Improving the detection of rare native fish species in environmental DNA metabarcoding surveys

1. The presence of threatened or endangered species often strongly influences management and conservation decisions. Within the Murray–Darling Basin (MDB), Australia, the presence of threatened native fish affects the management and allocation of water resources. In New South Wales, these decisions are currently based on traditional fisheries data and a predictive MaxEnt model. However, it is important to verify the model's predictive power given the implication it may have, but this requires methods with a high detection sensitivity for rare species.
2. Although the use of environmental DNA (eDNA) monitoring, in particular eDNA metabarcoding, achieves a higher detection sensitivity compared with traditional methods, earlier surveys in the MDB have shown that the highly abundant and invasive common carp (Cyprinus carpio) can reduce detection probabilities for rare species. Consequently, a polymerase chain reaction (PCR) blocking primer designed to block the amplification of carp eDNA could increase the detection probabilities for rare native species while simultaneously reducing the required sampling effort and survey costs. Although PCR blocking primers are often used in ancient DNA and dietary studies, no aquatic eDNA metabarcoding study to date has evaluated the potential benefits of using PCR blocking primers.
3. A laboratory and field-based pilot study was used to address this knowledge gap and assess the impact of a blocking primer, targeting cyprinid fishes (including carp), on the detection probabilities of native species and the minimum sampling effort required.
4. Results showed that the inclusion of the blocking primer increased the detection probabilities for native species by 10–20% and reduced the minimum required sampling effort by 25–50%. These findings provide important insights into possible methods for optimizing eDNA metabarcoding surveys for the detection of rare aquatic species.

     

Rhizosphere microbiota assemblage associated with wild and cultivated soybeans grown in three types of soil suspensions

Soil microbial community composition is determined by the soil type and the plant species. By sequencing the V3-V4 region of the bacterial 16S rRNA gene amplicons, the current study assessed the bacterial community assemblage in rhizosphere and bulks soils of wild (Glycine soja) and cultivated (Glycine max) soybeans grown in the suspensions of three important soil types in China, including black, red and soda-saline-alkali soils. The alpha-diversity of the bacterial community in the rhizosphere was significantly higher than that of the bulk soils suggesting that bulk soil lacks plant nurturing effect under the current study conditions. Black and red soils were enriched with nitrifying and nitrogen-fixing bacteria but the soda-saline-alkali soil suspension had more denitrifying bacteria, which may reflect agronomic unsuitability of the latter. We also observed a high abundance of Bradyrhizobium and Pseudomonas, enriched cellulolytic bacteria, as well as a highly connected molecular ecological network in the G. soja rhizosphere soil. Taken all, the current study suggest that wild soybeans may have evolved to recruit beneficial microbes in its rhizosphere that can promote nutrients requisition, biostasis and disease-resistance, therefore ecologically more resilient than cultivated soybeans.     

环境DNA宏条形码技术应用于溪流和水库鱼类物种多样性监测的敏感性和有效性评估

环境DNA宏条形码(Environmental DNA metabarcoding, eDNA metabarcoding)技术在调查鱼类物种丰度和多样性方面具有快速、高效、环境友好等优势,但其敏感性和有效性需进一步评估。本研究选择位于瓯江水系上游一级支流松荫溪发源地的高碧溪和成屏水库作为调查水域,分别利用传统捕捞方法和eDNA宏条形码技术对2个水域的鱼类物种进行调查。结果表明：捕捞方法在2个水域共发现鱼类24种,88%鱼类物种（21种）在瓯江水系有记录,其中高碧溪采集到鱼类12种,隶属于4目8科11属,成屏水库采集到鱼类16种,隶属于3目4科14属。eDNA宏条形码技术在2个水域共检测到鱼类31种,有84%的物种（26种）在瓯江水系有记录,其中高碧溪共检测出28种,隶属于5目12科25属,成屏水库共检测出27种,隶属于6目11科25属。然而,2种调查方法在溪流中共同发现的鱼类仅1种,在水库中共同发现的鱼类也只有5种。此外,高碧溪Shannon多样性指数、Simpson多样性指数、Margalef丰度指数以及Pielou均匀度指数均高于成屏水库,且2种调查方式得出的结论较为一致。由此可...     

人工模拟条件下环境DNA宏条形码技术的定量分析初探

近年来,环境DNA宏条形码技术(eDNA metabarcoding)在水生生态系统生物多样性评估等相关领域中得到广泛应用,因其具有快速测算群落中物种丰度的潜能,eDNA宏条形码技术成为资源保护和管理中颇具应用前景的调查工具。虽然大量证据表明eDNA高通量测序获得的reads数与自然环境中生物相对数量具有相关性,但一直不能得到明确的量化关系结果。eDNA的富集、扩增过程中的偏倚等诸多不确定因素,制约了该技术在生物资源调查领域的推广应用。假定水体中的eDNA全部回收,且PCR扩增时不存在引物偏倚性,这种理想状态下的水体中eDNA组成与其高通量测序reads数是否存在线性关系？为此,本研究在实验室可控条件下,选择2个同属近缘的凡纳滨对虾(Penaeus vannamei)和墨吉对虾(Penaeus merguiensis),对其DNA样品进行不同比例混合,模拟从自然水体中富集到的eDNA复合样品,既保证了样品的回收率,又降低了引物偏倚的干扰。以此为模板,探究eDNA宏条形码技术检测种群相对数量的准确性。结果显示,当2个物种DNA模板浓度比例为1∶1时,高通量测序结果注释得到的2个物种reads数比值为13/24(墨吉对虾/凡纳滨对虾),可见,即使是同属近缘种间依然存在轻微的引物偏倚现象,引物偏移率为1.5%。同时,根据7个实验组获得的高通量测序结果注释得到的2个物种reads数比值与对应模板中2个物种DNA浓度比值之间的线性回归分析表明,水体中eDNA组成与其高通量测序reads数间呈明显线性关系,即y=0.0716x+0.7043 (r²=0.9824)。综上所述,本研究为验证eDNA宏条形码技术监测水生生物资源量的可行性提供了直接证据,也为后续DNA宏条形码技术的定量研究提供了思路。     

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。     

Combining permanent aerobiological networks and molecular analyses for large-scale surveillance of forest fungal pathogens: A proof-of-concept

Forest disease management relies principally on a preventive approach in which epidemiological surveillance plays a crucial role. However, efficient and cost-effective surveillance methods are not currently available for large spatial scales. Nevertheless, aerobiological networks have been set up for several decades in many countries to monitor pollen dispersal and provide real-time assessments of allergenic risk. Here, we suggest that the same approach could be used for the surveillance of forest pathogens. Using molecular methods, we analysed samples from 12 sites of the French aerobiological network, at different dates. Both metabarcoding by high-throughput sequencing (using two markers and two different bioinformatics approaches) and real-time PCR targeting eight important forest pathogens were conducted. To validate the approach, temporal and spatial trends of spore detection were compared with field disease data. The metabarcoding approach demonstrated that many fungal plant pathogens could be found in aerobiological samples. Moreover, five of the eight targeted forest pathogens were detected by real-time PCR, with temporal and spatial trends of spore capture consistent with field data. In particular, Hymenoscyphus fraxineus was detected at high frequency in aerobiological samples in the areas where ash dieback has been present for the longest period of time, and at lower frequency in areas with more recent invasion. Spore detection of seasonal pathogens showed a temporal pattern similar to that of disease reports. Overall, our study provides a proof of concept that permanent aerobiological networks combined with molecular methods may provide a useful tool for large-scale surveillance of forest pathogens.     

Metabarcoding and development of new real‐time specific assays reveal Phytophthora species diversity in holm oak forests in eastern Spain

The evergreen holm oaks (Quercus ilex subsp. ilex and Q. ilex subsp. ballota) are the most representative tree species in the Iberian peninsula and the main tree species in oak‐rangeland ecosystems (dehesas). Oak decline in western, central and southern parts of Spain has been associated with root rot caused by Phytophthora cinnamomi for decades. However, Phytophthora species such as P.  quercina and P. psychrophila have recently been found associated with Quercus decline in eastern Spain where calcareous soils are predominant. Soil and root samples from two Quercus forests presenting decline symptoms in two different geographical areas in eastern Spain (Carrascar de la Font Roja and Vallivana) were analysed by amplicon pyrosequencing. Metabarcoding analysis showed Phytophthora species diversity, and revealed that an uncultured Phytophthora taxon, named provisionally Phytophthora taxon ballota, was the predominant species in both areas. In addition, a real‐time PCR assay, based on the pyrosequencing results, was developed for the detection of this uncultured Phytophthora taxon, and also for the detection of P. quercina. TaqMan assays were tested on soil and root samples, and on Phytophthora pure cultures. The new assays showed high specificity and were consistent with metabarcoding results. A new real‐time PCR protocol is proposed to evaluate the implication of different Phytophthora spp. in oak decline in eastern Spain.     

Molecular analysis of Phytophthora diversity in nursery‐grown ornamental and fruit plants

The genetic diversity of Phytophthora spp. was investigated in potted ornamental and fruit tree species. A metabarcoding approach was used, based on a semi‐nested PCR with Phytophthora genus‐specific primers targeting the ITS1 region of the rDNA. More than 50 ITS1 sequence types representing at least 15 distinct Phytophthora taxa were detected. Nine had ITS sequences that grouped them in defined taxonomic groups (P. nicotianae, P. citrophthora, P. meadii, P. taxon Pgchlamydo, P. cinnamomi, P. parvispora, P. cambivora, P. niederhauserii and P. lateralis) whereas three phylotypes were associated to two or more taxa (P. citricola taxon E or III; P. pseudosyringae, P. ilicis or P. nemorosa; and P. cryptogea, P. erythroseptica, P. himalayensis or P. sp. ‘kelmania’) that can be challenging to resolve with ITS1 sequences alone. Three additional phylotypes were considered as representatives of novel Phytophthora taxa and defined as P. meadii‐like, P. cinnamomi‐like and P. niederhauserii‐like. Furthermore, the analyses highlighted a very complex assemblage of Phytophthora taxa in ornamental nurseries within a limited geographic area and provided some indications of structure amongst populations of P. nicotianae (the most prevalent taxon) and other taxa. Data revealed new host–pathogen combinations, evidence of new species previously unreported in Italy (P. lateralis) or Europe (P. meadii) and phylotypes representative of species that remain to be taxonomically defined. Furthermore, the results reinforced the primary role of plant nurseries in favouring the introduction, dissemination and evolution of Phytophthora species.     

Novel tools for early detection of a global aquatic invasive,the zebra mussel Dreissena polymorpha

1. This study presents a species‐specific DNA‐based marker for detection of the zebra mussel Dreissena polymorpha, recognized as one of the worst invasive species worldwide.
2. The marker was developed in silico and experimentally tested on environmental samples. Gel and capillary electrophoreses for visualization of the PCR products were compared.
3. Marker specificity and sensitivity were assessed in vitro by cross‐amplifications and serial dilutions, respectively. The method allows detecting at least 0.7 ng of Dreissena DNA per μL and cross‐species amplification was not found in any case.
4. Next‐generation sequencing (NGS) metabarcoding (PCR amplification and massive sequencing of a DNA barcode) was used as an independent method for verifying presence of Dreissena DNA molecules in environmental plankton samples collected from the south‐eastern Baltic Sea.
5. The consistency between NGS results reporting presence of Dreissena and positive PCR amplification of the marker from the plankton samples confirmed the efficacy of this highly reproducible, fast, cheap and technically easy method.

Copyright © 2016 John Wiley & Sons, Ltd.