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64Russian Journal of Nematology, 2013, 21 (2), 93-115
Morphological and molecular characterisation of
Panagrolaimus Fuchs, 1930 (Nematoda, Rhabditida, Panagrolaimidae) species from Iran
12 2 3
Sara Mehdizadeh ' , Ebrahim Shokoohi and Joaquín Abolafia
1Young Researchers Society, Shahid Bahonar University of Kerman, Iran; 2Department of Plant Protection, College of Agriculture, Shahid Bahonar University of Kerman, Iran,
e-mail: [email protected]; 3Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén. Campus "Las Lagunillas" s/n. 23071-Jaén, Spain.
Accepted for publication 23 September 2013
Summary. Seven species of the family Panagrolaimidae are described from natural areas in Kerman province, Iran: Panagrolaimus concolor; P. facetus; P. cf. labiatus; P. cf. papillosus; P. cf. subelongatus; P. superbus and P. trilabiatus were recovered. Descriptions, measurements, illustrations and light microscopic photographs are provided for all species. Molecular analysis of three out of seven species based on sequences of the 18S rDNA places P. facetus close to P. davidi (HQ270131) and an unidentified Panagrolaimus (EU040129), only in the Maximum Likelihood (ML) tree. In the Bayesian Inference (BI) tree it groups with an unidentified Panagrolaimus (KC522709). Panagrolaimus trilabiatus groups with P. subelongatus (AY284681) and an unidentified Panagrolaimus (FJ590963) in BI tree. Panagrolaimus cf. papillosus groups with P. davidi (HQ270131) and is placed close to P. facetus (KF011488) in the BI tree. In the ML tree it groups with two unidentified Panagrolaimus (KC522708; FJ590961). Molecular data of 18S rDNA of P. facetus, P. cf. papillosus and P. trilabiatus are reported for the first time. In addition, Maximum Likelihood and Bayesian analyses revealed the paraphyly of the genus Panagrolaimus. Key words: Iran, morphology, Panagrolaimidae, Panagrolaimus, 18S rDNA, phylogeny, taxonomy.
The genus Panagrolaimus Fuchs, 1930 are bacterial feeding nematodes that occupy different niches, including the cold soil from Antarctica, temperate and semi-arid soils, and mosses (Shannon et al., 2005). These worms are used in developmental studies because they have three reproductive modes (i.e., gonochoristic, hermaphroditic and parthenogenetic) (Lewis et al, 2009). The main study on this group of nematodes has been done by Abolafía & Peña-Santiago (2006) who described several Iberian species and provided a key to species identification. Panagrolaimus comprises 44 nominal species (Abolafía & Peña-Santiago, 2006). So far, two species of this genus, namely P. dendroctoni (Fuchs, 1932) Rühm, 1956 and P. rigidus (Schneider, 1866) Thorne, 1937 were reported from Iran (Shokoohi et al, 2007).
Identification of the species of this genus is frequently very difficult (e.g. molecular analyses executed by Lewis et al, 2009) due to the high intraspecific variation of their size and morphology, and because sometimes the original descriptions of species are very poor or inexact. The present paper
is one of series on nematodes of the order Rhabditida from Iran (province of Kerman) and deals with seven known species belonging to the genus Panagrolaimus collected in natural areas. These species are reported for the first time from Iran.
MATERIALS AND METHODS
Studies on morphology. Nematodes were extracted from soil samples by Baermann's (1917) funnel technique. Specimens were fixed with hot 4% formaldehyde solution and processed to anhydrous glycerin by the method of De Grisse (1969). Measurements were taken directly using an ocular micrometer and/or a curvimeter upon drawing the corresponding organ or structure. Drawings were made using a drawing tube attached to the microscope Olympus CH2. LM pictures were made with a Nikon Eclipse 80i microscope equipped with a Nikon Digital Sight DS-5M camera.
Females of P. trilabiatus were cultured on water-agar 2% in Petri plates to which ten gravid females were added to each plate. The terminology used to
describe the morphology of the stoma and spicules follows the proposals by De Ley et al. (1995) and Abolafia & Peña-Santiago (2006), respectively.
Phylogenetic analysis. The sequences of species belonging to the family Panagrolaimidae used for phylogenetic analysis were obtained from the GenBank (73 sequences). 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 pl double distilled water. The tube was frozen in liquid nitrogen and the nematodes were crushed with vortex; 200 pl Tissue Lysis buffer (TL) and 20 pl proteinase K (20 mg ml-1) was 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'- GCT TGT CTC AAA GAT TAA GCC-3') and the reverse primer SSU_R_26 (5'- CAT TCT TGG CAA ATG CTT TCG-3') (Blaxter et al, 1998) were used in the PCR reactions for amplification of the partial 18S region (~900bp). PCR was conducted with 10 pl of the extracted DNA, 4 pl of PCR Master Mix (Kawsar Biotech company, Iran), 1 pl of each primers (10 pmol pl-1) and ddH2Ö to a final volume of 25 pl. The amplification was carried out using an Eppendorf master cycler gradient (Eppendorf, Hamburg, Germany), with 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 pi of product was loaded on a 1% agarose gel (40 mM Tris, 40 mM boric acid, and 1 mM EDTA) for DNA checking. 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 using the primers that were used in the amplification step. Sequencing reactions were performed by Bioneer company (South Korea) (http://eng.bioneer.com). Sequencing was performed in both directions. The DNA sequence was edited using Chromas version 1.45 (McCarthy, 1997). Primers for the sequencing reaction were those used in the amplification step. The sequence was confirmed in both directions and repeated. Sequences for the ingroups and outgroups were obtained from GenBanks. The ribosomal SSU sequences were analysed and aligned using the program BioEdit (Hall, 1999). The length of alignment is 1857 bp.
Maximum Likelihood (ML) and Bayesian Inference were used to reconstruct the phylogeny. The Maximum Likelihood tree was constructed using the program Mega 5 (Tamura et al, 2011).
The analyses were run for 10000 bootstrap replicates. The Bayesian tree was generated using the Bayesian inference method as implemented in the program MrBayes 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 model of sequence evolution and bootstrap analysis was implemented with 10000 replicates. The tree was visualised with TreeView program. For phylogenic relationships analysis of 18S rDNA, Caenorhabditis japonica Kiontke, Hironaka & Sudhaus, 2002 (AY602182) was chosen as outgroup. This selection was based on Lewis et al. (2009) and van Megen et al. (2009) studies. The original partial 18S sequences of Panagrolaimus species are deposited in the GenBank under the accession numbers: KF011487, KF011488, KF011489 for P. trilabiatus (881 bp), P. facetus (915 bp) and P. cf. papillosus (906 bp), respectively.
DESCRIPTIONS
Panagrolaimus concolor Massey, 1964 (Figs 1 & 2A-D)
Measurements. See Table 1.
Population collected in Kerman, province of Kerman (3$$, 1$).
Female. Body length 0.82-1.15 mm, cylindrical, slightly curved ventrad after fixation. Cuticle smooth; having 3.8 pm thickness, annuli 1 pm wide. Lateral field with two incisures occupying 12% of midbody diameter, fading out near phasmids. Lip region continuous with neck, having six small lips amalgamated in pairs, and a circular amphid opening. Stoma panagrolaimoid, with distinct cheilo-, gymno- and stegostom. Cheilostom without refringent rhabdia. Gymnostom longer than cheilostom, having cuticularised rhabdia differentiated in a shorter anterior part (corresponding with the anterior arcade epidermis), and a longer posterior part (posterior arcade epidermis). Stegostom having four distinct parts, bearing a minute tooth. Pharyngeal corpus cylindrical, 2.1-2.4 times isthmus length, with procorpus longer than metacorpus. Isthmus slenderer than corpus and distinctly separated from metacorpus. Basal bulb spheroid, with valvular apparatus. Cardia conoid, surrounded by intestinal tissue. Nerve ring at isthmus level, at 85-90% of
Fig. 1. Panagrolaimus concolor Massey, 1964. A: Neck. B: Anterior end. C: Female reproductive system. D: Entire female. E: Entire male. F: Male posterior end. G: Female posterior end.
neck length. Excretory pore opening at bulb level, at 98% of neck length. Deirid not visible. Reproductive system monodelphic-prodelphic, located on right side of intestine. Vulva protruding, located posterior to middle part of body. Ovary without flexure posterior to vulva level. Oviduct short, less than a half of the corresponding body diameter and well developed. Uterus tubular, about four times the corresponding body diameter long. Post-uterine sac 0.4-0.5 times the corresponding body diameter long. Vagina extending inward less than a half of the body width. Rectum 1.0-1.1 times the anal body diameter. Tail conical-elongate, with pointed tip. Phasmid at 53-57% of tail length.
Male. Body curved ventrally after fixation. Reproductive system monorchic, testis reflexed dorsad anteriorly. Tail conical, distally curved ventrad. Two pairs of precloacal genital papillae. Five pairs of caudal genital papillae are present along the tail comprising one ventral pair, one lateral pair, one dorsal pair, and two pairs near tail terminus. Spicules curved ventrally: manubrium rounded; calamus short, straight; lamina dorsally curved, with pointed terminus, having two longitudinal lines. Gubernaculum well developed, 15 pm long or less than half of the spicule length.
Locality and habitat. Material has been found in Mahan (province of Kerman), in association with Haloxylon persicum L.
Remarks. The material examined is similar to the original population of P. concolor described by Massey (1964) in having slightly rounded lips, minute denticle on stegostom, conoid female tail, lateral field with two incisures, corpus/isthmus ratio (2.1-2.4 vs 2.3), shape of spicules and gubernaculum. However, they differ in nerve ring position (near bulb level vs at middle length of isthmus), excretory pore position (near base of bulb level vs slightly more anterior), female tail slightly longer (61-76 vs 48 pm), and precloacal genital papillae (two pairs vs only one pair was observed in original description). Other similar species are P. artyukhovskii Blinova & Mishina, 1975, P. detritophagus Fuchs, 1930 and P. rigidus (Schneider, 1866) Thorne, 1937. Our population differs from P. artyukhoskii in having two lateral field (vs three), shorter corpus/isthmus ratio (2.1-2.4 vs 2.9) and spicules shape (lamina not pointed at ventral side vs lamina pointed at ventral side in original description) and female tail (61-76 vs 51 pm in original description). It differs from P. detritophagus in having slightly rounded lips (vs conical lips), two lateral fields (vs three lateral field) and shape of spicules (lamina not pointed at ventral side vs lamina pointed at ventral side in original
description). Also it differs from P. rigidus in fused lips (vs lips duplex), two lateral field (vs three lateral field).
This species is reported for the first time from Iran.
Panagrolaimus facetus Massey (1971) Andrassy, 1984 (Figs 2E-H & 3)
Measurements. See Table 1.
Population collected in Kerman, province of Kerman (3$$, 1$).
Female. Body length 0.73-0.78 mm, cylindrical, curved ventrad after fixation. Cuticle smooth; having 3 pm thickness, annuli 1 pm wide. Lateral field with two incisures occupying 13-17% of midbody diameter. Lip region continuous with neck, having six lips amalgamated in pair and a circular amphid opening. Stoma panagrolaimoid, with distinct cheilo-, gymno- and stegostom. Cheilostom without refringent rhabdia. Gymnostom longer than cheilostom, having cuticularised rhabdia. Stegostom having four distinct parts. Pharyngeal corpus cylindrical, 2.2-3.2 times isthmus length, with procorpus longer than metacorpus. Isthmus slenderer than corpus and separated with metacorpus distinctly. Basal bulb spheroid, with valvular apparatus. Cardia conoid, surrounded by intestinal tissue. Nerve ring at isthmus level, at 6678% of neck length. Excretory pore opening at isthmus level, at 76-84% of neck length. Deirid at 95% of neck length, at bulb level. Reproductive system monodelphic-prodelphic, located on right side of intestine. Ovary without flexure posterior to vulva level. Oviduct short, less than a half of the corresponding body diameter. Uterus tubular, about five times the corresponding body diameter long. Postuterine sac 0.6-0.8 times the corresponding body diameter long. Vagina extending inward less than a half of the body width. Vulva protruding, located posterior to middle part of body. Rectum 0.8-1.3 times the anal body diameter. Tail conical-elongate, with pointed end and mucro. Phasmid at 30-40% of tail length.
Male. Body curved ventrally after fixation. Reproductive system monorchic, testis reflexed dorsad anteriorly. Tail conical, distally curved ventrad, bearing a thin mucro. Two pairs of precloacal genital papillae. Five pairs of caudal genital papillae are present along the tail comprising one ventral pair, one lateral pair, one dorsal pair, and two pairs near tail terminus. Spicules curved ventrally: manubrium rounded, straight; calamus as wide as manubrium; lamina ventrally curved, with
Fig. 2. Panagrolaimus concolor Massey, 1964 (LM). A, B: Anterior end. C: Vagina region. D: Female posterior end (arrow points at phasmid). Panagrolaimus facetus (Massey, 1971) Andrassy, 1984 (LM). E: Anterior end. F: Lateral field. G: Vagina region. H: Female posterior end. Panagrolaimus cf. labiatus (Kreis, 1929) Andrassy, 1960 (juvenile, LM). I: Anterior end. J: Lateral field. K: Vagina region. L: Posterior end. Panagrolaimus cf. papillosus Loof, 1971 (LM). M: Anterior end. N: Lateral field. O: Vagina region. P: Female posterior end. Q: Male posterior end.
pointed terminus and three longitudinal lines. Gubernaculum well developed, 14 pm long or about 50% of the spicule length.
Locality and habitat. Material has been found in Kerman (province of Kerman), in soil.
Remarks. Our population is similar to P. facetus in having high lips, two lateral fields, location of nerve ring and excretory pore (both at isthmus level), conical elongated female tail. However, the material examined compared with the original description of this species provided by Massey (1971), differs in having slightly rounded lips (vs slightly acute lips), slightly shorter body (0.73-0.78 mm in females and 0.55 mm in males vs 0.83-0.86 mm in females and 0.78-0.81 mm in males in the original description), and slightly longer spicules (29 vs 23 pm in the original description). On the other hand, Andrássy (1984) reported this species with longer body (vs 0.80-0.90 mm in females and 0.80 mm in males), and shorter c value (c=13-14 vs 16-21).
This species also slightly resembles P. magnivulvatus Bostrom, 1995. However, it differs in lateral field (two incisures vs three incisures) and longer female tail (c' = 4 vs c' = 2-3) (see Abolafia & Peña-Santiago, 2006). Massey (1971) mentioned P. margaretae (Massey, 1964) Andrássy, 1984 as similar species with P. facetus. However, their lips are very different (more or less acute in P. facetus vs acute and prominent in P. margaretae), and lateral field (two vs three incisures).
This species is reported for the first time from Iran.
Panagrolaimus cf. labiatus (Kreis, 1929) Andrássy, 1960 (Figs 2I-L & 4)
Measurements. See Table 1.
Population collected in Kerman, province of Kerman (3$$, 2$$).
Female. Body length 0.92-0.97 mm, cylindrical, curved ventrad after fixation. Cuticle smooth; having 1 pm thickness, annuli 1 pm wide. Lateral field with two incisures occupying 8-10% of midbody diameter, fading out near tail terminus. Lip region continuous with neck, having six small lips amalgamated in pairs, and a circular amphid opening. Labial papillae well observed. Stoma panagrolaimoid, with distinct cheilo-, gymno- and stegostom. Cheilostom without refringent rhabdia. Gymnostom longer than cheilostom, having cuticularised rhabdia. Stegostom having four distinct parts. Pharyngeal corpus cylindrical, 2-3 times isthmus length, with procorpus longer than
metacorpus. Isthmus slender than corpus 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 65-80% of neck length. Excretory pore opening at bulb level, at 67-97% of neck length. Deirid at 88-94% of neck length, at bulb level. Reproductive system monodelphic-prodelphic, located on right side of intestine. Ovary without flexure posterior to vulva level. Oviduct short, less than a half of the corresponding body diameter. Uterus tubular, about five times the corresponding body diameter long, consisting of a long proximal tubular part and a short distal part with thinner walls. Post-uterine sac 0.8-0.9 times the corresponding body diameter long. Vagina extending inward less than a half of the body width. Vulva protruding, located posterior to middle part of body. Rectum 1.3-1.5 times the anal body diameter. Tail conical-elongate, with pointed end. Phasmid at
17-31% of tail length.
Male. Body curved ventrally after fixation. Reproductive system monorchic, testis reflexed dorsad anteriorly. Tail conical, distally curved ventrad. Two pairs of precloacal genital papillae. Five pairs of caudal genital papillae are present along the tail comprising one ventral pair, one lateral pair, one dorsal pair, and two pairs near tail terminus. Spicules curved ventrally: manubrium rounded, straight; calamus slenderer than manubrium; lamina ventrally curved, with rounded terminus and three longitudinal lines. Gubernaculum well developed, 14-16 pm long or about 50% of the spicule length.
Locality and habitat. Material has been found in Kerman (province of Kerman), in association with Haloxylon persicum L.
Remarks. Our specimens are similar to P. labiatus (Kreis, 1929) Andrassy, 1960 in rounded lip and conical female tail with convex in dorsal and ventral side. This species resembles in the measurements with P. labiatus. However, it differs from that species by having longer females (925972 vs 480-680 pm in the original description), and female tail tip (not differentiated at tip vs with a marked narrowing at its posterior half in the original description). From its junior synonym P. burdwanensis Chaturvedi and Khera, 1979 it differs by longer body (vs 0.74-0.78 mm in females and 0.56-0.62 mm in males), longer spicules (31-34 vs
18-20 pm), longer gubernaculum (14-15 vs 8-10 pm) and fewer precloacal genital papillae (one vs two precloacal genital papillae). Previous descriptions of P. labiatus are incomplete, and therefore it is impossible to be absolutely sure about the identity
Table 1. Measurements of Panagrolaimus concolor Massey, 1964, P. facetus (Massey, 1971) Andrassy, 1984 and P. cf. labiatus (Kreis, 1929) Andrassy, 1960 from Iran. All measurements in |im, and in the form; mean ± standard error (range).
Species P. concolor P. facetus P. cf. labiatus
Province, Locality Habitat Kerman, Mahan Haloxylon persicum Kerman, Kerman Soil Kerman, Kerman Haloxylon persicum
n 3ÇÇ 1$ 3ÇÇ 1$ 3ÇÇ 2$$
Body length 963.6±170.3 (827-1154) 936 766.7±26.2 (736-782) 554 943.4±25.0 (925-972) 811, 783
a 18.9±2.6 (16.7-21.8) 20.9 26.5±3.3 (23.0-29.6) 25.6 24.3±2.1 (22.1-26.3) 28.7, 24.6
b 6.2±1.1 (5.4-7.5) 6.6 4.5±0.5 (4.2-5.1) 3.9 5.3±0.0 (5.2-5.3) 4.0, 5.0
c 14.4±0.8 (13.5-15.1) 19.9 13.0±0.5 (12.6-13.6) 15.1 23.5±1.4 (22.6-25.1) 18.9, 22.08
c' 2.4±0.4 (2.1-2.9) 1.8 3.0±0.7 (2.2-3.6) 1.9 1.7±0.0 (1.7-1.8) 1.8, 1.6
V 55.1±4.9 (50-60) - 58.1±1.2 (57-59) - 60.7±2.4 (58.6-63.3) -
Lip region width 10.5±1.1 (9-12) 9 8.8±0.5 (8-9) 7 10.3±0.5 (10-11) 9, 10
Stoma 11.3±0.8 (10-12) 12 12.6±0.5 (12-13) 9 14.1±1.1 (13-16) 12, 13
Pharyngeal corpus 81.6±3.2 (79-85) 78 102.2±7.3 (96-110) 78 97.3±2.7 (94-99) 97, 87
Isthmus 36.1±1.8 (35-38) 28 39.9±9.2 (29-45) 39 34.5±4.9 (31-40) 26, 30
Bulb 26.5±0.03 (26-26) 26 24.5±0.9 (24-25) 20 29.8±2.7 (28-33) 26, 23
Pharynx length 144.2±2.7 (141-147) 132 166.7±16.2 (149-181) 137 163.1±5.9 (158-169) 158, 146
Neck 156.2±4.8 (153-162) 141 171.0±23.8 (143-186) 143 177±6 (171-183) 202, 155
Nerve ring-ant. end 134.5±4.9 (131-138) 116 121.7±25.5 (95-146) 103 134.9±13.0 (120-144) 127, 125
Excretory pore-ant. end 155.5±6.3 (151-160) 141 135.8±24.1 (109-157) 112 152.6±25.1 (124-167) 114, 97
Deirid-ant. end - - 185.8±0.0 (185.8) 127 169±1.6 (159-162) 144, 143
Cuticle thickness 3.8±0.00 (3.8) - 2 1.5 1.4±0.6 (1-2) 1.5
Body width: neck base 33.0±1.3 (32-34) 35 27.0±2.7 (25-30) 22 13.0±1.4 (12-15) 10, 12
Body width: midbody 50.8±1.8 (49-53) 45 29.2±4.1 (26-34) 22 39.1±3.3 (36-42) 28, 32
Body width: anus 27.4±1.7 (26-29) 26 20.4±5.3 (16-26) 20 23.0±1.4 (22-25) 23, 21
Lateral field - - 4.7±0.0 (4.7) 1.9 3.6 (n=1) 4
Vagina 40.6±2.1 (38-42) - 13.8±0.5 (13-14) - 13±2.9 (11-16) -
Ovary 570.2±54.7 (511-618) - 288.6±34.1 (254-322) - 316.4±59.3 (259-377) -
Postuterine sac 23.3±2.6 (21-26) - 22.6±0.9 (22-24) - 33.9±2.3 (32-36) -
Rectum 30.0±2.09 (28-32) - 21.4±2.7 (20-24) - 32.1±0.5 (31.8-32.7) -
Tail 66.5±8.6 (61-76) 47 58.8±1.4 (57-60) 37 40.3±2.8 (37-42) 43, 35
Vulva- anterior end 525.1±46.6 (496-579) - 445.5±24.1 (418-464) - 572.3±21.8 (547-585) -
Spicules - 37 - 29 - 31, 34
Gubernaculum - 15 - 14 - 14, 15
Fig. 3. Panagrolaimus facetus (Massey, 1971) Andrássy, 1984. A: Neck. B: Anterior end. C: Lateral field. D: Female reproductive system. E: Entire Female. F: Entire male. G: Male posterior end. H, I: Female posterior end.
Fig. 4. Panagrolaimus cf. labiatus (Kreis, 1929) Andrassy, 1960. A: Neck. B: Anterior end. C: Female reproductive system. D: Entire male. E: Entire female. F: Lateral field. G, I: Female posterior end. H: Male posterior end.
of our population. In view of close similarity of our specimens with P. labiatus, we consider it closely related to it and identify it as P. cf. labiatus.
Furthermore, this material is also similar to P. goodeyi Rühm, 1956. However they differ in phasmid position (near tail terminus in our population vs near mid tail in P. goodeyi in the original description).
Panagrolaimus cf. papillosus Loof, 1971 (Figs 2M-Q & 5)
Measurements. See Table 2.
Population collected in Kerman, province of Kerman (3$$, 1$).
Female. Body length 0.75-0.80 mm, cylindrical, curved ventrad after fixation. Cuticle annulated, having 1 ^m thickness, annuli 1 ^m wide. Lateral field with three incisures occupying 17-30% of midbody diameter. Lip region continuous with neck, having six rounded lips amalgamated in pairs, and a circular amphid opening. Labial papillae well observed. Stoma panagrolaimoid, with distinct cheilo-, gymno- and stegostom. Cheilostom without refringent rhabdia. Gymnostom longer than cheilostom, having cuticularised rhabdia. Stegostom having four distinct parts. Pharyngeal corpus cylindrical, 2.5-3.0 times isthmus length, with procorpus longer than metacorpus. Isthmus slenderer than corpus distinctly separated from metacorpus. Basal bulb ovoid to spheroid, with valvular apparatus. Cardia conoid, surrounded by intestinal tissue. Nerve ring at isthmus level, at 7076% of neck length. Excretory pore opening at isthmus level, near to bulb, at 81-84% of neck length. Deirid at 88-97% of neck length, at bulb level. Reproductive system monodelphic-prodelphic, located on right side of intestine. Ovary without flexure posterior to vulva level. Oviduct short, less than a half of the corresponding body diameter and well developed. Uterus tubular, about five times the corresponding body diameter long, consisting of a long proximal tubular part and a short distal part with thinner walls. Post-uterine sac 0.7-0.8 times the corresponding body diameter long. Vagina extending inward less than a half of the body width. Vulva protruding, located posterior to middle part of body. Rectum 1.1-1.4 times the anal body diameter. Tail conical. Phasmids at 65-80% of tail length.
Male. Body curved ventrally after fixation. Reproductive system monorchic, testis reflexed dorsad anteriorly. Tail conical, distally curved ventrad, having different colour and distinguished tip. Two pairs of precloacal genital papillae. Five
pairs of caudal genital papillae are present along the tail comprising one ventral pair, one lateral pair, one dorsal pair, and two pairs near tail terminus. Spicules curved ventrally: manubrium rounded; calamus as wide as manubrium, straight; lamina ventrally curved, with dorsally hump proximally, pointed terminus, and having two longitudinal lines. Gubernaculum well developed, 14 ^.m long or less than half of the spicule length.
Locality and habitat. Material has been found in Andoohjerd (province of Kerman), in association with Vitis vinifera L.
Remarks. This material is similar to P. papillosus described by Loof (1971) from Spitzbergen, in having prominent lips with conspicuous papillae, three lateral field, protruded vulva, symmetrical uterus, large posterior anal lips; however, it differs from that by having longer female body (754-800 vs 510-690 ^m in the original description), although this difference may be due to geographical variations. According to Loof (1971), this material also is similar to P. subelongatus; however they differ in conspicuous labial papillae and location of phasmid (near tail terminus in P. papillosus vs located in posterior third of tail in P. subelongatus).
Panagrolaimus cf. subelongatus (Cobb, 1914) Thorne, 1937 (Figs 7A-E & 6)
Measurements. See Table 2.
Population collected in Shahdad, province of Kerman (3$$, 2$$).
Female. Body length 709-764 ^m, cylindrical, curved ventrad after fixation. Cuticle smooth; having 2.8 ^m thickness, annuli 1.9 ^m wide. Lateral field with three incisures occupying 20-24% of midbody diameter, fading out near tail terminus. Lip region continuous with neck, having three lips and a circular amphid opening. Labial papillae well developed. Stoma panagrolaimoid, with distinct cheilo-, gymno- and stegostom. Cheilostom without refringent rhabdia. Gymnostom longer than cheilostom, having cuticularized rhabdia. Stegostom having four distinct parts. Pharyngeal corpus cylindrical, 2.8-3 times isthmus length, with procorpus longer than metacorpus. Isthmus slenderer than corpus distinctly separated from metacorpus. Basal bulb ovoid to spheroid, with valvular apparatus. Cardia conoid, surrounded by intestinal tissue. Nerve ring at isthmus level, at 6580% of neck length. Excretory pore opening at bulb level, at 80-90% of neck length. Deirid at 90% of neck length, at bulb level. Reproductive system
Fig. 5. Panagrolaimus cf. papillosus Loof, 1971. A: Neck. B: Anterior end. C: Female reproductive system. D: Entire male. E: Lateral field. F: Entire female. G-I: Female posterior end. J: Male posterior end.
Fig. 6. Panagrolaimus cf. subelongatus (Cobb, 1914), Thome, 1937. A: Neck. B: Anterior end. C: Lateral field. D: Female reproductive system. E: Male posterior end. F: Entire female. G: Entire male. H, I: Female posterior end.
monodelphic-prodelphic, located in right side of intestine. Ovary without flexure posterior to vulva level. Oviduct short, less than a half of the corresponding body diameter. Uterus tubular, about five times the corresponding body diameter long, consisting of a long proximal tubular part and a short distal part with thinner walls. Post-uterine sac 0.4-0.6 times the corresponding body diameter long. Vagina extending inward less than a half of the body width. Vulva protruding, located posterior to middle part of body. Rectum 0.5-0.7 times the anal body diameter. Tail conical, with pointed end. Phasmid at 21-23% of tail length.
Male. Body curved ventrally after fixation. Reproductive system monorchic, testis reflexed dorsad anteriorly. Tail conical, distally curved ventrad, with a mucro less than half of tail length. Two pairs of precloacal genital papillae and five pairs of caudal genital papillae are present along the tail comprising one ventral pair, one lateral pair, one dorsal pair, and two pairs near tail terminus. Spicules curved ventrally: manubrium rounded, straight; calamus slender than manubrium; lamina ventrally curved, with somewhat rounded terminus and two longitudinal lines. Gubernaculum well developed, 11-13 pm long or about 30-40% of the spicule length.
Locality and habitat. Material has been found in Shahdad (province of Kerman), in association with Citrus sinensis (L.).
Remarks. Our specimens are similar to P. subelongatus, especially the material described by Steiner (1924) and that described as P. obesus by Thorne (1937), its junior synonym, in rounded lips, gymnostom shape, female tail, phasmid location (at posterior third of tail), two precloacal papillae, arrangement of poscloacal papillae (four near tail terminus), spicules and gubernaculum shape. However, it differs from that by having longer female body length (709-764 vs 650 pm), and male tail with longer mucro (vs very short). Cobb (1914) and Andrássy (2005) described populations of this species with shorter female body (vs 600 pm in Cobb (1914) and 600-680 pm in Andrássy (2005)), and pharynx (155-158 vs 138 pm in Cobb (1914) and 130-140 pm in Andrássy (2005)).
This material also resembles to P. labiatus in having rounded lips and phasmid location (posterior third part of tail in both species); however, it differs in longer ovary (vs shorter ovary), and female tail (tapering gradually to the end vs marked narrowing near its terminus, see Abolafia & Peña-Santiago, 2006).
Panagrolaimus superbus Fuchs, 1930 (Figs 7F-J & 8)
Measurements. See Table 2.
Population collected in Kerman and Gharyatolarab, province of Kerman (39$, 2$3 and 3$$, 2^, respectively).
Female. Body length 0.86-0.89 mm, cylindrical, curved ventrad after fixation. Cuticle smooth; having 1-3 pm thickness, annuli 1 pm wide. Lateral field with three incisures occupying 19-22% of midbody diameter. Lip region continuous with neck, having six lips amalgamated in pairs, and a circular amphid opening. Stoma panagrolaimoid, with distinct cheilo-, gymno- and stegostom. Cheilostom without refringent rhabdia. Gymnostom longer than cheilostom, with cuticularised rhabdia. Stegostom having four distinct parts. Pharyngeal corpus cylindrical, 2.3-3.0 times isthmus length, with procorpus longer than metacorpus. Isthmus slenderer than corpus and distinctly separated from metacorpus. Basal bulb ovoid, with valvular apparatus. Cardia conoid, surrounded by intestinal tissue. Nerve ring at isthmus level, at 71-75% of neck length. Excretory pore opening at bulb level, at 86-87% of neck length. Deirid at 87-90% of neck length, at bulb level. Reproductive system monodelphic-prodelphic, located on right side of intestine. Ovary without flexure posterior to vulva level. Oviduct short, less than a half of the corresponding body diameter. Uterus tubular, about four times the corresponding body diameter long. Post-uterine sac 0.3-0.4 times the corresponding body diameter long. Vagina extending inward less than a half of the body width. Vulva protruding, located posterior to middle part of body. Rectum 1.2-1.3 times the anal body diameter. Tail conical, with pointed or rounded terminus. Phasmid at 50% of tail length.
Male. Body curved ventrally after fixation. Reproductive system monorchic, testis reflexed dorsad anteriorly. Tail conical, distally curved ventrad. Three pairs of precloacal genital papillae. Five pairs of caudal genital papillae are present along the tail comprising one ventral pair, one lateral pair, one dorsal pair, and two pairs near tail terminus. Spicules curved ventrally: manubrium rounded, straight; calamus as wide as manubrium; lamina ventrally curved, with dorsal hump proximally, pointed terminus, and two longitudinal lines. Gubernaculum well developed, 15 pm long or about less than half of the spicule length.
Locality and habitat. Material has been found in Kerman and Gharyatolarab (province of Kerman), in association with Haloxylon persicum L.
Fig. 7. Panagrolaimus cf. subelongatus (Cobb, 1914), Thome, 1937 (LM). A: Anterior end. B: Lateral field. C: Vagina region. D: Female posterior end. E: Male posterior end. Panagrolaimus superbus Fuchs, 1930 (LM). F: Anterior end. G: Lateral field. H: Vagina region. I: Female posterior end. J: Male posterior end. Panagrolaimus trilabiatus Zell, 1987 (LM). K: Anterior end. L: Lateral field. M: Vagina region. N: Female posterior end. O: Male posterior end.
Fig. 8. Panagrolaimus superbus Fuchs, 1930. A: Neck. B: Anterior end. C: Female reproductive system. D: Entire male. E: Lateral field. F: Entire Female. G: Male posterior end. H, I: Female posterior end.
Fig. 9. Panagrolaimus trilabiatus Zell, 1987. A: Neck. B, C: Anterior end. D: Entire male. E: Entire female. F: Female reproductive system. G: Lateral field. H: Female posterior end. I: Male posterior end.
Remarks. Our population is similar to material of P. superbus previously studied (Fuchs, 1930; Bostrom, 1989; Abolafia & Peña-Santiago, 2006) in having six free and separated lips, three lateral field, corpus/ isthmus ratio (2.3-3.0 vs 2.0-3.1 in Abolafia & Peña-Santiago, 2006), spicules shape and length (35-37 pm), gubernaculum shape and length (13-15 pm), and phasmid position (mid tail) and similar tail in both sexes. However, it differs with the material examined by Fuchs (1930) in having shorter body (0.73-0.89 mm in females and 0.62-0.88 mm in males vs 1.20 mm in female and 1.06 mm in male). In comparison with the material examined by Bostrom (1989) it differs by having shorter body (vs 0.86-1.32 mm in females and 0.74-1.05 mm in males). In addition, Abolafia & Peña-Santiago (2006) reported this species with longer body (vs 0.75-0.95 mm in females and 0.74-0.86 mm in males). Abolafia and Peña-Santiago (2006) studied this species with more posterior phasmids position (50% vs 62-69% oftail length). Compared with specimens studied by Thorne (1937) and Rühm (1956) as P. subelongatus, it differs slightly in body length range (vs 0.7-1.0 mm in females and 0.7-0.90 mm in males reported by Thorne, 1937; and 0.981.22 in females and 0.96-1.18 mm in males reported by Rühm, 1956).
This species is reported for the first time from Iran.
Panagrolaimus trilabiatus Zell, 1987 (Figs 7K-O & 9)
Measurements. See Table 2.
Population collected in Tehran, province of Tehran (5$$, 3^).
Female. Body length 0.85-1.12 mm, cylindrical, slightly curved ventrad after fixation. Cuticle smooth; having 4-5 pm thickness, annuli 1 pm wide. Lateral field with five incisures occupying 10% of midbody diameter, fading out near the phasmid. Lip region continuous with neck, having three lips and a circular amphid opening. Labial papillae distinct. Stoma panagrolaimoid, with distinct cheilo-, gymno- and stegostom. Cheilostom without refringent rhabdia. Gymnostom longer than cheilostom, with well cuticularised rhabdia. Stegostom having four distinct parts. Pharyngeal corpus cylindrical, 1.8-3.2 times isthmus length, with procorpus longer than metacorpus. Isthmus slenderer than corpus 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 6575% of neck length. Excretory pore opening at isthmus level, near the bulb, at 75-85% of neck
length. Deirid at 90-95% of neck length, at bulb level. Reproductive system monodelphic-prodelphic, located on right side of intestine. Ovary without flexure posterior to vulva level. Oviduct short, less than a half of the corresponding body diameter. Uterus tubular, about five times the corresponding body diameter long. Post-uterine sac 0.2-0.4 times the corresponding body diameter long. Vagina extending inward less than a half of the body width. Vulva protruding, located posterior to middle part of body. Rectum 0.8-0.9 times the anal body diameter. Tail conical, with lateral field ending at phasmid level. Phasmid at 40-50% of tail length.
Male. Body curved ventrally after fixation. Reproductive system monorchic, testis reflexed dorsad anteriorly. Tail conical, distally curved ventrad. Two pairs of precloacal genital papillae. Five pairs of caudal genital papillae are present along the tail comprising: one ventral pair, one lateral pair, one dorsal pair, and two pairs near tail terminus. Spicules curved ventrally: manubrium rounded; calamus as wide as manubrium, straight; lamina ventrally curved, very wide proximally and pointed terminus, having two longitudinal lines. Gubernaculum well developed, 15 pm long or less than half of the spicule length.
Locality and habitat. Material has been found in Kerman (province of Kerman), in association with seed coat of wheat (Triticum aestivum L.).
Remarks. This species description agrees with the material of P. trilabiatus described by Zell (1987) in rounded lips, lateral field with five incisures, corpus/isthmus ratio (1.8-3.2 vs 3.0), vulval position (56-62 vs 54-64) (see Zell, 1987; Abolafia & Peña-Santiago, 2006). However, it differs from that by having slightly longer body (0.85-1.12 mm in females and 0.76-0.79 mm in males vs 0.55-0.90 mm in females and 0.49-0.78 mm in males), tail slightly less slender (c'=2.1-2.7 in females and 1.7-2.1 in males vs c'=2.4-3.1 in females and 2.0-2.7 in males in type specimens), and spicules longer (36 vs 23-24 pm). This species with its robust body and five lateral field incisures could be recognized from other species of the Panagrolaimus.
This species is reported for the first time from Iran.
DISCUSSION
On the phylogeny of the family Panagrolaimidae. In this study, we tried to analyse the 18S rDNA to understand the evolutionary relationships of Panagrolaimus species. Relating to the family Panagrolaimidae, the consensus tree inferred from 18S rDNA (Figs 10 & 11) appears to
Table 2. Measurements of Panagrolaimus cf. papillosus Loof, 1971, P. cf. subelongatus (Cobb, 1914) Thorne, 1937, P. superbus Fuchs, 1930 and P. trilabiatus Zell, 1987 from Iran. All measurements in pm, and in the form; mean ± standard error (range).
Species P. cf. papillosus P. cf. subelongatus P. superbus P. trilabiatus
Locality Anduhjerd Shahdad Kerman Gharyatolara Kerman
Province Kerman Kerman Kerman Kerman Kerman
Habitat Vitis vinifera Soil Haloxylon persicum Seed coat of wheat (Triticum aestivum)
n 3?? 13 3?? 233 3?? 233 3?? 233 5?? 333
Body length 772.7±24.1 (754-800) 791 733.3±27.7 (709-764) 627, 682 875±14.1 (864-891) 882, 828 800±80.8 (736-891) 628, 709 1001.7±96.2 (854-1118) 778.8±13.8 (764-791)
a 23.4±0.7 (22.9-24.2) 22.7 23.5±3.8 (19.8-27.5) 26.7, 31.4 19.8±1.2 (18.5-21.0) 24.0, 21.9 20.3±3.01 (17.7-23.6) 22.3, 22.1 19.9±1.1 (18.8-21.6) 20.3±1.1 (19.3-21.5)
b 4.6±0.3 4.4 4.3±0.2 4.3, 5.0±0.1 4.9, 5.4±0.5 5.1, 6.6±0.3 5.4±0.2
(4.3-4.9) (4.1-4.6) 4.6 (4.8-5.2) 5.1 (5.0-6.0) 5.1 (6.1-7.2) (5.2-5.7)
19.8±0.6 18.6 20.5±1.09 14.8, 20.4±1.4 19.5, 23.3±2.9 19.6, 15.3±1.4 15.8±2.4
(19.0-20.2) (19.3-21.4) 15.4 (18.8-21.8) 17.5 (20.0-25.2) 19.3 (13.5-16.8) (13.1-17.6)
1.8±0.1 1.6 1.8±0.2 2.1, 1.8±0.1 1.7, 1.7±0.2 1.5, 2.3±0.2 1.9±0.2
(1.7-2.0) (1.6-2.1) 1.9 (1.7-2.0) 1.7 (1.5-2.0) 1.4 (2.1-2.7) (1.7-2.1)
V 63.8±2.4 62.2±3.6 64.0±6.9 58.6±4.01 59.0±2.4
(62-67) (58-66) (58-72) (54-62) (56-62)
Lip region width 11.0±0.5 (10.4-11.3) 10 11.3±0.9 (10-12) 7, 7 11.0±0.5 (10-11) 11 10.4±0.9 (9-11) 9, 10 8.9±0.8 (8-10) 7.9±0.5 (7-8)
Stoma 12.3±0.9 (11-13) 13 11.9±1.08 (11-13) 10, 12 14.8±1.0 (14-16) 14, 13 12.9±0.5 (12-13) 11, 10 10.9±0.8 (10-12) 9.4±0.0 (9.4)
Pharyngeal 97.2±1.4 40 97.2±1.4 73, 98.1±2.4 103, 78.6±2.6 71, 85.1±10.2 81.4±1.1
corpus (96-99) (96-99) 73 (95-100) 87 (75-80) 76 (68-95) (80-82)
Isthmus 35.3±2.3 (33-38) 13 33.6±1.1 (33-35) 39, 39 35.8±5.7 (32-42) 39, 35 30.3±2.8 (27-33) 24, 25 34.0±4.6 (26-39) 31.1±3.4 (28-35)
Bulb 29.4±1.1 (28-31) 11 26.1±2.3 (23-28) 18, 24 29.2±4.1 (24-32) 32, 31 27.0±1.4 (25-28) 21, 26 26.2±1.6 (24-28) 22.6±0.9 (22-24)
Pharynx 161.9±1.6 160 156.8±1.3 129, 163.2±3.7 173, 136.8±1.7 121, 145.8±12.5 134.2±6.4
length (160-163) (155-158) 135 (159-167) 153 (136-139) 128 (129-162) (127-140)
Neck 166.7±7.5 (162-175) 181 170.0±3.3 (167-174) 147, 148 174.6±4.8 (172-180) 181, 162 147.8±1.4 (146-149) 122, 139 152.5±14.8 (132-169) 143.5±4.7 (139-148)
Nerve ring- 124.2±0.5 118 130.7±2.09 112, 128.0±3.8 136, 107.5±4.0 89, 109.6±8.8 103.5±8.5
ant. end (124-124) (128-132) 116 (124-130) 124 (105-110) 93 (99-119) (97-113)
Excretory pore-ant. end 138.1±5.1 (133-143) 129 141.1±3.4 (139-143) 123, 126 152.2±3.9 (149-157) 157, 141 139.6±0.0 (139.6) n=1 110 121.9±6.3 (112-128) 119.3±4.6 (116-123)
Deirid-ant. End 154.7±9.1 (144-161) 158 161.3±0.0 (161) 119 155.7±2.8 (153-158) 145 132.1±0.0 (132.1) n=1 110 139.6±20 (125-154) 142.5±0.0 (142.5)
Cuticle thickness 2 2 2.8±0.0 (2.8) 2.8 1.5 1.6 1.4 1 3 3
Body width: 33.3±1.4 27.4±2.8 (24- 23, 35.8±1.8 32, 30.8±0.5 26 38.7±1.4 31.8±3.1
neck base (32-35) 30) 20 (34-38) 32 (30-31) (37-41) (28-34)
Body width: 33.0±1.8 35 31.8±4.8 (27- 23, 44.3±3.4 37, 39.6±1.8 28, 50.2±3.4 38.4±1.4
midbody (31-35) 36) 22 (41-48) 38 (38-41) 32 (45-55) (37-40)
Body width: 21.4±1.4 26 19.8±1.8 (18- 20, 23.6±0.0 27, 20.1±0.5 22, 28.7±3.1 26.1±1.1
anus (20-23) 22) 24 (23.6) 28 (20-21) 25 (23-32) (25-27)
Lateral field 7.5±2.6 (6-9) 7 7.1±0.6 (6.6-7.5) 3.8 9.4±0.0 (9.4) - 4.7±0.0 (4.7) 6.6 3.8±0.0 (3.8) 4.2±0.6 (3.8-4.7)
Vagina 13.8±0.5 11.3±0.9 15.7±3.8 10.7±0.5 13.6±2.2
(13-14) (10-12) (12-20) (10-11) (10-16)
Ovary 354.0±9.1 334.9±52.92 369.4±56.5 304.3±67.1 457.1±26.7
(345-364) (275-376) (329-409) (228-355) (428-491)
Table 2. Measurements of Panagrolaimus cf. papillosus Loof, 1971, P. cf. subelongatus (Cobb, 1914) Thorne, 1937, P. superbus Fuchs, 1930 and P. trilabiatus Zell, 1987 from Iran. All measurements in |im, and in the form; mean ± standard error (range) (continued).
Species P. cf. papillosus P. cf. subelongatus P. superbus P. trilabiatus
Locality Anduhjerd Shahdad Kerman Gharyatolara Kerman
Province Kerman Kerman Kerman Kerman Kerman
Seed coat of wheat
Habitat Vitis vinifera Soil Haloxylon persicum (Triticum aestivum)
n 3ÇÇ 13 3ÇÇ 233 3ÇÇ 233 3ÇÇ 233 5ÇÇ 333
Postuterine sac 24.0±3.5 15.1±0.9 1S.9±0.9 11.9±0.5 ^.б±2.4
_ _ _ _ _
(22-2S) (14-16) (1S-20) (11-12) (15-21)
2б.4±1^ 18.6±0.5 29.2±1.S 25.5±0.9 23.S±1.5
Rectum _ _ _ _ _
(24-2S) (18-19) (27-31) (24-2б) (22-25)
Tail 39.0±1.1 35.8±1.6 43.1±3.3 45, 34.б±4^ 32, б5.7±3.2 50.0±7.4
42
(3S-40) (34-37) (40-4б) 47 (3б-39) 37 (б1-б9) (44-5S)
Vulva- anterior 493.2±20.1 455.3±10.23 559.4±5б.3 4бб.7±13^ 590.7±б4.5
end (471-509) (445-466) (5^-б23) (454-4S2) (50S-673)
Spicules 29, 35, 30, 2S.9±3.3
— 32 — — — —
30 37 33 (25-32)
11, 15, 11, 11.3±0.9
Gubernaculum — 14 — — — —
13 13 14 (10-12)
be divided into five and six main clades based on the Bayesian tree and Maximum Likelihood tree respectively. According to the Bayesian tree clades are: I) Panagrolaimus spp. which form a robust clade (1.00 posterior probability); II) Halicephalobus spp., P. detritophagus, P. paetzoldi and two unidentified Panagrolaimus spp.; III) Panagrellus redivivus, Baujardia mirabilis and P. paetzoldi which is very consistent (1.00 posterior probability); IV) Turbatrix aceti; and V) Plectonchus spp. and Panagrobelus stammeri, set near the base of the tree.
The consensus tree obtained by ML is similar to that elaborated by BI (Bayesian Inference), however two populations of unidentified Panagrolaimus (FJ590956; FJ590959) form a separate clade in ML tree.
The genetic distance among the identified species of Panagrolaimus ranges from 0.00-0.542 for S SU rDNA gene. Genetic distance using Maximum Composite Likelihood method among Panagrolaimus species showed that P. trilabiatus (KF011487) and P. subelongatus (AY284681) have no genetic distance. The maximum genetic distance was observed between two populations of P. paetzoldi (FJ040414; FJ590979).
On Panagrolaimus species phylogeny. The phylogenetic tree inferred from SSU sequences of Panagrolaimus species indicated that this genus is a paraphyletic group, agreeing with Shannon et al. (2005; ITS and D3 genes), van Megen et al. (2009; SSU rDNA gene) and Lewis et al. (2009; 18S rDNA, 28S rDNA, ND5 genes). Within the family Panagrolaimidae, a well-supported clade was
formed by members of the genus Panagrolaimus with weak bootstrap support (64%) in ML tree, and 1.00 posterior probability in Bayesian Inference tree, while the rest of Panagrolaimus species including P. paetzoldi (FJ590979) and P. detritophagus (FJ590980) groups with Halicephalobus species. In addition, P. paetzoldi (FJ040414) groups with Panagrellus species and Baujardia mirabilis. The Bayesian tree analysis of 18S rDNA sequence places P. facetus (KF011488) close to an unidentified Panagrolaimus (KC52709) and to P. davidi (HQ270131) and an unidentified Panagrolaimus (EU040129) in ML tree. The unidentified Panagrolaimus (U81579) and P. subelongatus (AY284681) places close to P. trilabiatus (KF011487) in ML and Bayesian tree, respectively. P. trilabiatus and P. subelongatus; both species have three lips. Finally, P. cf. papillosus places close to P. davidi (HQ270131) in Bayesian tree and two unidentified Panagrolaimus (KC522708; FJ590961) in ML tree. The ML and Bayesian tree topology for Panagrolaimus species obtained from 18S ribosomal DNA is consistent with the topology given by Shannon et al. (2005), van Megen et al. (2009) and Lewis et al. (2009). Although the results indicate a single well-supported monophyletic clade (referred to herein as Clade I, see Figs 10 & 11) that exclusively contained most of the Panagrolaimus species, the rest of Panagrolaimus species (P. paetzoldi, FJ040414, FJ590979; P. detritophagus, FJ590980; Panagrolaimus
FJ590978 Panagrolaimus sp. FJ590997 Panagrolaimus sp. FJ590981 Panagrolaimus davidi FJ590983 Panagrolaimus sp. FJ590984 Panagrolaimus sp. FJ590985 Panagrolaimus sp. FJ590986 Panagrolaimus sp. FJ590987 Panagrolaimus sp. FJ590988 Panagrolaimus sp. FJ590989 Panagrolaimus sp. FJ590990 Panagrolaimus sp. KC522709 Panagrolaimus sp. KC522710 Panagrolaimus sp. KC522711 Panagrolaimus sp. KC522712 Panagrolaimus sp. KC522713 Panagrolaimus sp. KC522714 Panagrolaimus sp. KC522716 Panagrolaimus sp.
- FJ590976 Panagrolaimus sp. FJ590971 Panagrolaimus sp. FJ590970 Panagrolaimus sp. FJ590969 Panagrolaimus sp. FJ590968 Panagrolaimus sp. FJ590963 Panagrolaimus sp. FJ590958 Panagrolaimus sp. FJ590957 Panagrolaimus sp. GUO14546 Panagrolaimus detritophagus AJ567385 Panagrolaimus davidi AY284681 Panagrolaimus subelongatus HQ270130 Panagrolaimus davidi EU543176 Panagrolaimus detritophagus AF430487 Panagrolaimus sp. AF430486 Panagrolaimus sp. AF430485 Panagrolaimus sp. U81579 Panagrolaimus sp. KF011487 Panagrolaimus trilabiatus —EU040129 Panagrolaimus sp. KF011488 Panagrolaimus facetus
— HQ270131 Panagrolaimus davidi KC522706 Panagrolaimus rigidus KC522707 Panagrolaimus super bus FJ590974 Panagrolaimus rigidus FJ590973 Panagrolaimus superbus FJ590972 Panagrolaimus sp. FJ590962 Panagrolaimus sp. FJ590960 Panagrolaimus sp. AF430484 Panagrolaimus sp. AF430483 Panagrolaimus sp. DQ285636 Panagrolaimus cf. rigidus
—KC522715 Panagrolaimus sp. .FJ590961 Panagrolaimus sp. y .KC522708 Panagrolaimus sp. 361— KF011489 Panagrolaimus ci. papillosus
-AF202165 Turbatrix aceti
4|-FJ590980 Panagrolaimus detritophagus
~I_FJ590979 Panagrolaimus paetzoldi -AF202156 Halicephalobus gingivalis
65|HQ697250 Halicephalobus cf. gingivalis ' JF706244 Halicephalobus cf. gingivalis FJ590967 Halicephalobus sp. 9 FJ590966 Halicephalobus sp. FJ590965 Halicephalobus sp. FJ590964 Halicephalobus sp. IAF547385 Baujardia mirabilis ^¡-1 I— FJ040414 Panagrolaimus paetzoldi 6UaF083007 Panagrellus redivivus 'AF036599 Panagrellus redivivus
.FJ590959 Panagrolaimus sp.
99IFJ590956 Panagrolaimus sp. AF202153 Panagrobelus stammen ■
AY593920 Plectonchus sp. I yi 921AF202154 Plectonchus sp. |
-AY602182 Caenorhabditis japonica I
outgroup
Fig. 10. The Maximum Likelihood tree of new sequences of Panagrolaimus species from Iran and closely related species belonging to the family Panagrolaimidae based on 18S rDNA region from GenBank.
0.61
FJ590983 Panagrolaimus sp. HQ270130 Panagrolaimus davidi AJ567385 Panagrolaimus davidi FJ590976 Panagrolaimus sp. FJ590981 Panagrolaimus davidi KC522716 Panagrolaimus sp. U81579 Panagrolaimus sp. EU543176 Panagrolaimus detritophagus GUO14546 Panagrolaimus detritophagus FJ590957 Panagrolaimus sp. FJ590958 Panagrolaimus sp. FJ590968 Panagrolaimus sp. 100 FJ590969 Panagrolaimus sp. FJ590970 Panagrolaimus sp. FJ590971 Panagrolaimus sp. FJ590977 Panagrolaimus sp. FJ590984 Panagrolaimus sp. FJ590985 Panagrolaimus sp. FJ590988 Panagrolaimus sp. FJ590989 Panagrolaimus sp. KC522710 Panagrolaimus sp. KC522711 Panagrolaimus sp. KC522713 Panagrolaimus sp. KC522714 Panagrolaimus sp. j oqFJ590986 Panagrolaimus sp. _r KC522712 Panagrolaimus sp. ~ FJ590978 Panagrolaimus sp. 0.92. KC522707 Panagrolaimus superbus nn FJ5 90973 Panagrolaimus superbus KC522707 Panagrolaimus superbus FJ590974 Panagrolaimus rigidus 1.001 DQ285636 Panagrolaimus cf. rigidus KC522715 Panagrolaimus sp. 1.001- FJ590961 Panagrolaimus sp.
AF430487 Panagrolaimus sp. AF430485 Panagrolaimus sp. AF430486 Panagrolaimus sp. FJ590972 Panagrolaimus sp. FJ590962 Panagrolaimus sp. FJ590960 Panagrolaimus sp. AF430484 Panagrolaimus sp. 001 AF430483 Panagrolaimus sp. rFJ590990 Panagrolaimus sp. T-FJ590987 Panagrolaimus sp. FJ590963 Panagrolaimus sp.
-KF011487 Panagrolaimus trilabiatus
-AY284681 Panagrolaimus subelongatus KC522709 Panagrolaimus sp.
KF011488 Panagrolaimus facetus HQ270131 Panagrolaimus davidi
KF011489 Panagrolaimus cf. papillosus KC522708 Panagrolaimus sp.
EU040129 Panagrolaimus sp.
0.99
1.00
FJ040414 Panagrolaimus paetzoldi AF036599 Panagrellus redivivus AF083007 Panagrellus redivivus AF5473 85 Baujardia mirabilis AF202165 Turbatrix aceti | jy ¿ÜÜI AY593920 Plectonchus sp. ■
J 1 AF202154 Plectonchus sp. I y
1 '— AF202153 Panagrobelus stammeri
AY602182 Caenorhabditis japónica
l oo, FJ590967 Halicephalobus sp. Ann FJ590966 Halicephalobus sp. l.ooii FJ590964 Halicephalobus sp. FJ590965 Halicephalobus sp. 1 HQ697250 Halicephalobus cf. gingivalis 1JF/06244 Halicephalobus cf. gingivalis ■ AF202156 Halicephalobus gingivalis i— FJ590980 Panagrolaimus detritophagus '— FJ590979 Panagrolaimus paetzoldi
_r FJ590959 Panagrolaimus sp.
HoP FJ590956 Panagrolaimus sp.
II
III
outgroup
Fig. 11. The Bayesian Inference tree of new sequences of Panagrolaimus species from Iran and closely related species belonging to the family Panagrolaimidae based on 18S rDNA region from GenBank.
sp., FJ590956, FJ590959) were included within a separate clade, referred to herein as Clade III, IV. Although our analyses agrees with the previously published 18S rDNA tree of Panagrolaimomorpha (van Megen et al, 2009; Lewis et al, 2009), in the fact that the 18S rDNA gene does provide resolution within the panagrolaimids for the phylogenetic analysis within this group of nematodes, more rDNA genes are needed to understand more fully the relationships of the described species of Panagrolaimus.
ACKNOWLEDGEMENT
We thank Dr. Fasihi and Mr. Mohammadi (Kerman Medical University) for the opportunity to work in the Parasitology Lab. of the Medical School. Thanks also are extended to Mr. Ghafouri and Mr. Alirezaei for sampling.
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Mehdizadeh, S., Shokoohi, E. and Abolafia, J. Морфологическая и молекулярная характеристика видов Panagrolaimus Fuchs, 1930 (Nematoda, Rhabditida, Panagrolaimidae) из Ирана. Резюме. Представители семи видов семейства Panagrolaimidae описаны из природных экосистем провинции Керман, Иран: Panagrolaimus concolor; P. facetus; P. cf. labiatus; P. cf. papillosus; P. cf. subelongatus; P. superbus и P. trilabiatus. Приведены описания, измерения, рисунки и свето-микроскопические фотографии для всех видов. Проведен молекулярный анализ трех из семи выявленных видов. Лишь филогенетический анализ последовательностей 18S rDNA методом максимального правдоподобия (ML) показал близость P. facetus к P. davidi (HQ270131) и не определенному до вида Panagrolaimus (EU040129). В деревьях, построенных по результатам Байесова анализа (BI), этот вид объединяется с Panagrolaimus sp. (KC522709). Panagrolaimus trilabiatus близок у P. subelongatus (AY284681) и к Panagrolaimus sp. (FJ590963). В этом же древе Panagrolaimus cf. papillosus объединяется с P. davidi (HQ270131) и показывает родство к P. facetus (KF011488). В ML-дереве этот вид объединяется с двумя не определенными Panagrolaimus (KC522708; FJ590961). Нуклеотидные последовательности 18S rDNA для P. facetus, P. cf. papillosus и P. trilabiatus приводятся впервые. Как метод максимального правдоподобия (ML), так и Байесов анализ (BI) показали парафилию рода Panagrolaimus.
