A new entomopathogenic nematode, Steinernema colombiense n. sp. (Nematoda: Steinernematidae), from Colombia

Nematology, 2008, Vol. 10(4), 561-574

A new entomopathogenic nematode, Steinernema colombiense n. sp. (Nematoda: Steinernematidae), from Colombia Juan Carlos L ÓPEZ -N ÚÑEZ 1 , Kathryn P LICHTA 2 , Carmenza E. G ÓNGORA -B OTERO 1 and S. Patricia S TOCK 2,∗ 1

Disciplina de Entomología, Centro Nacional de Investigaciones de Café, Cenicafé, Cenicafé Planalto, Kilómetro 4, Vía Antigua a Manizales, Chinchiná, Caldas, Colombia 2 Department of Entomology, The University of Arizona, Forbes 410, 1140 E, South Campus Drive, Tucson, AZ 85721-0036, USA Received: 26 October 2007; revised: 17 December 2007 Accepted for publication: 19 December 2007

Summary – A new entomopathogenic nematode, Steinernema colombiense n. sp., is described from Colombia. Morphological, molecular (28S and ITS rDNA sequence data) and cross-hybridisation studies were used for diagnostics and identification purposes. In addition, 28S and ITS rDNA sequence data were used to assess evolutionary relationships of the new species with other Steinernema spp. Morphological diagnostic features for S. colombiense n. sp. include morphometric features of the third-stage infective juvenile, including body length of 636 (549-732) µm, narrow body diam. (31 (22-36) µm), position of the excretory pore (35 (31-40) µm), tail length (41 (32-53) µm), D% = 29 (25-33) and E% = 205 (138-284). In addition, males of first and second generations are characterised by the morphology of the spicules and gubernaculum, the number and arrangement of the genital papillae and the excretory pore position (at 67 (56-76) and 54 (46-63) µm, for first and second generations, respectively). In addition to these traits, 28S and ITS rDNA sequences analyses both showed this species to be a distinct and unique entity. Keywords – description, molecular, morphology, morphometrics, new species, phylogeny, South America, taxonomy.

Public awareness of the problems developed from the use of chemical pesticides (i.e., adverse impact on human health, wildlife, environmental pollution, pesticide resistance and pest resurgence) has dramatically increased over the past year in most Latin American countries, including Colombia. Environmentalists, scientists and labour groups from various countries not only demand laws to regulate chemical pesticide use but also to disseminate and sponsor the search and consideration of natural enemies and entomopathogens to control a wide array of invertebrate pests of agricultural forestry and urban significance. Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) are one of the beneficial organisms currently considered in biological control programmes in Colombia (López et al., 2007). Emphasis has been place on the discovery of native species and strains, as well as on the introduction of exotic species, particularly, currently available EPN formulations. ∗ Corresponding

EPN have been considered for control of several noxious agricultural pests, including the cassava bug (Cyrtomenus bergi Froeschner), oil palm borer (Sagalassa valida Walker), coffee berry borer (Hypothenemus hampei (Ferrari)) and white grub complex (Phyllophaga and Anomala spp.) (see Caicedo & Belloti, 1996; Saenz, 1998; López, 2002, 2003; Caicedo et al., 2004; Lara et al., 2004; Mello & Gail, 2004; Sáenz et al., 2005; López et al., 2007). In one of the earliest surveys in this country, a steinernematid nematode was isolated from a coffee plantation (Maracay Experimental Station, National Federation of Colombian Coffee Growers) on the western slopes of the Central Andean range (López, 2002). This uncharacterised Steinernema sp. was designated as isolate SNI0198. Morphological and molecular characterisation indicated that this nematode was a new, undescribed, species. We herein provide a formal description of this species using a combination of morphological and molecular methods.

author, e-mail: [email protected]

© Koninklijke Brill NV, Leiden, 2008 Also available online - www.brill.nl/nemy

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Materials and methods I SOLATION AND NEMATODE PROPAGATION Isolate SNI-0198 was recovered from soil samples in a coffee plantation at Maracay Experimental Station (National Federation of Colombian Coffee Growers) on the western slopes of the Central Andean mountain range. The insect-baiting method described by Bedding and Akhurst (1975) was used for isolation of this nematode. Baits (ten, last instar Galleria mellonella (L.) larvae) were placed in 250 ml plastic containers (five containers per sample) with moistened soil obtained from each sample. Containers were covered with a lid, turned upside down and kept at room temperature (20 ± 3◦ C). Galleria mellonella larvae were checked every 2-3 days and dead larvae were replaced by fresh ones. After 7 days, dead insects were removed from the baiting containers, thoroughly rinsed in distilled water and placed in modified White traps (Kaya & Stock, 1997) until emergence of third-stage infective juveniles. M ORPHOLOGICAL OBSERVATIONS Specimens for morphological studies were obtained from last instar G. mellonella larvae exposed to ca 100 third-stage infective juveniles (IJ) per larva on moistened filter paper in Petri dishes and incubated at 25◦ C in the dark. First and second generation adults and third-stage infective juveniles were randomly collected from infected cadavers following procedures described by Kaya and Stock (1997). Twenty-five randomly selected specimens of each nematode stage were examined either live or heatrelaxed and relaxed in Ringer’s solution (60◦ C). Killed specimens were fixed in triethanolamine formalin (TAF) at 50-60◦ C (Courtney et al., 1965), slowly dehydrated and processed to anhydrous glycerin (Seinhorst, 1959). Nematodes were mounted on glass slides with glass fibre used as cover slip supports to avoid flattening of the specimens. Quantitative measurements of each specimen were made using an Olympus BX81 microscope equipped with differential interference contrast optics and Olympus Microsuite software (Soft Imaging System, Lakewood, CO, USA). Illustrations were prepared from digitised camera lucida images. Morphological characters measured were based on the recommendations of Hominick et al. (1997). The following abbreviations have been used in the text or tables: D = (head to excretory pore/pharynx length) × 100; E = (head to excretory pore/tail length) × 100; 562

GS = gubernaculum length/spicule length; H = length of hyaline portion of tail; H% = H as % of tail length; SW = spicule length/anal body diam. Spicule length was measured along the curved median line. S CANNING ELECTRON MICROSCOPY (SEM) Adults were dissected from G. mellonella larvae in Ringer’s solution (pH 7.3). They were rinsed three times for 5 min in Ringer’s solution. Three-day-old IJ were rinsed for 3 × 15 min in 0.05% NaCl. All nematodes were relaxed and killed by heating in a water bath (60◦ C) for 2-3 min and were then fixed in 8% glutaraldehyde/25% EM grade (diluted in Ringer’s solution) for 2 h at room temperature. Fixed nematodes were rinsed three times in distilled water, post-fixed in OsO4 for 1 h, rinsed in distilled water and dehydrated at 15 min intervals through 30, 50, 70, 90, 95 and 100% ethanol. They were then critical point-dried in liquid CO2 , mounted on SEM stubs, coated with gold and scanned using a SES DS-130 at 15 kV accelerating voltage. M OLECULAR CHARACTERISATION AND PHYLOGENETIC ANALYSIS

Total genomic DNA isolation, PCR amplification (reaction, cycling conditions and primers) and sequence analysis followed protocols described by Stock et al. (2001) and Nguyen et al. (2001). LSU and ITS rDNA sequences and phylogenetic relationships with other Steinernema species were compared using an existing library (P. Stock’s laboratory, University of Arizona) of more that 50 Steinernema spp. and available sequences deposited in GenBank. Phylogenetic analyses (maximum parsimony analysis) of sequence data were made using PAUP* v 4.0b (Swofford, 2001) following criteria described by Stock et al. (2001) and Nadler et al. (2006). Caenorhabditis elegans was considered in both phylogenetic analyses as the outgroup taxon according to criteria described by Nadler et al. (2006). Ribosomal DNA sequences for the new Steinernema spp. were deposited in GenBank with accession numbers EU345420 and EU345421 for 28S and ITS rDNA sequences, respectively. C ROSS - HYBRIDISATION Reproductive isolation of the new species was tested using the modified hanging-blood assay as described by Kaya and Stock (1997). Steinernema carpocapsae (Weiser, 1955), S. monticolum Stock, Choo & Kaya, Nematology

A new Steinernema from Colombia

1997 and S. siamkayai Stock, Somsook & Kaya, 1998, depicted as close relatives to the new species from the phylogenetic analyses, were used for these experiments. Controls consisted of crosses with male and single female nematodes of the same species and single female. There were ten replicates per cross and tests were repeated twice.

Steinernema colombiense* n. sp. (Figs 1-3) M EASUREMENTS See Tables 1 and 2. D ESCRIPTION First generation male Body slender, ventrally curved posteriorly, J-shaped when heat-relaxed. First generation male larger (average = 1542 µm) than second generation male (average = 870 µm). Cuticle smooth under light microscopy. Lateral field and phasmids inconspicuous. Head truncate to slightly rounded, continuous with body. Six lips, amalgamated, but tips distinct, bearing one labial papilla each. Four cephalic papillae present. Amphidial apertures small, located posterior to lateral labial papillae. Stoma reduced (cheilo-, gymno- and stegostom vestigial), short and wide, with inconspicuous sclerotised walls. Pharynx muscular; procorpus cylindrical; metacorpus slightly swollen, non-valvate; indistinct isthmus followed by pyriform basal bulb containing reduced valve. Pharynx set off from intestine. Nerve-ring usually surrounding isthmus or anterior part of basal bulb. Excretory pore opening circular, located anterior to nerve ring at anterior one-third of metacorpus. Testis single, reflexed, consisting of germinal growth zone leading to seminal vesicle. Vas deferens with inconspicuous walls. Spicules paired, symmetrical, curved, ochre-brown in colour. Manubrium rhomboidal, with a rounded, ‘knob-like’, protrusion. Calomus (shaft) distinct. Lamina with rostrum or retinaculum and two internal ribs. Velum present, narrow. Blade terminus blunt. Gubernaculum arcuate, ca 75% of spicule length. Manubrium of gubernaculum curved ventrally and bifurcate. Tail conoid and non-mucronate. Gubernaculum more slender and longer than that of first generation male. * Specific epithet named after the country in which the species was found.

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Twenty-five genital papillae (12 pairs and one single) arranged as follows: five subventral precloacal pairs, single midventral precloacal papilla (located between precloacal pairs 4 and 5); one pair of cloacal papillae (located laterally on cloacal opening); two pairs adcloacal (one pair subventral, one pair subdorsal); four pairs postcloacal (one pair subventral, two pairs ventral, one pair subdorsal). Second generation male General morphology similar to that of first generation males, but smaller in size. Tail with or without mucro. Spicules with manubrium morphology slightly different to that of first generation male. Manubrium wider than long, with two small ribs. Calomus indistinct, continuous with shaft. Lamina with a small rostrum, velum present. First generation female Cuticle, lip region, stoma and pharyngeal region as in male. Body C-shaped when heat-relaxed. First generation females larger (average = 4608 µm) than second generation females (average = 1470 µm). Excretory pore located about mid-procorpus level. Ovaries opposed, reflexed dorsally; oviduct well developed; glandular spermatheca and uterus in ventral position. Vagina short, with muscular walls. Vulva located near mid-body, with protruding asymmetric lips (posterior lip larger than anterior). Tail blunt, conoid, lacking mucro. Anal lips usually protruding, asymmetric with anterior lip smaller than posterior. Second generation female Body open C-shaped when heat-relaxed. Similar to first generation female, but smaller. Vulva with protruding, symmetrical lips. Tail conoid, with post-anal swelling. Third-stage infective juvenile Body of heat-relaxed specimens almost straight, slender, gradually tapering posteriorly. Cuticle with fine transverse striae. Lateral field distinct with six longitudinal ridges (i.e., seven lines/incisures) in mid-body region. Head region continuous with body, slightly truncate. Six distinct labial papillae and four cephalic papillae present. Amphidial apertures pore-like. Lip region smooth, continuous; stoma closed. Pharynx long, narrow, with slightly expanded procorpus, narrower isthmus and pyriform basal bulb with valve. Cardia present. Nerve-ring located at isthmus level. Excretory pore located ca mid-corpus level. Hemizonid not observed. Anterior portion of intestine with bacterial receptacle. Intestine filled with numerous 563

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Fig. 1. Steinernema colombiense n. sp. A: Anterior end of first generation female, lateral view; B: Vulva of first generation female, lateral view; C: Vulva of second generation female, lateral view; D: Tail of first generation female, lateral view; E: Tail of second generation female, lateral view; F: First generation male, in toto, lateral view; G: First generation male spicule, lateral view; H: Second generation male spicule, lateral view; I: First generation male gubernaculum, dorsal view; J: Anterior end of third-stage infective juvenile, lateral view; K: Tail of third-stage infective juvenile, lateral view. (Scale bars: A, J = 23 µm; B-D = 35 µm; E, G-I, K = 15.5 µm; F = 170 µm.) 564

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Fig. 2. Light and scanning electron microscope photographs of male specimens of Steinernema colombiense n. sp. A: First generation male, in toto; B: Anterior end, showing excretory pore location (arrow); C, D: Scanning electron microscopy images of tail showing distribution of several genital papillae (pr = precloacal, c = cloacal, ad = adcloacal, po = postcloacal, v = length from anterior end to vulva opening); E: First generation male spicule, lateral view showing ‘knob-like’ manubrium (arrow); F: First generation male gubernaculum, ventral view showing bifurcate head and cuneus (arrow); G: Second generation male spicule, lateral view. (Scales are based on scale bar in A: A = 100 µm; B, D, E = 16 µm; C, F, G = 12 µm.) Vol. 10(4), 2008

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Table 1. Morphometrics of Steinernema colombiense n. sp. All measurements are in µm and in the form: mean ± s.d. (range). Male 1st generation

Female 2nd generation

1st generation

2nd generation

Infective juvenile

Holotype

Paratypes

Paratypes

Paratypes

Paratypes

Paratypes

n L

– 1678

a

–

19 1542 ± 226 (1209-2040) –

20 870 ± 61 (762-980) –

19 4608 ± 102 (2796-6752) –

20 1470 ± 211 (996-2123) –

b

–

–

–

–

–

V

–

–

–

Max. body diam.

95

Pharynx length

158

Head to excretory pore

70

Head to nerve ring

134

Tail length

40

Anal body diam.

38

Spicule length

70

31 ± 4 (22-36) 118 ± 6 (106-128) 35 ± 4 (31-40) 83 ± 5 (73-92) 41 ± 5 (32-53) 11.5 ± 2 (9-14) –

61

–

–

–

SW

1.8

–

–

–

GS

0.9

–

–

–

D%

44

–

–

E%

57

–

–

H

–

56 ± 6 (46-68) 122 ± 7 (105-140) 54 ± 4 (46-63) 92 ± 6 (80-105) 25 ± 4 (21-35) 32 ± 5 (23-43) 54 ± 5 (43-62) 35 ± 5 (23-43) 1.7 ± 0.4 (1.0-2.4) 1.6 ± 0.2 (1.3-2.3) 44 ± 4 (39-49) 218 ± 3 (166-263) –

59 ± 2 (55-64) 75 ± 18 (51-116) 147 ± 12 (111-179) 67 ± 11 (52-115) 120 ± 12 (102-155) 45 ± 5 (28-60) 36 ± 7 (38-62) –

Gubernaculum length

102 ± 20 (58-121) 156 ± 15 (123-199) 67 ± 5 (56-76) 121 ± 14 (96-155) 31 ± 6 (23-41) 37 ± 6 (26-49) 71 ± 4 (64-77) 50 ± 8 (47-63) 2.0 ± 0.3 (1.5-2.7) 1.5 ± 0.3 (1.1-1.8) 40 ± 4 (30-50) 52 ± 9 (35-69) –

52 ± 2 (49-55) 140 ± 15 (115-163) 185 ± 17 (155-215) 76 ± 7 (62-89) 137 ± 14 (112-173) 48 ± 10 (33-74) 64 ± 11 (47-81) –

20 636 ± 15 (549-732) 21 ± 3 (18-28) 5.4 ± 0.6 (4.4-6.4) –

–

–

H%

–

–

–

–

–

29 ± 2 (25-33) 205 ± 31 (138-284) 14 ± 2 (11-19) 35 ± 7 (27-50)

Fig. 3. Light and scanning electron microscope photographs of female and third-stage infective juvenile specimens of Steinernema colombiense n. sp. A: Anterior end of female, lateral view showing position of excretory pore (arrow); B: Vulva, lateral view of first generation female; C: Posterior end of first generation female, lateral view; D: Vulva, lateral view of second generation female; E: Posterior end of second generation female, lateral view; F-H: Third-stage infective juvenile. F: Anterior end, lateral view, showing position of excretory pore (arrow); G: Posterior end, lateral view, showing anus position (arrow); H: Bacterial receptacle; I: Lateral field pattern at mid-body level. (Scales are based on scale bar in A: A, G, H = 24 µm; B, E, F = 32 µm; C = 38.5 µm; D = 9 µm; I = 4.5 µm.) Vol. 10(4), 2008

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Table 2. Comparison of morphometric traits of infective juveniles of Steinernema colombiense n. sp. with other morphologically similar Steinernema spp. All measurements in µm and in the form: mean (range). Species S. colombiense n. sp. S. scapterisci1 S. carpocapsae2 S. riobrave3 S. siamkayai4

L

Max. body diam.

Excretory pore

Tail

D%

E%

636 (549-732) 572 (517-609) 558 (438-650) 622 (561-701) 446 (398-495)

31 (22-36) 24 (18-30) 25 (25-30) 28 (26-30) 21 (18-24)

35 (31-40) 39 (36-38) 38 (30-60) 56 (51-64) 35 (29-38)

41 (32-53) 54 (48-60) 53 (46-61) 54 (46-59) 36 (31-41)

29 (25-33) 31 (27-40) 26 (23-28) 49 (45-55) 37 (31-43)

205 (138-284) 73 (60-80) 60 (54-66) 105 (93-111) 96 (95-112)

1 After

Nguyen and Smart (1992). Poinar (1990). 3 After Cabanillas et al. (1994). 4 After Stock et al. (1998). 2 After

fat globules, narrow lumen. Rectum long, straight; anus distinct. Genital primordium evident. Tail conoid with pointed terminus. Hyaline portion occupying ca 25-50% of tail length.

T YPE HOST AND LOCALITY The natural host is unknown as S. colombiense n. sp. (isolate SNI-0198) was collected by baiting from soil samples from a coffee plantation (Coffea arabica var. Castillo) in Maracay Experimental Station, Quimbaya County, Quindío department, Colombia. Elevation of this region is 1402 m a.s.l. and specific geographic coordinates where EPN-positive samples were obtained are: 04◦ 36 N, 75◦ 44 W. Soil in this region is acidic (pH 4.9) with low organic matter (5.9%) and N content (0.29%).

T YPE MATERIAL Holotype first generation male, five first generation paratype males, five first generation paratype females and five paratype third-stage infective juveniles deposited in the USDA Nematode Collection, Beltsville, MD, USA, and five first generation paratype males, five first generation paratype females and five paratype third-stage infective juveniles deposited in the University of California Davis Nematode Collection, Davis, CA, USA. 568

D IAGNOSIS AND RELATIONSHIPS Steinernema colombiense n. sp. is characterised by the morphometrics of the third-stage infective juvenile which has a small body length of 636 (549-732) µm, narrow body diam. (31 (22-36) µm), the position of the excretory pore (35 (31-40) µm) and a short tail (41 (32-53) µm). Additional diagnostic traits include D% = 29 (25.533), E% = 205 (138-284) and H% = 35 (27-50). First generation males can be distinguished by the morphology of the spicules and gubernaculum, the values of ratios SW = 2.0 (1.5-2.7) and GS = 1.5 (1.1-1.8) and the number and arrangement of the genital papillae. First generation females can be recognised by the presence of protruding and asymmetric vulval lips. Phylogenetic analysis of LSU sequences data placed S. colombiense n. sp. in a clade that includes four species: S. carpocapsae, S. monticolum, S. siamkayai and S. scapterisci Nguyen & Smart, 1990, all of which are characterised by having the IJ with a short body length (average = 572 µm). However, the new species can be differentiated from these taxa by several morphometric differences of the IJ and first generation male. For example, third-stage infective juveniles of the new species have a larger body size (average = 636 µm) and higher value of E% (average = 205) than S. carpocapsae, S. scapterisci and S. monticolum. First and second generation males of S. colombiense n. sp. also differ from those of S. carpocapsae, S. scapterisci and S. monticolum by the number and distribuNematology

A new Steinernema from Colombia

Fig. 4. Evidence of large subunit (LSU) ribosomal DNA lineage independence for Steinernema colombiense n. sp. based on maximum parsimony analysis. Clades are in roman numbers. Number in bold indicates bootstrap value. Numbers after species names correspond to GenBank accessions. Vol. 10(4), 2008

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Fig. 5. Evidence of internal transcribed spacer region (ITS) of ribosomal DNA lineage independence for Steinernema colombiense n. sp. based on maximum parsimony analysis. Numbers at each node indicate bootstrap values. Numbers after species names correspond to GenBank accessions. 570

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Table 3. Adjusted character distance matrix based on 28S rDNA sequence comparison between Steinernema spp. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1. S. affine – 2. S. intermedium 6 – 3. S. anatoliense 66 70 – 4. S. websteri 63 67 2 – 5. S. bicornutum 77 81 50 49 – 6. S. ceratophorum 78 80 51 50 19 – 7. S. abbasi 68 68 58 56 49 48 – 8. S. riobrave 71 75 53 53 42 47 53 – 9. S. hermaphroditum 64 70 45 43 38 43 53 40 – 10. S. scarabaei 65 71 46 44 39 44 54 41 2 – 11. Steinernema sp. 62 68 43 41 36 41 51 44 12 12 – CFVII 12. S. karii 65 69 46 44 41 42 52 41 19 19 19 – 13. S. cubanum 64 64 48 46 45 46 60 49 27 28 27 24 – 14. S. longicaudum 64 64 48 46 45 46 60 49 27 28 27 27 27 24 – 15. S. glaseri 65 67 49 47 42 45 59 46 24 25 24 23 7 7 – 16. S. arenarium 63 65 47 45 36 43 55 42 16 16 14 17 16 17 12 – 17. S. diaprepesi 59 63 42 40 35 38 50 41 13 13 11 18 20 20 17 9 – 18. S. puertoricense 52 56 43 41 34 41 51 42 16 16 16 21 19 19 18 10 9 – 19. S. kraussei 63 65 43 41 35 41 48 44 28 28 30 29 30 30 30 24 25 22 – 20. S. oregonense 63 65 43 41 35 40 48 44 27 27 27 26 28 28 27 23 24 21 3 21. S. feltiae 62 64 44 42 32 39 51 43 24 24 24 25 27 27 24 20 21 18 6 22. S. kushidai 61 65 40 38 33 38 48 39 21 21 27 26 28 28 27 21 22 19 9 23. S. rarum 55 59 47 45 45 54 52 55 41 41 41 40 40 41 39 37 36 31 31 24. S. monticolum 154 150 161 161 156 155 156 153 156 156 155 154 154 151 148 147 147 151 153 25. S. scapterisci 153 149 160 160 155 154 155 152 155 155 154 152 153 153 150 147 146 150 152 26. S. carpocapsae 153 149 160 160 155 154 155 152 155 155 154 152 153 152 150 147 146 150 147 27. S. siamkayai 157 153 164 164 159 158 159 154 159 159 158 156 157 157 154 151 150 154 156 28. S. colombiense 157 153 164 164 159 160 157 160 159 159 158 156 157 154 150 151 150 154 155 n. sp. 29. C. elegans 173 175 166 165 178 173 173 170 166 166 161 163 159 158 160 162 162 163 166

Vol. 10(4), 2008 – 7 33 153 152 150 156 155

– 30 154 154 152 157 156

– 158 – 153 157 1 – 153 152 153 1 – 161 5 4 4 – 157 17 16 16 20

– 166 167 166 168 112 111 111 115 116

– 3 8 30 154 153 146 157 156

−

20 21 22 23 24 25 26 27 28 29

A new Steinernema from Colombia

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Table 4. Adjusted character distance matrix based on ITS rDNA sequence comparison between Steinernema spp. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1. S. tami – 2. S. siamkayai 5 – 3. S. colombiense 10 8 – n. sp. 4. S. carpocapsae 11 9 3 – 5. S. scapterisci 15 14 7 8 – 6. S. pakistanense 40 40 37 37 37 – 7. S. riobrave 41 36 35 35 36 37 – 8. S. abbasi 41 38 35 35 37 35 31 – 9. S. ceratophorum 31 28 26 26 29 29 27 21 – 10. S. bicornutum 30 27 25 25 25 28 28 28 25 9 – 11. S. sichuanense 42 39 36 34 38 37 36 30 28 31 – 12. S. affine 42 37 36 36 38 37 36 34 26 29 6 – 13. S. intermedium 41 36 35 35 37 38 37 34 28 31 9 5 – 14. S. beddingi 42 39 36 36 38 42 39 40 31 35 12 10 11 – 15. S. neocurtillae 37 32 31 31 32 37 34 38 25 26 33 32 31 34 – 16. S. cubanum 35 33 28 28 29 35 35 30 22 24 29 30 26 29 28 – 17. S. glaseri 33 31 26 26 27 35 35 28 20 22 29 30 26 29 26 2 – 18. S. arenarium 34 32 27 27 28 36 35 28 20 22 27 28 24 28 26 6 4 – 19. S. guangdongense 35 33 28 28 25 34 33 27 19 21 29 28 28 29 26 8 6 6 – 20. S. longicaudum 32 30 25 25 25 33 32 25 18 20 28 28 28 29 26 7 5 7 2 – 21. S. diaprepesi 36 34 29 29 26 33 35 29 19 21 29 30 28 29 24 9 7 8 8 7 – 22. S. khoisanae 39 37 32 32 29 35 32 31 24 25 30 29 27 30 27 9 7 7 7 7 8 23. S. aciari 34 32 26 26 27 31 33 28 18 20 28 28 27 28 25 9 7 10 10 8 6 24. S. karii 33 31 26 26 27 35 34 28 20 22 28 29 27 26 21 13 11 13 12 11 7 25. S. hebeiense 30 26 23 22 25 29 24 22 18 18 31 26 26 27 22 22 20 18 14 15 19 26. S. sangi 30 29 21 20 24 29 29 23 17 19 31 29 28 27 21 14 12 14 9 8 11 27. S. feltiae 30 26 23 23 23 31 28 25 17 17 34 29 29 30 20 19 17 17 13 12 16 28. S. kraussei 31 29 24 24 25 31 31 23 19 19 32 31 31 30 24 16 16 18 12 10 17 29. S. oregonense 31 27 24 24 25 33 30 27 19 19 34 29 29 30 21 18 16 16 10 11 15 30. S. monticolum 37 33 30 29 31 35 32 34 27 26 39 36 36 38 26 27 25 26 22 21 23 31. S. rarum 35 35 29 29 31 33 35 33 29 28 37 37 35 37 34 26 23 22 22 23 23 32. C. elegans 135 137 141 137 144 134 144 133 136 141 154 159 160 155 141 139 137 140 135 134 135

572 – 12 11 17 13 16 17 13 23 20 138

– 6 17 10 15 15 14 22 22 136

– 20 11 18 16 17 25 24 133

– 9 – 6 5 – 9 4 6 6 6 4 12 9 16 26 23 25 109 110 134

– 6 18 23 135

– 16 – 22 23 – 134 135 117 –

22 23 24 25 26 27 28 29 30 31 32

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A new Steinernema from Colombia

tion of genital papillae and by the overall morphology of the spicules and gubernaculum (see Table 2). In the new species the spicules are characterised by having a rhomboidal manubrium, with a rounded, ‘knob-like’, protrusion. The gubernaculum is also unique in its morphology with the manubrium curved ventrally and bifurcate. In addition to these qualitative differences, males of the new species can be differentiated by the SW value (average = 2.0 and 1.7 for first and second generation males, respectively; see Table 2). Both first and second generation females of S. colombiense n. sp. are characterised by having protruding vulval lips. This feature is shared with females of S. carpocapsae. Females of S. monticolum lack protruding vulval lips and S. siamkayai and S. scapterisci have only slightly protruding vulval lips. Both first and second generation females of the new species lack a tail mucro, whereas S. carpocapsae, S. scapterisci and S. monticolum may or may not have a mucro.

C ROSS - HYBRIDISATION TESTS Cross-hybridisation assays between males and females of S. colombiense n. sp., with S. carpocapsae, S. siamkayai and S. scapterisci yielded no progeny. In the controls, offspring were produced in all self-crossed species. No progeny were observed in the single female control plates.

Acknowledgements We are thankful to Y. Flores-Lara for assistance with the scanning electron microscopy images. The authors also express their gratitude to Cenicafé research leaders G. Cadena-Gómez, A.E. Bustillo-P., D. Rodríguez and C. Quintero for their support on the EPN project. This study was funded in part by a Colombia Ministry of the Environment grant (Project ENT1801) and The National Federation of Colombian Coffee Growers, Coffee Research Centre (Cenicafé).

M OLECULAR CHARACTERISATION AND PHYLOGENETIC ANALYSIS

Maximum parsimony (MP) analysis of the 28S rDNA sequences set yielded 399 parsimony informative characters and produced 15 equally parsimonious trees (Fig. 4) with a tree length of 1096 steps (CI = 0.64). MP analysis placed S. colombiense n. sp. as a relative of Clade I. This clade comprises Steinernema spp. known to have infective juveniles with small body size (average < 600 µm). Association of the new species with this clade (Fig. 5) was strongly supported by bootstrap resampling (100%). Pairwise distances (Table 3) also shows that S. colombiense n. sp. differs from closely related species by 16-20 base pairs (numbers in bold font). Analysis of ITS rDNA sequences yielded two equally parsimonious trees with a tree length of 3358 steps (CI = 0.45) (Fig. 5). A total of 615 parsimony informative traits was depicted for this analysis. Steinernema colombiense n. sp. was placed as a member of a clade encompassing species with small IJ, including S. tami Luc, Nguyen, Reid & Spiridonov, 2000, S. carpocapsae, S. scapterisci and S. siamkayai. The new species was considered to be a sister taxon of S. carpocapsae from which it differs by three base pairs (numbers in bold font). Affiliation of these two species was strongly supported by bootstrap resampling (100%). These observations and those from the 28S rDNA analysis provide further evidence for the distinctness of this species. Vol. 10(4), 2008

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