Species-level monitoring of rare and invasive fishes using eDNA metabarcoding in the middle and upper Yarlung Zangbo River, Tibet

Fish diversity of the Yarlung Zangbo River is very sensitive and vulnerable to biological invasion,anthropogenic activities and climate change,especially in the upper and middle reaches where several endemic fishes have become endangered and nearly ten invasive fishes have been established.Here,we used environmental DNA(eDNA)metabarcoding to monitor rare and invasive fishes,and to assess diversity in 25 sites from two wetlands(Lalu and Chabalang)and the main channel(YT),within the upper and middle reaches.To obtain a species-level resolution,we evaluated species discrimination potentials of three mitochondrial markers and found Cytb had the highest average genetic distance at each taxonomic level followed by COI and 12S.The 12S was unqualified for species assignment,as two species shared identical haplotypes.The newly designed Cytb primers used for metabarcoding showed an average mismatch of 0.28 and amplified well across species.In total,8942 operational taxonomic units(OTUs)were obtained based on a 100%identity threshold,among which 98.1%were assigned to 24 fishes based on our custom-made database and the remaining were assigned to six fishes based on the NCBI nt database.Almost all captured fishes were detected by the eDNA method except for two species.However,12 fishes detected by the eDNA method were not listed in catch data for several sites,including one endangered species(Oxygymnocypris stewartii),four previously recorded non-native species and two unrecorded non-native species(Monopterus albus and Siniperca chuatsi).The alpha diversities estimated by eDNA and capture-based methods were correlated for sites at Lalu.Both methods revealed significant differences in community composition between YT and the wetlands.Our results provide both basic information for conservation and management of rare and invasive fishes in the Yarlung Zangbo River and a framework of fish eDNA metabarcoding with a species-level resolution.

Are phylogenies resolved at the genus level appropriate for studies on phylogenetic structure of species assemblages?

Phylogenies are essential to studies investigating the effect of evolutionary history on assembly of species in ecological communities and geographical and ecological patterns of phylogenetic structure of species assemblages.Because phylogenies well resolved at the species level are lacking for many major groups of organisms such as vascular plants,researchers often generate a species-level phylogenies using a phylogeny well resolved at the genus level as a backbone and attaching species to their respective genera in the phylogeny as polytomies or by using a megaphylogeny well resolved at the genus level as a backbone and adding additional species to the megaphylogeny as polytomies of their respective genera.However,whether the result of a study using species-level phylogenies generated in these ways is robust,compared to that based on phylogenies fully resolved at the species level,has not been assessed.Here,we use 1093 angiosperm tree assemblages(each in a 110110 km quadrat)in North America as a model system to address this question,by examining six commonly used metrics of phylogenetic structure(phylogenetic diversity and phylogenetic relatedness)and six climate variables commonly used in ecology.Our results showed that(1)the scores of phylogenetic metrics derived from species-level phylogenies resolved at the genus level with species being attached to their respective genera as polytomies are very strongly or perfectly correlated to those derived from a phylogeny fully resolved at the species level(the mean of correlation coefficients is 0.973),and(2)the relationships between the scores of phylogenetic metrics and climate variables are consistent between the two sets of analyses based on the two types of phylogeny.Our study suggests that using species-level phylogenies resolved at the genus level with species being attached to their genera as polytomies is appropriate in studies exploring patterns of phylogenetic structure of species in ecological communities across geographical and ecological gradients.