Engineering sub-organelles of a diploid Saccharomyces cerevisiae to enhance the production of 7-dehydrocholesterol.

7-Dehydrocholesterol (7-DHC) is widely present in various organisms and is an important precursor of vitamin D3. Despite significant improvements in the biosynthesis of 7-DHC, it remains insufficient to meet the industrial demands. In this study, we reported high-level production of 7-DHC in an industrial Saccharomyces cerevisiae leveraging subcellular organelles. Initially, the copy numbers of DHCR24 were increased in combination with sterol transcriptional factor engineering and rebalanced the redox power of the strain. Subsequently, the effects of compartmentalizing the post-squalene pathway in peroxisomes were validated by assembling various pathway modules in this organelle. Furthermore, several peroxisomes engineering was conducted to enhance the production of 7-DHC. Utilizing the peroxisome as a vessel for partial post-squalene pathways, the potential of yeast for 7-dehydrocholesterol production was demonstrated by achieving a 26-fold increase over the initial production level. 7-DHC titer reached 640.77 mg/L in shake flasks and 4.28 g/L in a 10 L bench-top fermentor, the highest titer ever reported. The present work lays solid foundation for large-scale and cost-effective production of 7-DHC for practical applications.

Augmented Electrochemical Oxygen Evolution by d-p Orbital Electron Coupling.

While high-entropy alloys (HEAs), high-entropy oxides (HEOs), and high-entropy hydroxides (HEHs), have been advanced as a novel frontier in electrocatalytic oxygen evolution, their inherent activity deficiency poses a major challenge. To achieve the unlimited goal to tailor the structure-activity relationship in multicomponent systems, entropy-driven composition engineering presents substantial potential, by fabricating high-entropy anion-regulated transition metal compounds as sophisticated oxygen evolution reaction (OER) electrocatalysts. Herein, we developed a versatile two-dimensional high-entropy metal phosphorus trisulfides (HEPS3) as a promising and adjustable platform. Leveraging the multiple electron-coupling and d-p orbital hybridization induced by the cocktail effect, we disclose the exceptional oxygen evolution catalytic activity upon van der Waals material (MnFeCoNiZn)PS3, exhibiting an impressively low overpotential of 240 mV at a current density of 10 mA cm-2, a minimal Tafel slope of 32 mV dec-1, and negligible degradation under varying current densities for over 96 hours. Density functional theory (DFT) calculations further offer insights into the correlation between orbital hybridization and catalytic performance within high-entropy systems, underscoring the contribution of active phosphorus centers on the substrate to performance enhancements. Moreover, by achieving electron redistribution to optimize the electron coordination environment, this work presents an effective strategy for advanced catalysts in energy-related applications. This article is protected by copyright. All rights reserved.

Efficient production of l-glutathione by whole-cell catalysis with ATP regeneration from adenosine.

l-glutathione (GSH) is an important tripeptide compound with extensive applications in medicine, food additives, and cosmetics industries. In this work, an innovative whole-cell catalytic strategy was developed to enhance GSH production by combining metabolic engineering of GSH biosynthetic pathways with an adenosine-based adenosine triphosphate (ATP) regeneration system in Escherichia coli. Concretely, to enhance GSH production in E. coli, several genes associated with GSH and  l-cysteine degradation, as well as the branched metabolic flow, were deleted. Additionally, the GSH bifunctional synthase (GshFSA) and GSH ATP-binding cassette exporter (CydDC) were overexpressed. Moreover, an adenosine-based ATP regeneration system was first introduced into E. coli to enhance GSH biosynthesis without exogenous ATP additions. Through the optimization of whole-cell catalytic conditions, the engineered strain GSH17-FDC achieved an impressive GSH titer of 24.19 g/L only after 2 h reaction, with a nearly 100% (98.39%) conversion rate from the added  l-Cys. This work not only unveils a new platform for GSH production but also provides valuable insights for the production of other high-value metabolites that rely on ATP consumption.

Advances in biotin biosynthesis and biotechnological production in microorganisms.

Biotin, also known as vitamin H or B7, acts as a crucial cofactor in the central metabolism processes of fatty acids, amino acids, and carbohydrates. Biotin has important applications in food additives, biomedicine, and other fields. While the ability to synthesize biotin de novo is confined to microorganisms and plants, humans and animals require substantial daily intake, primarily through dietary sources and intestinal microflora. Currently, chemical synthesis stands as the primary method for commercial biotin production, although microbial biotin production offers an environmentally sustainable alternative with promising prospects. This review presents a comprehensive overview of the pathways involved in de novo biotin synthesis in various species of microbes and insights into its regulatory and transport systems. Furthermore, diverse strategies are discussed to improve the biotin production here, including mutation breeding, rational metabolic engineering design, artificial genetic modification, and process optimization. The review also presents the potential strategies for addressing current challenges for industrial-scale bioproduction of biotin in the future. This review is very helpful for exploring efficient and sustainable strategies for large-scale biotin production.

Museum skins enable identification of introgression associated with cytonuclear discordance.

Increased sampling of genomes and populations across closely related species has revealed that levels of genetic exchange during and after speciation are higher than previously thought. One obvious manifestation of such exchange is strong cytonuclear discordance, where the divergence in mitochondrial DNA (mtDNA) differs from that for nuclear genes more (or less) than expected from differences between mtDNA and nuclear DNA (nDNA) in population size and mutation rate. Given genome-scale datasets and coalescent modelling, we can now confidently identify cases of strong discordance and test specifically for historical or recent introgression as the cause. Using population sampling, combining exon capture data from historical museum specimens and recently collected tissues we showcase how genomic tools can resolve complex evolutionary histories in the brachyotis group of rock-wallabies (Petrogale). In particular, applying population and phylogenomic approaches we can assess the role of demographic processes in driving complex evolutionary patterns and assess a role of ancient introgression and hybridisation. We find that described species are well supported as monophyletic taxa for nDNA genes, but not for mtDNA, with cytonuclear discordance involving at least four operational taxonomic units (OTUs) across four species which diverged 183-278 kya. ABC modelling of nDNA gene trees supports introgression during or after speciation for some taxon pairs with cytonuclear discordance. Given substantial differences in body size between the species involved, this evidence for gene flow is surprising. Heterogenous patterns of introgression were identified but do not appear to be associated with chromosome differences between species. These and previous results suggest that dynamic past climates across the monsoonal tropics could have promoted reticulation among related species.

The Effect of Enzymatic Hydrolysis and Maillard Reaction on the Flavor of Chicken Osteopontin.

To reveal the changes in the flavor quality of chicken osteopontin (CO) before and after enzymatic hydrolysis and a thermal reaction, the present study was carried out to evaluate the volatile compounds and non-volatile compounds in CO. The results show that the chicken boneset enzymatic solution (CBES) presented a notably richer aroma after the enzymatic hydrolysis treatment. At the same time, the concentrations of the total free amino acids (FAAs) and 5'-nucleotides in the CBES increased dramatically. The ERP (enzymatic reaction paste) scores and the ORC (osteopontin reactive cream) scores were exceptionally high in terms of the umami and salty flavor profiles. As precursors, FAAs and 5'-nucleotides also boosted the Maillard reaction, leading to the generation of wide volatile compounds. Compared to CO, CBES, and ORC, the sensory evaluation showed that ERP scored the highest. In summary, the enzymatic hydrolysis treatment coupled with the Maillard reaction significantly enhanced the flavor profile of CO. These findings offer valuable insights into the high-value utilization of bone by-products, making a significant advancement in the field.

Progress in the study of intestinal microbiota involved in morphine tolerance.

Morphine is a widely used opioid for treatment of pain. The attendant problems including morphine tolerance and morphine dependence pose a major public health challenge. In recent years, there has been increasing interest in the gastrointestinal microbiota in many physiological and pathophysiological processes. The connectivity network between the gut microbiota and the brain is involved in multiple biological systems, and bidirectional communication between them is critical in gastrointestinal tract homeostasis, the central nervous system, and the microbial system. Many research have previously shown that morphine has a variety of effects on the gastrointestinal tract, but none have determined the function of intestinal microbiota in morphine tolerance. This study reviewed the mechanisms of morphine tolerance from the perspective of dysregulation of microbiota-gut-brain axis homeostasis, by summarizing the possible mechanisms originating from the gut that may affect morphine tolerance and the improvement of morphine tolerance through the gut microbiota.

N-acetylcysteine attenuates the incidence of phlebitis induced by carbomer/vinorelbine gel.

Background: The high incidence and severe clinical manifestations of phlebitis pose a complex and urgent clinical challenge. The rapid and simple establishment of animal phlebitis models and the development of preventive strategies are crucial to resolving this problem. Methods: In this study, we established such models by mixing vinorelbine ditartrate (VNR) and carbomer to form a sustained-release gel carrier, and then injected it around the veins rather than inside the vessels. Furthermore, we analyzed the efficacy of the carbomer/VNR gel in inducing phlebitis by monitoring the morphology of the veins using HE staining, immunohistochemical and immunofluorescence staining, and western blotting. Reactive oxygen species (ROS) and lipid peroxidation levels were determined using flow cytometry. Finally, we evaluated the inhibitory effect of N-acetylcysteine (NAC) on VNR-induced phlebitis in rabbits and rats. Results: Our findings suggested that the carbomer/VNR gel rapidly and easily induced phlebitis due to by retention of the gel in situ, wrapping the veins, and the prolonged release of VNR. NAC alleviated the VNR-induced oxidative stress response and expression of inflammatory cytokines by attenuating mitochondrial damage in venous endothelial cells, thereby preventing the occurrence of phlebitis in rabbits and rats. Conclusion: The in situ carbomer/VNR gel provides a rapid and simple method for establishing an animal model to study the pathogenesis of phlebitis. Furthermore, the observed therapeutic effect of NAC highlights its novel and efficacious role in preventing and treating phlebitis.

Ketogenic diet: a potential adjunctive treatment for substance use disorders.

Substance use disorders (SUD) can lead to serious health problems, and there is a great interest in developing new treatment methods to alleviate the impact of substance abuse. In recent years, the ketogenic diet (KD) has shown therapeutic benefits as a dietary therapy in a variety of neurological disorders. Recent studies suggest that KD can compensate for the glucose metabolism disorders caused by alcohol use disorder by increasing ketone metabolism, thereby reducing withdrawal symptoms and indicating the therapeutic potential of KD in SUD. Additionally, SUD often accompanies increased sugar intake, involving neural circuits and altered neuroplasticity similar to substance addiction, which may induce cross-sensitization and increased use of other abused substances. Reducing carbohydrate intake through KD may have a positive effect on this. Finally, SUD is often associated with mitochondrial damage, oxidative stress, inflammation, glia dysfunction, and gut microbial disorders, while KD may potentially reverse these abnormalities and serve a therapeutic role. Although there is much indirect evidence that KD has a positive effect on SUD, the small number of relevant studies and the fact that KD leads to side effects such as metabolic abnormalities, increased risk of malnutrition and gastrointestinal symptoms have led to the limitation of KD in the treatment of SUD. Here, we described the organismal disorders caused by SUD and the possible positive effects of KD, aiming to provide potential therapeutic directions for SUD.

Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli.

While there are several genome editing techniques available, few are suitable for dynamic and simultaneous mutagenesis of arbitrary targeted sequences in prokaryotes. Here, to address these limitations, we present a versatile and multiplex retron-mediated genome editing system (REGES). First, through systematic optimization of REGES, we achieve efficiency of ∼100%, 85 ± 3%, 69 ± 14% and 25 ± 14% for single-, double-, triple- and quadruple-locus genome editing, respectively. In addition, we employ REGES to generate pooled and barcoded variant libraries with degenerate RBS sequences to fine-tune the expression level of endogenous and exogenous genes, such as transcriptional factors to improve ethanol tolerance and biotin biosynthesis. Finally, we demonstrate REGES-mediated continuous in vivo protein evolution, by combining retron, polymerase-mediated base editing and error-prone transcription. By these case studies, we demonstrate REGES as a powerful multiplex genome editing and continuous evolution tool with broad applications in synthetic biology and metabolic engineering.

Bewildering biogeography: Waves of dispersal and diversification across southern Wallacea by bent-toed geckos (genus: Cyrtodactylus).

Bent-toed Geckos, genus Cyrtodactylus, are one of the most diverse terrestrial vertebrate groups, and their range extends from South Asia into Australo-Papua and adjacent Pacific islands. Given the generally high faunal endemism on Wallacean islands, it is rather paradoxical that the diversity in these geckos appears to be so low (21 species in Wallacea, 15 in the Philippines) compared with continental shelf assemblages (>300 species on Sunda + Sahul Shelves + adjacent islands). To determine whether this shortfall was real or an artifact of historical undersampling, we analyzed mitochondrial DNA sequences of hundreds of southern Wallacean samples (Lesser Sundas + southern Maluku). After screening to guide sample selection for target capture data collection, we obtained a 1150-locus genomic dataset (1,476,505 bp) for 119 samples of southern Wallacean and closely related lineages. The results suggest that species diversity of Cyrtodactylus in southern Wallacea is vastly underestimated, with phylogenomic and clustering analyses suggesting as many as 25 candidate species, in contrast to the 8 currently described. Gene exchange between adjacent candidate species is absent or minimal across the archipelago with only one case of > 0.5 migrants per generation. Biogeographical analysis suggests that the hitherto unrecognized diversity is the result of at least three independent dispersals from Sulawesi or its offshore islands into southern Wallacea between 6 and 14 Ma, with one invasion producing small-bodied geckos and the other two or three producing larger-bodied geckos. The smaller-bodied laevigatus group appears to be able to coexist with members of either larger-bodied clade, but we have yet to find members of the two larger-bodied clades occurring in sympatry, suggesting that ecological partitioning or competitive exclusion may be shaping individual island assemblages.

Impact of a novel prognostic model on allogeneic hematopoietic stem cell transplantation outcomes in patients with CMML.

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell malignancy, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curable treatment. The outcomes after transplant are influenced by both disease characteristics and patient comorbidities. To develop a novel prognostic model to predict the post-transplant survival of CMML patients, we identified risk factors by applying univariable and multivariable Cox proportional hazards regression to a derivation cohort. In multivariable analysis, advanced age (hazard ratio [HR] 3.583), leukocyte count (HR 3.499), anemia (HR 3.439), bone marrow blast cell count (HR 2.095), and no chronic graft versus host disease (cGVHD; HR 4.799) were independently associated with worse survival. A novel prognostic model termed ABLAG (Age, Blast, Leukocyte, Anemia, cGVHD) was developed and the points were assigned according to the regression equation. The patients were categorized into low risk (0-1), intermediate risk (2, 3), and high risk (4-6) three groups and the 3-year overall survival (OS) were 93.3% (95%CI, 61%-99%), 78.9% (95%CI, 60%-90%), and 51.6% (95%CI, 32%-68%; p < .001), respectively. In internal and external validation cohort, the area under the receiver operating characteristic (ROC) curves of the ABLAG model were 0.829 (95% CI, 0.776-0.902) and 0.749 (95% CI, 0.684-0.854). Compared with existing models designed for the nontransplant setting, calibration plots, and decision curve analysis showed that the ABLAG model revealed a high consistency between predicted and observed outcomes and patients could benefit from this model. In conclusion, combining disease and patient characteristic, the ABLAG model provides better survival stratification for CMML patients receiving allo-HSCT.

Sponge diversification in marine lakes: Implications for phylogeography and population genomic studies on sponges.

The relative influence of geography, currents, and environment on gene flow within sessile marine species remains an open question. Detecting subtle genetic differentiation at small scales is challenging in benthic populations due to large effective population sizes, general lack of resolution in genetic markers, and because barriers to dispersal often remain elusive. Marine lakes can circumvent confounding factors by providing discrete and replicated ecosystems. Using high-resolution double digest restriction-site-associated DNA sequencing (4826 Single Nucleotide Polymorphisms, SNPs), we genotyped populations of the sponge Suberites diversicolor (n = 125) to test the relative importance of spatial scales (1-1400 km), local environmental conditions, and permeability of seascape barriers in shaping population genomic structure. With the SNP dataset, we show strong intralineage population structure, even at scales <10 km (average F ST = 0.63), which was not detected previously using single markers. Most variation was explained by differentiation between populations (AMOVA: 48.8%) with signatures of population size declines and bottlenecks per lake. Although the populations were strongly structured, we did not detect significant effects of geographic distance, local environments, or degree of connection to the sea on population structure, suggesting mechanisms such as founder events with subsequent priority effects may be at play. We show that the inclusion of morphologically cryptic lineages that can be detected with the COI marker can reduce the obtained SNP set by around 90%. Future work on sponge genomics should confirm that only one lineage is included. Our results call for a reassessment of poorly dispersing benthic organisms that were previously assumed to be highly connected based on low-resolution markers.

Discovery of inhibitors against SARS-CoV-2 main protease using fragment-based drug design.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease 2019 (COVID-19), in which the main protease (Mpro) plays an important role in the virus's life cycle. In this work, two representative peptide inhibitors (11a and PF-07321332) were selected, and their interaction mechanisms of non-covalently bound with Mpro were firstly investigated by means of molecular dynamical simulation. Then, using the fragment-based drug design method, some fragments from the existing SARS-CoV and SARS-CoV-2 inhibitors were selected to replace the original P2 and P3 fragments, resulting in some new molecules. Among them, two molecules (O-74 and N-98) were confirmed by molecular docking and molecular dynamics simulation, and ADMET properties prediction was employed for further verification. The results shown that they presented excellent activity and physicochemical properties, and had the potential to be new inhibitors for SARS-CoV-2 main protease.

Combined treatment of ε-polylysine and heat damages protective structures and spore inner membranes to inactivate Bacillus subtilis spores.

The sterilizing effect of a combination of heat (80, 90, and 100 °C) and ε-polylysine (ε-PL, 0.25 and 1 g/L) treatments on Bacillus subtilis spores was investigated and compared with that of conventional heat sterilization. The inactivation rate of spores and changes in their protective structure were evaluated using different methods and techniques. Changes in cell membrane's fatty acids, cell walls, proteins and nucleic acids were also analyzed. The results showed that the combined heat and ε-PL treatments significantly (p < 0.05) inactivated the Bacillus subtilis spores compared with the single heat treatment. Besides, the inactivation of spores was enhanced as the temperature and ε-PL concentration of combined treatments increased. The inactivation rate was found to be 2.18 log after heating at 90 °C for 60 min combined with the addition of 1 g/L ε-PL. Additionally, the electrical conductivity of spores' suspension and the positive region of flow cytometry significantly (p < 0.05) increased depending on temperature and ε-PL concentration of a combination treatment, indicating significant damage in the cell membranes and increased permeability. Significant changes in the spore morphology were also observed by the microscopy analysis after a combination treatment. Furthermore, the Fourier transform infrared spectra indicated a phase change in the inner membrane and alteration in the structure of peptidoglycan layer, as well as protein and nucleic acids denaturation after combined treatments. Therefore, the combined heat and ε-PL treatments can be suggested as sterilizing alternative to conventional heat sterilization in the food industry.

A promising research direction for colorectal cancer immunotherapy: The regulatory mechanism of CCL5 in colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide, with high morbidity and mortality rates worldwide. Therefore, there is an urgent need to develop more effective treatments for CRC patients. In recent years, there has been some success in the immunotherapy of tumors, and immunotherapy has been used in many solid tumors including CRC. To date, the clinical efficacy of immunotherapy for CRC is limited, so more effective immunotherapy methods need to be explored. In patients with CRC, the CC chemokine CCL5 plays a role in the development of CRC and the recruitment and activation of immune cells, suggesting that it has potential for immunotherapy. This review mainly introduces the latest advances in the study of CCL5 acting as a marker of CRC and related mechanisms of immunotherapy, as well as the latest understanding of how CCL5 is involved in the invasion and development of CRC.

Effects of Nanofibers Orientation and Aspect Ratio on Dielectric Properties of Nanocomposites: A Phase-Field Simulation.

Polymer-based dielectrics with high energy storage density are attracting increasing attention due to their wide applications in pulsed-discharge and power conditioning electronic fields. Despite some numerical simulation about effects of horizontally arranged and vertically arranged fibers on dielectric properties of composites already studied, the influence mechanism of the specific orientation and aspect ratio still remains to be studied. In this work, the effects of orientation angles and aspect ratios of nanofiber fillers on breakdown behavior and dielectric properties of composites are theoretically analyzed by the finite element and phase-field method. The results show that the more inclined the nanofiber fillers is, the higher the nominal breakdown strength is, which benefits from the obstruction of the conductive channels by the nanofibers. However, the dielectric constant shows the opposite law, which is the result of the decreased polarization along the electric field direction of the nanofiber fillers. Besides this, by modulating the distribution of local electric field, a higher aspect ratio of nanofiber fillers helps to achieve a much higher breakdown strength with a slight sacrifice of dielectric constant. The present work provides a comprehensive and quantitative understanding of the orientation and aspect ratio of nanofiber fillers on the dielectric and breakdown properties of composites, providing important guidance on optimizing the energy storage performance.

Glucose protects the cell membrane, Na+/K+-ATPase, nucleic acids, and proteins in Bacillus subtilis spores under high pressure thermal sterilization.

The extreme resistance of bacterial spores to sterilization makes them a major concern to the food industry and consumers. In this study, the effect of glucose on the inactivation of Bacillus subtilis spores by high pressure thermal sterilization (HPTS) was evaluated. The results showed that the protective effects of glucose increased with the increase in its concentration. Compared with the HPTS control (no addition of glucose), the activity of Na+/K+-ATPase was increased, the leakage of proteins and the release of 2,6-pyridine dicarboxylic acid (DPA) was decreased, and the vibrational strength of the functional group P = O was reduced by the addition of glucose. At the same time, glucose treatment increased the content of α-helix by 6%-22%, while decreased the random coil content by 5%-13% of the cellular protein. In conclusion, the addition of glucose protected the cell membrane, Na+/K+-ATPase, cellular nucleic acids and proteins of B. subtilis under HPTS treatment.

Diverge and Conquer: Phylogenomics of southern Wallacean forest skinks (Genus: Sphenomorphus) and their colonization of the Lesser Sunda Archipelago.

The archipelagos of Wallacea extend between the Sunda and Sahul Shelves, serving as a semipermeable two-way filter influencing faunal exchange between Asia and Australo-Papua. Forest skinks (Genus Sphenomorphus) are widespread throughout southern Wallacea and exhibit complex clinal, ontogenetic, sexual, and seasonal morphological variation, rendering species delimitation difficult. We screened a mitochondrial marker for 245 Sphenomorphus specimens from this area to inform the selection of 104 samples from which we used targeted sequence capture to generate a dataset of 1154 nuclear genes (∼1.8 Mb) plus complete mitochondrial genomes. Phylogenomic analyses recovered many deeply divergent lineages, three pairs of which are now sympatric, that began to diversify in the late Miocene shortly after the oldest islands are thought to have become emergent. We infer a complex and nonstepping-stone pattern of island colonization, with the group having originated in the Sunda Arc islands before using Sumba as a springboard for colonization of the Banda Arcs. Estimates of population structure and gene flow across the region suggest total isolation except between two Pleistocene Aggregate Island Complexes that become episodically land-bridged during glacial maxima. These historical processes have resulted in at least 11 Sphenomorphus species in the region, nine of which require formal description. This fine-scale geographic partitioning of undescribed species highlights the importance of utilizing comprehensive genomic studies for defining biodiversity hotspots to be considered for conservation protection.

Phylogeography and population genetics of a widespread cold-adapted ant, Prenolepis imparis.

As species arise, evolve and diverge, they are shaped by forces that unfold across short and long timescales and at both local and vast geographical scales. It is rare, however, to be able document this history across broad sweeps of time and space in a single species. Here, we report the results of a continental-scale phylogenomic analysis across the entire range of a widespread species. We analysed sequences of 1402 orthologous ultraconserved element (UCE) loci from 75 individuals to identify population genetic structure and historical demographic patterns across the continent-wide range of a cold-adapted ant, the winter ant, Prenolepis imparis. We recovered five well-supported, genetically isolated clades representing lineages that diverged from 8.2-2.2 million years ago. These include: (i) an early diverging lineage located in Florida, (ii) a lineage that spans the southern United States, (iii) populations that extend across the midwestern and northeastern United States, (iv) populations from the western United States and (v) populations in southwestern Arizona and Mexico. Population genetic analyses revealed little or no gene flow among these lineages, but patterns consistent with more recent gene flow among populations within lineages, and localized structure with migration in the western United States. High support for five major geographical lineages and lack of evidence of contemporary gene flow indicate in situ diversification across the species' range, producing relatively ancient lineages that persisted through subsequent climate change and glaciation during the Quaternary.