River Eco-health Assessment Based On Invertebrate Of The Hai River Basin And Phylogeny For 9 Viviparidae Species

Healthy river ecosystems are an important prerequisite for a significant proportion of life on this planet,we well as one of the basic conditions for the existence and development of human society.The Hai River Basin is a part of the largest alluvial plain in China,the North China Plain,which is one of the main agricultural and industrial areas in the country.As the basin also comprises two megacities,Beijing and Tianjin,the prominent contradiction between the water demand for agriculture,industry and public supply,and limited water resources,puts further stress on the importance of regular monitoring of the health status of this basin.Hence,in the first two parts of this study,status,structure and dynamics of macrozoobenthos in the Hai River Basin are investigated and discussed.Sampling took part from May to November 2013,and tolerance values(TV)of benthic macroinvertebrates were calculated.A health assessment system was also established on the basis of these results.Meanwhile,phylogenetic analysis using all available Viviparidae mitogenomes and adaptive evolution study of five Viviparidae snail species were conducted as well.The main results are shown below:Among 408 sampling sites setted up in the Hai River Basin,230 valid sites were used in calculated finally.Altogether,323 species belonging to 76 families and 178 genera were recorded from nine river systems.Among these,there were 254 species of Arthropoda,40 species of Annelida,27 species of Mollusca and two other species.Seasonal differences were not prominent: 263 species were found during the summer months,and 236 during the autumn months.In summer,the highest number of species was found in the Ziya River system(102),and the lowest in the Tuhaimajia River(67)and Heilonggangyundong River(68)systems.In autumn,the highest number of species was found in the Luanhe River system(111),and the lowest in the Tuhaimajia River system(53).The largest seasonal discrepancy was observed in the Daqing River system,where the number of species was reduced by 35% in autumn(compared with summer).In terms of functional feeding groups,collector gatherers were the most abundant and shredders the least abundant.In terms of lifestyle,burrowers were the most abundant and swimmers the least abundant.The most prominent seasonal differences in the compostion of lifestyle groups were observed in Daqing and Tianjin River systems.Seasonal pollution TV values were obtained for 53 taxonomic groups of benthic fauna,including 36 species in the summer and 35 in the fall.Species exhibiting TV values below 2 were designated as sensitive,and those with TV values > 8 as tolerant species.Species belonging to the genera exhibiting low TV values can be used as indicators of clean water: Anodonta,Caenis,Ephemera,Heptagenia,Coenagrion and Caridina.Species belonging to the genera exhibiting high TV values can be used as indicators of heavy water pollution: Radix,Hippeutis,Hirudo,Cricotopis,Cladopelma.And finally,species belonging to the genera exhibiting very high TV values can be used as indicators of severe water pollution: Tubifex,Chironomus,Limnodrilus,Glyptotendipes.The river ecosystem health of The Hai River Basin is assessed using Benthic-Index of Biotic Integrity.According to the benthic index of biotic integrity(B-IBI)values,among the 230 sites sampled in the Hai River Basin in summer,68 sites could be described as healthy,whereas the health status of the remaining 162 sampling sites can not be described as satisfactory.The results also indicate that the health of lower reaches of rivers in the Hai River Basin is better that the health of upper river reaches.The investigation result of macrozoobenthos in the Hai River Basin has shown that freshwater snails belonging to the Viviparidae family were an abundant component of macrozoobenthos in the Hai River Basin.Freshwater snails play an important role in the health of river ecosystems,but the phylogeny of Viviparidae remains unresolved,and the status of many species questionable.Therefore,the remaining two parts of this study focus on the mitochondrial genomics,phylogeny,and adaptive evolution(based on comprehensive transcriptome data)of snail species belonging to Viviparidae family,which is distributed throughout most of China.Mitochondrial genome is one of the important means to study the molecular phylogeny of animals.The complete mitogenomes of nine Chinese viviparid snails,Viviparus chui,Cipangopaludina chinensis,C.ussuriensis,C.dianchiensis(endangered),Margarya melanioides(endangered),M.monodi(critically endangered),Bellamya.aeruginosa,B purificata and B quadrata,were sequenced and compared to almost all of the available caenogastropod mitogenomes.Viviparidae possess the largest mitogenomes(16 392 to 18 544 bp),exhibit the highest A+T bias(72.5% on average),and some exhibit unique gene orders(a rearrangement of the standard MYCWQGE box),among the Caenogastropoda.Apart from the Vermetidae family and Cerithioidea superfamily,which possessed unique gene orders,the remaining studied caenogastropod mitogenomes exhibited highly conserved gene order,with minimal variations.Maximum likelihood and Bayesian inference analyses,used to reconstruct the phylogenetic relationships among 48 almost complete(13 protein coding genes and all 37 coding genes)caenogastropod mitogenomes of 46 species,produced almost identical tree topologies.Viviparidae were divided into three clades: a)Margarya and Cipangopaludina(except C.ussuriensis),b)Bellamya and C.ussuriensis,c)Viviparus chui.Our results present evidence that some Cipangopaludina species(dianchiensis and cathayensis)should be renamed into the senior genus Margarya.The phylogenetic resolution obtained in this study is insufficient to fully resolve the relationships within the ‘b' clade,C.ussuriensis may have to be reassigned a different genus(possibly Bellamya,or even a new genus).Non-monophyly also remains pervasive among the higher(above the family-level)Caenogastropod taxonomic classes.We sampled foot muscle tissues and conducted transcriptome analyses on five Viviparidae species: V.chui,C.chinensis,C.ussuriensis,M.melanioides and B.aeruginosa.The selected species are distributed across a wide geographical range,from lakes on the Yunnan plateau(M.melanoides)to the northeast corner of China where winters are harsh and summers short(V.chui).A total of 57254802 ~ 68596882 sequence raw reads were generated from each of the five species by Illumina sequencing.After filtering,de novo assembly of cleaned reads was performed using Trinity.By defining the longest transcript for each gene as unigene,108101~155758 unigenes were acquired for each species.Seven different databases(Nr,Nt,Pfam,KOG/COG,Swiss-prot,KEGG and GO)were used to annotate these unigenes and predict proteins.The highest success rate of annotation was obtained using the GO database,and the lowest using the NT database.Among the five species,the highest unigene annotation success rate was obtained for C.ussuriensis(27.48%)and the lowest for B.aeruginosa(23.11%).Finally,orthologous genes of the five species were screened by Ortho MCL.Phylogenetic analysis was performed using 525 concatenated genes.The topology obtained was identical to the one obtained using mitochondrial genomes: C.ussuriensis and B.aeruginosa formed one clade,C.chinensis and M.melanioides formed annother clade,and V.chui was basal to the remaining four species.The branch-sites model was used to detect positively selected genes.Among the five positively selected genes detected in M.melanioides,OSBP1,Syt7,RAB37 and Rock2 genes are related to the metabolic regulation of Ca2+ or Ca2+-dependent proteins,whereas TP53I11 is involved in anti-UV radiation mechanisms and DNA repair.Among the 18 positively selected genes detected in V.chui,Rock2,ATP7 A,Eif2ak3,PARP14,EIF2 D,BDH1 and Kyat3 were related to energy metabolism,and TTPAL to protection of cells from lipid peroxidation at low temperatures.