CC BY
25Russian Journal of Nematology, 2012, 20 (1), 15 - 36
Two new species of Steinernema Travassos, 1927 from the humid forest of southern Cameroon
Françoise Ngo Kanga1,2, Phap Quang Trinh3, Lieven Waeyenberge4, Sergei E. Spiridonov5, Stefan Hauser1 and Maurice Moens2,4
'International Institute of Tropical Agriculture (ETA) Cameroon, P.O. Box: 2008 Messa, Yaoundé, Cameroon 2 Ghent University, Laboratory for Agrozoology, Coupure 555, 9000, Gent, Belgium 3 Institute of Ecology and Biological Resources, 18 Hoang Quoc Viet Rd., Hanoi, Vietnam
4 Institute for Agricultural and Fisheries Research, (ILVO), Crop Protection Department, Burg. Van Gansberghelaan 96,
9820 Merelbeke, Belgium, e-mail: [email protected]
5 Centre of Parasitology of A.N. Severtsov Institute of Ecology and Evolution of Russian Academy of Sciences, Leninskii
prospect, 33, Moscow, 119071, Russia
Accepted for publication 14 December 2011
Summary. Steinernema cameroonense sp. n. and S. nyetense sp. n. are two new species isolated from soil samples in Minkama, Obala and Nkontangan, Nyete, respectively. Steinernema cameroonense sp. n. is characterised by infective juvenile body length averaging 622 (490-693) ^m; pharynx length of 108 (100114) ^m; tail length of 76 (52-107) ^m; D and E values of 48 (42-56) and 75 (48-116), respectively. The lateral field pattern is 2, 4, 5, 6, 3, and 2. First generation males can be recognised by spicule length of 69 (51-85) ^m; GS = 64 (47-76); SW = 170 (131-200); D 64 (48-76). Steinernema nyetense sp. n. is characterised by infective juvenile body length averages 648 (565-708) ^m; pharynx length is 114 (104127) ^m; tail length is 82 (54-112) ^m; D and E values are 46 (37-50) and 66 (43-89), respectively. The lateral field pattern is 2, 4, 5, 4, 3, and 2. First generation males can be recognised by spicule length of 80 (67-98) ^m; GS = 66 (51-77); SW = 198 (124-283); D = 55 (40-70). As inferred from the sequence analysis of ITS rDNA S. cameroonense n. sp. and S. nyetense sp. n. represent separate group in the genus Steinernema distantly related to 'monticolum' and 'feltiae-kraussei-oregonense' clades. According to D2D3 LSU rDNA sequences the two new Steinernema species from Cameroon constitute a separate group related to inner nodes of Steinernema phylogeny.
Key words: molecular phylogeny, morphology, morphometrics, rDNA, SEM, Steinernema, taxonomy.
Entomopathogenic nematodes (EPN) of the genera Steinernema Travassos, 1927 and Heterorhabditis Poinar, 1976 are effective biological control agents for a wide variety of soil-inhabiting insects (Kaya & Gaugler, 1993). For a high efficiency as biological control agents against insect pests, EPN should be adapted to local environmental conditions (Bedding, 1990). However, for many regions where the use of EPN as biological control agents may be of importance, only limited information is available on their presence and taxonomy. Therefore, the isolation and the correct identification of entomopathogenic nematode species are critical to the success of their use as biopesticides for controlling insect pests (Qiu et al., 2004).
In a first survey of entomopathogenic nematodes conducted in various habitats of three agro-ecological zones of southern Cameroon in 2007 and 2008, eighteen isolates of Heterorhabditis baujardi and two isolates of Steinernema were collected (Kanga et al., 2012). These latter isolates did not correspond with any of the current species descriptions of the genus. Morphological characters, morphometrics, molecular data, and cross hybridisation tests support the contention that these nematodes are new species, herein described as Steinernema cameroonense sp. n. and S. nyetense sp. n.
MATERIALS AND METHODS
Nematode source. The isolates were obtained from soil samples by trapping with Galleria
mellonella (L.) larvae (Bedding & Akhurst, 1975). The samples were collected from a teak tree plantation at Obala in the centre region or from a forest habitat at Nyete in the south region, both of southern Cameroon. The newly extracted nematodes were collected alive in deionised water with 0.1% formalin and stored at 15°C in an incubator. They were maintained by recycling through G. mellonella (Dutky et al., 1964) every 2 months. Coordinates and altitudes of the sampling sites were registered using a GARMIN GPS eTrex Legend.
Morphological observations. Third-stage infective juveniles (IJ) were obtained by infecting G. mellonella larvae with 200 IJs ml-1 in a 9-cm diam Petri dish lined with filter paper. First and second generation females and males were obtained from the cadavers at 6-7 and 8-9 days after infection, respectively. Nematodes were prepared by heating and fixing in triethanolamine-formalin (TAF) fixative heated to 100°C and processed to anhydrous glycerin for slide mounting according to De Grisse (1969). For each isolate at least 20 females, males and infective juveniles were observed and measured. Observations were made using an Olympus BX51 light microscope equipped with an Olympus U-TV 0.5xC-3 digital camera. Specimen measurements were made using image software (CellD Soft Imaging System, Tokyo, Japan).
Scanning Electron Microscopy (SEM). For
scanning electron microscopy, both males and females of the first and second generation, as well as IJ, were fixed in TAF. The specimens were washed three times with 4% glutaraldehyde (buffered with cacodylate, pH 7.2) and cleaned again by ultrasonic treatment for 8 min. They were dehydrated in a graded ethanol series (30, 50, 75, 95% (45 min each) and 100% (overnight) at 25°C). They were critical point dried with liquid CO2, mounted on SEM stubs, coated with gold and smdied using a scanning electron microscope JEOL LSM-840. Spicules and gubernacula were prepared as described by Nguyen and Smart (1995).
Cross-hybridisation. The reproductive compatibility of the different species was tested using two techniques. In the first technique as in Stokwe et al. (2010), a Galleria larva was double folded and a sterile insulin needle was used to prick the skin close to a leg, with a drop of haemolymph being placed in a sterile Petri dish (35^10 mm). Drops of water were placed on the side of the dish to maintain high humidity levels. One IJ of S. cameroonense sp. n. and one of S. nyetense sp. n., S. feltiae or S. everestense were added to the dish. Similarly, one IJ of S. nyetense sp. n. and one of S.
feltiae or S. everestense were added to the dish. As a control, crosses between the IJ of the same species were conducted. The treatment was replicated 20 times. The Petri dishes were placed in closed plastic containers with tissue paper saturated with water to prevent the haemolymph from drying out, and stored in the dark at 25°C. The development of the inoculated IJ into adults and the reproduction of the nematodes were observed and recorded.
In the second technique, a drop of haemolymph was placed in a Petri dish and 20 IJ of S. cameroonense sp. n., S. nyetense sp. n., S. feltiae or S. everestense were transferred to that drop. The dishes were maintained as for the first technique. The treatment was replicated 10 times for each species and the nematodes were observed daily. When the sexes could be distinguished in the pre-adult stage, males and females of each species were placed separately in new drops. Then the pre-adult males and females of S. cameroonense sp. n. were transferred to the drop of blood containing the opposite sex of S. nyetense sp. n., S. feltiae or S. everestense. Similarly, the pre-adult males and females of S. nyetense sp. n. were transferred to the drop of blood containing the opposite sex of S. feltiae or S. everestense. Controls consisted of crosses of the same isolate. The nematodes were observed after 10 days for the presence of juveniles. Steinernema cameroonense n. sp. and S. nyetense sp. n. were primarily selected because they form a monophylectic group in the ITS and D2D3 phylogenetic trees. Steinernema feltiae and S. everestense were chosen because they are included in the 'feltiae-kraussei-oregonense' assemblage which is a sister group (Figs 9 & 10). Other closely related species were not available for the tests.
Molecular characterisation. For each of the isolates, DNA was extracted from three IJ. The IJ were cut in two or more parts in a 40 ^l drop of double distilled water on a glass slide and transferred to 8 ^l of worm lysis buffer (50 mM KCl, 10 mM Tris-Cl pH 8.3, 1.5 mM MgCfe, 1 mM DTT, 0.45% Tween 20) contained in an Eppendorf tube. Two ^l of protease K (600 ^g ml-) were added and nematodes lysates were frozen at - 70°C for a minimum of 15 min. After freezing, the lysis mix was incubated at 65°C for 1 h. After centrifugation (1 min; 16.000 g) of the tubes, 5 ^l of the DNA suspension were added to a PCR reaction mixture containing 5 ^l 10* PCR Buffer, 2 ^l MgCl2 (25 mM), 200 ^M ofeach dNTP, 1 ^M forward and reverse primer, 2 U Taq Polymerase (Invitrogen, Merelbeke, Belgium) and double distilled water to a final volume of 50 ^l. An rDNA fragment containing the internal transcribed spacer
regions ITS1 and ITS2, and the 5.8S rRNA gene was amplified by PCR using the forward primer TW81: 5'-GTT TCC GTA GGT GAA CCT GC-3' and the reverse primer AB28: 5'-ATATGCTTAAGTTCAGCGGGT-3' (Joyce et al., 1994). A second rDNA fragment containing the D2D3 expansion segment of the 28S rRNA gene was amplified using the forward primer D2A: 5'-ACA AGT ACC GTG AGG GAA AGT TG-3' and the reverse primer D3B: 5'-TCG GAA GGA ACC AGC TAC TA-3' (De Ley et al, 1999). The PCR reactions amplifying the ITS and D2D3 regions were done using the cycling profile described by Joyce et al. (1994) and De Ley et al. (1999), respectively. After DNA amplification, 5 ^l product were loaded on a 1.5% TAE buffered agarose gel (1 h, 100 V) for DNA checking. The gel was stained with ethidium bromide, visualised and photographed under UV. In case of a positive result, the remainder of the amplified product was purified in a 1% TAE buffered agarose gel (1 h, 100 V) following the instructions included in the Wizard SV Gel and PCR Clean-Up System kit (Promega Benelux, Leiden, The Netherlands). Subsequently, concentrations of the purified PCR products were measured using a UV spectrophotometer (Nanodrop ND-1000, Isogen Life Sciences, Sint-Pieters-Leeuw, Belgium). Purified PCR products were sequenced (Macrogen Inc, Seoul, Korea) in both directions to obtain overlapping sequences of the forward and reverse DNA strand. Finally, the sequences were visualised, edited and analysed using the software packages Chromas 2.00 (Technelysium, Helensvale, QLD, Australia) and BioEdit 7.0.4.1 (Hall, 1999), and deposited in GenBank (http://www.ncbi.nlm.nih.gov). The sequences were compared with sequences of Steinernema species available in Genbank (NCBI). Sequence alignments were made in Clustal X v. 1.61 (Thompson et al., 1997) using the default parameters. Phylogenetic trees inferred from maximum parsimony (MP) and neighbour joining (NJ) analyses were obtained using PAUP*4.0b10 (Swofford, 1998) and MEGA version 5 (Tamura et al., 2011). As the topology of trees was mainly the same only phylograms obtained with MEGA5 are presented in the paper. The GTR+G+I model was selected for two methods of analysis using models of evolution: maximum likelihood (ML) and Bayesian analysis (BI) with the use of ModelTest 5.0, when HKY+G model was selected in MEGA5, and used for ML analysis with this package. The programme MtGui by Pablo Nuin was used as an interface to prepare Modeltest results for ML analysis in PAUP* 4.0b10. The programme MrBayes 3.2 was used for Bayesian analysis
(Ronquist et al., 2011). In agreement with Nguyen et al. (2010), Caenorhabditis elegans Maupas, 1900 (EU131007) and Cervidellus alutus (Siddiqi, 1993) Shahina and De Ley, 1997 (AF331911) were applied as outgroup during calculation of the trees based on ITS sequences and on D2D3 sequences, respectively.
DESCRIPTIONS
Steinernema cameroonense sp. n. (Figs 1-3)
Measurements: See Table 1.
First generation male. Body curved ventrally posteriorly, broadly J-shaped when heat-killed. Cuticle with faint transverse striations, poorly seen under light microscope, but visible with SEM. Lateral fields present in mid-body with one narrow ridge. Phasmids not observed. Head rounded, gently tapering toward anterior end, slightly swollen. Lip region subtriangular with six unfused lips, each with one labial papilla at its tip, and four more protruding cephalic papillae at its base; former smaller than latter ones. Two amphidial pore-like apertures located behind labial lateral papillae. Perioral disc not observed. Cheilostom cuticle connected to a thick strongly cuticularised 1.9-2.5 ^m long ring of 1.1-2.6 ^m thick, forming the middle part of stoma walls. Additional thinner cuticular ring situated behind thicker one, being embedded into pharynx tissue. Excretory pore anterior to nerve ring, slightly posterior to middle of pharynx. Excretory channel strongly cuticularised. Pharynx corpus cylindrical, with visible metacorpal swelling and swelling around basal part of stoma. Nerve ring surrounding isthmus or at anterior slope of basal bulb. Cardia prominent and 5.8-11.7 ^m protruding into intestine lumen. Deirids not observed. Testis reflexed, comprising germinal zone with spermatocytes poorly distinguishable at the level of texture, growth zone with posteriorly filled with spermatids in testis middle, vacuolated vas deferens, and paired spicules. Spicules moderately curved (approx. 144°); with 15 ^m long and 12 ^m wide head (Fig. 2E, F). Velum 5 ^m wide, ending 2.5-12 ^m from rounded tip of spicules (Fig. 2F). Manubrium slightly longer than wide (spicule head length/width = 0.9-1.5), shaft very short. Gubernaculum boat-shaped in lateral view, anterior end rounded, needle-shaped cuneus present (Fig. 2G). Copulatory papillae totalling 22+1, comprising six pairs precloacal subventral papillae, single precloacal midventral papilla, one pair lateral, one pair adcloacal; 3 pairs postcloacal papillae including two subterminal pairs
Fig. 1. Steinernema cameroonense sp. n. First generation male. A: Head and pharynx region; B: Spicule; C: Spicule and gubernaculum; D: Gubernaculum; E: Close-up of tail region. First generation female. F: Head region; G: Vulva region; H: Tail region. I, J: Infective juvenile. I: Head and pharynx region; J: Tail region.
Table 1. Morphometries of Steinernema cameroonense n. sp. All measurements are in ^m and in the in the form: mean
± standard deviation (range).
Male Male (Paratypes) Female (Paratypes) Infective juveniles
Characters Holotype First generation Second generation First generation Second generation
n 1 20 20 20 20 20
Body length (L) 1373 1331±193 1018±119 3168±606 2236 ± 223 622 ± 61
(1019-1718) (813-1193) (2423-4607) (1790-2539) (490-694)
Body diameter (W) 103 90 ± 21 (65-124) 60 ± 7.6 (5184) 172±25 (110231) 102 ± 9.7 (83120) 30 ± 2.8 (2435)
Excretory pore (EP) 102 93 ± 11.5 (65109) 85 ± 10.8 (69115) 104 ± 21.0 (74140) 84 ± 11.6 (64106) 54 ± 4.9 (4564)
Nerve ring (NR) 121 114 ± 7.3 (104126) 100 ± 10.5 (86-127) 135 ± 9.8 (111152) 125 ± 11.6 (107148) 85 ± 8.3 (69100)
Pharynx length (ES) 148 146 ± 8.8 (127160) 125± 11.4 (114-158) 173 ± 11.9 (152-198) 155 ± 11.8 (131-179) 113 ± 5.6 (105-125)
Tail length (T ) 42 38 ± 2.9 (34-43) 34 ± 2.7 (2639) 38 ± 5.2 (30-52) 58 ± 14.9 (1981) 76 ± 18.7 (52-107)
Anal body diameter (ABD) 46 41 ± 3.3 (36-48) 38 ± 2.5 (3545) 44 ± 7.8 (30-60) 42 ± 5.4 (30-52) 15 ± 1.5 (1218.7)
Testis flexure 276 211 ± 33.9 150 ± 17.2
(156-282) (124-186)
Spicule length (SP) 75 69 ± 7.9 (51-85) 70 ± 5.3 (5682) - - -
Spicule width (SW) 20 15 ± 2.1 (12-20) 14 ± 10.3 (1018) - - -
Gubernaculum length (GL) 50 45 ± 6.4 (37-57) 39 ± 5.7 (2954) - - -
Manubrium length 16 15 ± 1.8 (12-19) 13 ± 1.5 (1117) - - -
Manubrium width 12 12 ± 1.2 (9-14) 14 ± 2.7 (920) - - -
Vulva (V) - - 1655±292 (1284-2350) 1186± 132 (831-1359) -
Hyaline region (H) - - - - 31± 4.8 (22-38)
a (L / W) 30 32 ± 4.1 (26-40) 27 ± 2.7 (2231) 18 ± 2.0 (15-22) 22 ± 1.8 (18-25) 21 ± 1.9 (1725)
b (L / ES) 9 9 ± 1.2 8 ± 1.0 18 ± 2.7 (14- 14 ± 1.4 (11- 6 ± 0.5 (5-
(7-12) (7-10) 24) 17) 6)
c (L / T) 33 35 ± 4.5 (28-43) 30 ± 3.6 (2438) 85 ± 17.4 (58112) 43 ± 20 (25-107) 9 ± 1.6 (6-12)
V% (V / L x 100) - 52 ± 2.0 (4857) 53 ± 2.5 (4658) -
D% = (EP/ES) x 100 68.72 64 ± 6.3 (48-76) 68 ± 6.5 (5782) 60 ± 10.6 (4379) 54 ± 7.5 (41-66) 48 ± 4.0 (4256)
E% = (EP/T) x 100 - - 75 ± 18.3 (48-116)
SW% = (GL/ ABD) x 100 162 170 ± 16.8 (131201) 182 ± 10.3 (159-199) - - -
GS% = (H / T) x 100 65.87 64 ± 6.8 (47-76) 56 ± 5.0 (4566) - - -
H% = (H / T) x 100 - - - - 50 ± 4.7 (4059)
and one subdorsal pair (Fig. 2B). Tail conoid with mucron usually present (Figs 1E & 2A). Phasmids inconspicuous.
Second generation male. Main morphological features similar to those of first generation males, except body length shorter and body diameter less.
First generation female. Body C-shaped when
killed by gentle heat. Cephalic end rounded, diam approx. 26 ^m. Lip region subtriangular with six unfused lips each with one labial papilla at its tip and four more protruding cephalic papillae at its base, former ones smaller than latter. Two amphidial apertures pore-like located behind labial lateral papillae (Fig. 2C). Stoma shallow with buccal
Fig. 2. Steinernema cameroonense sp. n. SEM and LM of first generation male. A: Close-up of tail end showing posterior genital papillae (6-11; s = midventral papilla); B: Posterior part showing genital papillae (1-11); E, F: Spicule; G: Gubernaculum. SEM of first generation female. C: En face view; D: Vulva region double flapped epiptygma surrounded by two lamella-like structures on both sides with surrounding annules; (Scale bars: A-B, D-G: 10 pm; C: 1 pm).
cavity 7-11 pm wide (Fig. 1F). Cuticle smooth under light microscope but with fine annulations visible with SEM, well discernible near vulva lips. Corpus of pharynx increasing in diameter from 16 pm at stoma to 30 pm before isthmus. Isthmus 20 pm wide. Basal bulb 37 pm wide, with transparent 11 pm long lobes of cardium. Excretory duct 2-6 pm wide. Excretory gland not observed. Lateral field with one ridge. Vulva a transverse slit with lips protruding up to 9-27 pm, unequal in size, with a long double flapped epiptygma surrounded by two lamella-like structures on both sides and with surrounding annules (Figs 1G & 2D). Tail conical and pointed with 3-9 pm long mucron on the tip (Fig. 1H). Visible post-anal swelling in fully mature females (Fig. 1H).
Second generation female. Similar to first generation female, but smaller and more slender (length = 2236 pm, maximum diam = 102 pm vs 3168 pm and 172 pm for first-generation female). Tail, tapering gradually to tail terminus, longer than anal body diameter.
Infective juveniles. Body elongate, almost straight or slightly curved, gradually tapering towards anterior and posterior ends. Exsheathed juvenile
with four cephalic papillae. Labial region smooth, continuous with body. Amphidial apertures cephalic papillae prominent (like setae) (Fig. 3B). Cuticle marked with prominent transverse striations. Excretory pore anterior to nerve ring. Hemizonid distinct, located anterior to base of basal bulb. Lateral field beginning anteriorly with one line at the fifth annule, two additional lines appearing at annules 15 to 16 (Fig. 3A). At fourth annule posterior to excretory pore, number of lateral lines increasing to form four ridges, with a fifth additional central line appearing at annules 11 to 12 to excretory pore. The central line divides into two, making a total of six ridges at mid-body. The two central ridges not as raised as others, weakly separated (Fig. 3C). In region of anus, ridges decreasing to three, ending with two towards tail end. With above arrangement, formula for lateral field is 2, 4, 5, 6, 3, and 2. Cardia present (Fig. 1I). Bacterial pouch located just posterior to cardia. Hemizonid distinct. Hemizonion not observed. Tail five times as long as anal body diameter and attenuate. Phasmid present near mid-tail, just ventral to lateral field, sometimes swollen (Figs 3D, E). Hyaline portion occupying 28 (19-35) pm of tail length.
Fig. 3. Steinernema cameroonense sp. n. SEM of infective juvenile. A: Anterior part showing the change in lateral field ridges, expansion from two to five ridges; B: Anterior end showing a cephalic papilla (cp); C: Lateral field in mid-body, six ridges; D: Ventral region of tail showing phasmids (arrow); E: Tail region showing the change in lateral field ridges, phasmid pore visible (arrow) (Scale bars: A, C-E: 10 pm; B: 1 pm).
Diagnosis and relationships. Steinernema cameroonense sp. n. is characterised by a combination of features of the various developmental stages of the nematode. The IJ have a body length averaging 622 (490-693) pm; distance from anterior end to excretory pore of 54 (45-64) pm, from anterior end to the base of pharynx of 108 (100-114) pm; tail length 76 (52-107) pm; D and E values of 48 (42-56) and 75 (48-116), respectively. The lateral field pattern of the new species is 2, 5, 6, 3, and 2. The first generation males of the new species can be recognised by a spicule length of 69 (51-85) pm; GS = 64 (47-76); SW = 170 (131-200); D = 64 (48-76). Females of S. cameroonense sp. n. are recognised by a conical and pointed tail with mucron on the tip and a protruding asymmetrical vulva with a long double flapped epiptygma surrounded two lamella-like structures at both sides and with surrounding annules.
The IJ of S. cameroonense sp. n. are closely related to those of S. monticolum Stock, Choo & Kaya, 1997, S. robustispiculum Phan, Subbotin, Waeyenberge & Moens, 2005, S. ashinuense Phan, Takemoto & Futai, 2006 and S. rarum (Doucet, 1986) Mamiya, 1988. The body length of the IJ of S. cameroonense sp. n. (622 (490-694) pm) is longer than that of S. rarum (510 (446-578) pm) but shorter than that of S. nyetense sp. n., S. monticolum, S. robustispiculum and S. ashiuense (648 (565-708), 706 (612-821), 712 (642-778) and 768 (720-800) pm, respectively) (Table 3). Its tail length (76 (52107) pm) is longer than that of S. rarum and S.
ashinuense (48 (42-52) and 71 (66-76), respectively), almost similar to that of S. robustispiculum and S. monticolum (75 (68-92) and (77 (71-95) pm, respectively), but shorter than that of S. nyetense sp. n. (82 (54-113) pm). Its E% (75 (48-116)) is lower than that of S. rarum (81 (67-91) pm), almost similar of that of S. robustispiculum, S. monticolum, S. ashinuense (75 (67-87), 76 (63-86) and 78 (70-85) pm, respectively), but higher than that of S. nyetense sp. n. (66 (44-89) pm). Steinernema cameroonense sp. n. also differs from S. nyetense sp. n. by a larger body diameter ((37-46) vs (25-35) pm) and a longer length of the pharynx of the IJ (124 (120-131) vs (108 (101-114) pm).
The D% of the first generation male of S. cameroonense sp. n. (64 (48-76) pm) is higher than that of S. rarum, S. ashiuense, S. monticolum, S. nyetense sp. n. and S. robustispiculum (50 (44-51), 50 (44-56), 55 (49-61), 55 (40-70) and 56 (50-63) pm, respectively). Manubrium shapes and length are different between S. cameroonense sp. n. and S. nyetense sp. n. The manubrium of S. cameroonense sp. n. is as long as wide with a very short shaft (calomus), whereas the manubrium of S. nyetense sp. n. is longer than wide with elongated shaft. Steinernema cameroonense sp. n. differs from S. nyetense sp. n. by the anterior part of the gubernaculum rounded with no projections from the neck toward the head (Fig. 1D).
The first generation female of S. cameroonense sp. n. differs from S. ashiuense and S. rarum by the presence of epiptygma, epiptygma being absent in S.
ashiuense and S. rarum. First generation females also differ from those of S. monticolum by having a protuberant vulva (Table 4). Steinernema cameroonense sp. n. differs from S. nyetense sp. n. by an asymmetrical vulva with a long double flapped epiptygma surrounded by two lamella-like structures on both sides and with surrounding annules, the vulva being symmetrical with a double flapped epitygma in S. nyetense sp. n. (Fig. 4C).
Type host and locality. Steinernema cameroonense sp. n. was collected from a soil sample at Obala, in the centre region of southern Cameroon. The nematode was collected in a teak plantation at Latitude 4°30'N, Longitude 12°01'E. The possible hosts in nature are unknown.
Etymology. The specific epithet is derived from Cameroon, the country of origin.
Type specimens. Holotype male, 5 male paratypes, 4 female paratypes and 5 infective juvenile paratypes of S. cameroonense sp. n. are deposited in the nematode collection of the Institute of Zoology, Ghent University, Ghent, Belgium.
Steinernema nyetense sp. n.
(Figs 4-6)
Measurements. See Table 2.
First generation male. Body curved ventrally posteriorly, broadly C-shaped when heat killed. Cuticle with faint transverse striations, poorly seen under light microscope, visible with SEM. Lateral field present at mid-body with one narrow ridge. Phasmids not observed. Head rounded, gently tapering toward anterior end, slightly swollen. Lip region rounded with six lips each with one labial papilla at the tip and four more protruding cephalic papillae at its base, former papillae smaller than latter ones. Two pore-like amphidial apertures located behind labial lateral papillae. Anterior end with a perioral disc around stoma. Cheilostom cuticle connected to a thick strongly cuticularised 1.9-2.8 pm long ring of 1.6-2.6 pm thick, forming the middle part of stoma walls. Additional thinner cuticular ring situated behind thicker one, being embedded in pharynx tissue. Excretory pore anterior to nerve ring, slightly posterior to middle of pharynx. Excretory channel strongly cuticularised. Pharynx corpus cylindrical, with visible metacorpal swelling. Nerve ring surrounding isthmus or on anterior slope of basal bulb. Cardia prominent and 6.6-10.8 pm protruding into intestine lumen. Deirids not observed. Intestinal lumen open throughout body length. Testis reflexed, comprising germinal zone with spermatocytes poorly distinguishable at the level of texture, growth zone with posteriorly
filled with spermatids in testis middle, vacuolated vas deferens, and paired spicules. Spicules moderately curved (approx. 144°); with 19 pm long and 12 pm wide head (Fig. 5E). Velum 6 pm wide, ending at 7-13 pm from rounded tip of spicules (Fig. 5F). Manubrium longer than wide (spicule head length/width = 1.4-2.0), shaft elongate. Gubernaculum boat-shaped in lateral view, anterior end rounded with two to three projections from the neck toward the head, needle-shaped cuneus present (Fig. 5G). Copulatory papillae totalling 22+1, comprising seven pairs precloacal subventral papillae, single precloacal midventral papilla, one pair adcloacal; three pairs postcloacal papillae including two subterminal and one subdorsal pairs. Tail conoid with mucron usually present. Phasmids inconspicuous.
Second generation male. Main morphological features similar to those of first generation males, except shorter body length and smaller body diameter.
First generation female. Body C-shaped when killed by gentle heat. Cephalic end rounded, diameter approx. 40 pm, continuous with body, with six labial papillae and four cephalic papillae (Fig. 5B). Cuticle smooth under light microscope, fine annulations visible with SEM. Stoma shallow with buccal cavity 11-20 pm wide. Cheilorhabdions prominent, well sclerotized, posterior part funnel-shaped (Fig. 4E). Pharynx procorpus cylindrical, muscular; metacorpus swollen, isthmus distinct, basal bulb enlarged, valvate. Nerve ring surrounding isthmus. Basal bulb 37 pm wide, with transparent 11 pm long lobes of cardium. Excretory pore anterior to nerve ring, well cuticularised; excretory gland not observed. Lateral field with one ridge. Vulva a transverse slit with lips protruding up to 9-25 pm, equal in size, with a long double flapped epiptygma (Fig. 4F). Tail conoid and pointed with 3-8 pm long mucron on the tip. Prominent post-anal swelling in fully mature females. Tail shorter than anal body diameter.
Second generation female. Similar to first generation female, but smaller. Body length half that of first generation (length = 1296 pm, maximum diameter = 113 pm vs 3615 pm and 219 pm). Tail, tapering gradually to tail terminus, longer than anal body diameter.
Infective juveniles. Body elongate, almost straight or slightly curved, gradually tapering towards anterior and posterior ends. Sheath (second-stage cuticle) present immediately after harvesting, but many IJ losing sheath during storage. Exsheathed juvenile with four cephalic papillae.
Fig. 4. Steinernema nyetense sp. n. A-D: First generation male. A: Head and pharynx region; B: Spicule; C: Spicule and gubernaculum; D: Gubernaculum; E: Close-up of tail region. E-G: First generation female. E: Head region; F: Vulva region with a double flapped epiptygma; G: Tail region. H, I: Infective juvenile. H: Head and pharynx region; I: Tail region.
Table 2. Morphometries of Steinernema nyetense n. sp. All measurements are in pm and in the in the form: mean ±
standard deviation (range).
Characters Male Male (Paratypes) Female (Paratypes) Infective juveniles
Holotype First generation Second generation First generation Second generation
n 1 20 20 20 20 20
Body length (L) 1887 1460 ± 264 678 ±149 3615± 856 1296 ± 249 648 ± 36
(1081-1887) (414-941) (2570-5257) (825-1758) (565-708)
Body diameter (W) 151 106 ± 27.4 (62- 51 ± 6.3 (37- 219 ± 41.7 (153- 113 ± 29.1 (42- 32 ± 2.5
159) 66) 310) 164) (25-37)
Excretory pore (EP) 103 88 ± 16.6 (57- 68 ± 12.2 (40- 124 ± 29.4 (72- 80 ± 12.5 (58- 52. ± 2.8
121) 85) 205) 103) (46-57)
Nerve ring (NR) 144 134 ± 22.4 (77- 116 ± 17.3 (84- 217 ± 25.7 (180- 134 ± 22.5 (87- 85 ± 9.6
197) 156) 284) 178) (72-102)
Pharynx length (ES) 177 158 ± 22.7 (98- 156 ± 23.9 251 ± 37.5 (207- 194 ± 22.5 114 ± 6.6
203) (103-196) 384) (141-221) (104-128)
Tail length (T ) 48 35 ± 6.0 35 ± 5.3 (25- 42 ± 5.9 55 ± 9.0 (33- 82 ± 19.1
(26-48) 49) (33-56) 77) (54-113)
Anal body diameter (ABD) 34 42 ± 6.9 (30-63) 37 ± 4.0 (31- 63 ± 6.3 (54-79) 41 ± 6.0 (29- 16 ± 1.5
44) 54) (14-20)
Testis flexure 240 176 ± 32.9 (128- 124 ± 25.4 (65- - - -
243) 165)
Spicule length (SP) 89 80 ± 7.6 57 ± 9.4 (36- - - -
(67-98) 75)
Spicule width (SW) 10 12 ± 1.6 10 ± 2.1 (6-14) - - -
(9-16)
Gubernaculum length (GL) 57 53 ± 6.4 (40-62) 34 ± 7.9 (24- - - -
52)
Manubrium length 22 19 ± 2.0 (16-23) 12 ± 2.4 (7-16) - - -
Manubrium width 12 12 ± 1.1 (10- 9 ± 2.1 - - -
14) (5-13)
Vulva (V) - - - 1919 ± 406 714± 148 -
(1274-2354) (472-973)
Hyaline region (H) - - - - - 28 ± 3.9
(20-35)
a (L / W) 55 36 ± 7.8 (22- 18 ± 4.3 (10- 16 ± 2.6 (11-22) 12 ± 2.6 (8- 21 ± 1.5
55) 28) 20) (19-26)
b (L / ES) 11 9 ± 1.1 4 ± 0.7 14 ± 2.6 (11-19) 7 ± 0.9 6 ± 0.4 (5-
(8-11) (3-6) (5-8) 6)
c (L / T) 39 42 ± 6.0 (28-56) 20 ± 4.3 (14- 87 ± 26.5 (53- 24 ± 7.1 (14- 8 ± 1.6 (6-
31) 150) 44) 11)
V% (V / L x 100) - - м 53 ± 3 55 ± 5 (40- -
(47-58) 63)
D% = (EP/ES) x 100 58.07 55 ± 6.8 (40-70) 43 ± 5 (35-52) 49 ± 9.0 (30-62) 42 ± 8.0 (28- 46 ± 3.1
55) (37-50)
E% = (EP/T) x 100 - - - - - 66 ± 14.2
(44-89)
SW% = (GL/ SP) x 100 262 199 ± 41.2 (125- 154 ± 26.3 - - -
283) (115-211)
GS% = (H / T) x 100 64.22 66 ± 7.3 (51-77) 61 ± 9.1 (43- - - -
79)
H% = (H / T) x 100 - - - - - 50 ± 5.5
(35-57)
Labial region smooth, continuous with body. Amphidial apertures prominent and sometimes swollen (Fig. 6B). Cuticle marked with prominent transverse striations. Excretory pore anterior to nerve ring. Hemizonid distinct, located anterior to base of basal bulb. Lateral field beginning anteriorly with one line at the sixth annule, two additional lines appearing at annules 15 or 16. At fourth annule
posterior to excretory pore, number of lateral lines increasing to form four ridges, a fifth additional line appearing at 7 or 8 annules posterior to excretory pore. This fifth ridge rises in the middle of two marginal ones making a total of five equally developed ridges at mid-body. Near region of anus, ridges decreasing to three, ending with two towards tail end. With the above arrangement, the formula for
Fig. 5. Steinernema nyetense sp. n. SEM and LM of a first generation male. A: Close-up of tail end showing posterior genital papillae (5-11; s = midventral papilla); D: Posterior part showing genital papillae (1-11); E, F: Spicule; G: Gubernaculum. SEM of a first generation female. B: En face of view; C: Vulva region; H: Tail region (Scale bars: A-H: 10 pm).
lateral field is 2, 4, 5, 4, 3, 2 (Fig. 3C). Pharynx long, narrow; isthmus distinct, surrounded by nerve ring; basal bulb elongate with visible valve. Cardia present. Bacterial pouch located just posterior to cardia. Hemizonid distinct. Hemizonion not observed. Tail five times as long as anal body diameter and attenuate. Phasmid present near mid-tail, just ventral to lateral field (Fig. 3D). Hyaline portion occupying 36 (20-54) pm of tail length.
Diagnosis and relationships. Steinernema nyetense sp. n. is characterised by combination of features of the various developmental stages of the nematode. The IJ are characterised by an average body length of 648 (565-708) pm, the distance from anterior end to excretory pore of 52 (46-57) pm, distance from anterior end to the base of pharynx of 114 (104-127) pm, a tail length of 82 (54-112) pm, and D and E values of 46 (37-50) and 66 (43-89), respectively. The lateral field pattern of the species is 2, 4, 5, 4, 3, and 2. The first generation males of the new species have a spicule length of 80 (67-98) pm, GS = 66 (51-77), SW = 198 (124-283), and D = 55 (40-70). Females of S. nyetense n. sp. are recognised by a conoid and pointed tail with a mucron on the tip and a protruding and symmetrical
vulva with a double flapped epiptygma.
Steinernema nyetense sp. n. is closely related to S. cameroonense sp. n., S. monticolum, S. robustispiculum, S. ashiuense and S. rarum. The body length of the IJ of S. nyetense sp. n. (648 (565708) pm) is longer than that of S. rarum and S. cameroonense sp. n. (510 (446-578) and 622 (490694) pm, respectively), but shorter than that of S. monticolum, S. robustispiculum and S. ashiuense ((706 (612-821), 712 (642-778), and 768 (720-800) pm, respectively)). The tail length of the IJ (82 (54113) pm) is the highest among these species; its E% of 66 (44-89) is the lowest among these species (Table 3). Steinernema nyetense sp. n. also differs from S. cameroonense sp. n. by the body diameter of the IJ ((25-35) vs (37-46) pm) and the length of the pharynx (108 (101-114) vs 124 (120-131) pm).
The D% of the first generation male of S. nyetense sp. n. (55 (40-70) pm) is higher than that of S. rarum and S. ashiuense, (50 (44-51) and 50 (4456) pm, respectively, almost similar to that of S. monticolum and S. robustispiculum (55 (49-61) and 56 (50-63) pm, respectively), but shorter than that of S. cameroonense sp. n. (64 (48-76) pm; Table 3). The spicule length of S. nyetense sp. n. (80 (67-98) pm)
Fig. 6. Steinernema nyetense sp. n. SEM of infective juvenile. A: Anterior part showing the change in lateral field ridges, expansion from two to five ridges; B: Anterior end; C: Lateral field in mid-body, five ridges; D: Posterior part showing the change in lateral field ridges, with a phasmid visible (arrow); E: Tail region showing lateral field with two ridges and a phasmid (arrow). (Scale bars: A, C-E: 10 pm; B: 1 pm).
is higher than that of S. rarum, S. robustispiculum, S. ashiuense, S. cameroonense sp. n. and S. monticolum (47 (42-52), 58 (51-65), 59 (50-65), 69 (51-85) and 70 (61-80) pm, respectively). The manubrium of S. cameroonense sp. n. is as long as wide with a very short shaft (calomus) whereas for S. nyetense sp. n., the manubrium is longer than wide with an elongated shaft. Steinernema nyetense sp. n. differs from S. cameroonense sp. n. by the anterior part of the gubernaculum rounded with two to three projections from the neck toward the head, the gubernaculum neck being without projections in S. cameroonense sp. n. (Fig. 4C).
The first generation females of S. nyetense sp. n. differ from that of S. ashiuense and S. rarum by the absence of an epiptygma. They also differ from S. monticolum by having a protuberant vulva (Table 4). Steinernema nyetense sp. n. differs from S. cameroonense sp. n. by an asymmetrical vulva with a symmetrical vulva with a double flapped epiptygma, the vulva being asymmetrical vulva with a long double flapped epitygma surrounded by two lamella-like structures on both sides and with surrounding annules in S. cameroonense sp. n. (Fig. 4F).
Type host and locality. Steinernema nyetense sp. n. was collected from a soil sample taken at Nyete, in the south region of southern Cameroon, in a forest at latitude 2°49'N, longitude 10°07'E. The possible hosts in nature are unknown.
Etymology. The specific epithet is derived from Nyete, the type locality.
Type specimens. Holotype male, 5 male paratypes, 4 female paratypes and 5 infective juvenile paratypes of S. nyetense sp. n. are deposited in the nematode collection of the Institute of Zoology, Ghent University, Ghent, Belgium.
Molecular characterisation. Steinernema cameroonense sp. n. and S. nyetense sp. n. are both characterised genetically by the ITS and D2D3 regions. The ITS sequence length of S. cameroonense sp. n. is 769 bp with a composition A = 0.22756, C = 0.19375, G = 0.25357, T = 0.32509. The D2D3 region is 593 bp long with composition A = 0.23946, C = 0.19392, G = 0.32377, T = 0.24283. The ITS sequence length of S. nyetense sp. n. is 735 bp with composition A = 0.22448, C = 0.19591, G = 0.24217, T = 0.33741. Its D2D3 region is 592 bp long with composition A = 0.24324, C = 0.19256, G = 0.32432, T = 0.23946. The pairwise distance comparison of the ITS and D2D3 regions is presented in Table 5 and 6.
For ITS regions, maximum parsimony analysis shows that the alignment resulted in 920 characters of which 161 are constant, 148 are parsimony-uninformative and 611 parsimony-informative. Parsimony and distance based tree-building approaches produce almost identical trees. The phylogenetic relationships between 54 species of Steinernema are presented in Figs 7 and 8 (MP analysis
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C. elegans (EU 131007) S. intermedium (AY171290) S. affine (AY171298) S.beddingi (AY603397) S. sichuanense (DQ884965) S. neocurtillae (AF122018) S. scapterisci (AY230183) S. backanense{AY487918) S. carpocapsae (AY17128) S. sasonense (AY487919) S. eapokense (AY487921) S. siamkayai (AF331917) S. cumgarense (AY487920) S. tom(AY171280) S. rarum (DQ221116) S. khoisanae (DQ314287) S. boemarei(Fjl52414) S. arenarium (AY230160) S.glaseri( AY171288) S. cubanum (AY230166) S. brazilense (FJ410325) S. diaprepesi (AF122021) S. australe (FJ235125) S. guangdongense (AY170341) S. longicaudum (AY230177) S. aciari (AY787660) S. karii (AY230173) S. sangi (AY355441) S. texanum (EF152568) S. cholashanense (EF431959) S. xueshanense (FJ666052) S. oregonense (AF 122019) S. akhursti (DQ375757) S. kraussei (AY171264) S. feltiae (DQ310469) S. citrae (EU740970) S. ichnusae (EU421129) S. litorale (AB243441) S. hebeiense (Dql05794)
S. kushidai (AB243440) S. everestense (HM000103)
S. schliemanni (HM778112)
S. robustispiculum (AY355442) S. ashiuense (DQ3 54694)
S. monticolum (AF 122017) S. monticolum (AB698757) S.nyetense sp.n. S.cameroonense sp.n. S. yirgalemense (ÀY748450) S. thermophilum (EF431958) S.*«(AY230158) S. pakistanense ( AY23 0181) S. riobrave (DQ835613) S. ceratophorum (AY230165) S. bicornutum (AY171279)
Fig. 7. Phylogenetic relationships of Steinernema cameroonense sp. n and Steinernema nyetense sp. n. with 50 Steinernema spp. based on ITS-rDNA sequences as inferred from MP and NJ analyses. Caenorhabditis elegans (EU131007) was used as outgroup. Support values are presented near the nodes in the form: bootstrap in MP/NJ/ME analyses.
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S. schliemanni (HM778112) S. monticolum (AF122017) S. robustispiculum (AY355442) S. ashiuense (DQ354694)
S. sangi (AY355441) S. texanum (EF152568) S. oregonense (AF122019) S. akhursti (DQ375757) S. cholashanense (EF431959) S. xueshanense (FJ666052) S. Araussei (AY171264) S. citrae (EU740970) S. hebeiense (DQ105794) S. feltiae (DQ310469) S. ichnusae (EU421129) S. litorale (AB243441) S. kushidai (AB243440) S. everestense (HM000103) S. nyetense sp.n. S.cameroonense sp.n. S. ceratophorum (AY230165) S. bicornutum (AY171279) S. riobrave (DQ835613) S. pakistanense (AY230181) S. yirgalemense (AY748450) S. thermophilum (EF431958) S. abbasi (AY230\58) S.rarum (DQ221116) S. karii (AY230173) S. aciari (AY787660) S. khoisanae (DQ314287) S. guangdongense (AY170341) S. longicaudum (AY230177) S. diaprepesi (AF 122021) S. brazilense (FJ410325) S. australe (FJ235125) S. boemarei (FJ152414) S. arenarium (AY230160) ,S'. glaseri (AY171288) S. cubanum (AY230166) S. intermedium (AY 171290) S. affine (AY171298) S. beddingi (AY603397) S. sichuanense (DQ884965) S. neocurtillae (AF 122018) S. scapterisci (AY230183) S. backanense (AY487918) S. carpocapsae (AY17128) S. sasonense (AY487919) S. eapokense (AY487921) S. siamkayai (AF331917) S. cumgarense (AY487920) S. tow (AY171280)
Fig. 8. Phylogenetic relationships of Steinernema cameroonense sp. n and Steinernema nyetense sp. n. with 50 Steinernema spp. based on ITS-rDNA sequences as inferred from BI and ML analyses. Bayesian analysis parameters (2000000 generations, burn-in 20000) Caenorhabditis elegans (EU131007) was used as outgroup. Support values are presented near the nodes in the form: posterior probability in BI analysis/bootstrap in ML analysis.
Table 3. Comparative morphometries of IJ and first generation males of Steinernema cameroonense sp. n. and Steinernema nyetense sp. n. and closely related species. All
measurements are in ¡im and in the in the form: mean (range). Data for new species in bold.
Species* Juveniles Male I References
L W EP T D% E% SP GL D% SW% GS%
S. ashiuense 768(720-800) 30(28-33) 55(51-59) 71(66-76) 46(43-50) 78(70-85) 59(50-65) 37(25-43) 50(44-56) 149(128-167) 63(43-73) Phan et al., 2006
S. robustispiculum 712(642-778) 30(28-33) 56(50-68) 75(68-92) 46(43-59) 75(67-87) 58(51-65) 41(36-44) 56(50-63) 129(120-150) 70(64-79) Phan et al., 2005
S. monticolum 706(612-821) 28(26-35) 58(54-62) 77(71-95) 47(44-50) 76(63-86) 70(61-80) 45(35-54) 55(49-61) 140(111-150) 60(50-70) Stock et al., 1997
S. nyetense sp. n. 648(565-708) 30(25-35) 52(46-57) 82(54-113) 76(52-107) 48(42-52) 46(37-50) 66(44-89) 80(67-98) 53(40-62) 55(40-70) 199(125-283) 66(51-77) Present study
S. cameroonense sp. n. S. rarum 622(490-694) 510(446-578) 32(37-46) 32(25-37) 54(45-64) 37(33-40) 48(42-56) 41(36-43) 75(48-116) 81(67-91) 69(51-85) 47(42-52) 45(37-57) 34(23-38) 64(48-76) 50(44-51) 170(131-201) 94(91-105) 64(47-76) 71(55-73) Present study Doueet et al., 2003
Table 4. Comparative morphology of Steinernema cameroonense sp. n. and Steinernema nyetense sp. n and closely related species.
Male 2nd
Species* IJ Male 1st generation generation Female 1st generation
Lateral line Spicule Gubernaculum papillae Mucron Mucron Vulva Tail swelling
S. ashiuense 5 equal ridges in midbody slightly yellowish, velum large, not covering spicule tip boat-shaped, cuneus long, needle-shaped, wing of corpus expanding laterally 20+1 P P protruding, no epiptygma tail dome-shaped, with terminal peg na
S. robustispiculum 8 ridges in midbody yellow-brown, prominent rostrum, velum large boat-shaped, cuneus long 22+1 P na protruding, with epiptygma tail dome-shaped, with terminal peg P
S. monticolum 8 unequal ridges in midbody brown-orange, velum present, pointed arcuate, large, posterior end forked 21/23 ± 1 P P not protruding tail short, blunt, with mucron A
S. nyetense sp. n. 2, 4, 5, 4, 3, 2 yellow-brown, velum large, boat-shaped in lateral view, needle-shaped 22+1 P P protruding, with epiptygma tail conoid and pointed tail with a short mucron on the tip P
S. cameroonense sp. n. 2, 4, 5, 6, 3, 2 yellow-brown, velum present, short calomus boat-shaped in lateral view, needle-shaped 22+1 P P protruding, with epiptygma tail conical pointed P
S. rarum 2, 8, 10, 6, 2 velum thin, spicule tip usually blunt. cuneus rod-like 21/23 +1 P P protruding, no epiptygma tail bluntly conical to dome-shaped with a short spine on the tip P
P: present, A: absent, na: not available * Descriptions from references cited in Table 3.
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Cervidellus alutus AF331911 S. intermedium AF331909 S.affine AF331899 S.sichuanense DQ884966 S.rarum AF331905 S.kushidai AF331897 S.everestense HM000104 S.cholashanense EF520284 S.kraussei AF331896 S.oregonense AF331891 S.texanum EF152569 S.xueshanense FJ666053 S.silvaticum DQ399663 S ichnusaeElJ421130. S.feltiae AF331906 S.puntauvense EF 187018 S.monticolum GU395647 S.schliemanni HM778113 S.nyetense sp.n. S.cameroonense sp.n S.karii AF331902 S.puertoricense AF331903 S.diaprepesi DQ849320 S.australe FJ235126 S.brazilense FJ410326 S.boemarei FJ152415 S.arenarium AF331892 S.cubanum AF331889 S.glaseri AF331908 S.khoisanae DQ314289 S.aciari AY787661 S.hermaphroditum AY598358 S.longicaudum AF331901 S.scarabaei AY172023 S.riobravis AF331893 S.yirgalemense AY748451 S.Asi AF331890 S.ceratophorum AF331888 S. bicornutum AF331904 S.websteri AYU1162 S.anatoliense GU569043 S. carpocapsae AF331900 S.siamkayai AF331907 S.scapterisci AF331898
Fig. 9. Phylogenetic relationships of Steinernema cameroonense n. sp. and Steinernema nyetense n. sp. with 45 Steinernema spp. based on D2D3 region sequences of the 28S rRNA gene as inferred from maximum parsimony and minimum evolution analyses. Cervidellus alutus (AF331911) was used as outgroup. Support values are presented near the nodes in the form: bootstrap in MP analysis/ bootstrap in ME analysis.
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Fig. 10. Phylogenetic relationships of Steinernema cameroonense n. sp. and Steinernema nyetense n. sp. with 45 Steinernema spp. based on D2D3 region sequences of the 28S rRNA gene as inferred from maximum parsimony and minimum evolution analyses. Cervidellus alutus (AF331911) was used as outgroup. Support values are presented near the nodes in the form: posterior probability in BI analysis/bootstrap in ML analysis.
of ITS region: tree length = 3997, CI = 0.4003, RI = 0.6770, RC = 0.2710, HI = 0.6277). In the MP, NJ and ME of ITS regions consensus trees, the two new species cluster in a subgroup standing apart, and related to the 'feltiae-kraussei-oregonense' (Spiridonov et al,. 2004) and 'monticolum' clade sensu Spiridonov et al. (2010). Bootstrap support over 70%
for this monophyletic assemblage of new Cameroonian species, 'monticolum' clade and the species of the feltiae-kraussei-oregonense-group was observed only in methods based on distance as optimality criterion: 70% and 93% for NJ and ME, respectively.
The maximum parsimony analysis of the D2D3 domain shows that the alignment resulted in 706
K>
Table 5. Pairwise distances of the ITS regions of Steinernema species in the feltiae-kraussei-oregonense-group. Below diagonal: total character differences, above diagonal: mean
character differences. Data for new species in bold.
►Tj
of o
w g
OQ P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1 S. cameroonense sp. n. - 0.051 0.212 0.202 0.218 0.205 0.243 0.253 0.254 0.246 0.253 0.255 0.285 0.254 0.269 0.264 0.274 0.287
2 S. nyetense sp. n. 32 - 0.212 0.217 0.231 0.216 0.243 0.252 0.250 0.239 0.252 0.249 0.2814 0.241 0.250 0.260 0.271 0.267
3 S. monticolum 114 122 - 0.039 0.158 0.053 0.194 0.196 0.200 0.192 0.210 0.196 0.198 0.208 0.202 0.202 0.227 0.211
4 S. robustispiculum 116 126 23 - 0.184 0.015 0.211 0.214 0.209 0.207 0.228 0.210 0.214 0.216 0.204 0.222 0.240 0.229
5 S. schliemanni 125 127 87 102 - 0.189 0.194 0.193 0.205 0.187 0.193 0.196 0.190 0.201 0.196 0.196 0.215 0.209
6 S. ashiuense 117 124 31 15 104 - 0.207 0.210 0.206 0.201 0.224 0.204 0.214 0.216 0.206 0.220 0.237 0.226
7 S. citrae 139 139 109 119 105 116 - 0.055 0.100 0.083 0.064 0.075 0.071 0.126 0.136 0.09 0.108 0.07
8 S. litorale 144 143 109 120 104 117 32 - 0.096 0.075 0.039 0.073 0.066 0.115 0.137 0.096 0.096 0.048
9 S. texanum 143 141 110 116 110 114 58 121 - 0.083 0.091 0.072 0.066 0.086 0.112 0.119 0.149 0.10
10 S. xueshanense 140 136 107 116 100 112 47 43 47 - 0.071 0.017 0.048 0.099 0.113 0.068 0.124 0.088
11 S. ichnusae 146 144 118 129 105 126 38 23 53 41 - 0.068 0.058 0.112 0.136 0.092 0.109 0.049
12 S. cholashanense 148 144 111 121 107 116 44 43 42 10 45 - 0.039 0.085 0.102 0.057 0.129 0.084
13 S. oregonense 146 140 111 121 102 120 41 38 38 28 34 23 - 0086 0.104 0.056 0.109 0.073
14 S. sangi 146 137 116 121 107 120 73 66 49 57 65 50 50 - 0.114 0.097 0.162 0.127
15 S. everestense 158 145 114 116 106 116 79 79 64 65 79 60 60 67 - 0.119 0.165 0.159
16 S. kraussei 154 151 115 127 107 125 53 56 47 40 54 34 33 57 70 - 0.137 0.098
17 S. hebeiense 155 154 126 134 116 132 63 56 86 71 64 75 63 93 95 80 - 0.105
18 S. feltiae 168 156 119 130 114 128 41 28 58 51 29 50 43 74 94 58 61 -
Table 6. Pairwise distances of the D2D3 regions of Steinernema species of arenarium-glaseri-karii-longicaudum-group and two new Cameroonian species. Below diagonal: total character differences, above diagonal: mean character differences. Data for new species in bold.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1 S. cameroonense sp. n. - 0.008 0.062 0.062 0.066 0.068 0.075 0.070 0.081 0.073 0.077 0.077 0.083 0.077 0.089 0.085 0.107
2 S. nyetense sp. n. 4 - 0.072 0.072 0.071 0.066 0.069 0.064 0.075 0.069 0.073 0.073 0.077 0.073 0.092 0.088 0.104
3 S. scarabaei 33 28 - 0.0 0.044 0.059 0.064 0.044 0.061 0.051 0.057 0.061 0.057 0.059 0.072 0.072 0.070
4 S. hermaphroditiim 33 28 0 - 0.044 0.059 0.064 0.044 0.061 0.051 0.057 0.061 0.057 0.059 0.072 0.072 0.070
5 S. longicaudatum 32 30 20 20 - 0.044 0.048 0.048 0.061 0.055 0.057 0.062 0.064 0.052 0.068 0.068 0.079
6 S. apuliae 31 30 27 27 20 - 0.033 0.042 0.051 0.044 0.048 0.048 0.048 0.035 0.051 0.052 0.091
7 S. boemarei 34 31 29 29 22 15 - 0.046 0.053 0.051 0.051 0.044 0.048 0.015 0.046 0.041 0.084
8 S.khoisanae 32 29 20 20 22 21 21 - 0.035 0.037 0.039 0.046 0.046 0.046 0.051 0.051 0.068
9 S. aciari 37 34 28 28 28 26 26 16 - 0.046 0.053 0.064 0.059 0.052 0.068 0.068 0.081
10 S. puertoricense 33 31 23 23 25 20 23 17 21 - 0.019 0.022 0.015 0.039 0.048 0.051 0.072
11 S. diaprepesi 35 33 26 26 26 22 23 17 24 9 - 0.033 0.031 0.044 0.051 0.051 0.079
12 S. australe 35 33 28 28 28 22 20 10 29 10 15 - 0.019 0.037 0.052 0.051 0.086
13 S. braziliense 38 35 26 26 29 22 22 21 27 10 14 9 - 0.039 0.054 0.052 0.079
14 S. arenarium 35 33 271 27 24 16 7 21 24 18 20 17 18 - 0.041 0.037 0.084
15 S. cubanum 41 42 33 33 31 23 21 23 31 22 23 24 25 19 - 0.051 0.079
16 S. glaseri 39 40 33 33 31 24 19 23 29 23 23 23 24 17 2 - 0.079
17 S. karii 49 47 32 32 36 41 38 31 37 33 36 39 26 38 36 36 -
O
o
characters of which 390 are constant, 65 are parsimony-uninformative and 251 characters are parsimony-informative. Parsimony and distance based tree-building approaches showed almost similar topology. The phylogenetic relationships between 47 species of Steinernema are presented in Figs 9 and 10 (for MP: tree length = 831, CI = 0.5475, RI = 0.7926, RC = 0.4340, HI = 0. 4525). In the MP and the NJ consensus trees, the two new species grouped in a subgroup standing apart, are related 'arenarium-glaseri-karii-longicaudum' group sensu Spiridonov et al. (2004). Bootstrap support in the monophyletic assemblage with the two species and the species of the arenarium-glaseri-karii-longicaudum-group is low in all analyses with only exception of NJ tree (97%). Affiliation of the two species as a sub-clade is supported by a bootstrap value of 100%, in all trees.
The nucleotide differences in ITS sequences between S. cameroonense sp. n. and S. nyetense sp. n. with the species of the 'monticolum' were lower than differences with species of feltiae-kraussei-oregonense' clade (Table 5). The nucleotide differences between S. cameroonense sp. n. and S. nyetense sp. n. were much lower than differences with other Steinernema species. The nucleotide differences of the D2D3 sequences between S. cameroonense sp. n. and S. nyetense sp. n. and other steinernematid species were on the level of 6-10% of this domain (Table 6).
Cross-hybridisation tests. In the cross hybridisation experiments, no progeny was observed between males and females of S. cameroonense sp. n. and S. feltiae or S. everestense. Equally, no progeny was observed between males and females of S. nyetense sp. n. and S. cameroonense sp. n. or S. feltiae or S. everestense. In the controls, offspring was observed in all self-crossed species. No progeny were observed in the single female control Petri dishes.
DISCUSSION
Morphological and molecular data provide evidence that S. cameroonense sp. n. and S. nyetense sp. n. are distinct from all other described Steinernema species, indicating that they are new species of steinernematids. Based on the ITS rDNA trees inferred from MP, NJ and ME analyses, S. cameroonense sp. n. and S. nyetense sp. n. are related to the species of 'monticolum' and feltiae-kraussei-oregonense' groups. When available D2D3 sequences were used for the analysis, a sub-clade composed of the two new Cameroonian species demonstrated weakly supported relationships with the arenarium-glaseri-karii-longicaudum-group. Members of the feltiae-kraussei-oregonense-group
are characterised by an average body length of IJ between 612 and 980 pm (Stock et al., 1997; Khatri-Chhetri et al., 2011a). IJ of members of the arenarium-glaseri-karii-longicaudum-group are characterised by a longer average body length (8321217 pm) (Khatri-Chhetri et al., 2011b). The body length of IJ of S. cameroonense sp. n. and S. nyetense sp. n. (622 (490-694) and 648 (565-708) pm, respectively) correspond more to the characteristics of the second group, the feltiae-kraussei-oregonense-group. At the same time Steinernema rarum, which is also related to the clade consisting of ' arenarium-glaseri-karii-longicaudum' clade and the two new Cameroonian species, is characterised by quite short IJs - 446-578 pm (Nguyen et al, 2007). On the basis of both morphological and molecular features it can be concluded that the subclade consisting of the two new species clusters with inner nodes of Steinernema phylogenetic trees, and probably constitutes separate evolutionary line of the genus.
ACKNOWLEDGEMENTS
The authors thank the Flemish Inter-university Council - University Development Co-operation (VLIR-UOS), Belgium for the PhD scholarship awarded to the senior author. They also thank Ms Nancy de Sutter at ILVO, Merelbeke, Belgium for her technical assistance. We acknowledge Ms Marjolein Couvreur, Department of Biology, Faculty of Sciences, Ghent University for her support at the scanning electron microscope. S.E. Spiridonov participation in this work was supported by RFBR (11-04-00590a).
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ноых вида рода Steinernema Travassos, 1927 из влажных тропических лесов Южного Камеруна . Резюме. Представители двух новых видов Steinernema cameroonense sp. n. и S. nyetense sp. n. были изолированы из почвенных проб, собранных близ населенного пункта Minkama округа Obala и Nkontangan, округа Nyete, соответственно. Steinernema cameroonense sp. n. характеризуется длиной тела инвазионной личинки 622 (490-693) мкм, длиной пищевода 108 (100-114) мкм, длиной хвостового конца инвазионных личинок 76 (52-107) мкм, значениями индексов D и E - 48 (42-56) и 75 (48-116), соответственно. Число ребер латерального поля составляет 2, 4, 5, 6, 3 и 2 на разных уровнях тела личинки. Самцы первого поколения могут быть идентифицированы по спикуле длиной 69 (51-85) мкм, значениям индексов GS = 64 (47-76), SW = 170 (131-200) и D = 64 (48-76). Steinernema nyetense sp. n. характеризуется длиной тела инвазионных личинок, составляющей в среднем 648 (565-708) мкм, длиной пищевода 114 (104-127) мкм, длиной хвостового конца 82 (54112) мкм, значениями индексов D и E - 46 (37-50) и 66 (43-89), соответственно. Число ребер латерального поля составляет на разных уровнях тела лиичнки 2, 4, 5, 4, 3 и 2. Самцы первого поколения могут быть идентифицированы по длине спикулы 80 (67-98) мкм и индексам GS = 66 (51-77), SW = 198 (124-283) и D = 55 (40-70). Как показал филогенетический анализ последовательностей ITS рибосомальной ДНК, S. cameroonense n. sp. и S. nyetense sp. n. составляют отдельную группу в роде Steinernema, отдаленно связанную с эволюционными линиями 'monticolum' и 'feltiae-kraussei-oregonense'. Анализ по последовательностям D2D3 большой субъединицы рибосомальной ДНК этих двух новых видов Steinernema из Камеруна показал, что они составляют единую группу, связанную с внутренними узлами филогении Steinernema.
