The auxin-responsive CsSPL9-CsGH3.4 module finely regulates auxin levels to suppress the development of adventitious roots in tea (Camellia sinensis).

The cutting technique is extensively used in tea breeding, with key emphasis on promoting the growth of adventitious roots (ARs). Despite its importance in tea cultivation, the mechanisms underlying AR development in tea remain unclear. In this study, we demonstrated the essential role of auxins in the initiation and progression of AR and established that the application of exogenous 1-naphthaleneacetic acid-enhanced AR formation in tissue-cultured seedlings and cuttings. Then, we found that the auxin-responsive transcription factor CsSPL9 acted as a negative regulator of AR development by reducing the levels of free indole-3-acetic acid (IAA) in tea plants. Furthermore, we identified CsGH3.4 as a downstream target of CsSPL9, which was activated by direct binding to its promoter. CsGH3.4 also inhibited AR development and maintained low levels of free IAA. Thus, these results revealed the inhibitory effect of the auxin-responsive CsSPL9-CsGH3.4 module on AR development by reducing free IAA levels in tea. These findings have significant theoretical and practical value for enhancing tea breeding practices.

Identification and profile of phenolamides with anthracnose resistance potential in tea (Camellia sinensis).

Tea anthracnose is a prevalent disease in China that can lead to reduced tea production and lower quality, yet there is currently a lack of effective means for controlling this disease. In this study, we identified 46 phenolamides (including 27 isomers) in different tissues and organs of tea plants based on a developed workflow, and the secondary mass spectra of all these compounds have been documented. It was revealed that tea plants predominantly accumulate protonated aliphatic phenolamides, rather than aromatic phenolamides. The profile of phenolamides indicate that their buildup in tea plants is specific to certain tissues and acyl-acceptors, and this distribution is associated with the extent of phenolamide acyl-modification. Additionally, it was observed that N-Feruloylputrescine (Fer-Put, a type of phenolamides) was responsive to the stimulated accumulation of the tea anthracnose pathogen. The findings of anti-anthracnose experiments in vitro and on tea leaf demonstrated that Fer-Put was capable of significantly inhibiting the growth of anthracnose pathogen colony, effectively prevented tea leaf disease. Furthermore, it was observed that Fer-Put treatment can enhance the antioxidant enzyme activity of tea leaves. TEA002780.1 and TEA013165.1 gene may be responsible for the biosynthesis of Fer-Put in the disease resistance process in tea plants. Through these studies, the types and distribution of phenolamides in tea plants have been elucidated, and Fer-Put's ability to resist anthracnose has been established, providing new insights into the resistance of tea anthracnose.

Delayed Chronic Acidic Postconditioning Improves Poststroke Motor Functional Recovery and Brain Tissue Repair by Activating Proton-Sensing TDAG8.

Acidic postconditioning by transient CO2 inhalation applied within minutes after reperfusion has neuroprotective effects in the acute phase of stroke. However, the effects of delayed chronic acidic postconditioning (DCAPC) initiated during the subacute phase of stroke or other acute brain injuries are unknown. Mice received daily DCAPC by inhaling 5%/10%/20% CO2 for various durations (three cycles of 10- or 20-min CO2 inhalation/10-min break) at days 3-7, 7-21, or 3-21 after photothrombotic stroke. Grid-walk, cylinder, and gait tests were used to assess motor function. DCAPC with all CO2 concentrations significantly promoted motor functional recovery, even when DCAPC was delayed for 3-7 days. DCAPC enhanced the puncta density of GAP-43 (a marker of axon growth and regeneration) and synaptophysin (a marker of synaptogenesis) and reduced the amoeboid microglia number, glial scar thickness and mRNA expression of CD16 and CD32 (markers of proinflammatory M1 microglia) compared with those of the stroke group. Cerebral blood flow (CBF) increased in response to DCAPC. Furthermore, the mRNA expression of TDAG8 (a proton-activated G-protein-coupled receptor) was increased during the subacute phase of stroke, while DCAPC effects were blocked by systemic knockout of TDAG8, except for those on CBF. DCAPC reproduced the benefits by re-expressing TDAG8 in the peri-infarct cortex of TDAG8-/- mice infected with HBAAV2/9-CMV-TDAG8-3flag-ZsGreen. Taken together, we first showed that DCAPC promoted functional recovery and brain tissue repair after stroke with a wide therapeutic time window of at least 7 days after stroke. Brain-derived TDAG8 is a direct target of DCAPC that induces neuroreparative effects.

Exploring the RNA Editing Events and Their Potential Regulatory Roles in Tea Plant (Camellia sinensis L.).

RNA editing is a post-transcriptional modification process that alters the RNA sequence relative to the genomic blueprint. In plant organelles (namely, mitochondria and chloroplasts), the most common type is C-to-U, and the absence of C-to-U RNA editing results in abnormal plant development, such as etiolation and albino leaves, aborted embryonic development and retarded seedling growth. Here, through PREP, RES-Scanner, PCR and RT-PCR analyses, 38 and 139 RNA editing sites were identified from the chloroplast and mitochondrial genomes of Camellia sinensis, respectively. Analysis of the base preference around the RNA editing sites showed that in the -1 position of the edited C had more frequent occurrences of T whereas rare occurrences of G. Three conserved motifs were identified at 25 bases upstream of the RNA editing site. Structural analyses indicated that the RNA secondary structure of 32 genes, protein secondary structure of 37 genes and the three-dimensional structure of 5 proteins were altered due to RNA editing. The editing level analysis of matK and ndhD in six tea cultivars indicated that matK-701 might be involved in the color change of tea leaves. Furthermore, 218 PLS-CsPPR proteins were predicted to interact with the identified RNA editing sites. In conclusion, this study provides comprehensive insight into RNA editing events, which will facilitate further study of the RNA editing phenomenon of the tea plant.

Transcriptome analysis reveals the promotive effect of potassium by hormones and sugar signaling pathways during adventitious roots formation in the apple rootstock.

Apples are economically valuable and widely consumed fruits. The adventitious roots (ARs) formation is gridlock for apple trees mass propagation. The possible function of multiple hormones and sugar signaling pathways regulating ARs formation has not been completely understood in apple. In this study, B9 stem cuttings were treated with KCl treatment, where the highest root numbers (220) and maximum root length of 731.2 cm were noticed in KCl-treated cuttings, which were 98.2% and 215% higher than control cuttings. The content of endogenous hormones: IAA, ZR, JA, GA, and ABA were detected higher in response to KCl at most time-points. To figure out the molecular mechanisms underlying this effect, we investigated transcriptome analysis. In total, 4631 DEGs were determined, from which about 202 DEGs were considerably enriched in pathways associated with hormone signaling, sugar metabolism, root development, and cell cycle-related and were thereupon picked out on their potential involvements in ARs formation. Though, IAA accumulation and up-regulation of various genes contribute to induce AR formation. These results suggest that AR formation is a complex biological process in apple rootstocks, influenced mainly by the auxin signaling pathway and sugar metabolism.

Genome-Wide Identification and Characterization of the CsFHY3/FAR1 Gene Family and Expression Analysis under Biotic and Abiotic Stresses in Tea Plants (Camellia sinensis).

The FHY3/FAR1 transcription factor family, derived from transposases, plays important roles in light signal transduction, and in the growth and development of plants. However, the homologous genes in tea plants have not been studied. In this study, 25 CsFHY3/FAR1 genes were identified in the tea plant genome through a genome-wide study, and were classified into five subgroups based on their phylogenic relationships. Their potential regulatory roles in light signal transduction and photomorphogenesis, plant growth and development, and hormone responses were verified by the existence of the corresponding cis-acting elements. The transcriptome data showed that these genes could respond to salt stress and shading treatment. An expression analysis revealed that, in different tissues, especially in leaves, CsFHY3/FAR1s were strongly expressed, and most of these genes were positively expressed under salt stress (NaCl), and negatively expressed under low temperature (4 °C) stress. In addition, a potential interaction network demonstrated that PHYA, PHYC, PHYE, LHY, FHL, HY5, and other FRSs were directly or indirectly associated with CsFHY3/FAR1 members. These results will provide the foundation for functional studies of the CsFHY3/FAR1 family, and will contribute to the breeding of tea varieties with high light efficiency and strong stress resistance.

Melatonin Ameliorates Inflammation and Oxidative Stress by Suppressing the p38MAPK Signaling Pathway in LPS-Induced Sheep Orchitis.

Gram-negative bacterial infections of the testis can lead to infectious orchitis, which negatively influences steroid hormone synthesis and spermatogenesis. Lipopolysaccharide (LPS), a major component of the Gram-negative bacterial cell wall, acts via toll like receptors 4 (TLR4) to trigger innate immune responses and activate nuclear factor kappa B signaling. The protective mechanisms of melatonin on LPS-induced infectious orchitis have not been reported. Herein, we developed an LPS-induced sheep infectious orchitis model. In this model, the phagocytic activity of testicular macrophages (TM) was enhanced after melatonin treatment. Moreover, we found that melatonin suppressed secretion of TM pro-inflammatory factors by suppressing the p38MAPK pathway and promoting Leydig cell testosterone secretion. Expressions of GTP cyclohydrolase-I and NADPH oxidase-2 were reduced by melatonin while heme oxygenase-1 expression was up-regulated. Thus, melatonin reduced the severity of LPS-induced orchitis by stimulating antioxidant activity. The results of this study provide a reference for the treatment of acute infectious orchitis.

Chitosan derived glycolipid nanoparticles for magnetic resonance imaging guided photodynamic therapy of cancer.

Currently, the development of polysaccharide, especially chitosan (CS), based drug delivery system to afford magnetic resonance imaging (MRI) guided theranostic cancer therapy remains largely unexplored. Herein, we successfully developed a CS derived polymer (Gd-CS-OA) through chemical conjugation of CS, octadecanoic acid (OA) and gadopentetic acid (GA). After self-assemble into glycolipid nanoparticles to loaded chlorin e6 (Ce6), the resulted Gd-CS-OA/Ce6 was able to realize MRI guided photodynamic therapy (PDT) of cancer. Our results revealed that Gd-CS-OA was able to increase the MRI sensitivity as compared to Gd-DTPA with decent residence time and preferable excretion behavior in vivo. Moreover, the Gd-CS-OA/Ce6 showed negligible hemolysis, satisfactory ROS generation and stability in physiological environments with preferable cellular uptake and enhanced in vitro cytotoxicity (through elevated ROS generation) on 4T1 cells. Most importantly, Gd-CS-OA/Ce6 demonstrated promising in vivo tumor targetability (enhanced penetration and retention effect) and powerful MRI guided tumor ablation through PDT on in situ 4T1 tumor model.

Size-Controlled Preparation and Behavior Study of Phospholipid-Calcium Carbonate Hybrid Nanoparticles.

BACKGROUND: Calcium carbonate (CC) nanoparticles have broad biomedical utilizations, owing to their multiple intrinsic merits. However, bare CC nanoparticles do not allow for the development of multifunctional devices suitable for advanced drug delivery in cancer therapy. METHODS: Phospholipid-modified phospholipid-CC hybrid nanoparticles were prepared in our study using a combination of vapor-diffusion and solvent-diffusion methods to offer optimized pharmaceutical capabilities. RESULTS: Considering that particle size is a critical parameter that plays an important role in both in vitro and in vivo behaviors of nanoparticles, we here for the first time a present detailed protocol for the size-controlled preparation of hybrid nanoparticles, as well as analysis of the in vitro/in vivo behaviors of differently sized hybrid nanoparticles. CONCLUSION: Our results might significantly advance the application of this promising material in more varied fields.

A novel biocompatible, simvastatin-loaded, bone-targeting lipid nanocarrier for treating osteoporosis more effectively.

An insufficient drug concentration at the target site and drug efflux resulting in poor efficacy are recognized as important obstacles in osteoporosis treatment. Simvastatin (SIM), which can treat osteoporosis by promoting osteoblast differentiation and mineralization through the bone morphogenetic proteins (BMP)-Smad signaling pathway, has lower bioavailability, and less bone tissue distribution. Herein, novel lipid nanoparticles (LNPs) delivering SIM (SIM/LNPs) for osteoporosis therapy were developed with aspartic oligopeptide (ASP n , here ASP6)-based bone-targeting moieties grafted to the nanoparticles (SIM/ASP6-LNPs) in an attempt to increase the concentration of SIM in bones with a relatively low dose to minimize adverse effects. In vivo experiments indicated that the ASP6-LNPs exhibited ideal bone-targeting characteristics, and in vitro cell evaluation experiments showed LNPs have good biocompatibility with MC3T3-E1 cells. The cell mineralization experiment revealed that the SIM-loaded LNPs induced osteoblast differentiation and the formation of mineralized nodules in MC3T3-E1 cells, achieving the same efficacy as that of SIM. Pharmacodynamic experiments revealed that SIM/ASP6-LNPs improved the efficacy of SIM on the recovery of bone mineral density when compared to SIM/LNPs or to SIM alone. Therefore, SIM/ASP6-LNPs may represent a potential bone-targeting drug delivery system (DDS) that contributes to the development of a novel osteoporosis treatment.

Correction: A novel biocompatible, simvastatin-loaded, bone-targeting lipid nanocarrier for treating osteoporosis more effectively.

[This corrects the article DOI: 10.1039/D0RA00685H.].

Diagnosis and treatment of novel coronavirus pneumonia based on the theory of traditional Chinese medicine / 中西医结合学报

Since the outbreak of novel coronavirus pneumonia (coronavirus disease 2019, COVID-19), it has rapidly spread to 187 countries, causing serious harm to the health of people and a huge social burden. However, currently, drugs specifically approved for clinical use are not available, except for vaccines against COVID-19 that are being evaluated. Traditional Chinese medicine (TCM) is capable of performing syndrome differentiation and treatment according to the clinical manifestations of patients, and has a better ability of epidemic prevention and control. The authors comprehensively analyzed the etiology and pathogenesis of COVID-19 based on the theory of TCM, and discussed its syndrome differentiation, treatment and prevention measures so as to provide strategies and reference for the prevention and treatment with TCM.

Review of Eerdun-Wurile clinical research / Монголын эм зүй, эм судлал

Objective@#To summarize the clinical application of Zhenbao pills (Eerdun-Wurile) in recent years, so as to clarify its clinical efficiency and provide an effective basis for future research.@*Methods@#The clinical application of Zhenbao pills (Eerdun-Wurile) was classified and summarized by consulting the relevant literature.@*Results@#Mongolian medicine Eerdun-Wurile consists of many active ingredients such as <i>Sandalwood Padauk</i>, pearl, <i>Myristica fragrans, Calculus bovis</i>, Bufallo horn concentrated powder, <i>Gardenia jasminoides Ellis, Glycyrrhiza uralensis Fisch, Euphorbia humifusa, Catsia tora Linn, Chingma Abutilon Seed</i>, Resina Liquidambaris, <i>Carthamus tinctorius L, Eugenia caryophyllata Thunb, Cardamoj amomum, Semen Nigellae, Cuminum cyminum L, Piper longum Linn, Lygodium japonicum</i>, crab, medicine terminalia fruit, <i>MeLia toosendan Sieb.etZucc</i>, musk, white sandalwood, <i>Cinnamomum cassia Presl, Aucklandia lappa Decne, Inula helenium L, Amomum tsao-ko Crevostet Lemaire etc</i>. Mongolian medicine Eerdun-Wurile used for treat cardiovascular and cerebrovascular diseases, cerebral infarction, cerebral infarction and ischemic stroke, rheumatic heart disease, coronary heart disease, angina pectoris, rheumatism, kind rheumatism, cervical spondylosis, arthritis, insomnia, high blood pressure, epilepsy, skin diseases, diabetes, eye diseases.@*Conclusion@#Mongolian Medicine Eerdun-Wurile has remarkable curative effect on cardiovascu- lar and cerebrovascular diseases, brain infarction, cerebral infarction, ischemic stroke, diabetic diseases, rheumatism, bone diseases and so on. In this paper, the curative effect of this drug was summarized to provide reference for its clinical application.

Melatonin Reduces Androgen Production and Upregulates Heme Oxygenase-1 Expression in Granulosa Cells from PCOS Patients with Hypoestrogenia and Hyperandrogenia.

BACKGROUND/AIMS: Polycystic ovary syndrome (PCOS) is an endocrine disorder characterized by abnormal hormone levels in peripheral blood and poor-quality oocytes. PCOS is a pathophysiological syndrome caused by chronic inflammation and oxidative stress. The aim of this study was to investigate the mechanism of melatonin regulation on androgen production and antioxidative damage in granulosa cells from PCOS patients with hypoestrogenia and hyperandrogenia. METHODS: Cumulus-oocyte complexes were collected from PCOS patients who had low levels of estrogen in follicular fluids. RESULTS: Melatonin triggered upregulation of cytochrome P450 family 19 subfamily A member 1 (CYP19A1) expression via the extracellular signal-regulated kinase pathway in luteinized granulosa cells. As a result, conversion of androgen to 17ß-estradiol was accelerated. We also found that melatonin significantly reduced the levels of inducible nitric oxide (NO) synthetase and NO in luteinized granulosa cells. Levels of transcripts encoding NF-E2-related factor-2 and its downstream target heme oxygenase-1 were also increased, leading to anti-inflammatory and antioxidant effects. We also found that melatonin could improve oocyte development potential. CONCLUSION: Our preliminary results showed that melatonin had a positive impact on oocyte quality in PCOS patients with hypoestrogenia and hyperandrogenia.

Free-electron-driven beam-scanning terahertz radiation.

A beam-scanning terahertz (THz) radiation mechanism in a free-electron-driven grating system is proposed for THz applications. By loading a period-asynchronous rod array above the grating, the spoof surface plasmon (SSP) originally excited by the electron changes its radiation characteristics owing to the rod-induced Brillouin zone folding effect. The rod array functions as an antenna and converts the SSP into a spatial coherent THz radiation. The radiation frequency and direction can be precisely controlled by the electron energy. The field intensity of the radiation is increased approximately 20 times compared with that of the conventional Smith-Purcell radiation in the same frequency range. In addition, a microwave-band scaling prototype is fabricated and the frequency-controlled radiation is measured. Excellent agreement between the experimental and simulated results is obtained. This study paves the way for the development of on-chip THz sources for advanced communication and detection applications.

Membrane metallo-endopeptidase is dispensable for repair after nerve injury.

Membrane metallo-endopeptidase (MME), also known as neprilysin (NEP), has been of interest for its role in neurodegeneration and pain due to its ability to degrade ß-amyloid and substance-P, respectively. In addition to its role in the central nervous system, MME has been reported to be expressed in the peripheral system, specifically in the inner and outer border of myelinating fibers, in the Schmidt-Lantermann cleft and in the paranodes. Recently, mutations of this gene have been associated with Charcot-Marie-Tooth Type 2 (CMT2). Peripheral nerve morphometry in mice lacking MME previously showed minor abnormalities in aged animals in comparison to CMT2 patients. We found that MME expression was dysregulated after nerve injury in a Neuregulin-1 dependent fashion. We therefore explored the hypothesis that MME may have a role in remyelination. In the naïve state in adulthood we did not find any impairment in myelination in MME KO mice. After nerve injury the morphological outcome in MME KO mice was indistinguishable from WT littermates in terms of axon regeneration and remyelination. We did not find any difference in functional motor recovery. There was a significant difference in sensory function, with MME KO mice starting to recover response to mechanical stimuli earlier than WT. The epidermal reinnnervation, however, was unchanged and this altered sensitivity may relate to its known function in cleaving the peptide substance-P, known to sensitise nociceptors. In conclusion, although MME expression is dysregulated after nerve injury in a NRG1-dependent manner this gene is dispensable for axon regeneration and remyelination after injury.

In-vitro differentiation of early pig spermatogenic cells to haploid germ cells.

Spermatogonial stem cells (SSCs) self-renew and contribute genetic information to the next generation. Pig is wildly used as a model animal for understanding reproduction mechanisms of human being. Inducing directional differentiation of porcine SSCs may be an important strategy in exploring the mechanisms of spermatogenesis and developing better treatment methods for male infertility. Here, we established an in-vitro culture model for porcine small seminiferous tubule segments, to induce SSCs to differentiate into single-tail haploid spermatozoa. The culture model subsequently enabled spermatozoa to express the sperm-specific protein acrosin and oocytes to develop to blastocyst stage after round spermatid injection. The addition of retinoic acid (RA) to the differentiation media promoted the efficiency of haploid differentiation. RT-PCR analysis indicated that RA stimulated the expression of Stra8 but reduced the expression of NANOS2 in spermatogonia. Genes involved in post-meiotic development, transition protein 1 (Tnp1) and protamine 1 (Prm1) were upregulated in the presence of RA. The addition of an RA receptor (RAR) inhibitor, BMS439, showed that RA enhanced the expression of cAMP responsive-element binding protein through RAR and promoted the formation of round spermatids. We established an efficient culture system for in-vitro differentiation of pig SSCs. Our study represents a model for human testis disease and toxicology screening. Molecular regulators of SSC differentiation revealed in this study might provide a therapeutic strategy for male infertility.

miRNAs associated with auxin signaling, stress response, and cellular activities mediate adventitious root formation in apple rootstocks.

Adventitious root (AR) formation is essential for the vegetative propagation of apple rootstocks. miRNAs play a significant role in regulating AR development, however, large-scale transcriptomic data on miRNA mediated AR formation in apple rootstocks is lacking. Therefore, in order to identify the molecular mechanisms underlying AR formation in 'M9-T337' apple rootstocks, transcriptomic changes occurring during key time points of AR formation (0, 3, and 16 days) were analyzed using high-throughput sequencing with a focus on miRNAs. A total of 84 known miRNAs and 56 novel miRNAs have differentially expressed were identified. Additionally, a total of 88 target genes of known miRNAs and 76 target genes of novel miRNAs were identified by degradome sequencing. The expression levels of the miRNAs and target genes were quantified by RT-qPCR. Results indicate that miRNAs and their target genes are associated with auxin signal-related (miR160 and miR390), stress response-related (miR398, miR395 and miR408), cell fate transformation-, proliferation- and enlargement-related (miR171, miR156, miR166, miR319 and miR396). These all involve pathways that participate in AR formation in 'M9-T337' apple rootstock. In addition, hormones (AUX, CTK, GA3, BR, JA, and ABA) are also involved in regulating AR formation. The candidate genes belonging to pathways associated with AR formation exhibited significantly higher expression levels, providing evidence that they may be involved in the regulation of AR development. The collective results of the present study indicate that the developmental process associated with AR formation in apple rootstock is extremely complex. The known and novel miRNAs and target genes that were identified by high-throughput and degradome sequencing, respectively, provide a framework for the future analysis of miRNAs associated with AR development in apple rootstocks, and provide new information that can be used to better understand AR development in woody plants.

Mitochondrial Ferritin Is a Hypoxia-Inducible Factor 1α-Inducible Gene That Protects from Hypoxia-Induced Cell Death in Brain.

Aims: Mitochondrial ferritin (protein [FtMt]) is preferentially expressed in cell types of high metabolic activity and oxygen consumption, which is consistent with its role of sequestering iron and preventing oxygen-derived redox damage. As of yet, the mechanisms of FtMt regulation and the protection FtMt affords remain largely unknown. Results: Here, we report that hypoxia-inducible factor 1α (HIF-1α) can upregulate FtMt expression. We verify one functional hypoxia-response element (HRE) in the positive regulatory region and two HREs possessing HIF-1α binding activity in the minimal promoter region of the human FTMT gene. We also demonstrate that FtMt can alleviate hypoxia-induced brain cell death by sequestering uncommitted iron, whose levels increase with hypoxia in these cells. Innovation: In the absence of FtMt, this catalytic metal excess catalyzes the production of cytotoxic reactive oxygen species. Conclusion: Thus, the cell ability to increase expression of FtMt during hypoxia may be a skill to avoid tissue damage derived from oxygen limitation.

The complete mitochondrial genome of Eterusia aedea (Lepidoptera, Zygaenidae) and comparison with other zygaenid moths.

Zygaenidae comprises >1036 species, including many folivorous pests in agriculture. In the present study, the complete mitochondrial genome (mitogenome) of a major pest of tea trees, Eterusia aedea was determined. The 15,196-bp circular genome contained the common set of 37 mitochondrial genes (including 13 protein-coding genes, two rRNA genes, and 22 tRNA genes) and exhibited the similar genomic features to reported Zygaenidae mitogenome. Comparative analyses of Zygaenidae mitogenomes showed a typical evolutionary trend of lepidopteran mitogenomes. In addition, we also investigated the gene order of lepidopteran mitogenomes and proposed that the novel gene order trnA-trnR-trnN-trnE-trnS-trnF from Zygaenidae and Gelechiidae and most other gene rearrangements of this tRNA cluster evolved independently. Finally, the mitogenomic phylogeny of Lepidoptera was reconstructed based on multiple mitochondrial datasets. And all the phylogenetic results revealed the sister relationships of Cossoidea and Zygaenoidea with both BI and ML methods, which is the first stable mitogenomic evidence for this clade.