Morphological and molecular characterisation of Steinernema hermaphroditum n. sp. (Nematoda: Steinernematidae), an entomopathogenic nematode from Indonesia, and its phylogenetic relationships with other members of the genus

Nema ology, 2004, Vol. 6(3), 401-412 Mo phological and molecula cha ac e isa ion o S eine nema he maph odi um n. sp. (Nema oda: S eine nema idae), an en omopa hogenic nema ode om Indonesia, and i s phylogene ic ela ionships wi h o he membe s o he genus S. Pa icia STOCK 1,∗, Ch is ine T. GRIFFIN 2and Rani CHAERANI 3 1Depa men o Plan Pa hology, Uni e si y o A izona, Fo bes 204. 1140 E., Sou h Campus D i e, Tucson, AZ 85721-0036, USA 2Depa men o Biology, Na ional Uni e si y o I eland Maynoo h, Maynoo h, Co. Kilda e, I eland 3BORIF, 3A Jalan Ten a a Pelaja , Bogo 16114, Indonesia Recei ed: 16 Feb ua y 2004; e ised: 22 Ap il 2004 Accep ed o publica ion: 26 Ap il 2004 Summa y – S eine nema he maph odi um n. sp., a new en omopa hogenic nema ode isola ed om soil samples in he Moluccan islands, Indonesia, is desc ibed. Mo phological obse a ions as well as biological e idence (c oss-hyb idisa ion s udies) indica e he dis inc ness o S. he maph odi um n. sp. om o he S eine nema spp. This new species is cha ac e ised by he p esence o he maph odi es in he i s adul gene a ion. Key mo phological diagnos ic cha ac e s include: a digi a e ail wi h a muc o and a glandula spe ma heca illed wi h spe m in he i s gene a ion he maph odi e; he alue o D%; he mo phology o he male spicules and gube naculum and he numbe and a angemen o he geni al papillae; he alues o D%, E% and he pa e n o he la e al ield o he hi d-s age in ec i e ju enile. Addi ionally, molecula e idence ob ained om ITS DNA RFLP p o iles, 28S DNA sequence analyses, and phylogene ic econs uc ion p o ide u he e idence o es ablish his nema ode as a new species. Keywo ds – desc ip ion, he maph odi ism, molecula , phylogeny, axonomy. En omopa hogenic nema odes (EPN) in he amily S eine nema idae Chi wood & Chi wood a e insec pa - asi es capable o in ec ing a b oad ange o insec species. They ha e been used as biological con ol agen s o in- sec pes s in a a ie y o c ops (Gaugle & Kaya, 1990; Kaya & Gaugle , 1993). The hi d-s age ju enile (IJ) is he only ee-li ing s age o hese nema odes and is com- monly ound in soil and epigeal habi a s by bai ing wi h li e insec s (Bedding & Akhu s , 1975). The IJ en e a li ing hos and elease symbio ic bac e ia o he genus Xeno habdus Poina & Thomas. The bac e ia kill he hos and he de eloping nema odes eed on he insec cada e and diges ed issues. The nema odes go h ough wo o mo e gene a ions, p oducing new IJ which eme ge in o he soil as hos esou ces a e deple ed. The S eine nema idae cu en ly comp ises wo gene a, S eine nema T a assos, 1927 wi h mo e han 35 ecog- nised species, and Neos eine nema Nguyen & Sma , ∗Co esponding au ho , e-mail: sps [email protected] izona.edu 1994, wi h only one species, N. longicu icauda Nguyen & Sma , 1994. The mos upda ed biogeog aphic accoun indica es ha hese nema odes ha e been isola ed om all con inen s (excep An a c ica) and almos all egions o he wo ld (Hominick, 2002). The numbe o EPN su - eys in opical and sub opical egions has signi ican ly inc eased du ing he las decade. T opical sou heas coun- ies in Asia, such as S i Lanka (Ama asinghe e al., 1994), Malaysia (Masson e al., 1996), Vie nam (Nguyen e al., 1999; Phan e al., 2001a, b) and Indonesia (G i in e al., 2000), ha e been ex ensi ely su eyed o EPN. As a esul , se e al new species ha e been eco e ed and de- sc ibed (S ock e al., 1998; Luc e al., 2000; Phan e al., 2001a, b). In a su ey conduc ed by G i in e al. (2000) in Indonesia, se e al S eine nema isola es we e isola ed in many o he islands ha comp ise he Moluccan a chipelago. The T87 s ain, o which se e al isola es ©Koninklijke B ill NV, Leiden, 2004 401 Also a ailable online - www.b ill.nl S.P. S ock e al. we e collec ed, mainly om he islands o Ambon and Se am, displayed an RFLP p o ile ha clea ly iden i ied hem as a S eine nema sp. The RFLP p o ile o his species mos closely esembled ha o S. longicaudum Shen & Wang (G i in e al., 2000). Howe e , du ing c oss-b eeding s udies o he T87 isola es, unipa en al ep oduc ion e idence was ound. The p esence o spe m in he ep oduc i e ac o unma ed i s gene a ion emales indica ed ha he mode o ep oduc ion o his species is by he maph odi ism (G i in e al., 2001). Acco ding o his s udy, a single IJ o he T87 isola e can colonise an insec and p oduce p ogeny. App oxima ely 1% o he IJ de eloped in o males and males we e also p esen in he second adul gene a ion, bu a a e y low le el (1-6%) (G i in e al., 2001). In addi ion o hese obse a ions, subsequen mo pho- logical s udies and molecula cha ac e isa ion based on sequence analysis om he la ge subuni (28S) o DNA p o ided u he e idence o suppo he T87 isola e as being a new S eine nema species. I is desc ibed and il- lus a ed below. Ma e ials and me hods ORIGIN AND PROPAGATION OF THE ISOLATES Isola es we e eco e ed om soil samples unde di - e en ege a ion co e s (i.e., sc ub and ees) along he coas al inge o he islands o Se am (Kamal) and Am- bon (Waai) in he Indonesian a chipelago (G i in e al., 2000). Nema odes we e p opaga ed in las -ins a la ae o he wax mo h, Galle ia mellonella (L.) a 28-30◦C. Eme ging IJ we e ha es ed in modi ied Whi e aps ollowing p o- cedu es desc ibed by Kaya and S ock (1997). Ha es ed IJ we e washed h ee imes by sedimen a ion in dis illed wa e and s o ed in wa e a 20◦C. MORPHOLOGICAL CHARACTERISATION Ligh mic oscopy s udies Twen y specimens om each s age (adul s and IJ) we e andomly collec ed om en G. mellonella cada - e s. Nema odes we e examined li e o hea - elaxed in Ringe ’s solu ion a 60◦C.Nema odeswe e ixedin i- e hanolamine o malin (TAF) (Cou ney e al., 1955) and p ocessed o anhyd ous glyce ine o moun ing (Sein- ho s , 1959). Specimens we e moun ed on glass slides, he co e slip being suppo ed by glass ods o a oid la en- ing. Obse a ions we e made om li e and moun ed spec- imens using an OlympusBX60 mic oscope equipped wi h di e en ial in e e ence con as op ics. Specimen mea- su emen s we e made using Scion Image so wa e (F ed- e ick, MD, USA) calib a ed wi h a s age mic ome e . Se- lec ion o mo phome ic cha ac e s was done acco ding o Hominick e al. (1997). Illus a ions we e p epa ed om digi ised came a lucida images. The ollowing abb e ia ions ha e been used in he ex o ables, ABD =anal o cloacal body diame e , EP = exc e o y po e posi ion, ES =pha ynx leng h, GS = GuL/SpL, GuL =gube naculum leng h, H =hyaline ail leng h; H% =H as % o TL; MBD =maximum body diame e , ML =muc o leng h, NR =ne e ing posi ion, a io a =L/MBD, a io b =L/ES, a io c =L/TL, D% = EP/ES ×100, E% =EP/TL ×100, SpL =spicule leng h (measu ed along he cu ed median line), S L =s oma leng h, S W =s oma diame e , SW =SpL/ABD, TL = ail leng h. Scanning elec on mic oscopy s udies Adul s we e dissec ed om G. mellonella la ae in Ringe ’s solu ion (pH 7.3). They we e insed h ee imes o 5 min in Ringe ’s solu ion. Th ee-day-old IJ we e insed o h ee imes 15 min in 0.05% NaCl. All ne- ma odes we e elaxed and killed by hea ing in a wa e - ba h (60◦C) o 2-3 min and we e hen ixed in 8% glu- a aldehyde – 25% EM g ade (dilu ed in Ringe ) o 2 h a oom empe a u e. Fixed nema odes we e insed in dis- illed wa e h ee imes, pos - ixed in OsO4 o 1 h, insed in dis illed wa e again and dehyd a ed a 15 min in e als h ough 30, 50, 70, 90, 95 and 100% e hanol. They we e hen c i ical poin d ied in liquid CO2, moun ed on SEM s ubs, coa ed wi h gold and scanned using an SES DS- 130 equipped wi h a digi al image came a and Imagecap 1000™ so wa e (Woods ock, GA, USA). An accele a ing ol age o 15 kV was used o all obse a ions. HYBRIDISATION TESTS Rep oduc i e compa ibili y o he new species was es ed using he ollowing S eine nema spp.: S. sca abaei S ock & Koppenhö e , 2003; S. longicaudum Shen & Wang, 1992; and an unknown S eine nema sp. om In- donesia (isola e T29). Thi d-s age ju eniles p oduced by i s gene a ion adul s (ei he by amphimixis o pa heno- genesis) we e su ace s e ilised in 0.4% hyamine and washed i e imes in s e ile dis illed wa e . Single ju e- niles we e ans e ed o hanging d ops o haemolymph 402 Nema ology S eine nema he maph odi um n. sp. om Indonesia acco ding o p ocedu es desc ibed by Kaya and S ock (1997).A o al o 20 pai s/nema odecombina ion we e es- ablished by ans e ing one male in o a d op con aining a mo phological emale once hey could be di e en ia ed o each o he es ed combina ions. Con ols consis ed o hanging d ops wi h adul s o he same species. MOLECULAR CHARACTERISATION AND PHYLOGENETIC ANALYSIS Molecula cha ac e isa ion o he new species was done by analysis o la ge-subuni o ibosomal DNA (LSU o 28S DNA) sequences. An exis ing lib a y o mo e ha 30 S eine nema spp. was used o sequence compa isons and phylogene ic in e p e a ion. To al genomic DNA iso- la ion, PCR ampli ica ion ( eac ion, cycling condi ions and p ime s) and sequence analysis ollowed p o ocols desc ibed by S ock e al. (2001). Phylogene ic analysis (maximum pa simony analysis) o LSU sequences was conduc ed using PAUP* e - sion 4.0 (Swo o d, 2001) ollowing c i e ia desc ibed by S ock e al. (2001). Ribosomal sequences o S. he - maph odi um n. sp. we e deposi ed in GenBank unde he accession numbe AY598358. S eine nema he maph odi um*n. sp. (Figs 1-3) MEASUREMENTS See Table 1. DESCRIPTION He maph odi e Body C-shaped when hea - elaxed. Cu icle smoo h un- de ligh mic oscopy, bu wi h ine ans e se s iae isi- ble unde SEM. La e al ield and phasmids inconspicuous. Head unca ed o sligh ly ound, con inuous wi h body. Six lips uni ed bu wi h ips dis inc , each bea ing one labial papilla. Fou cephalic papillae. Amphidial ape u es small, loca ed pos e io o la e al labial papillae. S oma educed (cheilo, gymno- and s egos om es igial), sho and wide, wi h inconspicuous scle o ised walls. Pha ynx se o om in es ine, wi h a cylind ical p oco pus, a non- al a ed and sligh ly swollen me aco pus, a dis inc is h- mus and a py i o m basal bulb con aining educed al e. *The speci ic epi he is de i ed om he p esence o he maph odi es in he i s adul gene a ion. Ne e- ing usually su ounding is hmus o an e io pa o basal bulb. Exc e o y po e opening ci cula , loca ed a pos e io hi d o me aco pus, jus an e io o ne e- ing. O a ies opposed, e lexed in do sal posi ion; o iduc well de eloped; glandula spe ma heca illed wi h spe ma o- zoa; u e us in en al posi ion. Vagina sho , wi h mus- cula walls. Vul a mo e o less median in posi ion wi h p o uding and asymme ic ul a lips (an e io lip la ge han he pos e io one). Epip ygma absen . Tail digi a e, muc o p esen in 60% o he examined specimens. Pos - anal swelling p esen . Male Cu icle, lip egion, s oma and pha yngeal egion as in i s gene a ion he maph odi e. Body cu ed pos e i- o ly, J-shaped when hea - elaxed. Single e lexed es is, consis ing o ge minal g ow h zone leading o seminal esicle. Vas de e ens wi h inconspicuous walls. Spicules pai ed,symme ical, cu ed,wi h och e-b owncolo a ion. Manub ium ec angula , wide han long, calomus sho , lamina wide wi h wo ibs, os um p esen . Velum sho , ex ending om os um o dis al en al opening. Gube - naculum boa -shaped in la e al iew, ca wo hi ds leng h o spicules. Cuneus long, needle-shaped, poin ed pos e- io ly. Geni al papillae numbe ing 21 ( en pai s and one single): six p ecloacal sub en al pai s, one single en- al p ecloacal papilla (loca ed be ween p ecloacal pai s 5 and 6), one subdo sal p ecloacal pai , one pos cloacal sub- do sal pai , one sub en al pos cloacal pai , one e minal pos cloacal pai . Second gene a ion males a e simila o i s gene a ion males, excep o body leng h and diame- e , posi ion o exc e o y po e (mo e an e io ly loca ed in second gene a ion), and size o spicules and gube nacu- lum. Female (second gene a ion) Body C-shaped when hea - elaxed. Cu icle, lip egion, s oma and pha yngeal egion as in i s gene a ion he - maph odi e. O a ies pai ed, opposed and e lexed in do - sal posi ion; o iduc well de eloped; spe ma heca absen ; u e us in en al posi ion. Vagina sho , wi h muscula walls. Vul a mo e o less median in posi ion wi h non- p o uding o sligh ly p o uding ul a lips. Epip ygma absen . Tail ound-conoid, wi hou a muc o. Pos -anal swelling p esen o absen . Thi d-s age in ec i e ju enile Body slende , ape ing egula ly om base o pha ynx o an e io end and om anus o e minus. Lip egion Vol. 6(3), 2004 403 S.P. S ock e al. Fig. 1. S eine nema he maph odi um n. sp. A-D: Fi s -gene a ion he maph odi e. A: In o o; B: An e io end, la e al iew; C: Vul al lips, la e al iew; D: Tail, la e al iew. E, F: Second-gene a ion emale. E: Tail, la e al iew; F: Vul al lips, la e al iew – G-J: Fi s gene a ion male: G-J. G: In o o; H: Spicule, la e al iew; I: Gube naculum, la e al iew; J: Tail, la e al iew – K, L: Thi d-s age in ec i e ju enile. K: An e io end, la e al iew; L: Tail, la e al iew. (Scale ba s: A, G =100 µm; B-E, J, K, L =50 µm; F, H, I =25 µm.) 404 Nema ology S eine nema he maph odi um n. sp. om Indonesia Fig. 2. S eine nema he maph odi um n. sp. A-D: He maph odi e. An e io end (la e al iew) showing exc e o y po e loca ion (a ow); B: An e io pa o ep oduc i e (o : o a y, sp: spe ma heca, u: u e us); C: Vul a (al: an e io lip, pl: pos e io lip); D: Tail (la e al iew) – E: Second gene a ion emale ul a (la e al iew). (All scales a e based on he scale ba in A. A =43 µm, B =17 µm, C = 10 µm, D =32 µm, E =15 µm.) Vol. 6(3), 2004 405 S.P. S ock e al. Fig. 3. S eine nema he maph odi um n. sp. A-D: Fi s gene a ion male. A: Pos e io end showing dis ibu ion o geni al papillae (p : p ecloacal, : en al, po: pos cloacal; B: Dis ibu ion o geni al papillae; C: Spicule (la e al iew); D: Gube naculum (do sal iew) – E, F: Thi d-s age in ec i e ju enile. E: Tail ( en al iew) showing anus posi ion (a ow); F: La e al ield pa e n (midbody le el). (All scales a e based on he scale ba in A. A =35 µm, B =33 µm, C =22 µm, D =20 µm, E =23 µm, F =10 µm.) 406 Nema ology S eine nema he maph odi um n. sp. om Indonesia Table 1. Mo phome ics o S eine nema he maph odi um n. sp. All measu emen s a e in µm and a e in he o m: mean ±s anda d de ia ion ( ange). Male He maph odi e Female Thi d-s age in ec i e ju enile 1s 2nd 1s 2nd gene a ion gene a ion gene a ion gene a ion Holo ype Pa a ypes Pa a ypes Allo ype Pa a ypes Pa a ypes Pa a ypes n1920192020 L 1550 1481 ±19 845 ±11 6345 6550 ±497 5129 ±125 928.5±98.5 (1115-1560) (698-975) (5843-7154) (3874-5623) (700-1100) a– – – – – –29±3.5 (23-35) b– – – – – –6.2±0.5 (5.6-7.1) c– – – – – –10.6±0.7 (9.3-11.7) V – – – 52.5 55.5±352±0.5– (51-61) (50-53) EP 75 75 ±553±4 125 124 ±887±765±2 (65-80) (49-60) (110-134) (79-98) (62.5-68) NR 117 119.5±4 116 ±4 141 147 ±6 126 ±5 102.5±2 (114-125) (100-119) (134-154) (115-133) (95-112.5) TL 32.5 33.5±221.5±1.566 69±258±1.577±6 (28-35) (19-25) (65-72) (50-62) (65-82.5) ML – – – 1.5 2 ±2.5– – 1.5-2.5 ABD 45 44 ±1.527±16870±257±219±2 (40-51) (28-35) (66-72) (48-61) (16-24) SpL 66 68 ±260±1– – – (65-70) (55-64) GuL 50 48 ±1.541±1.5– – – – (47-50) (39-44) SW 1.33 1.5±0.51.8±0.5– – – – (1.4-1.6) (1.7-1.9) GS 0.75 0.71 ±0.01 0.65 ±0.02 – – – – (0.70-0.75) (0.60-0.70) D% 48 ±540±2––50±2.5 (44-50) (39-43) (47-55) E% – – – – – 85 ±7 (76-100) H– – – – – –33.4±2 (31-40) H% – – – – – – 51 ±1 (49-53) smoo h; mou h closed. Cu icle wi h ans e se s iae; la e al ield dis inc wi h eigh equidis an longi udinal idges (i.e., nine lines) in mid-body egion. Pha ynx long, na ow. Ne e- ing loca ed is hmus le el. Exc e o y po e loca ed ca middle o pha ynx. Basal bulb al a e. Ca dia p esen . An e io po ion o in es ine wi h do sally displaced pouch con aining symbio ic bac e ia. Lumen o in es ine na ow; ec um long; anus dis inc . Geni al p imo dium e iden . Tail conoid wi h poin ed e minus. Hyaline comp ising ca hal he ail leng h. Vol. 6(3), 2004 407 S.P. S ock e al. Table 2. Compa ison o mo phome ics (mean and ange) o hi d-s age in ec i e ju eniles and i s gene a ion males o S eine nema he maph odi um n. sp. and o he mo phologically simila S eine nema spp. All measu emen s a e in µm. Species Thi d-s age in ec i e ju enile Fi s gene a ion males L MBD EP TL D% E% LF SPL GUL SW D% M S. he maph odi um 928.5 28.5 65 77 50 85 8 68 48 1.5 48 A n. sp. (700-955) (25-32.5) (62.5-68) (65-82.5) (47-55) (76-100) (65-70) (47-50) (1.4-1.6) (44-50) S. el iae 1849 26 62 81 45 78 8 70 41 1.13 60 P (736-950) (22-29) (53-67) (70-92) (42-51) (69-86) (65-77) (34-47) (0.99-1.3) NA S. hanhi 2851 31 75 63 58 119 8 72 49 1.8 73 A (720-960) (27-39) (68-84) (52-72) (52-67) (101-138) (67-78) (40-56) (1.5-2.1) (64-82) S. neocu illae 3885 34 18 80 12 23 6 58 52 1.43 19 P (741-988) (28-42) (14-22) (64-97) (10-15) (18-30) (52-64) (44-59) (1.18-1.64) (13.26) S. ka ii 4932 33 74 74 57 96 8 83 57 NA 66 A (876-982) (31-35) (68-80) (64-80) NA NA (73-91) (42-64) (57-78) S. k aussei 5957 33 63 79 47 80 8 55 33 1.10 53 P (797-1102) (30-36) (50-66) (63-86) NA NA (52-57) (23-38) NA NA S. o egonense 6980 34 66 70 50 100 6-8 71 56 1.51 73 A (820-1110) (28-38) (60-72) (64-78) (40-60) (90-110) (65-73) (52-59) NA (64-75) S. loci 2986 37 80 75 57 107 8 71 46 1.9 73 A (896-1072) (30-45) (71-86) (66-83) (52-63) (94-120) (60-80) (40-52) (1.7-2.1) (61-80) S. sca abaei 7918 31 77 76 60 100 8 74 42 1.7 64 A (890-956) (25-37) (72-81.5) (71-80) (50-65) (90-110) (63-79) (33-47) (1.5-2.0) (51-75) A: absen ; LF: la e al ield; M: muc o; NA: no a ailable; P: p esen . 1a e Poina , 1990; 2a e Phan e al., 2001a; 3a e Nguyen & Sma , 1992; 4a e Wa u u e al., 1997; 5a e M ᡠcek e al., 1994; 6a e Liu & Be y, 1996; 7a e S ock & Koppenhö e , 2003. TYPE HOST AND LOCALITY Hos unknown in na u e as ma e ial collec ed om bai -insec (G. mellonella la ae). The ype isola e was eco e ed om sc ubs and ees along he coas al s ip o Kamal, Se am island, Indonesia (G i in e al., 2000). OTHER LOCALITIES Banana/he bs and g ass/he bs/coconu habi a s in Waai, Ambon island, Indonesia (G i in e al., 2000). TYPE MATERIAL Holo ype male second gene a ion, allo ype he maph o- di e i s gene a ion, i e pa a ype he maph odi es i s gene a ion, i e pa a ype second gene a ion emales, ou pa a ype second gene a ion males, i e pa a ype hi d s age in ec i e ju eniles deposi ed in he Uni e si y o Cali o nia Nema ode Collec ion, Da is, CA, USA; i e pa a ype i s gene a ion males, i e pa a ype i s gene a- ion emales; i e pa a ype hi d-s age in ec i e ju eniles deposi ed in USDA Nema ode Collec ion, Bel s ille, MD, USA. HYBRIDISATION TESTS A emp s o c oss-hyb idise S. he maph odi um sp. n. wi h S. sca abaei,S. longicaudum and S eine nema sp. (T29 isola e) yielded no p ogeny. Con ol c osses using indi iduals o he same species p oduced iable o sp ing. Only 60% o he d ops (12 ou o 20) wi h couples o S. he maph odi um n. sp. p oduced p ogeny. MOLECULAR CHARACTERISATION AND PHYLOGENETIC RELATIONSHIPS Maximum pa simony (MP) analysis o LSU sequence da a yielded 371 pa simony in o ma i e cha ac e s and p oduced wo mos pa simonious ees wi h a ee leng h o 1080 s eps (CI =0.60). A s ic consensus (50% majo i y- ule) ee wi h he co esponding boo s ap al- ues is p esen ed in Figu e 4. 408 Nema ology S eine nema he maph odi um n. sp. om Indonesia Fig. 4. E idence o la ge subuni (LSU) ibosomal DNA lineage independence o S eine nema he maph odi um n. sp. based on maximum pa simony analysis. Numbe s in bold e e o boo s ap alues. S eine nema he maph odi um n. sp. was placed in a clade ha includes long-IJ S eine nema species also known as he glase i-g oup. This clade comp ises S. glase i (S eine , 1929); S. a ena ium (A yukho sky, 1967); S. pue o icense Román & Figue oa, 1994; S. cubanum M ᡠcek, He nandez & Boema e, 1994; S. longi- caudum;S. ka ii Wa u u, Hun & Reid, 1997; S. sca abaei, and wo uniden i ied S eine nema species: T29 om In- donesia and, CF VII om Cali o nia, USA. The new S eine nema species was depic ed as a sis e axon o S. sca abaei, a species isola ed om sca ab bee le la ae in New Je sey, USA (S ock & Koppenhö e , 2003). E olu- iona y ela ionships be ween hese wo axa was s ongly suppo ed by boo s ap esampling (100%). Pai wise dis- ance ma ix indica ed ha S. he maph odi um n. sp. and S. sca abaei di e ed in one cha ac e (Table 3). DIAGNOSIS AND RELATIONSHIPS S eine nema he maph odi um n. sp. is cha ac e ised by he p esence o he maph odi es in he i s adul gene a ion, and he low p opo ion o males in bo h adul gene a ions (a e age: 1 and 5-6%, espec i ely (G i in e al., 2001)). He maph odi es o he new species a e cha ac e ised by ha ing asymme ic ul al lips and he p esence o a spe ma heca illed wi h spe m. Males a e cha ac e ised by he numbe and dis ibu ion o he male geni al papillae (21 papillae: se en p ecloacal pai s, h ee pos cloacal pai s and a single en al papilla), by ha ing a non-muc ona e ail and by he mo phology o he spicules and gube naculum. Thi d-s age in ec i e ju eniles a e dis inguished by he la e al ield pa e n (eigh equidis an idges a he midbody le el) and he hyaline po ion, which occupies ca 50% o he ail leng h. S eine nema he maph odi um n. sp. is mo phologically mos simila o a g oup o species placed in he el iae- g oup (Table 2) which a e cha ac e ised by he size o he hi d-s age in ec i e ju eniles (a e age be ween 800-1000 µm), and by he p esence o eigh idges a he midbody le el (excep o S. neocu illae and S. o egonense Liu & Be y, 1996). Howe e , he new species can be sepa a ed om o he membe s o he el iae-g oup by a combina ion o mo phological ai s as well as by molecula and biological cha ac e is ics. In ec i e ju eniles o S. he maph odi um n. sp. esem- ble h ee species o he el iae-g oup iz.S. el iae (Filip- je , 1934), S. o egonense and S. k aussei (S eine , 1923) in he mo phome ic alues o all diagnos ic ea u es (see Table 2). Howe e , males o he new species can be sep- a a ed om hese h ee species by he mo phology o he spicules and gube naculum. Fo example, when com- pa ed o S. el iae and S. k aussei, he spicules in S. he maph odi um n. sp. ha e a s ou e and mo e ec an- gula manub ium and a sho e and wide lamina. Ad- di ionally, he gube naculum in he new species has a longe cuneus han ha p esen in hese wo species. The spicules in S. o egonense ha e a homboid-shaped and longe manub ium han ha o S. he maph odi um n. sp. Also, he spicule lamina in S. o egonense is na owe and wi h a mo e p onounced cu a u e han ha o he new species. In addi ion o his, males o S. he maph odi um n. sp. lack a ail muc o which is p esen in S. el iae and S. k aussei. The new species can be sepa a ed om S. hanhi Phan, Nguyen & Moens, 2001 by ha ing in ec i e ju eniles wi h a longe ail (see Table 2) and by he lowe mean alue o E% which is 85 ±7 (76-100) s 119 ±8 (101- Vol. 6(3), 2004 409