Coccidioides fungi are widely distributed in the American continents, with an expanding western range documented by a recently discovered cryptic population of Coccidioides immitis in Washington State. The assembled and annotated reference genome sequence of the soil-derived C. immitis strain WA_211 will support population and functional genomics studies. Coccidioides fungi are widely distributed in the American continents, with an expanding western range documented by a recently discovered cryptic population of Coccidioides immitis in Washington State. The assembled and annotated reference genome sequence of the soil-derived C. immitis strain WA_211 will support population and functional genomics studies. Coccidioides immitis and Coccidioides posadasii are fungal species found in desert-like areas of the American continents  using “–minlen 500 –trim” and shovill defaults for downstream tools. The shovill pipeline trimmed reads for adaptors and low quality using Trimmomatic v.0.39 (C. immitis RS genome (GenBank accession number AAEC00000000) . This scaffolding assumes the colinearity of WA_211 and RS, but no breakpoints were observed within contigs in a comparison of a dotplot by D-GENIES   with Rag0000000) , with deD-GENIES .Coccidioides repeat element library  (ab initio predictors Augustus (The genome contains 15% repetitive sequences masked by RepeatMasker v.open-4.0.7 using a  library . Genes w library . Gene prRNA-seq) . Gene prAugustus , SNAP v.Augustus , CodingQAugustus , and GenAugustus  using exAugustus , 23, 24,Augustus . ConsensAugustus . FunannoAugustus , eggNOG Augustus , MEROPS Augustus , and dbCAugustus  databaseAugustus , comprisAugustus .RHJW00000000. The version described in this paper is version RHJW02000000. The genomic sequence reads used in this assembly were previously deposited under SRA project accession number SRR1292227, and the RNA-seq reads used had been deposited under BioProject number PRJNA169242 and SRA accession number SRP013923.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number


As biocatalysts, CESHs are unique epoxide hydrolases because their substrate is a small, mirror-symmetric, highly hydrophilic molecule, and their products show very high enantiomeric purity with nearly 100% enantiomeric excess. In this paper, we review over forty years of the history, process and mechanism studies of CESHs as well as our perspective on the future research and applications of CESH in enantiomeric tartaric acid production.Tartaric acid is an important chiral chemical building block with broad industrial and scientific applications. The enantioselective synthesis of  BK-58, have also been sequenced and cloned ; specifically Nocardia tartaricans nov. sp. was identified as a preferred natural species . Its molar conversion rate is over 90% and the CESH[L] formation is effectively induced by sodium cis-epoxysuccinate during fermentation [Rhodococcus ruber M1 isolated from soil was the first strain in Rhodococcus reported to produce CESH[L] [Klebsiella sp. BK-58 can produce a novel CESH[L] with good thermal and pH stability, which is a promising biocatalyst for the industrial production of l(+)-TA [More than ten species with CESH[L] activity have been reported since the 1970s. Miura et al. discovered that microorganisms of the genus  species . Kamatanhizobium . Two strh as 96% ,28. Screentation . In addi CESH[L] . Further l(+)-TA .Achromobacter and two novel species of Alcaligenes with CESH[D] activity  producing bacteria, Bordetella sp. strain 1-3 has also been reported to have the ability to degrade d(−)-TA as its carbon source, so some measures should be adopted to stop this degradation process to accumulate d(−)-TA. Furthermore, the molecular weight of CESH[D] from Bordetella sp. strain 1-3 is the same as the beta subunit of the previously reported CESH[D] from Alcaligenes sp. The eight amino acid sequence of the N-terminal region of CESH[D] from Bordetella sp. strain 1-3 has also been shown to have the same sequence as the beta subunit from Alcaligenes sp. [Ten species in four genera have been reported to have CESH CESH[D] activity. Sato et al. first isolated four novel species of activity . A strai d(−)-TA ,33. The  MCI3611 . Two str d(−)-TA ,36. Unlienes sp. .Rhodococcus opacus, Nocardia tartaricans CAS-52, and Klebsiella sp. BK-58, and CESH[D] genes from Alcaligenes sp. MCI3611 and Bordetella sp. BK-52  from Klebsiella sp. BK-58. The two CESH[D]s from Alcaligenes sp. MCI3611 and Bordetella sp. BK-52 have identical amino acid sequences. The amino acid sequences of CESHs from most bacteria in The gene sequences of several CESH-producing bacteria have been reported, including CESH[L] genes from p. BK-52 ,37,40,43hia coli ,19,37,43Rhodococcus rhodochrous showed that the effect of the large increase in stability at a lower temperature was much more important than the decrease in activity  and CESH[D] show different stabilities. CESH[D] from d pH 6.5 . A compad pH 6.5 . However and 8.5 ,39. TherRhodococcus opacus has been fused with a carbohydrate binding module (CBM30), and the resulting fusion enzyme (CBM30-CESH) exhibited improved temperature and pH adaptability than free native CESH[L]  gene from  CESH[L] . The CES5.0–10.0 . Random e enzyme .Since the discovery of CESH activity in the 1970s, CESH-producing bacteria and CESHs have been utilized to produce TA with high enantiopurity. The cell lysate or crude enzyme solution is not suitable for TA production because the cellular protease degrades CESH rapidly. Therefore, significant effort has been made to optimize TA production using CESHs or CESH-producing bacteria. TA production was established in the 1970s including the surfactant addition and recovery of TA from the media ,25. SubsNocardia tartaricans, Corynebacterium, and Rhizobium are gelatin, κ-carrageenan, and sodium alginate, respectively   .E. coli successfully [The genes of CESH[L] and CESH[D] have been cloned and expressed in essfully ,40,41,43essfully , and theessfully . The preessfully . Still, l-2-haloacid dehalogenase, of which the structure is known. Therefore, homology modeling has been performed to elucidate the catalytic mechanism of CESH[L] [18O experiments for both CESH[L]s . Only two CESH[L] sequences have been reported and they share a 36% sequence identity ,37,43. B CESH[L] ,42,44. L CESH[L] . Therefo CESH[L] . The twoAs CESH[L] and L-2-haloacid dehalogenase only have about a 30% sequence identity and different substrates, the catalytic residues cannot be deduced from homology modeling. Two mutagenesis analyses revealed that D18, H190, and D193 are essential for the activity; therefore, they were proposed to be a catalytic triad, with D18 activating the attacking water molecule ,44. HoweBordetella sp. BK-52 and Alcaligenes sp. MCI3611 have about 30% sequence identity to the Kce enzyme, whose function is totally different [18O labeling experiment indicated that CESH[D] hydrolyzes CES through a one-step mechanism [Although CESH[D] and its gene sequence were reported earlier than CESH[L], knowledge of CESH[D] catalysis was not obtained until very recently. The protein sequences of CESH[D]s from ifferent ,40,41. Hifferent . The metifferent , which iifferent . The thiechanism  instead d(−)-TA. In the complex structure, three oxygen atoms of TA occupy the positions of the three coordinative water molecules in the substrate-free CESH[D] structure. The identification of the structure of the product–enzyme complex provided the details of substrate binding and positioning, from which the key catalytic residues and substrate recognition residues were elucidated. Instead of the previously proposed catalytic residue D251 [The catalytic mechanism of CESH[D] was not elucidated in detail until our recent report on high-resolution CESH[D] structures . The strdue D251 , the catdue D251 . The structure of CESH[D] has some distinct features in comparison with known EHs. In contrast with the α/β hydrolase fold or LEH fold adopted by most EHs, except for LA4H, ChEH, and FosX, CESH[D] adopts a TIM-barrel fold . Also, CCESHs are unique among the known EHs because the CES substrate is highly hydrophilic and mirror-symmetric. The structural features of CES suggest that CESHs have specific substrates; in other words, the CES molecule is fixed in CESH with an exact position, which leads to the high stereoselectivity and regioselectivity of CESHs. This feature is of great interest for enantioselective synthesis. Although CESHs and their host bacteria have been successfully applied in industry for TA production, there are still many questions to be addressed, and there is a lot of room for growth in the production of TA using CESHs. Currently, the structure and the catalytic mechanism of CESH[L] are still not fully understood. Determination of the high-resolution structure of CESH[L], particularly of complexes with a substrate or product, is necessary to elucidate its catalytic mechanism. With this structure, rational engineering to enhance the stability will be possible. Furthermore, CESHs could also potentially be engineered as biocatalysts to catalyze different substrates, but this potential has not been explored in past studies. Although many microorganisms have been reported to have CESH activity, only very few of them have been sequenced. Therefore, much work is still needed to isolate the CESHs and analyze their protein/gene sequences. The new sequences of CESHs may provide other new features that will help us to understand these enzymes and promote their applications.E. coli cells, and it will improve the production of enantiomerically pure TA using CESHs. Currently, the production of TA using whole cell catalysts is performed by wild strains. No metabolic engineering of these microorganisms has previously been reported. Therefore, understanding the features of these microorganisms and the development of genetic engineering are important topics for future studies. CESHs are intracellular enzymes that are present in certain microorganisms, which cause the activity of whole cell catalysts to depend on the cell permeability. If the CESHs could be engineered as a secretive protein, or immobilized on the cell surface, their activity would be greatly enhanced. This engineering could be done in either the original species or the recombinant


Justicia tranquebariesis on thioacetamide (TAA)-induced oxidative stress and hepatic fibrosis. Male Wister albino rats (150–200 g) were divided into five groups. Group 1 was normal control. Group 2 was J. tranquebariensis (400 mg/kg bw/p.o.)-treated control. Group 3 was TAA (100 mg/kg bw/s.c.)-treated control. Groups 4 and 5 were orally administered with the leaf extract of J. tranquebariensis (400 mg/kg bw) and silymarin (50 mg/kg bw) daily for 10 days with a subsequent administration of a single dose of TAA (100 mg/kg/s.c.). Blood and livers were collected and assayed for various antioxidant enzymes . Treatment with J. tranquebariensis significantly reduced liver TBARS and enhanced the activities of antioxidant enzymes in TAA-induced fibrosis rats. Concurrently, pretreatment with J. tranquebariensis significantly reduced the elevated liver markers  in the blood. In addition, J. tranquebariensis- and silymarin- administered rats demonstrated the restoration of normal liver histology and reduction in fibronectin and collagen deposition. Based on these findings, J. tranquebariensis has potent liver protective functions and can alleviate thioacetamide-induced oxidative stress, hepatic fibrosis and possible engross mechanisms connected to antioxidant potential.The present study aims to examine the protective effect of  Liver fibrosis is a scarring mechanism of the liver related to prominent accretion of an extracellular matrix. Without efficient treatment at an early stage, reversible liver fibrosis develops into irreversible cirrhosis leading to liver failure, portal hypertension and the need for liver transplantation in many cases . These pJusticia tranquebariensis L. (Family: Acanthaceae), a common shrub is broadly scattered in various regions of India, Malaysia and Sri Lanka. The fresh leaves are coolant and aperient, generally used in jaundice, liver diseases, and smallpox [J. tranquebariensis revealed that the leaves contain adequate quantities of phytosterols, flavonoids, glycosides, triterpenoids, alkaloids, saponins, and tannins [O-glucoside, brassicasterol, campesterol, stigmasterol, sitosterol, and spinasterol [J. tranquebariensis leaf extract on liver fibrosis. Thus, this current investigation aimed to observe the antifibrotic and hepatoprotective effect of J. tranquebariesis on TAA-intoxicated rats.smallpox . Bruisedsmallpox . The jui tannins ,15. Aeri tannins . In addinasterol . Furthernasterol , antipyrnasterol , and antnasterol  potentiaJusticia tranquebariensis L.  were acquired from the central animal house in Management and Science University, Shah Alam, Selangor, Malaysia. Animals were kept in animal cages under standard lab conditions . Rats were adapted to the laboratory settings for a week prior to the initiation of experimental treatments. Rats were supplied with free access to standard pellet food and water. The experimental studies were carried out based on the ethical approval and the protocol was permitted by the Institutional Ethical Committee of Management and Science University, Malaysia .TAA, thiobarbituric acid (TBA), 1-chloro-2,4-dinitrobenzene (CDNB), and nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) were procured from Sigma-Aldrich Co., St. Louis, MO, USA. Reagents and chemicals used in the studies were of the analytical grade.J. tranquebariensis was given at various doses of 100, 200, 400, 800 mg/kg bw to different groups of rats. Among the doses, the 400 mg/kg bw showed more effectiveness than the other doses. Hence 400 mg/kg bw was used in this investigation. The dosage of TAA (100 mg/kg bw) and standard drug silymarin (50 mg/kg bw) used in the present study were chosen according to a previous study [A preliminary study was carried out to validate the optimal dose of plant extract by examining serum hepatic marker enzymes in TAA-intoxicated rats. Administration of aqueous extract from the leaves of us study .J. tranquebariensis control): Rats of this group were pretreated with J. tranquebariensis (400 mg/kg bw p.o./day) for 10 days. Group 3 (toxic control): Rats of this group were treated with a single dose of TAA (100 mg/kg bw/s.c.) on the 10th day. Group 4 (J. tranquebariensis plus TAA): Rats of this group were pretreated with J. tranquebariensis (400 mg/kg bw p.o./day) for 10 days and subsequent administration of a single dosage of TAA (100 mg/kg/s.c.). Group 5 (Silymarin plus TAA): Rats of this group were pretreated with silymarin (50 mg/kg bw/p.o./day) for 10 days followed by a single dose subcutaneous injection of TAA (100 mg/kg). TAA hepatotoxicity induction was followed our previous investigation [Following laboratory adaptation, animals were randomly divided into five groups, each group comprising of six rats. All rats were kept fasting for 24 h before the experiment. Group 1 : Rats of this group received 5 mL of distilled water/kg bw. Group 2 . In the liver tissue homogenates, LPO was calculated using TBARS based on the method of Ohkawa et al. . SOD andAfter sacrificing the animals, the livers were quickly removed and preserved in 10% formosaline and processed for paraffin embedding following the standard micro techniques. Sections (5 µm thick) of liver tissues stained with hematoxylin and eosin (H&E) were evaluated for histopathological under a light microscope. p < 0.05 was considered to indicate a significant difference between groups.All data obtained in the study were expressed as mean ± standard deviation (S.D.). The data of the groups were statistically done using one-way analysis of variance (ANOVA) and the individual comparison was obtained by Duncan’s Multiple Range Test (DMRT) by the SPSS software for Windows Version 20.0 . A value of p < 0.05), whereas tissue TBARS level significantly increased (p < 0.05) when compared with the normal control group. However, pretreatment administration of J. tranquebariensis (400 mg/kg bw) and silymarin (50 mg/kg bw) in TAA-induced rats have significantly altered to the above changes by regulating the TBARS level which subsequently increased those antioxidant enzymes.The levels of tissue TBARS formation and the activities of antioxidant enzymes SOD, CAT, GPx, GR, GST and total reduced GSH in the liver of normal control and experimental rats are demonstrated in p < 0.05) in TAA-intoxicated rats when compared with the normal control group. However, pretreatment of J. tranquebariensis (400 mg/kg bw) significantly decreased (p < 0.05) the above liver marker enzymes and total bilirubin levels observed in TAA-intoxicated rats.The activities of liver marker enzymes such as ALT, AST, ALP, GGT and the content of TB in the serum of control and experimental groups are shown in J. tranquebariensis and silymarin-treated rats are exhibited in J. tranquebariensis (400 mg/kg bw) and silymarin (50 mg/kg bw) showed a normal lobular pattern with sinusoidal spaces, moderate swelling, and restoration of normal liver histology and thereby a decrease in collagen and fibronectin deposition.The histology of normal control, TAA control, J. tranquebariensis on TAA-induced liver fibrosis in rats. A noticeable indication of liver injury is the release of cytoplasmic cellular enzymes into the blood due to the obstructions caused by chemicals in the transport functions of liver cells [J. tranquebariensis with a healing of hepatic parenchyma and regeneration of hepatocytes [J. tranquebariensis prevented the TAA toxicity effect on ALP activity in serum. In addition, the normalization of serum TB levels is done by the administration of J. tranquebariensis, which shows an indication of the normal functions of the hepatocyte.In the present study, we investigated the effect of an aqueous extract from the leaves of er cells . The incatocytes ,30. The atocytes ,32. PretJ. tranquebariensis clearly enhanced GSH levels and facilitated the rapid and efficient consumption of ROS generation in TAA-intoxicated rats. GST is a soluble enzyme located in the cytosol, which plays a significant function in the detoxification of xenobiotics [J. tranquebariensis can, therefore, reduce TAA hepatotoxicity. There was a decrease in GPx activity in animals administered with TAA, which could be due to the higher production of toxicity. In the presence of J. tranquebariensis, GPx levels were restored back to control levels. The increase in hepatic GR activities was shown in J. tranquebariensis administered rats, as compared with the liver of TAA-induced rats. Elevated levels of SOD and CAT are insightful indexes into liver damage as they eradicate ROS, thereby reducing the harmful effects. On the contrary, an earlier report showed that TAA induction raises the TBARS levels, which instigated to reduce the levels of SOD and CAT [J. tranquebariensis and silymarin significantly decreased TBARS and elevated antioxidant enzymes in TAA-induced rats. Earlier studies have also suggested that J. tranquebariensis has strong antioxidant potential [GSH in the cytosolic pool consists of 85% hepatocellular GSH and 15% mitochondrial GSH. The reductions of hepatic GSH provide valuable evidence of the defensive role of GSH against noxious foreign materials . Therefoobiotics . The incotential  and prevotential .J. tranquebariensis (400 mg/kg bw). This degree of hepatoprotection could be based on its various properties of anti-oxidation, inhibition of lipid peroxidation, regeneration of intracellular glutathione content, protein synthesis and improvement of liver regeneration from hepatocellular necrosis [The degree of protection was maximally observed in silymarin (50 mg/kg bw/p.o)-treated groups when compared with necrosis ,42,43. Fnecrosis ,45,46,47J. tranquebariensis showed a normal lobular pattern with minimal pooling of blood in the sinusoidal spaces, moderate swelling, condense collagen and fibronectin deposition and re-establishment of liver architecture to normal. In conclusion, the result of this study revealed that pretreatment of J. tranquebariensis has a strong hepato-protective effect on TAA-induced liver oxidative stress and liver fibrosis in rats owing to its strong antioxidant properties. However, further evidence is needed to establish the possible mechanisms of the hepatoprotection in order to widely use J. tranquebariensis as an antihepatotoxic agent.In the present study, we induced hepatic fibrosis by subcutaneous injection of a single dose of TAA. TAA is normally metabolized by CYP2E1 releasing ROS, which attacks DNA, lipid, and protein . This ma


Symplocarpus, a skunk cabbage genus, includes two sister groups, which are drastically different in life history traits and thermogenesis, as follows: The nonthermogenic summer flowering S. nipponicus and thermogenic early spring flowering S. renifolius. Although the molecular basis of thermogenesis and complete chloroplast genome (plastome) of thermogenic S. renifolius have been well characterized, very little is known for that of S. nipponicus. We sequenced the complete plastomes of S. nipponicus sampled from Japan and Korea and compared them with that of S. renifolius sampled from Korea. The nonthermogenic S. nipponicus plastomes from Japan and Korea had 158,322 and 158,508 base pairs, respectively, which were slightly shorter than the thermogenic plastome of S. renifolius. No structural or content rearrangements between the species pairs were found. Six highly variable noncoding regions  were identified between S. nipponicus and S. renifolius and 14 hot-spot regions were also identified at the subfamily level. We found a similar total number of SSR (simple sequence repeat) motifs in two accessions of S. nipponicus sampled from Japan and Korea. Phylogenetic analysis supported the basal position of subfamily Orontioideae and the monophyly of genus Symplocarpus, and also revealed an unexpected evolutionary relationship between S. nipponicus and S. renifolius. Symplocarpus Salisb. ex W.P.C.Barton belongs to the basal lineage known as Proto-Araceae of the arum family Araceae. It specifically belongs to the subfamily Orontioideae, which includes two other genera, Orontium L. and Lysichiton Schott [Orontium occurs exclusively in the eastern United States, two genera, Lysichiton and Symplocarpus, are found in both eastern Asia (EAS) and western North America (WNA)/eastern North America (ENA), making these two genera an ideal system to study intercontinental disjunction in the Northern Hemisphere. In particular, genus Symplocarpus is of great interest for pollination biology and biogeography given its distribution pattern and thermogenesis that is characteristic of certain species. It includes five species  Salisb. ex W.P.C.Barton, S. nabekuraensis Otsuka & K.Inoue, and S. egorovii N.S.Pavlova & V.A.Nechaev) and usually occurs in wet places and forest swamps [Symplocarpus foetidus is the only species widely distributed in ENA and the remaining four species are found in EAS. Of the five species, S. renifolius and S. foetidus share flowering time in the early spring before the emergence of leaves and fruit ripening in the fall of the same year. The two species, especially the eastern Asian skunk cabbage S. renifolius, have been a subject of intensive study in thermogenesis. Blooming in early spring, often under snow, the plants can maintain a spadix temperature above 20 °C, despite ambient temperatures down to −14 °C [S. renifolius, its sister species, S. nipponicus, is not thermogenic and has a more spherical spadix while lacking precocious flowering. Its flowering season is in summer and its fruits ripen in the following spring [Symplocarpus suggested that S. nipponicus is the earliest divergent lineage within the genus [S. foetidus  and S. nipponicus , with a basic chromosome number of x = 15 [S. renifolius was most likely evolved during or after polyploidization of nonthermogenic S. nipponicus. The divergence between S. nipponicus and S. renifolius was estimated to be 20.65 ± 6.44 million years ago (mya) based on the Bayesian molecular dating method [The skunk cabbage genus n Schott ,2. Whilet swamps ,3,4. Symo −14 °C ,7,8,9,10o −14 °C ,12,13. Uhe genus ,16. The f x = 15 . The cytS. renifolius sampled from Korea [Symplocarpus and compared it to those of other Araceae species. Since the first whole chloroplast (cp) genome sequence was reported in the late 1980s, hundreds of plastomes have been characterized and utilized at various levels for phylogenetic analyses as well as DNA barcoding studies ,19,20. Tom Korea . This stS. nipponicus sampled from Japan and Korea and compared them with that of its thermogenic sister species S. renifolius, sampled from Korea. By comparing the thermogenic and nonthermogenic types of plastome sequences, we identified changes in the organization and structure of the cp genome between the two sister species, which diverged during the early Miocene and have drastically different life history traits and thermogenesis during flowering. In addition, we aimed to identify mutational hotspot regions and plastome-based simple sequence repeat (SSR) markers, which can be useful for improving the resolution of phylogenetic relationships at lower taxonomic levels.In the present study, we determined the complete plastome sequences of nonthermogenic eastern Asian Symplocarpus nipponicus sampled from Japan was 158,322 base pairs (bp), with a large single copy (LSC) region of 86,433 bp, small single copy (SSC) region of 20,271 bp, and two inverted repeat (IR) regions (IRA and IRB) of 25,809 bp each. Symplocarpus nipponicus sampled from Korea was slightly longer, a total of 158,508 bp, with large single copy (LSC) region of 86,595bp, small single copy (SSC) region of 20,309 bp, and two inverted repeat (IR) regions of 25,802 bp  and 37.3% , respectively, and contained 130 genes, including 85 protein-coding, eight rRNA, and 37 tRNA genes  and the S. nipponicus plastome sequence from Korea was 186 bp longer than that of S. nipponicus from Japan. The complete plastome sequences of S. nipponicus from Japan and S. renifolius from Korea showed 99.7% similarity  and the thermogenic S. renifolius plastome sequence from Korea was 199 bp longer than that of nonthermogenic S. nipponicus from Japan , four rRNA genes , and six protein-coding genes . Fifteen genes  contained one intron, while clpP and ycf3 each contained two introns  and S. nipponicus contained the smallest genome within the family [Symplocarpus represents the earliest diverged lineage within the family, i.e., the proto-Araceae, followed by sequential divergence of Lemnoideae  and Aroideae (Dieffenbachia and Colocasia) [Symplocarpus nipponicus  to Spirodela polyrhiza  found in Araceae, any general trend in either an increase or decrease in genome size could not be postulated. Within proto-Araceae, genus Symplocarpus contained relatively smaller cp genome sizes compared to other members such as Gymnostachys and Orontium [n = 2x = 30; S. nipponicus) and polyploids  within Symplocarpus require further study. Finally, it has been shown that the functional gene infA is highly variable as a pseudogene or missing gene within the family Araceae. However, like S. renifolius, infA of S. nipponicus was located in the LSC as an intact gene. In addition, psbZ was located between trnS-UGS and trnG-UCC in the LSC. When the two plastomes of thermogenic S. renifolius and nonthermogenic S. nipponicus were compared, the average value of nucleotide diversity (PI) was 0.00054, and six highly variable regions based on a significantly higher PI values of > 0.004 were identified. The regions that varied the most between S. nipponicus and S. renifolius were psbC/trnS and petA/psbJ intergenic regions, with a PI value of 0.0067 and 0.005 each, respectively. Four additional regions, including trnS/trnG, trnC/petN, ycf4/cemA, and rpl3/rpl22 intergenic regions were also highly variable, with a PI value of 0.00417. Future chloroplast-based phylogeographic studies would be greatly benefited by the application of these highly variable markers (The complete chloroplast genome of e family . The genlocasia) ,36. GiveOrontium . It is y markers .Symplocarpus (two accessions of S. nipponicus from Japan and Korea and one accession of S. renifolius from Korea) showed the same frequency of codon usage. The usage of AUG codon was especially shared with trnfM-CAU and trnI-CAU. The codon usage was biased toward a high representation of A and U at the third codon position, which was similar to the phenomenon observed in most land plant cp genes [The frequency of codon usage was calculated for the cp genome based on the sequences of protein coding genes and tRNA genes, which are summarized in cp genes .S. nipponicus from Korea, while similar numbers (83 sites) were found in S. nipponicus from Japan , followed by the ndhD gene (8). In accordance with previous reports [petB gene in S. nipponicus from Japan than individual from Korea.A total of 82 editing sites in 20 protein-coding genes were identified in om Japan . The ndh reports ,40, we opsaI), two photosystem II subunit gene (psbB and psbM), three NADP dehydrogenase , one ribosome large subunit gene (rpl23), and one cytochrome c biogenesis maturase (matK). However, the majority (62 of 71 genes) had a Ka/Ks ratio of > 1, which suggests that these genes were positively selected within the Araceae family. Alocasia macrorhizos, Lemna minor, and Woffia australiana showed a high value of Ka/Ks > 1 in all 14 taxa comparison. In general, previous studies showed that Ka/Ks values are usually less than one [Overall, the average Ka/Ks ratio of the 71 common protein-coding genes analyzed in the 14 plastomes was 0.14. For each conserved gene, the total of nine (out of 71 genes) had an average Ka/Ks ratio below one for the 14 comparison groups, suggesting that these genes were subject to strong purifying selection pressures in Araceae chloroplasts. Those genes included one photosystem I subunit gene , whereas 22 (17.7%) were located in introns, 26 (21%) in protein coding genes, and 5 (4%) in tRNA and rRNA genes . Therefore, 75% of the SSRs were located in intergenic and intron regions, while only 25% were distributed in conserved genic regions. In addition, mononucleotide, dinucleotide, trinucleotide, and pentanucleotide repeats were detected in 71 (57.3%), 47 (37.9%), five (4%), and one (0.8%) unique consensus SSRs, respectively. No tetranucleotides and hexanucleotides were found. Of a total of 88 unique consensus SSRs identified from S. nipponicus in Korea , whereas 14 (15.9%) were located in introns, 15 (17.1%) in protein coding genes, and three (3.4%) in 23S rRNA genes. Therefore, 79.5% of the SSRs were located in intergenic and intron regions, while only 20.5% were distributed in conserved genic regions. In addition, mononucleotide, dinucleotide, and trinucleotide repeats were detected in 52 (59.09%), 34 (38.63%), and two (2.27%) unique consensus SSRs, respectively. No tetranucleotide, pentanucleotide, and hexanucleotides were found. Given the similar parameter settings of the MISA program to identify SSR motifs, we found slightly higher numbers of SSRs in the LSC region in S. nipponicus from Japan  than in that of S. renifolius , but significantly more than S. nipponicus from Korea  [S. nipponicus  than in that of S. renifolius (30 SSRs), but significantly higher numbers in the IR regions . The total number of SSR motifs found in these two congeneric species (Symplocarpus nipponicus and S. renifolius) was significantly higher than that in the members of Lemnoideae  [S. nipponicus can be useful for future population-level studies of individual species or for comparative population genetic studies between intercontinental disjunct species pairs in eastern Asia (S. renifolius and S. nipponicus) and in eastern North America (S. foetidus).Most of the SSRs were located in intergenic regions ( 67.04%) . Howevern Lemna) . It appeSymplocarpus nipponicus, S. renifolius, Lemna minor, Spathiphyllum kochii, Alocasia macrorrhizos, Dieffenbachia seguine, Spirodela polyrhiza, Wolffia australiana, and Colocasia esculenta were plotted using mVISTA analysis, using annotated S. renifolius plastome as a reference (The complete plastome sequences of two accessions of eference . Based otrnS/trnA intergenic region (PI = 0.24358), followed by the other two regions, psbI/trnS (PI = 0.21919) and ndhF gene (PI = 0.2105). Other variable regions were also identified, including ycf1 gene (PI = 0.19811) and ten intergenic regions (trnQ/psbK (PI = 0.19756), rpl32/trnL (PI = 0.19697), trnC/petN (PI = 0.19256), trnT/trnL (PI = 0.18911), rps16/trnQ (PI = 0.18875), trnT/psbD (PI = 0.18861), rpoB/trnC (PI = 0.18628), trnL/ccsA (PI = 0.18428), psbK/psbI (PI = 0.18322), and petA/psbJ (PI = 0.1825)). When these hotspot regions based on the representative lineages within the family Araceae were compared to those from two congeneric species of Symplocarpus, we found that highly variable regions based on the family level are not necessarily the same as those of within the genus level Symplocarpus, with the exception of petA/psbJ and trnC/petN regions. Compared to highly variable noncoding cp regions identified by Shaw et al. [rpl32/trnL, trnT/trnL, rps16/trnQ, trnT/psbD, and petA/psbJ) in Araceae are consistently and highly variable regardless of taxonomic level. The remaining nine regions in Araceae were not identified as hotspot regions in angiosperms, suggesting no universal “best” region. Overall, we identified several highly variable plastome regions in Araceae, which may help resolve phylogenetic relationships and could potentially be used as effective markers for barcoding and phylogenetic studies of higher taxonomic levels. Sliding window analysis revealed highly variable regions in the Araceae plastome . The avePI = 0.188, psbK/p PI = 0.258, folloSymplocarpus (subfamily Orontioideae) within Araceae, (2) the monophyly of Lemnoideae and its sister relationship to the rest of Araceae, (3) relationships among four genera of Lemnoideae, and (4) the close relationship between Colocasia and Pinellia. Few unexpected relationships based on the current phylogeny included the position of Spathiphyllum, Zantedeschia, and Epipremnum. Within the subfamily Monsteroideae, the previous studies suggested that Epipremnum (Rhaphidophora clade) is related to Spathiphyllum (Monsteroideae). However, our phylogeny suggested that Epipremnum is closely related to Dieffenbachia (Spathicarpeae). The previous studies suggested that Dieffenbachia is more closely related to Zantedeschia (Zantedeschia clade) than it is to Epipremnum. One most surprising and unexpected finding in this study was the position of Alocasia macrorrhizos (KR 296655). Our phylogenetic tree based on the complete cp genome sequence and 85 concatenated coding genes suggested that Alocasia macrorrhizos is not closely related to other members of the same family Araceae. Rather, it came out as a sister to the clade containing Musaceae, Arecaceae, and Typhaceae  and biological factors  cause phylogenetic incongruence  outside of its own family Araceae, it may be caused by either long branch attraction or simply a taxon misidentification issue. It appears that Alocasia macrorrhizos (KR 296655) shows somewhat longer branch length compared to that of other lineages, but other factors  also likely contributed to its unusual position. The optimality criteria as a contributing factor, however, can be ruled out given the same tree topology found between the ML and BI (Bayesian Inference) tree (with unspecified model selection) based on the complete plastid sequences of Alismatales [Alocasia has been sequenced and reported, we could not confirm any of these possibilities [Alocasia macrorrhizos by sampling additional species of Alocasia. The ML (maximum likelihood) tree confirmed previously known phylogenetic relationships within the family Araceae based on earlier studies, while unexpected relationships and positions of certain taxa were also revealed in this study ,44,45,46yphaceae . On the ia clade ,44, or Ana clade . Therefoeference  and refesmatales . It is abilities ,34. It rSymplocarpus, we found that S. nipponicus is sister to the clade containing S. foetidus and S. renifolius, revealing overall phylogenetic relationships among the three common species [S. nipponicus accessions (S. nipponicus sampled from Korea and Japan. The complete plastid sequence phylogeny showed that S. nipponicus sampled from Korea is more closely related to S. renifolius sampled from Korea than to its conspecific population sampled from Japan (55% BS support). On the other hand, the 85 concatenated coding genes data set suggested that two accessions of S. nipponicus are sister to each other (99% BS support). The complete plastid sequence-based phylogeny was unexpected given drastically different morphological, cytological, and life history traits between thermogenic S. renifolius and nonthermogenic S. nipponicus. However, our broader phylogenetic study based on extensive sampling of proto-Araceae found that S. renifolius in Korea is diploid, unlike known tetraploid conspecific populations in Japan, and that populations of S. renifolius in Korea are more closely related to S. nipponicus in Korea than to conspecific populations in Japan [S. renifolius are morphologically distinctive from those of Japan, warranting new taxonomic recognition. Therefore, the relationship suggested based on the complete plastid sequence in this study is not completely unexpected, perhaps reflecting true evolutionary relationship among those taxa. It requires further confirmation based on different types of molecular markers, especially from nuclear single-copy or low-copy genes.Within the genus  species ,49. As s species , one impcessions . In thisin Japan . In addiSymplocarpus nipponicus leaves from a single plant were collected from the natural population in Japan (voucher specimen: SKKU_Kim 1608001) and Korea . Total DNA was isolated using the DNeasy plant Mini Kit  and sequenced using the Illumina HiSeq 4000  at Macrogen Corporation . The resulting paired-end reads were assembled de novo using Velvet v. 1.2.10 with multiple k-mers [Fresh e k-mers . The tRNe k-mers . Annotate k-mers  and the e k-mers .Symplocarpus nipponicus sampled from Japan and Korea was compared to that of S. renifolius (NC 003970), a thermogenic sister species, using mVISTA [S. nipponicus and S. renifolius cp genome sequences and eight additionally related plastome  sequences were aligned using MAFFT v. 7 [S. nipponicus from Korea and Japan, protein-coding genes were conducted using the online program predictive RNA editor for plants (PREP) suite [Symplocarpus cp genomes (S. renifolius and S. nipponicus from Korea and S. nipponicus from Japan) compared to 11 closely related species in the family of Araceae .The complete plastome of nonthermogenic g mVISTA  in Shuffg mVISTA . The twoFFT v. 7  and adjuFFT v. 7 . DnaSP vFFT v. 7  using a FFT v. 7  with relFFT v. 7 . To predP) suite  with 22 P) suite , using GP) suite . Using DP) suite , we calcSimple sequence repeat (SSR) markers were identified in the plastome sequence using MISA  with minLiriodendron chinense (Magnoliids) was used as the outgroup taxon and nonparametric bootstrap analysis was performed with 1000 replicates. For the phylogenetic analysis, 17 representative species from the Mesangiospermae , and one species from Magnoliaceae in Magnoliids) were aligned using MAFFT v. 7 in Geneious . MaximumSymplocarpus nipponicus sampled from Japan and Korea and compared it to the plastomes of thermogenic sister species S. renifolius and several other representative species from two subfamilies (Lemnoideae and Aroideae) of Araceae. Comparative phylogenomic analysis between thermogenic and nonthermogenic Symplocarpus sister species revealed highly conserved plastomes despite drastically different life history traits and thermogenesis. We found six highly variable regions that could be used as powerful molecular markers for future phylogeographic and population genetic studies , Aroideae (Colocasia), Mosteroideae, Zamioculcadoieae, and Spathicarpeae, revealed 14 highly variable regions with high PI values . These mutation hotspots could be used as useful markers to improve the resolution of unresolved phylogenetic relationships within Araceae as well as effective barcoding markers for the identification of species.We determined the complete plastome sequence of nonthermogenic eference ). Moreov


Bacillus anthracis, is widely used in China to identify anthrax bacteria. In this study, we report the complete AP631 phage genome sequence as well as comparative genomic analysis with other bacteriophages of B. cereus and related species. The double-stranded circular DNA genome of phage AP631 was 39,549 bp in length with 35.01% G + C content. The phage genome contained 56 putative protein-coding genes but no rRNA or tRNA genes. Comparative phylogenetic analysis of the phage major capsid proteins and terminase large subunits showed that phage AP631 belongs to the B. cereus sensu lato phage clade II. Comparative genomic analysis revealed a high degree of sequence similarity between phage AP631 and B. anthracis phages Wbeta, Gamma, Cherry, and Fah, as well as three AP631-specific genes bearing no significant similarity to those of other phages.AP631, a virulent bacteriophage of  Bacillus anthracis phage from crude sewage [B. anthracis have been isolated from sources worldwide [B. anthracis from other B. cereus group members [B. anthracis phage from crude sewage and designated it AP631, indicating that this was the first B. anthracis phage isolated in China [B. anthracis and form clear plaques, it is now widely used in China to identify anthrax bacteria. Under the electron microscope, AP631 has a hexagonal head around 50 nm in diameter and a long noncontractile tail around 180 nm in length [Siphoviridae in the order Caudovirales. In 2016, Zhang et al. found that AP631 could nonspecifically lyse eight isolates of B. cereus preserved in their laboratory, forming turbid plaques [B. cereus group. These data are useful for extending our understanding of the genomic characteristics of this group of phages and for enabling various applications related to the identification and control of B. anthracis.Phages that infect prokaryotes were first reported by Frederick W. Twort in 1915 , 3. In 1e sewage . Since torldwide , includi members . Phage Fin China . BecauseB. anthracis strain A16R by plating on BHI agar, followed by amplification in BHI nutrient broth. Bacterial cells were removed from culture supernatants by filtration through a 0.22-μm syringe filter prior to isolation of bacteriophage genomic DNA. Genomic DNA was purified using a QIAGEN Lambda DNA Extraction Kit  following the manufacturer’s instructions.Phage AP631 was obtained from the Lanzhou Institute of Biological Products Co., Ltd., Gansu Province, China, and propagated on de novo using SOAP [http://soap.genomics.org.cn/soapdenovo.html). Potential open reading frames (ORFs) were identified using PHASTER (http://phaster.ca) [http://lowel.ab.ucsc.edu//tRNAscan-SE/). Comparative analysis of phage AP631 nucleotide and amino acid sequences with other known sequences was performed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Comparative phylogenetic analysis was conducted using the neighbor-joining method in MEGA6 [http://easyfig.sourceforge.net/).Whole-genome sequencing was performed at the Beijing Novogene Bioinformatics Technology Co., Ltd. on the Illumina HiSeq PE150 platform with approximately 1340-fold coverage. High-quality paired-end reads were assembled ing SOAP , 11 (httster.ca) . Potentiin MEGA6  and Clusin MEGA6   were continuous and adjacent to the attR site. ORF44–ORF46 showed sequence similarity to genes of three different Bacillus species , which probably indicates the evolutionary history and former hosts of phage AP631. The endolysin sequence of phage AP631 was identical to that of the B. anthracis phages Wbeta, Gamma, Cherry, and Fah.The genome of phage AP631 is 39,549 bp in length with a G + C content of 35.01%, similar to that of its host B. cereus group phages were obtained from the NCBI in GenBank format. A phylogenetic analysis of the major capsid proteins and the terminase large subunits of all 14 phages (including AP631) was conducted using the neighbor-joining method in MEGA6 [B. anthracis phages Cherry, Gamma, Wbeta and Fah, which all belong to evolutionary group II B. cereusB. anthracis phages Cherry, Gamma, Wbeta and Fah, which range from 36,615 bp to 40,867 bp. The AP631 genome is colinear with the other four B. anthracis phage genomes and shows a similar cassette organization. The left end of the genome contains genes related to DNA packaging, the phage head protein, the head-tail adaptor protein, and tail proteins, and the central region of the genome contains genes related to host cell lysis, lysogeny control, DNA replication and gene regulation. These regions were found to be almost identical to those of the phage Fah genome and showed a high degree of similarity to the genomes of phages Gamma, Cherry and Wbeta. By contrast, the right end of the AP631 genome was highly divergent when compared with the other four B. anthracis phage genomes, and most of the genes in this region had no known function. Three ORFs (ORF44–ORF46), which showed no significant similarity to any proteins in the other four phage genomes, were located in the highly variable right genomic region (Fig. The genome size of phage AP631 is similar to those of the Nucleotide sequence accession number: The GenBank accession number for phage AP631 is MK085976.


Dadih samples from two different origins (Kamang and Gadut in West Sumatra) manufactured with different methods (back-slopping or spontaneous fermentation) were evaluated for the diversity of lactic acid bacteria (LAB).Four dadih samples manufactured with two different fermentation methods were obtained from Kamang and Gadut regions. Both genotypic and phenotypic characteristic  were used to analyze the diversity of dadih LAB population.Lactococcus, Lactobacillus, and Pediococcus. Sequencing results showed that 41.6% (five isolates) were identified as Lactococcus lactis ssp. lactis, 25% (three isolates) were identified as Lactobacillus plantarum ssp. plantarum, 16.6% (two isolates) were identified as L. lactis ssp. cremoris, and 8.3% (one isolate each) were identified as Pediococcus pentosaceus and Lactobacillus pentosus.This study showed that LAB count in back-slopping fermented dadih was one log cycle higher than spontaneous fermented dadih. LAB isolates from the two regions were divided into three genera, namely L. lactis ssp. lactis, L. lactis ssp. cremoris, L. plantarum ssp. plantarum, L. pentosus, and P. pentosaceus. On the other hand, spontaneous fermented dadih only contained three different species, namely L. lactis ssp. lactis, L. lactis ssp. cremoris, and L. plantarum ssp. plantarum. This research showed that back-slopping fermentation offers greater abundance and diversity compared to spontaneous fermentation in dadih.Five species were determined in back-slopping fermented dadih, i.e.,  Dadih is naturally fermented buffalo milk from West Sumatra and has become an integral part of Minangkabau diet. The dadih has “yogurt-like” consistency, i.e. soft texture, whitish cream color, sour, and pleasant taste. Dadih surface is smooth and shiny, clean without air bubbles in the middle of the product . The ferLeuconostoc paramesenteroides, Lactobacillus casei ssp. rhamnosus, L. casei ssp. casei, Lactococcus lactis ssp. lactis, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus fermentum, Pediococcus pentosaceus, L. rhamnosus, and Enterococcus faecium [Various types of indigenous microflora dominated by lactic acid bacteria (LAB) in spontaneous fermentation cannot be fully controlled or correctly predicted, even though this process has been practiced for many years . Microor faecium -9.Spontaneous and back-slopping fermentation methods are still performed during dadih production in Gadut and Kamang regions, West Sumatra. No study has been conducted on comparing these two fermentation methods that are applied during dadih production. We assume that LAB population is affected by the different fermentation methods. The aim of this study was, therefore, to highlight the diversity of LAB isolated from dadih in Gadut and Kamang, West Sumatra.No live animals were used in the present study. Samples were purchased from herdsmen from Kamang and Gadut region in West Sumatra.Kamang is located in Agam District; meanwhile, Gadut is located in Limapuluhkota District. Their distance from Bukittinggi city is 16 km and 42.4 km, respectively.Four dadih samples were purchased from two regions in West Sumatra, namely Kamang and Gadut. Dadih sample from Kamang was processed with back-slopping fermentation by adding approximately 50 g (1 tablespoon) of previous dadih into 1600 ml of fresh milk and then placed into bamboo tube. Fermentation process occurred for 48 h at room temperature. Spontaneous fermentations were applied in dadih from Gadut. Fresh buffalo milk was inserted into the bamboo tube and let it fermented for 48 h at room temperature. pH of dadih sample was measured using Hanna HI8424 microcomputer pH meter and then 5 g were aseptically removed in a sterile tube and transferred to the laboratory under cool condition (4°C) for further analyses.7-108) was made and inoculated on de Man-Rogosa-Sharpe (MRS) Agar (Merck) + 0.5% CaCO3 medium by double layer technique and incubated at 37°C for 48 h. The number of LAB colonies was expressed as log unit colony count (log cfu) per gram sample. Ten colonies with the surrounding clear zone were randomly selected in each plate. Non-motile rod and coccus, Gram-positive and catalase-negative isolates were plated on fresh MRS plates to obtain a pure culture. LAB isolates were stored in 10% (v/v) of sterile glycerol at −20°C [About 1 g of each dadih sample was mixed with 45 ml of sterile NaCl 0.85%. An appropriate dilution 107-108 was™ Mini gDNA Bacteria Kit (Geneaid). The DNA pellets were suspended in 50 µL TE buffer (Tris-EDTA) and stored at −20°C. LAB 16S rRNA gene was amplified using a universal primer 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) [st BASE for sequencing 16S rDNA gene. The sequencing results were subjected into National Center for Biotechnology Information (NCBI) GenBank (www.ncbi.nlm.nih.gov) and basic local alignment search tool (BLAST) was adopted to search GenBank database for sequence homology analysis to determine the genera of LAB. Species with similarity more than 97% was considered as the same. Phylogenetic tree of LAB was constructed with MEGA 10 software, , and neighbor-joining methods were performed to test confidence with bootstrap data set of 1000 times [The genome DNA of the selected LAB isolates was extracted using PrestoACTT-3’) . A total00 times .Ò 50 CHL kit . The fermentation profile of isolates was determined using APILAB Plus software version 3.3.3. from bioMérieux [Identification of LAB isolates was carried out through observation of carbohydrate fermentation patterns using APIoMérieux .Fifty-three presumptive LAB colonies with clear zone around the colony were randomly selected. The morphological test results showed 31 isolates that were categorized as non-motile rod and coccus, Gram-positive bacteria, and negative to catalase . Based oLactococcus, Lactobacillus, and Pediococcus. Sequencing results showed that 41.6% (5 isolates) were identified as L. lactis ssp. lactis, 25% (three isolates) were identified as L. plantarum ssp. plantarum, 16.6% (two isolates) were identified as L. lactis ssp. cremoris, and 8.3% (one isolate each) were identified as P. pentosaceus and Lactobacillus pentosus  origin had greater diversity than that originated from Gadut . It consLactococcus genus, which was composed of one species, namely L. lactis. The second cluster belonged to Lactobacillus and Pediococcus genera. Lactobacillus genus consisted of two species, i.e. L. plantarum ssp. plantarum and L. pentosus, while Pediococcus genus was identified as P. pentosaceus.The neighbor-joining method and p-distance parameter model were applied to construct the phylogenetic tree among the aligned 16S rRNA sequences of all LAB isolates and reference strains . In the Fermentation profile of LAB isolates was carried out based on the ability to ferment various carbohydrates using API 50 CHL Kit test . API 50 Dadih fermentation methods in the West Sumatra are generally classified as spontaneous. The product quality and safety are associated with the diversity and the population of microorganisms contained in the buffalo milk. Quality limitations from spontaneous fermentation caused a shift to back-slopping fermentation, which has been proven to accelerate the fermentation rate and increase safety, sensory quality, and product nutrition content -15. DadiThe results of the study  showed tLactococcus (seven isolates), Lactobacillus (four isolates), and Pediococcus (one isolate). Seven Lactococcus isolates were identified as L. lactis ssp. lactis and L. lactis ssp. cremoris; four Lactobacillus isolates were identified as L. pentosus and L. plantarum ssp. plantarum; and there was only one isolate identified as P. pentosaceus. All isolates had the same homology ≥97% with those in the reference sequence GenBank database at NCBI  [Lactobacillus spp. and Pediococcus spp. that are both acid tolerance. The existence of these species in the final product is predominantly caused by the presence of other species who grown first and provide a suitable environment for their growth [remoris) -34. Whenr growth . During r growth ,37.Lactococcus and Leuconostoc genera grown at the beginning and in the middle of fermentation (0-8 h). Isoelectric pH (4.6) will be reached by L. lactis after 8 h of fermentation, while Leuconostoc spp. need more time, approximately 14 h to reach the same pH value [th h fermentation. Acid tolerance is the property that enables that Lactobacilli to survive in the final product [This succession phenomenon is a process that takes place synergistically during dadih fermentation. This same phenomenon also occurs in Kivuguto, a traditional fermented milk from East Africa. LAB succession in African fermented milk mostly initiates when pH value . The acc product .Lactobacillus and Pediococcus genera were stand in same cluster, distinct from Lactococcus genus. Genus Pediococcus is an integral, not peripheral, part of the genus Lactobacillus [Pediococcus was reported to fall within Lb. casei branch of LAB [According to 16S rRNA gene sequence, a phylogenetic tree was constructed to describe a genetic relationship between isolates. The result  showed tbacillus  since thh of LAB . The pheThe diversity of LAB in the samples manufactured by the two examined methods was influenced by many factors. More extensive in-depth research is necessary to evaluate the contributing factor that affects the diversity of LAB in dadih. The different methods, recipes, raw material, environmental temperature, and location difference, may also cause some variation in strain . FurtherL. pentosus and P. pentosaceus). Understanding the role of complex, bacterial population and diversity could be useful to adjust the fermentation methods from spontaneous to back-slopping in dadih manufacturing.The different methods in dadih fermentation significantly affected LAB diversity. The back-slopping fermentation method provided greater diversity of LAB than spontaneous fermentation in dadih (two different species had been isolated, namely MBS supervised the research. CUW and EAS carried out the analyses. MBS, DWL, IW, and EAS supervised laboratory work. CUW wrote the manuscript with the help of MBS, DWL, and IW. All authors read and approved the final manuscript.


Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common and essential serotype that causes salmonellosis in Guizhou province. This study aimed to investigate the antimicrobial resistance (AMR) and molecular genotyping of 79 S. Enteritidis clinical isolates from 2011 to 2016 in Guizhou, China. Antimicrobial resistance and minimum inhibitory concentrations (MICs) of S. Enteritidis clinical isolates were detected by micro broth dilution method against ten classes 16 antimicrobial agents, and molecular genotyping were examined by pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA). All (100%) isolates showed resistance to at least one antimicrobial. Resistance to nalidixic acid (98.7%) was the highest, followed by sulfamethoxazole (87.3%) and ampicillin (77.2%). The majority of isolates (92.4%) showed decreased susceptibility to ciprofloxacin. Resistance to the third and fourth-generation cephalosporins was observed. Twenty-six AMR profiles were observed, and the predominant AMR profile was ampicillin-streptomycin-sulfamethoxazole-amoxicillin/clavulanic acid-nalidixic acid. A high burden of multidrug resistance (MDR) (81.0%) was found. Seventy-nine S. Enteritidis isolates were divided into 33 different pulsotypes (PTs), and the most frequent PT was PT18. Twenty-six different MLVA types (MTs) were generated with seven VNTR loci analysis of these isolates. The dominant PTs and MTs were persistent during 2011–2016. S. Enteritidis clinical isolates showed higher genetic diversity using PFGE combined with MLVA grouped into 60 PT-MT genotypes. No correlation was observed between genotypes, AMR profiles and geographic location. These data revealed the characteristics of AMR and molecular genotyping of S. Enteritidis clinical isolates in Guizhou province. These results highlight that strengthening the AMR and molecular genotyping surveillance is essential to prevent and control salmonellosis in Guizhou. PFGE combined with MLVA should be powerful tools for the molecular genotyping of S. Enteritidis isolates. Salmonella (NTS) is one of the most common pathogens that contaminate food and causes diarrhea in humans. 93.8 million cases of gastroenteritis due to Salmonella species occur each year globally, with 155,000 deaths [Salmonella each year [Salmonella infection sometimes occurred [Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common serotype among human isolates globally [S. Enteritidis was the most common serotype [Nontyphoidal ach year , and outglobally . A recenglobally , and of serotype .Salmonella strains and often used for surveillance and outbreak investigations [Salmonella, it is time-consuming, labor-intensive and offers low throughput [S. Enteritidis [S. Enteritidis strains and provides crucial epidemiological information [Molecular genotypes can be helpful to active outbreak investigation and surveillance of pathogen, especially same serotype isolates. PFGE is a standard and international molecular subtyping method to discern igations . Althougroughput . The diseritidis . MLVA iseritidis , 11. Theormation .S. Enteritidis was the most common serotype [S. Enteritidis had been reported in Guizhou [Guizhou is a multinational and mountainous area in the southwest of China with 34.75 million permanent residents, which is known as a “natural encyclopedia” of the karst landform. Recent years, rich natural, cultural and environmental resources have been to boost tourism in Guizhou, which may permit the dissemination of diarrhea pathogens and AMR. Our previous surveillance on bacterial pathogens of diarrheal patients in Guizhou province showed that NTS was the primary pathogen caused infectious diarrhea and serotype –15. Rece Guizhou –18. Wide Guizhou –21.S. Enteritidis clinical isolates from Guizhou have lacked so far. This study aimed to perform AMR, PFGE and MLVA of S. Enteritidis clinical isolates from Guizhou for better understanding the AMR and molecular epidemiologic characteristics and revealing associations between AMR profiles, genotypes and geographic location.However, data on AMR and molecular genotyping of The present study was reviewed and approved by the Ethics Review Committee of Guizhou Provincial Center for Disease Control and Prevention. All data/isolates were analyzed anonymously.S. Enteritidis strains were isolated from eight cities . All isolates were stored in 20% glycerol in -80°C, which were revived by inoculation into Luria-Burtani (LB) agar plates. These isolates were confirmed as S. Enteritidis with API20E identification kits  and serotyped by slide agglutination with commercial Salmonella poly and monovalent O and H antisera .Bacterial isolates used in this study were derived from clinical patients (stool and blood) from 2011 to 2016 in Guizhou province, Southwest of China. All 79 S. Enteritidis isolates were determined using the micro broth dilution method according to the manufacturer’s instructions . It contained 16 animicrobial agents including ampicillin (AMP), amoxicillin/clavulanic acid (AMC), ceftriaxone (CRO), cefepime (FEP), cefoxitin (FOX), imipenem (IPM), gentamicin (GEN), streptomycin (STR), nalidixic acid (NAL), ciprofloxacin (CIP), sulfamethoxazole (SOX), trimethoprim /sulfamethoxazole (SXT), azithromycin (AZM), chloramphenicol (CHL), doxycycline (DOX) and tetracycline (TET) that are in ten classes of drugs . Briefly, freshly culture bacteria were suspended in saline and adjusted to a 0.5 Mcfarland turbidity standard concentration. The 60 μl bacterial suspension was absorbed into a disposable aseptic groove with a pipettor, then 12 ml nutrient broth was added into the groove and mixed intensively. 100 μl of the mixture was added into each hole of 96 holes microporous plate except a negative hole. The 100 μl nutrient broth was added into the negative hole. The microporous plates were incubated at 35°C for 18h. Isolates were classified as susceptible, intermediate or resistant according to the Clinical Laboratory Standards Institute guidelines  except streptomycin. The interpretive standards of streptomycin used were the National Antimicrobial Resistance Monitoring System for enteric bacteria (NARMS) established breakpoints for Salmonella isolates (https://www.cdc.gov/narms/antibiotics-tested.html). The MIC of streptomycin used susceptible ≤16 μg/mL, no MIC range of intermediate susceptibility exists, and resistant ≥32 μg/mL. Decreased susceptibility to ciprofloxacin (MIC ≥0.12 μg/mL) includes isolates with MICs categorized as intermediate or resistant. Escherichia coli ATCC25922 was used as a control strain. Multidrug-resistant (MDR) was identified as resistance to three or more classes of drugs.The MICs for 79 Salmonella serotypes (https://www.cdc.gov/pulsenet/pathogens/pfge.html) with CHEF DRIII . All isolates, including the reference strain-Salmonella serovar Braenderup H9812 in this study, were digested with XbaI enzyme . The cluster analysis of PFGE was performed using BioNumerics software , and the Dice coefficient was determined using the unweighted pair group method with arithmetic averages (UPGMA). Band comparison was performed using dice coefficient with 1.50% optimization and 1.50% position tolerance. If the PFGE patterns of isolates have the same numbers of bands and the same apparent size, isolates are designated genetically indistinguishable[PFGE was performed according to the standard operating procedure for pulsenet PFGE of guishable.S. Enteritidis was accessible on the website (https://www.cdc.gov/pulsenet/pathogens/mlva.html). The PCR primers of seven VNTR loci  were synthesized according to the procedure by Biotechnology Corporation . Capillary electrophoresis was performed for PCR products on an Applied Biosystems Genetic Analyzer 3730xl. The data were clustered with the categorical coefficient and generated a minimum spanning tree (MST) with BioNumerics software basing on seven VNTR loci to know the genetic relationship, which followed the laboratory standard operating procedure.Laboratory standard operating procedure for pulsenet MLVA of S. Enteritidis isolates exhibited resistance to at least one class antimicrobial in Guizhou , followed by the resistance to sulfamethoxazole , ampicillin , streptomycin  and amoxicillin/ clavulanic acid   and cefepime . None of these isolates was resistant to cefoxitin. Only 3.8% (3/79) of S. Enteritidis isolates were resistant to ciprofloxacin. However, 92.4% (73/79) of isolates were shown to be decreased susceptibility to ciprofloxacin (MIC ≥0.12 μg/mL)  . Further2 μg/mL) . AdditioS. Enteritidis isolates were observed. 81.0% (64/79) of S. Enteritidis isolates were resistant to three or more classes of antimicrobial agents. Resistance to five classes antibiotics was the most frequent, accounting for 38.0% (30/79) of S. Enteritidis isolates, whereas exhibited MDR to at most eight classes antibiotics . Meanwhile, 44.3% (35/79) of S. Enteritidis isolates were resistant to at least the combination of traditional antimicrobials, including ampicillin, streptomycin, sulfamethoxazole and amoxicillin/ clavulanic acid (S. Enteritidis isolates were shown resistance to ampicillin-chloramphenicol-streptomycin-sulfamethoxazole-tetracycline (ACSSuT).In all isolates, high-level MDR ibiotics . Furthernic acid . Only 2.S. Enteritidis isolates were analyzed by PFGE using enzyme XbaI, which generated 33 different XbaI pulsotypes (PT1-PT33) with similarity indices ranged from 60.1% to 100% (S. Enteritidis isolates were grouped into two clusters (cluster A and cluster B) (The 79  to 100% . Among tuster B) . The majuster B) .S. Enteritidis isolates further. Among these isolates, 26 different MLVA types (MTs) were discriminated  were performed for the characteristics of 79 iminated . Nine MTiminated . MT5 incenerated . A majorngletons .S. Enteritidis isolates were also determined using PFGE combined with MLVA. If the isolates have the same PTs and the same MTs, isolates are designated genetically indistinguishable. The 79 S. Enteritidis clinical isolates were divided into 60 unique PT-MT genotypes  S. Enteritidis isolates were resistant to at least one antimicrobial agent, which was higher than those reported from the Midwestern United States (50%), Spanish (61.7%), Serbia (22%) and Shanghai (77.3%) [S. Enteritidis isolates in our study was observed (S. Enteritidis was reported in pork (88.46%) and chicken (94.1%) in Guizhou [S. Enteritidis infection. Therefore, controlling the application of antibiotics for clinical patients and agriculture was essential, since the antibiotics abuse may further accelerate the accumulation and spread of antimicrobial resistance. More importantly, the routine AMR surveillance of Salmonella isolates was crucial to early warn the spread of MDR [In the present study, the AMR and MICs of 79  (77.3%) –26. Furtobserved , which w Guizhou , 29. Mead of MDR –32.S. Enteritidis clinical isolates from Western China (20%), Thailand (51.1%) and Iran (90.9%) [S. Enteritidis infection may lead to treatment failure. Meanwhile, in our study, almost all isolates (except one isolate) were observed resistance to nalidixic acid  –36. Howe (90.9%) –39. More (90.9%) . Due to xic acid , which wS. Enteritidis isolates exhibited resistance to the third-cephalosporin (ceftriaxone), of which five isolates showed co-resistance to the fourth-generation cephalosporin (cefepime) and decreased susceptibility to ciprofloxacin  to prevent and control S. Enteritidis infection in our future studies, which would provide multiple genetic data, including revealing genetic relatedness with isolates from other counties or continents, helping with source trace-back investigations and predicting antimicrobial resistance.Although the genotyping of PFGE and MLVA can provide important molecular epidemiological information for S. Enteritidis clinical isolates from 2011 to 2016 in Guizhou, China. High-level resistance to antimicrobials was observed. All isolates were resistant to at least one antimicrobial. A high burden of MDR was found. Co-resistance to the third and fourth-generation cephalosporins and a high proportion of decreased susceptibility to ciprofloxacin exhibited. It is essential to control the abuse of antimicrobial agents and strengthen the surveillance of AMR. PFGE analysis with XbaI enzyme digestion divided the 79 S. Enteritidis isolates into 33 different PTs and MLVA analysis of these isolates based on seven VNTR loci obtained 26 distinct MTs.This study investigated the AMR, PFGE and MLVA molecular genotyping of 79 S. Enteritidis isolates revealed higher diversity than single PFGE or MLVA analysis. No correlation was observed between these genotypes, AMR profiles and geographic location. The genotyping of S. Enteritidis using PFGE combined with MLVA can provide crucial epidemiological information. To our knowledge, this is the first study that we reported the AMR and the molecular genotyping of S. Enteritidis clinical isolates from Guizhou province of Southwestern China. These results might be useful to provide a scientific basis for control and prevention of salmonellosis in Guizhou province.Analysis of PFGE combined with MLVA for 79 S1 Table(XLSX)Click here for additional data file.


Odorrana is widely distributed in the mountains of East and Southeastern Asia. An increasing number of new species in the genus have been recognized especially in the last decade. Phylogenetic studies of the O. schmackeri species complex with wide distributional range also revealed several cryptic species. Here, we describe a new species in the species complex from Guizhou Province of China. Phylogenetic analyses based on mitochondrial DNA indicated the new species as a monophyly clustered into the Odorrana clade and sister to O. schmackeri, and nuclear DNA also indicated it as an independent lineage separated from its related species. Morphologically, the new species can be distinguished from its congeners based on a combination of the following characters: (1) having smaller body size in males (snout-vent length (SVL) <43.3 mm); (2) head longer than wide; (3) dorsolateral folds absent; (4) tympanum of males large and distinct, tympanum diameter twice as long as width of distal phalanx of finger III; (5) two metacarpal tubercles; (6) relative finger lengths: II < I < IV < III; (7) tibiotarsal articulation reaching to the level between eye to nostril when leg stretched forward; (8) disks on digits with circum-marginal grooves; (9) toes fully webbed to disks; (10) the first subarticular tubercle on fingers weak; (11) having white pectoral spinules, paired subgular vocal sacs located at corners of throat, light yellow nuptial pad on the first finger in males.The genus  OdorranaFrogs of the genus O. schmackeri was firstly found in Gaojiayan Town, Changyang County (Co.), Hubei province (Prov.), China, and named by O. schmackeri based on some specimens from Daba Mountains and Xiushan Co. in Chongqing City of China near to the type locality of the species, and presented the diagnosis characters for the species, such as having great circle brown spots on dorsa and the outer vocal sac below pharynx in males. Afterward, based on mass of reports and investigations, the species has been suggested to be distributed in Henan, Shaanxi, Gansu, Sichuan, Guizhou, Hubei, Anhui, Jiangsu, Zhejiang, Hunan, Guangdong and Guangxi provinces of China  from Wuyi Mountains in Fujian Prov., Nanling Mountains in the border of Guangdong and Hunan provinces and mountains in eastern Guizhou Prov.; Clade B was O. tianmuii occurring from Huangshan Mountains in Anhui Prov. and Tianmu Mountains in Zhejiang Prov.; Clade C was O. schmackeri sensu stricto occupying a more narrow distribution area in Funiu Mountains in Henan and northern Hunan provinces and Daba Mountains in Hubei Prov.; Clade D was proposed as a cryptic species from mountains in Jiangxi Prov.; Clade E was the second cryptic species from mountains of northwestern Guizhou Prov.; Clade F was the third cryptic species from Funiu Mountains in Henan Prov. and being sympatric with Clade C; and Clade G was the fourth cryptic species from Daba Mountains in Hubei Prov. and also being sympatric with O. schmackeri sensu stricto  had a large range across central Guizhou and Guangxi provinces; and another cryptic species (Odorrana sp2 defined by them) occupied a large range around the southern part of Sichuan Basin  whether Clade G from Daba Mountains in Odorrana sp2 in Recently, many studies have paid attention to the phylogenetic relationships and diversification of the  complex . Li et a stricto . Also, Zan Basin . HoweverOdorrana superficially resembling O. schmackeri. As noted, some of them were collected from the population in the Lengshuihe Reserve in Jinsha Co., Guizhou Prov., China .O. schmackeri specimens from two localities, 23 O. huanggangensis specimens from three localities, three O. yizhangensis specimens from two localities and two O. lungshengensis specimens from one locality . Stages of tadpoles were identified following O. schmackeri including two topotypes, three of O. huanggangensis, three of O. yizhangensis including one topotype, and two of O. lungshengensis  and Bayesian Inference (BI) methods, implemented in PhyML v. 3.0  and MrBa studies . To avoi studies , for the studies . In thisO. schmackeri species complex recognized by To further visualize the degree of genetic splits among the new taxon and its related species especially members of the In addition, to access the genetic divergence between the new taxon and its related species on nuclear DNA, haplotype networks for DOLK and KCNF gene datasets were constructed, respectively, using the maximum parsimony method in TCS v. 1.21 .p-distance on the 16S rRNA gene were estimated using MEGA v. 6.06  was used to test the significance of differences on morphometric characters between males and females and between different species. The significance level was set at 0.05. To show the spatial distribution of different species on the morphometric characters, principal component analyses (PCA) were performed. These analyses were carried out in the R .Odorrana species on morphology. Comparative morphological data were obtained from literatures for O. absita  and three female specimens  of the new taxon were scanned in the high-resolution X-ray scanner . The specimens were scanned along the coronal axis at an image resolution of 2,000 × 2,000. Segmentation and three-dimensional reconstruction of the CT images were made using VG57 Studio Max 2.2 .The Animal Care and Use Committee of Chengdu Institute of Biology, CAS provided full approval for this purely observational research (Number: CIB2013041102). Field work was approved by the Management Office of the Lengshuihe Nature Reserve (project number: LSH-201304003).http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:E98B65CB-E8E3-4412-9613-D9DD32A77B99. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central, and CLOCKSS.The electronic version of this article in portable document format will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix Odorrana and sister to the O. schmackeri clade. ND2 splitstree also strongly supported the splits between the new taxon and its related species . More significantly, it was higher or even much higher than the interspecific genetic distance between many sister species, for example, that between O. huanggangensis and O. tianmuii (0.014), O. hainanensis and O. fengkaiensis (0.013), O. nasuta and O. versabilis (0.013), O. versabilis and O. exiliversabilis (0.017), O. morafkai and O. banaorum (0.013), and O. grahami and O. jingdongensis , 14 specimens collected by S. Z. Li from the Lengshuihe Nature Reserve in Jinsha Co., Guizhou Prov., China. Six males: CIBjs20150803001, CIBjs20150803003, CIBjs20150803004, CIBjs20150803005 collected on August 3, 2015 and CIBjs20150804002 on August 4, 2015; eight females: CIBjs20150803006, CIBjs20150803007, CIBjs20150803008 collected on August 3, 2015, CIBjs20150804001, CIBjs20150804003, CIBjs20150804004, CIBjs20150804005 on August 4, 2015 and CIBjs20150808020 on August 8, 2015. Seven specimens collected by S. Z. Li and J. C. Lv from Meitan Co. of Guizhou Prov., China. Three males: CIBGYU20130917004, CIBGYU20130917005, and CIBGYU20130917007 collected by J. C. Lv on September 17, 2013; four females: CIBGYU20130917001, CIBGYU20130917002, and CIBGYU20130917003 by S. Z. Li on September 17, 2013, CIBGYU20130917006 by J. C. Lv on September 17, 2013. Three females: GYU20130921001, CIBGYU20130921002, and CIBGYU20130921003 collected by S. Z. Li on September 21, 2013. A total of 10 tadpoles (CIBjs20171014001–CIBjs20171014010) collected by S. Z. Li on October 10, 2017.Odorrana kweichowensis sp. nov. is assigned to genus Odorrana based upon molecular phylogenetic analyses and the following morphological characters: dorsum is green; tips of digits dilated, tapering, disks with circum-marginal grooves, and vertical diameter longer than horizontal diameter in the disks; supernumerary tubercle below the base of fingers III and IV; feet fully webbed to disks, without tarsal fold; the first finger thick and nuptial pad distinct.Odorrana kweichowensis sp. nov. could be distinguished from its congeners by a combination of the following characters: (1) having smaller body size in males (SVL <43.3 vs. SVL >48 mm in many other species); (2) head longer than wider; (3) dorsolateral folds absent; (4) tympanum of males large and distinct, tympanum diameter in males twice as long as width of distal phalanx of finger III; (5) two metacarpal tubercles; (6) relative finger lengths: II < I < IV < III; (7) tibiotarsal articulation reaching to the level between eye to nostril when leg stretched forward; (8) disks on digits with circum-marginal grooves; (9) toes fully webbed to disks; (10) the first subarticular tubercle on fingers weak; (11) having white pectoral spinules, paired subgular vocal sacs located at corners of throat, light yellow nuptial pad on the first finger in males.Head longer than wide (HDL/HDW = 1.08); top of head flat; snout obtusely rounded in dorsal view (SL/HDL = 0.41), rounded in profile, projecting beyond lower jaw; eye large and convex, ED 0.83 times of SL; IND = 5.1 mm, larger than IOD = 3.2 mm; NED = 3.4 mm larger than NSD = 2.9 mm; tympanum circular, large and distinct, twice as long as width of distal phalanx of finger III (TYD/FDW = 2.84); vomerine teeth on well-developed ridges; tongue deeply notched posteriorly; paired gular pouches at corners of throat. Forelimbs moderately robust (LW/SVL = 0.11); lower arm and hand beyond a half of body length (LAL/SVL = 0.53); the first finger slightly longer than the second; finger tips on I–IV dilated to wide, tapering, disks with circum-marginal grooves; nuptial pad on the inner of first finger from base to subarticular tubercles; subarticular tubercles relatively prominent; inner metacarpal tubercle oblong and outer metacarpal tubercle indistinct; no finger webbing.Hindlimbs long; tibiotarsal articulation reaching to the level between eye to nostril when hindlimb adpressed along the side of body; heels overlapping when hindlimbs positioned at right angle to body; singular longer than thigh length; FL 0.85 times SVL; toes slender, relative toe lengths: I < II < III < V < IV; toes tapering dilated, disks with circum-marginal grooves; feet fully webbed to disks; web becoming narrower and continuing to the disks as lateral fringe on toes II, III, and IV; outer metatarsal tubercle absent; inner metatarsal tubercle present.Skin shagreened, a number of pustules scattered on dorsum of trunk and flanks; several irregular tubercles scattered on flanks; dorsolateral folds absent; two large glands on the place between below tympanum and above arm; white spinules on throat, chest, fore abdomen, inner side of forearms and upper and lower lips.In life, dorsum with bright green network; large black spots in the center on dorsum, continuing onto dorsal portion of legs to form dark counter-point to bright green irregular bands; flank light yellow with several black spots; tympanum brown–black; upper and lower lip with vertical black bars; arms brown with black transverse bands, thighs with five brown bands and tibias with six .On dorsum color fades to dark olive with dark brown blotches , upper aBasic statistics for measurements were presented in The skull morphology of the five scanned specimens were almost identical, and thus, only one representative (voucher number: CIBjs20150803001) was presented  and descBody and tail yellowish-brown , at GosnSnout-vent length in females approximately 1.8 times that in males ; paired subgular vocal sacs located at corners of throat, pinkish lineae musculinae on dorsal side, nuptial pad light yellow on the inner of first finger from base to subarticular tubercles in males  and 6.Odorrana kweichowensis sp. nov. differs from O. amamiensis, O. andersonii, O. bacboensis, O. cangyuanensis, O. chapaensis, O. geminata, O. graham, O. hmongorum, O. ishikawae, O. jingdongensis, O. junlianensis, O. kuangwuensis, O. macrotympana, O. margaretae, O. mawphlangensis, O. mutschmanni, and O. wuchuanensis, by having smaller body size .Odorrana kweichowensis sp. nov. differs from O. absita, O. aureola, O. banaorum, O. bolavensis, O. exiliversabilis, O. gigatympana, O. graminea, O. hosii, O. heatwolei, O. indeprensa, O. khalam, O. leporipes, O. livida, O. monjerai, O. narina, O. nasica, O. nasuta, O. orba, O. supranarina, O. tormota, O. trankieni, O. utsunomiyaorum, O. versabilis, O. yentuensis, and O. zhaoi, by lacking dorsolateral folds (vs. present in other species).Odorrana kweichowensis sp. nov. differs from O. fengkaiensis by tibiotarsal articulation reaching the level between eye to nostril when leg stretched forward (vs. reaching the level below nostril in the latter); from O. rotodoar by tibiotarsal articulation reaching the level between eye to nostril when leg stretched forward (vs. reaching the level beyond eye in the latter); from O. lipuensis  and O. hainanensis  by paired external vocal sacs located at corners of throat; from O. chloronota by having head longer than wide (vs. head wider than long in the latter); from O. morafkai and O. sinica by having white pectoral spinules in mature males (vs. absence in the latter); from O. arunachalensis, by the relative finger lengths I < II < IV < III (vs. II < I < IV < III in the latter).O. schmackeri group , O. kweichowensis sp. nov. differs from O. anlungensis by having white pectoral spinules in mature males (vs. absence in the latter), tibiotarsal articulation reaching the level between eye to nostril when leg stretched forward (vs. reaching the nostril in the latter), web becoming narrower and continuing to the disks of toe IV (vs. web continuing to the subarticular tubercles of toe IV in the latter); from O. hejiangensis by the disks of fingers with circum-marginal grooves , two metacarpal tubercles ; from O. lungshengensis by the body size of males with maximum SVL 43.3 mm , tympanum large and distinct, and beyond twice as long as width of distal phalanx of finger III , two metacarpal tubercles , tibiotarsal articulation reaching the level between eye to nostril when leg stretched forward (vs. reaching the tip of snout in the latter); from O. nanjiangensis by the body size in males with maximum SVL 43.3 mm , two metacarpal tubercles , tibiotarsal articulation reaching the level between eye to nostril when leg stretched forward (vs. reaching the tip of snout in the latter); from O. swinhoana by the body size in males with maximum SVL 43.3 mm , tibiotarsal articulation reaching the level between eye to nostril when leg stretched forward (vs. reaching the tip of snout); from O. tianmuii by the relative finger lengths II < I < IV < III (vs. I < II < IV < III in the latter), two metacarpal tubercles ; O. tiannanensis by the body size in males with maximum SVL 43.3 mm , tibiotarsal articulation reaching the level between eye to nostril when leg stretched forward (vs. reaching the level beyond the tip of snout in the latter); O. yizhangensis by the body size in males with maximum SVL 43.3 mm , two metacarpal tubercles , tibiotarsal articulation reaching the level between eye to nostril when leg stretched forward (vs. reaching the tip of snout in the latter); from O. huanggangensis by the relative finger lengths II < I < IV < III (vs. I < II < IV < III in the latter), tibiotarsal articulation reaching the level between eye to nostril when leg stretched forward (vs. reaching the nostril in the latter); from O. schmackeri, by the first subarticular tubercle on fingers and supernumerary tubercles being weak  in Jinsha Co., Meitan Co.  and Zheng’an Co.  in Guizhou Prov. of China. Geographical distances between these localities were from 89 to 173 km. Population from the Lengshuihe Nature Reserve inhabited broad streams, and near the riparian areas, surrounded by evergreen broadleaved forests  recognized by O. hejiangensis in a narrow area along the boundary between northern Guizhou Prov. and Sichuan Prov. and Chongqing City, China  from Guizhou Prov. of China, and provide evidence for its phylogenetic allocations. O. kweichowensis sp. nov. was only known from a narrow range in the northwestern part of Guizhou Prov. of China, and occurred from mountain streams at mid and low elevations similar to most odorous frogs. In our fieldwork, the new species was found to be seriously threatened by local villagers and construction of dams and roads. Thus, further more detailed investigations on the species are urgent to ascertain its distributional range and population status. With our description, we contributed to a better knowledge of the diversity of the genus Odorrana in the southwestern China, and thus suggested that more comprehensive phylogeographic studies would highlight radiation patterns of the group.We described a new species of the odorous frog genus 10.7717/peerj.5695/supp-1Supplemental Information 1Unit: mm. Abbreviations for the morphometric characters refer to Methods section.Click here for additional data file.10.7717/peerj.5695/supp-2Supplemental Information 2“*” denoted the sequences downloaded from GenBank.Click here for additional data file.10.7717/peerj.5695/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.5695/supp-4Supplemental Information 4Unit: mm. Abbreviations for the morphometric characters refer to Methods section.Click here for additional data file.10.7717/peerj.5695/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.5695/supp-6Supplemental Information 6Unit: mm. Abbreviations for the morphometric characters refer to Methods section.Click here for additional data file.10.7717/peerj.5695/supp-7Supplemental Information 7Voucher information for each sequence refer to Click here for additional data file.