Dimethyl itaconate inhibits antigen-specific Th17 cell responses and autoimmune inflammation via modulating NRF2/STAT3 signaling.

Pathogenic Th17 cells play a crucial role in autoimmune diseases like uveitis and its animal model, experimental autoimmune uveitis (EAU). Dimethyl itaconate (DMI) possesses potent anti-inflammatory effects. However, there is still a lack of knowledge about the role of DMI in regulating pathogenic Th17 cells and EAU. Here, we reported that intraperitoneal administration of DMI significantly inhibited the severity of EAU via selectively suppressing Th17 cell responses. In vitro antigen stimulation studies revealed that DMI dramatically decreased the frequencies and function of antigen-specific Th17, but not Th1, cells. Moreover, DMI hampered the differentiation of naive CD4+ T cells toward pathogenic Th17 cells. DMI-treated DCs produced less IL-1ß, IL-6, and IL-23, and displayed an impaired ability to stimulate antigen-specific Th17 activation. Mechanistically, DMI activated the NRF2/HO-1 pathway and suppressed STAT3 signaling, which subsequently restrains p-STAT3 nuclear translocation, leading to decreased pathogenic Th17 cell responses. Thus, we have identified an important role for DMI in regulating pathogenic Th17 cells, supporting DMI as a promising therapy in Th17 cell-driven autoimmune diseases including uveitis.

AS101 regulates the Teff/Treg balance to alleviate rabbit autoimmune dacryoadenitis through modulating NFATc2.

Sjögren's syndrome (SS) dry eye can cause ocular surface inflammation and lacrimal gland (LG) damage, leading to discomfort and potential vision problems. The existing treatment options for SS dry eye are currently constrained. We investigated the possible therapeutic effect and the underlying mechanism of AS101 in autoimmune dry eye. AS101 was injected subconjunctivally into a rabbit model of autoimmune dacryoadenitis and its therapeutic effects were determined by evaluating clinical and histological scores. The expressions of effector T cells (Teff)/regulatory T cells (Treg)-related transcription factors and cytokines, inflammation mediators, and transcription factor NFATc2 were measured by quantitative real-time PCR and/or Western blot both in vivo and in vitro. Additionally, the role of NFATc2 in the immunomodulatory effects of AS101 on T cells was explored by co-culturing activated peripheral blood lymphocytes (PBLs) transfected with NFATc2 overexpression lentiviral plasmid with AS101. AS101 treatment potently ameliorated the clinical severity and reduced the inflammation of LG. Further investigation revealed that AS101 treatment led to decreased expression of Th1-related genes (T-bet and IFN-γ) and Th17-related genes (RORC, IL-17A, IL-17F, and GM-CSF) and increased expression of Treg-related gene Foxp3 in vivo and in vitro. Meanwhile, AS101 suppressed the expression of TNF-α, IL-1ß, IL-23, IL-6, MMP-2, and MMP-9. Mechanistically, AS101 downregulated the expression of NFATc2 in inflamed LGs. Overexpression of NFATc2 in activated PBLs partially blunted the effect of AS101 on Teff suppression and Treg promotion. In conclusion, AS101 is a potential regulator of Teff/Treg cell balance and could be an effective treatment agent for SS dry eye.

METTL3 inhibits autoreactive Th17 cell responses in experimental autoimmune uveitis via stabilizing ASH1L mRNA.

Methyltransferase like 3 (METTL3), a primary N6-methyladenosine (m6A) methyltransferase, has been implicated in various biological and pathological processes including immune responses. However, the functions and mechanisms of METTL3 in pathogenic T helper (Th)17 cells are poorly understood. Here we found significantly decreased METTL3 expression along with reduced m6A levels in eyeballs and T cells of experimental autoimmune uveitis (EAU). Overexpression of METTL3 ameliorated the development of EAU and suppressed pathogenic Th17 cell responses in vivo and in vitro. Mechanistically, METTL3 promoted the expression of absent, small, or homeotic-like 1 (ASH1L) via enhancing its stability in a YT521-B homology domain containing 2 (YTHDC2)-dependent manner, which further decreased the expression of IL-17 and IL-23 receptor (IL-23R), resulting in reduced pathogenic Th17 responses. Together, our data reveal a pivotal role of METTL3 in regulating pathogenic Th17 responses, which may contribute to human uveitis therapy.

Mettl3 induced miR-338-3p expression in dendritic cells promotes antigen-specific Th17 cell response via regulation of Dusp16.

Pathogenic Th17 cells are critical drivers of multiple autoimmune diseases, including uveitis and its animal model, experimental autoimmune uveitis (EAU). However, how innate immune signals modulate pathogenic Th17 responses remains largely unknown. Here, we showed that miR-338-3p endowed dendritic cells (DCs) with an increased ability to activate interphotoreceptor retinoid-binding protein (IRBP)1-20 -specific Th17 cells by promoting the production of IL-6, IL-1ß, and IL-23. In vivo administration of LV-miR-338-infected DCs promoted pathogenic Th17 responses and exacerbated EAU development. Mechanistically, dual-specificity phosphatase 16 (Dusp16) was a molecular target of miR-338-3p. miR-338-3p repressed Dusp16 and therefore strengthened the mitogen-activated protein kinase (MAPK) p38 signaling, resulting in increased production of Th17-polarizing cytokines and subsequent pathogenic Th17 responses. In addition, methyltransferase like 3 (Mettl3), a key m6A methyltransferase, mediated the upregulation of miR-338-3p in activated DCs. Together, our findings identify a vital role for Mettl3/miR-338-3p/Dusp16/p38 signaling in DCs-driven pathogenic Th17 responses and suggest a potential therapeutic avenue for uveitis and other Th17 cell-related autoimmune disorders.

Sex identification of birds in Taipei Zoo.

As a conservation and breeding institution for birds, Taipei Zoo plays an important role in restoring endangered species. As approximately half of all bird species are monomorphic, precisely confirming the sex of individuals is critical for the management of ex-situ conservation breeding populations, as well as for understanding the sex ratio of those in the wild. Generally, PCR is used more reliably for sex determination versus traditional methods such as plumage, behavior or hormone levels. Nevertheless, the various primer sets and annealing temperatures vary between species, and so inaccurate sexing can occasionally happen due to inadequate PCR conditions. To reduce the probability of misidentification, and to establish a PCR condition database for sex determination across the diverse range of avian taxa, we tested multiple primer sets and annealing temperatures for amplification of the bird sex-specific gene fragments (CHD1) for each captive or rescued avian species held at Taipei Zoo since 2014. A total of 162 species across 22 orders were tested using one or two primer sets. One hundred and fifty-five species were successfully sexed by the primer set 2550F/2718R and the success rate of sex typing reached over 90% of species tested in each order. Most species have suitable PCR annealing temperatures between 45°C and 55°C, and the species in the same avian taxa showed similar results in temperature. This indicates that it is possible to select the annealing temperature of other species in the same family when the species had not been tested before. We expect this study will improve the success rate of identifying sex by using applicable PCR conditions and reduce the time for searching references every time before attempts to PCR sex birds.

[Effects of propiconazole on physiological and biochemical properties of Panax notoginseng and dietary risk assessment].

To study the residue and dietary risk of propiconazole in Panax notoginseng and the effects on physiological and bioche-mical properties of P. notoginseng, we conducted foliar spraying of propiconazole on P. notoginseng in pot experiments. The physiolo-gical and biochemical properties studied included leaf damage, osmoregulatory substance content, antioxidant enzyme system, non-enzymatic system, and saponin content in the main root. The results showed that at the same application concentration, the residual amount of propiconazole in each part of P. notoginseng increased with the increase in the times of application and decreased with the extension of harvest interval. After one-time application of propiconazole according to the recommended dose(132 g·hm~(-2)) for P. ginseng, the half-life was 11.37-13.67 days. After 1-2 times of application in P. notoginseng, propiconazole had a low risk of dietary intake and safety threat to the population. The propiconazole treatment at the recommended concentration and above significantly increased the malondialdehyde(MDA) content, relative conductivity, and osmoregulatory substances and caused the accumulation of reactive oxygen species in P. notoginseng leaves. The propiconazole treatment at half(66 g·hm~(-2)) of the recommended dose for P. ginseng significantly increased the activities of superoxide dismutase(SOD), peroxidase(POD), and catalase(CAT) in P. notoginseng leaves. The propiconazole treatment at 132 g·hm~(-2) above inhibited the activities of glutathione reductase(GR) and glutathione S-transferase(GST), thereby reducing glutathione(GSH) content. Proconazole treatment changed the proportion of 5 main saponins in the main root of P. notoginseng. The treatment with 66 g·hm~(-2) propiconazole promoted the accumulation of saponins, while that with 132 g·hm~(-2) and above propiconazole significantly inhibited the accumulation of saponins. In summary, using propiconazole at 132 g·hm~(-2) to prevent and treat P. notoginseng diseases will cause stress on P. notoginseng, while propiconazole treatment at 66 g·hm~(-2) will not cause stress on P. notoginseng but promote the accumulation of saponins. The effect of propiconazole on P. notoginseng diseases remains to be studied.

Rapid determination and dietary intake risk assessment of 249 pesticide residues in Panax notoginseng.

UPLC-MS/MS and GC-MS/MS were used to establish a method to simultaneously determine various pesticide residues in Panax notoginseng. Results showed that the limits of detection of 249 pesticides were all 5-10 µg/kg. The detection rate of pesticides in 121 P. notoginseng samples was 93.39%, and 19 pesticides were detected. According to the US Code of Federal Regulations, the Chinese Pharmacopoeia recommended algorithm, and the Japanese "positive list system", the pass rates of pesticide residues were 100%, 99.17%, and 89.26%, respectively. The chronic risk quotient (ADI%) and acute risk quotient (ARfD%) of P. notoginseng were 0.00-0.12% and 0.00-0.15%, respectively. In summary, the detection method established in this study can be used for routine analysis of various P. notoginseng pesticide residues. The pesticide residues in the main root samples of P. notoginseng were at a safe level and unlikely pose health risks to consumers.

Optimal gestational weight gain in Chinese pregnant women by Chinese-specific BMI categories: a multicentre prospective cohort study.

OBJECTIVE: To establish optimal gestational weight gain (GWG) in Chinese pregnant women by Chinese-specific BMI categories and compare the new recommendations with the Institute of Medicine (IOM) 2009 guidelines. DESIGN: Multicentre, prospective cohort study. Unconditional logistic regression analysis was used to evaluate the OR, 95 % CI and the predicted probabilities of adverse pregnancy outcomes. The optimal GWG range was defined as the range that did not exceed a 1 % increase from the lowest predicted probability in each pre-pregnancy BMI group. SETTING: From nine cities in mainland China. PARTICIPANTS: A total of 3731 women with singleton pregnancy were recruited from April 2013 to December 2014. RESULTS: The optimal GWG (ranges) by Chinese-specific BMI was 15·0 (12·8-17·1), 14·2 (12·1-16·4) and 12·6 (10·4-14·9) kg for underweight, normal weight and overweight pregnant women, respectively. Inappropriate GWG was associated with several adverse pregnancy outcomes. Compared with women gaining weight within our proposed recommendations, women with excessive GWG had higher risk for macrosomia, large for gestational age and caesarean section, whereas those with inadequate GWG had higher risk for low birth weight, small for gestational age and preterm delivery. The comparison between our proposed recommendations and IOM 2009 guidelines showed that our recommendations were comparable with the IOM 2009 guidelines and could well predict the risk of several adverse pregnancy outcomes. CONCLUSIONS: Inappropriate GWG was associated with higher risk of several adverse pregnancy outcomes. Optimal GWG recommendations proposed in the present study could be applied to Chinese pregnant women.

Genomic targets for high-resolution inference of kinship, ancestry and disease susceptibility in orang-utans (genus: Pongo).

BACKGROUND: Orang-utans comprise three critically endangered species endemic to the islands of Borneo and Sumatra. Though whole-genome sequencing has recently accelerated our understanding of their evolutionary history, the costs of implementing routine genome screening and diagnostics remain prohibitive. Capitalizing on a tri-fold locus discovery approach, combining data from published whole-genome sequences, novel whole-exome sequencing, and microarray-derived genotype data, we aimed to develop a highly informative gene-focused panel of targets that can be used to address a broad range of research questions. RESULTS: We identified and present genomic co-ordinates for 175,186 SNPs and 2315 Y-chromosomal targets, plus 185 genes either known or presumed to be pathogenic in cardiovascular (N = 109) or respiratory (N = 43) diseases in humans - the primary and secondary causes of captive orang-utan mortality - or a majority of other human diseases (N = 33). As proof of concept, we designed and synthesized 'SeqCap' hybrid capture probes for these targets, demonstrating cost-effective target enrichment and reduced-representation sequencing. CONCLUSIONS: Our targets are of broad utility in studies of orang-utan ancestry, admixture and disease susceptibility and aetiology, and thus are of value in addressing questions key to the survival of these species. To facilitate comparative analyses, these targets could now be standardized for future orang-utan population genomic studies. The targets are broadly compatible with commercial target enrichment platforms and can be utilized as published here to synthesize applicable probes.

Human umbilical cord mesenchymal stem cells alleviate ongoing autoimmune dacryoadenitis in rabbits via polarizing macrophages into an anti-inflammatory phenotype.

Mesenchymal stem cells (MSCs) exhibit beneficial effects on autoimmune dacryoadenitis. However, the underlying mechanisms are not fully understood. In this study, we investigated the therapeutic effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) on rabbit autoimmune dacryoadenitis, an animal model of Sjögren's syndrome (SS) dry eye, and explored whether the effects of MSCs were related to their modulation on macrophage polarization. We have showed that systemic infusion of hUC-MSCs after disease onset efficiently diminished the chronic inflammation in diseased LGs and improved the clinical symptoms. Further analysis revealed that hUC-MSC treatment significantly inhibited the expression of pro-inflammatory M1 macrophage markers iNOS, TNF-α and IL-6, and promoted the expression of anti-inflammatory M2 macrophage markers Arg1, CD206, IL-10, IL-4 and TGF-ß in LGs. Mechanistically, hUC-MSCs activated AKT pathway in macrophages, resulting in upregulation of M2-associated molecule Arg1, which was partly abolished by PI3K inhibitor, LY294002. Together, our data indicated that hUC-MSCs can skew macrophages into an M2 phenotype via affecting AKT pathway. These data may provide a new insight into the mechanisms of hUC-MSCs in the therapy of SS dry eye.

miR-223-3p promotes autoreactive Th17 cell responses in experimental autoimmune uveitis (EAU) by inhibiting transcription factor FOXO3 expression.

Pathogenic T helper (Th)17 cells are key mediators of autoimmune diseases such as uveitis and its animal model, experimental autoimmune uveitis (EAU). However, the contribution of microRNAs (miRs) to the intrinsic control of pathogenic Th17 cells in EAU remains largely unknown. Here, we have reported that miR-223-3p was significantly up-regulated in interphotoreceptor retinoid-binding protein-specific Th17 cells, and its expression was enhanced by IL-23-signal transducer and activator of transcription 3 signaling. Knockdown of miR-223-3p decreased the pathogenicity of Th17 cells in a T-cell transfer model of EAU. Mechanistic studies showed that miR-223-3p directly repressed the expression of forkhead box O3 (FOXO3), and FOXO3 negatively regulated pathogenic Th17 cell responses partially via suppression of IL-23 receptor expression. Thus, our results reveal an important role for miR-223-3p in autoreactive Th17 cell responses and suggest a potential therapeutic avenue for uveitis.-Wei, Y., Chen, S., Sun, D., Li, X., Wei, R., Li, X., Nian, H. miR-223-3p promotes autoreactive Th17 cell responses in experimental autoimmune uveitis (EAU) by inhibiting transcription factor FOXO3 expression.

MicroRNA expression profile of Lacrimal Glands in rabbit autoimmune dacryoadenitis model.

Purpose: To identify the differential expression of microRNAs (miRs) and the related gene networks and signal pathways in lacrimal glands (LGs) of rabbit autoimmune dacryoadenitis. Methods: Autoimmune dacryoadenitis in rabbits was induced by transferring activated peripheral blood lymphocytes (PBLs). The LGs of normal and model group rabbits were collected for small RNA sequencing. The most differentially expressed miRs were validated by quantitative real time-polymerase chain reaction (qRT-PCR). Further, bioinformatics analysis including target gene prediction, Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. Results: A total of 15 miRs were differentially expressed in the LGs of rabbit autoimmune dacryoadenitis relative to normal controls. GO and KEGG analysis revealed that most target genes of these dysregulated miRs were implicated in MAPK signaling pathway. Conclusion: Our results showed for the first time the differentially expressed miRs and the related pathways involved in the pathogenesis of rabbit autoimmune dacryoadenitis. These results may contribute to elucidating molecular pathogenesis of Sjögren's syndrome (SS) dry eye.

Evaluation of tear film and the morphological changes of meibomian glands in young Asian soft contact lens wearers and non-wearers.

BACKGROUND: The aim of this study was to explore the differences in terms of tear film and meibomian glands (MGs) between young Asian soft contact lens (CL) wearers and non-wearers. METHODS: A prospective, cross-sectional observational study was conducted using 148 subjects (63 non-wearers, and 85 soft CL wearers who had been wearing CLs for more than 1 year) recruited from a clinic in Tianjin, China. All subjects first responded to an Ocular Surface Disease Index (OSDI) questionnaire and then underwent a standardized dry eye examination, which included measuring tear meniscus height (TMH), non-invasive tear breakup time (NIBUT), and corneal fluorescein staining (CFS). The MGs were evaluated via ImageJ, distorted MG count and the MG dropout were recorded. RESULTS: Compared to the control group (non-wearers), the CL group recorded higher OSDI and CFS scores, lower TMH and NITBUT values, a larger distorted MG count, and larger MG dropout (all P < 0.05). Pearson correlation analysis found a correlation between MG dropout and the duration of CL use (r = 0.440, P < 0.001), OSDI (r = 0.298, P = 0.006), and CFS scores (r = 0.442, P < 0.001). CONCLUSION: CL wearers showed higher MG dropout and reduced TMH and NITBUT, which likely contributes to severe CL-related dry eye symptoms. CL use may lead to a higher MG dropout rate, and the extent of the MG dropout presumably influences the tear film status in CL wearers.

Functional Conversion and Dominance of γδ T Subset in Mouse Experimental Autoimmune Uveitis.

We have previously shown that activated γδ T cells have a much stronger proinflammatory effect in the development of experimental autoimmune uveitis than their nonactivated counterparts. Our present study explored γδ T cell subsets are functionally distinct in autoimmune pathogenesis and determined the pathogenic contribution of biased Vγ4+ γδ T cell activation in this disease. By systematically comparing two major peripheral γδ T cell subsets, the Vγ1+ and the Vγ4+ cells, we found that the Vγ4+ cells were readily activated in B6 mice during experimental autoimmune uveitis development, whereas Vγ1+ cells remained nonactivated. Cytokines that were abundantly found in the serum of immunized mice activated Vγ4+, but did not activate Vγ1+, cells. The Vγ4+ cells had a strong proinflammatory activity, whereas the Vγ1+ cells remained nonactivated when tested immediately after isolation from immunized mice. However, when the Vγ1+ cells were activated in vitro, they promoted inflammation. Our results demonstrated that activation is a major factor in switching the enhancing and inhibiting effects of both Vγ1+ and Vγ4+ γδ T cell subsets, and that γδ T cell subsets differ greatly in their activation requirements. Whether the enhancing or inhibiting function of γδ T cells is dominant is mainly determined by the proportion of the γδ T cells that are activated versus the proportion not activated.

DNA barcoding reveals CITES-listed species among Taiwanese government-seized chelonian specimens.

Compared to traditional morphological identification, DNA barcoding-molecular identification based on sequencing of a segment of mitochondrial cytochrome c oxidase subunit I (COI)-provides a shortcut to authenticating chelonian identifications. Here, we selected 63 government-seized chelonian specimens deposited at Taipei Zoo for DNA barcoding analysis. DNA barcoding and subsequent phylogenetic analysis successfully authenticated 36 chelonian species, including five that are listed in CITES Appendix I. Approximately 90% (57/63) of the specimens were successfully authenticated by our molecular approach, but lack or error of BOLD reference sequences, biological processes such as hybridization, and uncertain species delimitation all reduced the accuracy of DNA barcoding. To increase the accuracy of DNA barcoding, Taipei Zoo will continue to enrich the BOLD database and also establish a genetic database, to include additional genetic markers, by using government-seized chelonian specimens. A fast and accurate method to authenticate seized samples could assist law enforcement agencies to prosecute criminals and restrict illegal exploitation of wild chelonian resources.

TLR7 Engagement on Dendritic Cells Enhances Autoreactive Th17 Responses via Activation of ERK.

In this study, we showed that TLR7 activation significantly promoted interphotoreceptor retinoid-binding protein (IRBP)-specific Th17 responses by upregulating RORγt, IL-17, GM-CSF, and IL-23R expression in experimental autoimmune uveitis mice. In vivo administration of CL097 activated dendritic cells (DCs) and endowed them with an increased ability to activate IRBP-specific Th17 cells. CL097-treated DCs (CL097-DCs) formed a cytokine milieu that favored the generation and maintenance of Th17 cells by stimulating IL-1ß, IL-6, and IL-23 expression. Furthermore, IRBP-specific T cells from immunized mice injected with CL097-DCs produced more IL-17 and transferred more severe experimental autoimmune uveitis than did those from mice injected with DCs. The enhanced immunostimulatory activities of CL097-DCs depended on JNK, ERK, and p38 activation. Blockade of ERK, but not p38 or JNK, completely abolished the Th17 responses induced by CL097-DCs. Collectively, our findings suggest that CL097 treatment significantly promotes autoreactive IL-17+ T cell responses through enhancing DC activation, which is mediated, at least in part, via the activation of ERK signaling.

Characterization of calcineurin from Cryptococcus humicola and the application of calcineurin in aluminum tolerance.

BACKGROUND: Calcineurin (CaN) is a Ca2+- and calmodulin (CaM)-dependent serine/threonine phosphatase. Previous studies have found that CaN is involved in the regulation of the stress responses. RESULTS: In this study, the growth of Cryptococcus humicola was inhibited by the CaN inhibitor tacrolimus (FK506) under aluminum (Al) stress. The expression of CNA encoding a catalytic subunit A (CNA) and its interaction with CaM were upregulated when the concentration of Al was increased. A CaM-binding domain and key amino acids responsible for interaction with CaM were identified. ∆CNAb with a deletion from S454 to A639 was detected to bind to CaM, while ∆CNAa with a deletion from R436 to A639 showed no binding to CaM. The binding affinities of CNA1 and CNA2, in which I439 or I443 were replaced by Ala, were decreased relative to wild-type CNA. The phosphatase activities of ∆CNAa, CNA1 and CNA2 were lower than the wild-type protein. These results suggest that the region between R436 and S454 is essential for the interaction with CaM and I439, I443 are key amino acids in this region. The ability of the CNA transgenic yeast to develop resistance to Al was significantly higher than that of control yeast. Residual Al in the CNA transgenic yeast culture media was significantly lower than the amount of Al originally added to the media or the residual Al remaining in the control yeast culture media. These findings suggest that CNA confers Al tolerance, and the mechanism of Al tolerance may involve absorption of active Al. CONCLUSIONS: Al stress up-regulated the expression of CNA. CaM-binding domain and key amino acids responsible for interaction with CaM were identified and both are required for phosphatase activities. CNA conferred yeast Al resistance indicating that the gene has a potential to improve Al-tolerance through gene engineering.

Spatiotemporal Diversification of the True Frogs (Genus Rana): A Historical Framework for a Widely Studied Group of Model Organisms.

True frogs of the genus Rana are widely used as model organisms in studies of development, genetics, physiology, ecology, behavior, and evolution. Comparative studies among the more than 100 species of Rana rely on an understanding of the evolutionary history and patterns of diversification of the group. We estimate a well-resolved, time-calibrated phylogeny from sequences of six nuclear and three mitochondrial loci sampled from most species of Rana, and use that phylogeny to clarify the group's diversification and global biogeography. Our analyses consistently support an "Out of Asia" pattern with two independent dispersals of Rana from East Asia to North America via Beringian land bridges. The more species-rich lineage of New World Rana appears to have experienced a rapid radiation following its colonization of the New World, especially with its expansion into montane and tropical areas of Mexico, Central America, and South America. In contrast, Old World Rana exhibit different trajectories of diversification; diversification in the Old World began very slowly and later underwent a distinct increase in speciation rate around 29-18 Ma. Net diversification is associated with environmental changes and especially intensive tectonic movements along the Asian margin from the Oligocene to early Miocene. Our phylogeny further suggests that previous classifications were misled by morphological homoplasy and plesiomorphic color patterns, as well as a reliance primarily on mitochondrial genes. We provide a phylogenetic taxonomy based on analyses of multiple nuclear and mitochondrial gene loci. [Amphibians; biogeography; diversification rate; Holarctic; transcontinental dispersal.

Complete Mitochondrial Reveals a New Phylogenetic Perspective on the Brackish Water Goby Mugilogobius Group (Teleostei: Gobiidae: Gobionellinae).

The Mugilogobius group consists of brackish water gobionellines widely distributed in the Indo-West Pacific region. Complete mitochondrial genome and morphological evidence was collected to estimate their phylogenetic relationship and taxonomic status. A total of 11 genera were sampled, including Brachygobius, Calamiana, Hemigobius, Mugilogobius, Pandaka, Pseudogobiopsis, Pseudogobius, Redigobius, Rhinogobius, Stigmatogobius, and Wuhanlinigobius, five of which were sequenced for the first time. A morphological phylogenetic tree was also reconstructed based on 35 characters. The molecular phylogenetic trees reveal that the Mugilogobius group contains four major clades. The present study also reveals that the adult male mouth size and forked sensory papillae row d can be considered as synapomorphies, and that the head pores on inter-orbital, anterior oculoscapular, and preopercular regions can be regarded as derived features among the Mugilogobius group. Furthermore, the absence of posterior oculoscapular pores may provide a clue for understanding the evolutionary history of the Mugilogobius group.