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The widely used small subunit 18S rDNA molecule greatly underestimates true diversity in biodiversity surveys of the meiofauna.

ABSTRACT: Molecular tools have revolutionized the exploration of biodiversity, especially in organisms for which traditional taxonomy is difficult, such as for microscopic animals (meiofauna). Environmental (eDNA) metabarcode surveys of DNA extracted from sediment samples are increasingly popular for surveying biodiversity. Most eDNA surveys use the nuclear gene-encoding small-subunit rDNA gene (18S) as a marker; however, different markers and metrics used for delimiting species have not yet been evaluated against each other or against morphologically defined species (morphospecies). We assessed more than 12,000 meiofaunal sequences of 18S and of the main alternatively used marker [Cytochrome c oxidase subunit I (COI) mtDNA] belonging to 55 datasets covering three taxonomic ranks. Our results show that 18S reduced diversity estimates by a factor of 0.4 relative to morphospecies, whereas COI increased diversity estimates by a factor of 7.6. Moreover, estimates of species richness using COI were robust among three of four commonly used delimitation metrics, whereas estimates using 18S varied widely with the different metrics. We show that meiofaunal diversity has been greatly underestimated by 18S eDNA surveys and that the use of COI provides a better estimate of diversity. The suitability of COI is supported by cross-mating experiments in the literature and evolutionary analyses of discreteness in patterns of genetic variation. Furthermore its splitting of morphospecies is expected from documented levels of cryptic taxa in exemplar meiofauna. We recommend against using 18S as a marker for biodiversity surveys and suggest that use of COI for eDNA surveys could provide more accurate estimates of species richness in the future.

SUBMITTER: Tang CQ

PROVIDER: S-EPMC3479535 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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The widely used small subunit 18S rDNA molecule greatly underestimates true diversity in biodiversity surveys of the meiofauna.

Tang Cuong Q CQ Leasi Francesca F Obertegger Ulrike U Kieneke Alexander A Barraclough Timothy G TG Fontaneto Diego D

Proceedings of the National Academy of Sciences of the United States of America 20120917 40

Molecular tools have revolutionized the exploration of biodiversity, especially in organisms for which traditional taxonomy is difficult, such as for microscopic animals (meiofauna). Environmental (eDNA) metabarcode surveys of DNA extracted from sediment samples are increasingly popular for surveying biodiversity. Most eDNA surveys use the nuclear gene-encoding small-subunit rDNA gene (18S) as a marker; however, different markers and metrics used for delimiting species have not yet been evaluate ...[more]

PMID: 22988084

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Project description:BACKGROUND: Microeukaryotes are an effective indicator of the presence of environmental contaminants. However, the characterisation of these organisms by conventional tools is often inefficient, and recent molecular studies have revealed a great diversity of microeukaryotes. The full extent of this diversity is unknown, and therefore, the distribution, ecological role and responses to anthropogenic effects of microeukaryotes are rather obscure. The majority of oil from oceanic oil spills (e.g., the May 2010 accident in the Gulf of Mexico) converges on coastal ecosystems such as mangroves, which are threatened with worldwide disappearance, highlighting the need for efficient tools to indicate the presence of oil in these environments. However, no studies have used molecular methods to assess the effects of oil contamination in mangrove sediment on microeukaryotes as a group. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the population dynamics and the prevailing 18S rDNA phylotypes of microeukaryotes in mangrove sediment microcosms with and without oil contamination, using PCR/DGGE and clone libraries. We found that microeukaryotes are useful for monitoring oil contamination in mangroves. Our clone library analysis revealed a decrease in both diversity and species richness after contamination. The phylogenetic group that showed the greatest sensitivity to oil was the Nematoda. After contamination, a large increase in the abundance of the groups Bacillariophyta (diatoms) and Biosoecida was detected. The oil-contaminated samples were almost entirely dominated by organisms related to Bacillariophyta sp. and Cafeteria minima, which indicates that these groups are possible targets for biomonitoring oil in mangroves. The DGGE fingerprints also indicated shifts in microeukaryote profiles; specific band sequencing indicated the appearance of Bacillariophyta sp. only in contaminated samples and Nematoda only in non-contaminated sediment. CONCLUSIONS/SIGNIFICANCE: We believe that the microeukaryotic targets indicated by our work will be of great applicability in biomonitoring hydrocarbons in mangroves under oil contamination risk or during recovery strategies.

Project description:Many studies have now confirmed that sperm DNA fragmentation (SDF) is associated with a poorer outcome of some forms of assisted reproduction technology. For this reason, SDF is an important parameter to evaluate in male fertility assessment. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay coupled to flow cytometry is one of the most promising methods for SDF quantification. Several kits for the detection of DNA fragmentation are currently available on the market and all are recommended as equally appropriate to quantify SDF. In this work we compared for the first time the efficacy of two different types of TUNEL kits for SDF quantification: one using an indirect antibody-based labeling system (BrdUTP/fluorescein-anti-BrdUTP) and another using a direct labeling system (fluorescein-dUTP). We demonstrated that TUNEL indirect labeling system largely underestimates SDF when compared with the direct labeling, the differences ranging from 19.2% to 85.3% (p<0.05, n = 22). We observed that these differences were most pronounced among dead spermatozoa where indirect labeling stained 40.1% [23.6%, 58.2%] and the direct system 65.7% [36.5%, 90.9%] (n = 10, p<0.05). Interestingly, we found that both systems stained the living spermatozoa with the same efficiency. We showed that the differences are due to the steric hindrance of the antibody during its binding to the BrdUTP. Indeed, after sperm DNA decondensation, the percentages of TUNEL positivity increased significantly from 46.3% [31.8%, 61.7%] to 97.5% [96.1%, 98.8%] (p<0.05, n = 5). Our results are important for future use of TUNEL in clinical practice. Laboratories relying on the use of an antibody-based system heavily underestimate SDF, most particularly in infertile patients with reduced sperm motility. As a consequence, the kit using BrdUTP/fluorescein-anti-BrdUTP should not be recommended as a method to assay DNA damage in semen. This study represents one further step in the standardization of TUNEL among laboratories.

Dataset Information

The widely used small subunit 18S rDNA molecule greatly underestimates true diversity in biodiversity surveys of the meiofauna.

Publications

The widely used small subunit 18S rDNA molecule greatly underestimates true diversity in biodiversity surveys of the meiofauna.

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