Integration of Gut Microbiota with Transcriptomic and Metabolomic Profiling Reveals Growth Differences in Male Giant River Prawns (Macrobrachium rosenbergii).

The giant freshwater prawn (GFP; Macrobrachium rosenbergii), a tropical species cultured worldwide, has high market demand and economic value. Male GFP growth varies considerably; however, the mechanisms underlying these growth differences remain unclear. In this study, we collected gut and hemolymphatic samples of large (ML), medium (MM), and small (MS) male GFPs and used the 16S rRNA sequencing and liquid chromatography-mass spectrometry-based metabolomic methods to explore gut microbiota and metabolites associated with GFP growth. The dominant bacteria were Firmicutes and Proteobacteria; higher growth rates correlated with a higher Firmicutes/Bacteroides ratio. Serum metabolite levels significantly differed between the ML and MS groups. We also combined transcriptomics with integrative multiomic techniques to further elucidate systematic molecular mechanisms in the GFPs. The results revealed that Faecalibacterium and Roseburia may improve gut health in GFP through butyrate release, affecting physiological homeostasis and leading to metabolic variations related to GFP growth differences. Notably, our results provide novel, fundamental insights into the molecular networks connecting various genes, metabolites, microbes, and phenotypes in GFPs, facilitating the elucidation of differential growth mechanisms in GFPs.

Identification and characterization of olfactory gene families in Macrobrachium rosenbergii based on full-length transcripts and genome sequences.

The olfactory gene families include odorant binding proteins (OBPs), chemosensory proteins (CSPs), olfactory receptors (ORs), ionotropic receptors (IRs) and gustatory receptors (GRs). To investigate the molecular function of olfactory perception in Macrobrachium rosenbergii, we integrated the full-length transcripts and whole-genome sequences to identify the olfactory gene families. In this study, a total of 38,955 full-length transcripts with an N50 length of 3383 bp were obtained through PacBio SMRT sequencing. Through the annotation of full-length transcripts and whole-genome sequences, several olfactory gene families were identified, including 18 MrORs, 16 MrIRs, 151 MrIGluRs (ionotropic glutamate receptors), 2 MrVIGluRs (variant ionotropic glutamate receptors) and 3 MrCRs (chemosensory receptors). Notably, the CRs were first identified in prawns and shrimps. Additionally, the olfactory gene families in M. nipponense were identified, comprising 4 MnORs, 21 MnIRs, 79 MnIGluRs, 5 MnVIGluRs, 1 MnGR and 1 MnOBP, using the available whole-genome sequences. Meanwhile, the external morphology of the chemical sensory organs of M. rosenbergii was explored, and the presence of plumose setae (PS), hard thorn setae (HTS), bamboo shoot setae (BSS), soft thorn setae (STS) and aesthetascs (AE) on the antennules, HTS and BSS on the second antennae, and PS on the pereiopods were observed by scanning electron microscope. This study provides valuable insights for future functional studies into the olfactory perception of crustaceans and establishes a theoretical basis for molecular design breeding in M. rosenbergii.

Histopathological observation and comparative transcriptome analysis reveal immune response mechanisms to Aeromonas dhakensis infection in Macrobrachium rosenbergii.

The Macrobrachium rosenbergii industry is threatened by various Aeromonas, resulting in high mortality of adult prawns. However, there are few studies on the immune response of M. rosenbergii infected with Aeromonas dhakensis. In this study, we observed the hepatopancreas and gills histopathologically, performed a comparative transcriptome analysis of the hepatopancreas, and analyzed the candidate gene expression of immune-related genes in the hemolymph, hepatopancreas, and gills of M. rosenbergii that had been infected with A. dhakensis. Histopathology revealed the hepatopancreas was successively inflamed, followed by cellular vacuolation, lumen deformation, and finally tissue erosion; partial and severe inflammation of the gills occurred successively, and eventually the gill tissue atrophy and the gill filaments detached from the gill arch. Transcriptome analysis showed that a total of 77,742 unigenes and 8664 differentially expressed genes (DEGs), and the immune-related DEGs were mainly enriched in lysosome and phagosome pathways. In addition, 4 immune-related candidate genes (RhoA, CASP9, PKC, and DSCIGN) based on KEGG and PPI analysis were monitored at 6, 12, and 24h post injection (hpi) in hepatopancreas, hemolymph and gills. Their spatio-temporal expression results indicated that A. dhakensis have activated the immune system of M. rosenbergii. The present study may provide new information on the complex immune mechanism of M. rosenbergii.

The Dynamics of Gene Expression Unraveling the Immune Response of Macrobrachium rosenbergii Infected by Aeromonas veronii.

To further investigate the immune response of Macrobrachium rosenbergii against Aeromonas veronii, comparative transcriptomic analyses of the M. rosenbergii hepatopancreas were conducted on challenge and control groups at 6, 12, and 24 h post-infection (hpi), independently. A total of 51,707 high-quality unigenes were collected from the RNA-seq data, and 8060 differentially expressed genes (DEGs) were discovered through paired comparisons. Among the three comparison groups, a KEGG pathway enrichment analysis showed that 173 immune-related DEGs were considerably clustered into 28 immune-related pathways, including the lysosome, the phagosome, etc. Moreover, the expression levels of the four key immune-related genes (TOLL, PAK1, GSK3ß, and IKKα) were evaluated at various stages following post-infection in the hepatopancreas, hemolymph, and gills. Both PAK1 and GSK3ß genes were highly up-regulated in all three tissues at 6 hpi with A. veronii; TOLL was up-regulated in the hepatopancreas and hemolymph but down-regulated in the gill at 6 hpi, and IKKα was up-regulated in hemolymph and gill, but down-regulated in the hepatopancreas at 6 hpi. These findings lay the groundwork for understanding the immune mechanism of M. rosenbergii after contracting A. veronii.

Alterations of the Gut Microbiota and Metabolomics Associated with the Different Growth Performances of Macrobrachium rosenbergii Families.

To investigate the key gut microbiota and metabolites associated with the growth performance of Macrobrachium rosenbergii families, 16S rRNA sequencing and LC-MS metabolomic methods were used. In this study, 90 M. rosenbergii families were bred to evaluate growth performance. After 92 days of culture, high (H), medium (M), and low (L) experimental groups representing three levels of growth performance, respectively, were collected according to the weight gain and specific growth rate of families. The composition of gut microbiota showed that the relative abundance of Firmicutes, Lachnospiraceae, Lactobacillus, and Blautia were much higher in Group H than those in M and L groups. Meanwhile, compared to the M and L groups, Group H had significantly higher levels of spermidine, adenosine, and creatinine, and lower levels of L-citrulline. Correlation analysis showed that the abundances of Lactobacillus and Blautia were positively correlated with the levels of alpha-ketoglutaric acid and L-arginine. The abundance of Blautia was also positively correlated with the levels of adenosine, taurine, and spermidine. Notably, lots of metabolites related to the metabolism and biosynthesis of arginine, taurine, hypotaurine, and fatty acid were upregulated in Group H. This study contributes to figuring out the landscape of the gut microbiota and metabolites associated with prawn growth performance and provides a basis for selective breeding.

Integrated Transcriptomic and Metabolomic Analyses Reveal Low-Temperature Tolerance Mechanism in Giant Freshwater Prawn Macrobrachium rosenbergii.

Water temperature, as an important environmental factor, affects the growth and metabolism of aquatic animals and even their survival. The giant freshwater prawn (GFP) Macrobrachium rosenbergii is a kind of warm-water species, and its survival temperature ranges from 18 °C to 34 °C. In this study, we performed transcriptomic and metabolomic analyses to clarify the potential molecular mechanism of responding to low-temperature stress in adult GFP. The treatments with low-temperature stress showed that the lowest lethal temperature of the GFP was 12.3 °C. KEGG enrichment analyses revealed that the differentially expressed genes and metabolites were both enriched in lipid and energy metabolism pathways. Some key genes, such as phosphoenolpyruvate carboxykinase and fatty acid synthase, as well as the content of the metabolites dodecanoic acid and alpha-linolenic acid, were altered under low-temperature stress. Importantly, the levels of unsaturated fatty acids were decreased in LS (low-temperature sensitive group) vs. Con (control group). In LT (low-temperature tolerant group) vs. Con, the genes related to fatty acid synthesis and degradation were upregulated to cope with low-temperature stress. It suggested that the genes and metabolites associated with lipid metabolism and energy metabolism play vital roles in responding to low-temperature stress. This study provided a molecular basis for the selection of a low-temperature tolerant strain.

Combined transcriptomics and metabolomics analysis reveals lipid metabolic disruption in swamp eel (Monopterus albus) under chronic waterborne copper exposure.

Excessive copper can induce many adverse effects although it's an essential trace element in organisms. The effects of copper on the lipid metabolism have aroused increasing attention. This study investigated the liver lipid metabolism in swamp eel (Monopterus albus, M. albus) chronically exposed to 0, 10, 50, and 100 µg/L Cu2+ for 56 days. The results showed that copper increased the contents of triglyceride (TG), total cholesterol (T-CHO), non-esterified fatty acids (NEFA), and lipid droplets. Transcriptomic analysis found 1901 differentially expressed genes (DEGs) and 140 differential alternative splicing (DAS) genes in the 50 µg/L Cu2+ group, and 1787 DEGs and 184 DAS genes in the 100 µg/L Cu2+ group, respectively, which were enriched in peroxisome proliferator-activated receptor (PPAR), adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), and other signaling pathways. The expression levels of key genes related to PPAR and AMPK signaling pathways were significantly down-regulated after chronic exposure to Cu2+. Meanwhile, metabolomics analysis showed that 52 and 110 differentially expressed metabolites (DEMs) were identified, which were mainly enriched in glycerophospholipids metabolism and steroid synthesis. Moreover, combined analysis of transcriptome and metabolome showed that glycerophospholipid metabolism co-enriched 19 down-regulated DEGs and 4 down-regulated DEMs. Taken together, our results suggested that chronic waterborne copper exposure promoted lipid synthesis, disrupted the metabolic homeostasis of glycerophospholipid, and led to excessive hepatic lipid deposition in M. albus. The combined omics approach enhanced our understanding of copper pollution to lipid metabolism.

The complete mitogenome of giant freshwater prawn (Macrobrachium rosenbergii) from two different selective breeding populations in China.

The giant freshwater prawn (GFP), Macrobrachium rosenbergii, is one of the largest freshwater shrimps in the world, being widely cultured because of its high economic value. In this study, complete mitogenomes of two GFP individuals from different selective breeding populations, 'South Taihu No.2' (ST) and 'Shufeng' (SF), were newly sequenced, compared with each other, and with those of other published Macrobrachium species. The total length is 15,767 bp (ST) and 15,766 bp (SF), including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and one control region. The phylogenetic analyses based on whole mitogenome sequences suggest that 'Shufeng' has a slightly distant relationship with 'South Taihu No.2', with a pairwise genetic distance of 0.011. This study can provide a genetic background for the GFP selective breeding, and add significantly to the knowledgebase regarding crustacean biology and aquaculture as well.

Molecular identification and functional analysis of MyD88 in giant freshwater prawn (Macrobrachium rosenbergii) and expression changes in response to bacterial challenge.

Myeloid differentiation factor 88 (MyD88) is a crucial adaptor protein for Toll-like receptor (TLR)-mediated signaling pathways and plays an important role in immune response. In this study, the full-length cDNA of MyD88 from Macrobrachium rosenbergii (MRMyD88) was cloned. The MRMyD88 cDNA is 1758 bp long and contains a 1398-bp open reading frame. Multiple sequence alignment and phylogenetic analysis revealed that the amino acid sequence of MRMyD88 shared high identity with the known MyD88 proteins. The MRMyD88 mRNA was widely expressed in all examined tissues, with highest level in intestine, followed by gonad and pleopod. Furthermore, the MRMyD88 promoter region, spanning 1622 bp, contains several transcription factor-binding sites, including nine GATA-1 box motifs. Electrophoretic mobility shift assay showed that Gfi-1, SRF, and Oct-1 bind to the upstream region of MRMyD88. Additionally, the results showed that the expression levels of TLR1, TLR2 and TLR3 were different in response to Vibrio anguillarum, Lactobacillus plantarum and Aeromonas hydrophila infections. However, these bacteria significantly increased the expression levels of MyD88 and prophenoloxidase. These data suggest that the TLR-mediated signaling pathway is MyD88-dependent in response to pathogenic and probiotic bacteria in M. rosenbergii.

The complete mitochondrial genomes of Parabotia kiangsiensis (Cypriniformes: Botiidae).

Parabotia kiangsiensis is an endemic species of genus Parabotia in China. In this study, we sequenced the complete mito-genome of P. kiangsiensis. The genome is 16,592 base pair (bp) in length, encoding 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and one non-coding control region (D-loop). The nucleotide composition is A: 30.66%, T: 25.13%, G: 16.3%, and C: 27.92% (AT content:55.79%). The complete mitogenome of P. kiangsiensis provides basic data for the genetic diversity conservation of this species.

Molecular characterization of TLR3 and TRIL in silvery pomfret (Pampus argenteus) and their expression profiles in response to bacterial components.

Toll-like receptors (TLRs) play important roles in the innate system by recognizing pathogen-associated molecular patterns derived from various microbes. In this study, we reported the cloning and identification of paTLR3 and paTLR4 interactor with leucine rich repeats (TRIL) cDNA from silvery pomfret (Pampus argenteus). The full-length paTLR3 and paTRIL cDNA were 2996 and 3163 bp long, respectively. Both of the two proteins contained many LRR domains, one LRR-C terminal domain and one transmembrane region, which fits with the characteristic TLR and its analogue domain architecture. Phylogenetic analyses revealed that paTLR3 and paTRIL shared the closest relationship with Lateolabrax japonicas and Notothenia coriiceps, respectively. The expression levels of paTLR3 and paTRIL varied greatly among the examined tissues with the highest expression both in liver. Following exposure to V. anguillarum flagellin, A. hydrophila lipopolysaccharide (LPS) and L. plantarum lipoteichoic acid (LTA), paTLR3 and paTRIL were all up-regulated. V. anguillarum flagellin induced the highest expression levels of paTLR3 and paTRIL. A. hydrophila flagellin and A. hydrophila LPS induced the highest expression levels of IL-1ß and IL-8, respectively. The present results will provide the valuable information for understanding the structure, function and the immune defense process of paTLR3 and paTRIL in silvery pomfret.

Genetic parameters and selection response for the harvest body weight of the giant freshwater prawn (Macrobrachium rosenbergii) in a breeding program in China.

A multi-trait selective breeding program of Macrobrachium rosenbergii was initiated in China in 2015. In this program, the M. rosenbergii resources were widely collected from four countries, the origin of the founders was verified with 16 microsatellites and the pedigree was reconstructed, and the optimum contribution selection was used to make the mating design. In this study, we evaluated the genetic parameters and selection response for the harvest body weight (HBW) of M. rosenbergii after being communally reared for 95-109 days. The data were collected from two generations that comprised 25,212 progenies from 150 sires and 198 dams. The residual maximum-likelihood methodology was employed to evaluate the variance components, by fitting an animal model. The accuracy of estimated breeding values increased by 0.38% after pedigree reconstruction using microsatellite markers. The estimated heritability (h2) for HBW was moderate (0.212 ± 0.049) and the common environmental coefficient (c2) was low (0.063 ± 0.017) when all the data were used for the analysis. Within generations, h2 was moderate to high (0.198 ± 0.080 to 0.338 ± 0.049). c2 could only be estimated in G1, which was 0.055 ± 0.030. The average HBW of males was significantly larger than that of females (P < 0.01). h2 estimated for female HBWs were higher than that for males within generations, while h2 estimated for female HBWs were lower than that for males across generations. But they were not significantly different (P > 0.05). The genetic correlations between sexes were moderate to high within each generation (0.529 to 0.763). Two methods were used to estimate the realized response. One method was calculated from the differences between the least squares means of the selected population HBW and that of control population HBW, which was 14.01%. The other method was calculated from the differences between the EBVs of the selected population HBW and that of control population HBW, which was 11.52%. The predicted responses derived from two sets of genetic parameters acquired from within- and across- generation datasets were 11.68% and 10.67%, respectively. The present study provides valuable information for breeding programs of M. rosenbergii.

Evolution and phylogenetic application of the MC1R gene in the Cobitoidea (Teleostei: Cypriniformes).

Fish of the superfamily Cobitoidea sensu stricto (namely loaches) exhibit extremely high diversity of color patterns, but so far little is known about their evolutionary mechanism. Melanocortin 1 receptor gene (MC1R) plays an important role during the synthesis of melanin and formation of animal body color patterns. In this study, we amplified and sequenced the partial MC1R gene for 44 loach individuals representing 31 species of four families. Phylogenetic analyses yielded a topology congruent with previous studies using multiple nuclear loci, showing that each of the four families was monophyletic with sister relationships of Botiidae+ (Cobitidae+(Balitoridae+Nemacheilidae)). Gene evolutionary analyses indicated that MC1R in loaches was under purifying selection pressure, with various sites having different dN/dS values. Both Botiidae and Cobitidae had lower dN/dS values than those of background lineages, suggesting their evolution might be strongly affected by purifying selection pressure. For Balitoridae and Nemacheilidae, both had larger dN/dS values than those of background lineages, suggesting they had a faster evolutionary rate under more relaxed selection pressure. Consequently, we inferred that the relatively stable color patterns in Botiidae and Cobitidae might result from the strong purifying selection pressure on the MC1R gene, whereas the complicated and diverse color patterns in Balitoridae and Nemacheilidae might be associated with the relaxed selection pressure. Given the easy experimental procedure for the partial MC1R gene and its excellent performance in reconstructing phylogeny, we suggest this gene could be used as a good molecular marker for the phylogenetic study of fish species.

The complete mitochondrial genome sequence of Jinshaia abbreviata (Cypriniformes, Balitoridae).

Jinshaia abbreviata, belonging to the family Balitoridae in Cypriniformes, is endemic to the Upper Yangtze River with most population distributed in the Jinsha River. In this study, the complete mitochondrial genome of J. abbreviata was sequenced with its structure analyzed. The mitochondrial genome of J. abbreviata is similar to those of the typical vertebrates, 16,567 bp in length, including 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and one non-coding control region (D-loop). The D-loop of J. abbreviata was characterized by one termination-associated sequence with two pair of motifs and six conserved sequence blocks (CSB-F, CSB-E, CSB-D, and CSB I-III). The mitogenome sequence of J. abbreviata could contribute to estimate the phylogenetic relationship of the Balitoridae. And further investigations with more Jinshaia species and individuals included need to be performed to better understand the speciation process and evolutionary history of the genus Jinshaia.

The complete mitochondrial genome sequence of Sinogastromyzon sichangensis (Cypriniformes, Balitoridae).

Sinogastromyzon sichangensis, belonging to the family Balitoridae in Cypriniformes, is endemic to the Upper Yangtze River and the Qing River. In this study, the complete mitochondrial genome of S. sichangensis was sequenced with its structure analyzed. The mitochondrial genome of S. sichangensis is similar to those of the typical vertebrates, 16,567 bp in length, including 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and one non-coding control region (D-loop). The D-loop of S. sichangensis was characterized by one termination-associated sequence and seven conserved sequence blocks (CSB-F, CSB-E, CSB-D, CSB-B and CSB I-III). The mitogenome sequence of S. sichangensis could contribute to estimate the phylogenetic relationship of the Balitoridae. Further investigations with more Sinogastromyzon species and other balitorid fishes included should be performed to better understand the evolutionary history of this unique group of fishes which can successfully adapt to mountain torrents.

Geological events and Pliocene climate fluctuations explain the phylogeographical pattern of the cold water fish Rhynchocypris oxycephalus (Cypriniformes: Cyprinidae) in China.

BACKGROUND: Rhynchocypris oxycephalus is a cold water fish with a wide geographic distribution including the relatively warm temperate regions of southern China. It also occurs in second- and third-step geomorphic areas in China. Previous studies have postulated that high-altitude populations of R. oxycephalus in southern China are Quaternary glacial relics. In this study, we used the mitochondrial gene Cytb and the nuclear gene RAG2 to investigate the species phylogeographical patterns and to test two biogeographic hypotheses: (1) that divergence between lineages supports the three-step model and (2) climatic fluctuations during the Quaternary resulted in the present distribution in southern China. RESULTS: Phylogenetic analysis detected three major matrilines (A, B, and C); with matrilines B and C being further subdivided into two submatrilines. Based on genetic distances and morphological differences, matriline A potentially represents a cryptic subspecies. The geographic division between matrilines B and C coincided with the division of the second and third geomorphic steps in China, suggesting a historical vicariance event. Pliocene climatic fluctuations might have facilitated the southwards dispersal of R. oxycephalus in matriline C, with the subsequent warming resulting in its split into submatrilines C1 and C2, leaving submatriline C2 as a relic in southern China. CONCLUSIONS: Our study demonstrates that geological events (three steps orogenesis) and climate fluctuations during the Pliocene were important factors in shaping phylogeographical patterns in R. oxycephalus. Notably, no genetic diversity was detected in several populations, all of which possessed unique genotypes. This indicates the uniqueness of local populations and calls for a special conservation plan for the whole species at the population level.

Molecular phylogeny and conservation priorities of the subfamily Acheilognathinae (Teleostei: Cyprinidae).

It is increasingly accepted that conservation work should consider the evolutionary history of target species. Fishes in the subfamily Acheilognathinae, family Cyprinidae, are, with the exception of three species exclusively distributed in Europe, restricted to Asia and show a distinct spawning behavior in laying their eggs in gill chambers of freshwater mussels. At present, many of the 70 species recognized in this group are facing with serious population decline in China and Japan, and their phylogenetic relationships are not well resolved. In the present study, based on mtDNA cyt b and 12S rRNA gene sequences, we reconstructed a more detailed species-level phylogenetic tree of this group, and assessed species conservation priorities based on their evolutionary distinctiveness. Molecular phylogenetic analyses showed that the Acheilognathinae contains two major clades: Acheilognathus clade and Tanakia-Rhodeus clade. Based on this phylogenetic result, conservation priority analyses were conducted using ED (evolutionary distinctiveness)/HED (heightened evolutionary distinctiveness), and EDGE (evolutionary distinctiveness and global endangeredness)/HEDGE (heightened evolutionary distinctiveness and global endangeredness) methods. The results suggested that T. himantegus, T. lanceolata, A. gracilis, A. imberbis, T. tanago, and A. longipinnis should be ranked as the top-priority species for conservation. According to our results, we also discussed the current conservation efforts of the bitterling fishes and gave suggestions for future work.

[Phylogeographic analyses of Sarcocheilichthys nigripinnis(Teleostei: Cyprinidae)based on mitochondrial DNA Cyt b gene sequences].

Sarcocheilichthys nigripinnis is a small cyprinid fish widely distributed in East Asia, and it has been widely used in biogeographic analyses of freshwater fishes in China. In the present study, 142 S. nigripinnis individuals from 20 sampling sites in eight river systems were collected to investigate its phylogeography and genetic variations.. Populations from the Yellow River represent northern clade and all others represent southern clade. The results showed that 56 haplotypes were identified as mitochondrial DNA (mtDNA) Cyt b gene of 1 140 bp length. Relatively high haplotype diversity (h=0.971) and low nucleotide diversity (π=0.0212) were detected, and the estimated average genetic distance was 2.2%. Moreover, a neighbor-joining(NJ)tree revealed seven strongly supported lineages. Populations from the Yellow River were located at the basal position, whereas the remaining populations were more derived. Our results indicate that S. czerskii might have been evolved from S. nigripinnis in the Yellow River ~1.03 Ma. Based on the phylogeographical analysis of S. nigripinnis, we also suggest that vicariance, following mountain uplift and drainage isolation, plays an important role in producing evolutionary lineage differentiations. Moreover, molecular dating estimated that the divergence time of S. czerskii could be dated back to 0.95~3.92 Ma. Mismatch distribution analysis and neutrality tests also suggested the recent demographic expansions of S. nigripinnis populations, and that the effects of Pleistocene climatic changes could be a vital factor of the population dynamics of S. nigripinnis.

Phylogenetic relationships of Pseudorasbora, Pseudopungtungia, and Pungtungia (Teleostei; Cypriniformes; Gobioninae) inferred from multiple nuclear gene sequences.

Gobionine species belonging to the genera Pseudorasbora, Pseudopungtungia, and Pungtungia (Teleostei; Cypriniformes; Cyprinidae) have been heavily studied because of problems on taxonomy, threats of extinction, invasion, and human health. Nucleotide sequences of three nuclear genes, that is, recombination activating protein gene 1 (rag1), recombination activating gene 2 (rag2), and early growth response 1 gene (egr1), from Pseudorasbora, Pseudopungtungia, and Pungtungia species residing in China, Japan, and Korea, were analyzed to elucidate their intergeneric and interspecific phylogenetic relationships. In the phylogenetic tree inferred from their multiple gene sequences, Pseudorasbora, Pseudopungtungia and Pungtungia species ramified into three phylogenetically distinct clades; the "tenuicorpa" clade composed of Pseudopungtungia tenuicorpa, the "parva" clade composed of all Pseudorasbora species/subspecies, and the "herzi" clade composed of Pseudopungtungia nigra, and Pungtungia herzi. The genus Pseudorasbora was recovered as monophyletic, while the genus Pseudopungtungia was recovered as polyphyletic. Our phylogenetic result implies the unstable taxonomic status of the genus Pseudopungtungia.

[Skeletal anatomy and phylogenetic position analysis of Gobiocypris rarus].

The phylogenetic position of Gobiocypris rarus, a small cyprinid fish of interest in many biological areas due to its unique characteristics, is still under debate. At the morphological view, it belongs to the Danioninae subfamily of Cyprinidae; however, recent molecular research recognizes it as a member of the Gobioninae subfamily. To investigate the phylogenetic position of Gobiocypris rarus, we prepared transparent skeleton specimens, selected 47 characteristics and reconstructed the phylogenetic tree using PAUP. The results indicated that Gobiocypris rarus was clustered with Gobioninae, which was in agreement with recent molecular phylogenetic conclusions.