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61Russian Journal of Nematology, 2014, 22 (1), 11 - 22
Description of Ektaphelenchoides poinari sp. n. (Nematoda: Ektaphelenchinae) from Iran with a compendium of the valid species of the genus Ektaphelenchoides Baujard, 1984
Farzad Aliramaji1, Ebrahim Pourjam1, Mohammad Reza Atighi1, Weimin Ye2, Ali Roshan-Bakhsh1 and Majid Pedram1
1 Department of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran
e-mail: pourjame@modares.ac.ir 2 Nematode Assay Section, North Carolina Department of Agriculture, Raleigh, NC 27607, USA
Accepted for publication 18 November 2013
Summary. Ektaphelenchoides poinari sp. n. is described and illustrated based on morphological, morphometric and molecular data. The new species is characterised by females with 477-565 ^m long body, offset lip region 7.5-9.5 ^m wide, separated from the rest of the body by a shallow constriction, 1823 ^m long stylet lacking knobs at base, position of excretory pore at the level to slightly posterior of the metacorpus base, three incisures in lateral field, short post-uterine sac (PUS) (0.3-0.4 corresponding body width), posterior end of the body conical, males rare, spicules having rounded condylus, pointed rostrum and blunt end. The new species is close to E. attenuata, E. kelardashtensis, E. musae, E. pini, E. sylvestris and E. winteri, but differs from them by its shorter PUS, shape of posterior body end in females and males, position of vulva and excretory pore and molecular characters. Ektaphelenchoides poinari sp. n. was easily differentiated from other sequenced species by the partial small subunit rRNA gene (SSU), D2D3 expansion segment of the large subunit rRNA gene (LSU) and internal transcribed spacer 1 (ITS1). Phylogenetic analysis with these sequences suggests that E. poinari sp. n. is close to other Ektaphelenchoides species and Devibursaphelenchus. A compendium for valid species based on morphological and morphometric characters is also given.
Key words: internal transcribed spacer 1, large subunit rRNA gene, molecular, morphology, morphometrics, new species, phylogeny, small subunit rRNA gene, taxonomy, Tehran.
Hunt (2008) listed the valid species of aphelenchid genera. The genus Ektaphlenchoides Baujard, 1984 currently contains ten valid species namely E. andrassyi Atighi, Pourjam, Pedram, Ye & Aliramaji, 2013, E. attenuata (Massey, 1974) Baujard, 1984, E. compsi Baujard, 1984, E. hunti Atighi, Pourjam, Pedam, Ye & Robbins, 2012, E. kelardashtensis Atighi, Pourjam, Pedram, Ye, Robbins & Namjou, 2013, E. musae Baujard, 1984, E. pini (Massey, 1966) Baujard, 1984, E. ruehmi Yaghoubi, Pourjam, Atighi & Pedram, 2014, E. spondylis Kanzaki, Giblin-Davis & Center 2009, E. sylvestris Pedram, Pourjam, Atighi, Ye & Houshmand, 2012 and E. winteri Hooper, 1995. A review of related hosts and locality of the above mentioned species is given in Atighi et al. (2012). There is little information on the biology of the genus. According to Hooper (1995), the adults and
juveniles of E. winteri were firmly attached to their insect host and the contents of the nematode intestine were pink, the same colour as the haemolymph of the insect host. This observation was similar to those reported for some other aphelenchid genera, e.g. Acugutturus Hunt, 1980 and Noctuidonema Remillet & Silvain, 1988 (Hunt, 1993). Kanzaki et al. (2009) recovered E. spondylis from the body cavity of Spondylis buprestoides. The present authors recovered several species on bark of different trees in Iran (some were not reported). For most of them, rearing on fungus plates and fungus plates with some other rhabditid and aphelenchid nematodes was not successful. The only successful culture was for E. sylvestris (Pedram et al., 2012) on co-cultures with aphelenchid and rhabditid nematodes, although its predatory behaviour was not observed during occasional observations.
Recetly, Kanzaki (2014) showed predatory feeding habit of E. spondylis Kanzaki, Giblin-Davis & Center, 2009 on Pseudodiplogasteroides sp. The current information leads us to propose a wide range of host association types for the members of the genus. During nematode surveys in the north of Iran, a new species of Ektaphelenchoides was recovered from bark samples of a dead pine tree having galleries of bark beetles and was described in the present paper as E. poinari sp. n. This is the fifth species originally described from Iran.
MATERIALS AND METHODS
Several wood samples were collected from different regions in the north of Iran during 2010 to 2012. Bark samples from a dead European red pine (Pinus sylvestris L.), showing bark beetles galleries, were collected from the city of Tehran and yielded an aphelenchid nematode, belonging to Ektaphelenchoides. The nematode was also recovered from the soil around the trunk of the host tree. The second population with few individuals was recovered from decaying dung samples collected from the city of Shahr-e-Rey. They were extracted from the wood samples by soaking a small | amount of wood in water for 48 h and were hand picked under a Nikon stereomicroscope, model SMZ1000. To extract nematodes from soil and dung, the tray method (Whitehead & Hemming, 1965) was used. The nematodes were heat-killed by adding boiling 4% formalin solution and then transferred to the anhydrous glycerin and mounted on permanent slides according to De Grisse (1969). Permanent slides were made and examined under a Nikon Eclipse E600 light microscope. Photographs were taken using an Olympus DP72 digital camera attached to an Olympus BX51 microscope powered with differential interference contrast (DIC).
Ten nematodes were picked into distilled water and their identity was confirmed with light microscopy before being placed into 50 ^l AE buffer (10mM Tris-Cl, 0.5mM EDTA; pH 9.0) and crushed with a pipette tip. DNA samples were stored at -20°C until used as a PCR template. Primers for SSU amplification were forward primer 18S965 (5'-GGCGATCAGATACCGCCCTAGTT-3') and reverse primer 18S1573R (5'-TACAAAGGGCAGGGACGTAAT-3') (Mullin et al, 2005), forward primer SSUF07 (5'-AAAGATTAAGCCATGCATG-3') and reverse primer SSUR26 (5'-CATTCTTGGCAAATGCTTT CG-3') (Floyd et al., 2002), and forward primer 18 SnF (5' -TGGATAACTGTGGTAATTCTAGAG C-3') and reverse primer 18SnR (5'-TTACGACTTTTGCCCGGTTC-3') (Kanzaki &
Futai, 2002). Primers for ITS1 amplification were forward primer rDNA2 (5'-TTGATTACGTTCC CTGCCCTTT-3') (Vrain et al, 1992) and reverse primer rDNA1.58S (5'-ACGAGCCGAGTGATCC ACCG-3') (Cherry et al, 1997). Primers for 28S D2D3 amplification were forward primer D2a (5'-ACAAGTACCGTGAGGGAAAGT-3') and reverse primer D3b (5'-TGCGAAGGAACCAGCTACTA-3') (Nunn, 1992). The 25 ^l PCR was performed using Apex Taq Red Master Mix DNA polymerase (Genesee Scientific Corporation, San Diego, CA, USA), according to the manufacturer's protocol. The thermal cycler program for PCR was as follows: denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s, and extension at 72°C for 2 min. A final extension was performed at 72°C for 10 min (Ye et al, 2007). PCR products were cleaned using ExoSap-IT (Affymetrix, Inc., Santa Clara, CA, USA) according to the manufacturer's protocol and were sequenced by Genomic Sciences Laboratory in North Carolina State University using an Applied Biosystems 3730 XL DNA Analyzer (Life Technologies, Carlsbad, CA). The resulted DNA sequences were compared with other nematode species in GenBank using the BLAST homology search program. The most similar sequences were downloaded for phylogenetic analysis. The DNA sequences were aligned by Clustal W (http://workbench.sdsc.edu, Bioinformatics and Computational Biology group, Dept. Bioengineering, UC San Diego, CA). The model of base substitution in the SSU and LSU sets were evaluated using MODELTEST version 3.07 (Posada & Crandall, 1998). The Akaike-supported model, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates were used in phylogenetic analyses. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003) running the chain for 1,000,000 generations and setting the 'burn in' at 1,000. We used MCMC (Markov Chain Monte Carlo) methods within a Bayesian framework to estimate the posterior probabilities of the phylogenetic trees (Larget & Simon, 1999) using the 50% majority-rule.
DESCRIPTION
Ektaphelenchoides poinari sp. n.
(Figs 1-3)
Measurements. Table 1.
Female. Body cylindrical, tapering gradually to both ends, open J-shaped when heat-killed. Cuticle finely annulated. Lateral field with three incisures
Table 1. Morphometrics of Ektaphelenchoides poinari sp. n All measurements in ^m and in the form: mean ± s.d. (range).
Character Original population from city of Tehran Shahr-e-Rey population
Female Male Female
Holotype Paratypes Paratype
n - 15 1 4
L 506 517.0 ± 25.4 (477-565) 527 513.0±76.5 (410-595)
a 29.8 28.5 ± 1.4 (25.1-31.1) 32.9 29.7±4.0 (25-35)
b 6.1 7.4 ± 0.6 (6.1-8.4) 8 8.0±0.7 (7.0-8.5)
b' 2.9 3.0 ± 0.2 (2.5-3.4) 3.4 3.2±0.5 (2.7-3.8)
c - - 15.5 -
c' - - 2.0 -
T or V 78.9 79.3 ± 0.7 (77.8-80.4) 63.9 77.8±1.0 (76.5-78.7)
Head height 3.5 4.0 ± 0.5 (3.5-5.0) 4 3.8±0.5 (3-4)
Head width 7.5 8.0 ± 0.6 (7.5-9.5) 8 8.5±1.3 (7-10)
Stylet 18 20.0 ± 1.3 (18-23) 20 20.3±1.5 (19-22)
Stylet conus 8 8.0 ± 0.7 (7-9) 8 6.3±0.5 (6-7)
m 44.4 39.9 ± 3.2 (33.3-46.2) 40 31.0±4.3 (27.3-36.8)
Median bulb from anterior end 62 59.5 ± 2.9 (56-67) 56 56.5±5.0 (49-60)
Excretory pore from anterior end 67 68.0 ± 2.2 (65-72) 70 64.5±3.0 (63-69)
Hemizonid from anterior end 90 88.0 ± 2.4 (83-93) 88 79.5±6.0 (74-86)
Pharynx base from anterior end 83 70.5 ± 4.7 (65-83) 66 64±4 (59-69)
Nerve ring from anterior end 82 71.0 ± 3.6 (65-78) 68 84±6 (78-91)
Median bulb width 10 11.0 ± 0.5 (10-12) 10.5 11±1 (10-12)
Median bulb length 17 18 ± 1 (16-19) 17 15.5±1.5 (13-17)
Pharyngeal overlapping 92 102.0 ± 8.6 (89-117) 90 96±11 (88-111)
Maximum body width 17 18.0 ± 1.1 (17-20) 16 18.6±1.1(17.5-20)
Vulval body width (VBW) 16 17.0 ± 0.9 (15.5-19) - 17.3±1.3 (16-19)
Body width at MB 14.5 15.0 ± 0.8 (14-17) 15 15.5±0.5 (16-19)
PUS 7 7.0 ± 1.1 (5-8) - 8.0±0.8 (7-9)
Vulva - body end 107 107.0± 7.4 (96-121) - 114±15 (94-127)
Testis or ovary length 241 246 ± 25 (187-293) 337 231±23 (197-250)
Anal (cloacal) body width - - 12 -
Tail - - 34 -
Spicule length (arc line) - - 15 -
Capitulum - - 6.5 -
(Figs 1P, 3C). Head set off from body contour by a shallow constriction, width ca 2 times height. Stylet without basal knobs, conus ca 44.5% total stylet length. Procorpus cylindrical, 1.6-2.2 times longer than stylet, connected to a muscular, rectangular metacorpus with the granular part ca 30% of its length and the posterior part weakly muscular. Pharyngo-intestinal junction one metacorpal valve width posterior to metacorpus. Pharyngeal glands well developed, overlapping intestine dorsally ca 1.4-1.7 times the distance from anterior end to the base of median bulb or ca 5.8-8.3 times body width
at median bulb level. Nerve ring surrounding pharyngeal glands and intestine at ca 3.7-4.9 metacorpus length posterior to the base of metacorpus. Excretory pore with slight variation in position, i.e. at the level of the metacorpus base to slightly posterior. Hemizonid 17.6-25.2 times metacorpus length from anterior end. Reproductive system monodelphic-prodelphic, gonad outstretched, occupying 38-54% of the body length, located at left of intestine. Oocytes mostly in two rows. Oviduct small, spermatheca oval to irregular, filled with rounded sperm (Figs 1I & J, 2E). Crustaformeria
Fig. 1. Ektaphelenchoides poinari sp. n. A: Female entire body; B: Female pharyngeal region; C: Female anterior end; D: Male anterior end; E: Male pharyngeal region; F: Male entire body; G & H: Variation in position of excretory pore; I & J: Part of female reproductive tract; K: Male tail region; L-O: Variation in female posterior body end; P: Lateral field with three incisures.
Fig. 2. Ektaphelenchoides poinari sp. n. A: Female, part of posterior part showing PUS and blind end of intestine; B: Anterior end in detail; C: Lip region in detail; D: Anterior region; E: Spermatheca with fine rounded sperm cells and PUS (arrow); F: Male posterior end; G: Spicule; H-J: Variation in position of E-pore (arrows); K: Three lines in lateral field; L-O: Variation in female posterior body end. All scale bars = 10 ^m.
obscure, details hardly visible, uterus thick-walled, vagina not sclerotised, straight, slightly inclined anteriorly. Post-uterine sac short, less than half body width at vulva region (0.3-0.4 corresponding body width) or ca 8.8 % of the vulva to end of intestine distance. Intestine ends in a blind sac, vulva to end of intestine distance 64-90 ^m. Anus and rectum indistinct. Distance between vulva and body end 6-7 times longer than body width at vulva. Posterior body end conical, ending to a rounded to pointed tip (Fig. 1 L-O).
Male. Rare. Body slender, tapering gradually to both ends, J-shaped after heat relaxation. General morphology similar to that in female, except sexual characters and stronger ventrally bent posterior body end. Testis single, expanded anteriorly. Spicules arcuate, separate, ca 2.2 times longer than capitulum width, having well developed condylus with rounded tip, pointed rostrum, lamina/calomus complex smoothly rounded and no cucullus. The single midventral precloacal papilla (P1) and the distal subventral pair of papillae (P4) not observed. One pair (P2) just before cloacal opening and another pair (P3) at ca 34% of tail length posterior to cloacal aperture (Fig. 3 A & B). Tail conoid with rounded terminus.
Type habit and locality. The original population was recovered from the bark of a dead Pinus sylvestris L. and soil around its trunk in a small park, Poonak square, Tehran, Iran in February 2011.
Other locality. The second population (few individuals) was recovered from decaying dung samples collected from the city of Shahr-e-Rey.
Type material. Holotype female, five paratype females and one paratype male deposited at Nematode Collection of the Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran. Three female paratypes deposited at each of the following collections: CABI Europe-UK, Egham, Surrey, UK;
USDA Nematode Collection, Beltsville, MD, USA and Department of Nematology, Agricultural University, Wageningen, the Netherlands.
DIAGNOSIS AND RELATIONSHIPS
Ektaphelenchoides poinari sp. n. is characterised by females with 477-565 ^m long body, the offset lip region 7.5-9.5 ^m wide, separated from the rest of the body by a shallow constriction, 18-23 ^m long stylet lacking knobs at the base, the position of an excretory pore at the level to slightly posterior of the metacorpus base, three incisures in the lateral field, the short PUS (ca 0.4 corresponding body width), the posterior end of the body conical, rare males with spicules having the rounded condylus, the pointed rostrum and the blunt tip. By having a short PUS, the new species comes close to six known species of the genus, namely E. attenuata, E. kelardashtensis, E. musae, E. pini, E. sylvestris and E. winteri. Compared to E. attenuata, the new species has a shorter body (477-565 vs 870-950 ^m), a posteriorly located vulva (V = 77.8-80.4 vs 60-63), an anteriorly located excretory pore (65-72 vs 97-100 ^m from anterior), the shorter PUS (5-8 vs 23-28 ^m) or 0.3-0.4 vs 1/2 vulval body diameter and a conical tail with the rounded tip vs filiform. Compared to E. kelardashtensis, the new species has a longer stylet (18-23 vs 13-16 ^m), a posteriorly located vulva (V = 77.8-80.4 vs 61.568.0) and a conical tail with the rounded tip vs filiform. Compared to E. musae, the new species has a posteriorly located vulva (V = 77.8-80.4 vs 64-69), the shorter PUS (5-8 vs 9-19 ^m long) and a conical tail with the rounded tip vs filiform. Compared to E. pini, the new species has an anteriorly located excretory pore (65-72 vs 94-123 ^m from anterior end), a hemizonid posterior to
Fig. 3. Ektaphelenchoides poinari sp. n. A: The subventral papillae pair (P2) just before cloacal opening (arrow); B: The other pair of subventral papillae (P3) (arrow); C: Cross section showing lateral field with three incisures. All scale bars = 10 ^m.
100
88
52
100
80
100
100
100 I-AB368532 Ektaphelenchus obtusus
-1_| AY284651 Seinura sp.
I FJ969140 Seinura demani AB470969 Noctuidonema s p.
KC881252 Ektaphelenchoides poinari sp. n.
100
EU 287588 Cryptaphelenchus sp
- FJ040413 Anomyctus xenurus
50i-AY508024 Bursaphelenchus gerberae
1— AY508027 Bursaphelenchus paracorneolus r AB368529 Bursaphelenchus rufipennis AY508028 Bursaphelenchus poligraphi 55(1 OCk AM397020 Bursaphelenchus vallesianus i- AY508031 Bursaphelenchus sexdentati I AY508032 Bursaphelenchus sexdentati AY508012 Bursaphelenchus borealis gg. AM397013 Bursaphelenchus hildegardae nj AY508033 Bursaphelenchus tusciae gl AY508013 Bursaphelenchus eggersi TOO- AB299221 Bursaphelenchus clavicauda AM 397023 Bursaphelenchus yongensis JQ765873 Bursaphelenchus comeclus -4 J Q765872 Bursaphelenchus corneolus i HQ407406 Bursaphelenchus corneolus
CG Q421483 Bursaparaparvis picularis AB218829 Burs aphelenchus parvispicularis — AB232162 Bursapnelenchus sinensis
1|00
80 100
100|
Ei"
70
AM397016 Bursaphelenchus pinasteri —|r AY508018 Bursaphelenchus hofmanni
70'-J N153102 Bursaphelenchus mazandaranense
100 r- AM279709 Bursaphelenchus antoniae AY508019 Bursaphelenchus hyloblanum ■\ oo . AM397017 Bursaphelenchus rainulfi
I- AY508017 Bursaphelenchus hellenicus
1001— AY508011 Bursaphelenchus abietinus
- JF317266 Bursaphelenchus africanus
- AB358983 Bursaphelenchus okinawaensis j 4.— AM397019 Bursaphelenchus thailandae
- AY508026 Bursaphelenchusplatzeri
'-AY509153 Bursaphelenchus cocophilus
AY508016 Bursaphelenchus fungivorus 100 i— AM397021 Bursaphelenchus willibaldi ~L GQ845409 Bursaphelenchus braaschae
82r
90
100
54
101
97
AY508030 Bursaphelenchus seani gc — 1 AY508029 Bursaphelenchus seani I AB067757 Bursaphelenchusconicaudatus "L AM397011 Bursaphelenchus conicaudatus _ HQ699855 Bursaphelenchuspopuli EEAY508015 Bursaphelenchus fraudulentus | AY508014 Bursaphelenchus fraudulentus ' AB067758 Bursaphelenchus fraudulentus AM397022 Bursaphelenchus xyhphilus AY508021 Bursaphelenchus mucronatus AY508020 Bursaphelenchus mucronatus
- AY284648 Bursaphelenchus mucronatus AB067760 Bursaphelenchus xylophilus
- AB097864 Bursaphelenchus luxuriosae AY508034 Bursapnelenchus xylophilus GU206792 Bursaphelenchus xylophilus AB067759 Bursaphelenchus mucronatus AM397015 Bursaphelenchus mucronatus F J 768947 Bursaphelenchus xyhphilus AB299224 Bursaphelenchus doul F J 501985 Bursaphelenchus doui AM397012 Bursaphelenchus doui AB299223 Bursaphelenchus doui
-AY508025 Bursaphelenchus anatolius
100 . AY508010 Bursaphelenchus abruptus I AB067756 Bursaphelenchus abruptus
-AB36&535Aphelenchoides stammeri
■ EU 287590 Laimaphelenchus preissii
G U190764 Schistonchus caprifici
95
100
97
97
FN 564938 Schistonchus caprifici H M151003 Schistonchus sp.
FN564940 Schistonchus caprifici G U190763 Schistonchus caprifici 100 f EU306346 Laimaphelenchus penardi
I— AY593919 Laimaphelenchus penardi AY508035Aphelenchoides besseyi
. AY284644 Aphelenchoides blastophthorus
5 changes
Fig. 4. The 10001st Bayesian tree inferred from SSU under GTR+I+G model (lnL = 4913.3193; freqA = 0.2563; freqC = 0.1919; freqG = 0.2626; freqT = 0.2892; R(a) = 1.3177; R(b) = 3.8058; R(c) = 1.1678; R(d) = 0.2231; R(e) = 8.3565; R(f) = 1; Pinva = 0.4171; Shape = 0.4241). Posterior probability values exceeding 50% are given on appropriate clades.
Fig. 5. The 10001st Bayesian tree inferred from LSU D2D3 under TVM+I+G model (-lnL = 16729.0039; freqA = 0.1695; freqC = 0.19; freqG = 0.3218; freqT = 0.3186; R(a) = 1.1046; R(b) = 4.0379; R(c) = 1.4553; R(d) = 0.58; R(e) = 4.0379; R(f) = 1; Pinva = 0.1688; Shape = 0.7095). Posterior probability values exceeding 50% are given on appropriate clades.
excretory pore vs immediately posterior and a conical tail with the rounded tip vs filiform. Compared to E. sylvestris, the new species has a shorter body (477-565 vs 644-843 pm), a posteriorly located vulva (V = 77.8-80.4 vs 75.0-76.5) and a different position of the excretory pore (at the level of the metacorpus base to slightly posterior to the metacorpus base vs posterior to metacorpus). Compared to E. winteri, the new species has a shorter body (477-565 vs 993-1460 pm), an anteriorly located vulva (V = 77.8-80.4 vs 78-85) and the shorter PUS (0.3-0.4 vs 0.4-0.8 corresponding body width).
Remark. Description of the new species is based on the original population. The data of the second population is given in Table 1.
Molecular characterisation and phylogeny of Ektaphelenchoides poinari sp. n. For molecular analyses, the 690-bp ribosomal DNA small subunit (SSU) (KC881252), the 744-bp ribosomal DNA large subunit (LSU) D2D3 (KC881254) and the 889-bp ribosomal DNA internal transcribed spacer 1 (ITS1) (KC881253) were sequenced. A Blastn search of these sequences revealed the strongest match to aphelenchids, but nothing was identical. The species with close identity were included in the phylogenetic analyses.
Figure 4 presents a phylogenetic tree based on the SSU of many aphelenchids. Using Aphelenchoides blastophtorus Franklin, 1952 as the outgroup, Ektaphelenchoides poinari sp. n. is in the 100%-supported clade which includes Ektaphelenchus obtusus Thorne & Malek, 1968, Seinura demani (T. Goodey, 1928) J.B. Goodey, 1960, Seinura sp., Noctuidonema sp., Cryptaphelenchus sp. and Anomyctus xenurus Allen, 1940. Based on currently valid sequences of SSU for the mentioned genera, it would be difficult to resolve the phylogenetic relations between them. Further sequences of morphologically similar genera like that group of aphelenchids that lack a functional rectum are needed to enable assessment of the usefulness of 18S rDNA sequences to infer the relationships between them.
Figure 5 presents the phylogenetic tree based on the LSU of many aphelenchids. Using Aphelenchus avenae Bastian, 1865 as the outgroup, Ektaphelenchoides poinari sp. n. is in a 100%-supported clade which includes many Ektaphelenchoides species, namely E. pini, E. andrassyi, E. compsi, E. spondylis, E. hunti, E. kelardashtensis and Ektaphelenchus obtusus. Ektaphelenchoides poinari sp. n. is unique and different from three other described species from Iran. This clade also contains five species of the
genera Devibursaphelenchus Kakulia, 1967, Aphelenchoides Fischer, 1894 and Cryptaphelenchus Fuchs, 1937, and Aphelenchoides stammeri Körner, 1954 is in the basal position. All genera in this fully supported clade except A. stamerri, are members of Ektaphelenchinae Paramonov, 1964 (Braasch, 2009). All of them lack a functional rectum and anus.
Figure 6 presents a phylogenetic tree based on the ITS region of some aphelenchids. Using Aphelenchoides sp. as the outgroup, Ektaphelenchoides poinari sp. n. is closest to E. pini, the only sequenced Ektaphelenchoides species for this region and some species in Devibursaphelenchus.
Etymology. The new species named in honour of Prof. George O. Poinar Jr, the well-known pioneer scientist in systematics of insect associated nematodes.
List of the species of Ektaphelenchoides Baujard, 1984
Type species. Ektaphelenchoides pini (Massey, 1966) Baujard, 1984
= Seinura pini Massey, 1966
E. pini was described as Seinura pini by Massey in association with Dendroctonus adjunctus in a ponderosa pine.
Other valid species:
E. andrassyi Atighi, Pourjam, Pedram, Ye & Aliramaji, 2013;
E. andrassyi was isolated from bark samples of a beech tree (Fagus orientalis Lipsky) with bark beetle galleries from Kelardasht forests, Mazandaran province, northern Iran.
E. attenuata (Massey, 1974) Baujard, 1984;
= Seinura attenuate Massey, 1974
E. attenuata was described as Seinura attenuata by Massey (1974) in association with Dendroctonus terebrans in a loblolly pine.
E. compsi Baujard, 1984;
E. compsi was isolated from xylem of Pinus laricio Poir, Loiret, France.
E. hunti Atighi, Pourjam, Pedram, Ye & Robbins, 2012;
E. hunti was isolated from bark samples of a beech tree (Fagus orientalis Lipsky) with bark beetle galleries from Kelardasht forests, Mazandaran Province, northern Iran.
E. kelardashtensis Atighi, Pourjam, Pedram, Ye, Robbins & Namjou, 2012;
E. kelardashtensis was isolated from bark samples of an unidentified tree with bark beetle galleries from Kelardasht forests, Mazandaran province, northern Iran.
Table 2. Diagnostic morphometric data and morphological characters of species of Ektaphelenchoides.
Species L a b V Stylet Lateral lines Position of excretory pore End of body shape Spicule Reference
E. andrassyi 480-621 34.9-43.0 6.5-8.7 71.9-75.0 12.5-16.0 3 Base of metacorpus to posterior Conical 13-17 Atighi et al., 2013a
E. attenuata 870-880 34-42 12-13 61-63 17-19 Obscure Posterior to base of metacorpus Filiform - Massey, 1974, Baujard,1984
E. compsi 700-900 38-49 8-10 73.5-77.0 18-24 Obscure Posterior to base of metacorpus Conical 19-24 Baujard, 1984
E. hunti 711-929 35.9-44.5 7.7-10.0 70.4-72.6 16-23 3 At the middle of metacorpus to slightly posterior and at 2-8 ^m distances from the base of metacorpus. Conical 12.5-17.5 Atighi et al., 2012
E. kelarda-shtensis 433-577 34.7-44.4 8.0-11.2 61.5-68.0 13-16 Obscure Posterior to base of metacorpus Filiform 8-10 Atighi et al., 2013b
E. musae 500-710 28-33 7.4-9.8 64-69 18.5-22.0 Obscure Posterior to base of metacorpus Filiform - Baujard, 1984
E. pini 720 33 9 74 23 3 Posterior to base of metacorpus Filiform 22-28 Massey, 1966, Baujard, 1984
E. ruehmi 355-487 31.3-38.0 5.3-7.0 67.3-73.3 9.0-12.5 3 Posterior to base of metacorpus Filiform 13 Yaghoubi et al., 2014
E. poinari sp. n. 477-565 25.1-31.1 6.3-8.4 77.8-80.4 18-26 Obscure Level of the metacorpus base to slightly posterior Conical 15 Present study
E. spondylis 595-710 28.6-47.3 7.0-8.7 71.2-72.3 19-23 Obscure Posterior to base of metacorpus Conical 23-26 Kanzaki et al, 2009
E. sylvestris 644-843 25-32 8-11 75.0-76.5 18-23 3 Posterior to base of metacorpus Conical - Pedram et al., 2012
E. winteri 993-1460 25-35 9.0-16.6 78-85 19-26 Obscure Posterior to base of metacorpus Conical 23-27 Hooper, 1995
Fig. 6. The 10001st Bayesian tree inferred from ITS under Chi-square = 76.809094 (df = 24), P = 0.00000019 model (lnL = 5384.542; freqA = 0.00284; freqC = 0.001759; freqG = 0.002223; freqT = 0.003177; R(a) = 1.295; R(b) = 9.6763; R(c) = 2.0628; R(d) = 0.9699; R(e) = 12.2474; R(f) = 1; Pinva = 0; Shape = 0.7401). Posterior probability values exceeding 50% are given on appropriate clades.
E. musae Baujard, 1984;
E. musae was isolated from banana corn tissue from the Ivory Coast.
E. poinari sp. n. - present study. E. spondylis Kanzaki, Giblin-Davis & Center, 2009;
E. spondylis was isolated from body cavity of Spondylis buprestoides from Japan.
E. sylvestris Pedram, Pouijam, Atighi, Ye & Houshmand, 2012;
E. sylvestris was isolated from the bark of a dead Pinus sylvestris L. in a small park, Poonak square, Tehran, Iran.
E. winteri Hooper, 1995;
E. winteri was found attached to larvae of Xylodiplosis sp. (Diptera: Cecidomyidae) emerging from logs of oak, Quercus robur L., cut from trees at Crickley Hill, Gloucestershire, England.
Identification of Ektaphelenchoides species. The diagnostic morphometric data and morphological characters of ten valid species and one new species of the genus are given in Table 2. The morphometric data are given according to the original descriptions and any subsequent redescriptions of the species. The characters most useful for separating species delimitation include length of body, PUS and stylet, the position of an excretory pore and vulva (V), the number of lines in a lateral field and shape of body end in females, and length and shape of spicules in males.
ACKNOWLEDGMENTS
We acknowledge the Iranian National Science Foundation (INSF) and Tarbiat Modares University (Iran) for financial support and Azam Houshmand for providing the studied material.
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Резюме. Приведено описание и иллюстрации для нового вида Ektaphelenchoides poinari sp. n., сопровождающееся данными по морфометрии и молекулярно-таксономическим особенностям. Новый вид характеризуется длиной тела у самок 477-565 мкм; обособленной губной областью шириной 7.5-9.5 мкм, отделенной от остального тела неглубоким сужением; стилетом длиной 18-23 мкм, не имеющим утолщений при основании; положением экскреторной поры на уровне или несколько сзади от основания метакорпуса; тремя инцизурами в латеральном поле; коротким рудиментом задней матки - задним маточным мешком (PUS), составляющим 0.3-0.4 ширины тела в этом месте; коническим задним концом тела; редко встречающимися самцами; спикулами с округлым кондилюсом, заостренным рострумом и тупой дистальной оконечностью. Новый вид близок к E. attenuata, E. kelardashtensis, E. musae, E. pini, E. sylvestris и E. winteri, но отличается от них более коротким PUS, формой задней оконечности тела у самок и самцов, положением вульвы, а также молекулярно-таксономическими особенностями. Ektaphelenchoides poinari sp. n. отличается от других видов, изученных молекулярными методами, по последовательностям малой субъединицы рибосомы, D2D3-сегмента последовательности большой субъединицы и ITS 1-участка. Филогенетический анализ всех этих последовательностей показывает, что E. poinari sp. n. близок к другим видам родов Ektaphelenchoides и Devibursaphelenchus. Дана сводка по всем валидным видам рода, основанная на морфологических и морфометрических критериях.
