study aimed to provide a high quality photographic documentation of important morphological characters of S. siamkayai to which some notes on its life cycle are added. Furthermore we complete its molecular characterisation with the analysis of the D2-D3region of 28S rDNA. Because the natural
position for the D2-D3region marker. The morphological and morphometric variability between some of the species have not been tested molecularly and some taxa, such as H. femina and H. silvaticus Bernard & Niblack, 1982 , have been separated mainly by the presence of a spermatheca filled with sperm
10 min. Four nematodes were used for molecular analysis. Two sets of primers (synthesised by Invitrogen) were used in the PCR analyses to amplify the 28S D2-D3 regions and ITS of rDNA. The 28S D2-D3region was amplified with the forward primer D2A (5′-ACA AGT ACC GTG AGG GAA AGT TG-3′) and the
F194 (Ferris et al ., 1993) and the reverse primer 5368r (Vrain, 1993), and the 28S D2-D3region was amplified with the forward primer D2A and the reverse primer D3B (De Ley et al ., 1999). PCR conditions were as described by Ye et al . (2007) and Li et al . (2008). PCR products were separated
sediments in the Sea of Japan. For both species, partial sequences of cytochrome oxidase c subunit I ( COI ) and D2-D3region of the 28S rDNA are provided.
Material and methods
Samples were collected from two subtidal localities on the coast of Vladivostok, Peter the Great Bay (the Sea of Japan
surveyed. The species were identified based on morphology and morphometrics and further characterised based on fragment sequences of the 28S rRNA of the D2-D3region. The region revealed variations in sequencing information that supports the morphological identification. In this work, six Pratylenchus spp
, 1971 (Zhuo et al. , 2010). The new species differs from A. menthae mainly by the number of lateral lines (4 vs 2).
Molecular characterisation and phylogeny
The sequences of full length ITS (1069 bp, isolate 8503, GenBank accession number FJ768943) and partial region 28S D2/D3region
well resolved and the phylogenetic analyses did not
support it as a sister group to Longidorus as previously inferred from morphology.
Secondary structure models were constructed for the D2/D3region of LSU rRNA for all
studied species. It was found that sequence-based and structural
three molecular markers were able to discriminate all Hirschmanniella species. Nevertheless, in this study, the D2-D3region was the easiest, fastest and most successful region to be amplified, a finding which is in agreement with De Ley et al. (2005).
A survey of
placement of R. arabocoffeae and R. duriophilus
among R. similis suggests that the burrowing nematode could be a species complex.
Limited information on the variability of the 18S and D2D3region gives an indication of
their possible value, besides ITS1-5.8-ITS, as molecular markers within