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44Russian Journal of Nematology, 2012, 20 (2), 157-166
Insulanema longispiculum gen. n., sp. n. a new
Carnoyidae (Nematoda: Rhigonematomorpha)
from Phu Quoc Island, Vietnam
12 1 Svetlana V. Malysheva , Pham Van Luc and Sergei E. Spiridonov
1Centre of Parasitology, A.N. Severtsov's Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii
prospect, 33, Moscow, 119071, Russian Federation; e-mail: [email protected] 2Vietnam National Museum of Nature, Hoang Quoc Viet Rd, Cau Giay, Hanoi, Viet Nam
Accepted for publication 25 September 2012
Summary. Insulanema longispiculum, a new genus and species of Carnoyidae (Rhigonematomorpha), is described from the hind gut of diplopod Apeuthes Attems, 1937 (Spirobolida: Trigoniulidae), collected on Phu Quoc Island in the Kien Giang Province, Vietnam. The new genus is characterised by the absence of any spines or other cuticular formations in the cervical region of both male and female, the presence of six cuticular pieces around the oral opening, the presence of a smooth cephalic cap and area rugosa in the male, very long isomorphic and isometric spicules, boat-shaped gubernaculum with a dorsal hole and 14 genital papillae with two postcloacal pairs subventral and one posteriormost pair subdorsal in the middle of conical part of the tail. Insulanema gen. n. could be easily distinguished from African and Australasian Carnoyidae by the position of the last postcloacal pair of genital papillae and enormously long spicules in the male.
Key words: description, diplopods, Insulanema, morphology, new species, Ransomnematoidea, SEM, taxonomy, Vietnam.
In the traditional classification of nematodes, the orders Ascaridida, Oxyurida, Rhigonematida and Spirurida exclusively consist of parasitic forms. Molecular analysis of the class Nematoda (Blaxter et al., 1998) demonstrated that these four groups formed a single monophyletic clade (Clade III). Sampling of this clade for phylogenetic analysis based on molecular data increased the number of species from eight studied by Blaxter et al. (1998), to 32 in Holterman et al. (2006) and up to 113 studied by Nadler et al. (2007). Molecular data persuaded De Ley and Blaxter (2002) to change the system of nematodes in total and those of Clade III in particular. However, the parasitic nematodes of this clade have not been studied in equal detail. For example, the infraorder Rhigonematomorpha of De Ley and Blaxter, 2002 (Rhigonematida of traditional classification) was represented with only two taxa: one species of Rhigonematoidea and one species of Ransomnematoidea. We present here the description of a new Carnoyidae from a Vietnamese diplopod and additional molecular data for the nematodes of the infraorder Rhigonematomorpha.
MATERIALS AND METHODS
Nematode material. Nematodes were obtained from a single diplopod specimen Apeuthes Attems, 1937 (Spirobolida: Trigoniulidae) collected by Dr Pham Van Luc on Phu Quoc Island, Kien Giang Province, Vietnam in July of 2010.
The sequences of SSU rDNA and D1-D3 LSU rDNA were obtained for this species. For the phylogenetic analysis, molecular data for three other Carnoyidae were also obtained: 1) Cattiena trachelomegali Hunt & Spiridonov, 2001 recovered from Thyropygus Pocock, 1894 (Spirostreptida: Harpagophoridae), collected by O. O. Tolstenkov, May 2011, Cat Tien National Park, Lam Dong Province, Vietnam (11o 27' N, 107o 26' E); 2) Cattiena fansipanis Malysheva & Pham Van Luc, 2012 recovered (Malysheva & Pham Van Luc, 2012) from an unidentified diplopod (Spirobolida: Pseudospirobolellidae), collected by S. E. Spiridonov, August 2009 near Sa Pa, Lao Cai Province, Vietnam (22o 20' N, 103o 51' E); and 3) Brumptaemilius justini Adamson & Anderson, 1984 recovered from the diplopod Archispirostreptus
gigas Peters, 1855 (Spirostreptida: Spirostreptidae), collected by D. V. Karelin, May, 2011, Dar-es-Salaam, Tanzania (6o 47' S, 16o 46' E). Molecular data were also obtained for three representatives of Hethidae: 1) Heth sp. 1 recovered from an unidentified diplopods (Spirostreptida:
Iulomorphidae) collected by T. Cribb and S. E. Spiridonov in July, 2008 at Mount Glorious, Queensland, Australia (27o 21' S, 152o 46' E), 2) Heth sp. 2 recovered from a diplopod Salpidobolus Silvestri, 1897 (Spirobolida: Rhinocricidae), collected by D. V. Karelin, February 2011, Sumba Island, Indonesia (9° 41' S, 119° 59' E), and 3) Heth taybaci Malysheva & Spiridonov, 2010 recovered from an unidentified diplopod (Spirostreptida: Harpagophoridae), collected by S. E. Spiridonov, November 2007, near Sin Ho, Lai Chau Province, Vietnam (22o 27' N, 103o 18' E). Molecular data for one Rhigonematidae recovered from diplopod Thyropygus Pocock, 1894 (Spirostreptida: Harpagophoridae), collected by S. E. Spiridonov, July 2007 in Cat Tien National Park forest, Lam Dong Province, Vietnam (11° 27' S, 107° 26' E) and identified as Rhigonema ingens Hunt, 1997 were also obtained.
Voucher specimens No. 1190 for Brumptaemilius justini, No. 1191 for Cattiena trachelomegali, No. 1184 for Cattiena fansipanis, No. 1193 for Heth taybaci, No. 1194 for Heth sp. 1, No. 1195 for Heth sp. 2 and No. 1185 for Rhigonema ingens were deposited in the collection of the Centre of Parasitology, A. N. Severtsov's Institute of Ecology and Evolution, Russian Academy of Sciences. All diplopods were identified by Dr Sergei I. Golovatch, A. N. Severtsov's Institute of Ecology and Evolution, Russian Academy of Sciences.
Morphological observation. The diplopod was narcotised with chloroform and then decapitated. The hind gut was cut off and dissected in NaCl 0.9% solution. Nematodes were fixed in hot (60-70°C) 4% formaldehyde and processed to glycerol according to Seinhorst (1959). Measurements and drawings were made with the aid of a camera lucida. Some nematodes were prepared for SEM by dehydration through a graded ethanol series and acetone and dried in a critical point drier. After coating with gold/palladium they were examined in a Jeol JSM-6380LA electron microscope.
Molecular profiles. Some specimens were stored in 95% ethanol and kept at -18°C prior to DNA extraction. The DNA was extracted from all samples using methods detailed by Holterman et al. (2006). The worm-lysis solution was prepared immediately before DNA extraction containing the
950 ^l of mixture of 2 ml of 1M NaCl, 2 ml of 1M Tris-HCl (pH 8) plus 5.5 ml of deionized water plus 10 ^l of mercaptoethanol and 40 ^l of proteinase K (20 mg ml-1). Single nematodes were each transferred to 25 ^l of sterile water and after addition of 25 ^l of worm-lysis solution the tube was incubated at 65°C for 90 min. The tubes with homogenate were then incubated at 99°C for 5 min to deactivate proteinase K and 0.8-1.2 ^l of the obtained homogenate was used as template for PCR reactions. The PCR reactions were performed using PCR kits of different producers according to manufacturers' instructions.
Two pairs of primers were used to amplify D1-D3 expansion segment of large subunit of ribosomal DNA (LSU rDNA). Primers fw1 (5'-AGC GGA GGA AAA GAA ACT A-3') and rev1 (5'-TAC TAG AAG GTT CGA TTA GTC-3') were used to amplify D1-D2 segment of LSU rDNA (Sonnenberg et al., 2007). PCR cycling parameters included primary denaturation at 94°C for 4 min followed by 40 cycles of 94°C for 20 s, 52°C for 20 s and 72°C for 1 min 30 s, followed by post-amplification extension at 72°C for 8 min. Primer pairs D2A (5'-ACA AGT ACC GTG AGG GAA AGT TG-3') and D3B (5'-TCG GAA GGA ACC AGC TAC TA-3') (Nunn, 1992) were used to amplify D2-D3 segment of LSU rDNA. PCR cycling parameters included primary denaturation at 94°C for 5 min followed by
34 cycles of 94°C for 60 s, 50°C for 60 s and 72°C for 1 min, followed by post-amplification extension at 72°C for 10 min.
Nematode-specific primers nem18SF (5'-CGC GAA TRG CTC ATT ACA ACA GC-3') and nem18SR (5'-GGG CGG TAT CTG ATC GCC-3') (Floyd et al., 2005) were used to amplify a portion of the small subunit of ribosomal DNA (SSU rDNA). PCR cycling parameters included primary denaturation at 95oC for 5 min followed by 5 cycles of 94oC for 30 s, 47oC for 30 s and 72oC for 40 s and
35 cycles of 94oC for 25 s, 54oC for 30c and 72oC for 40 s, followed by post-amplification extension at 72oC for 5 min.
Primers 24F (5'-AGR GGT GAA ATY CGT GGA CC-3') and Q39 (5'-TAA TGA TCC WTC YGC AGG TTC ACC TAC-3') were used to obtain the remaining 3' end of SSU rDNA (Blaxter et al., 1998). PCR cycling parameters included primary denaturation at 95oC for 5 min followed by 35 cycles of 94oC for 60 s, 53oC for 90 s and 72oC for 90 s, followed by post-amplification extension at 72oC for 6 min.
The PCR reaction products were visualized in agarose gel and bands were excised for DNA extraction with Promega columns (Wizard® SV Gel
and PCR Clean-Up System). Samples were directly sequenced using the same primers as were used for primary PCR reactions.
The nematode sequences were deposited in NCBI GenBank as: JX999732 (Brumptaemilius justini), JX419378 (Cattiena trachelomegali), JX436470 (Cattiena fansipanis), JX436471 (Insulanema longispiculum gen. n., sp. n.), JX946704 (Heth taybaci), JX443484 (Heth sp. 1), JX443483 (Heth sp. 2), JX131616 (Rhigonema ingens) for LSU rDNA and JX999733 (Brumptaemilius justini), JX982117 (Cattiena trachelomegali), JX982118 (Cattiena fansipanis), JX982119 (Insulanema longispiculum gen. n., sp. n.), JX987085 (Heth taybaci), JX987086 (Heth sp. 1), JX987087 (Heth sp. 2) for SSU rDNA.
For comparative purposes and phylogeny construction, other sequences from NCBI GenBank were also used (deposition numbers are indicated on the trees). Sequence alignments were generated using Clustal X (Thompson et al., 1997) under default values for gap opening and gap extension penalties. MEGA 5 was used for the search of proper model of evolution and to obtain phylogenies based on maximum parsimony (MP), neighbour joining (NJ) and maximum likelihood (ML) (Tamura et al, 2011). Modeltest 3.5 was also used
to find the optimal model of evolution for the obtained data. Mr Bayes v3.2.1. (Ronquist et al, 2012) was used for Bayesian analysis (Bayesian Inference - BI). Bayesian analyses were run for 2 * 106 generations under GTR+G+I model using relative burn-in (discarding the first 25% of samples). At the end of the analyses, average standard deviation of split frequencies was lower than 0.01. Tracer vi.3 (Rambaut & Drummond, 2007) was used to estimate convergence statistics.
DESCRIPTION
Insulanema gen. n. (Figs 1-4)
Carnoyidae. Small to medium sized nematodes; females 2800-4600 ^m long and males 2000-3000 ^m long. Strongly marked sexual dimorphism apparent in stoma and pharynx structure.
Female. Cephalic extremity narrow, bearing four cephalic papillae and amphids. Lateral alae absent. Six cuticular pieces arranged as two dorsal and four subventral surround oral opening. Body cuticle with fine annulation free of any formations. Buccal cavity long, consisting of anterior capsule with tree cuticular
Table 1. Insulanema longispiculum gen. n., sp. n. Morphometric data in the form: mean ± s.d. (range).
All linear measurements are in цт.
Character Females Males
Holotype Paratype Paratype
n - 15 8
L 4220 3870 i 501 (2842-4600) 2370 i 350 (2000-2970)
L' 2720 2545 i 310 (1970-3020) 2012 i 293 (1700-2500)
a 18.3 16 i 2.7 (12-21) 18 i 3.2 (15-24)
b 9 9 i 0.9 (7-11) 4.2 i 0.8 (2.7-5.4)
c 2.8 3 i 0.2 (3-3.4) 6.7 i 0.8 (6-8.6)
c' 15 12 i 1.8 (9-16) 4.5 i 0.8 (3.6-5.6)
V 34 36 i 3.2 (31-43) -
v' 52.2 54 i 3.2 (50-60) -
Total pharynx length 470 425 i 27 (370-480) 570 i 67 (512-725)
Max. body diam. 230 250 i 44 (150-300) 133 i 25 (100-180)
Anal/cloacal body diam. 100 112 i 19 (80-150) 81 i 15 (58-100)
Spicule length (arc) - - 502 i 33 (475-575)
Gubernaculum length - - 90 i 4.3 (85-99)
Tail length 1500 1324 i 230 (830-1600) 356 i 65 (275-450)
Table legend. n: number of specimens examined; L: total body length (head to tail tip); L': body length from head to anal/ cloacal aperture; a: total body length divided by maximum body diameter; b: total body length divided by pharyngeal length (the pharynx is defined as head end to the pharyngo-intestinal junction); c: total body length divided by tail length; c': tail length divided by body diameter at the anal/ cloacal aperture; V: position of vulva from anterior end expressed as percentage of body length; V': position of vulva from anterior end expressed as percentage of distance from head to anal aperture.
Fig. 1. Insulanema longispiculum gen. n. sp. n., female (A-F). A: total view; B: optical section through buccal cavity, lateral view; C: optical section through buccal cavity, ventral view; D: anterior end, lateral view; E: superficial structures of vulva region, lateral view; F: anal region, lateral view. (G-K) male. G: total view; H, I: optical section through buccal cavity, lateral view; J: posterior end, ventral view; K: spicules and gubernaculum. (Scale bar for A = 400 |um, B, C = 30 |um, D, H = 60 |m, E, F, K = 100 |m, G = 200 |m, I = 40 |m, J = 175 |m).
Fig. 2. SEM micrographs of Insulanema longispiculum gen. n. sp. n., female. A: anterior end, lateral view; B: head, lateral view; C; head, apical view; D: feather-like lamellae in oral opening, apical view. (Scale bars are in ^m).
feather-like projections and numerous small setaceous processes and posterior tubular part, surrounded by pharyngeal tissue. Pharynx narrow anteriorly, then widens abruptly after nerve ring level into subcylindrical muscular corpus. Isthmus long, narrow, leading to the spheroid basal bulb. Vulva median in position. Vagina long, muscular, follows anteriorly for some distance then turns posteriorly before joining uterus. Uterus runs
posteriorly to vulva level before dividing into two oviducts. Seminal receptacle present. Ovaries extend over the basal bulb level. Eggs large in size, with thin shells. Tail long, initially conoidal, with long subulate tip.
Male. Cephalic region with four cephalic papillae and amphids. Lateral alae absent. Body cuticle with fine annulation free of any formations. Buccal cavity divided into three parts: short anterior
part lined by thin layer of cuticle, longer middle part bearing small, anteriorly directed setaceous processes, and posterior part with one dorsal and two subventral feather-like cuticular lamellae projecting into lumen. Pharyngeal corpus long, subcylindrical, leading to a well defined isthmus and spheroid basal bulb. Spicules paired, isometric and isomorphic. Gubernaculum boat-shaped, with distinct dorsal hole in proximal body third. Caudal alae absent. Fourteen genital papillae present and distributed as: four precloacal pairs with posteriormost at cloacal level; three postcloacal pairs with two subventral pairs just under posterior cloacal lip and one pair subdorsal at the conical part of the tail. Area rugosa present in cloacal region. Tail initially convex conoidal with long distal tip.
Type and only species. Insulanema longispiculum gen. n., sp. n.
Distribution. Vietnam.
Etymology. The genus name derives from the Latin "Insula" (island) and refers the collection site of the host in Vietnam. It is to be regarded as neuter in gender.
Insulanema longispiculum gen. n., sp. n. (Figs 1-4)
Measurements: See Table 1.
Female. Cephalic extremity narrow, body widening gradually posterior to cephalic cap, reaching max. diam. near mid-body then slowly tapering posterior to vulva towards anus and terminating in long subulate tail. Cephalic cap comprise 4-5 cuticular rings. Oral aperture triangular in shape; each side of triangle underlined by two cuticular pieces making six in total. Four cephalic papillae nearly connected with four long cuticular ridges. Amphidial openings rounded, slightly prominent. Body cuticle with fine annulation free of any formations, except somatic papillae dispersed all over the body. Lateral alae absent. Buccal cavity long, consisting of anterior, thick-walled broad capsule ca 22-30 ^m in length with tree cuticular feather-like lamellae one dorsal and two subventral, and numerous small setaceous processes, and posterior, ca 55-75 ^m long, tubular part with thickened, striated walls. Pharynx narrowed anteriorly, widens abruptly into subcylindrical muscular corpus. Corpus 183 ± 14 (155-210) ^m long and 50-62 ^m wide passes posteriorly into narrow isthmus. Basal bulb ca 85-89 ^m in diam. with three cardia lobes projecting into a gut lumen. Nerve ring 83 ± 7.7 (70-95) ^m from anterior end, located at level of junction of buccal cavity and corpus. Excretory pore 180 ± 6.4 (165-
187) ^m from anterior extremity. Intestine long, narrowed anteriorly, with mixed bacterial content. Vulva in the form of transverse slit located 1360 ± 154 (1170-1700) ^m from anterior end and 1166 ± 185 (800-1400) ^m anterior to anus. Vagina long, muscular, follows anteriorly from vulva, forming a spiral loop and then continues anteriorly parallel to body wall for some distance then turns posteriorly before joining common uterus. Uterus runs posteriorly to vulva level before dividing into two oviducts. Each oviduct forms an inflation (seminal receptacle) containing sperm cells. Ovaries extend over the basal bulb level. Distal part of ovaries strongly curved. Eggs 177 ± 8.8 (160-195) x 80 ± 10 (60-95) ^m in size, with thin flexible shell. Tail initially conoidal, narrowing rapidly to form very long subulate tip.
Male. Cephalic extremity narrow, body widening posterior to cephalic cap, reaching max. diam. near mid-body, then narrowing towards anus and terminating with long subulate tail. Cephalic cap rounded, smooth with one dorsal and two subventral lips bearing four cephalic papillae. Oral aperture triangular in shape. Amphidial openings rounded, approximated to lip region. Body cuticle with fine annulation free of any formations. Somatic papillae dispersed all over the body. Lateral alae absent. Buccal cavity 70-77 ^m long, consisting of three parts: short anterior part (about 7-10 ^m long) lined by thin layer of cuticle, forming serrate margin inside mouth opening, longer middle part (about 2530 ^m long) bearing small, anteriorly directed setaceous processes, and posterior part with one dorsal and two subventral feather-like cuticular lamellae projecting into lumen. Pharyngeal corpus long, subcylindrical, with max. diam. of 35-40 ^m at middle then gradually narrowing to 17-22 ^m at union with isthmus. Basal bulb 67-85 ^m in diam. Nerve ring 176 ± 14 (150-187) from anterior extremity, surrounding pharyngeal corpus. Excretory pore 194 ± 15 (167-210) ^m from anterior end. Flexure of testis 315 ± 54 (250-400) ^m posterior to basal bulb. Spicules paired, ventrally arcuate, distally curved under acute angle, closely adpressed, equal in size and shape. Spicules heads elongate, slightly inclined ventrally. Terminal ends of spicules with knob-like tips. Cuticular punctation present in the middle part of the shaft. Gubernaculum boat-shaped, with distinct dorsal hole in proximal body third. Caudal alae absent. Fourteen genital papillae present: anteriormost pair ventro-sublateral, second pair subventral, third pair lateral and fourth pair situated near anterior precloacal lip. Three postcloacal pairs arranged as subventral pair on posterior cloacal lip, subventral pair just under posterior cloacal lip and
Fig. 3. SEM micrographs of Insulanema longispiculum gen. n. sp. n., male. A: anterior end, lateral view; B: anterior end, apical view; C: spicules distal tip; D: cloacal region, ventral view (same scale as E); E: cloacal region, lateral view. (Scale bars are in ^m).
subdorsal pair in the middle of conical part of the tail. Area rugosa present in cloacal region, consisting of two subventral fields of minute dots arranged in irregular transverse rows. Each field begins just before the level of the last precloacal pair of papillae and extends behind the second pair of postcloacal papillae. Cuticularised median duct discernible under light microscope, appears as mid-ventral papilla on anterior cloacal lip. Tail initially convex conoidal with long distal tip.
Type host and locality. Diplopod Apeuthes Attems, 1937 (Spirobolida: Trigoniulidae), collected in Kien Giang Province, Phu Quoc Island, Vietnam (10° 17' N, 104° 00' E).
Type material. Holotype female No. 1183 and paratype male No. 1182 of I. longispiculum gen. n., sp. n. deposited in the collection of the Centre of Parasitology, Institute of Ecology and Evolution, Russian Academy of Sciences. Paratype male UGMD 104248 and paratype female UGMD 104249
deposited in the nematological collection of the Museum voor Dierkunde, Ghent University, Belgium.
Etymology. The specific name reflects the peculiarity of the male's morphology.
Differential diagnosis. Insulanema
longispiculum gen. n., sp. n. is characterised by absence of any cuticular formations as spine rows or spine collars in cervical region in both sexes; presence of six cuticular pieces surrounding female oral opening; presence of smooth cephalic cap and area rugosa in the male; enormously long isomorphic and isometric spicules; boat-shaped gubernaculum with dorsal hole and 14 genital papillae with two postcloacal pairs subventral and one posteriormost pair subdorsal in position.
Lack of any cuticular formations in the cervical region in both males and females and length of spicules make the new genus unique between Indo-Asian and Australasian Carnoyidae, whose females and often males bear well developed cuticular armature at the anterior end. Cuticular formations in the cervical region are also absent in Brumpt-aemilius sensu stricto (Hunt, 1997), representative of African Carnoyidae.
Insulanema gen. n., could be easily distinguished from Pygecantha Hunt, 1997, Zyxispinifer Hunt, 2000 and Hekarella Hunt & Mohotti, 2000 by lack of lateral alae in both sexes; last postcloacal pair of genital papillae displaced anteriorly onto conical part of the tail vs last precloacal pair position on the tail spike; length of both spicules. In addition, Insulanema gen. n. has digonant female genital tract vs monogonant in Zyxispinifer and Hekarella.
The presence of six cuticularised plates surrounding the female oral aperture, narrow boat-shaped gubernaculum with dorsal hole, not reported for the Indian genus Raonema Kloss, 1965, but found in Australasian genera (Pygecantha, Cattiena Hunt & Spiridonov, 2001, Zyxispinifer and Hekarella) and development of area rugosa in males, reported until now only for Brumptaemilius-group, make Insulanema gen. n. similar with this African Carnoyidae group. A smooth cephalic cap present in Insulanema gen. n. was reported for Afrocarnoya / Martadamsonius / Waerebekeia -another group of African Carnoyidae and standing apart African genus Abirovulva Hunt, 2002. Thus, Insulanema gen. n. combines morphological features of all African Carnoyidae lineages and could be easily distinguished from all Indo-Asian, Australasian and African Carnoyidae by the position of the last postcloacal pair of genital papillae and enormously long spicules in the male.
Phylogenetic relationships. Insulanema longispiculum gen. n., sp. n. differs from other
Carnoyidae in 13-17 bp of SSU rDNA, and 44-62 bp of D2-D3 segment of LSU rDNA. When D1-D3 segments of LSU rDNA were used for comparison the differences were in the range of 51-71 bp.
Phylogenetic analysis of SSU and LSU rDNA was made with four different methods (MP, NJ, ML and BI). The topology of all trees obtained with these four methods was similar, thus, only BI trees are presented (Fig. 4.). Monophyletic status was demonstrated in all analyses of SSU rDNA for families Hethidae and Carnoyidae, but not for Ransomnematoidea (Carnoyidae + Hethidae) or Rhigonematomorpha (Rhigonematidae + Carnoyidae + Hethidae). Phylogenetic analysis of D2-D3 segment of LSU rDNA demonstrated monophyletic status of families Carnoyidae and Hethidae. The monophyly of the Ransomnematoidea (Carnoyidae + Hethidae) was also strongly supported (Fig. 4, B). The sister relationships of Ransomnematoidea with Rhigonematoidea (Rhigonematidae) were not observed.
DISCUSSION
Australasian representatives of Carnoyidae have several common features with African carnoyids of the Brumptaemilius-group: presence of six cuticular pieces around oral opening and narrow boat-shaped gubernaculum with dorsal hole. Between other Australasian Carnoyidae Insulanema longispiculum gen. n. sp. n. is even more similar to Brumptaemilius-group because of the absence of cuticular formations in the cervical region of both sexes and the presence of area rugosa in males. In the LSU rDNA tree Insulanema longispiculum gen. n. sp. n. is placed between African Brumptaemilius Dollfus, 1952 and Australasian Cattiena. LSU rDNA analysis also demonstrates that families Hethidae and Carnoyidae are sister groups with 100% support, which could be considered as an indication of the monophyletic status of Ransomnematoidea, although molecular data for Ransomnematidae are needed to prove it for the entire superfamily. SSU rDNA tree is based on much lower amount of nucleotide differences and in general does not resolve inner nodes of Ransomnematoidea phylogeny, but in this tree Insulanema longispiculum gen. n. sp. n. is also a separate clade between African and Australasian taxa.
In addition to the phylogenetic information, the LSU rDNA sequences provide additional taxonomic features for distinguishing species and taxa of Ransomnematoidea. The sequences of SSU rDNA are less informative as distinguishing characters at the level of genera and species, although they could still be
Fig. 4. Phylogenetic relationships among the studied species of superfamily Ransomnematoidea and related groups based on the partial sequences of SSU and LSU rDNA. Posterior probability values are given near the nodes. Consensus 50% majority rule trees; Scale - expected changes per site.
A: Bayesian inference of the SSU rDNA sequences; B: Bayesian inference of the LSU rDNA sequences.
used for the first identification of material because of the possibility of using universal primers and better representation of nematode SSU rDNA sequences in NCBI GenBank. Erroneous morphological identification of the sequenced material can significantly diminish the efficiency of sequencing-aided identification. Thus, the sequence deposited in NCBI GenBank as Thelastoma gueyei
(AM260939) from Senegal in fact represents that of the nematode belonging to the genus Brumptaemilius and most probably to the species Brumptaemilius justini (AF036589) as these two sequences differ in 4 bp. We also obtained partial sequence of SSU rDNA of Brumptaemilius justini collected in Tanzania (JX999732), which differs in 4 bp from the known sequence of this nematode
from Malawi (AF036589). Such differences apparently represent normal intraspecific variability of SSU rDNA for B. justini.
Our data on SSU and LSU rDNA supports the observation of Nadler et al. (2007) based on SSU rDNA only, which did not reveal the monophyletic status of Rhigonematomorpha (Rhigonematoidea + Ransomnematoidea). Additional sampling is needed to clarify the status of this taxon.
ACKNOWLEDGEMENT
The authors are grateful to Dr S.I. Golovatch for the identification of the diplopod and to the Laboratory of the Electron Microscopy of the Moscow State University for technical assistance. This research was supported by grants 11-04-00590a and 12-04-31402 mol_a from the Russian Foundation of Basic Researches.
REFERENCES
Blaxter, M.L., De Ley, P., Garey, J.R., Liu, L.X., SCHELDEMANN, P., VlERSTRAETE, A., VANFLETEREN, J.R., Mackey, L.Y., Dorris, M., Frisse, L.M., Vida, J.T. & Thomas, W.K. 1998. A molecular evolutionary framework for the phylum Nematoda. Nature 392: 71-75. De Ley, P. & Blaxter, M. 2002. Systematic position and phylogeny. In: Lee D.L. (Ed.). The Biology of Nematodes. London, Taylor and Francis, pp. 1-30. Floyd, R.M., Rogers, A.D., Lambshead, P.J.D & Smith, C.R. 2005. Nematode-specific PCR primers for the 18S small subunit rRNA gene. Molecular Ecology Notes 5: 611-612. HOLTERMAN, M., VAN DER WURFF, A., VAN DEN ELSEN, S., VAN MEGEN, H., Bongers, T., Holovachov, O., Bakker, J. & HELDER, J. 2006. Phylum-Wide Analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution towards crown clades. Molecular Biology and Evolution 23: 1792-1800. Hunt, D.J. 1997. A synopsis of the African genera of the Carnoyidae with proposal of Afrocarnoya fmbriifer gen.
n., sp. n. and Gilsonema gen. n. (Nematoda: Rhigonematida), parasites in diplopods from Ivory Coast. International Journal of Nematology 7: 201-212.
Malysheva, S.V. & Pham Van Luc. 2012. Cattiena fansipanis sp. n. (Nematoda: Rhigonematida: Carnoyidae) from a millipede (Myriapoda: Diplopoda: Spirobolida) in North Vietnam. Systematic Parasitology 81: 135-146.
Nadler, S.A., Carreno, R.A., MejIa - Madrid, H., Ullberg, J., Pagan, C., Houston, R. & Hugot, J.-P. 2007. Molecular phylogeny of clade III nematodes reveals multiple origins of tissue parasitism. Parasitology 134: 1421-1442.
Nunn, G.B. 1992. Nematode molecular evolution. Ph.D. Dissertation, University of Nottingham, Nottingham, UK, 187 pp.
Rambaut, A. & Drummond, A.J. 2007. Tracer v1.4. <http://beast.bio.ed.ac.uk/Tracer>.
Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Aaron, D., Hohna, S., Larget, B., Liu Liang, Suchard, M.A. & Huelsenbeck, J.P. 2012. MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice across a Large Model Space. Systematic Biology doi: 10.1093/sysbio/sys029.
Seinhorst, J.W. 1959. A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4: 67-69.
SONNENBERG, R., Nolte, A.W. & Tautz, D. 2007. An evaluation of LSU rDNA D1-D2 sequences for their use in species identification. Frontiers in Zoology doi: 10.1186/1742-9994-4-6.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731-2739.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24: 4876-4882.
S. V. Malysheva, Pham Van Luc, S. E. Spiridonov. Insulanema longispiculum gen. n., sp. n. новый вид Carnoyidae (Nematoda: Rhigonematomorpha) с острова Фу Куок во Вьетнаме.
Резюме. Дано описание нового рода и вида семейства Carnoyidae (Rhigonematomorpha) - Insulanema longispiculum gen. n., sp. n. из задней кишки диплоподы рода Apeuthes Attems, 1937 (Spirobolida: Trigoniulidae). Новый род характеризуется отсутствием шипиков или других кутикулярных структур на переднем конце тела самцов и самок, шестью кутикулярными пластинками вокруг ротового отверстия, гладкой головной капсулой, наличием area rugosa у самцов, очень длинными спикулами равной длины, ладьевидным рульком с отверстием, и 14-ю генитальными папиллами, из которых две постклоакальные пары расположены субвентрально, а самая задняя пара расположена в середине конической части хвостового конца. От всех других карнойид новый род отличается очень длинными спикулами и расположением последней пары генитальных папилл.
