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81Russian Journal of Nematology, 2014, 22 (1), 49 - 66
Four new geographical records of rhabditid nematodes (Nematoda: Rhabditida: Rhabditomorpha) from Iran with a note on the phylogenetic position of Pelodera
112 3
Ebrahim Shokoohi , Sara Mehdizadeh , Negin Amirzadi and Joaquín Abolafia
1 Department of Plant Protection, College of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran
e-mail: [email protected] 2 Young Researchers and Elite Club, Damghan Branch, Islamic Azad University, Damghan, Iran 3 Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén. Campus "Las Lagunillas" s/n. 23071-
Jaén, Spain
Accepted for publication 18 March 2014
Summary. During a survey of soil nematodes in Iran, four new records of rhabditids, namely Cuticularia oxycerca (de Man, 1895) Andrassy, 1983; Mesorhabditis acuticauda Ahmad, Shah & Mahamood, 2010; Pelodera pseudoteres Schulte, 1989 and Protorhabditis oxyuroides Sudhaus, 1974 were recovered. Molecular analysis of an Iranian population of P. pseudoteres based on 18S rDNA showed three nucleotide differences compared with a population from the USA (strain SB116; 1648 bp). By contrast, a strain of phylogenetically close species, P. teres, also from the USA (strain EM437; 1646 bp), showed 19 nucleotide differences. Phylogenetic analysis also places the Iranian strain closest to P. pseudoteres from the USA and confirms that molecularly characterised Pelodera species form a monophyletic clade. In addition, illustrations and measurements for all species and SEM observations for M. acuticauda are provided. All species are recorded for the first time from Iran.
Key words: Cuticularia, Iran, Mesorhabditis, Pelodera, Protorhabditis, phylogeny, SEM, taxonomy, 18S rDNA.
The nematodes of Rhabditina Andrassy, 1974 are largely free-living but ecologically diverse, including multiple transitions to parasitism (Sudhaus, 2010). The latest revision of this family was provided by Andrassy (2005) and Sudhaus (2011), who listed all genera and their species.
The genus Mesorhabditis Osche, 1952 is distributed all over the world (Andrassy, 2005). The members of this genus are mainly terrestrial but could be found in other habitats (e.g. fallen leaves, plant residues and fresh water; Andrassy, 2005). Sudhaus (1976) considered sixteen species of this genus as valid. However, the first deep revision on this genus was provided by Sudhaus (1978), who revised six species and described three as new species. Andrassy (1983, 1984, 2005) assumed 21 species as valid within the genus Mesorhabditis. Finally, Sudhaus (2011) divided this genus into two groups including Monhystera and Spiculigera, having 34 valid species in total. The most recent
species from this genus was described by Ahmad et al. (2010).
The genus Pelodera Schneider, 1866 has been studied by some scientists (Sudhaus, 1991; Andrassy, 2005; Shokoohi & Abolafia, 2011). Members of this genus are mainly terrestrial; however, some species may be found in decayed and freshwater habitats (Andrassy, 2005). One species of the genus Pelodera was transferred to the genus Rhomborhabditis Andrassy, 1983, as Pelodera pseudoteres Schulte, 1989. Later, Sudhaus (2011) put this species as valid within the Teres-group of the genus Pelodera.
The genus Cuticularia van der Linde, 1938 is less studied, being a rare taxon within the Rhabditina. The first revision of this genus was provided by Eroshenko (2002), describing C. annulata as a new species. This genus comprises seven species (Andrassy, 2005), with C. oxycerca being the species with the most distribution worldwide.
Finally, the genus Protorhabditis (Osche, 1952) Dougherty, 1953 has been revised by Sudhaus (1991) and Andrássy (2005). The last revision on this genus including morphometric table and key to species was provided by Abolafia & Peña-Santiago (2007), while Sudhaus (2011) provided a list of species, considering 15 valid species under this genus.
Concerning the Rhabditina in Iran, some species of this group have been reported previously (Shokoohi & Abolafia, 2011). This paper documents four new reports of rhabditid species from Iran, Cuticularia oxycerca (de Man, 1895) Andrássy, 1983, Mesorhabditis acuticauda Ahmad, Shah & Mahamood, 2010, Pe lode ra pseudoteres Schulte, 1989 and Protorhabditis oxyuroides Sudhaus, 1974. Phylogenetic placement of the Iranian P. pseudoteres is given based on 18S rDNA sequences. The classification of Andrássy (2005) is followed herein.
MATERIALS AND METHODS
Studies on Morphology. The nematodes were extracted from soil samples by the Baermann funnel technique (Baermann, 1917) and a centrifugation method according to Jenkins (1964). Worms were fixed with hot 4% formaldehyde solution and processed to anhydrous glycerin according to De Grisse (1969). Measurements were taken using an ocular micrometer and drawings were made using a drawing tube attached to the microscope (Olympus CH-2). For SEM, fixed specimens were hydrated (1 day), dehydrated in a graded ethanol series (25, 30, 50, 70, 95, 100%) and finally in acetone (100%), critical point dried, coated with gold and observed with a JEOL JSM-5800 microscope operating at 4kV. The terminology used for morphology of stoma and spicules follows the proposals by De Ley et al. (1995) and Abolafia & Peña-Santiago (2006), respectively.
Phylogenetic analysis. The sequences of several species belonging to the family Rhabditidae used for phylogenetic analysis were found in the GenBank. DNA extraction was done using an AccuPrep Genomic DNA Extraction Kit (Bioneer Corporation, Korea) (http://www.bioneer.com) according to the manufacturer's instructions. Specimens were picked into 1.5 ml tube containing 5 ^l of double distilled water. The tube was frozen in liquid nitrogen and was pulverized using a vortex; 200 ^l Tissue Lysis buffer (TL) and 20 ^l proteinase K (20mg ml-1) were then added. The homogenate was incubated at 60°C for 2 h. The supernatant was extracted and
stored at -20°C. The forward primer SSU_F_04 (5'-GCTTGTCTCAAAGATTAAGCC-3') and the reverse primer SSU_R_26 (5'-CATTCTTGGCAAA TGCTTTCG-3') (Blaxter et al, 1998) were used in the PCR reactions for amplification of the partial 18S region (~900bp). PCR was conducted with 10 ^l of the extracted DNA, 4 ^l of PCR Master Mix (Kawsar Biotech Co., Iran), 1 ^l of each primer (10 pmol ^l-1) and ddH2O to a final volume of 25 ^l. The amplification was carried out using an Eppendorf master cycler gradient (Eppendorf, Hamburg, Germany), which was 3 min at 94°C, 37 cycles of 45 s at 94°C, 45 s at 56°C and 1 min at 72°C, and finally one cycle of 6 min at 72°C followed by a holding temperature of 4°C. After DNA amplification, 5 ^l of product was loaded on a 1% agarose gel (40 mM Tris, 40 mM boric acid, and 1mM EDTA) to check the quality of the DNA product. The bands were stained with 50 mM ethidium bromide and visualised and photographed on 1% agarose gel under a UV transilluminator. Product was stored at -20°C prior to sequencing. PCR product was purified for sequencing and sequenced with the primers that were used for the amplification. Sequencing was performed in both directions. The DNA sequence was edited using Chromas version 1.45 (McCarthy, 1997). Sequencing reactions were performed by the Bioneer Co. (South Korea) (http://eng.bioneer.com). Primers for the sequencing reaction were those used in the amplification step. The sequence was confirmed in both directions and repeated. Available sequences for other Rhabditomorpha and outgroup were obtained from NCBI GenBank. The ribosomal SSU sequences were aligned using BioEdit (Hall, 1999). Phylogenetic trees were generated using the Bayesian inference method as implemented in the program Mr Bayes 3.1.2 (Ronquist & Huelsenbeck, 2003). The analysis under GTR model was initiated with a random starting tree and run with the Markov chain Monte Carlo (MCMC) for 106 generations. The distance matrix option of Mega 5 (Tamura et al., 2011) was used to calculate genetic distances according to the Maximum composite Likelihood of sequence evolution. The Bayesian tree was visualised with the TreeView program.
For phylogenetic analysis of 18S rDNA, Panagrolaimus detritophagus (EU543176) was used as outgroup. This selection was based on earlier studies (De Ley & Blaxter, 2002; Kiontke & Fitch, 2005; van Megen et al., 2009). The original partial 18S sequence of P. pseudoteres is deposited in the GenBank under accession number KC509908.
Table 1. Measurements of Cuticularia oxycerca (de Man, 1895) Andrassy, 1983 and Mesorhabditis acuticauda Ahmad, Shih & Mahamood, 2010. All measurements in |im in the form: mean ± standard deviation (range).
Species C. oxycerca M. acuticauda
Locality Shahrood Northern Tehran
Province Semnan Tehran
Rhizosphere Mallow Grass
n 6$$ 9ââ 10$$ 10^
L 613.7 ± 44.7 (574-677) 546 ± 61.4 (487-675) 404±35.2 (353-467) 288±75.1 (167-357)
a 14.5 ± 1.5 (12.1-15.9) 14.7 ± 0.8 (14-15.8) 15.6±1.8 (12.8-18.5) 13.9±3.9 (7.2-16.9)
b 3.5 ± 0.2 (3.1-3.8) 3.3 ± 0.2 (3-3.6) 4.6±0.4 (3.9-5.0) 3.6±1.0 (1.9-4.6)
c 37.3 ± 6.7 (29.2-45.8) 20.8 ± 2.2 (18-25) 10.4±1.2 (8.3-12.1) 18.5±6.4 (8.2-24.6)
c' 0.8 ± 0.1 (0.7-1) 1 ± 0.0 (1-1.1) 3.8±0.5 (3.2-4.7) 1.6±0.2 (1.1-1.9)
V 57.4 ± 1.7 (55-57) - 77.8±5.8 (66-85) -
Lip region diameter 11.8 ± 0.8 (11-13) 11 ± 0.7 (10-12) 6.0±0.8 (5-7) 5.3±0.6 (5-6)
Stoma 20.7 ± 1.2 (20-23) 20.8 ± 1.3 (19-23) 14.4±1.0 (13-16) 13.2±1.4 (11-15)
Pharyngeal corpus 97.2 ± 3.3 (93-103) 93.8 ± 7.5 (86-107) 44.3±2.7 (40-48) 41.0±4.2 (32-44)
Isthmus 45.7 ± 2.8 (43-51) 41.8 ± 3.2 (34-45) 21.0±2.8 (15-25) 21.0±3.5 (17-28)
Bulb 34 ± 1.7 (31-35) 31 ± 2.4 (28-36) 20.7±1.2 (19-23) 18.4±1.9 (15-20)
Pharynx length 176.7 ± 8.9 (165-187) 165.6 ± 12.4 (151-190) 87.6±3.9 (82-94) 81.7±5.3 (70-88)
Nerve ring - ant. end 110 ± 7.6 (102-120) 103 ± 11 (92-124) 69.5±4.4 (66-78) 63.2±5.8 (54-72)
Excretory pore - ant. end 113 ± 10.3 (103-128) 110 ± 9.2 (102-127) 80.9±5.8 (74-869 73.5±8.0 (65-84)
Deirid - ant. end 147 ± 9.9 (140-154) 135 ± 5.7 (131-139) ? 88.5±11.6 (80-97)
Annuli width - - 0.7±0.2 (0.7-1.3) 0.7±0.2 (0.7-1.3)
Cuticle thickness 6.5 ± 0.5 (6-7) 5.7 ± 0.5 (5-6) 0.7±0.2 (0.7-1.3) 0.7±0.2 (0.7-1.3)
Body diameter: neck base 19.3 ± 2.1 (17-23) 18 ± 1.3 (16-20) 7.6±0.7 (7-9) 6.5±0.7 (6-8)
Body diameter: midbody 42.7 ± 5.1 (36-49) 37 ± 3.9 (32-44) 26.1±2.1 (22-28) 20.9±1.3 (19-23)
Body diameter: anus 20 ± 1.1 (18-21) 25 ± 1.7 (22-28) 10.4±0.9 (9-11) 10.5±1.7 (9-13)
Anterior genital branch 211, 243 377.0 ±74.4 (319-461) 165, 142 212, 249
Posterior genital branch 200, 215 - - -
Rectum 27.3 ± 1.5 (25-29) 25.4 ± 2.4 (21-29) 19.6±2.8 (16-24) ?
Tail 16.8 ± 2.6 (13-20) 26.4 ± 1.3 (24-28) 39.7±4.5 (33-46) 16.2±2.4 (13-20)
Vulva-anterior end 352.7 ± 33.1 (321-396) - 317.0±25.9 (283-360) -
Spicules - 31.9 ± 2.4 (30-35) - 34.6±1.8 (33-37)
Gubernaculum - 9 ± 0.5 (9-10) - 14.9±1.3 (12-16)
RESULTS
Cuticularia oxycerca (de Man, 1895) Andrassy, 1983
(Figs 1 & 2)
Measurements. Table 1.
Population from province of Semnan (6 females, 9 males).
Female. Body almost straight, slightly curved ventrad after fixation. Cuticle loose, thick. Lateral field not visible. Lip region offset from the neck, having six rounded lips, bearing small papillae. Stoma rhabditoid, 20-23 ^m long, with distinct cheilo-, gymno- and stegostom. Cheilostom finely cuticularised. Gymnostom longer than cheilostom, having well cuticularised walls. Stegostom having glottoid apparatus with three denticles. Pharyngeal collar present, covers less than half of the stoma. Pharyngeal corpus 1.8-2.2 times isthmus length, with procorpus longer than metacorpus. Metacorpus distinct, swollen. Isthmus robust and distinctly separated from metacorpus. Basal bulb ovoid, with valvular apparatus. Cardia conoid, surrounded by
intestinal tissue. Nerve ring at isthmus level, at 4961% of neck length. Excretory pore opening at isthmus level, at 50-62% of neck length. Deirid at isthmus level, at 73-78% of neck length. Intestine without distinct specialisation. Reproductive system didelphic-amphidelphic. Ovaries straight. Oviducts short. Uteri differentiated in a distal part swollen (spermatheca) and a proximal part tubular. Vagina with fine walls, extending inward one-third of the body width. Vulva not protruding, located slightly posterior to middle part of body. Rectum 1.2-1.5 times anal body diameter. Tail almost dome-shaped or cupola-shaped with short conoid, pointed distal part. Phasmid at 16-20% of tail length.
Male. General morphology similar to female. Body length 0.48-0.67 mm, curved ventrally after fixation. Genital system monorchic, with testis reflexed ventrad anteriorly. Tail cupola-shaped, with pointed tip and mucro. Bursa peloderan, opening anteriorly with eight pairs of papillae, three precloacal and five postcloacal, arranged in 1+2/5 pattern (according to Andrassy, 1983), the GP 7th is shorter. Spicules with rounded manubrium; calamus
Fig. 1. Cuticularia oxycerca (de Man, 1895) Andrassy, 1983. A: Neck. B-D: Lip region. E: Female reproductive system. F: Entire male. G: Entire female. H: Male posterior end. I-L: Female posterior end.
Fig. 2. Cuticularia oxycerca (de Man, 1895) Andrassy, 1983 (LM). A: Neck. B: Lip region. C: Female reproductive system. D: Excretory pore. E, F: Female posterior end.
Fig. 3. Mesorhabditis acuticauda Ahmad, Shah & Mahamood, 2010. A: Neck. B, C: Lip region. D: Female reproductive system. E: Entire male. F, H: Female tail. G: Entire female. I: Male tail.
short and offset; and lamina curved ventrad and thinner distally. Gubernaculum curved ventrad.
Locality and habitat. The specimens were found in Shahrood (province of Semnan), in association with common mallow (Malva sylvestris L.).
Remarks. Morphology and measurements of the Iranian population agree with those of previous material examined (de Man, 1895; Andrassy, 1983; Eroshenko, 2002; Tahseen et al., 2009); however, it differs in female tail length (13-20 vs 30-60 pm), and gubernaculum length (9-10 vs 10-16 pm). Especially from Eroshenko's (2002) population, our material differs slightly in body length ratio (vs 6001800 pm in females and 500-1500 pm in males). From Tahseen et al. (2009) population it differs by having slightly shorter female (574-677 vs 602-815 pm).
This genus and species is reported for the first time from Iran.
Mesorhabditis acuticauda Ahmad, Shah & Mahamood, 2010
(Figs 3-5)
Measurements. Table 1.
Population from Tehran, province of Tehran (10 females, 6 males).
Female. Body slightly curved ventrad after fixation. Cuticle annulated; annuli 0.7-1.3 pm. Lateral field with four grooves. Six rounded lips, offset, each ending in a setiform papilla with acute terminus. Stoma rhabditoid, 13-16 pm long, with distinct cheilo-, gymno- and stegostom. Cheilostom finely cuticularised. Gymnostom longer than cheilostom, having well cuticularised lumen. Stegostom having glottoid apparatus without denticles. Pharyngeal collar present, but short. Pharyngeal corpus 1.9-2.7 times isthmus length, with procorpus longer than metacorpus. Pharyngeal corpus lumen zipper-like. Metacorpus distinct, swollen. Isthmus robust, distinctly separated from metacorpus. Basal bulb ovoid, with valvular apparatus. Cardia conoid, surrounded by intestinal tissue. Nerve ring at isthmus level, at 80-83% of neck length. Excretory pore opening at isthmus level, at 90-91% of neck length. Deirid not visible. Intestine without distinct specialisation. Reproductive system monodelphic-prodelphic, located at the right side of intestine. Ovary straight. Oviduct long, tubular. Uterus differentiated in a distal part swollen (spermatheca) having sperm and a proximal part more or less tubular. Two small postvulval lateral sacs present, sometimes not clearly visible. Vagina with fine walls, extending inward one-third of the body width. Vulva not protruding, posteriorly located (at approx. 80% of
body length). Rectum 1.8-2.1 times anal body diameter. Vulva-anus/tail distance is 1.1-1.3 anal body diameters. Tail conical, with acute terminus. Phasmid at anus level.
Male. General morphology similar to female. Body curved ventrally after fixation. Genital system monorchic. Tail conical, curved ventrad. Bursa peloderan, opening anteriorly with ten pairs of papillae, two precloacal and eight postcloacal, arranged in 2/4+4 pattern (according to Andrassy, 1983), the GP 8th and GP 9th are joined at their base in some specimens. Spicules fused at about one third of their length; manubrium rounded; calamus short and offset; lamina curved ventrad with hump and zigzag-shaped at its tip. Gubernaculum curved ventrad, well expanded in its anterior part.
Locality and habitat. The specimens were found in Tehran (province of Tehran), in association with carpet grass (Axonopus sp.).
Remarks. This population from Iran is very similar to the original description ofM. acuticauda. However, two morphological characters appear different: the presence of two postvulval lateral sacs (vs lacking sacs), and the shape of the spicule tip (zigzag-shaped vs straight). Although these features were not described by Ahmad et al. (2010), it may depend on the fact that the postvulval lateral sacs are sometimes not clearly visible, while only the median part between both sacs can be seen; in this case the genital system seems to lack sacs. The other character, the presence of spicule with zigzag-shaped tip is not always apparent or it could be a geographical variability.
This genus and species is reported for the first time from Iran.
Pelodera pseudoteres Schulte, 1989
(Fig. 6)
Measurements. Table 2.
Material from province of Yazd (one female and one male).
Female. Body slightly curved ventrad after fixation. Cuticle annulated; annuli 1.7 pm. Lateral field not visible. Lip region offset, having six rounded lips, bearing small papillae. Stoma rhabditoid, 25 pm long, with distinct cheilo-, gymno- and stegostom. Cheilostom finely cuticularised. Gymnostom longer than cheilostom, having well cuticularised lumen. Stegostom having glottoid apparatus with three denticles. Pharyngeal collar present, covers half of the stoma. Pharyngeal corpus 7 times isthmus length, with procorpus longer than metacorpus. Metacorpus distinct, swollen. Isthmus robust, distinctly separated from metacorpus. Basal bulb ovoid, with valvular apparatus.
Table 2. Measurements of Pelodera pseudoteres Schulte, 1989 and Protorhabditis oxyuroides Sudhaus, 1974. All measurements in |im in the form: mean ± standard deviation (range).
Species P. pseudoteres P. oxyuroides
Locality Meybod Damghan
Province Yazd Semnan
Rhizosphere Grape Willow
n 2 s Juvenile 1122 6SS
L 1179 934 659 587.9 ± 64.4 (452-668) 511.8 ± 36 (454-541)
a 15.7 18.9 16.3 24.4 ± 2.7 (19.7-27.7) 21.7 ± 2.7 (19.1-26.8)
b 5.5 4.9 3.9 4.5 ± 0.5 (3.3-4.9) 4 ± 0.2 (3.6-4.2)
c 23.5 25.2 15.6 8.6 ± 1.7 (6.5-12.6) 17.6 ± 0.8 (17.1-19.1)
c' 1.77 1.1 1.9 5.1 ± 0.8 (3.4-6.1) 1.6 ± 0.1 (1.3-1.7)
V 56 - - 57.2 ± 5.9 (53-73) -
Lip region diameter 20 19 13 7.2 ± 0.8 (6-8) 7.7 ± 0.5 (7-8)
Stoma 25 21 23 21.2 ± 1.7 (19-24) 22 ± 1.7 (19-23)
Pharyngeal corpus 129 104 69 75.4 ± 4 (68-83) 82.3 ± 16.5 (74-116)
Isthmus 19 21 43 35.2 ± 2.9 (31-40) 33.5 ± 1.6 (31-35)
Bulb 37 36 34 21.3 ± 1.5 (20-25) 21 ± 1.8 (19-23)
Pharynx length 186 162 147 131.2 ± 7.6 (118-141) 128.2 ± 6.3 (116-133)
Nerve ring-ant. end 160 143 121 96.5 ± 5.9 (87-105) 94 ± 3.7 (87-97)
Excretory pore-ant. end 197 152 141 95.3 ± 11 (73-111) 97.4 ± 5.1 (92-103)
Deirid-ant. end ? ? ? 112 (n = 1) 112 (n = 1)
Annuli width 1.7 1.3 1.0 1 1
Cuticle thickness 4 3 2 1 1
Body diameter: neck base 23 19 35 10.3 ± 1 (9-12) 10.3 ± 0.5 (10-11)
Body diameter: midbody 75 49 40 24.2 ± 2.3 (22-30) 23.2 ± 3.1 (20-28)
Body diameter: anus 28 36 22 13.9 ± 1.8 (12-16) 17.8 ± 0.8 (17-19)
Anterior genital branch 575 725 - 157.0 ± 18.4 (144-178) 354
Posterior genital branch 591 - - 157.3±21.6 (137-180) -
Rectum 37 - 23 21.5 ± 2.2 (19-26) 22 ± 3.6 (18-25)
Tail 50 37 42 70.5 ± 14.7 (44-94) 28.3 ± 2.3 (24-30)
Vulva-anterior end 660 - - 333 ± 19.2 (292-357) -
Spicules - 55 - - 25 ± 2 (22-28)
Gubernaculum - 29 - - 14.3 ± 3.1 (10-17)
Cardia conoid, surrounded by intestinal tissue. Nerve ring at isthmus level, at 74% of neck length. Excretory pore opening at isthmus level, at 91% of neck length. Deirid not visible. Intestine without distinct specialisation. Reproductive system didelphic-amphidelphic. Ovaries straight. Oviducts long, tubular. Uteri differentiated in a distal part swollen (spermatheca) having sperm, a middle part tubular with narrow lumen and a proximal part more or less swollen, these two later parts separated by a sphincter-like structure. Vagina with fine walls, extending inwards one-third of the body width. Vulva protruding, located posterior to middle part of body. Rectum 1.3 times anal body diameter. Tail conical, with acute end; it tapers more or less suddenly but distinctly in its distal part. Phasmid at 34% of tail length from the anus.
Male. General morphology similar to female. Body curved ventrally after fixation. Genital system monorchic, with testis reflexed ventrad anteriorly; genital tract lacking needle-like crystalline fibres.
Tail conical, curved ventrad, with pointed tip. Bursa peloderan, opened anteriorly with ten pairs of papillae, three precloacal and seven postcloacal, arranged in 1+2/7 pattern (according to Andrassy, 1983), the GP 9th and GP 10th shorter. All papillae swollen at tip. Spicules fused at about two-thirds of their length; manubrium rounded; calamus short and offset; lamina curved ventrad and expanded with rounded tip. Gubernaculum curved ventrad, expanded in its anterior part.
Juvenile. Body slightly curved ventrad after fixation. Cuticle clearly annulated, with annuli 1 pm. Lateral field not visible. Lip region offset, having six rounded lips, bearing small papillae. Stoma rhabditoid, 23 pm long, with distinct cheilo-, gymno- and stegostom. Cheilostom finely cuticularised. Gymnostom longer than cheilostom, having well cuticularised lumen. Stegostom having glottoid apparatus with three denticles. Pharyngeal collar present, wraps around half of the stoma. Pharyngeal corpus 1.5 times isthmus length, with
procorpus longer than metacorpus. Metacorpus distinct, swollen, 25 ^m long. Isthmus robust, distinctly separated from metacorpus. Basal bulb ovoid, 34 ^m long, with valvular apparatus. Cardia conoid, 3 ^m long, surrounded by intestinal tissue. Nerve ring at isthmus level, at 72% of neck length. Excretory pore opening at bulb level, at 84% of neck length. Deirid not visible. Intestine without distinct specialization. Rectum about one anal body diameter long. Tail conical, with acute end. Phasmid at 21% of tail length.
Locality and habitat. The specimens were found in Meybod (province of Yazd), in association with grape (Vitis vinifera L.).
Remark. The Iranian specimens of P. pseudoteres are almost identical to the original description of this species presented by Schulte (1989). According to the original description, the male lacks needle-like crystalline fibers in the genital tract, a character differentiating P. pseudoteres from P. teres Schneider, 1866; such structures are also lacking in the Iranian specimens. Other characters defined by Schulte (1989) are clearly visible in the Iranian population (e.g. offset lips, pharyngeal collar, conical tail and arrangement of bursal rays on male tail). However, the Iranian specimens differ in having a somewhat shorter stoma (21-25 vs 23-31 ^m), and longer spicules (55 vs 36-48 ^m).
This species is reported for the first time from Iran.
DNA Characterisation. The 18S rRNA gene sequence of P. pseudoteres amplified by the two primers SSU-F-04 and SSU-R-26 is 808 base pairs (bp) long. Within this fragment, only three base pair differences distinguish the Iranian population from the American population of P. pseudoteres (accession nr. EU196023; 99% identity) and 19 bp differences in comparison to P. teres (accession number AF083002; 97% identity).
Pairwise Maximum Composite Likelihood distance among the 18S rDNA region of Pelodera species showed that P. pseudoteres populations from Iran and USA have identical sequences at this genetic marker (see Table 3) where, among the molecularly characterised species of Pelodera, P. pseudoteres was closest to P. teres.
Protorhabditis oxyuroides Sudhaus, 1974
(Figs 7 & 8)
Measurements. Table 2.
Population from province of Semnan (11 females, 6 males).
Female. Body slightly curved ventrad after fixation. Cuticle annulated; annuli 1 ^m. Lateral field not visible. Lip region continuous with body,
having six rounded to conoid lips, bearing small papillae. Stoma rhabditoid, 19-24 ^m long, with distinct cheilo-, gymno- and stegostom, the latter lacking glottoid apparatus. Cheilostom with weakly refractive walls. Buccal prism with straight walls, very long and narrow. Pharyngeal collar present, very short. Pharyngeal corpus 2.0-2.1 times isthmus length, with procorpus longer than metacorpus. Metacorpus distinct, swollen. Isthmus robust and distinctly separated from metacorpus. Basal bulb ovoid to spheroid, with valvular apparatus. Cardia conoid, surrounded by intestinal tissue. Nerve ring at isthmus level, at 73-74% of neck length. Excretory pore opening at isthmus level, at 62-79% of neck length. Deirid not visible. Intestine without distinct specialisation. Reproductive system didelphic-amphidelphic. Ovaries reflexed and continued with their corresponding oviducts. Uteri differentiated in a distal part swollen (spermatheca) and a proximal part more or less tubular. Vagina with thin walls, extending inwards less than half of the corresponding body diameter. Vulva protruding, located posterior to middle part of body. Rectum 1.5-1.6 times anal body diameter. Tail conical elongate. Phasmid at 34-48% of tail length.
Male. General morphology similar to female. Body curved ventrally after fixation. Genital system monorchic, with testis reflexed ventrad anteriorly. Tail elongate-conoid, curved ventrad, with pointed tip. Bursa peloderan, opening anteriorly with nine pairs of papillae, two precloacal and seven postcloacal, arranged in 1+1/4+3 pattern (according Andrássy, 1983), the GP 1st anterior to bursa, and GP 7 , GP 8th and GP 9th are shorter. Spicules with rounded manubrium, calamus short and offset, and lamina curved ventrad and expanded with pointed tip. Gubernaculum curved ventrad, expanded in its anterior part.
Locality and habitat. The specimens were found in Damghan (province of Semnan), in association with willow (Salix sp.).
Remarks. The specimens examined fit well with those studied by Sudhaus (1974), but the females are shorter (452-668 vs 595-872 ^m) and the gubernaculum longer (10-17 vs 4-12 ^m). Compared to the material examined by Andrássy (1983), the Iranian specimens have a smaller range in the body size (452-668 ^m vs 590-870 ^m in females and 454-541 ^m vs 320-720 ^m in males). For key to species identification see Abolafia & Peña-Santiago (2007).
This genus and species is recorded for the first time from Iran.
On the phylogenetic position of Pelodera. According to Sudhaus (2011), the genus Pelodera is divided into three monophyletic groups based on
Fig. 4. Mesorhabditis acuticauda Ahmad, Shah & Mahamood, 2010 (LM). A: Neck. B, C: Lip region. D: Female reproductive system. E, F: Male posterior end. G: Postvulval sac. H: Vagina. I: Uterus.
Fig. 7. Protorhabditis oxyuroides Sudhaus, 1974. A: Neck. B: Lip region. C: Female posterior end. D: Female reproductive system. E: Entire male. F: Entire female. G: Male posterior end.
Fig. 8. Protorhabditis oxyuroides Sudhaus, 1974 (LM). A: Neck. B: Stoma. C: Lip region. D: Female reproductive system. E: Female posterior end. F: Vagina and postvulval sac. G: Male posterior end. H: Bursa and papillae.
1.00
1.00
1.00
1.011
EU543176 Panagrolaimus detritophagus | panagr0LA)M0M0RPHA (out-group) — AF082996 Rbabditoides inermis || Rhabditidae
1,00 [
1.00
1.00
1.001
1.00
AF083002 Pelodera teres EU 196023 Pelodera pseudoteres KC509908 Pelodera pseudoteres Iran M
- EU 196021 Pelodera cylindriea
_rooj- AF083018 Pelodera punctata
1.00
1.00
U13932 Pelodera strongyloides - AF083013 Mesorhabditis anisomorpba
— AF083014 Crustorhabditis transita
— AF083016 Mesorhabditis spiculigera
- EU003189 Parasitorhabditis obtusa 1.00 h- AF082999 Distolabrellus veeehi
olí— EF990714 Teratorhabditis synpapillata i.oôL EF990716 Teratorhabditis mariannae
— AF083004 Heterorhabditis marelatus JQ237848 Metarhabditis rainai - RBU13935 Metarhabditis blumi
i oo i— EU 196006 Rhabditis brass icae L AY284653
1.00
1.00
1.00
1.00
1.00
Peloderidae
Peloderinae
Mesorhabditidae
Peloderidae, Heterorhabditinae
■n
1.001
-| 1.00 1.00
1.01
1.001
Rhabditis cf. terrícola DQ639980 Pellioditis hennaphrodita DQ639982 Pellioditis neopapillosa FJ516754 Pellioditis neopapillosa EU 196005 Cepbaloboides cf. anna ta AY284654 Rhabditella axei RSU13936 Oscheius myriophilus n ! oni— EU 196010 Dolichorhabditis doliehura
HQ 130502 Dolichorhabditis tipulae I, ,W1 BUI96009 Dolichorhabditis tipulae ir AF083021 Litoditis marina
AF083020 Litoditis mediterránea EU 196020 Buetschlinenia nidrosiensis EU 196012 Cruzncma tripartitum CTU73449 Cruznema tripartitum
Rhabditidae
öl
i.oor
EU 196013 Ablechroiulus lacustris ~~1L AF083012 Ablechroiulus dudichi — AF083026 Caenorbabditis sonorae
1.00
roof 0 501
1.00
Tool
U13929 Caenorbabditis briggsae AY602182 Caenorbabditis japónica AY284652 Caenorbabditis elegans
--AY602179 Prodontorhaßditis wirthi
AY593921 Diploscapter coronatus
00]_|-
Tool
TÍ
EU 196002 Protorbabditis sp. AF083017 Haematozoon subulatum AF083023 Cuticularia oxycerca AF083022 Fbikilolaimus regenfussi AY593925 ßunonenia ret ¡cu latum
\ Peloderidae, peiiioditinae Rhabditidae
Peloderidae, Peiiioditinae
I Mesorhabditidae I Rhabditidae
Peloderidae, Peiiioditinae
Protorhabditidae-Diploscapteridae
roo)- EU I96017 Bunonema retícula tum 1-001 U8I584 Pristionchus pacitieus
O.l
AF0830I0 Pristionchus pa ci tic us
Rhabditidae
BUNONEMATOMORPHA DIPLOGASTEROMORPHA
Fig. 9. The Bayesian tree inferred from the analysis of 18S rRNA gene sequence alignment of known and newly sequenced Pelodera pseudoteres (square marked) from Iran and other related taxa.
Table 3. Pairwise genetic distance of the 18S rDNA region among Pelodera and closely species studied formed a clade.
Species Fragment length 1 2 3 4 5 6 7 8 9 10 11 12
1 Pelodera teres AF083002 1646
2 Pelodera pseudoteres EU196023 1648 0.027
3 Pelodera cylindrica EU196021 1606 0.925 0.984
4 Pelodera punctata AF083018 1649 0.941 0.998 1.287
5 Mesorhabditis spiculigera AF083016 1362 0.340 0.343 0.779 0.843
6 Mesorhabditis anisomorpha AF083013 1647 0.371 0.386 0.770 0.908 0.110
7 Teratorhabditis synpapillata EF990714 1635 0.329 0.352 0.759 0.822 0.079 0.120
8 Teratorhabditis mariannae EF990716 1683 0.325 0.345 0.768 0.837 0.078 0.125 0.027
9 Parasitorhabditis obtusa EU003189 1685 0.325 0.333 0.795 0.853 0.054 0.115 0.055 0.043
10 Pelodera pseudoteres KC509908 614 0.027 0.000 0.984 0.998 0.343 0.386 0.352 0.345 0.333
11 Pelodera strongyloides U13932 1613 0.920 0.975 1.308 0.011 0.832 0.908 0.819 0.827 0.842 0.975
12 Crustorhabditis transita AF083014 1670 0.318 0.324 0.800 0.873 0.073 0.117 0.065 0.057 0.049 0.324 0.862
13 Distolabrellus veechi AF082999 1697 0.328 0.340 0.767 0.827 0.055 0.125 0.047 0.039 0.045 0.340 0.816 0.050
several phenotypic traits derived from evolution, mainly tail morphology: the Teres-group is distinguished by having cupola-shaped or short conoid female tail and bursa with three precloacal papillae; the Coarctata-group is distinguished by having cupola-shaped female tail and bursa with two precloacal papillae; and the Strongyloides-group is distinguished by having cupola-shaped or conical female tail and bursa with two precloacal papillae. These groups correspond to different genera according to Andrassy (2005): Rhomborhabditis Andrassy, 1983, Coarctadera Dougherty, 1953 and Pelodera Schneider, 1866 sensu stricto, respectively.
In our phylogenetic analysis using 18S rDNA (Fig. 9), this morphological classification scheme is supported by molecular data. Thus, the molecularly characterised species of Pelodera form a monophyletic group with 100% posterior probability and are divided into the three groups indicated by Sudhaus (2011). This result suggests that the phenotypic characters are also informative for phylogenetic relationships. However, more taxon sampling within Pelodera is needed to understand the full phylogenetic status of the species within this genus.
In addition, our analysis places the Pelodera species in a clade together with Distolabrellus Anderson, 1983, Mesorhabditis, Parasitorhabditis Fuchs, 1937 and Teratorhabditis Osche, 1952, agreeing with Kiontke & Fitch (2005) and van Megen et al. (2009). Fitch (1997) demonstrated with the analysis of the SSU rDNA that Pelodera (using P. strongyloides Schneider, 1860) and Teratorhabditis (using T. palmarum Gerber & Giblin-Davis, 1990) are within the same monophyletic group, this being consistent with the current study.
ACKNOWLEDGMENTS
We thank and appreciate Dr Zolala and Dr Fasihi for the opportunity to work in the Molecular Laboratory and Prof. Dr David Fitch for revising the manuscript. We appreciate financial support for this research by the Research deputy of Shahid Bahonar University of Kerman and the assistance of Research Technical Services of University of Jaén (Spain) for SEM study.
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E. Shokoohi, S. Mehdizadeh, N. Amirzadi, J. Abolafia. Четыре новых сообщения о находках рабдитид (Nematoda: Rhabditida: Rhabditomorpha) в Иране и оценка филогенетических связей рода Pelodera. Резюме. Обнаружены четыре новых для фауны Ирана вида почвенных рабдитид: Cuticularia oxycerca (de Man, 1895) Andrassy, 1983; Mesorhabditis acuticauda Ahmad, Shah & Mahamood, 2010; Peloderapseudoteres Schulte, 1989 и Protorhabditis oxyuroides Sudhaus, 1974. Проведенный по последовательностям 18S рДНК филогенетический анализ положения иранской популяции P. pseudoteres показал различия в трех позициях по сравнению с популяцией этого вида из США (изолят SB116; 1648 п.н.). От филогенетически близкого вида P. teres (также из США, изолят EM437; 1646 п.н.) отличия составили 19 нуклеотидов. Филогенетический анализ показывает близость иранской популяции P. pseudoteres к представителям этого вида из США и подтверждает монофилетический характер линии, составленной видами Pelodera. Приводятся иллюстрации и измерения для вида M. acuticauda.
