Mechanism of Transcription Anti-termination in Human Mitochondria.

In human mitochondria, transcription termination events at a G-quadruplex region near the replication origin are thought to drive replication of mtDNA by generation of an RNA primer. This process is suppressed by a key regulator of mtDNA-the transcription factor TEFM. We determined the structure of an anti-termination complex in which TEFM is bound to transcribing mtRNAP. The structure reveals interactions of the dimeric pseudonuclease core of TEFM with mobile structural elements in mtRNAP and the nucleic acid components of the elongation complex (EC). Binding of TEFM to the DNA forms a downstream "sliding clamp," providing high processivity to the EC. TEFM also binds near the RNA exit channel to prevent formation of the RNA G-quadruplex structure required for termination and thus synthesis of the replication primer. Our data provide insights into target specificity of TEFM and mechanisms by which it regulates the switch between transcription and replication of mtDNA.

Structural Basis of Mitochondrial Transcription Initiation.

Transcription in human mitochondria is driven by a single-subunit, factor-dependent RNA polymerase (mtRNAP). Despite its critical role in both expression and replication of the mitochondrial genome, transcription initiation by mtRNAP remains poorly understood. Here, we report crystal structures of human mitochondrial transcription initiation complexes assembled on both light and heavy strand promoters. The structures reveal how transcription factors TFAM and TFB2M assist mtRNAP to achieve promoter-dependent initiation. TFAM tethers the N-terminal region of mtRNAP to recruit the polymerase to the promoter whereas TFB2M induces structural changes in mtRNAP to enable promoter opening and trapping of the DNA non-template strand. Structural comparisons demonstrate that the initiation mechanism in mitochondria is distinct from that in the well-studied nuclear, bacterial, or bacteriophage transcription systems but that similarities are found on the topological and conceptual level. These results provide a framework for studying the regulation of gene expression and DNA replication in mitochondria.

Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase.

Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production, yet understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We report structures of two transcription initiation intermediate states of yeast mtRNAP that explain promoter melting, template alignment, DNA scrunching, abortive synthesis, and transition into elongation. In the partially melted initiation complex (PmIC), transcription factor MTF1 makes base-specific interactions with flipped non-template (NT) nucleotides "AAGT" at -4 to -1 positions of the DNA promoter. In the initiation complex (IC), the template in the expanded 7-mer bubble positions the RNA and NTP analog UTPαS, while NT scrunches into an NT loop. The scrunched NT loop is stabilized by the centrally positioned MTF1 C-tail. The IC and PmIC states coexist in solution, revealing a dynamic equilibrium between two functional states. Frequent scrunching/unscruching transitions and the imminent steric clashes of the inflating NT loop and growing RNA:DNA with the C-tail explain abortive synthesis and transition into elongation.

Structural basis of how MGME1 processes DNA 5' ends to maintain mitochondrial genome integrity.

Mitochondrial genome maintenance exonuclease 1 (MGME1) helps to ensure mitochondrial DNA (mtDNA) integrity by serving as an ancillary 5'-exonuclease for DNA polymerase γ. Curiously, MGME1 exhibits unique bidirectionality in vitro, being capable of degrading DNA from either the 5' or 3' end. The structural basis of this bidirectionally and, particularly, how it processes DNA from the 5' end to assist in mtDNA maintenance remain unclear. Here, we present a crystal structure of human MGME1 in complex with a 5'-overhang DNA, revealing that MGME1 functions as a rigid DNA clamp equipped with a single-strand (ss)-selective arch, allowing it to slide on single-stranded DNA in either the 5'-to-3' or 3'-to-5' direction. Using a nuclease activity assay, we have dissected the structural basis of MGME1-derived DNA cleavage patterns in which the arch serves as a ruler to determine the cleavage site. We also reveal that MGME1 displays partial DNA-unwinding ability that helps it to better resolve 5'-DNA flaps, providing insights into MGME1-mediated 5'-end processing of nascent mtDNA. Our study builds on previously solved MGME1-DNA complex structures, finally providing the comprehensive functional mechanism of this bidirectional, ss-specific exonuclease.

Mechanism of transcription initiation and primer generation at the mitochondrial replication origin OriL.

The intricate process of human mtDNA replication requires the coordinated action of both transcription and replication machineries. Transcription and replication events at the lagging strand of mtDNA prompt the formation of a stem-loop structure (OriL) and the synthesis of a ∼25 nt RNA primer by mitochondrial RNA polymerase (mtRNAP). The mechanisms by which mtRNAP recognizes OriL, initiates transcription, and transfers the primer to the replisome are poorly understood. We found that transcription initiation at OriL involves slippage of the nascent transcript. The transcript slippage is essential for initiation complex stability and its ability to translocate the mitochondrial DNA polymerase gamma, PolG, which pre-binds to OriL, downstream of the replication origin thus allowing for the primer synthesis. Our data suggest the primosome assembly at OriL-a complex of mtRNAP and PolG-can efficiently generate the primer, transfer it to the replisome, and protect it from degradation by mitochondrial endonucleases.

Helix unwinding and base flipping enable human MTERF1 to terminate mitochondrial transcription.

Defects in mitochondrial gene expression are associated with aging and disease. Mterf proteins have been implicated in modulating transcription, replication and protein synthesis. We have solved the structure of a member of this family, the human mitochondrial transcriptional terminator MTERF1, bound to dsDNA containing the termination sequence. The structure indicates that upon sequence recognition MTERF1 unwinds the DNA molecule, promoting eversion of three nucleotides. Base flipping is critical for stable binding and transcriptional termination. Additional structural and biochemical results provide insight into the DNA binding mechanism and explain how MTERF1 recognizes its target sequence. Finally, we have demonstrated that the mitochondrial pathogenic G3249A and G3244A mutations interfere with key interactions for sequence recognition, eliminating termination. Our results provide insight into the role of mterf proteins and suggest a link between mitochondrial disease and the regulation of mitochondrial transcription.

Mammalian RNase H1 directs RNA primer formation for mtDNA replication initiation and is also necessary for mtDNA replication completion.

The in vivo role for RNase H1 in mammalian mitochondria has been much debated. Loss of RNase H1 is embryonic lethal and to further study its role in mtDNA expression we characterized a conditional knockout of Rnaseh1 in mouse heart. We report that RNase H1 is essential for processing of RNA primers to allow site-specific initiation of mtDNA replication. Without RNase H1, the RNA:DNA hybrids at the replication origins are not processed and mtDNA replication is initiated at non-canonical sites and becomes impaired. Importantly, RNase H1 is also needed for replication completion and in its absence linear deleted mtDNA molecules extending between the two origins of mtDNA replication are formed accompanied by mtDNA depletion. The steady-state levels of mitochondrial transcripts follow the levels of mtDNA, and RNA processing is not altered in the absence of RNase H1. Finally, we report the first patient with a homozygous pathogenic mutation in the hybrid-binding domain of RNase H1 causing impaired mtDNA replication. In contrast to catalytically inactive variants of RNase H1, this mutant version has enhanced enzyme activity but shows impaired primer formation. This finding shows that the RNase H1 activity must be strictly controlled to allow proper regulation of mtDNA replication.

Structure and dynamics of the mitochondrial DNA-compaction factor Abf2 from S. cerevisiae.

Mitochondria are essential organelles that produce most of the energy via the oxidative phosphorylation (OXPHOS) system in all eukaryotic cells. Several essential subunits of the OXPHOS system are encoded by the mitochondrial genome (mtDNA) despite its small size. Defects in mtDNA maintenance and expression can lead to severe OXPHOS deficiencies and are amongst the most common causes of mitochondrial disease. The mtDNA is packaged as nucleoprotein structures, referred to as nucleoids. The conserved mitochondrial proteins, ARS-binding factor 2 (Abf2) in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and mitochondrial transcription factor A (TFAM) in mammals, are nucleoid associated proteins (NAPs) acting as condensing factors needed for packaging and maintenance of the mtDNA. Interestingly, gene knockout of Abf2 leads, in yeast, to the loss of mtDNA and respiratory growth, providing evidence for its crucial role. On a structural level, the condensing factors usually contain two DNA binding domains called high-mobility group boxes (HMG boxes). The co-operating mechanical activities of these domains are crucial in establishing the nucleoid architecture by bending the DNA strands. Here we used advanced solution NMR spectroscopy methods to characterize the dynamical properties of Abf2 together with its interaction with DNA. We find that the two HMG-domains react notably different to external cues like temperature and salt, indicating distinct functional properties. Biophysical characterizations show the pronounced difference of these domains upon DNA-binding, suggesting a refined picture of the Abf2 functional cycle.

Multiple introgression events during the diversification history of the edible Mexican grasshopper genus Sphenarium (Orthoptera: Pyrgomorphidae).

Speciation with gene flow often leads to ambiguous phylogenetic reconstructions, reticulate patterns of relatedness and conflicting nuclear versus mitochondrial (mt) lineages. Here we employed a fragment of the COI mtDNA gene and nuclear genome-wide data (3RAD) to assess the diversification history of Sphenarium, an orthopteran genus of great economic importance in Mexico that is presumed to have experienced hybridisation events in some of its species. We carried out separate phylogenetic analyses to evaluate the existence of mito-nuclear discordance in the species relationships, and also assessed the genomic diversity and population genomic structure and investigated the existence of interspecific introgression and species limits of the taxa involved based on the nuclear dataset. The species delineation analyses discriminated all the currently recognised species, but also supported the existence of four undescribed species. The mt and nuclear topologies had four discordant species relationships that can be explained by mt introgression, where the mt haplotypes of S. purpurascens appear to have replaced those of S. purpurascens A and B, S. variabile and S. zapotecum. Moreover, our analyses supported the existence of nuclear introgression events between four species pairs that are distributed in the Sierra Madre del Sur province in southeast Mexico, with three of them occurring in the Tehuantepec Isthmus region. Our study highlights the relevance of genomic data to address the relative importance of allopatric isolation versus gene flow in speciation.

Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues.

Methylation on CpG residues is one of the most important epigenetic modifications of nuclear DNA, regulating gene expression. Methylation of mitochondrial DNA (mtDNA) has been studied using whole genome bisulfite sequencing (WGBS), but recent evidence has uncovered technical issues which introduce a potential bias during methylation quantification. Here, we validate the technical concerns of WGBS, and develop and assess the accuracy of a new protocol for mtDNA nucleotide variant-specific methylation using single-molecule Oxford Nanopore Sequencing (ONS). Our approach circumvents confounders by enriching for full-length molecules over nuclear DNA. Variant calling analysis against showed that 99.5% of homoplasmic mtDNA variants can be reliably identified providing there is adequate sequencing depth. We show that some of the mtDNA methylation signal detected by ONS is due to sequence-specific false positives introduced by the technique. The residual signal was observed across several human primary and cancer cell lines and multiple human tissues, but was always below the error threshold modelled using negative controls. We conclude that there is no evidence for CpG methylation in human mtDNA, thus resolving previous controversies. Additionally, we developed a reliable protocol to study epigenetic modifications of mtDNA at single-molecule and single-base resolution, with potential applications beyond CpG methylation.

MitoPhen database: a human phenotype ontology-based approach to identify mitochondrial DNA diseases.

Diagnosing mitochondrial disorders remains challenging. This is partly because the clinical phenotypes of patients overlap with those of other sporadic and inherited disorders. Although the widespread availability of genetic testing has increased the rate of diagnosis, the combination of phenotypic and genetic heterogeneity still makes it difficult to reach a timely molecular diagnosis with confidence. An objective, systematic method for describing the phenotypic spectra for each variant provides a potential solution to this problem. We curated the clinical phenotypes of 6688 published individuals with 89 pathogenic mitochondrial DNA (mtDNA) mutations, collating 26 348 human phenotype ontology (HPO) terms to establish the MitoPhen database. This enabled a hypothesis-free definition of mtDNA clinical syndromes, an overview of heteroplasmy-phenotype relationships, the identification of under-recognized phenotypes, and provides a publicly available reference dataset for objective clinical comparison with new patients using the HPO. Studying 77 patients with independently confirmed positive mtDNA diagnoses and 1083 confirmed rare disease cases with a non-mitochondrial nuclear genetic diagnosis, we show that HPO-based phenotype similarity scores can distinguish these two classes of rare disease patients with a false discovery rate <10% at a sensitivity of 80%. Enriching the MitoPhen database with more patients will improve predictions for increasingly rare variants.

Discovery of highly reactive self-splicing group II introns within the mitochondrial genomes of human pathogenic fungi.

Fungal pathogens represent an expanding global health threat for which treatment options are limited. Self-splicing group II introns have emerged as promising drug targets, but their development has been limited by a lack of information on their distribution and architecture in pathogenic fungi. To meet this challenge, we developed a bioinformatic workflow for scanning sequence data to identify unique RNA structural signatures within group II introns. Using this approach, we discovered a set of ubiquitous introns within thermally dimorphic fungi (genera of Blastomyces, Coccidioides and Histoplasma). These introns are the most biochemically reactive group II introns ever reported, and they self-splice rapidly under near-physiological conditions without protein cofactors. Moreover, we demonstrated the small molecule targetability of these introns by showing that they can be inhibited by the FDA-approved drug mitoxantrone in vitro. Taken together, our results highlight the utility of structure-based informatic searches for identifying riboregulatory elements in pathogens, revealing a striking diversity of reactive self-splicing introns with great promise as antifungal drug targets.

Accurate mapping of mitochondrial DNA deletions and duplications using deep sequencing.

Deletions and duplications in mitochondrial DNA (mtDNA) cause mitochondrial disease and accumulate in conditions such as cancer and age-related disorders, but validated high-throughput methodology that can readily detect and discriminate between these two types of events is lacking. Here we establish a computational method, MitoSAlt, for accurate identification, quantification and visualization of mtDNA deletions and duplications from genomic sequencing data. Our method was tested on simulated sequencing reads and human patient samples with single deletions and duplications to verify its accuracy. Application to mouse models of mtDNA maintenance disease demonstrated the ability to detect deletions and duplications even at low levels of heteroplasmy.

Expression of Hairpin-Enriched Mitochondrial DNA in Two Hairworm Species (Nematomorpha).

Nematomorpha (hairworms) is a phylum of parasitic ecdysozoans, best known for infecting arthropods and guiding their hosts toward water, where the parasite can complete its life cycle. Over 350 species of nematomorphs have been described, yet molecular data for the group remain scarce. The few available mitochondrial genomes of nematomorphs are enriched with long inverted repeats, which are embedded in the coding sequences of their genes-a remarkably unusual feature exclusive to this phylum. Here, we obtain and annotate the repeats in the mitochondrial genome of another nematomorph species-Parachordodes pustulosus. Using genomic and transcriptomic libraries, we investigate the impact of inverted repeats on the read coverage of the mitochondrial genome. Pronounced drops in the read coverage coincide with regions containing long inverted repeats, denoting the 'blind spots' of short-fragment sequencing libraries. Phylogenetic inference with the novel data reveals multiple disagreements between the traditional system of Nematomorpha and molecular data, rendering several genera paraphyletic, including Parachordodes.

A report on the complete mitochondrial genome of the trematode Azygia robusta Odhner, 1911, its new definitive host from the Russian Far East, and unexpected phylogeny of Azygiidae within Digenea, as inferred from mitogenome sequences.

New data on the complete mitochondrial genome of Azygia robusta (Azygiidae) were obtained by the next-generation sequencing (NGS) approach. The mitochondrial DNA (mtDNA) of A. robusta had a length of 13 857 bp and included 12 protein-coding genes, two ribosomal genes, 22 transfer RNA genes, and two non-coding regions. The nucleotide sequences of the complete mitochondrial genomes of two A. robusta specimens differed from each other by 0.12 ± 0.03%. Six of 12 protein-coding genes demonstrated intraspecific variation. The difference between the nucleotide sequences of the complete mitochondrial genomes of A. robusta and Azygia hwangtsiyui was 26.95 ± 0.35%; the interspecific variation of protein-coding genes between A. robusta and A. hwangtsiyui ranged from 20.5 ± 0.9% (cox1) to 30.7 ± 1.2% (nad5). The observed gene arrangement in the mtDNA sequence of A. robusta was identical to that of A. hwangtsiyui. Codon usage and amino acid frequencies were highly similar between A. robusta and A. hwangtsiyui. The results of phylogenetic analyses based on mtDNA protein-coding regions showed that A. robusta is closely related to A. hwangtsiyui (belonging to the same suborder, Azygiida) that formed a distinct early-diverging branch relative to all other Digenea. A preliminary morphological analysis of paratypes of the two azygiid specimens studied showed visible morphological differences between them. The specimen extracted from Sakhalin taimen (Parahucho perryi) was most similar to A. robusta. Thus, we here provide the first record of a new definitive host, P. perryi, for A. robusta and also molecular characteristics of the trematode specimens.

mtDNA-Specific Ultrasensitive Near-Infrared Fluorescent Probe Enables the Differentiation of Healthy and Apoptotic Cells.

Mitochondrial DNA (mtDNA) as a class of important genetic material is easily damaged, which can result in a series of metabolic diseases, hereditary disease, and so on. mtDNA is an ultrasensitive indicator for the health of living cells due to the extremely short physiological response time of mtDNA toward damage (ca. 5.0 min). Therefore, the development of specific ultrasensitive fluorescent probes that can in real-time monitor mtDNA in vivo are of great value. With this research, we developed a near-infrared twisted intramolecular charge transfer (TICT) fluorescent probe YON. YON is a thread-like molecule with an A-π-D-π-A structure, based on the dicyanoisophorone fluorophore. The molecular design of YON enabled the specific binding with dsDNA (binding constant (K) = 8.5 × 105 M-1) within 1.3 min. And the appropriate water-oil amphiphilicity makes YON significantly accumulate in the mitochondria, enabling the specific binding to mtDNA. The fluorescence intensity at 640 nm of YON enhanced linearly with increasing concentrations of mtDNA. Dicyanoisophorone as the strong electron-withdrawing group that was introduced into both ends of the molecule resulted in YON being a classic quadrupole, so it could ultrasensitively detect trace mtDNA. The minimum detection limit was 71 ng/mL. Moreover, the large Stokes shift (λex = 435 nm, λem = 640 nm) makes YON suitable for "interference-free" imaging of mtDNA. Therefore, YON was used to monitor trace changes of mtDNA in living cells; more importantly, it could be used to evaluate the health of cells by monitoring microchanges of mtDNA, enabling the ultrasensitive evaluation of apoptosis.

Rapid diversification of the Variable Seedeater superspecies complex despite widespread gene flow.

Disentangling the evolutionary relationships of rapidly radiating clades is often challenging because of low genetic differentiation and potentially high levels of gene flow among diverging taxa. The genus Sporophila consists of small Neotropical birds that show, in general, relatively low genetic divergence, but particularly high speciation rates and pronounced variation in secondary sexual traits (e.g., plumage color), which can be important in generating premating reproductive isolation. In cases like these, the use of genome-wide sequence data can increase the resolution to uncover a clade's evolutionary history. Here, we used a phylogenomic approach to study the evolutionary history and genetic structure of the Variable Seedeater superspecies complex, which includes S. corvina, S. intermedia, and S. americana. Using ∼25,000 genome-wide single nucleotide polymorphisms (SNPs), we confirmed that the Variable Seedeater superspecies complex is monophyletic. However, a phylogenetic reconstruction based on a mitochondrial marker (ND2) resulted in a discordant tree topology, particularly in the position of Wing-barred Seedeater S. americana, which might be due to a mitochondrial capture event. Our results suggest historical gene flow among lineages, particularly between species with conflicting topologies. Among the four phenotypically variable S. corvina subspecies, our structure analyses identified three main distinct genetic groups (K = 3), and that the entirely black subspecies, S. c. corvina, is derived from within a pied-colored clade. Further, we inferred widespread gene flow across the whole species' distribution, including between subspecies. However, gene flow was about 100 times lower at the geographic boundaries of the entirely black and the pied subspecies, suggesting an important role for plumage divergence in limiting gene flow. Overall, our findings suggest that the early diversification of the Sporophila genus occurred rapidly despite historical gene flow between lineages and that divergence in plumage color possibly influences the extent of gene flow among taxa.

Comparative phylogenomic patterns in the Baja California avifauna, their conservation implications, and the stages in lineage divergence.

Comparative phylogeography explores the historical congruence of co-distributed species to understand the factors that led to their current genetic and phenotypic structures. Even species that span the same biogeographic barrier can exhibit different phylogeographic structures owing to differences in effective population sizes, genetic marker bias, and dispersal abilities. The Baja California peninsula and adjacent desert regions include several biogeographic barriers, including the Vizcaíno Desert and Sierra de la Laguna (Cape District), that have left phylogeographic patterns in some but not all species. We used genome-wide SNP data to test the hypothesis that the diverse phylogeographic patterns inferred from prior studies were supported. We found that mitochondrial DNA, single nuclear gene, and genome-wide SNP data show that the cactus wren and LeConte's thrasher have a concordant historical division at or near the Vizcaíno Desert in north-central Baja California, the Gila woodpecker is at an intermediate stage of divergence, and the California gnatcatcher lacks phylogeographic structure. None of these four species are classified taxonomically in a way that captures their evolutionary history with the exception of the LeConte's thrasher. We also analyzed mtDNA data on samples of nine other species that span the Vizcaíno Desert, with four showing no apparent division, and six additional species from the Sierra de la Laguna, all but one of which are differentiated. Reasons for contrasting phylogeographic patterns among these species should be explored further with genomic data to test the extent of concordant phylogeographic patterns. The evolutionary division at the Vizcaíno desert is well known in other vertebrate species, and our study further corroborates the extent, profound effect, and importance of this biogeographic boundary. The areas north and south of the Vizcaíno Desert, which contains considerable diversity, should be recognized as historically significant areas for conservation.

Population connectivity across a highly fragmented distribution: Phylogeography of the Chalcophaps doves.

Chalcophaps is a morphologically conserved genus of ground-walking doves distributed from India to mainland China, south to Australia, and across the western Pacific to Vanuatu. Here, we reconstruct the evolutionary history of this genus using DNA sequence data from two nuclear genes and one mitochondrial gene, sampled from throughout the geographic range of Chalcophaps. We find support for three major evolutionary lineages in our phylogenetic reconstruction, each corresponding to the three currently recognized Chalcophaps species. Despite this general concordance, we identify discordant mitochondrial and nuclear ancestries in the subspecies C. longirostris timorensis, raising further questions about the evolutionary history of this Timor endemic population. Within each of the three species, we find evidence for isolation by distance or hierarchical population structure, indicating an important role for geography in the diversification of this genus. Despite being distributed broadly across a highly fragmented geographic region known as a hotspot for avian diversification, the Chalcophaps doves show modest levels of phenotypic and genetic diversity, a pattern potentially explained by strong population connectivity owing to high overwater dispersal capability.

Geological and climatic influences on population differentiation of the Phrynocephalus vlangalii species complex (Sauria: Agamidae) in the northern Qinghai-Tibet Plateau.

Extremely heterogeneous topography and complex paleoclimatic history of the Qinghai-Tibet Plateau (QTP) have a key role in promoting genetic divergence among populations and lineage/species formation. Here, we sequenced one nuclear and three mitochondrial markers of 532 individuals from the entire range of the Phrynocephalus vlangalii species complex including two species, P. putjatai and P. vlangalii, endemic to the northern QTP. We integrated multilocus phylogeny, demographic analysis and geographic barrier detection to evaluate the population structure and dynamics. We found a new mitochondrial clade (PV-I) in the Gonghe County population of P. vlangalii, partial mitochondrial DNA replacement within P. vlangalii and complete mitochondrial DNA replacement between P. putjatai and P. vlangalii. Neutrality test, mismatch distribution analysis and Extended Bayesian Skyline Plot (EBSP) analysis all supported a significant expansion of the Qaidam Basin population of P. vlangalii (PV-II-2) from 0.091 to 0.026 Ma after Penultimate Glaciation. The uplift of the Arjin and Anyemanqen Mountains during the Kunhuang Movement (∼1.2 Ma) split populations of P. vlangalii in Akesai, Qaidam Basin and source of the Yellow River. The uplift of the Elashan Mountains during the second phase of the Qingzang Movement (∼2.5 Ma) contributed to the divergence of the Gonghe County population of P. vlangalii from other conspecific populations. The third phase of the Qingzang Movement (∼1.7 Ma) contributed to the divergence of the Xinghai population of P. vlangalii from P. putjatai and to the divergence of the northern populations of P. putjatai from the southern conspecific populations. Our data support the idea that the geological and climatic changes following the orogeny of the QTP may have promoted population differentiation and shaped the current population patterns of the P. vlangalii species complex in the northeastern QTP.