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A solution containing dimethyl sulphoxide, disodium EDTA, and saturated NaCl (abbreviated here as DESS) was tested for various applications in the preservation of nematodes for combined morphological and molecular analyses. The solution can be used to preserve individual nematodes, nematode extracts, or entire soil/sediment samples. Preserved material can be easily stored for months at room temperature, shipped by mail, or carried in luggage. Morphological features are usually well preserved; specimen quality being comparable to formalin-based fixatives and much better than ethanol fixation. Specimens can be transferred to glycerin with little or no modification of traditional protocols. Unlike formalin-preserved material, routine PCR can be performed on individual specimens after any of these procedures with success rates and amplification sizes comparable to PCR of fresh specimens. At this point we have no data on long-term preservation quality. Nevertheless, DESS solution clearly enhances and simplifies a wide range of nematological studies due to its combined suitability for morphological and molecular analyses, as well as its less hazardous chemical properties.

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.

Moens, T. (2010). Linking DNA sequences to morphology: cryptic diversity and population genetic structure in the marine nematode Thoracostoma trachygaster (Nematoda, Leptosomatidae).-Zoologica Scripta, 39, 276-289. Recent taxonomic and population genetic studies have revealed the presence of substantial cryptic diversity through sequence analysis of nematode morphospecies classified in different major clades. Correct interpretations of intra-and interspecific genetic variation require certainty about the conspecificity of the sequenced specimens, which in turn must depend on appropriate protocols with built-in verifiability procedures. In this study, we performed a population genetic study in the free-living marine nematode Thoracostoma trachygaster, a member of one of the earliest major clades to diverge in nematode phylogeny. We collected 367 nematodes from 11 populations located in the Californian Bight, all of which were video captured before DNA extraction to document and verify their individual morphology. Sequences for the cytochrome c oxidase subunit 1 (COI), D2D3 and 18S genes showed eight deeply divergent clades, and using a reverse taxonomy approach, six of these clades proved to be other morphospecies than T. trachygaster. Phylogenetic analyses of COI, internal transcribed spacer and D2D3 showed evidence for two sympatrically distributed cryptic species within the morphospecies T. trachygaster. Population genetic analyses of the most widespread cryptic species showed a moderate genetic structuring (F ST = 0.28), and 18% of this genetic variation was caused by differences between populations north and south of Point Conception. Within the southern Californian Bight, some genetic differentiation could be attributed to differences between populations north and south of Malibu, supporting the idea of a barrier to gene flow near Los Angeles region. The results for T. trachygaster support the contention that species diversity within freeliving nematodes is underestimated, and that dispersal of marine nematodes from tidal environments associated with kelp holdfasts is substantial at scales of a few 100 km.

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