Научная статья на тему 'Morphological and molecular analysis based on sequence of the 18S rDNA of Mononchoides adjunctus Massey, 1966 (Nematoda: Neodiplogasteridae) from Iran'

Morphological and molecular analysis based on sequence of the 18S rDNA of Mononchoides adjunctus Massey, 1966 (Nematoda: Neodiplogasteridae) from Iran Текст научной статьи по специальности «Биологические науки»

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description / Mononchoides adjunctus / phylogeny / taxonomy

Аннотация научной статьи по биологическим наукам, автор научной работы — Sara Mehdizadeh, Ebrahim Shokoohi, Joaquín Abolafia

Mononchoides adjunctus Massey, 1966 was isolated during a survey on free-living nematodes belonged to the family Neodiplogasteridae from a natural lawn in south-eastern Iran, near to Lout desert, in Kerman province. This population of Mononchoides is characterised by a dorsal claw-like tooth 2 μm long and 1 μm wide, 15 longitudinal ridges on the female body, a uterine sac associated with two dumbbellshaped pouches, small spicules (19-25 μm long), a short gubernaculum (11-14 μm or less than half of the spicule length), two pairs precloacal papillae, five pairs postcloacal papillae, papillae (GP5) comprising of three small papillae, and a long filiform tail (154-171 μm in females, 110-137 μm in males). Molecular analysis of M. adjunctus based on sequence of the 18S rDNA placed it together with Tylopharynx foetidus (EU306343) as sister group. Measurements, illustrations and phylogenetic position of M. adjunctus and closely related species are given.

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Анализ Mononchoides adjunctus Massey, 1966 (Nematoda: Neodiplogasteridae) из Ирана по результатам изучения морфологии вида и последовательности 18S rDNA.

Нематоды Mononchoides adjunctus Massey, 1966 обнаружены во время сбора нематод семейства Neodiplogasteridae в луговой почве близ пустыни Лут в провинции Керман (юговосточная часть Ирана). Особи этой популяции Mononchoides характеризуются наличием в стоме дорсального когтевидного зуба длиной 2 мкм и диаметром 1 мкм; 15-ю продольными ребрами на теле самок; матками, связанными с двумя карманами гантелевидной формы; небольшими спикулами (19-25 мкм в длину); коротким рульком (11-14 мкм или менее половины длины спикулы); двумя парами преклоакальных папилл; пятью парами постклоакальных папилл, (GP5) из трех мелких папилл; длинным нитевидным хвостовым филаментом (154-171 мкм у самок, 110-137 мкм у самцов). Молекулярный анализ M. adjunctus по последовательностям 18S rDNA показал близость их к Tylopharynx foetidus (EU306343). Приводятся результаты измерений, рисунки, фотографии, а также данные по филогенетическим связям M. adjunctus и близких видов

Текст научной работы на тему «Morphological and molecular analysis based on sequence of the 18S rDNA of Mononchoides adjunctus Massey, 1966 (Nematoda: Neodiplogasteridae) from Iran»

Russian Journal of Nematology, 2013, 21 (2), 63-71

Morphological and molecular analysis based on sequence of the 18S rDNA of Mononchoides adjunctus Massey, 1966 (Nematoda: Neodiplogasteridae) from Iran

12 2 3

Sara Mehdizadeh ' , Ebrahim Shokoohi and Joaquín Abolafia

1Young Researchers Society, Shahid Bahonar University of Kerman, Kerman, Iran; 2Department of Plant Protection, College of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran,

e-mail: eshokoohi@mail.uk.ac.ir; ^Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, Campus "Las Lagunillas" s/n. 23071-Jaén, Spain.

Accepted for publication 12 September 2013

Summary. Mononchoides adjunctus Massey, 1966 was isolated during a survey on free-living nematodes belonged to the family Neodiplogasteridae from a natural lawn in south-eastern Iran, near to Lout desert, in Kerman province. This population of Mononchoides is characterised by a dorsal claw-like tooth 2 ^m long and 1 ^m wide, 15 longitudinal ridges on the female body, a uterine sac associated with two dumbbell-shaped pouches, small spicules (19-25 ^m long), a short gubernaculum (11-14 ^m or less than half of the spicule length), two pairs precloacal papillae, five pairs postcloacal papillae, papillae (GP5) comprising of three small papillae, and a long filiform tail (154-171 ^m in females, 110-137 ^m in males). Molecular analysis of M. adjunctus based on sequence of the 18S rDNA placed it together with Tylopharynx foetidus (EU306343) as sister group. Measurements, illustrations and phylogenetic position of M. adjunctus and closely related species are given. Key words: description, Mononchoides adjunctus, phylogeny, taxonomy.

The genus Mononchoides was erected by Rahm in 1928. This genus belongs to the family Neodiplogasteridae sensu stricto according to Andrassy (1984, 2005), or to the family Diplogasteridae sensu lato according to Sudhaus & Fürst von Lieven, (2003). That first option is maintained in the present paper. The number of species and taxonomy of this genus has always been challenging according to different scientists (Calaway & Tarjan, 1973; Andrassy, 1984, 2005; Gagarin, 1998; Sudhaus & Fürst von Lieven, 2003), especially if members of genera Diplenteron Andrassy, 1964, Glauxinema Allgen, 1947, Pareudiplogaster Paramonov, 1952 are included or not under Mononchoides. In this respect, Gagarin (1998), and later Sudhaus and Fürst von Lieven (2003), considered the genus Glauxinema Allgen, 1947 as a junior synonym of the genus Mononchoides, although later Gagarin and Thanh (2006), considered this synonymisation as a mistake. On the other hand, Andrassy (2005)

maintained both genera could be separated on the basis of the morphology of the buccal cavity.

There are only a few studies about diplogasterid nematodes from Iran. The only diplogasterid reported recently has been Pristionchus pacificus Sommer, Carta, Kim & Sternberg, 1996, by Hasani-Kakhki et al. (2013). The present paper, part of a series on nematodes belonging to the order Rhabditida from the province of Kerman (Iran), deals with one new record on diplogasterids, this time of the genus Mononchoides collected in natural areas. In addition, SSU rDNA analysis and phylogenetic position of a species of this genus is given.

MATERIALS AND METHODS

Nematode materials. Nematodes were extracted from soil samples by Baermann's funnel technique (1917). They were fixed with hot 4% formaldehyde solution and processed to anhydrous glycerin by the method of De Grisse (1969). Measurements were taken

Fig. 1. Mononchoides adjunctus Massey, 1966. A: Neck. B: Anterior end at stoma level. C: Idem at amphid level. D: Female reproductive system. E: Entire female. F: Entire male. G: Male posterior end. H: Female posterior end. I: Spicules (ventral view).

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 an Olympus CH-2 microscope. LM pictures were made with a Nikon Eclipse 80i microscope equipped with a Nikon Digital Sight DS-5M camera. 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 several species of the superfamily Diplogasteroidea used for phylogenetic analysis were obtained from the GenBank. DNA extraction was done using an AccuPrep Genomic DNA extraction kit (Bioneer Corporation, Korea, http://www.bioneer.com) according to the manufacturer's instructions. Ten live individual specimens were picked into 1.5 ml tubes containing 5 ^l double distilled water. Each tube was frozen in liquid nitrogen and was crushed using a vortex; 200 ^l Tissue Lysis buffer (TL) and 20 ^l proteinase K (20 mg ml-1) were 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. PCR was conducted with 10 ^l of the extracted DNA, 4 ^l of PCR Master Mix (Kawsar Biotech company, Iran), 1 ^l of each primers (10 pmol ^l-1) and ddH2O to a final volume of 25 ^l. The amplification was carried out using an Eppendorf Mastercycler gradient (Eppendorf, Hamburg, Germany), with the following paramters: 3 min at 94°C, 37 cycles of 45 s at 94°C, 45 s at 56°C and 1 min at 72°C, and finally one cycle of 6 min at 72°C followed by a holding temperature of 4°C. After DNA amplification, 5 ^l of product was loaded on a 1% agarose gel (40 mM Tris, 40 mM boric acid, and 1 mM EDTA) to verify amplification. The bands were stained with 50 mM ethidium bromide and visualised and photographed on 1% agarose gel under a UV transilluminator. Product was stored at -20°C prior to sequencing. PCR product was purified for sequencing and sequenced with primers that were used for the amplification step. Sequencing was performed in both directions. The DNA sequence was edited using Chromas version 1.45 (McCarthy, 1997). Sequencing reactions were performed by the Bioneer Company (South Korea, http://eng.bioneer.com). Primers for the sequencing reaction were those used in the amplification step. All sequences were confirmed in both directions and repeated. The original partial 18S sequence of Mononchoides adjunctus Massey, 1966

was 711 bp. The sequence was deposited in GenBank under accession number KF151166. Additional sequences for the ingroups and outgroups were obtained from NCBI GenBank.

The ribosomal SSU sequences were analysed and aligned using BioEdit (Hall, 1999). The length of alignment was 1863 bp. Phylogenetic trees were generated using the Bayesian inference method as implemented in the program Mr Bayes 3.1.2 (Ronquist & Huelsenbeck, 2003). The analysis under GTR model was initiated with a random starting tree and run with the Markov chain Monte Carlo (MCMC) for 106 generations. The distance matrix option of Mega 5 (Tamura et al., 2011) was used to calculate genetic distances according to the Maximum Composite Likelihood model of sequence evolution and bootstrap analysis was implemented with 2000 replicates. Bunonema reticulatum Richters, 1905 (AY593925) and Rhabditoides inermis (Schneider, 1866) Dougherty, 1955 (AF082996) were used as outgroups for this phylogenic analysis. These selections were based on a study by Steel et al. (2011). The Bayesian tree was visualised with the TreeView program (Page, 1996).

DESCRIPTION

Mononchoides adjunctus Massey, 1966 (Figs 1 & 2)

Material examined. Four females and three males from one locality, in good state of preservation.

Measurements. See Table 1.

Female. Body 0.55-0.65 mm long. Habitus slightly curved ventrad after fixation. Cuticle annulated, lacking punctation. Annuli 1.5 |im wide. Lateral field not observed. Cuticle smoothly annulated, 1 pm at mid-body, with 15 conspicuous longitudinal ridges at midbody level. Lip region continuous with body contour, consisting of six lips, each with a small papilla, 1-2 ^m long. Stoma 1.5-2.0 times longer than wide. Cheilostom wide, walls heavily cuticularised. Cheilostom subdivided into several (about 10-12) narrow, rod-like plates. Bifurcated apex of cheilorhabdia extending near labial contour. Gymnostom wide, about 5 ^m. Second part of stoma consisting of gymnostom and stegostom, both anisotopic with subventral walls slightly longer than dorsal. Amphidial aperture oval shaped, located at base of dorsal tooth, about 3 ^m wide. Stegostom bearing a large, claw-like dorsal tooth, 2 ^m long and 1 ^m wide; with a duct of dorsal gland and pointed toward anterior part of stoma. Posterior part of stegostom (= meta- and telostegostom) forming a cylindrical tube, 34 ^m wide and 5-6 ^m long. Stegostom cylindrical, 58 ^m long and 3-5 ^m wide, about 1.5 times longer than

Fig. 2. Mononchoides adjunctus Massey, 1966. (LM, female). A: Neck. B, C: Stoma (at right and left sublateral view, respectively). D: Anterior genital branch. E: Cuticle. F: Posterior genital branch. G, H: Body posterior end at phasmids level (left and right phasmids, respectively).

wide. Pharynx diplogasteroid, having pharyngeal corpus 1.1-1.3 times longer than postcorpus (isthmus + basal bulb). Pharyngeal procorpus cylindrical, 1.5-2.0 times metacorpus length. Metacorpus swollen, 17-24 ^m long. Isthmus robust, 21-27 ^m long. Basal bulb ovoid, 20-24 ^m long and 13-14 ^m long. Cardia conoid, surrounded by intestinal tissue. Nerve ring at 67-75% of neck length, at isthmus level. Excretory pore at 71-80% of neck length, at isthmus level. Deirid not visible. Intestine without distinct specializations. Reproductive system didelphic amphidelphic with both branches equally developed. Ovaries long with oocytes arranged in one to two indistinct rows in germinal zone. Oviducts short, not well distinguished from the adjacent uterus. Uteri 4-5 times as long as the corresponding body diameter, sometimes having spermatozoa at distal part; a pair of dumb-bell-shaped pouches present at proximal part (ovijector), 17-20 pm long, connecting both uteri. Vagina with narrow lumen and extending inwards less than half of the corresponding body diameter. Vulval opening anterior to mid-body. Vulva lips weakly cuticularised, not protruding. Rectum 0.9-1.4 times anal body diameter long. Tail first conical then filiform. Phasmid at 1820% of tail length.

Male. Body J-shaped after fixation. Reproductive system monorchic, testis reflexed dorsad anteriorly. Tail first conical, then filiform, distally curved ventrad. Two pairs of precloacal genital papillae. Five pairs of caudal genital papillae are present along the tail. The GP5 (genital papillae) comprising three papillae close to each other. The GP6 is the shortest papillae. Spicules free, curved ventrally: manubrium almost straight and conoid, calamus with hump, and lamina ventrally curved. Gubernaculum well developed, 11-14 |im long or about 52-62% of the spicule length.

Locality and habitat. The species was collected in Andoohjerd (province of Kerman, Iran; N: 30°13'41.5"; E: 057°45'36.7"; date of sampling: 2012), associated with dominat grass species Axonopus sp.

Remarks. The Iranian material is similar with the original description of the M. adjunctus provided by Massey (1966). The Iranian material is similar to M. adjunctus by having small body length less than 1 mm (554-654 pm in females and 509-536 pm in males), stoma as long as lip region wide, pharyngeal corpus slightly longer than postcorpus, vulva pre-equatorial (V% = 43-45), tail with similar length (c' less than 15 in females).

However, it differs in body length (554-654 pm in females and 509-536 pm in males vs 780-870 pm in females and 700 pm in males), and c value in males (3.7-4.9 vs 3.2).

Other similar species are M. subdentatus (Gunhold, 1952) Andrassy, 1984 and M. pulcherrimus Andrassy,

Table 1. Measurements of Mononchoides adjunctus Massey, 1966. All measurements are in |m and in the form: mean + s.d. (range).

Province; Locality; Habitat Kerman; Andoohjerd; grass

n 4ÇÇ 3SS

L 611.4±42.2 521.2±13.9

(554-654) (509-536)

a 26.4±0.6 31.3±1.6

(25.7-27.0) (29.5-32.3)

b 5.4±0.1 (5.3-5.5) 5.6±0.2 (5.4-5.8)

c 3.7±0.1 (3.6-3.8) 4.4±0.6 (3.7-4.9)

c' 12.7±0.8 (12-14) 7.5±0.8 (7-8)

V 44.2±0.6 (43-45) -

Lip region width 10.8±0.3 (10.5-11.0) 8.3

Seta length 1.5±0.6 (1.0-2.0) 1.0

Amphid width 4.7±0.5 (4-5) 4.0

Stoma length 11.3±1.0 (10-12) 9.0±0.5 (8-9)

Cheilostom width 2.9 2.8

Stegostom length 6.1±1.5 (5-8) 5.6±0.9 (5-6)

Stegostom width 4.4±1.3 (3-6) 4.0±0.5 (4-5)

Procorpus length 33.8±0.6 (33-34) 29.3±1.4 (28-31)

Metacorpus length 20.8±2.7(18-23) 17.3±1.1 (17-18)

Isthmus length 23.8±2.1(21-26) 23.5±1.4 (22-25)

Bulb length 21.8±1.5 (21-23) 15.7±1.6 (15-18)

Neck length* 113.2±6.2 (105-119) 93.8±1.1 (93-94)

Excretory pore position 84.6±10.5 (75-96) 74.5±0.7 (74-75)

Nerve ring position 70.6±3.9 (67-74) 66.2±2.0 (65-68)

Body diameter at neck base level 20.0±1.6 (18-22) 16.0±1.4 (15-18)

Midbody diameter 23.2±1.5 (22-25) 16.7±0.9 (16-18)

Body diameter at anus/ cloaca level 12.9±0.7 (12-14) 14.8±1.3 (14-16)

Anterior genital 105.5±16.1

branch (83-117)

Posterior genital 111.6±12.2

branch (100-124)

Vagina 8.3±0.6 (8-9) -

Vulva to anus 201.8±15.1

distance (185-222)

Rectum length 14.2±2.3 (13-18) -

Tail length 163.4±7.2 (154-171) 119.9±15.0 (110-137)

Spicule length - 21.8±2.6 (19-24)

Gubernaculum length - 12.3±1.3 (11-14)

* Stoma + pharynx length

Table 2. Estimates of genetic distance of 18S rDNA region among Mononchoides and closely related species studied using Maximum Composite Likelihood method.

Species 1 2 3 4 5 6

1 Mononchoides striatus JF769024

2 Tylopharynx foetidus EU306343 0.124

3 Mononchoides composticola GU943511 0.116 0.064

4 Mononchoides composticola GU943512 0.116 0.064 0.000

5 Neodiplogaster sp. AB478640 0.120 0.074 0.064 0.064

6 Mononchoides adjunctus KF151166 0.205 0.147 0.147 0.147 0.143

FJ040449 Odontopharynx longicaudata | Odontopharyngidae

Diplogasteridae Tylopharyngidae

— HQ 130144 Acrostichus puri - EU306343 Tylopharynx foetidus

I I

1.00 , 1.00|

Neodiplogasteridae

0.62

■ KF151166 Mononchoides adjunctus

- AB478640 Neodiplogaster sp.

AY593924 Mononchoides striatus GU943511 Mononchoides composticola GU943512 Mononchoides composticola JX163981 Parapristionchus giblindavisi JX163977 Micoletzkya masseyi - FJ040437 Fictor sp.

- FJ040438 Demaniella sp. | Diplogasteroididae

JQ399907 Pristionchus arcanus - JQ699287 Pristionchus pacificus

II

i.oo[_

Neodiplogasteridae

1.00 1.00 0.99

JQ005869 Diplogasteroides magnus | Diplogasteroididae i— AB597237 Pseudodiplogasteroides compositus | Pseudodiplogasteroididae EU419759 Diplogastrellus metamasius | Diplogasteridae

- EU196024 Myctolaimus ulmi | Cylindrocorporidae

AB597233 Koerneria lucani | Neodiplogasteridae AB501144 Parasitodiplogaster sp. | Diplogasteridae - AF082996 Rhabditoides inermis i

outgroup

- AY593925 Bunonema reticulatum

III

Fig. 3. The Bayesian Inference tree of known and newly sequenced Mononchoides species from Iran based on sequence of the 18S rDNA region.

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1987. Our specimens compared with M. subdentatus has stoma slightly longer than wide (vs wider than long), longer female tail (154-171 vs 146-158 pm; the measurement taken from the original measurement). In comparison with the latter species it has longer female (554-654 vs 880-1020 pm), shorter female tail (vs 200215 pm), and spicules (19-24 vs 36-38 pm). Furthermore, Iranian specimens are similar to M. striatulus (Fuchs, 1933) Goodey in Goodey (1963); however, in our specimens spicules are free (vs spicules fused at them posterior half).

This species is reported for the first time from Iran.

DISCUSSION

The tree contains three main clades: I) Odontopharynx longicaudata de Man, 1912; II) Acrosticus puri Kanzaki, Giblin-Davis, Zeng, Ye, Center & Thomas, 2010; Tylopharynx foetidus (Bütschli, 1874) Goodey, 1928; Neodiplogaster sp.; Mononchoides spp.; Mikoletzkya masseyi Susoy, Kanzaki & Herrmann, 2013; Fictor sp.; Demaniella sp.; Pristionchus spp.; Parapristionchus giblindavisi Kanzaki, Ragsdale, Herrmann, Mayer, Tanaka & Sommer, 2012; Diplogasteroides magnus (Völk, 1950) Weingärtner, 1953; Pseudodiplogasteroides compositus Körner, 1954; Diplogastrellus metamasius Kanzaki, Giblin-Davis, Zeng, Ye & Center, 2008 and Myctolaimus ulmi (Goodey, 1930) Sudhaus & Fürst von Lieven, 2003; and III) Koerneria lucani (Körner, 1954) Meyl, 1960 and Parasitodiplogaster sp.

Figure 3 shows the evolutionary relationships of the material examined as derived from molecular analysis. The sequence of Iranian M. adjunctus is included, together with other two M. composticola and M. striatus sequences, in a clade dominated by long-tailed Diplogasteroidea taxa, confirming other recent studies, e.g., those by Steel et al. (2011) and Kanzaki et al. (2011). Genetic distance according Maximum Composite Likelihood among 18S rDNA region of Mononchoides species showed that M. adjunctus has the maximum genetic distance (0.205) with M. striatus (AY593924) from The Netherlands (Table 2). Furthermore, in comparison with M composticola (GU943511; GU943512) reported from Belgium, its genetic distance is 0.116.

The phylogenetic analysis places Mononchoides close to Tylopharynx foetidus, in agreement with Steel et al. (2011). However, our analysis agrees with the previously published 18S rDNA trees of Diplogasteromorpha (van Megen et al, 2009; Steel et al., 2011; Kanzaki et al. 2011), in the fact that the 18S rDNA gene does provide resolution within the diplogastrids for the phylogenetic analysis within this group of nematodes.

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Резюме. Нематоды Mononchoides adjunctus Massey, 1966 обнаружены во время сбора нематод семейства Neodiplogasteridae в луговой почве близ пустыни Лут в провинции Керман (юго-восточная часть Ирана). Особи этой популяции Mononchoides характеризуются наличием в стоме дорсального когтевидного зуба длиной 2 мкм и диаметром 1 мкм; 15-ю продольными ребрами на теле самок; матками, связанными с двумя карманами гантелевидной формы; небольшими спикулами (19-25 мкм в длину); коротким рульком (11-14 мкм или менее половины длины спикулы); двумя парами преклоакальных папилл; пятью парами постклоакальных папилл, (GP5) из трех мелких папилл; длинным нитевидным хвостовым филаментом (154-171 мкм у самок, 110-137 мкм у самцов). Молекулярный анализ M. adjunctus по последовательностям 18S rDNA показал близость их к Tylopharynx foetidus (EU306343). Приводятся результаты измерений, рисунки, фотографии, а также данные по филогенетическим связям M. adjunctus и близких видов.

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