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

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.

[1]  Alfried P Vogler,et al.  Sequence-based species delimitation for the DNA taxonomy of undescribed insects. , 2006, Systematic biology.

[2]  Mark L. Blaxter,et al.  Second-generation environmental sequencing unmasks marine metazoan biodiversity , 2010, Nature communications.

[3]  J. Vanfleteren,et al.  Mitochondrial DNA variation and cryptic speciation within the free-living marine nematode Pellioditis marina , 2005 .

[4]  N. Baeshen,et al.  Biological Identifications Through DNA Barcodes , 2012 .

[5]  J. Landry,et al.  A universal DNA mini-barcode for biodiversity analysis , 2008, BMC Genomics.

[6]  P. Francalacci,et al.  Population structure of the Monocelis lineata (Proseriata, Monocelididae) species complex assessed by phylogenetic analysis of the mitochondrial Cytochrome c Oxidase subunit I (COI) gene , 2009, Genetics and molecular biology.

[7]  Andrew P. Martin,et al.  Environmental DNA sequencing primers for eutardigrades and bdelloid rotifers , 2009, BMC Ecology.

[8]  J. Wiens Species delimitation: new approaches for discovering diversity. , 2007, Systematic biology.

[9]  Koen Martens,et al.  Lost sex : the evolutionary biology of parthenogenesis , 2009 .

[10]  A. Rogers,et al.  Development and evaluation of a DNA-barcoding approach for the rapid identification of nematodes , 2006 .

[11]  T. Schröder,et al.  Cryptic speciation in the cosmopolitan Epiphanes senta complex (Monogononta, Rotifera) with the description of new species , 2007, Hydrobiologia.

[12]  A. Lambert,et al.  ABGD, Automatic Barcode Gap Discovery for primary species delimitation , 2012, Molecular ecology.

[13]  O. Giere Meiobenthology: The Microscopic Motile Fauna of Aquatic Sediments , 1993 .

[14]  P. Taberlet,et al.  Environmental DNA , 2012, Molecular ecology.

[15]  W. Sung,et al.  Metagenetic community analysis of microbial eukaryotes illuminates biogeographic patterns in deep-sea and shallow water sediments. , 2012, Molecular ecology.

[16]  Holly M. Bik,et al.  Sequencing our way towards understanding global eukaryotic biodiversity. , 2012, Trends in ecology & evolution.

[17]  Diego Fontaneto,et al.  Independently Evolving Species in Asexual Bdelloid Rotifers , 2007, PLoS biology.

[18]  Edward Ayres,et al.  Molecular study of worldwide distribution and diversity of soil animals , 2011, Proceedings of the National Academy of Sciences.

[19]  P. Taberlet,et al.  Species detection using environmental DNA from water samples , 2008, Biology Letters.

[20]  H. Godfray Challenges for taxonomy , 2002, Nature.

[21]  U. Jondelius,et al.  Patterns of Diversity in Soft-Bodied Meiofauna: Dispersal Ability and Body Size Matter , 2012, PloS one.

[22]  P. Taberlet,et al.  DNA from soil mirrors plant taxonomic and growth form diversity. , 2012, Molecular ecology.

[23]  K. I. Ugland,et al.  Changes in the root‐associated fungal communities along a primary succession gradient analysed by 454 pyrosequencing , 2012, Molecular ecology.

[24]  Matthew J. Colloff,et al.  Ecological assessment of estuarine sediments by pyrosequencing eukaryotic ribosomal DNA , 2010 .

[25]  E. Herniou,et al.  Extreme levels of hidden diversity in microscopic animals (Rotifera) revealed by DNA taxonomy. , 2009, Molecular phylogenetics and evolution.

[26]  R. Giblin-Davis,et al.  Ultrasequencing of the meiofaunal biosphere: practice, pitfalls and promises , 2010, Molecular ecology.

[27]  J. Sites,et al.  Delimiting species: a Renaissance issue in systematic biology , 2003 .

[28]  K. Martens,et al.  Cryptic Species in Putative Ancient Asexual Darwinulids (Crustacea, Ostracoda) , 2012, PloS one.

[29]  S. Derycke,et al.  Integrative taxonomy in two free-living nematode species complexes , 2008 .

[30]  N. Yoccoz The future of environmental DNA in ecology , 2012, Molecular ecology.

[31]  P. Hebert,et al.  Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species , 2003, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[32]  S. Rice,et al.  An analysis of species boundaries and biogeographic patterns in a cryptic species complex: the rotifer--Brachionus plicatilis. , 2006, Molecular phylogenetics and evolution.

[33]  A. Chao Nonparametric estimation of the number of classes in a population , 1984 .

[34]  Joshua Adams,et al.  Using Population Genetic Theory and DNA Sequences for Species Detection and Identification in Asexual Organisms , 2010, PloS one.