Nitric Oxide Ameliorates the Effects of Hypoxia in Mice by Regulating Oxygen Transport by Hemoglobin.

Xiaoying Zhou, Wenting Su, Quanwei Bao, Yu Cui, Xiaoxu Li, Yidong Yang, Chengzhong Yang, Chengyuan Wang, Li Jiao, Dewei Chen, and Jian Huang. Nitric oxide ameliorates the effects of hypoxia in mice by regulating oxygen transport by hemoglobin. High Alt Med Biol. 00:00-00, 2024.-Hypoxia is a common pathological and physiological phenomenon in ischemia, cancer, and strenuous exercise. Nitric oxide (NO) acts as an endothelium-derived relaxing factor in hypoxic vasodilation and serves as an allosteric regulator of hemoglobin (Hb). However, the ultimate effects of NO on the hematological system in vivo remain unknown, especially in extreme environmental hypoxia. Whether NO regulation of the structure of Hb improves oxygen transport remains unclear. Hence, we examined whether NO altered the oxygen affinity of Hb (Hb-O2 affinity) to protect extremely hypoxic mice. Mice were exposed to severe hypoxia with various concentrations of NO, and the survival time, exercise capacity, and other physical indexes were recorded. The survival time was prolonged in the 5 ppm NO (6.09 ± 1.29 minutes) and 10 ppm NO (6.39 ± 1.58 minutes) groups compared with the 0 ppm group (4.98 ± 1.23 minutes). Hypoxia of the brain was relieved, and the exercise exhaustion time was prolonged when mice inhaled 20 ppm NO (24.70 ± 6.87 minutes vs. 20.23 ± 6.51 minutes). In addition, the differences in arterial oxygen saturation (SO2%) (49.64 ± 7.29% vs. 42.90 ± 4.30%) and arteriovenous SO2% difference (25.14 ± 8.95% vs. 18.10 ± 6.90%) obviously increased. In ex vivo experiments, the oxygen equilibrium curve (OEC) left shifted as P50 decreased from 43.77 ± 2.49 mmHg (0 ppm NO) to 40.97 ± 1.40 mmHg (100 ppm NO) and 38.36 ± 2.78 mmHg (200 ppm NO). Furthermore, the Bohr effect of Hb was enhanced by the introduction of 200 ppm NO (-0.72 ± 0.062 vs.-0.65 ± 0.051), possibly allowing Hb to more easily offload oxygen in tissue at lower pH. The crystal structure reveals a greater distance between Asp94ß-His146ß in nitrosyl -Hb(NO-Hb), NO-HbßCSO93, and S-NitrosoHb(SNO-Hb) compared to tense Hb(T-Hb, 3.7 Å, 4.3 Å, and 5.8 Å respectively, versus 3.5 Å for T-Hb). Moreover, hydrogen bonds were less likely to form, representing a key limitation of relaxed Hb (R-Hb). Upon NO interaction with Hb, hydrogen bonds and salt bridges were less favored, facilitating relaxation. We speculated that NO ameliorated the effects of hypoxia in mice by promoting erythrocyte oxygen loading in the lung and offloading in tissues.

Hypoxia-Induced Myocardial Hypertrophy Companies with Apoptosis Enhancement and p38-MAPK Pathway Activation.

Li, Xiaoxu, Zhijun Pu, Gang Xu, Yidong Yang, Yu Cui, Xiaoying Zhou, Chenyuan Wang, Zhifeng Zhong, Simin Zhou, Jun Yin, Fabo Shan, Chengzhong Yang, Li Jiao, Dewei Chen, and Jian Huang. Hypoxia-induced myocardial hypertrophy companies with apoptosis enhancement and p38-MAPK pathway activation. High Alt Med Biol. 00:00-00, 2024. Background: Right ventricular function and remodeling are closely associated with symptom severity and patient survival in hypoxic pulmonary hypertension. However, the detailed molecular mechanisms underlying hypoxia-induced myocardial hypertrophy remain unclear. Methods: In Sprague-Dawley rats, hemodynamics were assessed under both normoxia and hypobaric hypoxia at intervals of 7 (H7), 14 (H14), and 28 (H28) days. Morphological changes in myocardial tissue were examined using hematoxylin and eosin (HE) staining, while myocardial hypertrophy was evaluated with wheat germ agglutinin (WGA) staining. Apoptosis was determined through TUNEL assays. To further understand the mechanism of myocardial hypertrophy, RNA sequencing was conducted, with findings validated via Western blot analysis. Results: The study demonstrated increased hypoxic pulmonary hypertension and improved right ventricular diastolic and systolic function in the rat models. Significant elevations in pulmonary arterial systolic pressure (PASP), mean pulmonary arterial pressure (mPAP), right ventricular mean pressure (RVMP), and the absolute value of +dp/dtmax were observed in the H14 and H28 groups compared with controls. In addition, right ventricular systolic pressure (RVSP), -dp/dtmax, and the mean dp/dt during isovolumetric relaxation period were notably higher in the H28 group. Heart rate increased in the H14 group, whereas the time constant of right ventricular isovolumic relaxation (tau) was reduced in both H14 and H28 groups. Both the right heart hypertrophy index and the heart weight/body weight ratio (HW/BW) were elevated in the H14 and H28 groups. Myocardial cell cross-sectional area also increased, as shown by HE and WGA staining. Western blot results revealed upregulated HIF-1α levels and enhanced HIF-2α expression in the H7 group. In addition, phosphorylation of p38 and c-fos was augmented in the H28 group. The H28 group showed elevated levels of Cytochrome C (Cyto C), whereas the H14 and H28 groups exhibited increased levels of Cleaved Caspase-3 and the Bax/Bcl-2 ratio. TUNEL analysis revealed a rise in apoptosis with the extension of hypoxia duration in the right ventricle. Conclusions: The study established a link between apoptosis and p38-MAPK pathway activation in hypoxia-induced myocardial hypertrophy, suggesting their significant roles in this pathological process.

Morphological and genetic analysis of Ceratomyxa saurida Zhao et al. 2015 and Ceratomyxa mai sp. nov. (Myxozoa: Ceratomyxidae) from the East China Sea.

We describe Ceratomyxa saurida Zhao et al. 2015 and Ceratomyxa mai sp. nov. (Myxozoa: Ceratomyxidae) from the East China Sea. C. saurida was found in the gallbladders of 3/13 specimens of its type host, Saurida elongata Temminck and Schlegel 1846 (Aulopiformes). Myxospore characters were consistent with the original description to which we have added small subunit (SSU) rRNA gene data. C. mai sp. nov. was found in gallbladders of 3/13 specimens of S. elongata and 5/13 specimens of Neobythites sivicola Jordan and Snyder 1901 (Ophidiiformes). Mature myxospores of C. mai sp. nov. were crescentic in sutural view, with a deeply concave posterior angle 142.2±8.2° (125.8‒158.2°) and an arched anterior side. Shell valves were smooth and equal, 20.9±1.9 (17.3‒24.7) µm thick and 9.2±0.5 (8.1‒9.9) µm long, and joined at a straight, thin sutural plane passing between two nematocysts (polar capsules). The nematocysts were equal-sized, pyriform, 2.6±0.2 (2.4‒2.9) µm long and 2.7±0.2 (2.4‒3.3) µm wide, with their tapered ends pointed toward each other, located in the anterior third of the spore. Sequences of the SSU rRNA gene and internal transcribed spacer 1 showed that the isolates of C. mai sp. nov. obtained from S. elongata and N. sivicola were identical. The SSU rRNA gene sequence of C. mai sp. nov. was distinct from all known myxosporeans and clustered with C. saurida, and then with Ceratomyxa filamentosi Kalatzis, Kokkari and Katharios 2013, both of which also infect Aulopiformes fishes.

The complete mitogenome of the Mugil cephalus (Mugiliformes: Mugilidae) from the Yellow Sea, China.

Mugil cephalus Linnaeus, 1758 is a teleost fish widely distributed in coastal waters that plays an important role in commercial fisheries. In the present study, the complete mitogenome of M. cephalus from the Yellow Sea, China, was sequenced using Illumina Novaseq sequencing. The mitogenome of the M. cephalus was 16,744 bases in length (GenBank accession No. ON262567) including 2 rRNA genes, 22 tRNA genes, 13 protein-coding genes and a D-loop control region. The overall base composition of the genome was 28.2% A, 29.5% C, 26.9% T, and 15.4% G. The analysis of genetic similarity and phylogenetic relationship of M. cephalus from different geographic regions of the world indicated that the species from the Yellow Sea was most similar to NWP1 which is one of the three cryptic species of M. cephalus in Northwestern Pacific.

The complete mitochondrial genome of the Riparia riparia (Passeriformes: Hirundinidae).

The Sand Martin (Riparia riparia) belongs to Hirundinidae. In this study, the complete mitochondrial genome of R. riparia was sequenced and characterized. The genome was 17,963 bases in length (GenBank accession no. OK537984) including 13 protein-coding genes, two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and two control regions. The overall base composition of R. riparia mitogenome was 30.5% for A, 31.8% for C, 14.5% for G, and 23.2% for T. Phylogenetic analysis revealed that R. riparia was genetically closest to the species of genus Tachycineta. R. riparia mitogenome could contribute to our understanding of the phylogeny and evolution of this species.

Description of Myxidium pseudocuneiforme n. sp. (Myxosporea: Myxidiidae) from Cyprinus carpio in China, with the resolution on a taxonomic dilemma of Myxidium cuneiforme.

Investigations on myxozoan parasites of fish from Chongqing in China, revealed two Myxidium cuneiforme-like myxosporeans infecting the gallbladder of Cyprinus carpio carpio and Carassius auratus. We researched their myxospore morphology, and analyzed their genetic similarity and phylogenic relationships to other myxozoans based on small subunit ribosomal DNA (18S rDNA) sequences. Although both parasites recovered were morphologically similar, the myxosporean isolated from C. auratus was consistent in morphology to Myxidium cuneiforme, which was described from this host species. The parasite isolated from C. c. carpio had overlapping myxospore dimensions to M. cuneiforme, but on average, the polar capsules were not as long. More importantly, this parasite was genetically distinct from M. cuneiforme with 96.3% and 96.5% similarity in two sequences of 18S rDNA, and we propose the name Myxidium pseudocuneiforme n. sp. for this myxozoan from common carp. Its mature myxospores are ellipsoidal and asymmetric with pointed ends in valvular view, arc-shaped or fusiform in sutural view. The pyriform polar capsules are equal in size, and polar filament with 5-6 coils. This study highlights that molecular characteristics and host specificity are indispensable for myxozoan species identification when presented with the taxonomic dilemma of whether we are observing one species that exhibits slight morphological differences or multiple, but similar, species in different hosts.

Genome-Wide Association Study of Meat Quality Traits in a Three-Way Crossbred Commercial Pig Population.

Meat quality is an important trait for pig-breeding programs aiming to meet consumers' demands. Geneticists must improve meat quality based on their understanding of the underlying genetic mechanisms. Previous studies showed that most meat-quality indicators were low-to-moderate heritability traits; therefore, improving meat quality using conventional techniques remains a challenge. Here, we performed a genome-wide association study of meat-quality traits using the GeneSeek Porcine SNP50K BeadChip in 582 crossbred Duroc × (Landrace × Yorkshire) commercial pigs (249 males and 333 females). Meat conductivity, marbling score, moisture, meat color, pH, and intramuscular fat (IMF) content were investigated. The genome-wide association study was performed using both fixed and random model Circulating Probability Unification (FarmCPU) and a mixed linear model (MLM) with the rMVP software. The genomic heritability of the studied traits ranged from 0.13 ± 0.07 to 0.55 ± 0.08 for conductivity and meat color, respectively. Thirty-two single-nucleotide polymorphisms (SNPs) were identified for meat quality in the crossbred pigs using both FarmCPU and MLM. Among the detected SNPs, five, nine, seven, four, six, and five were significantly associated with conductivity, IMF, marbling score, meat color, moisture, and pH, respectively. Several candidate genes for meat quality were identified in the detected genomic regions. These findings will contribute to the ongoing improvement of meat quality, meeting consumer demands and improving the economic outlook for the swine industry.

Myxobolus jialingensis n. sp. (Myxozoa: Myxobolidae) infecting urinary bladder and hepatopancreas of yellowhead catfish Tachysurus fulvidraco from China.

In the present study, we described a novel myxosporean species, Myxobolus jialingensis n. sp. (Myxozoa: Myxobolidae), which infected the urinary bladder and hepatopancreas of yellowhead catfish Tachysurus fulvidraco in China. The mature spores of M. jialingensis n. sp. were pyriform with the length of 15.8 ± 0.7 (15.4-17.0) µm and width of 8.0 ± 0.3 (7.8-8.9) µm. Two pyriform polar capsules were slightly unequal in size: the larger polar capsule was 7.4 ± 0.3 (6.7-8.0) µm in length and 3.1 ± 0.2 (2.8-3.6) µm in width; and the smaller polar capsule measured 7.3 ± 0.3 (6.6-8.1) µm in length and 3.3 ± 0.2 (2.9-3.6) µm in width. The polar capsules were directed toward the apex of the spore, packing seven to eight spirals of the polar filaments. The small subunit ribosomal RNA gene (18S rDNA) sequence of M. jialingensis n. sp. was unique among all myxozoans, and the highest similarity was 96.1% with M. voremkhai. Phylogenetic analysis based on 18S rDNA sequences revealed that myxosporeans infecting the close host affinity (belonging to the same order) had close phylogenetic relationship and, some myxosporeans infecting the same host order might have multiple origins.

The first report of two Sphaeromyxa species (Myxozoa: Bivalvulida) from the South China Sea: Sphaeromyxa scorpaena n. sp. from long-fingered scorpionfish (Scorpaenodes albaiensis) and Sphaeromyxa theraponi from tiger perch (Terapon jarbua).

Two myxosporean species of the genus Sphaeromyxa were isolated from the gallbladders of marine fish in the South China Sea. Sphaeromyxa scorpaena n. sp. was collected from Scorpaenodes albaiensis Evermann and Seale, 1907. The mature myxospores were arcuate-shaped with tapered to pointed ends, and a length of 14.1 ± 0.7 (13.8-15.1) µm and a width of 5.2 ± 0.3 (4.9-5.8) µm. The polar capsules (PCs) were pyriform with a length of 3.2 ± 0.2 (3.1-3.5) µm and a width of 1.6 ± 0.1 (1.4-1.8) µm, and containing ribbon-like polar filaments irregularly folded 1.5-2.5 turns. Molecular characteristics and phylogenetic analysis based on 18S rDNA as well as morphological comparison confirmed that S. scorpaena n. sp. was a previously undescribed species. Sphaeromyxa theraponi, isolated from Terapon jarbua Forsskål, 1775, was reported for the first time from the South China Sea. The mature myxospores were slightly arched, tapering to bluntly rounded ends, with a length of 17.3 ± 0.9 (15.5-19.4) µm and a width of 4.8 ± 0.3 (4.1-5.3) µm. A sporoplasm was situated in the space between PCs in the myxospore. The PCs were pyriform, which contained ribbon-like polar filaments irregularly folded by 2-3 turns, with a length of 7.0 ± 0.5 (5.8-8.1) µm and a width of 2.6 ± 0.2 (2.2-3.0) µm. Our morphological and phylogenetic analyses suggest that the pointed ends of S. scorpaena n. sp. might be a secondarily acquired characteristic rather than an ancestral trait.

Taxonomy on three novel species of Sphaeromyxa Thélohan 1892 (Myxozoa, Bivalvulida, Sphaeromyxidae) with insight into the evolution of the genus.

Three new myxosporeans of the genus Sphaeromyxa Thélohan 1892 were discovered from the coastal waters off Xiamen in the East China Sea and characterized based on morphological and SSU rDNA data. Sphaeromyxa photopectoralis sp. n. was described from Photopectoralis bindus, and Sphaeromyxa sebastisca sp. n. was described infecting both Sebastiscus marmoratus (type-host) and Scorpaenopsis cirrosa. These two species are morphologically consistent with the "balbianii" group, possessing straight myxospores and truncated ends, but are distinct from one another genetically and by myxospore dimensions. A third myxosporean infecting Siganus fuscescens was described as Sphaeromyxa xiamenensis sp. n., and this species is morphologically consistent with the "incurvata" group, bearing arcuate myxospores with rounded ends. The molecular phylogeny and estimated rRNA secondary structure suggest that marine sphaeromyxids are probably derived from freshwater myxidiids, and "incurvata" and "balbianii" groups might each represent independent evolutionary lineages. The present study also shows that S. limocapitis phylogenetically nested in "incurvata" group.

Characterization of Myxidium spinibarba sp. nov. (Cnidaria, Myxosporea, Myxidiidae) from Spinibarbus sinensis (Bleeker, 1871) in Chongqing China.

In the present study, we described a new species of Myxidium Bütschli, 1882, obtained from the gallbladder of Spinibarbus sinensis (Bleeker, 1871) from the Jialing River in Chongqing, China. Myxidium spinibarba sp. nov. was identified based on morphological and SSU rDNA sequence data. The mature myxospores were fusiform in valvular view and ovoid in sutural view, with somewhat protrusive poles and mean dimensions (all in µm) of 11.8 ± 0.5 (10.6-12.4) in length and 6.1 ± 0.5 (5.5-7.2) in width. The polar capsules were pyriform and equal in size with mean dimensions of 3.6 ± 0.4 (3.0-4.4) in length and 3.0 ± 0.2 (2.7-3.2) in width. The new species was distinct from related species of Myxidium in its morphology and molecular characteristics. Phylogenetic analysis indicated the clustering of species based on the presence or absence of valvular striations. Moreover, myxospore morphology, rather than the host environment, played an important role in the partial phylogenetic clustering.

Morphological and molecular identification of Myxobolus parakoi sp. nov (Myxozoa: Myxobolidae), from Cyprinus carpio in Chongqing China.

During an ongoing investigation into the myxosporean diversity of common carp in Chongqing, China, Myxobolus parakoi sp. nov. was found to infect the gill lamellae of Cyprinus carpio (Linnaeus, 1758). Both morphological and SSU rDNA data revealed that M. parakoi sp. nov. was distinct from other myxosporeans. Mature myxospores of M. parakoi sp. nov. were pyriform in the frontal view. The spores were 15.98 ± 0.78 (14.59-17.72) µm in length and 7.84 ± 0.78 (6.66-9.75) µm in width. The two polar capsules were pyriform and equal in size, exhibiting 8.72 ± 0.50 (7.76-9.92) µm in length and 3.03 ± 0.23 (2.63-3.56) µm in width. Polar filaments within the polar capsules were coiled with 11 or 12 turns. Phylogenetic analysis revealed that M. parakoi sp. nov. and Myxobolus koi (FJ710800) were clustered together, forming a sister subclade with Myxobolus tanakai. This study also implied that the morphology of the myxospores was generally correlated with their phylogenetic relationship, while such correlation was not strong or consistent.

Morphological and Molecular Identification of a Novel Species, Ceratomyxa siganicola n. sp. (Myxozoa: Ceratomyxidae) from Siganus fuscescens, in East China Sea.

INTRODUCTION: Ceratomyxa Thélohan, 1892 is one of the largest genera under Myxosporea Butschli, 1881, and has a worldwide distribution, but little attention has been paid to myxosporean parasites from the Chinese seawaters, East China Sea. METHODS: Morphology and molecular biology methods were combined for species identification and phylogenetic analysis. RESULTS: A new coelozoic myxosporean species, Ceratomyxa siganicola n. sp., was found to infect the gallbladder of Siganus fuscescens (Houttuyn, 1782) (Perciformes, Siganidae) from coastal waters of Xiamen, East China Sea, China. Mature myxospores of the novel species exhibited the morphologically typical features of the genus Ceratomyxa. They were slightly crescent shaped with rounded ends, measuring 5.6 ± 0.5 (4.8-6.5) µm in length and 19.1 ± 1.8 (16.0-22.1) µm in thickness. The posterior angle was slightly convex to straight and measured 177.1 ± 0.5 (175.2-178.4)°. Spore valves were slightly unequal and smoothly ovoid in the lateral view. Two polar capsules were spherical, equal in size and measured 2.7 ± 0.2 (2.1-3.0) µm in diameter. The 18S rDNA sequence of C. siganicola n. sp. was unique among all myxozoans, and the highest similarity was 97.4% with Ceratomyxa barnesi. Phylogenetic analysis showed that C. siganicola n. sp. was clustered within the clade of siganid ceratomyxids. The present results also indicated that the species radiation of Ceratomyxa occurred not only within host affinity but also within locality.

The impacts of geographic and host species isolation on population divergence of Myxobolus lentisuturalis.

Samples of Myxobolus lentisuturalis were found in the gallbladder of Carassius auratus in Chongqing, China, without obvious disease symptoms, which were different from samples reported in Hubei, China, and Italy which were described as highly pathogenic muscle-infecting species. In order to improve our understanding of the relationships between these different samples, we analyzed geography, DNA sequence data, and site specificity. The results indicated that (1) the genetic relationship between Chongqing and Italy samples of M. lentisuturalis was much closer than relationship between each of them and the Hubei samples; (2) host species isolation was more important than the geographic isolation in divergence of M. lentisuturalis samples, and the species might be specialized among its different host species; and (3) geographic isolation and infection-site variation played a limited impact in genetic differentiation among different samples of M. lentisuturalis infecting the same host species.

Histone deacetylase inhibitors promote eNOS expression in vascular smooth muscle cells and suppress hypoxia-induced cell growth.

Hypoxia stimulates excessive growth of vascular smooth muscle cells (VSMCs) contributing to vascular remodelling. Recent studies have shown that histone deacetylase inhibitors (HDIs) suppress VSMC proliferation and activate eNOS expression. However, the effects of HDI on hypoxia-induced VSMC growth and the role of activated eNOS in VSMCs are unclear. Using an EdU incorporation assay and flow cytometry analysis, we found that the HDIs, butyrate (Bur) and suberoylanilide hydroxamic acid (SAHA) significantly suppressed the proliferation of hypoxic VSMC lines and induced apoptosis. Remarkable induction of cleaved caspase 3, p21 expression and reduction of PCNA expression were also observed. Increased eNOS expression and enhanced NO secretion by hypoxic VSMC lines were detected using Bur or SAHA treatment. Knockdown of eNOS by siRNA transfection or exposure of hypoxic VSMCs to NO scavengers weakened the effects of Bur and SAHA on the growth of hypoxic VSMCs. In animal experiments, administration of Bur to Wistar rats exposed to hypobaric hypoxia for 28 days ameliorated the thickness and collagen deposition in pulmonary artery walls. Although the mean pulmonary arterial pressure (mPAP) was not obviously decreased with Bur in hypoxic rats, right ventricle hypertrophy index (RVHI) was decreased and the oxygen partial pressure of arterial blood was elevated. Furthermore, cell viability was decreased and eNOS and cleaved caspase 3 were induced in HDI-treated rat pulmonary arterial SMCs. These findings imply that HDIs prevent hypoxia-induced VSMC growth, in correlation with activated eNOS expression and activity in hypoxic VSMCs.

Alveolar Hypoxia-Induced Pulmonary Inflammation: From Local Initiation to Secondary Promotion by Activated Systemic Inflammation.

Pulmonary hypertension (PH) is a pathological condition with high mortality and morbidity. Hypoxic PH (HPH) is a common form of PH occurring mainly due to lung disease and/or hypoxia. Most causes of HPH are associated with persistent or intermittent alveolar hypoxia, including exposure to high altitude and chronic obstructive respiratory disease. Recent evidence suggests that inflammation is a critical step for HPH initiation and development. A detailed understanding of the initiation and progression of pulmonary inflammation would help in exploring potential clinical treatments for HPH. In this review, the mechanism for alveolar hypoxia-induced local lung inflammation and its progression are discussed as follows: (1) low alveolar PO2 levels activate resident lung cells, mainly the alveolar macrophages, which initiate pulmonary inflammation; (2) systemic inflammation is induced by alveolar hypoxia through alveolar macrophage activation; (3) monocytes are recruited into the pulmonary circulation by alveolar hypoxia-induced macrophage activation, which then contributes to the progression of pulmonary inflammation during the chronic phase of alveolar hypoxia, and (4) alveolar hypoxia-induced systemic inflammation contributes to the development of HPH. We hypothesize that a combination of alveolar hypoxia-induced local lung inflammation and the initiation of systemic inflammation ("second hit") is essential for HPH progression.

Genetic variation and population history of three Carassius auratus populations in Huaihe River, China.

In order to investigate the relationships of drainage history of Huaihe River with the genetic history of Carassius auratus along the river, we examined the genetic variations and population histories of three wild C. auratus populations in Huaihe River based on the D-loop gene. The results showed that their nucleotide and haplotype diversities were ranged from 0.00268 to 0.00651 and from 0.863 to 0.902, respectively, and their genetic distance was quite small. The analysis of molecular variance demonstrated that a frequent inter-population connection and large historic gene flows occurred among the three populations. Demographic analysis indicated that expansions had been happened in three populations. After investigating the historic process of the Huaihe River, we presumed that both nature and artificial factors may play important roles in shaping the genetic structure of the three populations. The present study also provided genetic information of C. auratus for further conservation of its germplasm resources.

DNA barcoding and molecular phylogeny in Ranidae.

The family Ranidae has the widest distribution compared with other frog family. The phylogeny of the Ranidae is still a matter of dispute. In the present study, we analyzed the COI barcodes of 29 species from six genera belonging to the family Ranidae. Twenty-seven species (93.10% of all the species) were correctly identified by their DNA barcodes. Pelophylax lessonae and Pelophylax ridibundus shared the same one barcode sequence. Kimura two-parameter distances were calculated between barcodes. Pair-wise comparisons among-species were distributed from 0.16% (between Pelophylax lessonae and Pelophylax esculenta) to 29.13% (between Rana warszewitschii and Rana dybowskii). The average genetic distance between species was 28 times higher than the average genetic distance within species. The neighbor-joining method was used to construct a phylogenetic tree, which grouped all the genera into two divergent clades. The results indicated that some currently recognized genera of Ranidae may not be monophyletic. COI gene data supported the hypothesis of polyphyly for Rana, Amolops, Babina, and Hylarana. DNA barcoding is an effective molecular tool for Ranidae species identification and phylogenetic inference.

DNA barcoding and phylogenetic relationships in Anatidae.

Mitochondrial cytochrome c oxidase subunit I (COI) has been used as a powerful marker in a variety of phylogenetic studies. According to studies of bird species, the 694-bp sequence of the mitochondrial gene encoding COI is extremely useful for species identification and phylogeny. In the present study, we analyzed the COI barcodes of 79 species from 26 genera belonging to the Anatidae family. Sixty-six species (83.54%) of the species were identified correctly from their DNA barcodes. The remaining 13 species shared barcodes sequences with closely related species. Kimura two-parameter (K2P) distances were calculated between barcodes. The average genetic distance between species was 41 times higher compared to the average genetic distance within species. Neighbor-joining method was used to construct a phylogenetic tree, which grouped all of the genera into three divergent clades. Dendrocygna and Nomonyx + Oxyura were identified as early offshoots of the Anatidae. All the remaining taxa fell into two clades that correspond to the two subfamilies Anserinae and Anatiane. Based on our results, DNA barcoding is an effective molecular tool for Anatidae species identification and phylogenetic inference.

DNA barcoding revises a misidentification on musk deer.

As an endangered animal group in China, musk deer (genus Moschus) have attracted the attention of deer biologists and wildlife conservationists. Clarifying the taxonomic status and distribution of musk deer species is important to determine the conservation status for each species and establish appropriate conservation strategies. There remains some uncertainty about the species determination of the musk deer in the Guandi Forest District of Shanxi Province, China. The musk deer in Shanxi would appear to represent an extension of the geographical distribution of either the Forest Musk Deer from the southwest or the Siberian Musk Deer from the northeast, or possibly both. The musk deer population in Shanxi Province provides an interesting and significant case to test the value of applying molecular methods to make a genetic species identification. In order to clarify the species status of the Shanxi musk deer, we sequenced 627 bp of the COI gene and ≈723 bp of the D-loop gene in 12 musk deer samples collected from the Guandi Forest District, and the two reference samples collected from Sichuan. Genetic analyses from the data suggest that all of the samples from the Guandi Forest District are M. berezovskii rather than M. moschiferus. It is most likely that the most previous studies had wrong species identification. And it is the first time we use DNA barcoding to prove that Shanxi is a new distribution of M. berezovskii.