Epiphytic zooplankton community profiles in a typical urban wetland as revealed by DNA metabarcoding

Zooplankton,a crucial component of urban wetland,are one of the effective bioindicators for monitoring the feeding stocks of organisms at higher trophic levels and assessing the ecological quality of ecosystems.However,information about the characteristics of epiphytic zooplankton community structure resulted from traditional methods is limited and hindered by the large amount of detritus and sludge attached to the macrophytes.We investigated the epiphytic zooplankton communities associated with macrophytes(Vallisneria,Nymphaea,and Thalia dealbata)in a subtropical wetland using as DNA markers of the 18 S rRNA gene and the mitochondrial cytochrome c oxidase subunit I(COI)gene.A total of 241 OTUs of zooplankton were obtained from COI amplicons,including 194 OTUs of Rotifera,22 of Cladocera,and 25 of Copepoda,while only 62 OTUs of zooplankton were obtained from 18 S rDNA amplicons including 34 OTUs of Rotifera and 28 of Copepoda.The zooplankton communities associated with the three macrophytes were similar,but they differed significantly from those in the open waters.However,there were no significant temporal differences among the zooplankton communities.Epiphytic zooplankton communities were dominated by littoral zooplankton such as Testudinella,Lecane,and Philodina.Microzooplankton,especially littoral species,utilize macrophytes as food sources and as refuges against predation.This further led to an increase inαandβdiversity of zooplankton communities in urban wetlands.Our result suggests that the joint use of multiple molecular markers could improve the taxonomic resolution and generate a comprehensive biodiversity profile of zooplankton.

基于环境DNA metabarcoding的舟山及其邻近海域鱼类空间分布格局的初步研究

为了解舟山及其邻近海域主要鱼类群落的种类组成,监测和保护其多样性,本研究于2019年5月在舟山及其邻近海域9个站位共采集了27个水样,采用环境DNA(environmental DNA,eDNA)metabarcoding技术确定了各站位鱼类群落组成和生物多样性。结果显示,舟山及其邻近海域共检出52种鱼类,隶属于18目37科49属,有4种鱼类仅注释到属级分类阶元。其中,鲈形目和鲭形目占比最高,分别为28.85%和15.38%,不同海域优势种存在较大差异。多样性(Shannon指数、Simpson指数和Pielou指数)表现趋势基本相同,均表现为舟山近海海域>长江河口海域>舟山外海海域。各鱼种eDNA在不同水层变化趋势大致可以分为3种,且多数鱼种序列丰度在水层间的变化趋势与其栖息水层偏好高度吻合。此外,通过与其他学者的研究结果进行比较分析,发现同一时间相同海域的eDNA metabarcoding研究结果差异较大,表明目前eDNA技术仍不能完全替代传统调查方法。未来可以将eDNA metabarcoding技术作为一种辅助手段应用于渔业资源监测,提高检测效率并减少对生态系统的干扰。本研究可为岛礁海域的鱼类群落调研提供新的思路和方法。

基于环境DNA metabarcoding的中街山列岛鱼类物种多样性

岛礁生境支撑着极高的水生生物多样性,而鱼类物种多样性是水生生物多样性的重要组成部分。为准确了解岛礁海域的鱼类时空分布格局和多样性特征,依靠传统的渔业资源调查方式存在诸多的困难与挑战,亟需建立灵敏、高效的生物多样性监测方法。本研究于2019年在中街山列岛海域设置了4个采样站位,并在2、5、8和11月等4个时间采集海水表层样品,采用环境DNA metabarcoding技术进行高通量测序。结果显示,中街山列岛近岸海区共检测出鱼类37种,隶属于10目26科36属;鱼类种数呈现夏季>冬季>秋季>春季的趋势;四季共有种仅2种,约占鱼类总种数的5.41%;只在一个季节被检测出的鱼类约占总种数的54.05%。结合NMDS分析和ANOSIM检验的结果显示,鱼类群落在不同季节间有显著差异,而在不同站位间无显著差异。鱼类群落的均匀度较为稳定,多样性和丰富度指数在站位间无明显变化,但在季节间表现为夏季最高、春季最低。研究表明,中街山列岛近岸海区的鱼类分布受季节影响较大,而在不同采样站位的差异不大,可能受站位较为集中的影响。

基于环境DNA metabarcoding的西轩岛近海鱼类多样性研究

鱼类多样性的保护对于生态系统的科学管理和资源的可持续利用至关重要。环境DNA metabarcoding技术的出现和应用为水生生物的调查与监测带来了强有力的技术革新。本研究以浙江舟山近海岛屿——西轩岛为例,设计了4个不同采样站位,先后于2019年2月(冬季)、5月(春季)和11月(秋季)共采集水样12个,通过环境DNA提取、扩增、高通量测序以及生物信息学分析,对西轩岛近海鱼类多样性进行了分析,同时评估了鱼类多样性的时空差异。结果显示,共监测到鱼类33种,隶属于12目26科32属,其中,鲈形目(Perciformes)种类最多,共19种,约占所有种类的57.6%。不同采样季节的多样性指数和均匀度指数均存在显著差异,表明季节可能是影响西轩岛近海鱼类多样性的因素之一。综合时间和空间分析的结果显示,在繁殖季节且远离舟山本岛一侧的采样点监测到的鱼种数量更多。通过比对之前传统渔业资源调查的结果发现,不同季节优势种存在较大变化,可能与采样点数量较少且集中有关。进化树富集结果显示,各季节的优势鱼种与传统调查手段的结果有较大差异,表明目前环境DNA仍不能完全替代传统调查方法,但可以将环境DNA方法与传统的调查方法相结合,以确保监测结果的准确性和可靠性。

基于环境DNA metabarcoding和底拖网调查的南黄海西部鱼类多样性比较

为探究环境DNA metabarcoding(eDNA metabarcoding)和底拖网在陆架海域环境下获取鱼类多样性和分布的差异,基于2020年夏季南黄海西部13个站位的环境DNA采样和底拖网调查数据,使用Alpha多样性指数和Beta多样性分析,比较了环境DNA metabarcoding和底拖网检测鱼类生物多样性的表达程度,探讨了使用环境DNA metabarcoding技术监测海洋生物多样性和空间分布的效果。研究显示:环境DNA metabarcoding检测出45种鱼类,底拖网调查检测出32种鱼类,重叠种类23种;在环境DNA metabarcoding结果中,小黄鱼(Larimichthys polyactis,27.20%)、方氏云鳚(Pholis fangi,21.16%)、黄鮟鱇(Lophius litulon,18.67%)、日本鳀(Engraulis japonicus,8.59%)、小眼绿鳍鱼(Chelidonichthys spinosus,4.06%)占有相对较高的读数;底拖网调查的结果中,小黄鱼(L.polyactis,16.14%)、日本带鱼(Trichiurus japonicus,16.13%)、黄鮟鱇(Lophius litulon,12.95%)、方氏云鳚(P.fangi,12.80%)、细纹狮子鱼(Liparis tanakae,12.45%)占有较高的资源量。研究结果表明:环境DNA metabarcoding和底拖网调查检测到的鱼种存在部分重叠,且环境DNA metabarcoding获取的鱼类种数高于底拖网调查(p<0.05);垂直多层采样能够进一步提高环境DNA metabarcoding检测对鱼类多样性的解释效果。环境DNA metabarcoding技术为传统的渔业资源评估模式提供了一种新思路,可作为传统渔业资源调查的有效补充。

Fecal DNA metabarcoding reveals the dietary composition of wintering Red-crowned Cranes(Grus japonensis)

Understanding the diet of threatened wildlife is vital for species-specific conservation and habitat management measures.The Red-crowned Crane(Grus japonensis)is a vulnerable bird distributed in Northeast Asia.Previous dietary studies of this bird focused mainly on its plant food composition based on field observations and microhistological identification.Herein,a total of 45 fecal samples were collected in November,December and January(15 fecal samples each month)from wintering cranes,and then subjected to a high throughput sequencing meta-barcoding approach to determine the primary plant(rbcL)and animal(COI)food items in their diet.A total of 230 operational taxonomic units(OTUs)of plant foods and 371 OTUs of animal foods were obtained.The main plant foods in the wintering period were Miscanthus,Zea,and Hordeum genera,which were similar to those in the breeding and the migration periods.Both agricultural and natural plants were detected,indicating a relatively broad dietary niche for this crane species.However,the main animal foods were representatives of Theridiidae,Megascolecidae,and Agelenidae,in sharp contrast to previous studies.The higher number of small terrestrial arthropods in animal foods might be due to the indirect intake of plants.The composition of both plant and animal foods in the diet showed the highest diversity in December,while it was homogeneous in January.The plant of Zea genus became the main source of nutrition in late winter,as supplementary feeding was performed in the reserve,which could help Red-crowned Cranes to get through the cold season.The results obtained in this work would contribute to the development of effective conservation strategies for the Red-crowned Crane.

环境DNA metabarcoding及其在生态学研究中的应用

环境DNA metabarcoding(eDNA metabarcoding)是指利用环境样本(如土壤、水、粪便等)中分离的DNA进行高通量的多个物种(或高级分类单元)鉴定的方法。近年来,该方法引起了学者的广泛关注,逐渐应用于生物多样性研究、水生生物监测、珍稀濒危物种和外来入侵物种检测等生态学领域。介绍环境DNA metabarcoding的含义和研究方法;重点介绍环境DNA metabarcoding在物种监测、生物多样性研究和食性分析等生态学领域中的应用;总结环境DNA metabarcoding应用于生态学研究领域面临的挑战并对该方法的发展进行展望。

基于宏基因组(metabarcoding)技术的橡胶种植对林下节肢动物多样性的影响

地处热带边缘的西双版纳地区近几十年来土地利用方式发生巨变,热带森林逐渐被单一种植橡胶林所替代。探讨森林-橡胶林镶嵌格局对节肢动物群落组成和多样性的影响,分析节肢动物群落分布格局的驱动因素,以期为残存森林的保护提供相关科学数据。以西双版纳纳板河流域不同景观梯度下的片段森林和橡胶林为研究样地,采用马氏网诱集法,结合宏基因组(metabarcoding)技术调查林下节肢动物多样性和群落组成。结果表明:(1)基于metabarcoding技术,从橡胶林和片段森林林下马氏网中共得到397个可操纵的分类单元(OTUs),其中,片段森林中共收集到节肢动物18目347个OTUs;橡胶林收集到节肢动物14目136个OTUs。(2)片段森林中节肢动物物种多样性显著高于橡胶林;非度量多维尺度分析(NMDS)结果显示,橡胶林和片段森林中节肢动物群落组成具有显著差异,片段森林节肢动物群落beta多样性大于橡胶林。(3)在景观尺度上,影响节肢动物物种多样性和群落分布的主要因素为森林覆盖率;在样方尺度上,影响橡胶林节肢动物物种多样性的主要因素为胶树年龄和到森林边缘距离;影响森林节肢动物物种多样性的主要因素为片段森林面积、树种多样性、到森林边缘距离、坡向和海拔。森林覆盖率、树种多样性、橡胶林到森林边缘距离和胶树年龄等因素都与节肢动物多样性呈显著相关性,因此,在选择橡胶林种植区时需考虑森林覆盖率、树种多样性及橡胶林到森林边缘距离等因素,且森林覆盖率达50%以上显著有利于生物多样性保护。该研究结果可为森林和橡胶林的管理提供数据资料和科学依据。

Revealing an Invasion Risk of Fish Species in Qingdao Underwater World by Environmental DNA Metabarcoding

Environmental DNA(eDNA)metabarcoding has emerged as a potentially powerful tool to monitor invasive fish species.As an alternative(or complementary)tool for biodiversity monitoring,e DNA metabarcoding had been used to detect species in aquariums,which represents an important transit avenue for introducing non-indigenous species with high population densities.In this study,eDNA metabarcoding as well as morphological characterization were used to reveal the diversity of non-indigenous species in a large aquarium at Qingdao Underwater World.Environmental DNA metabarcoding of 14 water samples at five locations from the Big Water Tank detected 24 non-indigenous species and four putative non-indigenous operational taxonomic units(OTUs).In contrast,only 20 non-indigenous species were observed by morphological characterization.Some species undetected by morphological characterization,such as Oreochromis niloticus(Linnaeus,1758),are highly adaptable to various environments and/or have invaded preferred regions where they threaten native aquatic species.eDNA metabarcoding also detected seven local fishes that were not identified by morphological characterization.However,analysis of OTU diversity among stations and sample replications revealed that eDNA varied within and/or between stations.Increasing sampling effort as well as negative controls are required to increase the detection rate of species and to eliminate false-positive OTUs.

Effects of sampling strategies and DNA extraction methods on eDNA metabarcoding: A case study of estuarine fish diversity monitoring

Environmental DNA(eDNA)integrated with metabarcoding is a promising and powerful tool for species composition and biodiversity assessment in aquatic ecosystems and is increasingly applied to evaluate fish diversity.To date,however,no standardized eDNA-based protocol has been established to monitor fish diversity.In this study,we investigated and compared two filtration methods and three DNA extraction methods using three filtration water volumes to determine a suitable approach for eDNA-based fish diversity monitoring in the Pearl River Estuary(PRE),a highly anthropogenically disturbed estuarine ecosystem.Compared to filtration-based precipitation,direct filtration was a more suitable method for eDNA metabarcoding in the PRE.The combined use of DNeasy Blood and Tissue Kit(BT)and traditional phenol/chloroform(PC)extraction produced higher DNA yields,amplicon sequence variants(ASVs),and Shannon diversity indices,and generated more homogeneous and consistent community composition among replicates.Compared to the other combined protocols,the PC and BT methods obtained better species detection,higher fish diversity,and greater consistency for the filtration water volumes of 1000 and 2000 mL,respectively.All eDNA metabarcoding protocols were more sensitive than bottom trawling in the PRE fish surveys and combining two techniques yielded greater taxonomic diversity.Furthermore,combining traditional methods with eDNA analysis enhanced accuracy.These results indicate that methodological decisions related to eDNA metabarcoding should be made with caution for fish community monitoring in estuarine ecosystems.

Seasonal variations in the plant diet of the Chinese Monal revealed by fecal DNA metabarcoding analysis

The Chinese Monal(Lophophorus thuysii)is an alpine-obligate galliform species of global conservation priority.It has been listed as a first class protected wildlife species in China,requiring conservation actions during the 14 th Five-Year Plan period.However,the diet composition of Chinese Monal and its seasonal variations have rarely been studied,constraining the effective conservation of the species.Here,we investigated the plant diet composition of the Chinese Monal and its seasonal variations using a DNA metabarcoding approach on fecal samples.We collected 190 fecal samples of the Chinese Monals from the central Qionglai Mountains located in China,and analyzed the plant diet of this species using a DNA metabarcoding approach.Taxonomic profiling of higher plants in the fecal samples was performed using the second internal transcribed spacer(ITS2)amplicon.Downstream analyses,including rarefaction curves,nonmetric multidimensional scaling(NMDS)and permutational multivariate analysis of variance(PERMANOVA),were used to explore the seasonal variations in diet composition.The Chinese Monal foraged a wide range of plant recipes composed of 35 families and 83 genera throughout the year,with Brassicaceae,Apiaceae,and Poaceae as the dominant families,and Cardamine as the dominant genus.The species consumed plants from 62 genera from 28 families during the breeding season(n=81)and 66 genera from 31 families during the non-breeding season(n=109).Further,the plant diet composition significantly varied between the breeding and non-breeding seasons,especially for the frequency of occurrence and relative read abundances at genus level.Our study analyzed the plant diet of the Chinese Monal at a high resolution for the first time,and the results revealed that the seasonal variations in its plant diet composition was adapted to plant phenology and foraging strategy.Fritillaria species,a previously confirmed important food resource for the Chinese Monal,were not detected in any fecal samples,potentially due to overharvesting of Fritillaria bulbs for Traditional Chinese Medicine.Therefore,we highly recommend further restriction of herb gathering in Chinese Monal habitats to facilitate the conservation of this endangered species.Altogether,our study enriches essential ecological information for the Chinese Monal and also provides insights into conservation management for this endangered species.

NGS-metabarcoding revealing novel foraminiferal diversity in the Western Pacific Magellan Seamount sediments

Seamount is a unique deep-sea ecosystem widely distributed in the world.Its biodiversity is vibrant due to its specific geographical and hydrological conditions.However,the diversity and features of foraminifera in such an environment have rarely been studied.We extracted environmental DNA(eDNA)in sedime nts and amplified the partial small subunit ribo somal DNA(SSU rDNA)of fo raminife ra to understand the foraminiferal diversity from four sites in Magellan Seamount(Western Pacific Ocean).Partial S SU rDNA sequencing was conducted and 912979 foraminiferal reads were obtained and gathered into 266 operational taxonomic units(OTUs).In the available dataset,a high proportion of rare OTUs and low identity OTUs in each studied sample showed that the Magellan Seamount foraminiferal community might have a high genetic novelty.The relative abundance of foraminifera varied between replicates probably due to the existence of bias in amplification process and patchiness of the deep-sea floor.It showed that the Magellan Seamount has a relatively high benthic foraminiferal diversity characterized mainly by monothalamiids(76.37%of total reads)in association with rotaliids(19.03%of total reads),including planktic foraminiferal sequences(38.58%of rotaliids;7.36%of total reads).The remaining reads were assigned to miliolids(0.83%of total reads)and textulariids(0.66%of total reads),and 3.11%of total reads are unassigned to a specific family.The co mparative analysis with foraminiferal assemblage s from coastal and deep-sea environme nts indicated that seamounts could aggregate species from a nearby deep-sea.

Application of eDNA Metabarcoding for Detecting Anura in North China

eDNA metabarcoding is an advanced method formonitoring biodiversityproposed in recent years.By analyzing DNA in water,soil and sediment samples,the technology obtains species distribution and population quantity information.It was found that macrobarcode technology is more accurate than the traditional method in measuring the species richness of some groups.In Europe,America and South America,the relia bility of this technology in monitoring amphibian diversity in the wild was studied,and it was found to be better than traditional biodiversity monitoring methods in detecting species diversity.At present,amphibian monitoring mainly depends on various traditional methods,such as transects,drift fence traps,artificial shelters and mark-recapture.These monitoring techniques have many shortcomings,such as low accuracy and strong subjectivity of study results.These technologies have poor effects on rare,invasive and endangered species with strong concealment ability,low density and strong seasonality and are difficult to implement in sites inaccessible to people.Traditional monitoring technology also requires considerable investment of human and material resources,and the economic cost is relatively high,while eDNA metabarcoding ismore efficient and less costly,so it is important to use eDNA meta barcoding in amphibian monitoring in China.In this study,the eDNA meta barcoding and traditional line transect method(TLTM)were used to study the characteristics of the two methods in the Beijing-Tianjin-Hebeiregion.Repeated samplingwas conducted on 58 waterbodies in July 2019 and June 2020.After sequencing the samples using highthroughput sequencing technology,the differences between metabarcoding and commonly used TLTM surveys in detecting the diversity of four amphibians in North China were assessed.Our results showed that eDNA meta barcoding is more sensitive to the detection of the four amphibian species in the sampling area,and the combined use of eDNA metabarcoding and TLTM can improve the survey results of amphibians in the survey area to the greatest extent.In addition,in the process of species classification and identification of metabarcoding results,7 species of reptiles were detected,indicating that eDNA metabarcoding is also useful to detect reptiles.The results of this study indicate that metabarcoding in combination with TLTM can accurately estimate the diversityof amphibians in a short-term survey in North China and is also useful in reptile species detection.

鱼类环境DNA metabarcoding片段的近缘物种识别差异

已知的鱼类环境DNA(eDNA)metabarcoding片段均未被针对性考察其对近缘物种的适用性,实际使用过程中存在“物种丢失”风险。为筛选出物种识别率最高的片段,本研究比较了15个主流片段对106属(共935种)鱼类的识别差异。研究结果如下:(1)蛋白质编码基因(COI,片段15)的物种识别率最高,但其引物通用性最差;片段09、片段11、片段07、片段03、片段12的引物序列总平均遗传距离也较大,均存在eDNA低效扩增的风险;(2)片段长度影响物种识别率,核糖体基因中片段05、片段06、片段01、片段02及片段13的物种识别率较高;(3)非度量多维尺度分析(NMDS)显示,不同基因、同一基因不同片段的识别结果存在较大差异,应考虑多片段、多基因组合应用;片段01与片段02、片段05与片段06等在NMDS图上距离较近,存在相互替代性;(4)物种类群影响识别结果,eDNA研究仍需要进一步开发高识别率片段。综合物种识别率、引物通用性、NMDS分析等多方面因素,本研究推荐2×150 bp测序平台使用片段01(Mi Fish-U)、2×250 bp测序平台使用片段05(Ac12S),辅以片段13(Vert-16S-eDNA)等进行近缘鱼类多样性调查。本研究旨在为提高鱼类eDNA调查结果准确性提供一定技术支撑。

Comparison in diversity of eukaryotic algae in surface sediments from different functional sea areas of Qingdao coast,the Yellow Sea,China:a metabarcoding approach

Surface sediment samples were collected in three different functional sea areas in Qingdao coast,East China,including the inner Jiaozhou Bay,the Laoshan Coast,and the Amphioxus Reserve area.Diversity and community structure of eukaryotic algae especially those of phytoplankton resting stages were assessed by metabarcoding V4 region of the 18S rDNA.Biogenic elements including total organic carbon(TOC),organic matter(OM),total nitrogen(TN),total phosphorus(TP),and biogenic silicon(BSi)were analyzed.A total of 1496 eukaryotic operational taxonomic units(OTUs)were measured,including 207algal OTUs,which contributed to 13.84%of the total OTUs.Ninety-eight species in 8 phyla,24 classes of eukaryotic algae were detected.Among them,47 species have been reported to form resting stages,and 12 species are firstly recorded in Chinese coastal waters.Dinofl agellates dominated in both DNA reads and OTU richness,which contributed to 73.02%and 61.35%of the eukaryotic algal sequences and OTU richness,respectively.DNA reads,OTU richness and alpha diversity indexes of eukaryotic algae were higher in the Laoshan Coast,and lower in Jiaozhou Bay.Eukaryotic algal community differed in the three sea areas,which was dominated by chrysophytes in Jiaozhou Bay,by dinofl agellates in the Laoshan Coast,and co-dominated by dinoflagellates and chrysophytes in the Amphioxus Reserve area.Clustering analysis showed that the Laoshan Coast and the Amphioxus Reserve area are clustered together,while Jiaozhou Bay is clustered separately.Thirty-six harmful algal bloom(HAB)species were detected,and 10 species have been reported to form blooms in Jiaozhou Bay and the Qingdao coast before.Some of these species occurred widely and dominantly in this study,suggesting high potential risk of HABs in the Qingdao coastal area.

Community Characteristics Analysis of Eukaryotic Microplankton via ITS Gene Metabarcoding Based on Environmental DNA in Lower Reaches of Qiantang River, China

Eukaryotic microplankton plays an important role in water biotic community and in maintaining the stability of water ecosystems. Environmental DNA metabarcoding provides the opportunity to integrate traditional and emerging approaches to discover more new species, and develop molecular biotic indices that can be more rapidly, frequently, and robustly used in water quality assessments. In order to examine assemblages of eukaryotic microplankton in lower reaches of Qiantang River, ITS gene metabarcoding technology based on environmental DNA was carried out. As a result, various species of phytoplankton, fungi and zooplankton were annotated on. More phylum, classes and specieses of eukaryotic phytoplankton and zooplankton were found after compared communities taxa based on metabarcoding with that obtained from morphological examination. Nevertheless, Chlorophyceae was the most common assemblage both identified by using these two methods, also Mesocyclops leuckarti and Acanthocyclops bicuspidatus were both found to be the dominant species of Cyclopoida in the river. Additionally, the reads proportions of phytoplankton and zooplankton at the three freshwater sampling sites (Tonglu, Fuyang and Wenyan) decreased as temperature drop. Meanwhile, twenty classes of fungi were annotated on, of which the community characteristic was first researched in the river. There were significant spatial differences in values of Chao1 index for eukaryotic microplankton. Cluster analysis and Non-metric multidimensional scaling ordination further confirmed that the community composition of eukaryotic microplankton at class level for Jiashao-September sample had the most dissimilarity with the others.

Application of plant DNA metabarcoding of lake sediments for monitoring vegetation compositions on the Tibetan Plateau

Benefiting from the rapid development of environmental DNA(eDNA) technologies, sedimentary DNA(sedDNA)emerges as a promising tool for monitoring plant compositions in remote regions. The Tibetan Plateau(TP), renowned for its harsh environment and numerous ponds and lakes, presents a potentially demanding region for the application of sedDNA on vegetation investigations. Here, we used the g and h universal primers for the P6 loop region of the chloroplast trn L(UAA)intron to amplify plant DNA in surface sediments from 59 ponds and small lakes on the southwestern TP. The applicability and limitations of using plant DNA metabarcoding for modern vegetation monitoring and palaeo-vegetation reconstructions have been assessed by comparing sedDNA, pollen, and vegetation survey data. Our results showed that plant DNA metabarcoding recorded 186 terrestrial taxa, of which 30.1% can be identified at the species level. The plant sedDNA approach can effectively disclose the dominant plant taxa(including Asteraceae, Cyperaceae and Poaceae) and significant vegetation assemblages in the vicinity of the investigated sites. The number of taxa and taxonomic resolution of plant sedDNA exceeded that of pollen analysis(75 taxa detected, 5.3% can be identified at species level). Unlike pollen that retains a broad spectrum of regional plant signals(including Pinus and Artemisia), plant sedDNA mirrors very local plants, underscoring its utility in local vegetation monitoring and reconstructions. To conclude, plant DNA metabarcoding of(small) lake sediments warrant increased attention in the future for local vegetation monitoring and reconstructions on the TP.

DNA metabarcoding analysis of fungal community on surface of four root herbs

Objective: Angelicae Sinensis Radix(ASR, Danggui in Chinese), Cistanches Herba(CH, Roucongrong in Chinese), Ginseng Radix et Rhizoma(PG, Renshen in Chinese), and Panacis Quinquefolii Radix(PQ,Xiyangshen in Chinese), widely used as medicine and dietary supplement around the world, are susceptible to fungal and mycotoxin contamination. In this study, we aim to analyze their fungal community by DNA metabarcoding.Methods: A total of 12 root samples were collected from three main production areas in China. The samples were divided into four groups based on herb species, including ASR, CH, PG, and PQ groups. The fungal community on the surface of four root groups was investigated through DNA metabarcoding via targeting the internal transcribed spacer 2 region(ITS2).Results: All the 12 samples were detected with fungal contamination. Rhizopus(13.04%-74.03%),Aspergillus(1.76%-23.92%), and Fusarium(0.26%-15.27%) were the predominant genera. Ten important fungi were identified at the species level, including two potential toxigenic fungi(Penicillium citrinum and P. oxalicum) and eight human pathogenic fungi(Alternaria infectoria, Candida sake, Hyphopichia burtonii, Malassezia globosa, M. restricta, Rhizopus arrhizus, Rhodotorula mucilaginosa, and Ochroconis tshawytschae). Fungal community in ASR and CH groups was significantly different from other groups,while fungal community in PG and PQ groups was relatively similar.Conclusion: DNA metabarcoding revealed the fungal community in four important root herbs. This study provided an important reference for preventing root herbs against fungal and mycotoxin contamination.

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.

DNA metabarcoding the diet of Podarcis lizards endemic to the Balearic Islands

Dietary studies are essential to unravel the functioning of ecosystems and ultimately to understand biodiversity.This task,which at first may seem simple,becomes especially complex in those cases of omnivorous species with highly variable diets.In this regard,the emergence of next-generation DNA sequencing methodologies represents a powerful tool to address the problem.Here we implement a high-throughput metabarcoding strategy based on the analysis of four molecular markers aimed at sequencing both mitochondrial(animal prey)and chloroplast(diet plants)genome fragments from fecal samples of two lizard species endemic to the Balearic Archipelago(Podarcis lilfordi and P.pityusensis)obtained through non-invasive methods.The results allowed for the characterization of their diets with a high degree of taxonomic detail and have contributed a large number of new trophic records.The reported diets are based mainly on the consumption of arthropods,mollusks and plants from a diversity of taxonomic orders,as well as carrion and marine subsidies.Our analyses also reveal inter-and intra-specific differences both in terms of seasonality and geographical distribution of the sampled lizard populations.These molecular findings provide new insights into the trophic interactions of these threatened endemic lizards in their unique and isolated ecosystems.