Steinernema schliemanni sp. n. (Steinernematidae; Rhabditida) – a new species of steinernematids of the ‘monticolum’ group from Europe Текст научной статьи по специальности «Биологические науки»

Russian Journal of Nematology, 2010, 18 (2), 175 - 190

Steinernema schliemanni sp. n. (Steinernematidae; Rhabditida) - a new species of steinernematids of the 'monticolum' group

from Europe

1 2 2 3

Sergei E. Spiridonov , Lieven Waeyenberge and Maurice Moens '

1 Centre of Parasitology of A.N. Severtsov Institute of Ecology and Evolution of Russian Academy of Sciences, Leninskii

prospect, 33, Moscow, 119071, Russia e-mail: [email protected] 2Institute of Agriculture and Fisheries Research, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium; 3Laboratory of Agrozoology, Department of Crop Protection, Ghent University, Coupure Links 653, 9000 Ghent, Belgium.

Accepted for publication 28 August 2010

Summary. Steinernema schliemanni sp. n. found on the surface of a cadaver of Osmoderma ceremita (Coleoptera) in Germany in April 2003 is described. The new species belongs to the group of species related to S. monticolum ('monticolum'-group). It differs from the other species of this group in the structure of the lateral field of infective juveniles, viz. 8 equal ridges at mid-body in S. schliemanni sp. n. vs unequal ridges in S. monticolum and S. robustispiculum and five ridges in S. ashiuense. Infective juveniles of S. schliemanni sp. n. are longer (842-1008 ^m) than those of all other species of the 'monticolum' group. Steinernema schliemanni sp. n. differs from the other species of the 'monticolum'-group in at least 115 positions of the ITS rDNA sequence (18%). The nucleotide difference of the D2D3 region of the LSU rDNA between S. schliemanni sp. n. and the closest species, S. monticolum, is 22 bp (3.9% of the compared sequences), which is higher than differences between species such as S. feltiae, S. kraussei and S. oregonense. The 16S rDNA sequence of the bacterium associated with S. schliemanni sp. n. demonstrates close relationships with several Xenorhabdus species; the nucleotide difference is the lowest with X. hominickii (28 bp or 2%), isolated from S. monticolum.

Key words: bacterial symbionts, molecular phylogeny, rDNA, SEM, Steinernematids, taxonomy.

The geographical distribution of at least some species of entomopathogenic steinernematid nematodes does not demonstrate clear patterns. When some cosmopolite species, like Steinernema feltiae (Filipjev, 1934) Wouts, Mracek, Gerdin & Bedding, 1982, can be found in a wide variety of habitats, the 'endemism' of other steinernematids might be explained by inadequate sampling. For example, S. glaseri (Steiner, 1929) Wouts, Mracek, Gerdin & Bedding, 1982, was described as a characteristic component of North American soil ecosystems but was later also reported from continental Europe, Iran and the Azores (Rosa et al., 2000; Ansari et al., 2005; Karimi et al., 2009). Some groups of steinernematid species demonstrate more or less pronounced patterns of geographical distribution. Steinernema monticolum Stock, Choo & Kaya, 1997 was described from Korea (Stock et al., 1997) and later reported from Japan (Kuwata et al., 2006). Two related species, S. robustispiculum

Phan, Subbotin, Waeyenberge & Moens, 2005 and S. ashiuense Phan, Takemoto & Futai, 2006, were described from Vietnam and Japan, respectively (Phan et al., 2005, 2006). These three species can be considered as forming a separate evolutionary line or sub-clade in the genus Steinernema ('monticolum'' group within the 'feltiae-kraussei-oregonense' group). A strain closely resembling S. ashiuense was found during a survey in the south of the Russian Far East (Vladivostok area), whereas another strain similar to S. monticolum was reported from Kamchatka peninsula (Spiridonov & Moens, 2005). Thus, until now all species and strains of the 'monticolum' group were only reported from Eastern Asia.

In 2003 a larva of the genus Osmoderma Lepeletier & Serville, 1828 (Coleoptera) naturally infected with entomopathogenic nematodes (EPNs) was found on the soil surface in Mecklenburg (North-East Germany). The cadaver with nematodes

was sent by Prof. Walter Sudhaus to the BBA Institute of Nematology in Münster, where a living culture was established on Galleria mellonella L. The morphology of these nematodes was studied under both the light and scanning electron microscope and sequences of the ITS rDNA and the D2D3 region of the LSU rDNA were obtained through direct sequencing or cloning of corresponding PCR-products with consecutive sequencing. It was found that the strain belonged to the 'monticolum' group and constituted a new Steinernema species. The morphology, morphometrics and molecular features of this European representative of the 'monticolum' group are herein described.

MATERIAL AND METHODS

Nematode culture and fixation. The juvenile suspension obtained in 2003 in Münster was used for re-inoculation of Galleria last-instar larvae. Initially, a stable laboratory culture could only have been established through injections in last-instar Galleria larvae. The nematodes obtained were used to infect Galleria caterpillars in Petri dishes lined with moist filter paper. Since then the nematodes were kept alive in G. mellonella. Adult stages of nematodes were recovered from insect cadavers at 3 (first generation) and 7 days (second generation) post inoculation. They were fixed in hot formaldehyde (4-6% formaline) and processed into glycerol for morphological study (Seinhorst, 1959). Drawings and measurements were made using a Karl Zeiss Jenaval light microscope.

Cross breeding. For cross-breeding experiments the nematodes were cultured in blood drops of Galleria haemolymph (Nguyen & Duncan, 2002). Only S. monticolum was available for crossbreeding tests. In a first experiment pairs of infective juveniles of both species were formed by carrying two juveniles into the hanging drop. A total of 60 pairs included 20 pairs of juveniles from both species, and 20 pairs of conspecific juveniles of each species. In a second experiment fourth-stage juveniles were dissected from Galleria cadavers and after identification of their sex were transferred to hanging drops (n=32). Conspecific pairs were also established (n=25). Twenty five single forth-stage female juveniles were also cultured in drops for each species. The formation of first- and second-stage juveniles in haemolymph or inside females was considered as positive results of mating.

SEM study. For scanning electron microscopy the nematodes were fixed in 4-6% formalin, dehydrated in ethanol solutions with gradient

concentrations (30%, 50%, twice 96% 1 h each at room temperature). They were critical point dried with liquid CO2 (HCP-2 HITACHI), mounted on aluminium stubs, coated with gold (BIO-RAD SC502) and studied using a scanning electron microscope JEOL-100 CX. Spicules and gubernacula were prepared as suggested by Nguyen and Smart (1994).

Molecular observations. DNA was obtained in two ways. Single juveniles were cut in two parts which were transferred to 8 ^l of worm-lysis buffer (100 mM KCl, 20 mM Tris-HCl pH 8.3, 3 mM MgCl2, 2 mM DDT and 0.9% Tween 20). This mixture was incubated for 1 h under 65°C followed by 10 min at 95°C. Alternatively, suspensions of living juveniles and adults were washed several times with sterile water through centrifugation in 1.5 ml Eppendorf tubes. DNA was then extracted with the use of columns from Wizard® SV Genomic DNA Purification System according to the producer's protocol (Promega Benelux, Leiden, The Netherlands).

Either 1-4 ^l of the homogenate in worm-lysis buffer or 1-2 ^l of DNA obtained from Wizard® columns, were used as template in a PCR reaction. For amplification of ITS rDNA two primer pairs were used: 18S (5'-TTG ATT AGG TCC CTG CCC TTT-3') and 26S (5-TTT CAC TCG CCG TTA CTA AGG-3') (Vrain et al., 1992), or TW81 (5'-GTT TCC GTA GGT GAA CCT GC-3') and AB28 (5'-ATA TGC TTA AGT TCA GCG GGT-3') (Curran & Driver, 1994). For amplification of the D2D3 region of the LSU rDNA the pair of primers D2A (5'-ACA AGT ACC GTG AGG GAA AGT -3') and D3B (5'-TGC GAA GGA ACC AGC TAC TA-3') was used (He et al., 2005). The PCR cycling parameters included a primary denaturation step at 94°C for 5 min followed by 34 cycles of 94°C for 1 min, 55°C (for amplification of ITS rDNA) or 50°C (for amplification of D2D3 region of LSU rDNA) for 1 min and 72°C for 1 min, followed by a postamplification extension step at 72°C for 6 min. Following electrophoresis (100V, 10mA) on a 0.8% agarose gel, PCR products were cleaned using Promega® Wizard SV Gel and PCR Clean-Up System, ligated into pGEM-T vector (both Promega Benelux) and used to transform JM 109 Escherichia coli competent cells (Promega Benelux) according to Promega protocols and application guide, third edition. Transformed colonies were subcultured in 1.5 ml of LB medium and incubated 16-22 h at 37°C. Plasmid DNA was obtained from transformed E. coli using Promega® SV Miniprep DNA purification system (Promega Benelux), according to the manufacturer's instructions and sent for

sequencing by commercial company with vector primers T7 and SP6.

Symbiotic bacteria. Symbiotic bacteria were isolated from a suspension of infective juveniles after 2 h incubation in 0.1% mertiolate. Surface-sterilised juveniles were homogenized in a glass micro-mortar; the mortar content was spread over the surface of NBTA agar (Koppenhofer, 2007). Bacteria from blue colonies were transferred to LB broth, which was shaken during 2-3 days at room temperature. After the centrifugation of the bacterial culture (2000 g, 5 min), the sediment of bacterial cells was transferred to 500 ^l of lysis buffer (10mM Tris-HCl, pH: 8.0; 1mM EDTA; 1% Triton X-100) and incubated at 95°C for 30 min. After centrifugation (7000 g, 2 min) 2 ^l of the supernatant was used for PCR (Babic et al., 2000) with following primer pair: sense - (5'-GAA GAG TTT GAT CAT GGC TC-3'), antisense - (5'-AAG GAG GTG ATC CAG CCG CA-3'). The obtained PCR product was directly sequenced using the same primers.

Phylogenetic analysis. For comparative purposes and construction of the phylogeny, the set of nematode and bacterial rDNA sequences deposited in GenBank was used. BLAST option was used to trace out related forms (Altschul et al., 1990). The NCBI accession numbers of used sequences are indicated on the trees near the appropriate specific names. Bootstrap values are presented near nodes. Sequence alignments were generated using Clustal X under default values for gap opening and gap extension penalties. Alignments were analysed using PAUP* 4.0b10 (Swofford, 1998) for maximum parsimony (MP), and maximum likelihood (ML). Steinernema affine (Bovien, 1937) Wouts, Mracek, Gerdin & Bedding, 1982 and S. intermedium (Poinar, 1985) were used as outgroups in all cladograms. The GTR+G+I model was selected for maximum likelihood analysis with the use of ModelTest 5.0 (Posada & Crandall, 1998). The programme MtGui by Pablo Nuin (2005) was used as an interface to prepare Modeltest results for ML analysis in PAUP* 4.0b10.

Table 1. Morphometries (mean ± SD (range), in цт of Steinernema schliemanni sp. n.

Character 1st generation 2nd generation 1st generation 2nd generation Infective juvenile

male (n=15) male (n=13) female (n=10) female (n=10) (n=25)

Body length 1463±368 1143±71 6408±1269 2497±278 934±54

(896-2213) (1059-1273) (4431-8340) (2112-2991) (842-1008)

Maximal body diameter 87±13 59±5.6 176±38 139±12.4 35±2.4

(76-120) (50-70) (134-261) (121-169) (30-38)

Pharynx total length 152±13 137±4.5 180±23 177±17.5 148±9.2

(121-170) (125-142) (152-221) (156-209) (127-162)

Distance from anterior end to 85±13 87±5.8 114±3.8 106±4.1 72±5.3

excretory pore (68-106) (79-98) (106-117) (102-112) (61-80)

Anal body width 36±3.6 35.7±3.7 60±5.6 42±2.7 17±0.8

(31-42) (30-42) (54-69) (37-47) (16-18)

Spicule length along chord 72±5.4 53±3.8 - - -

(61-81) (50-61)

Gubernaculum length 53±5.4 38±4.8 - - -

(43-64) (31-48)

Tail length 28±3.7 28±7.6 46±6.1 35±4.4 88±5.4

(22-32) (23-51) (36-55) (31-46) (76-95)

Hyaline portion of tail - - - - 55±7.3

(40-70)

Vulva (%) - - 54±2.5 56±1.2 -

(50.8-59.6) (54.1-58.1)

a 15.5±2.7 19.2±1.5 37.5±9.4 17.9±1.5 26±1.8

(10.3-20.5) (17.3-21.4) (25.6-50.4) (16.2-20.9) (23-30)

b 8.9±1.2 8.3±0.7 35.8±7.1 14.1±1.0 6.3±0.3

(7.4-11.8) (7.7-9.7) (24.3-44.4) (13.1-16.1) (5.6-6.7)

c 49.3±14.8 42.5±7.8 141.3±39.9 71.2±11.3 10.6±0.6

(35.9-87.2) (24.9-53.3) (91.4-204.6) (54.9-88.5) (9.6-12.2)

h% - - - - 66±0.2

(51-74)

D% (EP/Pharynx x 100) 54.4±3.1 63.3±6.2 61.4±4.9 59.6±5.2 47.6±3.4

(50-58) (58-74) (52-70) (50-66) (42-55)

Fig. 1. Steinernema schliemanni sp. n. morphology, lateral aspect. A-C: First generation female. A: total view; B: anterior end, C: spermatozoa in oviduct and uterus. D, E: first generation male. D: total view; E: posterior end; F, G: Second generation male. F: total view; G - posterior end; H-K: Infective juveniles. H: total view; I: head end; J: anterior end (vesicle in the intestine); K: posterior end. Scale bars: A = 1000|m, B, C = 250 |m; D, F = 300 |m; E, G, J = 100 |m; I = 50 |m; K = 90 |m.

Fig. 2. Steinernema schliemanni sp. n. Male morphology, SEM. A: posterior end, lateral view, (white arrowheads indicating position of genital papillae); B: posterior end of second specimen, lateral view; C: posterior end of third specimen, lateral view, D: posterior end, subventral view, E: posterior end, ventral view.

DESCRIPTION

Steinernema schliemanni sp. n. (Figs 1-7)

Measurements (Table 1).

Holotype (first generation male): Body length = 1140 ^m; Maximal body diameter = 77 ^m; Pharynx total length =139 ^m; Tail length = 26 ^m; Spicule length along the chord = 65; Gubernaculum length = 49 ^m.

First generation male. Body C- or J-shaped after fixation. Anterior end hemispherical, 20-25 ^m in diameter, with broad funnel-shaped buccal cavity. Strongly cuticularised stoma walls 2-3 ^m long, stegostom at least 5 ^m long. Pharynx 12-15 ^m wide at stegostom level, widening gradually up to 16-19 ^m at level of excretory pore, then narrowing to isthmus (11-13 ^m). Basal bulb 25-30 ^m wide, without conspicuous valves. Cardium round, 6-8 ^m long lobes protruding into intestinal lumen. Prominent deirids at cardium level. Excretory duct

1-1.5 ^m wide, curving backward 5 ^m from surface. Flexure of testis 50-60 ^m posterior to basal bulb. Prominent pseudoceolomocytes (15 ^m diam.) anterior to flexure. Testis filled with spermatocytes with round 4 ^m wide nuclei at level of flexure. Nuclei in spermatocytes disappear at mid-body level, with broader testis lumen posteriorly filled with 7-10 ^m spermatids with granular cytoplasm. Strongly vacuolated vas deferens, walls narrow to ejaculatory duct starting at level of spicule heads. Narrow posterior part of intestine squeezed between vas deferens and body wall. Spicules moderately curved (approx. 120°), with 12 ^m long and 10 ^m wide head and 7-8 ^m wide shaft (Fig. 3A). Anteriorward projection on ventral edge of spicule proximal end (Fig. 3B). Proximal end of spicules covered with thin cuticular membrane (Fig. 3D). Velum 4 ^m wide, ending at

2-12 ^m from rounded tip of spicules (Fig. 3A-C). Gubernaculum with protruding crurae, conspicuous median hole and proximal beak-like projection, directed toward ventral body surface. Cuneus absent (Fig. 3E).

Two pairs of postloacal papillae on ventral side near tail terminus. Tiny (1 ^m high) peg-like mucron. Pair of dorsolateral papillae near tail terminus. One pair of papillae at cloaca level and one pair situated in 8-10 ^m anterior to cloaca (Fig. 2C-E). Unpaired median papilla as flattened elevation (4 ^m in diam.) in 20 ^m anterior to cloaca. Five pairs of genital papillae discernible in precloacal area anterior to unpaired median papilla. A pair of post-deirids in dorsolateral position in front of anteriormost genital papillae. Total count of genital papillae: 22+1.

First generation female. Cephalic end rounded, diameter approx. 30 ^m. Bowl-shaped buccal cavity 12-15 ^m wide. Corpus of pharynx increasing in diameter from 20 ^m at stoma to 30 ^m before isthmus. Isthmus 20 ^m wide. Basal bulb 40 ^m wide, with transparent 10 ^m long lobes of cardium. Excretory duct 1-2 (im wide. Excretory channel lumen in lateral chords traced up to vulva, its walls collapsing at posterior. Intestinal cells at proventriculus level large, more than 50 ^m in size. Ovary flexures 150-200 ^m posterior to basal bulb and in 20-50 ^m before the anus level. Sometimes ovary forms additional loop. Spherical chamber at mid-oviduct, composed of radially arranged cells. Numerous spermatozoa (diameter of nucleus containing end: 7-9 ^m) forming chains of 3-8 cells scattered in lumen of spherical chamber and neighbouring parts of ovary and uterus. Anterior vulva lip 20-30 ^m high overhanging smaller (10 ^m high) posterior lip. Tail end cupola-shaped with acute 20 ^m long terminus.

Second generation males. Body rarely C-shaped, more often J-shaped after fixation. Main morphological features similar to those of first generation males. Strongly cuticularised stoma walls 2-3 ^m long.

Second generation female. No pronounced difference between first and second generation females in morphology and even in morphometrics of cephalic end, buccal cavity and pharynx. Basal bulb usually narrower (30-35 ^m wide), lobes of cardium of same size. Intestinal cells with thinner walls. Ovary flexures situated at longer distance from basal bulb or anal opening (300-400 and 100150 ^m, respectively). No morphologically distinct chambers or widened parts of oviduct. Vulva lips equal, 10 ^m high, usually covered with transparent substance. Tail end conical with rounded terminus.

Infective juveniles. Heat relaxed body broadly C-shaped. Anterior end slightly set-off from body contours (Fig. 4B). Amphidial opening near labial circle. Four cephalic papillae in 4-5 ^m from anterior end, just posterior to amphids. Lateral field starts in 60-70 ^m from anterior end (Fig. 4A and C) as four ridges. At 10 ^m from the beginning, the lateral field contains six ridges (Fig. 2C). Eight equal ridges discernible at mid-body (addition of two marginal ones). This pattern continues to mid-tail level.

Cross-breedings tests. The pairs of males and females of S. monticolum and S. schliemanni sp. n. were unable to produce progeny either the pair was composed of single infective juveniles of each species, or fourth-stage individuals of opposite sex from each species were combined. In conspecific pairs of juveniles two pairs produced

Fig. 3. Steinernema schliemanni sp. n. Morphology of male copulatory apparatus, SEM. A: pair of spicules; B: separate spicule with different shape of shaft and velum; C: ending of velum; D: proximal part of spicule, E: gubernaculum.

Table 2. Pairwise differences in ITS rDNA region between Steinernema schliemanni sp. n and related species. Below diagonal: total character differences, above diagonal: mean character differences.

Species (accession number in NCBI GenBank) 1 2 3 4 5 6

1 Steinernema schliemanni sp. n. (HM778112) - 18.5% 20.5% 20.1% 19.9% 23.6%

2 Steinernema monticolum (AF122017) 115 - 6.0% 4.5% 4.9% 20.8%

3 Steinernema ashiuense, (DQ354694) 128 39 - 2.7% 0.9% 21.2%

4 Steinernema robustispiculum, (AY355442) 126 30 18 - 1.8% 21.0%

5. Steinernema sp. MY5 (AB243437) 124 33 6 12 - 21.1%

6 Steinernema feltiae, (AY171246) 151 135 137 136 137 -

Fig. 4. Steinernema schliemanni sp. n. and S. monticolum. A-E: S. schliemanni sp. n. Cuticle of infective juvenile in lateral view, SEM. A: Anterior end; B: Head end; C: beginning of lateral field; D: Lateral field at mid-body; E: Lateral field at tail end; F: S. monticolum. Lateral field at mid-body.

progeny for S. schliemanni sp. n. and one for S. monticolum (n=50 for both species). Single nematodes of both isolates did not produce progeny. Conspecific pairs of pre-adult stages produced progeny in about 8-12% of mating experiments (2 and 3 cases for these two species correspondingly; n=25 for both species).

DISCUSSION

Molecular characterisation. Sequences of ITS rDNA for S. schliemanni sp. n. were obtained through direct sequencing in 2003 of nematodes originating from the first inoculation of Galleria caterpillars with newly obtained infective juveniles. Since then ITS rDNA sequences were obtained through cloning of PCR product in 2007 and in 2009. Sequences of the D2D3 region of LSU rDNA were obtained in 2007. Obtained sequences were deposited in GenBank (for S. schliemanni sp. n. -862 bp long ITS1-5.8S-ITS2 sequence: HM778112; partial 541 bp long D2D3 LSU rDNA sequence: HM778113; partial 510 bp long D2D3 LSU rDNA sequence for type culture of S. monticolum: HM778114). Phylogenetic relationships of S. schliemanni sp. n. with related Steinernema spp. are presented in Figs 5-7. In both the MP and ML ITS rDNA cladograms the new species is related to S. monticolum, S. ashiuense and S. robustispiculum (Figs 5-6) composing the 'monticolum' clade. Bootstrap support for this clade is moderate (89%) or high (100%) in MP and ML trees, respectively. The inner topology of the 'monticolum' clade is the same in both cladograms (S. schliemanni sp. n. (S. monticolum (S. ashiuense + S. robustispiculum))). The new species is positioned in the basal position in this lineage. The entire clade is a sister group for the 'feltiae-kraussei-oregonense' clade. The cladograms obtained from the MP and ML analysis of partial D2D3 LSU rDNA S. schliemanni sp. n. and S. monticolum form a single clade (Fig. 7) which is the sister one for the 'feltiae-kraussei-oregonense clade'. The nominal LSU rDNA sequence obtained for S. monticolum by Stock et al. (2002) and deposited in GenBank under AF 331895 is linking inside ' carpocapsae'-clade of Steinernema genus.

Molecular characterisation of symbiotic bacteria. The partial 16S sequence of symbiotic bacteria of S. schliemanni sp. n. was deposited in NCBI GenBank under accession number HQ122653. In the MP analysis, the Xenorhabdus sp. from S. schliemanni sp. n. clusters with a group composed ofX. bovienii (Akhurst, 1983) Akhurst & Boemare, 1993, X.

hominickii Tailliez, Pages, Ginibre & Boemare, 2006, X. japonica Niushimura, Hagiwara, Suzuki & Yamanaka, 1995, and X. koppenhoeferi Tailliez, Pages, Ginibre & Boemare, 2006. Xenorhabdus bovienii demonstrates a stable but weakly supported relationship with the Xenorhabdus sp. isolated from S. schliemanni sp. n. (Fig. 8A). Inner phylogenetic nodes of this Xenorhabdus group were not resolved in our analysis; in the strict consensus trees they were collapsing (data not shown). In the NJ tree, inner nodes of this group were collapsing in 50% majority rule consensus tree (Fig. 8B). Xenorhabdus hominickii is the closest Xenorhabdus species to the Xenorhabdus sp. from S. schliemanni sp. n. with only 28 bp difference (Table 4).

Differential diagnosis. In all phylogenetic trees, S. schliemanni sp. n. clusters with the species of the 'monticolum'-group. Apart of three described species (S. monticolum, S. ashiuense and S. robustispiculum), a strain identified as Steinernema sp. MY5 (Yoshida et al., 1998) is a permanent member of this lineage. The lateral field of infective juveniles of S. schliemanni sp. n. consists of eight ridges at mid-body (Fig. 4D). In two species of the aforementioned group, the lateral field has a completely different appearance at mid-body, viz. two well pronounced submarginal lines and two lower ridges in the centre of the field. Such lateral field morphology was described for both S. robustispiculum (Phan et al., 2005) and S. monticolum (Fig. 4F). The lateral field of S. ashiuense was described as containing five ridges (Phan et al., 2006). Inside the 'monticolum' clade, S. schliemanni sp. n. can be easily distinguished from the three other species by the morphometrical characters of infective juveniles. Infective juveniles of S. schliemanni sp. n. are significantly longer (842-1008 ^m) than those of all three other species. The ranges of the body length of infective juveniles in S. monticolum, S. ashiuense and S. robustispiculum are 612-821 ^m, 720-800 ^m and 642-778 ^m, respectively. The EP (distance from anterior end to excretory pore) and tail length are proportionally higher in the longer juveniles of S. schliemanni sp. n. The ratios D% and 'c' of infective juveniles are virtually the same in all four species under comparison. The size of spicules of first generation males of S. schliemanni sp. n. resembles that of the spicules of S. monticolum (6481 vs 61-80); spicules of first generation males of S. ashiuense and S. robustispiculum are shorter (not longer than 65 ^m). The nucleotide differences between the three known species of the 'monticolum'

87

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S. intermedium (AY230172) S. ajfine (AY230159) ,Y. varum (AY275273) S. glaseri (AY230172) S. khoisanae (DQ314287) S. guangdongense (AY170341) S. diaprepesi {AF122021) S. loci (AY355443) S. aciari (AY787660) S. kushidai (AB243440) ,Y. sangi (AY355441) Steinernema sp. MY6 (AB243443)

S. silvaticum (AY171255) S. krausse i (AY 171251)

S. weiseri (AY230167) Steinernema sp. 141-C (EU740970) S. litorale (AB243441) S.feltiae (AY 171246) S. cholashanense (EF431959) {j- S oregonense (AY230180) S akhursti (DQ375757)

S. schliemanni sp. n. (HM778112) S. robustispiculum (AY355442) S. ashiuense (DQ354694) Steinernema sp. MY5 (AB243437) S. monticolum AB243438 (Japan) S. monticolum AI 122017 (Korea)

scapterisci (AY230172) carpocapsae (AY230164) siamkayai (AF3 31917) tami (AY 171280) bicornutum (AF 121048) riobrave (AY230182) pakistanense (AY230181) yirgalemense (AY748450) abbasi (EF469773)

Fig. 5. Steinernema schliemanni sp. n. phylogenetic position inferred from analysis of ITS rDNA sequences. Bootstrap 50% majority-rule consensus tree. Criterion = parsimony. Of 892 total characters 570 are parsimony-informative. Gaps are treated as "missing". 10000 bootstrap replicates.

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S. intermedium (AY230172) S. affine (AY230159) S rarum (AY275273) S. khoiscincie (DQ314287) S. loci (AY355443) .V. aciari (AY787660) S. guangdongense (AY170341) S. diaprepesi (AF122021) .V. glaseri (AY230172) S. scapterisci (AY230172) S. carpocapsae (AY230164) S. siamkayai (AF331917) S. tami (AY171280) S. bicornutum (AF 121048) S. yirgalemense (AY748450) S. abbasi (EF469773) S. pakistanense (AY23 0181) .V. riobrave (AY230182) S. kushidai (AB243440) S. sangi (AY355441) S. cholashanense (EF431959) Steinernema sp. MY6 (AB243443) S. silvaticum (AY171255)

S. kraussei (AY171251)

S. weiseri (AY230167) ———I |—Steinernema sp. 141-C (EU740970)

100

£ ri

100 I

yJLr S' litorale (AB243441)

100

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100

98

LC

c

S. feltiae (AY171246) r-S. oregonense (AY230180) L S. akhursti (DQ375757)

— S. schliemanni sp. n. (HM778112)

- S. robustispiculum (AY355442) S. ashiuense (DQ354694) Steinernema sp. MY5 (AB243437) S. monticolum Japan (AB243438) S. monticolum Korea (AF122017)

Fig. 6. Steinernema schliemanni sp. n. phylogenetic position inferred from analysis of ITS rDNA sequences. Bootstrap 50% majority-rule consensus tree. Criterion = likelihood. GTR+G+I model. 100 bootstrap replicates.

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S. ajfine (AY33 1899) S. intermedium (AF3 31909) Steinernema sp. USA (AY171276) »S. abbasi (AF331890) »S. riobrave (GUI77834) .S'. bicornutum (AF3 31904) S. ceratophorum (AF33 1888) .V. kraussei (AF3 3 1 896) S. oregonense (AF3 31891) S.feltiae (AF33 1906) S. schliemanni sp. n. (HM778113) .S'. monticolum (HM778114) »S. rarum (AF33 1905) .V. karn (A 133 1 902) S. puertoricense (AF33 1903) S. arenarium (AF33 1892) »S. cubanum (AF3 31899) .V. glaseri (AF33 1908) .»S. scapterisci (AF3 31898) . Steinernema sp. (AF33 1895) ■ S. siamkayai (AF3 31907) • S. carpocapsae (AF331900)

affine (AY331899) S. intermedium (AF331909) ^ riobrave (GUI 77834)

bicornutum (AF331904) S. ceratophorum (AF331888) ^ abbasi (AF33 1890)

Steinernema sp. (Af33 1895)

scapterisci (AF 33 1898) S. siamkayai (AF33 1907) S. carpocapsae (AF33 1900) S.feltiae (AF 33 1906) S. oregonense (AF331891)

kraussei (AF33 1896) S. schliemanni sp.n. (IIIV1778 1 1 3 ) 5". monticolum (HM778114) ^ rarum (AF33 1905) Steinernema sp. USA (AY171276) ^ karii (AF33 1902) S. puertoricense (AF33 1903) S. arenarium (AF33 1892) »S. cubanum (AF33 1899) ^ glaseri (AF33 1908)

Fig. 7. Steinernema schliemanni sp. n. phylogenetic position inferred from analysis of D2D3 expansion segment sequences of LSU rDNA. A: bootstrap 50% majority-rule consensus tree. Criterion = parsimony. Of 595 total characters 232 are parsimony-informative. Gaps are treated as "missing". 100 bootstrap replicates. B: bootstrap 50% majority-rule consensus tree. Criterion = likelihood. GTR+G model. 100 bootstrap replicates.

Table 3. Pairwise differences in 16S DNA region betweenXenorhabdus sp. bacteria isolated from Steinernema schliemanni sp. n. and related species. Below diagonal: total character differences, above diagonal: mean character differences.

Species 1 2 3 4 5 6 7

1 Xenorhabdus sp. ex Steinernema schliemanni sp. n. - 2.4% 2.7% 2.2% 2.8% 2.0% 2.3%

2 Xenorhabdus bovienii 34 - 4.0% 3.7% 3.8% 4.1% 3.5%

3 Xenorhabdus budapestensis 39 56 - 2.1% 3.0% 3.0% 3.0%

4 Xenorhabdus ehlersii 31 52 - % 2.8% 3.3%

5 Xenorhabdus japonica 40 53 34 37 - 2.4% 2.4%

6 Xenorhabdus hominickii 28 42 42 39 42 - 2.4%

7 Xenorhabdus koppenhoeferi 32 49 42 47 34 34 -

Fig. 8. Phylogenetic position of Xenorhabdus sp. isolated from infective juveniles of Steinernema schliemanni sp. n. inferred from analysis of 16S DNA. 1000 bootstrap replicates. A: Bootstrap 50% majority-rule consensus tree. Criterion = parsimony. Of 1402 total characters 116 are parsimony-informative. Gaps are treated as "missing". B: Neighbour-joining bootstrap 50% majority-rule consensus tree. Tree length = 522.

clade varies between 30-39 bp; this corresponds to 4-6% of the ITS region. The nucleotide differences between the three species and S. schliemanni sp. n. are much greater (115 bp or 18%) (Table 2). Though very few sequences of the D2D3 region of the LSU rDNA are deposited in NCBI GenBank for species related to S. schliemanni sp. n., it is obvious that the level of nucleotide differences between the new species and the closest S. monticolum is higher than that between species of 'feltiae-kraussei-oregonense' group (Table 3). Remarkably, the difference with the sequence of the nominal S. monticolum from GenBank (AF 331895) is much higher, reaching 31% of the compared sequences.

Type locality. The forest in MecklenburgVorpommern province of Germany. Numerous juveniles and non-mature second generation individuals were found around the cadaver of Osmoderma eremita Scopoli, 1763 beetle larva on the soil surface in April, 2003.

Deposition of type material: Holotype - male (N°1109) and paratype - 1st generation female (N° 1110) are deposited in the collection of the Centre of Parasitology, A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Moscow. Paratype male is present in the collection of Julius Kühn-Institut in Münster, Germany. Paratypes (male and female) are also deposited in the collection of University of Ghent (Belgium).

Ethymology. The species is named after entrepreneur and scientist Heinrich Schliemann (1822-1890) who was born in Neubukow (Mecklenburg-Vorpommern) and used his fortune gained in Russia to conduct the famous excavations of Troy.

DISCUSSION

An analysis of the phylogenetic relationships of the species of the 'monticolum' sub-clade of the genus Steinernema inferred from the available ITS rDNA (ITS1+5.8S+ITS2) sequences and partial sequences of LSU rDNA, reveals contradiction in the position of S. monticolum. In the trees based on ITS rDNA sequences (Stock, 2001; Nguyen et al., 2001), S. monticolum clusters with the feltiae-kraussei-oregonense' clade; however, in the trees based on LSU rDNA the species is a member of 'carpocapsae' clade (Stock, 2001). In the course of our study we obtained a partial sequence of the D2D3 expansion segment of the LSU rDNA for the S. monticolum type strain. When this sequence was used for phylogenetic analysis, this species clustered with S. schliemanni sp. n. and showed relationships with the 'feltiae-kraussei-oregonense' clade (Fig. 7). When using the sequence deposited previously

under accession number AF 331895 for S. monticolum, the species always clustered with S. carpocapae and related species. It seems that the LSU rDNA sequence for S. monticolum deposited in NCBI GenBank represents a species of the 'carpocapsae' group, rather then S. monticolum, as was already reported by Nguyen et al. (2007). The congruence between ITS rDNA and LSU rDNA trees based on the newly obtained sequence for S. monticolum looks more logical.

Obviously, S. schliemanni sp. n. is a member of the 'monticolum" clade, i.e. a group of three described species and several isolates of unknown taxonomic status. The newly described species always occupied a basal position inside this clade; species found in Asia formed a strongly supported subgroup. The 16S rDNA sequence of bacteria associated with S. schliemanni sp. n. demonstrates close relationships with several Xenorhabdus species; the nucleotide difference is the lowest with X. hominickii (28 bp or 2%). This latter species was earlier isolated from both S. karii and S. monticolum (Koppenhofer, 2007). A level of nucleotide differences around 2% was reported for some pairs of independent Xenorhabdus species, but usually interspecific difference is higher (Table 3). Additional studies are needed to elucidate the taxonomic status of the Xenorhabdus species from S. schliemanni sp. n. In the meantime it can be concluded that the molecular characteristics of both the nematode and its associated bacterium confirm the affiliation of the nematode with the 'monticolum' group.

ACKNOWLEDGEMENT

We are grateful to Dr Nguyen Ba Khuong (University of Gainesville, Florida) for providing the S. monticolum type culture and his kind attention toward this study, and to Dr Dieter Sturhan, who provided the primary nematode culture and for his critical comments during the manuscript preparation.

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Резюме. Приводится описание Steinernema schliemanni sp. n. , личинки которых были обнаружены на поверхности трупов личинок жуков Osmoderma ceremita (Coleoptera) в Германии в апреле 2003. Новый вид относится к группе видов, близких к виду S. monticolum (т.н. группа 'monticolum'). Новый вид отличается от других видов этой группы строением латеральных полей инвазионных личинок: наличием восьми равных ребер в поле, в отличие от неравных по высоте ребер у S. monticolum и S. robustispiculum и пяти ребер у S. ashiuense. Инвазионные личинки S. schliemanni sp. n. имеют более длинное тело (842-1008 мкм), чем у остальных представителей группы 'monticolum'. Steinernema schliemanni sp. n. отличается от других видов группы 'monticolum' как минимум по 115 позициям в последовательности ITS rDNA (18%). Различия в нуклеотидном составе участка D2D3 домена LSU rDNA между S. schliemanni sp. n. и ближайшим видом S. monticolum составляет 22 пары нуклеотидов (3,9% сравниваемых последовательностей), что превышает различия по этому участку между такими видами, как S. feltiae, S. kraussei и S. oregonense. Последовательность 16S rDNA ассоциированной с S. schliemanni sp. n. бактерии близка по нуклеотидному составу к последовательностям нескольких видов Xenorhabdus. При этом наименьшие различия отмечены с X. hominickii (28 пары нуклеотидов или 2%), выделенными от S. monticolum.