

Pilot study of circulating cell-free mitochondrial DNA in relation to brain structure in youth bipolar disorder.

BACKGROUND: Mitochondrial dysfunction is implicated in the neuropathology of bipolar disorder (BD). Higher circulating cell-free mitochondrial DNA (ccf-mtDNA), generally reflecting poorer mitochondrial health, has been associated with greater symptoms severity in BD. The current study examines the association of serum ccf-mtDNA and brain structure in relation to youth BD. We hypothesized that higher ccf-mtDNA will be associated with measures of lower brain structure, particularly in the BD group. METHODS: Participants included 40 youth (BD, n = 19; Control group [CG], n = 21; aged 13-20 years). Serum ccf-mtDNA levels were assayed. T1-weighted brain images were acquired using 3T-MRI. Region of interest (ROI) analyses examined prefrontal cortex (PFC) and whole brain gray matter, alongside exploratory vertex-wise analyses. Analyses examined ccf-mtDNA main-effects and ccf-mtDNA-by-diagnosis interaction effects controlling for age, sex, and intracranial volume. RESULTS: There was no significant difference in ccf-mtDNA levels between BD and CG. In ROI analyses, higher ccf-mtDNA was associated with higher PFC surface area (SA) (ß = 0.32 p < 0.001) and PFC volume (ß = 0.32 p = 0.002) in the overall sample. In stratified analyses, higher ccf-mtDNA was associated with higher PFC SA within both subgroups (BD: ß = 0.39 p = 0.02; CG: ß = 0.24 p = 0.045). Higher ccf-mtDNA was associated with higher PFC volume within the BD group (ß = 0.39 p = 0.046). In vertex-wise analyses, higher ccf-mtDNA was associated with higher SA and volume in frontal clusters within the overall sample and within the BD group. There were significant ccf-mtDNA-by-diagnosis interactions in three frontal and parietal clusters, whereby higher ccf-mtDNA was associated with higher neurostructural metrics in the BD group but lower neurostructural metrics in CG. CONCLUSIONS: Contrasting our hypothesis, higher ccf-mtDNA was consistently associated with higher, rather than lower, regional neuralstructural metrics among youth with BD. While this finding may reflect a compensatory mechanism, future repeated-measures prospective studies evaluating the inter-relationship among ccf-mtDNA, mood, and brain structure across developmental epochs and illness stages are warranted.

Cross-disorder GWAS meta-analysis of endocannabinoid DNA variations in major depressive disorder, bipolar disorder, attention deficit hyperactivity disorder, autism spectrum disorder, and schizophrenia.

The endocannabinoid system (ECS) is implicated in multiple mental disorders. In this study, we explored DNA variations in the ECS across major depressive disorder (MDD), bipolar disorder, attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and schizophrenia by performing a cross-disorder genome-wide association study (GWAS) meta-analysis. We obtained six datasets from the Psychiatric Genomics Consortium containing GWAS summary statistics from European cohorts (284,023 cases and 508,515 controls). Effective sample size weighted meta-analysis was performed for 2241 single nucleotide polymorphisms (SNPs) pertaining to gene bodies of 33 endocannabinoid genes using METAL, where an overall z-statistic is calculated for each marker based on a weighted sum of individual statistics. Heterogeneity was examined with I2 and X2 tests. MAGMA gene-based analysis was also performed. We identified nine SNPs significantly associated with a change in risk of having a mental disorder. The lead SNP was rs12805732 (Gene: Diacylglycerol Lipase Alpha; DAGLA). Four SNPs had substantial heterogeneity (I2>60 %). DAGLA had the strongest association with disease risk in gene-based analysis. Our findings suggest that the ECS may be a shared pathway in mental disorders. Future studies validating these findings would contribute to the identification of biomarkers of disease risk across multiple mental disorders.

Association analyses of the autosomal genome and mitochondrial DNA with accelerometry-derived sleep parameters in depressed UK biobank subjects.

BACKGROUND: The bidirectional relationship between sleep disturbances and depression is well documented, yet the biology of sleep is not fully understood. Mitochondria have become of interest not only because of the connection between sleep and metabolism but also because of mitochondria's involvement in the production of reactive oxygen species, which are largely scavenged during sleep. METHODS: Genome-wide association studies (GWAS) of eight accelerometry-derived sleep measures were performed across both the autosomal and mitochondrial DNA (mtDNA) among two severity levels of depression in UK Biobank participants. We calculated SNP heritability for each of the sleep measures. Linear regression was performed to test associations and mitochondrial haplogroups. RESULTS: Variants included in the GWAS accounted for moderate heritability of bedtime (19.6%, p = 4.75 × 10-7), sleep duration (16.6%, p = 8.58 × 10-6) and duration of longest sleep bout (22.6%, p = 4.64 × 10-4). No variants passed genome-wide significance in the autosomal genome. The top hit in the severe depression sample was rs145019802, near GOLGA8B, for sleep efficiency (p = 1.17 × 10-7), and the top hit in the broad depression sample was rs7100859, an intergenic SNP, and nap duration (p = 1.25 × 10-7). Top mtDNA loci were m.12633C > A of MT-ND5 with bedtime (p = 0.002) in the severe sample and m.16186C > T of the control region with nap duration (p = 0.002) in the broad sample. CONCLUSION: SNP heritability estimates support the involvement of common SNPs in specific sleep measures among depressed individuals. This is the first study to analyze mtDNA variance in sleep measures in depressed individuals. Our mtDNA findings, although nominally significant, provide preliminary suggestion that mitochondria are involved in sleep.

Mitochondria's role in sleep: Novel insights from sleep deprivation and restriction studies.

OBJECTIVES/METHODS: The biology underlying sleep is not yet fully elucidated, but it is known to be complex and largely influenced by circadian rhythms. Compelling evidence supports of a link among circadian rhythms, sleep and metabolism, which suggests a role for mitochondria. These organelles play a significant role in energy metabolism via oxidative phosphorylation (OXPHOS) and several mitochondrial enzymes display circadian oscillations. However, the interplay between mitochondria and sleep is not as well-known. This review summarises human and animal studies that have examined the role of mitochondria in sleep. Literature searches were conducted using PubMed and Google Scholar. RESULTS: Using various models of sleep deprivation, animal studies support the involvement of mitochondria in sleep via differential gene and protein expression patterns, OXPHOS enzyme activity, and morphology changes. Human studies are more limited but also show differences in OXPHOS enzyme activity and protein levels among individuals who have undergone sleep deprivation or suffer from different forms of insomnia. CONCLUSIONS: Taken altogether, both types of study provide evidence for mitochondria's involvement in the sleep-wake cycle. We briefly discuss the potential clinical implications of these studies.

Corrigendum: Potential genetic overlap between insomnia and sleep symptoms in major depressive disorder: A polygenic risk score analysis.

[This corrects the article DOI: 10.3389/fpsyt.2021.734077.].

Melatonin's neuroprotective role in mitochondria and its potential as a biomarker in aging, cognition and psychiatric disorders.

Melatonin is an ancient molecule that is evident in high concentrations in various tissues throughout the body. It can be separated into two pools; one of which is synthesized by the pineal and can be found in blood, and the second by various tissues and is present in these tissues. Pineal melatonin levels display a circadian rhythm while tissue melatonin does not. For decades now, melatonin has been implicated in promoting and maintaining sleep. More recently, evidence indicates that it also plays an important role in neuroprotection. The beginning of our review will summarize this literature. As an amphiphilic, pleiotropic indoleamine, melatonin has both direct actions and receptor-mediated effects. For example, melatonin has established effects as an antioxidant and free radical scavenger both in vitro and in animal models. This is also evident in melatonin's prominent role in mitochondria, which is reviewed in the next section. Melatonin is synthesized in, taken up by, and concentrated in mitochondria, the powerhouse of the cell. Mitochondria are also the major source of reactive oxygen species as a byproduct of mitochondrial oxidative metabolism. The final section of our review summarizes melatonin's potential role in aging and psychiatric disorders. Pineal and tissue melatonin levels both decline with age. Pineal melatonin declines in individuals suffering from psychiatric disorders. Melatonin's ability to act as a neuroprotectant opens new avenues of exploration for the molecule as it may be a potential treatment for cases with neurodegenerative disease.

Increased levels of circulating cell-free mtDNA in plasma of late life depression subjects.

BACKGROUND: Oxidative stress (OS) has been implicated in the pathophysiology of late-life depression (LLD). Mitochondria are the primary source of oxidative stress and can be significantly damaged with increased OS. Circulating cell-free mtDNA (ccf-mtDNA) is a marker of cellular stress and mitochondria damage triggered by oxidative stress. METHODS: We evaluated the plasma levels of ccf-mtDNA in between 32 LLD and 21 never-depressed participants. We also investigated the association between ccf-mtDNA and the severity of depressive episodes and cognition performance. RESULTS: We found a higher ccf-mtDNA level in LLD cases compared with controls (t = -2.91, p = 0.005). Also, ccf-mtDNA was significantly correlated with the severity of depression (r = 0.42, p = 0.001). There was no significant correlation between ccf-mtDNA and measures of cognitive decline. LIMITATIONS: The small sample size and cross-sectional design were the main limitations of this study. CONCLUSION: Our results suggest that LLD is associated with elevated mitochondrial damage and cellular stress. If validated, the measurement of ccf-mtDNA in LLD can guide the development of novel treatments focused on cytoprotection and reduction of mitochondrial dysfunction for this condition.

Potential Genetic Overlap Between Insomnia and Sleep Symptoms in Major Depressive Disorder: A Polygenic Risk Score Analysis.

Background: The prevalence of insomnia and hypersomnia in depressed individuals is substantially higher than that found in the general population. Unfortunately, these concurrent sleep problems can have profound effects on the disease course. Although the full biology of sleep remains to be elucidated, a recent genome-wide association (GWAS) of insomnia, and other sleep traits in over 1 million individuals was recently published and provides many promising hits for genetics of insomnia in a population-based sample. Methods: Using data from the largest available GWAS of insomnia and other sleep traits, we sought to test if sleep variable PRS scores derived from population-based studies predicted sleep variables in samples of depressed cases [Psychiatric Genomics Consortium - Major Depressive Disorder subjects (PGC MDD)]. A leave-one-out analysis was performed to determine the effects that each individual study had on our results. Results: The only significant finding was for insomnia, where p-value threshold, p = 0.05 was associated with insomnia in our PGC MDD sample (R 2 = 1.75-3, p = 0.006). Conclusion: Our results reveal that <1% of variance is explained by the variants that cover the two significant p-value thresholds, which is in line with the fact that depression and insomnia are both polygenic disorders. To the best of our knowledge, this is the first study to investigate genetic overlap between the general population and a depression sample for insomnia, which has important treatment implications, such as leading to novel drug targets in future research efforts.

Overlapping mechanisms linking insulin resistance with cognition and neuroprogression in bipolar disorder.

Cognitive impairment is highly prevalent in the progression of both diabetes mellitus and bipolar disorder. The relationship between insulin resistance in diabetes and the risk of developing major neurocognitive disorders such as Alzheimer's disease has been well described. Insulin resistance and the associated metabolic deficiencies lead to biochemical alteration which hasten neurodegeneration and subsequent cognitive impairment. For bipolar disorder, some patients experience a cyclical, yet progressive course of illness. These patients are also more likely to have medical comorbidities such as cardiovascular disease and diabetes, and insulin resistance in particular may precede the neuroprogressive course. Diabetes and bipolar disorder share epidemiological, biochemical, and structural signatures, as well as cognitive impairment within similar domains, suggesting a common mechanism between the two conditions. Here we describe the association between insulin resistance and cognitive changes in bipolar disorder, as well as potential implications for therapeutic modulation of neuroprogression.

Association between the -2548G/A polymorphism of the leptin gene and antipsychotic-induced weight gain: Analysis of the CATIE sample and meta-analysis.

BACKGROUND: Antipsychotics, especially most of the second-generation antipsychotics, have a high risk for metabolic syndrome and antipsychotic-induced weight gain (AIWG). A promoter variant of the leptin (LEP) gene, -2548G/A (rs7799039), has been associated with AIWG in several studies. The aim of this study was to evaluate this association in the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) sample, followed by meta-analysis. METHODS: We investigated the association between rs7799039 and AIWG in a sub-sample of European (N = 164) individuals from the CATIE study. Body mass index (BMI) change and weight gain (presence or absence) was analyzed using ANCOVA and logistic regression, respectively. For the meta-analysis, a literature search was conducted using MEDLINE, Embase, and PsycINFO up to October 2019. The pooled odds ratio was calculated for presence or absence of weight gain (≥7% weight change) using a random effects model. RESULTS: We did not detect an association between rs7799039 and BMI change or weight gain (presence or absence) in the CATIE sample. As for the meta-analysis, we included 12 studies. No significant associations between the LEP rs7799039 polymorphism and AIWG were observed under the allelic genetic model (allele A vs. allele G) (OR = 1.10 [0.71, 1.70], p = .68). In the subgroup analyses of first-episode schizophrenia patients, a significant association between the A-allele and weight gain was observed, respectively (OR = 2.32 [1.41, 3.82], p = .0009). CONCLUSIONS: The present meta-analysis showed no significant effect of rs7799039 on AIWG. However, this variant may influence AIWG in first-episode schizophrenia patients. Further investigation of a larger and more homogenous sample is required to elucidate the role of the LEP gene in AIWG.

Genome-Wide Association Study of Sleep Disturbances in Depressive Disorders.

Sleep disturbance affects about 75% of depressed individuals and is associated with poorer patient outcomes. The genetics in this field is an emerging area of research. Thus far, only core circadian genes have been examined in this context. We expanded on this by performing a genome-wide association study (GWAS) followed by a preplanned hypothesis-driven analysis with 27 genes associated with the biology of sleep. All participants were diagnosed by their referring physician, completed the Beck Depression Inventory (BDI), and the Udvalg for Kliniske Undersogelser Side Effect Rating Scale at baseline. Our phenotype consisted of replies to 3 questions from these questionnaires. From standard GWAS chip data, imputations were performed. Baseline total BDI scores (n = 364) differed significantly between those with and those without sleep problems. We were unable to find any significant GWAS hits although our top hit was for changes in sleep and an intergenic marker near SNX18 (p = 1.06 × 10-6). None of the markers in our hypothesis-driven analysis remained significant after applying Bonferroni corrections. Our top finding among these genes was for rs13019460 of Neuronal PAS Domain Protein 2 with changes in sleep (p = 0.0009). Overall, both analyses were unable to detect any significant associations in our modest sample though we did find some interesting preliminary associations worth further exploration.

The phylogeography of African Brazilians.

BACKGROUND/AIMS: Approximately four million Africans were taken as slaves to Brazil, where they interbred extensively with Amerindians and Europeans. We have previously shown that while most White Brazilians carry Y chromosomes of European origin, they display high proportions of African and Amerindian mtDNA lineages, because of sex-biased genetic admixture. METHODS: We studied the Y chromosome and mtDNA haplogroup structure of 120 Black males from Sao Paulo, Brazil. RESULTS: Only 48% of the Y chromosomes, but 85% of the mtDNA haplogroups were characteristic of sub-Saharan Africa, confirming our previous observation of sexually biased mating. We mined literature data for mtDNA and Y chromosome haplogroup frequencies for African native populations from regions involved in Atlantic Slave Trade. Principal Components Analysis and Bayesian analysis of population structure revealed no genetic differentiation of Y chromosome marker frequencies between the African regions. However, mtDNA examination unraveled considerable genetic structure, with three clusters at Central-West Africa, West Africa and Southeast Africa. A hypothesis is proposed to explain this structure. CONCLUSION: Using these mtDNA data we could obtain for the first time an estimate of the relative ancestral contribution of Central-West (0.445), West (0.431) and Southeast Africa (0.123) to African Brazilians from Sao Paulo. These estimates are consistent with historical information.

Ancestral genomes, sex, and the population structure of Trypanosoma cruzi.

Acquisition of detailed knowledge of the structure and evolution of Trypanosoma cruzi populations is essential for control of Chagas disease. We profiled 75 strains of the parasite with five nuclear microsatellite loci, 24Salpha RNA genes, and sequence polymorphisms in the mitochondrial cytochrome oxidase subunit II gene. We also used sequences available in GenBank for the mitochondrial genes cytochrome B and NADH dehydrogenase subunit 1. A multidimensional scaling plot (MDS) based in microsatellite data divided the parasites into four clusters corresponding to T. cruzi I (MDS-cluster A), T. cruzi II (MDS-cluster C), a third group of T. cruzi strains (MDS-cluster B), and hybrid strains (MDS-cluster BH). The first two clusters matched respectively mitochondrial clades A and C, while the other two belonged to mitochondrial clade B. The 24Salpha rDNA and microsatellite profiling data were combined into multilocus genotypes that were analyzed by the haplotype reconstruction program PHASE. We identified 141 haplotypes that were clearly distributed into three haplogroups (X, Y, and Z). All strains belonging to T. cruzi I (MDS-cluster A) were Z/Z, the T. cruzi II strains (MDS-cluster C) were Y/Y, and those belonging to MDS-cluster B (unclassified T. cruzi) had X/X haplogroup genotypes. The strains grouped in the MDS-cluster BH were X/Y, confirming their hybrid character. Based on these results we propose the following minimal scenario for T. cruzi evolution. In a distant past there were at a minimum three ancestral lineages that we may call, respectively, T. cruzi I, T. cruzi II, and T. cruzi III. At least two hybridization events involving T. cruzi II and T. cruzi III produced evolutionarily viable progeny. In both events, the mitochondrial recipient (as identified by the mitochondrial clade of the hybrid strains) was T. cruzi II and the mitochondrial donor was T. cruzi III.