An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding

Abstract

Molecular surveys of meiofaunal diversity face some interesting methodological challenges when it comes to interstitial nematodes from soils and sediments. Morphology-based surveys are greatly limited in processing speed, while barcoding approaches for nematodes are hampered by difficulties of matching sequence data with traditional taxonomy. Intermediate technology is needed to bridge the gap between both approaches. An example of such technology is video capture and editing microscopy, which consists of the recording of taxonomically informative multifocal series of microscopy images as digital video clips. The integration of multifocal imaging with sequence analysis of the D2D3 region of large subunit (LSU) rDNA is illustrated here in the context of a combined morphological and barcode sequencing survey of marine nematodes from Baja California and California. The resulting video clips and sequence data are made available online in the database NemATOL (http://nematol.unh.edu/ ). Analyses of 37 barcoded nematodes suggest that these represent at least 32 species, none of which matches available D2D3 sequences in public databases. The recorded multifocal vouchers allowed us to identify most specimens to genus, and will be used to match specimens with subsequent species identifications and descriptions of preserved specimens. Like molecular barcodes, multifocal voucher archives are part of a wider effort at structuring and changing the process of biodiversity discovery. We argue that data-rich surveys and phylogenetic tools for analysis of barcode sequences are an essential component of the exploration of phyla with a high fraction of undiscovered species. Our methods are also directly applicable to other meiofauna such as for example gastrotrichs and tardigrades.

DNA Barcoding of Freshwater Fishes from Lake Laut Tawar, Aceh Province, Indonesia

“…DNA barcoding has already been applied in many animals such as birds (Hebert et al 2004b), springtails invertebrates (Hogg and Hebert 2004), skipper butterflies (Hebert et al 2004a), blowflies (Whitworth et al 2007), leaf beetles (Jurado-Rivera et al 2009), nematodes (De Ley et al 2005), amphibians (Vences et al 2005a), ants (Smith et al 2005), crustaceans (Lefébure et al 2006), and scuttle flies (Boehme et al 2010). In addition, DNA barcodes have been obtained for more than 8000 species of fish and the COI sequences deposited in the Barcode of Life Data Systems (BOLD) online workbench and repository (Ragnasingham and Hebert 2007).…”

The application of DNA sequence data for the identification of benthic nematodes from the North Sea

“…67 % in Pereira et al 2010). Earlier studies using the same fragment reported ''unreliable'' amplification results (Bhadury et al 2006a) as well as very high success rates (De Ley et al 2005). Due to the fact that a marker for molecular species identification should be easy to amplify, PCR optimization represents an essential task for further studies.…”

“…The primer pair D2A and D3B (De Ley et al 2005) was used for the amplification of the 28S rDNA: D2-D3 region. Amplicons were generated using PCR beads (illustra puReTaq Ready-To-Go PCR Beads, GE Healthcare UK Limited, Buckinghamshire, United Kingdom) in 25-ll reactions, filled up with 2 ll of DNA extract, 0.5 ll of primer D2A (20 pm/ll), 0.5 ll of primer D3B (20 pm/ll) and 22 ll of ddH 2 0.…”

“…However, the combination of molecular and morphological approaches has been shown to be most effective in species identification (De Ley et al 2005) as well as disentangling species complexes within the Nematoda in general (e.g. Derycke et al 2008;Gutiérrez-Gutiérrez et al 2013); for example, Pereira et al (2010) successfully identified nematodes from Baja California using partial 18S and 28S rDNA sequence data in combination with morphological analysis.…”

“…Litvaitis et al 2000;De Ley et al 2005;Pereira et al 2010) as well as internal transcribed spacers (ITS; Hugall et al 1999;Elbadri et al 2002;Félix et al 2014). Here, various studies showed that the D2-D3 region of the 28S rDNA represents one of the most suitable and promising markers for a valid nematode identification, which can be used to delimitate even closely related species (De Ley et al 2005;Pereira et al 2010).…”