Species Density Can Be Better Estimated From The Number Of Segregating Sites Using Environmental DNA(eDNA) & Genetic And Morphology Research Of The Seriola Lalandi In Bohai Sea Of China

Environmental DNA(e DNA)has been used successfully in detecting presence or absence of species in ecological studies,but applying e DNA in quantifying species abundance generated mixed results.Recent studies were focused on correlation between the number of copies of targeted e DNA and species biomass/density.The amount of DNA in water samples is jointly affected by many complicated factors,such as distance,body size,temperature,season,feeding and breeding behaviors.Differences in those factors among water samples could obscure the correlation between the number of DNA copies and species abundance.Here,we propose using the number of segregating sites as a proxy for species density.We examined relationship between the number of segregating sites and species abundance in vitro,in silico and in situ(mesocosm experiments)using two brackish goby species,Acanthogobius hasta and Tridentiger bifasciatus.Both analyses on the simulated data and the in vitro data with DNA mixed from known number of individuals showed strong correlation between the number of segregating sites and species density(R2 > 0.9;P < 0.01).Results from e DNA samples of the mesocosm experiment corroborated that the number of segregating sites significantly correlated with species density(P < 0.01),but was not affected by either body size or feeding behavior of the fish(P > 0.05).The results also showed that the number of segregating sites predicted species density more precisely(R2 = 0.70)than the number of DNA copies did(R2 = 0.46).Using the number of segregating sites extracted from e DNA data to quantify species density is a promising alternative to current methods based on DNA copy numbers.This innovation may truly empower e DNA technology to become quantitative.The ultimate goal of this study is to evaluate the species density in natural waters based on the number of segregating sites,we selected Seriola lalandi as target species,which is a kind of commercial oceanic fish.The commercial breeding of this species has been launched in China,nevertheless,the collection of basic genetic and morphological data in the early stage of breeding is not comprehensive enough.So,the present data did not provide adequate effective information for the quantitative research using environmental DNA based on the number of segregating sites.In this paper,the population structure and genetic diversity of the S.lalandi in China were studied by comparing the population samples of S.lalandi in Bohai Sea of China with the ones in southern Australia based on 17,690 nuclear loci.The genetic diversity was evaluated by expected heterozygosity,observed heterozygosity and nucleotide diversity.To find distinct morphology characters between China and Australia populations,we compared the shape difference between samples from two populations by constructing shape structure and countable characters,which guarantees the accuracy of subsequent sample collection.The result of STRUCTURE analysis showed that population of S.lalandi from China and Australia have completely distinct ancestries.The result of species delimitation also supported that S.lalandi from China and Australia are actually two species.But there was no significant difference between the genetic diversity of S.lalandi population in China(expected heterozygosity: 0.19,observed heterozygosity: 0.19,nucleotide diversity: 0.19 ± 0.09)and Australia(expected heterozygosity: 0.23,observed heterozygosity: 0.22,nucleotide diversity: 0.22 ± 0.11).We also investigated morphological characters of S.lalandi.Comparison of meristic characters between the Chinese population and the Australian population revealed that main shape difference were in the number of spinous dorsal(most individuals with 6 in the Chinese population vs.most individuals with 5 in the Australian population),the number of pectoral fins(most individuals with 22 in the Chinese population vs.most individuals with 20 in the Australian population),and the number of gill rakers(ranged as 4+15-10+19 in the Chinese population vs.4+13-8+17 in the Australian).Geometric morphology based on 8 landmarks also revealed significant difference between the two population: the distance between tip of snout and origin of pectoral fin was smaller in the Chinese population(0.28-0.33)than that of the Australia individuals(0.31-0.38);the distance between tip of snout to origin of pectoral fin also was smaller for the Chinese fishes(0.19-0.25)than that of the Australian fish(0.24-0.27).Finally,we developed 20 molecular markers from 17,690 nuclear genes as candidate genes for further study.This part of research was a prophase investigation for using segregating sites method evaluated the species density in water,the result showed the China S.lalandi as target species is feasible in theory because this species with abundant genetic diversity.And the data we obtained can provide basic information for genetic breeding of S.lalandi.It is convenient to develop genetic breeding potential of S.lalandi using these data and accelerate the pace of aquaculture of S.lalandi.