Irma Tandingan De Ley

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

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.

DESS: a versatile solution for preserving morphology and extractable DNA of nematodes

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.

Linking DNA sequences to morphology: cryptic diversity and population genetic structure in the marine nematode Thoracostoma trachygaster (Nematoda, Leptosomatidae)

Derycke, S., De Ley, P., De Ley, I.T., Holovachov, O., Rigaux, A. & 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.

Nematodes from vernal pools in the Santa Rosa Plateau Ecological Reserve, California I. Hirschmanniella santarosae sp. n. (Nematoda: Pratylenchidae), a cryptic sibling species of H. pomponiensis Abdel-Rahman & Maggenti, 1987.

Hirschmanniella santarosae sp. n. is described from the largest vernal pool in the Santa Rosa Plateau Ecological Reserve, Murrieta, California, USA. The cryptic new species is morphologically very close to H. pomponiensis and H. gracilis as it can be distinguished from the former only by a more anterior excretory pore position and by more flattened as well as laterally expanded stylet knobs, whilst it differs from the latter in the greater distance from phasmid to tail tip and in the bursal alae ending near the phasmids rather than near the tail tip. Analysis of the rDNA small subunit (SSU) and D2D3 expansion segments of the large subunit (LSU) shows that H. santarosae sp. n. is genetically distinct, having respective sequence homology of 98.89% and 95.9% with H. pomponiensis for these loci. Congruence in SSU and D2D3 gene trees as well as unambiguous character autapomorphies further support the new species status of H. santarosae sp. n. and sibling relationship with H. pomponiensis. Although many more isolates and species will need to be studied before informative biogeographic analyses can be performed, the presently available sequence data suggest that some Hirschmanniella lineages have diversified independently on either side of the Atlantic.

Biodiversity of littoral nematodes from two sites in the Gulf of California

The Gulf of California, Mexico, consists of unique environmental conditions resulting in a rich array of biological diversity. Nematodes are present in most marine sediments and are probably the most abundant metazoans. This research analyses and compares nematode biodiversity in two shallow, littoral locations of the Gulf and compares the results with other Pacific and Atlantic faunae. Samples collected in Punta Estrella and Santa Clara were processed for nematode extraction by standard methods. A total of eighty genera were identified, sixty-seven occurring in coarser sediments at Punta Estrella and fifty-five in more silty sediments at Santa Clara. Nematode abundance was higher at the latter location, on average. The region was not especially high or low in diversity compared to other littoral sites, and was most similar to locations in Europe with a corresponding ecology but at temperate latitudes.

Description of Phasmarhabditis californica n. sp. and first report of P. papillosa (Nematoda: Rhabditidae) from invasive slugs in the USA

A new species of Phasmarhabditis and the known species P. papillosa were isolated from cadavers of invasive slugs in California. Phasmarhabditis papillosa is the type of the genus and has not previously been reported from the Americas. Both species are characterised based on morphology, morphometrics and molecular data. Molecular phylogenies were inferred from concatenated DNA sequence alignments of nearly complete SSU and the D2-D3 domains of the LSU rDNA. Phasmarhabditis californica n. sp. is characterised by a robust body, mature egg-laying specimens almost spindle-shaped when relaxed, 1.5 (1.3-1.8) mm long, tapering to a bluntly rounded anterior end with stoma about as long as lip region diam., six inner labial papillae and four outer cephalic papillae, pharynx with rounded to pyriform basal bulb, vulva located halfway along the body, hermaphroditic, didelphic, amphidelphic, and short, conoid tail constricted at one-third its length with prominent phasmids. Not a single male was found among five strains. Phasmarhabditis papillosa is gonochoristic and has a longer isthmus, pyriform basal bulb and longer, dome-shaped spicate female tail constricted halfway along its length. Sequence analysis revealed unambiguous autapomorphies for P. papillosa and P. californica n. sp. Phylogenetic analyses placed these two species in a monophyletic clade comprising Phasmarhabditis species and other gastropod-parasitic taxa. Morphology, genetic distance, reproductive strategy and nucleotide autapomorphies support the new taxon.

Morphological, molecular characterisation and phylogenetic position of Longidorus mindanaoensis n. sp. (Nematoda: Longidoridae) from a Philippine Avicennia mangrove habitat.

Longidorus mindanaoensis n. sp. is described from samples associated with riverine mangroves in the Philippines and is the first Longidorus species collected from such a habitat. Its relationship within the genus is inferred from molecular and morphological data. Phylogenetic inferences were performed for D2-D3 and SSU rDNA using appropriate phylogenetic algorithms. The new species is characterised by a thickened basal layer of the body cuticle with prominent spiral fibres, a dome-shaped continuous lip region, an oval to elliptical fovea with pore-like opening, the guiding ring at 2.2-2.5 lip region diam. from the anterior end, very weakly developed odontophore, all three pharyngeal gland nuclei at about the mid-point of the basal bulb, vulva at 30-39%, tail convex-conoid to hemispherical, shorter than anal body diam., males with 19-24 ventromedian supplements, and four juvenile stages, all with the replacement odontostyle at a short distance from the functional one. On the basis of amphid shape and structure, shape of anterior body region and general body shape, L. mindanaoensis n. sp. comes close to L. caespiticola, L. eridanicus, L. helveticus, L. macrosoma, L. poessneckensis, L. orongorongensis, L. waikouaitii and L. belondiroides. D2-D3 analysis placed the new species as sister to L. pisi and the two combined are sister clade to a monophyletic group that includes (L. poessneckensis (L. caespiticola (L. helveticus, L. macrosoma))). However, on the basis of SSU phylogeny, L. mindanaoensis n. sp. is positioned within a clade that includes (Longidorus sp. (L. mindanaoensis n. sp. (L. poessneckensis (L. helveticus, L. macrosoma)))). The ‘close’ relationship of the new species with L. pisi in the D2-D3 tree is incongruent with the widely different morphology of the two species. The other species that are close to the new species in the cladogram are in agreement with the morphology.

Acromoldavicus mojavicus n. sp. (Nematoda: Cephaloboidea) from the Mojave Desert, California

Acromoldavicus (Cephalobina, Cephaloboidea) with its highly distinctive lip region has only a single species, Acromoldavicus skrjabini Nesterov & Lisetskaya, originally described from Moldova and subsequently also detected at sites in the Middle East and near the Mediterranean. Herein, Acromoldavicus mojavicus n. sp. is described from sandy soil surrounding a Joshua tree (Yucca brevifolia) in a remote area of the Mojave Desert, California, USA. The lip region of A. mojavicus n. sp. is bilaterally symmetrical with three triangular probolae surrounded by three pairs of plate-like lips. The lip region is organised along similar lines as that of A. skrjabini, but differs in several respects, such as its larger size, presence of elongate posterior processes on each lip and division of the lateral lips into two lobes (excluding the dorso-sublateral guard processes). In addition, phylogenetic interpretation of sequence data from the large-subunit of ribosomal DNA provides further evidence for autapomorphies and separate species status for A. skrjabini and A. mojavicus n. sp. Characteristics shared with Cephaloboidea include the offset spermatheca and males with eight pairs of genital papillae. Both species of Acromoldavicus have a buccal capsule with a reduced gymnostom, a character that seems to be shared with the cephalobid Elaphonema and in part is a basis for placement of both genera in Elaphonematidae. The species A. mojavicus n. sp. exhibits additional similarities with Elaphonema spp. that further support this placement.

Baujardia mirabilis gen. n., sp. n. from pitcher plants and its phylogenetic position within Panagrolaimidae (Nematoda: Rhabditida)

Measurements, line drawings and scanning electromicrographs are provided of Baujardia mirabilis gen. n., sp. n., isolated from pitcher fluid of Nepenthes mirabilis from Thailand. The new genus differs from all known nematodes in having two opposing and offset spermatheca-like pouches at the junction of oviduct and uterus. It also differs from most known Rhabditida in having four cephalic setae instead of papillae. Phylogenetic analysis of small subunit rDNA sequence data robustly places the new genus within Panagrolaimidae as a sister taxon to Panagrellus. These unusual nematodes resemble Panagrellus in body size (1.8-2.7 mm in females, 1.3-1.9 mm in males) and in the monodelphic, prodelphic female reproductive system with thickened vaginal walls and prominent postvulval sac. However, they differ from Panagrellus in the characters mentioned above, in their comparatively longer stegostom and in the shape of the male spicules. Because of its aberrant characters, inclusion of this new genus in Panagrolaimidae requires changes to the family diagnosis.

Morphology, molecular characterisation and systematic position of the genus Cynura Cobb, 1920 (Nematoda: Plectida)

Descriptions of three known species of Cynura, i.e., C. cerambus, C. klunderi and C. papillata, are given, including SEM micrographs of C. cerambus and a tabular compendium for all species of the genus. The phylogenetic relationships of C. klunderi are inferred from molecular data. Bayesian analyses of small subunit (SSU) of rRNA sequences support a position nested among the Plectidae suggesting the secondary simplification in the morphology of pharyngeal valvular apparatus in Cynura and the ‘return’ from a terrestrial to a marine environment in this genus.