01 nematodes 16.indd

J. Nematode Morphol. Syst., 16 (2): 113-129 (2013)

Two new diplogastrid nematodes from Iran

Description of two new species of diplogastrid nematodes (Rhabditida: Diplogastridae) from Iran

M. R. Atighi1, E. Pourjam1*, N. Kanzaki2, R. M. Giblin-Davis3, I. Tandingan De Ley4, M. Mundo-Ocampo5 and M. Pedram1

Summary.- Two new species of diplogastrid nematodes are described and illustrated based upon morphological, morphometric, SEM and molecular characters. Mononchoides iranicus sp. n. is characterized by its body length, number of longitudinal ridges (42 in female and 34 in male), shape and size of the stoma, anteriorly located vulva, shape of gubernaculum and arrangement of genital papillae in males. It is similar to M. adjunctus, M. americanus, M. andrassyi, M. aphodii, M. bicornis, M. filicaudatus, M. longicaudatus, M. megaonchus, M. parastriatus, M. splendidus, M. subamericanus and M. trichuris. Koerneria ruehmi sp. n. is distinguishable by its vulva position, funnel-shaped gubernaculum, arrangement of male ventral papillae and presence of small bursal flap. The new species is phylogenetically close to the ‘Japanese isolate of K. luziae’, and these two species form a small basal clade relative to the rest of the family. The morphological differences and the phylogenetic relations of both new species with other diplogastrid taxa are discussed. Keywords: Iran, Koerneria ruehmi sp. n., LSU-rDNA, molecular analysis, Mononchoides iranicus sp. n., morphology, morphometrics, SEM, taxonomy. Resumen.- Se describen e ilustran dos nuevas especies de nematodos diplogástridos a partir de rasgos morfológicos, morfométricos, MEB y moleculares. Mononchoides iranicus sp. n. se caracteriza por su longitud corporal, el número de crestas longitudinales (42 en la hembra y 34 en el macho), la forma y el tamaño del estoma, la posición anterior de la vulva, la forma del gubernáculo y la disposición de las papilas genitales en el macho. Es muy similar a M. adjunctus, M. americanus, M. andrassyi, M. aphodii, M. bicornis, M. filicaudatus, M. longicaudatus, M. megaonchus, M. parastriatus, M. splendidus, M. subamericanus y M. trichuris. Koerneria ruehmi sp. n. se distingue por la posición de la vulva, la forma de embudo que presenta el gubernáculo, la disposición de las papilas ventrales en el macho y la presencia de una pequeña solapa en la bursa. Es filogenéticamente muy próxima al ‘aislado japonés de K. luziae’, y ambas formas constituyen un pequeño clado basal próximo al resto de la familia. Se discuten las diferencias morfológicas y la relaciones filogenéticas de las dos nuevas especies con otros táxones diplogástridos. Palabras clave: Análisis molecular, Irán, Koerneria ruehmi sp. n., LSU-ADNr, MEB, Mononchoides iranicus sp. n., morfología, morfometría, taxonomía.

Introduction The diplogastrid nematodes are predominantly terrestrial, commonly occurring in saprobic habitats like dung, compost, slime flux, decomposing plants and other organic materials all over the world (Andrássy, 2005).

1

2

3

4

5 *

Department of Plant Pathology, College of Agriculture, Tarbiat Modares University, Tehran, Iran. Forest Pathology Laboratory, Forestry and Forest Product Research Institute, 1 Matsunosato, Tsukuba, Ibaraki, 305-8687, Japan. Fort Lauderdale Research and Education Center, University of FloridaIFAS, 3205 College Avenue, Fort Lauderdale, FL 33314-7719, USA. Department of Nematology, University of California, Riverside, CA 92507, USA. CIIDIR-IPN. Unidad Sinaloa, Mexico. Corresponding author. E-mail: [email protected]

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In other words, they are available in places or materials that are rich in bacteria and/or fungi. Further, many of them are phoretic associates or, in a few cases, parasites of insects (Sudhaus & Fürst von Lieven, 2003). During extensive surveys for diplogastrid nematodes in Iran, several samples from the bark of different trees and rotten wood were collected from Northern Iran. The study of these samples revealed the presence of two unknown forms of the genera Mononchoides Rahm, 1928 and Koerneria Meyl, 1960 which are described in the following. Sudhaus and Fürst von Lieven (2003) listed 43 nominal species in the genus Mononchoides. Since then, several new species have been added to the genus: M. gaugleri Siddiqi, Bilgrami & Tabassum, 2004, collected from agricultural farm soil near Rutgers University, New Brunswick, New Jersey, USA; M. megaonchus Mahamood, Ahmad & Shah, 2007, found in decaying banana rhizome


M. R. Atighi et al.

that was collected from F-Sector, Itanagar, Arunachal Pradesh, India; and, more recently, M. composticola Steel, Moens, Scholaert, Boshoff, Houthoofd & Bert, 2011, from a compost heap at the Institute for Agricultural and Fisheries Research in Merelbeke, Belgium. The genus Koerneria has 37 nominal species that have been recovered from various habitats and in association with different insect hosts (Sudhaus & Fürst von Lieven, op. cit.). Besides, Fürst von Lieven (2008) added K. sudhausi to its list.

Materials and Methods Sampling, extraction, mounting and drawing: The samples were collected from bark of a pine tree (Pinus sylvestris L.) and of an unknown dead tree, both with galleries of bark beetles. Pieces of bark were chopped to small pieces. To obtain a cleaner suspension of nematodes, the tray method was employed (Whitehead & Hemming, 1965). Nematodes were handpicked under a Nikon SMZ1000 stereomicroscope, heat-killed by adding boiling 4% formalin solution and then transferred to anhydrous glycerin and mounted in permanent slides according to De Grisse (1969). Permanent slides were made and examined using a Nikon Eclipse E600 light microscope. Photomicrographs were taken using an Olympus DP72 digital camera attached to an Olympus BX51 microscope with differential interference contrast (DIC). Drawings were made using a drawing tube attached to the microscope and were redrawn using CorelDRAW® software version 12. The terminology used for describing the stoma follows De Ley et al. (1995) and the description of the different elements of the buccal cavity follows Fürst von Lieven and Sudhaus (2000). For the male genital papillae, the nomenclature proposed by Sudhaus and Fürst von Lieven (2003) and Kazanki et al. (2012) was employed. Scanning Electron Microscopy (SEM): Previouslyfixed specimens were limited to a few females. These were repeatedly rinsed in distilled water for 5 minutes to remove all traces of formalin and then post-fixed for 4 h in 2.0% osmium tetroxide. Post-fixed specimens were dehydrated through a series of aqueous dilutions of 20100% ethanol. Dehydrated specimens were critical point dried in a Tousimis Autosamdri-810® critical point dryer. Specimens were mounted on double-sticky copper tape attached to aluminum stubs, coated for 1-3 minutes with a 25 nm layer of gold palladium in a Cressington 108 Auto®sputter coater, and then observed with a XL 30FEG Phillips 35® scanning electron microscope operating at 10 kV (Mundo-Ocampo et al., 2003). Cross sections: They were made after examining the fixed individuals and selecting a suitable specimen. The chosen nematode was put on a drop of pure glycerin on an inverted cover of a 6 cm Petri dish and sliced perpendicular to the longitudinal axis using a common razor. Each cross section was transferred to a small drop of melted glycerin-


gel (De Grisse, 1969) and covered with a cover slip before the drop congealed. The cover slip was then positioned with point pressure to reveal the full cross-section and sealed with colorless nail polish. Molecular and phylogenetic analyses: Ethanolpreserved nematodes were washed three times with sterile water. DNA extraction, amplification, cleaning and sequencing were performed as described by Tandingan De Ley et al. (2007). The molecular sequences of D2/D3 expansion segments of 28S ribosomal RNA gene (D2/D3 LSU) of both newly discovered species were compared with those of other nematode species available in GenBank using the BLAST homology search program. The selected sequences were aligned using the ClustalX2 (http://www.clustal.org/) software. The model of base substitution was evaluated using MrModeltest 2 (Nylander, 2004). The Akaike-supported model, the base frequencies, the proportion of invariable sites and the gamma distribution shape parameters and substitution rates were used in phylogenetic analyses. Bayesian analysis was performed to infer a phylogenetic tree using MrBayes v3.1.2 (Ronquist & Huelsenbeck, 2003) running the chain for 106 generations. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analyses. The Markov Chain Monte Carlo (MCMC) method within a Bayesian framework was used to determine equilibrium distribution and help estimate the posterior probabilities of the phylogenetic trees (Larget & Simon, 1999) using the 50% majority rule. A Maximum Likelihood tree was constructed by using RaxmlGUI 1.1 (Silvestro & Michalak, 2011) software and selecting the GTR model of nucleotide substitution with a gamma model of rate heterogeneity and invariable sites, in 1000 bootstrap replicates. For both phylogenetic analyses methods, Rhabditoides inermis (EU195981) was used as outgroup.

Results Mononchoides iranicus sp. n. (Figs 1-3)

Morphometrics: See Table I. Male: Body almost straight or slightly arcuate ventrad when heat-killed. Cuticle 2.0-3.5 µm thick, with 34 prominent longitudinal ridges at mid-body, all ridges equidistant and bearing fine transverse striation. Lip region continuous with body contour, consisting of six fused lips, edge of lip region with four additional cephalic setae. Amphidial opening appear as small oval slits, located laterally, at level of the base of cheilostom. When the stoma is retracted, the mouth becomes ‘zipper’-shaped along the dorsal-ventral axis and ‘sealed’ by the cheilorhabdions. Presumably, the mouth forms a cheilorhabdion-rimmed

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Figure 1. Mononchoides iranicus sp. n. A: Entire female. B: Entire male. C: Female, anterior region. D: Female, details of lip region. E: Serrated anterior edge of gymnostom and left subventral ridges. F: Female, cross section. G: Male cross section. H: Spicules. I-K: Gubernaculum. L: Male, caudal region in ventral view. M: Male, caudal region in lateral view.

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Figure 2. Mononchoides iranicus sp. n. A: Female, anterior region. B, C & D: Female, details of lip region. E: Amphidial fovea and opening. F: Female, lip region in face view. G: Male, lip region with four cephalic setae. H: Female, cross section. I: Male, cross section. J: Body surface. K: Part of female genital tract. L: Vulval glands. M: Vulva region in ventral view. N: Female, posterior body region with anus. O & P: Male, caudal region in lateral view showing gubernaculum and spicules. Q: Male, location of ‘ad’ papilla (= P5d) and phasmid. (All scale bars = 10 μm.) J. Nematode Morphol. Syst., 16 (2): 113-129 (2013)

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circle when the lower stomatal elements are protracted. Buccal cavity ca 2.5-3.6 times longer than wide. Cheilostom with 14-16 narrow rib-like plates (cheilorhabdions) that are bifurcate at the apex and extend beyond the labial contour when posterior stomatal elements are retracted; inner wall of cheilostom cuticularized. The gymnostom forms a cuticular anisomorphic cylinder with its anterior edge weakly serrated; its dorsal side strongly cuticularized, thickened at the base and about 20% shorter than the ventral side that remains more uniformly thin and longer. Pro-mesostegostom wide in dorsal sector and relatively thin in subventral sectors. Metastegostom bearing a prominent claw-like dorsal tooth and a smaller sickle-shaped right subventral tooth; dorsal tooth with a prominent duct of the dorsal pharyngeal gland opening just behind its tip; both teeth are movable when live specimens were examined with light microscopy; the left lateral sector of metastegostom bears a row of denticles, their number not easily quantified. Telostegostom a long tube (stegostome-cylinder), averaging 2.4-3.5 times longer than wide. Neck region comprising ca 19-22% of total body length. Pharynx divided into muscular procorpus slightly expanded posteriorly into an oval muscular metacorpus with prominent muscular valvular apparatus; median bulb 14-26 µm wide and located at 49-55% of pharyngeal length; isthmus non-muscular and ending in a non-muscular elongated oval basal bulb without valves. Nerve ring encircling the isthmus at its anterior half or at its middle. Excretory pore located at level of posterior part of isthmus, at 105-133 µm from anterior end. Hemizonid situated in front of the excretory pore. Cardia present, well-developed. Deirid small, located at level of the anterior part or the middle part of the terminal bulb. Postdeirid not found in light microscopic observation. Testis single, anteriorly reflexed, latero-ventral, always extending posterior to pharynx. Spicules stout, paired, separate in ventral view, smoothly ventrally arcuate in lateral view; rounded manubrium at anterior end; lamina/ calamus complex expanded just posterior to manubrium, then slightly tapering to smoothly pointed distal end. In lateral view, the gubernaculum is expanded proximally and tapers distally to a needle-like point. Nine pairs of genital papillae (P1-P9), a single ventral papilla (vs) and a pair of phasmids (Ph) present and arranged as < P1, (P2d, P3), (vs, C), P4, P5d, Ph, (P6, P7, P8), P9d> (= < v1, (v2d, v3), (vs, C), v4, ad, Ph, (v5, v6, v7>, pd > according to the nomenclature by Sudhaus and Fürst von Lieven (2003). All papillae are seta-like, and P1-P5 and vs are rather large and clear; P6 and P7 small, with bilobed bases; P8 and P9 are intermediate between P1-5 and P6, 7 in size; phasmid (Ph) and P6 are clearly separated; P9d posterior to P6, P7 and P8. Phasmid located at 0.9-1.1 anal body widths behind the cloacal aperture. Tail conical, posterior end filiform. Female: General morphology similar to that of males but slightly larger, tapering towards both extremities, slightly anteriorly and sharply posteriorly. Anterior end similar to that of males except lacking four cephalic setae. Cuticle 2.5-3.5 µm thick, with 42 prominent longitudinal ridges at mid-body. Buccal cavity 2.7-3.7 times longer

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than wide. Neck region comprising 16-21% of total body length. Median bulb located at 49-55% of pharyngeal length. Excretory pore situated at level of the posterior part of isthmus, at 124-145 µm from the anterior end. Hemizonid in front of excretory pore. Reproductive system amphidelphic, with both branches equally developed and reflexed terminus often reaching near the vulva level. Ovary totally reflexed (= antidromous flexion) with oocytes arranged in one row in the germinative zone; germinative and growth zones broad, distinctly separated from each other. Oviduct narrow, with the spermatheca not set off from the uterus, and its distal part filled with sperm cells in most individuals, i.e., serving as true spermatheca. Vagina perpendicular to body axis, muscular and with narrow lumen. Vulva pore-like, equatorial or slightly pre-equatorial. Two pairs of glands open at the vagina-uterus junction. Phasmids prominent, situated at 0.7-1.1 anal body diameters behind the anus. Tail filiform, 0.8-1.4 times the vulva-anus distance long. Molecular characterization: For molecular analyses, the partial sequences of LSU rDNA D2/D3 of two isolates of M. iranicus sp. n. (677-bp with accession numbers KF219667 and KF219668) were used. There were no differences between the sequences of two isolates of M. iranicus sp. n. A BlastN search of LSU sequences of M. iranicus sp. n. revealed an 86% identity match with six Mononchoides spp. (FJ661059, FJ661065, FJ661064, FJ661062, FJ661063 and FJ661081). Diagnosis: Mononchoides iranicus sp. n. is characterized by the combination of the following characters: body length 851-1205 μm, cuticle with fine transverse striation and equidistant longitudinal ridges (42 in female and 32 in male), presence of right subventral sickle-shaped tooth and a coarse row of denticles on left subventral sector of metastegostom, relative narrowness of the telostegostom, anterior position of vulva, length of filiform tail in female and male, shape and size of gubernaculum, spicules, arrangement of genital papillae in the male and presence of a single ventral papilla. Relationships: The new species is most similar to M. adjunctus Massey, 1966, M. americanus (Steiner, 1930) Chitwood, 1937, M. andrassyi (Timm, 1961) Gagarin, 1998, M. aphodii (Bövien, 1937) Sudhaus & Fürst von Lieven, 2003, M. bicornis (Rahm, 1928) Andrássy, 1984, M. filicaudatus (Allgén, 1947) Sudhaus & Fürst von Lieven, 2003, M. longicaudatus (Khera, 1965) Andrássy, 1984, M. megaonchus, M. parastriatus (Paesler, 1946) Andrássy, 1984 and M. splendidus (Körner, 1954) J. B. Goodey in T. Goodey, 1963, M. subamericanus (van der Linde, 1938) Calaway & Tarjan, 1973 and M. trichuris (Cobb, 1893) J. B. Goodey in T. Goodey, 1963 based on the combination of morphometrics and morphological characters such as the shape and dimensions of the stoma, shape of the gubernaculums, arrangement of genital papillae in male, and length of filiform tail in female and male. It differs from M. adjunctus by its longer body (851-


M. R. Atighi et al.

Figure 3. Mononchoides iranicus sp. n. A: Female, lip region. B: Body surface. C: Female, caudal region. D: Male, lip region. E: Female, anal region. F & G: Male, caudal region showing genital papillae. (Scale bar: A = 5 µm; B, D, F= 10 µm; C = 30 µm; E, G = 20 µm.)


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1205 vs 780-870 μm), shorter tail (215-334 vs 363 μm), longer stoma length (30-35 vs 13 μm), cuticle with weak transverse striations at anterior body region (vs prominent transverse striations), presence of right subventral sickleshaped tooth and a coarse row of denticles on left subventral sector of metastegostom (vs no apparent right subventral tooth and having finer denticles on left subventral metastegostmal sector), relative narrowness of the telostegostom (ca 4.0 vs 1.5 times longer than wide), shape of gubernaculum (sharp conical at proximal end vs rounded), number and arrangement of male genital papillae (three precloacal pairs and a single ventral papilla vs two precloacal pairs and no apparent single ventral papilla), and longer spicules (37-44 vs 35 μm). From M. americanus by the number of longitudinal ridges in the female (42 vs 24), metastegostom bearing a right subventral sickle-shaped tooth and a coarse row of denticles on left subventral sector (vs no apparent teeth or denticles on subventral metastegostmal sectors), relative narrowness of the telostegostom (ca 4.0 vs 1.8 times longer than wide), more anterior vulva (V = 40-46 vs V = 46), shape of gubernaculum (sharply conical proximal end vs re-curved), shorter gubernaculum (14-18 vs 17-22 μm) and number and arrangement of male genital papillae (nine and a single ventral papilla vs 10 without an apparent single ventral papilla or phasmids and with the I-V papillae pairs probably representing an overestimate of its number, based upon original drawings). From M. andrassyi by the number of longitudinal ridges in the female (42 vs 20), gymnostom thickness (thick vs thin), metastegostom with small right subventral sickle-shaped tooth and a coarse row of denticles on left subventral sector (vs a less protuberant right subventral rounded tooth and no apparent denticles on the subventral metastegostmal sector), relative narrowness of the telostegostom (ca 4.0 vs 2.0 times longer than wide), more posterior position of vulva (V = 40-46 vs V = 35.5-42), shape of gubernaculum (distally needle-like, sharp conical proximally vs triangular), number and arrangement of genital papillae in males (nine and a single ventral papilla vs 10 without an apparent single ventral papilla or phasmids). From M. aphodii in its metastegostom having a right subventral sickle-shaped tooth and a coarse row of denticles on left subventral sector (vs no apparent teeth or denticles on the subventral metastegostomatal sectors), the relative narrowness of the telostegostom (ca 4.0 vs ca 2.0 longer than wide), more anterior vulva (V = 40-46 vs V = 53), higher a-ratio (23-27 vs 15-17), lower c-ratio (2.9-4.1 vs 5.5-6.6), more anterior location of the excretory pore (opposite isthmus vs opposite terminal bulb), and lower number of male genital papillae (nine vs 10, based upon the original drawings with the phasmid pair probably having been omitted and the three ventral tail papillae having been overestimated at four). From M. bicornis by its shorter body length in both female (851-1205 vs 12001800 μm) and male (711-877 vs 2358, 2360 µm), higher a-ratio (23-27 vs 13-15), the relative narrowness of the telostegostom (ca 4.0 vs ca 2.0 times longer than wide), shape of dorsal tooth (sickle-shaped vs bar-like, as drawn),

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and shape of gubernaculum (proximal portion expanded into a cone vs proximal portion only barely expanded into cylinder). From M. filicaudatus by possessing a cuticle with longitudinal ridges (vs being apparently smooth), relative size and shape of the dorsal tooth (dorsal tooth extending ventrally about 20% beyond the anterior/ posterior midline vs not extending past the midline), relative narrowness of the telostegostom (ca 4.0 vs ca 2.0 times longer than wide), more posterior vulva (V = 40-46 vs V = 40), lower a-ratio (23-27 vs 30), higher c-ratio (2.94.1 vs 2.8), longer neck region (165-204 vs 148 μm) and shorter tail (215-334 vs 350 μm). The male of M. filicaudatus was not described. From M. longicaudatus by its higher c-ratio (2.9-4.1 vs 2.3-2.5), the relative narrowness of the telostegostom (ca 4.0 vs ca 2.0 longer than wide), shape of dorsal tooth (claw-like and simple vs claw-like and coarse, as drawn), metastegostom with a right subventral sickle-shaped tooth and a coarse row of denticles on left subventral sector (vs no apparent teeth or denticles on the subventral metastegostomatal sector), more posterior vulva (V = 40-46 vs V = 34-38), longer neck region in female (165-204 vs 163 µm), shorter female tail (215-334 vs 540 μm), greater number of longitudinal ridges in female (42 vs 16), cardia present and well developed (vs not observed), tips of ovaries not extending beyond the vulva (vs extending beyond the vulva), longer spicules and gubernaculum (37-44 vs 23-25 μm, 14-18 vs 12 μm, respectively), shape of gubernaculum (distally needle-like vs straight) and presence (vs absence) of a single ventral papilla. From M. megaonchus by having higher c-ratio (2.9-4.1 vs 1.8-2.6), more posterior vulva (V = 40-46 vs V = 30-40), shorter female tail (215-334 vs 422-873 μm), greater number of longitudinal ridges in female (42 vs 34), metastegostom with a right subventral sickle-shaped tooth and a coarse row of denticles on left subventral sector (vs right and left subventral walls without tooth, teeth or denticles), shorter gubernaculum (14-18 vs 19-21 μm), shape of gubernaculum (distally needle-like vs distally attenuated into a beak-like process), presence (vs absence) of a single ventral papilla, and arrangement of dorsal pre-cloacal papillae (dorsal papilla is located as second papillae vs as third one). From M. parastriatus by disproportionate sizing of the dorsal and right subventral teeth (dorsal tooth about 5.0 times larger than right subventral tooth vs being about equal in size), coarser row of denticles on left subventral sector of metastegostom, relative narrowness of the telostegostom (ca 4.0 vs ca 1.4 times longer than wide), more posterior vulva (V = 40-46 vs V = 33-41), greater number of longitudinal ridges in females (42 vs 20), lower value of b-ratio (4.8-6.3 vs 8.0-10.0), presence of a single ventral papilla (vs absence of ventral papilla based upon original drawings with the phasmid pair probably having also been omitted). From M. splendidus by its shorter body length (851-1205 vs 1145-1305 μm), lower a-ratio (23-27 vs 2736), length of stoma (2.7-3.7 vs less than 2.0 times longer than wide), relative narrowness of the telostegostom (ca 4.0 vs ca 2.2 times longer than wide), shape of gubernaculum (distally needle-like, sharp conical


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Table I. Morphometrics of Mononchoides iranicus sp. n. All measurements are in μm and in the form: mean ± s.d. (range).

Character

n

Holotype

Paratypes 18

Paratypes 20

L

846

784 ± 52 (711-877)

954 ± 84 (851-1205)

L’

664

614 ± 57 (519-708)

693 ± 62 (603-896)

a

24

24.6 ± 1.0 (22.2-26.5)

25.2 ± 1.2 (23.3-27.4)

a’

19

19.3 ± 1.1 (16.8-21.7)

18.3 ± 0.9 (16.8-20.4)

b

5

5.0 ± 0.2 (4.6-5.4)

5.3 ± 0.4 (4.8-6.3)

b’

4

3.9 ± 0.3 (3.5-4.4)

3.9 ± 0.3 (3.4-4.7)

c

4.5

4.6 ± 0.6 (3.7-5.5)

3.7 ± 0.3 (2.9-4.1)

c’

6.5

6.6 ± 0.8 (5.4-8.9)

10.2 ± 1.1 (8.4-12.8)

V or T

48

42.2 ± 4.1 (35-50)

43.8 ± 1.5 (41-46)

-

-

60.3 ± 1.6 (56-63)

16

14.9 ± 0.8 (13-16)

19.0 ± 1.0 (18-21)

Stoma width

9

8.5 ± 0.7 (8.0-10.0)

10.7 ± 0.9 (9.0-12.0)

Stoma length

26

25.4 ± 1.4 (24-28)

32.7 ± 1.5 (30-35)

Median bulb from anterior body

87

81.4 ± 3.7 (75-88)

94.6 ± 3.4 (88-100)

MB

51

51.6 ± 1.6 (49-55)

52.6 ± 1.7 (49-55)

Body width at MB

30

26.8 ± 2.2 (22-32)

31.7 ± 1.9 (29-36)

Median bulb width

16

16.3 ± 2.7 (14-16)

20.3 ± 0.9 (19-22)

Anterior end to nerve ring

110

99.7 ± 5.0 (90-107)

114 ± 5 (105-123)

Excretory pore

135

122 ± 9 (105-133)

136 ± 6 (124-145)

Hemizonid

127

115 ± 8 (100-128)

129 ± 6 (118-136)

Pharynx length

170

158 ± 7 (145-170)

180 ± 8 (165-204)

-

-

417 ± 30 (360-497)

Max. body width

35

31.9 ± 2.4 (28-36)

37.9 ± 2.3 (35-44)

Length of anterior gonad branch

405

332 ± 46 (250-406)

174 ± 23 (124-230)

G1

-

-

18.3 ± 2.0 (14.5-22.7)

Length of posterior gonad branch

-

-

186 ± 28 (142-233)

G2

-

-

19.6 ± 2.8 (14.6-25.1)

28

26.0 ± 1.7 (22-28)

25.6 ± 1.9 (22-29)

-

-

276 ± 35 (230-399)

182

170 ± 14 (140-195)

261 ± 35 (215-334)

-

-

1.0 ± 0.1 (0.8-1.4)

Spicules

42

39.6 ± 2.4 (37-44)

-

Gubernaculum

17

16.5 ± 1.2 (14-18)

-

V’ Lip region width

Head-vulva

Anal body width Vulva - anus Tail Tail/vulva-anus


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proximally vs proximally claw-shaped, distally forming a tube around the spicules), gubernaculum size (14-18 vs 19-22 μm) and presence (vs absence in original drawing) of a single ventral papilla. From M. subamericanus by having a greater number of longitudinal ridges in female (42 vs 24), relative narrowness of the telostegostom (ca 4.0 vs ca 2.2 times longer than wide), metastegostom with a right subventral sickle-shaped tooth and a coarse row of denticles on left subventral sector (vs no apparent teeth or denticles on the subventral metastegostomatal sectors), more posterior vulva (V= 40-46 vs V = 36) and shorter female tail (215-334 vs 550 μm), the male of M. subamericanus was not described. And from M. trichuris by its shorter body length (851-1205 vs 1500 μm), lower a-ratio (23-27 vs 32), higher c-ratio (2.9-4.1 vs 1.8), relative narrowness of the telostegostom (ca 4.0 vs ca 1.5 times longer than wide), metastegostom with a right subventral sickle-shaped tooth and a coarse row of denticles on left subventral sector (vs no apparent teeth or denticles on the subventral metastegostomatal sectors), more posterior position of vulva (V = 40-46 vs V = 28), shorter female tail (215-334 vs 810 μm), and shape of gubernaculum (distally needle-like, sharp conical proximally vs distally triangular and proximally rounded). Type locality and habitat: Northern Iran, Golestan province, Gorgan, collected from bark samples of a pine tree (Pinus sylvestris L.), in November, 2010. Type material: Male holotype, five male paratypes and five female paratypes deposited at Nematode Collection of the Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran. Three male and 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. Etymology: The species epithet refers to the geographical origin of the new species in Iran. Remarks: Close examination of the SEM pictures herein provided for ventral male tail papillae homologs (v5-7 =P6-8) for M. iranicus sp. n. (Fig. 3F-G) and those reported for M. composticola by Steel et al. (2011) suggest that these papillae may be difficult to interpret correctly using just light microscopy. For example, in the case of M. composticola, the v7 (P8) homologs are seta-like and v6 (P7) appear to comprise three papillae grouped together (or a single papilla that has been spatially divided into three components; an anteriorly directed microseta, a central papilla, and a posteriorly directed microseta). Interestingly, the v6 (P7) homolog in M. iranicus sp. n. appears as a tripartite ‘single’ papilla with a papillate core flanked by anterior and posterior microprojections (Fig. 3F-G). Unfortunately, in many of the original descriptions for members of the Mononchoides, it is difficult to know if the described and figured papillae have been correctly

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drawn and are therefore useful for species comparisons and determinations. Mononchoides americanus and M. aphodii come to mind in this instance, where it appears that the v6 (P7) homolog may have been interpreted to involve more than a single component (Bövien, 1937; Steiner, 1930). Also, in many of the older descriptions it is difficult to know whether the phasmid was omitted in the drawings and description or included as an additional pair of papillae. Further work on this group will require re-isolation and re-examination of authentic materials of these originally described isolates along with acquisition of molecular sequence data for more in depth and less speculative species comparisons.

Koerneria ruehmi sp. n. (Figs 4 & 5)

Morphometrics: See Table II. Male: Body straight or slightly arcuate ventrad when heat-killed. Cuticle with fine annuli and fine longitudinal striation, lacking lateral fields when examined under light microscope. Lip region continuous with the adjacent body; lips totally amalgamated, bearing six short, papilliform labial sensilla; edge of lip region with four cephalic setae. Amphidial apertures pore-like, located at level of the middle of cheilostom. Stomatal di- or polyphenism not observed; stoma stenostomatous. Cheilostom tube-like, possessing thick wall, widened posteriorly, its inner wall cuticularized. Gymnostom forming a cuticular ring, its anterior edge serrated, hardly visible in fixed specimens, but visible in live nematodes; margin strongly cuticularized. Metastegostom bearing a prominent claw-like dorsal tooth and dagger-like right subventral tooth; dorsal tooth about 5.0 times larger than the right subventral tooth and with a narrow duct of dorsal gland opening just behind its tip, hardly visible; both teeth moved when live specimens were examined. The left subventral sector of metastegostom bears two rows of denticles, one lateral and another subventral. A telostegostomatal apodeme protrudes subventrally on each side, being more distinguishable from each other in ventral view, and more visible in live specimens, especially in juveniles. Telostegostom a long tube. Neck region comprising ca 19-25% of total body length. Procorpus muscular, slightly expanding posteriorly into an oval muscular metacorpus with prominent muscular valvular apparatus, 14-17 µm wide, and located at 4851% of pharynx length. Isthmus non-muscular, grading into an elongated pyriform basal bulb. Excretory pore difficult to see in fixed individuals, clearly visible in live ones, located at about level of the anterior part of basal bulb. Hemizonid prominent, situated slightly in front of the excretory pore. Nerve ring enveloping the isthmus at the anterior half of its length. Cardia present, well-developed. Deirids located at level of the anterior half to middle of basal bulb. Postdeirid not observed. Testis ventral to intestine, not reflexed, in


M. R. Atighi et al.

Figure 4. Koerneria ruehmi sp. n. A: Female, entire. B: Male, entire. C: Female, lip region. D: Female, anterior region (arrow pointing at deirid). E: Female, genital tract. F: Male, caudal region in ventral view. G: Male, caudal region in lateral view. H-J: Spicules and gubernaculums. K: Vulval glands. L: Female, caudal region.


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Figure 5. Koerneria ruehmi sp. n. A: Female, anterior region; B: Female, details of lip region. C: Female, lip region in ventral view. D: Female, part genital tract. E: Male, lip region. F: Female, caudal region. G & H: Male, caudal region in ventral view showing small bursa. I: Vulval glands. J & K: Male, caudal region. L & M: Gubernaculum. N-R: Male, caudal region showing genital papillae. (Scale bars = 10 μm, except E = 2 μm.)

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some individuals reaching near the base of basal bulb. Spicules stout, in ventral view separate, in lateral view smoothly curved ventrad; manubrium rounded at its anterior end; lamina/calamus complex expanded just behind the manubrium, then smoothly tapering to smoothly-pointed distal end, and with its anterior part outgrowth smoothly-rounded; tip free. Gubernaculum conspicuous, its anterior half expanded and rounded, ear-shaped in lateral view, whereas the posterior half forms a tube-like process enveloping the spicules, the tip of spicules free. Nine pairs of genital papillae and a pair of phasmids present and arranged as (= < v1, v2, (C, v3d), v4, ad, Ph, (v5, v6), pd, v7 > in nomenclature of Sudhaus and Fürst von Lieven (2003). P1, P2 , P3d, P4 and P5d equal in size. P6 and P7 equal and larger than previous papillae. P8d larger than P9. Phasmid hardly visible. Cloacal aperture to phasmid distance 0.9-1.4 times the cloacal body diameter. Tail short, with slightly pointed tip. Small bursal flap explicitly visible when examined in ventral view. Female: General morphology similar to that of males, with slightly larger body. Anterior end similar to that of males except for lacking four cephalic setae situated on edge of lip region. Neck region comprising ca 16-20% of total body length. Median bulb 15-20 µm wide and located at 46-52% of pharynx length. Reproductive system amphidelphic with both branches equally developed, terminus reflexed (= antidromous flexion), often reaching near the vulva but never surpassing it. Oocytes arranged in one row in the germinal zone. Oviduct broad, spermatheca not set off from the uterus, filled with oval sperm cells in some individuals. Vagina perpendicular to body axis, extending inwards about onethird of the corresponding body diameter. Vulva pore-like. Phasmids prominent, 1.4-2.4 anal body diameters behind the anus. Tail long, with pointed or filiform tip terminus, 0.3-0.5 times the vulva-anus distance long. Molecular characterization: For molecular analyses, the partial sequences of LSU rDNA D2/D3 of two isolates (677-bp with accession numbers KF219669 and KF219670) were used. There were no differences between the sequences of both isolates. A BlastN search revealed a 93% identity with a ‘Japanese isolate of K. luziae’, the closest match in GenBank. The alignment of LSU rDNA D2/D3 partial sequences of K. ruehmi sp. n. and ‘K. luziae’ yielded 655 total characters (after manual editing of the alignment) with 42 variable characters and two insertions/ deletions. Diagnosis: Koerneria ruehmi sp. n. is characterized by the morphology of its stoma, shape of its gubernaculum (anterior half expanded and rounded, earshaped in lateral view, and posterior half forming a tubelike process enveloping the spicules, leaving the tip of the spicules free), vulva position, arrangement of genital papillae, presence of small bursa and shape of the tail tip in males.


Relationships: The new species comes morphologically closest to K. sudhausi Fürst von Lieven, 2008 and K. luziae (Körner, 1954) Meyl, 1960, based primarily on the shape of male tail. It differs from K. sudhausi in the absence of stomatal diphenism (no eurystomatous morph observed), form of reproduction (gonochoristic vs hermaphroditic), its smaller male body (539-721vs 755-1423 μm), lower c-value (15-17 vs 28-41), shorter spicules (22-29 vs 50-56 µm) and gubernaculum (10-15 vs 23-28 µm), gubernaculum shape (funnel-like in anterior half, enveloping the spicules in posterior half vs trough-like with a small hood), more anterior position of vulva (47-56 vs 55-60 µm), and tail length in both sexes (long vs short). And from the original description of K. luziae in telostegostom longer than wide (vs shallow and wider than long), male tail cuticle (thin, appearing a narrow terminal bursal flap vs thicker, appearing to envelop the tail tip starting at P5d when observed in ventral view), gubernaculum shape (anterior half ear-shaped in lateral view, posterior half forming a tube-like process enveloping the spicules, with the tip of the spicules becoming free vs not expanded and rounded in its anterior half), shape of male tail tip (acute vs rounded), shape of female tail (long, with pointed or filiform tip vs conoid, with pointed tip). Type locality and habitat: Northern Iran, Mazandaran province, Sisangan forest, bark of unknown dead tree with galleries of bark beetles. Collected in September 2010. Type material: Male holotype, five male and five female paratypes deposited at Nematode Collection of the Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran. Three male and three female paratypes 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. Etymology: The new species is named in honor of Walter Rühm, a pioneer in the taxonomy of bark-beetle associated nematodes. Remarks: The new species was distinguished from K. luziae using several morphological characters, e.g. stegostomatal shape and presence/absence of bursa. Nevertheless, these characters are difficult to observe correctly. For example, the thick cuticle around tail region sometimes appears like a narrow leptoderan bursa in ventral view, as drawn in the original description of K. luziae by Körner (1954). Therefore, some of the above diagnostic characters require re-examination using authentic materials of K. luziae. The presence of a small bursal flap in males of K. ruehmi sp. n. is recorded for the first time in the genus, and it is herein regarded as a diagnostic character to separate the new species from all of the other Koerneria species although, as stated above, detailed confirmation is necessary for the original K. luziae. This character has been described before in other genera such as Rhabditolaimus Füchs, 1914 by Susoy

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Table II. Morphometrics of Koerneria ruehmi sp. n. All measurements are in μm and in the form: mean ± s.d. (range). Holotype

Paratypes 15

Paratypes 20

L

585

607 ± 59 (539-721)

793 ± 72 (660-923)

L’

546

568 ± 56 (503-677)

689 ± 57 (585-803)

a

29.3

27.2 ± 2.0 (23.4-31.3)

27.6 ± 1.9 (23.6-31.5)

a’

27.3

25.4 ± 2.0 (22.0-29.5)

24 ± 1.5 (21.0-27.4)

b

4.4

4.7 ± 0.4 (4.0-5.3)

5.6 ± 0.3 (5.1-6.4)

b’

4

4.4 ± 0.4 (3.7-5.0)

4.9 ± 0.3 (4.4-5.7)

c

15

15.7 ± 0.7 (14.5-17.0)

7.8 ± 0.8 (6.4-9.1)

c’

2.4

2.1 ± 0.1 (2.0-2.3)

6.0 ± 0.8 (4.4-7.7)

V or T

60.3

57.7 ± 6.1 (45-68)

51 ± 2 (47-56)

V’

-

-

58.8 ± 1.9 (55-64)

Lip region width

9

9.0 ± 0.4 (8.5-10.0)

9.5 ± 0.8 (8.0-11)

Stoma width

4

3.5 ± 0.4 (3.0-4.0)

3.4 ± 0.5 (2.5-4.0)

Stoma length

2

1.9 ± 0.2 (1.5-2.0)

2.0 ± 0.4 (1.5-3.0)

Median bulb

67

64 ± 3.3 (57-68)

70.1 ± 4.2 (64-77)

MB

51

49.6 ± 0.8 (47.8-50.8)

49.5 ± 1.5 (46-52)

Body width at MB

17

18.9 ± 0.8 (18-20)

21.8 ± 1.9 (19-25)

Median bulb width

15

15.2 ± 0.8 (14-17)

17 ± 1.3 (15-20)

Nerve ring from anterior body

89

86 ± 6.5 (73-95)

95 ± 4.6 (86-102)

Character

n

Excretory pore

106

110 ± 8 (96-125)

118 ± 11 (96-135)

Hemizonid

102

104 ± 8 (92-120)

110 ± 9 (90-125)

Pharynx length

132

129 ± 7 (115-139)

141 ± 10 (125-158)

Head-vulva

-

-

405 ± 33 (338-461)

Body width

20

22.4 ± 2.3 (19-29)

28.8 ± 2.6 (25-35)

Length of anterior gonad branch

353

352 ± 60 (257-463)

216 ± 46 (154-322)

G1 or T

-

-

27.2 ± 4.7 (19.9-39.4)

Length of posterior gonad branch

-

-

201 ± 44 (147-312)

G2

-

-

25.3 ± 4.2 (19.5-33.8)

16

18.0 ± 0.7 (17-19)

17.3 ± 1.4 (15-20)

-

-

284 ± 30 (241-342)

Tail

39

38.7 ± 2.9 (35-44)

104 ± 18 (75-138)

Spicules

26

25.5 ± 1.5 (23-28)

-

Gubernaculum

10

12.1 ± 1.2 (9-13)

-

Anal body width Vulva - anus

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and Herrmann (2012). Morphologically and molecularly (see above), K. ruehmi sp. n. is closest to K. luziae which is a known phoretic associate of stag beetles (Dorcus parallelopipedus L.) in Germany (Körner, 1954). The insect host of K. ruehmi sp. n. was not discovered in this study, but lucanids, such as Lucanus ibericus Motschulsky, 1845 are known from Iran (Hajiqanbar et al., 2012) and it would be worth examining this species and others for their nematode associates to see if one or more of them are hosts of K. ruehmi sp. n. Recent work by Kanzaki et al. (2011) showed that lucanids share very interesting associations with a variety of nematodes, including ‘K. luziae’, in Japan.

Molecular phylogeny of the two Iranian diplogastrid species Figure 6 presents a phylogenetic Bayesian tree inferred from the multiple alignment of LSU rDNA D2/ D3 partial sequences of 32 diplogastrid taxa, one outgroup and four isolates of the two newly-discovered species. The dataset was composed of 725 total characters from which 537 characters were variable and 471 characters were parsimonious informative after manual editing. The average nucleotide composition was as follows: 19.2% A, 22.3% C, 32.9% G and 25.6 % T. Using Rhabditoides inermis (EU195981) as the outgroup, the two isolates of M. iranicus sp. n. formed a monophyletic group with high (1.00) posterior probability and formed a fully supported monophyletic group (1.00) with Neodiplogaster sp. (AB478641) and Neodiplogaster crenatae (AB326309). The six other ‘Mononchoides spp.’ available in GenBank formed a monophyletic group with Pristionchus sp. (DQ059064) and P. aerivorous (AB477071), suggesting that the genus Mononchoides is not a monophylectic taxon. Furthermore, the inferred position of the two Iranian isolates of M. iranicus sp. n. were quite divergent from the six other species of ‘Mononchoides’. In this tree, K. ruehmi sp. n. and K. luziae form a monophyletic group with 0.97 posterior probability. The other species of Koerneria (AB597244, AB440321, FJ661069, EU195999, JX163970 and AY840563) form another monophyletic group that is completely separated from the clade of the new species. According to our tree, the monophyly of the genus Koerneria is not supported. Phylogenetic analysis using Maximum Likelihood produced a tree with identical inferences to the Bayesian tree for all wellsupported lineages.

Discussion Remarks on the polyphyly of the genus Mononchoides Mononchoides spp. were inferred to be in two separate clades in the present phylogenetic analyses, i.e., J. Nematode Morphol. Syst., 16 (2): 113-129 (2013)

clear polyphyly was observed in the genus. However, this polyphyly is derived from the tentative identification of the species present in GenBank from previous studies, having a common ancestor with the genus Neodiplogaster. The Mononchoides species included in the Pristionchus clade were isolated by two of the authors (RGD and NK) and their colleagues from fig syconia in Australia (Accession number FJ661059) or bees in Turkey (FJ661062 - FJ661065, FJ661081) (see GenBank sequence information: http://www.genome.jp/dbget-bin/www_ bfind?genbank-today). The former was isolated on the basis of adult specimens, and were tentatively identified as Mononchoides sp. due to morphological characters (two large stegostomatal teeth and more than six rugae-like plates), and because molecular sequences were closest to Mononchoides at the time (Kanzaki et al., unpubl. records). Nevertheless, recently, several species in the Pristionchus/ Parapristionchus clade have been reported to have more than six cheilostomatal plates (Kanzaki et al., 2012; Ragsdale et al., 2013), i.e., the number of cheilostomatal plates is not a definitive character to separate these genera. Further, the molecular sequence information about diplogastrid nematodes in GenBank was not sufficient to identify these species correctly at the time. Therefore, the Ficus-associated Mononchoides sp. is considered to be an unidentified species of Pristionchus. Regarding the beeassociated isolates, these were isolated as dauer juveniles, which do not possess diagnostic generic morphological characters, and were identified solely on molecular sequences (Hazir et al., 2010; Giblin-Davis et al., unpubl. records). Therefore, as above, the molecular identification of these species needs to be corrected to Pristionchus spp., and further culture attempts or excavation of the soil nest of the Andrenid bee hosts should allow for reverse taxonomy to associate a morphospecies of Pristionchus with the sequence data.

Remarks on the genus Koerneria The genus Koerneria was clearly separated into two molecular clades (Fig. 6). This result was consistent with the previously suggested phylogenetic relationship inferred from near full length of 18S ribosomal RNA gene (Kanzaki et al., 2011, 2012). As the type species of the genus is K. goffarti (Körner, 1954) Meyl, 1960, it may eventually be necessary to pull K. luziae and K. ruehmi sp. n. into an new genus once more isolates can be discovered and sequenced and clear morphological autapomorphies can be found to support the action. One potential autapomorphy for this new lineage might be the paired ventral P6 and P7 male papillae followed at more than twice the distance by the P8 papillae which are more seta-like versus the more standard equidistant pairing of the papilla-like ventral P6-8 papillae in most diplogastrids. Furthermore, the species status of the Japanese isolate of ‘K. luziae’ requires further comment here. The new species K. ruehmi sp. n. is morphologically very similar to K. luziae, both being distinguished by some

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Figure 6. Bayesian 50% majority rule consensus tree inferred from 37 sequences of LSU rDNA D2/D3 under the GTR + I + G model. Posterior probabilities more than 50% are given on top and the score from Maximum Likelihood (ML) following 1000 bootstrap replicates are given on the bottom for appropriate clades. *These isolates with accession numbers FJ661059, FJ661065, FJ661064, FJ661062, FJ661063 and FJ661081 were identified using GenBank in Hazir et al., 2010 as “Mononchoides”. Our study has revealed that they were incorrectly designated as Mononchoides and should be shifted to Pristionchus.

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minor morphological differences. In a previous study, one of the present authors (NK), identified the Japanese isolated as ‘K. luziae’ based on general morphology (Kanzaki et al., 2011). Nevertheless, when considering the morphological similarity between K. ruehmi sp. n. and the original description of K. luziae by Körner (1954), it now appears possible that the ‘Japanese isolate of K. luziae’ is a morphologically similar third species in this clade. Herrmann et al. (2006) examined the reproductive mode and distribution patterns of Pristionchus spp., and reported that only hermaphroditic species can spread widely (becoming cosmopolitan species), while the distributions of gonochoristic species tend to be more localized or endemic. Thus, the distribution pattern and molecular differences between the Japanese isolate of ‘K. luziae’ and K. ruehmi sp. n. support the possibility that the Japanese isolate of ‘K. luziae’ is a separate species. Re-isolation and culturing of the authentic K. luziae from Dorcus parallelopipedus in Germany, followed by genetic and morphological characterization is necessary to better understand the genus Koerneria and its relatives for future reclassification efforts.

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