A genetic atlas for the butterflies of continental Canada and United States.

Multi-locus genetic data for phylogeographic studies is generally limited in geographic and taxonomic scope as most studies only examine a few related species. The strong adoption of DNA barcoding has generated large datasets of mtDNA COI sequences. This work examines the butterfly fauna of Canada and United States based on 13,236 COI barcode records derived from 619 species. It compiles i) geographic maps depicting the spatial distribution of haplotypes, ii) haplotype networks (minimum spanning trees), and iii) standard indices of genetic diversity such as nucleotide diversity (π), haplotype richness (H), and a measure of spatial genetic structure (GST). High intraspecific genetic diversity and marked spatial structure were observed in the northwestern and southern North America, as well as in proximity to mountain chains. While species generally displayed concordance between genetic diversity and spatial structure, some revealed incongruence between these two metrics. Interestingly, most species falling in this category shared their barcode sequences with one at least other species. Aside from revealing large-scale phylogeographic patterns and shedding light on the processes underlying these patterns, this work also exposed cases of potential synonymy and hybridization.

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.

Mitochondrial replication's role in vertebrate mtDNA strand asymmetry.

Mitogenomes are defined as compact and structurally stable over aeons. This perception results from a vertebrate-centric vision, where few types of mtDNA rearrangements are described. Here, we bring a new light to the involvement of mitochondrial replication in the strand asymmetry of the vertebrate mtDNA. Using several species of deep-sea hatchetfish (Sternoptychidae) displaying distinct mtDNA structural arrangements, we unravel the inversion of the coding direction of protein-coding genes (PCGs). This unexpected change is coupled with a strand asymmetry nucleotide composition reversal and is shown to be directly related to the strand location of the Control Region (CR). An analysis of the fourfold redundant sites of the PCGs (greater than 6000 vertebrates), revealed the rarity of this phenomenon, found in nine fish species (five deep-sea hatchetfish). Curiously, in Antarctic notothenioid fishes (Trematominae), where a single PCG inversion (the only other record in fish) is coupled with the inversion of the CR, the standard asymmetry is disrupted for the remaining PCGs but not yet reversed, suggesting a transitory state. Our results hint that a relaxation of the classic vertebrate mitochondrial structural stasis promotes disruption of the natural balance of asymmetry of the mtDNA. These findings support the long-lasting hypothesis that replication is the main molecular mechanism promoting the strand-specific compositional bias of this unique and indispensable molecule.

Molecular differentiation of Sarcocystis miescheriana and Sarcocystis suihominis using a new multiplex PCR targeting the mtDNA cox1 gene in wild boars in southern Italy.

The increase of wild boar populations density and their meat consumption across Europe could expose humans to a plethora of foodborne diseases as sarcocystosis, caused by the zoonotic protozoan Sarcocystis suihominis. Humans become infected by eating raw or undercooked pig (Sus scrofa domesticus) containing S. suihominis sarcocysts. Despite this, to date very few data are available on the risk of infection by this parasite to wild boar (Sus scrofa) meat consumers. Thus, the present study aimed to assess the occurrence of Sarcocystis spp. in wild boars from southern Italy, applying both histology and a new multiplex PCR assay targeting the cox1 gene. Between 2019 and 2020, 997 muscle tissues (i.e., n = 269 oesophagus, n = 277 diaphragms, n = 298 hearts, n = 153 tongues) from 311 wild boars were collected and screened by a combined histological and molecular approach. Overall, 251 (80.7%) animals tested were positive for Sarcocystis spp., and S. miescheriana whose definitive hosts are canids, was the only molecularly identified species. A statistically significant difference (p < 0.05) in the prevalence of Sarcocystis infection was found according to the wild boar age and muscle tissue. Findings outlined the low zoonotic potential of infection to humans via wild boar meat consumption in Italy and the importance of the application of new molecular methods in distinguishing different Sarcocystis species.

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.

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.

Genetic diversity and population structure of Capitulum mitella (Linnaeus, 1767) in Fujian (China) revealed by mtDNA COI sequences.

Background: Capitulum mitella is a widely distributed and ecologically important stalked barnacle that settles extensively on rocky shores. This species contributes to the structural complexity of intertidal habitats and plays a critical role in the marine ecosystem. This study aimed to reveal the genetic diversity and population structure of C. mitella by analyzing the mitochondrial cytochrome oxidase I (COI) gene. Methods: A 683bp fragment of the COI gene was sequenced from 390 individuals sampled from six localities in Fujian, China. Results: A total of 84 distinct haplotypes were identified through the analysis of 82 polymorphic sites, resulting in an average haplotype diversity (h) of 0.660 and nucleotide diversity (π) of 0.00182. Analysis of molecular variance (AMOVA) and pairwise F ST statistics showed no significant population structure. Neutrality tests and mismatch distributions provided evidence of recent population expansion for the species. Conclusions: We suggest that the species' high dispersal ability, and ocean currents coupled with limited physical barriers in the region, contribute to its current phylogeographic structure. These findings enhance our comprehension of the genetic diversity and population structure of C. mitella, providing valuable insights for future conservation efforts.

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.

The complete mitochondrial genome of Ogmocotyle ailuri: gene content, composition and rearrangement and phylogenetic implications.

Trematodes of the genus Ogmocotyle are intestinal flukes that can infect a variety of definitive hosts, resulting in significant economic losses worldwide. However, there are few studies on molecular data of these trematodes. In this study, the mitochondrial (mt) genome of Ogmocotyle ailuri isolated from red panda (Ailurus fulgens) was determined and compared with those from Pronocephalata to investigate the mt genome content, genetic distance, gene rearrangements and phylogeny. The complete mt genome of O. ailuri is a typical closed circular molecule of 14 642 base pairs, comprising 12 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions. All genes are transcribed in the same direction. In addition, 23 intergenic spacers and 2 locations with gene overlaps were determined. Sequence identities and sliding window analysis indicated that cox1 is the most conserved gene among 12 PCGs in O. ailuri mt genome. The sequenced mt genomes of the 48 Plagiorchiida trematodes showed 5 types of gene arrangement based on all mt genome genes, with the gene arrangement of O. ailuri being type I. Phylogenetic analysis using concatenated amino acid sequences of 12 PCGs revealed that O. ailuri was closer to Ogmocotyle sikae than to Notocotylus intestinalis. These data enhance the Ogmocotyle mt genome database and provide molecular resources for further studies of Pronocephalata taxonomy, population genetics and systematics.

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.

Signatures of purifying selection and site-specific positive selection on the mitochondrial DNA of dromedary camels (Camelus dromedarius).

The two species of the Old World Camelini tribe, dromedary and Bactrian camels, show superior adaptability to the different environmental conditions they populate, e.g. desert, mountains and coastal areas, which might be associated with adaptive variations on their mitochondrial DNA. Here, we investigate signatures of natural selection in the 13-mitochondrial protein-coding genes of different dromedary camel populations from the Arabian Peninsula, Africa and southwest Asia. The full mitogenome sequences of 42 dromedaries, 38 domestic Bactrian, 29 wild Bactrian camels and 31 samples representing the New World Lamini tribe reveal species-wise genetic distinction among Camelidae family species, with no evidence of geographic distinction among dromedary camels. We observe gene-wide signals of adaptive divergence between the Old World and New World camels, with evidence of purifying selection among Old World camel species. Upon comparing the different Camelidae tribes, 27 amino acid substitutions across ten mtDNA protein-coding genes were found to be under positive selection, in which, 24 codons were defined to be under positive adaptive divergence between Old World and New World camels. Seven codons belonging to three genes demonstrated positive selection in dromedary lineage. A total of 89 codons were found to be under positive selection in Camelidae family based on investigating the impact of amino acid replacement on the physiochemical properties of proteins, including equilibrium constant and surrounding hydrophobicity. These mtDNA variants under positive selection in the Camelidae family might be associated with their adaptation to their contrasting environments.

Legacies of domestication, Neolithic diffusion and trade between Indian subcontinent and Island Southeast Asia shape maternal genetic diversity of Andaman cattle.

Andaman cattle is a precious indigenous livestock species endemic to Andaman and Nicobar Islands, India. Till date, origin and genetic makeup of the breed which is warranted for breed conservation is not known. Moreover, the spread of zebu cattle from Indus valley to different parts of Island Southeast Asia (ISEA) is not properly understood. Here, we report the genetic diversity, population structure of Andaman cattle and their evolution in the context of epicentre of zebu domestication and ISEA. High genetic diversity in complete mitochondrial D-loop sequences indicated the ability of the breed to withstand impending climate change. Total 81 haplotypes were detected and all of them except three belonged to Bos indicus. The presence of taurine haplotypes in Andaman cattle indicate introgression by European-derived cattle. A poor phylogenetic signal of Andaman cattle with genetic affinities with cattle of Indian subcontinent and ISEA was observed. The poor phylogenetic structure may be due to multidirectional gene flow from Indian subcontinent and ISEA, with which Andaman shares a close cultural and trade relationship from Neolithic age. We hypothesize that Andaman cattle is the outcome of Neolithic diffusion from centre of zebu domestication along with multidirectional commercial exchange between Indian subcontinent and ISEA.

Non-B DNA conformations analysis through molecular dynamics simulations.

BACKGROUND: Non-B DNA conformations are molecular structures that do not follow the canonical DNA double helix. Mutagenetic instability in nuclear and mitochondrial DNA (mtDNA) genomes has been associated with simple non-B DNA conformations, as hairpins or more complex structures, as G-quadruplexes. One of these structures is Structure A, a cloverleaf-like non-B conformation predicted for a 93-nt (nucleotide) stretch of the mtDNA control region 5'-peripheral domain. Structure A is embedded in a hot spot for the 3' end of human mtDNA deletions revealing its importance in influencing the mutational instability of the mtDNA genome. METHODS: To better characterize Structure A, we predicted its 3D conformation using state-of-art methods and algorithms. The methodologic workflow consisted in the prediction of non-B conformations using molecular dynamics simulations. The conservation scores of alignments of the Structure A region in humans, primates, and mammals, was also calculated. RESULTS: Our results show that these computational methods are able to measure the stability of non-B conformations by using the level of base pairing during molecular dynamics. Structure A showed high stability and low flexibility correlated with high conservation scores in mammalian, more specifically in primate lineages. CONCLUSIONS: We showed that 3D non-B conformations can be predicted and characterized by our methodology. This allowed the in-depth analysis of the structure A, and the main results showed the structure remains stable during the simulations. GENERAL SIGNIFICANCE: The fine-scale atomic molecular determination of this type of non-B conformation opens the way to perform computational molecular studies that can show their involvement in mtDNA cellular mechanisms.

Population genetic structure of Neoschongastia gallinarum in South China based on mitochondrial DNA markers.

The genetic diversity and differentiation of four geographic populations of Neoschongastia gallinarum were evaluated using concatenated mitochondrial gene sequences (pCOI, pCOII, and pND5). Based on the results, the N. gallinarum populations had high genetic diversity and strong ecological adaptability. Genetic differentiation among paired populations calculated using concatenated mitochondrial gene sequences revealed that geographic isolation resulted in genetic differentiation among the populations of N. gallinarum, and gene flow between populations associated with human trade activities. Systematic development and molecular variance based on haplotypes revealed that genetic variation existed in different haplotypes; however, no clear rule related to geographic region was found. Further, genetic variation was mainly derived from individuals within the population. A neutral test based on concatenated mitochondrial gene sequences and nucleotide pair differences revealed that N. gallinarum did not experience an obvious population expansion in recent historical periods. Accordingly, the population size was relatively stable.

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.

Mitochondrial diversity and inter-specific phylogeny among dolphins of the genus Stenella in the Southwest Atlantic Ocean.

The genus Stenella is comprised of five species occurring in all oceans. Despite its wide distribution, genetic diversity information on these species is still scarce especially in the Southwest Atlantic Ocean. Some features of this genus can enhance opportunities for potential introgressive hybridization, e.g. sympatric distibution along the Brazilian coast, mixed known associations among species, karyotype uniformity and genome permeability. In this study we analyzed three genes of the mitochondrial genome to investigate the genetic diversity and occurrence of genetic mixture among eighty specimens of Stenella. All species exhibited moderate to high levels of genetic diversity (h = 0.833 to h = 1.000 and π = 0.006 to π = 0.015). Specimens of S. longirostris, S. attenuata and S. frontalis were clustered into differentiated haplogroups, in contrast, haplotypes of S. coeruleoalba and S. clymene were clustered together. We detected phylogenetic structure of mixed clades for S. clymene and S. coeruleoalba specimens, in the Southwest Atlantic Ocean, and also between S. frontalis and S. attenuata in the Northeast Atlantic Ocean, and between S. frontalis and S. longirostris in the Northwest Atlantic Ocean. These specimes were morphologically identified as one species but exhibited the maternal lineage of another species, by mitochondrial DNA. Our results demonstrate that ongoing gene flow is occurring among species of the genus Stenella reinforcing that this process could be one of the reasons for the confusing taxonomy and difficulties in elucidating phylogenetic relationships within this group.

Nuclear mitochondrial DNA sequences in the rabbit genome.

Numtogenesis is observable in the mammalian genomes resulting in the integration of mitochondrial segments into the nuclear genomes (numts). To identify numts in rabbit, we aligned mitochondrial and nuclear genomes. Alignment significance threshold was calculated and individual characteristics of numts were analysed. We found 153 numts in the nuclear genome. The GC content of numts were significantly lower than the GC content of their genomic flanking regions or the genome itself. The frequency of three mammalian-wide interspersed repeats were increased in the proximity of numts. The decreased GC content around numts strengthen the theory which supposes a link between DNA structural instability and numt integration.

Heteroplasmic mitochondrial genomes of a Raillietina tapeworm in wild Pangolin.

BACKGROUND: Raillietina species belong to the family Davaineidae, which parasitizes in a wide variety of mammals and birds, causing stunted growth, lethargy, emaciation, and digestive tract obstruction. However, only a limited number of Raillietina species have been identified in wild animals. METHODS: We analyzed and annotated the complete mitochondrial (mt) genome of a worm from the intestine of a wild pangolin using Illumina sequencing of whole genomic DNA. RESULTS: These findings showed the presence of two mtDNA sequences in Raillietina sp., designated as mt1 and mt2, with the lengths of 14,331 bp and 14,341 bp, respectively. Both the mts genomes of Raillietina sp. comprised 36 genes, containing 12 protein-coding genes (PCGs), 2 ribosomal RNAs, and 22 transfer RNAs. Gene arrangements of both mt genomes of Raillietina sp. were similar to those of most flatworms, except for taeniids, which shift positions between tRNAL1 and tRNAS2 genes. Twenty of 22 tRNA secondary structures of Raillietina sp. had a typical cloverleaf structure similar to Raillietina tetragona. Sequence differences between the mt1 and mt2 genomes were 4.4%, and this difference arises from the mtDNA heteroplasmic mutations. Moreover, heteroplasmic mtDNA mutations were detected in PCGs, tRNAs, rRNAs, NCRs, and intergenes, but the highest proportion of heteroplasmy of 79.0% was detected in PCGs, indicating the occurrence of mtDNA heteroplasmy in Raillietina sp. To our knowledge, this is the first report of mtDNA heteroplasmy in tapeworm parasites. Phylogenetic analyses of 18S rRNA, ITS2, and 12 PCG sequences demonstrated that the worm was clustered with other Raillietina species in the Davaneidae family. CONCLUSIONS: We found a novel Raillietina species in wild pangolin with the existence of mitochondrial DNA heteroplasmy. Thus, these findings provide insights into the heterogeneity of the mt genome in parasitic cestodes, and mt genome data contributes to the understanding of pangolin-parasitic cestodes in terms of their molecular biology, epidemiology, diagnosis, and taxonomy.

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.

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.