Morphological characterisation of three isolates of Heterorhabditis Poinar, 1976 from the "Irish group" (Nematoda: Rhabditida: Heterorhabditidae)and additional evidence supporting their recognition as a distinct species, H. downesi n. sp

Sys ema ic Pa asi ology 51: 95–106, 2002. © 2002 Kluwe Academic Publishe s. P in ed in he Ne he lands. 95 Mo phological cha ac e isa ion o h ee isola es o He e o habdi is Poina , 1976 om he ‘I ish g oup’ (Nema oda: Rhabdi ida: He e o habdi idae) and addi ional e idence suppo ing hei ecogni ion as a dis inc species, H. downesi n. sp. S. Pa icia S ock1∗, Ch is ine T. G i in2&AnnM.Bu nell 2 1Depa men o Nema ology, Uni e si y o Cali o nia Da is. One Shields A e., Da is, CA 95616-8668, USA 2Ins i u e o Bioenginee ing and Ag oecology, Depa men o Biology, Na ional Uni e si y o I eland, Maynoo h, Co. Kilda e, I eland ∗P esen add ess: Depa men o Plan Pa hology, Uni e si y o A izona, 1140 E. Sou h Campus D i e, Tucson, AZ 85721-0036, USA Accep ed o publica ion 19 h Ma ch, 2001 Abs ac The mo phological a ia ion o h ee ep esen a i e isola es o he ‘I ish g oup’ o He e o habdi is was examined. Fi s gene a ion he maph odi es we e cha ac e ised by ha ing a blun and muc ona e ail. Females (second gene - a ion) and hi d-s age in ec i e ju eniles we e also dis inguished by he mo phology o he ail and he p esence o a e ac ile p ojec ion in he ail ip. Males we e cha ac e ised by he posi ion o he exc e o y po e and by he alue o a io SW. These mo phological ea u es do no i he desc ip ion o cu en ly ecognised He e o habdi is species, and p o ide addi ional e idence in suppo o he conside a ion o he I ish g oup as a new species. A desc ip ion o his species, as H. downesi n. sp., is p o ided. In oduc ion En omopa hogenic nema odes o he amily He - e o habdi idae Poina , 1976 a e obliga e and le hal pa asi es o insec s. The only ee-li ing s age is he hi d-s age in ec i e ju enile (IJ), which is symbio - ically associa ed wi h en e ic bac e ia o he genus Pho o habdus Boema e e al., 1993. The IJs pene a e in o he haemocoel o he hos ei he om he gu o by ab ading he in e segmen al memb anes o he in- sec by using a do sal oo h. Once in he haemocoel o he insec , he IJs elease he bac e ial cells, which mul iply and diges he hos issues, he eby p o id- ing sui able nu ien condi ions o nema ode g ow h and de elopmen . The insec hos dies apidly, usually wi hin 2 days o he ini ial pene a ion o he nema- odes. The nema odes esume de elopmen , moul o he J4 s age and each adul hood wi hin h ee days (in H. bac e iopho a Poina , 1976) when cul u ed in i o in la ae o he g ea e wax mo h Galle ia mellonella a 23 ◦C (Wang & Bedding, 1996). Nema ode ep o- duc ion con inues o a leas wo gene a ions un il he nu ien s a us o he cada e de e io a es, whe eupon adul de elopmen is supp essed and IJs accumula e and eme ge in o he soil whe e hey may su i e o se e al mon hs in he absence o a sui able hos . He e o habdi ids a e e ec i e biological con ol agen s o se e al insec pes s (Be y e al., 1997; Ge - i sen e al., 1998; Shields e al., 1999; Long e al., 2000; Mannion e al., 2000). Thus, in e es in s udying hese nema odes has inc eased d ama ically in ecen yea s, no only o add ess aspec s ela ed o biological con ol, bu also ega ding basic esea ch a eas such as ecology, biodi e si y, e olu ion, biochemis y, sym- biosis and molecula gene ics ( e iew by Bu nell & S ock, 2000). The He e o habdi idae cu en ly comp ise one genus, He e o habdi is Poina , 1976, wi h nine ecog- nised species, H. bac e iopho a Poina , 1976 ( ype- species); H. megidis Poina , Jackson & Klein, 1987; H. zealandica Poina , 1990; H. indicus Poina , Ka unaka & Da id, 1992; H. a gen inensis S ock, 96 1993; H. hawaiiensis Ga dne , S ock & Kaya, 1994; H. b e icaudis Liu, 1994; H. ma ela us Liu & Be y, 1996 and H. poina i Kakulia & Mikaia, 1997. How- e e , he axonomic s a us o some o hese species is cu en ly being e iewed (S ock e al., unpublished). Su eys o he e o habdi ids, which ha e been conduc ed in many pa s o he wo ld, indica e ha hese nema odes ha e a global dis ibu ion (Hominick e al., 1996; G i in e al., 1999; S ock e al., 1999). S udies conduc ed in Eu ope indica e ha h ee He e o habdi is ypes a e p esen in his con inen , H. bac e iopho a, he no h-wes Eu opean (NWE) (conside ed o be H. megidis) and he ‘I ish g oup’ (Smi s e al., 1991; G i in e al., 1994). H. bac- e iopho a isola es ha e been eco e ed in Spain, sou he n F ance, I aly, cen al Ge many, Hunga y and Molda ia, sugges ing his species is dominan in sou he n and cen al Eu ope (Smi s e al., 1991; De Douce & Gaba a, 1994; G enie e al., 1996; G i in e al., 1999). NWE ype isola es ha e been epo ed om he Ne he lands, Poland, he sou h o England, Belgium, no he n Ge many, Denma k and Es onia, (Smi s e al., 1991; Hominick e al., 1995; Midu u i e al., 1996; G i in e al., 1999). I ish ype isola es we e o iginally eco e ed in I eland (G i in e al., 1991, 1994), bu we e subsequen ly epo ed in B i ain (G i in e al., 1994; Hominick e al., 1995), Hun- ga y and Denma k (G i in e al., 1999). Addi ionally, anewHe e o habdi is species, H. poina i, has been epo ed om Geo gia (Kakulia & Mikaia, 1997). Molecula e idence (RFLP p o iles and IEF p o- ein elec ophenog ams) s ongly indica es he I ish ype isola es ep esen a sepa a e species (Smi s e al., 1991; Joyce e al., 1994a, b). Mo eo e , c oss- hyb idisa ion es s ha e demons a ed ha he I ish ype isola es a e ep oduc i ely isola ed om o he He e o habdi is species (Dix e al., 1994; G i in e al., 1994; Joyce e al., 1994b). Likewise, a molecula phy- logene ic s udy, based on ITS-1 DNA sequence da a, by Adams e al. (1998) indica ed he I ish ype o He e o habdi is (isola e K122) was a dis inc species. In spi e o his cumula i e e idence, he axonomic s a us o he I ish g oup o He e o habdi is has no ye been implici ly se led. No s udies ha e been ca ied ou o desc ibe he mo phological a iabili y (i any) among hese isola es and hei di e ences o known He e o habdi is species. In his s udy we examined he mo phological a i- a ion o h ee ep esen a i e isola es o he I ish ype o He e o habdi is, p o iding addi ional e idence in suppo o hei conside a ion as a dis inc species. A desc ip ion o his new species is he ein p esen ed. Ma e ials and me hods Th ee isola es ep esen ing he I ish ype o He - e o habdi is we e selec ed o his s udy. One o hese isola es, K122, was he i s isola e eco e ed in his g oup; he e o e, i will be conside ed as he ype iso- la e. This isola e was eco e ed om g asslands in Wex o d, I eland (G i in e al., 1991, 1994). The e- maining isola es s udied a e om con inen al Eu ope. Isola e EU94 was collec ed om an open coni e ous o es wi h g ound oak, oses and g ass in Ho nbaek, Denma k (G i in e al., 1999), and isola e EU349 was collec ed om a oadside e ge wi h ees in Kesckeme , Hunga y (G i in e al., 1999). All isola es we e p opaga ed in i o wi h las in- s a la ae o Galle ia mellonella a 22 ±3◦C. Insec cada e s we e dissec ed on days 3-5 o eco e i s gene a ion he maph odi es and on days 7-9 o eco e second gene a ion adul s, ollowing Kaya & S ock (1997). Thi d-s age in ec i e ju eniles we e ha es ed du ing he i s 2 days a e ini ial eme gence (app ox- ima ely days 13-18) om he cada e , as sugges ed by Nguyen & Sma (1996). Fo each o he s udied isola es, 20 specimens om each s age ( i s -gene a ion he maph odi es, second- gene a ion males, second-gene a ion emales and hi d-s age in ec i e ju eniles) we e andomly col- lec ed om 10 G. mellonella cada e s (Hominick e al., 1997). Nema odes we e examined li e o hea - killed in 60 ◦C Ringe ’s solu ion. Nema odes we e ixed in ie hanolamine o malin (TAF) (Cou ney e al., 1955) and p ocessed o anhyd ous glyce ine o moun ing (Seinho s , 1959). Specimens we e moun ed on glass slides suppo ed wi h glass ods o a oid hei la ening. Obse a ions we e made om li e and moun ed specimens using a Nikon Eclipse E600 mi- c oscope equipped wi h di e en ial in e e ence con- as op ics. Specimen measu emen s we e made using Scion Image so wa e (F ede ick, Ma yland, USA) ha was calib a ed using a s age mic ome e . Selec- ion o mo phome ic cha ac e s was done acco ding o S ock & Kaya (1996) and S ock (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: L = o al body leng h; ABW =anal o cloacal body wid h; EP =exc e o y po e posi ion; ES =oesophagus leng h; GS =GuL/SpL; GuL =gu- 97 be naculum leng h; MBW =maximum body wid h; ML =muc o leng h; NR =ne e- ing posi ion; a io a=L/MBW; a io b =L/ES; a io c =L/TL; a io d=EP/ES; a io e =EP/TL; SpL =spicule leng h (measu ed along he cu a u e in a line along he cen- e o he spicule); S L =s oma leng h; S W =s oma wid h; SW =SpL/ABW; TL = ail leng h (measu ed wi hou conside ing he ex a cu icula shea h o he second s age ju enile); TRL = es is e lexion leng h; V= ul a posi ion om an e io end exp essed as pe cen age o L. He e o habdi is downesi n. sp. Type-hos : Unknown. Nema odes eco e ed by soil bai ing echnique (Bedding & Akhu s , 1975). Type-locali y: G assland a Cu acloe, Co. Wex o d, I eland. This ype-locali y co esponds o he sampling si e whe e isola e K122 ( he ype-isola e) was eco - e ed (see G i in e al., 1994). Type-specimens: Holo ype, second gene a ion male (UCDNC 3816), allo ype second-gene a ion e- male (UCDNC 3817), 15 pa a ype he maph odi es (UCDNC 3818-3820), 15 males (UCDNC 3821- 3823), 15 hi d-s age in ec i e ju enile (UCDNC 3824-3826) deposi ed a he Uni e si y o Cali o nia Da is Nema ode Collec ion. Addi ional pa a ype ma- e ial (5 he maph odi es, 4 second-gene a ion males, 4 second-gene a ion emales and 5 hi d-s age in ec i e ju eniles deposi ed a CABI Bioscience Nema ode Collec ion, UK Cen e (Egham), UK. E ymology: This species is named o P o esso Ma - in J. Downes, a colleague and ac i e collabo a o in he isola ion o he I ish ype o He e o habdi is. Desc ip ion (Figu es 1-3) The ollowing desc ip ion is based on he examina ion o mo phological and mo phome ic ea u es o 3 He - e o habdi is isola es conside ed in his s udy. Adul s. Head unca ed o sligh ly ounded (Fig- u e 1A,F). Six dis inc p o uding poin ed lips su - ounding o al ape u e. Each lip bea s one labial papilla. Amphidial ape u es inconspicuous, po e-like. S oma sho , wide. Cheilos om sho wi h e ac ile habdions; gymnos om and s egos om educed (Fig- u e 2A). Oesophagus habdi oid. Co pus cylind ical; me aco pus no di e en ia ed. Is hmus sho . Basal bulb py i o m wi h educed al e. Ne e- ing loca ed in middle o is hmus. Exc e o y po e loca ed a le el o basal bulb (Figu e 1A). He maph odi e ( i s gene a ion). Wi h o o es is. Vul a loca ed nea middle o body. Vul al lips p o- uding, wi h an e io lip la ge han pos e io lip (Figu es 1B, 2B). Pos anal swelling well de eloped (Figu es 1C, 2C). Tail e minus blun and muc ona e (Figu es 1C, 2C). Female (second gene a ion). Amphidelphic. Vul a si - ua ed nea middle o body. Vul al lips sligh ly p o ud- ing (Figu es 1D, 2D). Tail conoid, na owing owa ds end in o e ac ile spike-like s uc u e (Figu es 1E, 2E). Pos anal swelling sligh ly de eloped (Figu e 1E). Male (second gene a ion). Mono chic. Tes is an e i- o ly e lexed. Spicules pai ed, symme ical, s aigh o a cua e, wi h poin ed ips (Figu es 1G, 2F,H). Manub ium (head) squa e, well se o om lamina. Calamus dis inc . Lamina wi h one in e nal ib. Velum absen . Gube naculum hin, abou hal size o spicules. Bu sa open, pelode an, a ended by complemen o 9 pai s o bu sal ays (papillae). A angemen o papillae isas ollows:12()33(Figu e1G).Pai s4and7 ben do sally. Pai 8 does no each im o bu sa (Fig- u e 2G). Thi d-s age in ec i e ju enile (IJ). Enshea hed in cu i- cle o second-s age ju enile (J2) (Figu e 3A). Cu icle o J2 wi h longi udinal idges h oughou mos o body leng h and essella e pa e n in mos an e io pa o body (seen wi h DIC ligh mic oscopy). Body slende , g adually ape ing pos e io ly. IJ wi h p ominen cu- icula do sal oo h (seen wi h DIC ligh mic oscopy) (Figu e 3B,C). Amphidial opening small, po e-like. Ne e- ing loca ed in middle o is hmus. Exc e o y po e loca ed pos e o o basal bulb (Figu e 1H). La - e al ield wi h 2 idges (as seen wi h DIC mic oscopy and con i med by c oss-sec ions) (Figu e 3D). Tail sho , conoid, ape ing o small spike-like ip (Fig- u es 1I-K, 3E-G). Mo phology o ail ip a iable, wi h mos cha ac e is ic shape (80% o examined IJs om 3 s udied isola es) shown in Figu es 1I,J, 3E,F. Dimensions Holo ype male (isola e K122): L =877 µm; MBW = 33 µm; S L =7µm; S W =4.5 µm; ES =100 µm; NR =62.5 µm; EP =90 µm; TL =32 µm; ABW =23 µm; TRL =131 µm; SpL =42 µm; GuL = 19 µm; GS =0.45; SW =1.8. 98 Figu e 1. He e o habdi is downesi n. sp. A. An e io end o he maph odi e; B. Vul a o he maph odi e; C. Tail o he maph odi e in la e al iew; D. Vul a o emale; E. Tail o emale in la e al iew; F. Male; en i e wo m; G. Male ail in la e al iew; H. An e io end o hi d-s age in ec i e ju enile; I-K. Tail o hi d-s age in ec i e ju enile. Scale-ba s:A,D-F,50µm; B,C,G,H, 25 µm; I-K, 18 µm 99 Figu e 2. Adul s ages o He e o habdi is downesi n. sp. A. An e io egion o i s gene a ion he maph odi e showing s oma egion; B. Tail o i s gene a ion he maph odi e showing muc o (a ow); C. Vul al lips o second gene a ion emale; D. Tail o male in la e al iew; E. Tail o male in en al iew showing a angemen o bu sal ays; F. Spicule. Scale:1cm=A, 8 µm; B, 2 µm; C, 8 µm; D-F, 1 µm. 100 Figu e 3. Thi d-s age in ec i e ju enile o He e o habdi is downesi n. sp. A-C. An e io end showing cu icula oo h (a ows); D. Cu icle showing la e al ield pa e n (l ); E-F. Tail showing mos ypical shape o he ‘spike-like’ ip (a ows). G. A ypical ail ip. Scale-ba s: A-C,E-G, 15 µm; D, 10 µm. 101 Table 1. Mo phome ic cha ac e s (in µm) o adul s o He e o habdi is downesi n. sp. Means and s anda d de ia ion a e gi en in pa en heses. Pa a ypes K122 EU94 EU349 H(1)M(2)F(3)H(1)M(2)F(3)H(1)M(2)F(3) (n =19) (n =19) (n =20) (n =20) (n =20) (n =20) (n =20) (n =20) (n =20) L 3,050-5,051 669-876 1,231-2,728 3,180-5,019 830-1,042 1,200-2,800 3,100-5,025 837-1,066 1,157-2,700 (4,362 ±266) (800 ±76) (1,747 ±387) (4,795 ±121) (936 ±79) (1,810 ±394) (4,510 ±298) (946 ±68) (1,837 ±464) MBW 183-291 33-40 74-131 185-284 37-55 74-129 195-255 33-46 73-119 (254 ±84) (36 ±3) (102 ±11) (231 ±29) (46 ±7) (104 ±13) (225 ±33) (39 ±4) (109 ±16) STL 11-15 6.5-8 8-13 12.5-15.5 6.5-9.7 8-13 11-14 6-10 8-13 (13.5±0.6) (7.5±0.5) (10 ±2) (13 ±1.5) (8 ±1) (10 ±1.5) (12.5±1.5) (8 ±1.3) (10 ±1) STW 7-10 3-4.5 3.5-9 9-11 3-5 3.5-9 7-9.5 3-5 3.5-9 (8.5±1.4) (4.2±0.5) (8.5±2) (8 ±1.5) (3.8±0.6) (6 ±1.5) (8.5±1) (4 ±1) (6 ±2) NR 172-230 62-78 117-151 169-208 82-103 121-151 165-225 63-75 117-149 (200 ±19) (70 ±7) (131 ±11) (187 ±10) (94 ±8) (130 ±9) 195 ±23 (69 ±3) (126 ±8) EP 200-254 86-91 99-126 197-271 66-78 98-135 198-275 73-92 98-124 (235 ±19) (89 ±2) (112 ±7) (236 ±17) (72 ±4.5) (112 ±10) (241 ±25) (84 ±5) (109 ±8) ES 230-244 97-106 111-155 218-244 75-94 113-153 225-251 90-107 113-150 (240 ±11) (101 ±3) (131 ±13) (230 ±13) (87 ±6) (130 ±10) (247 ±9) (96 ±4) (119 ±9) TRL – 108-181 – – 107-125 – – 103-179 – (150 ±22) (113 ±10) (146 ±26) TL 60-70 29-34 70-122 55-75 31-39 70-115 61-72 28-42 71-122 (65 ±3) (32 ±2) (89 ±18) (70 ±5) (33 ±3) (85 ±12) (68 ±7) (36 ±3) (86 ±15) ML 1.5-2.5 – 1.5-6 1.5-2.5 – 1.5-5 1.5-2 – 1.7-6.5 (2 ±0.2) (3 ±1.5) (2 ±0.2) (3 ±1) (1.8±0.3) (3 ±1.5) ABW 57-65 21-28 25-38 55-68 20-27 27-37 57-70 21-27 24-37 (62 ±2) (24 ±2) (33 ±3.5) (59 ±4) (24 ±2) (33 ±3) (65 ±5) (23 ±2) (32 ±4) V 50-55 – 47-60 51-55 – 47-56 51-55 – 47-55 (52 ±0.5) (51 ±4) (53 ±1) (52 ±3) (52 ±1) (51 ±3) SpL – 41-47 – – 40-51 – – 41-53 – (43 ±2) (44 ±4) (48 ±3) GuL – 17-19.5 – – 15-20 – – 13-23 – (18 ±1) (17 ±2) (18 ±3) GS – 0.36-0.47 – – 0.35-0.42 – – 0.28-0.45 (0.43 ±0.04) (0.39 ±0.02) (0.37 ±0.05) SW – 1.7-2.2 – – 1.7-2.3 – – 1.6-2.4 (1.8±0.2) (1.8±0.2) (2.1±0.2) (1)He maph odi e ( i s gene a ion). (2)Male (second gene a ion). (3)Female (second gene a ion). 102 Allo ype second-gene a ion emale (isola e K122): L =1,500 µm; MBW =102 µm; S L =9µm; S W = 4µm; ES =128 µm; NR =110 µm; EP =131 µm; TL =76 µm; ML =4µm; ABW =29 µm; AE-V = 712 µm; V =47.5. Pa a ypes (he maph odi es, males and emales, and hi d-s age in ec i e ju enile): See Tables 1 and 2. Diagnosis and ela ionships He e o habdi is downesi n. sp. can be sepa a ed om o he species in he genus by a combina ion o mo - phological and mo phome ic ai s o all li e s ages (Tables 3, 4). He maph odi es o H. downesi n. sp. can be dis inguished om all cu en ly ecognised He - e o habdi is species by ha ing a blun and muc ona e ail. Males o he new species can be sepa a ed om o he He e o habdi is species by ha ing he na owes body wid h. Addi ionally, males o his species a e cha ac e ised by he posi ion o he exc e o y po e, which is mo e an e io ly loca ed han o he desc ibed He e o habdi is species. The spicules o H. downesi n. sp. esemble hose o H. ma ela us and H. megidis in ha ing a squa e-shaped manub ium (head), which is well se o om he lamina by a hin calamus (neck), bu can be sepa a ed om hese species by he alue o a io SW (mean: 1.8) (Table 3). The ail o he emales (second gene a ion) o H. downesi n. sp. esembles ha o H. ma ela us. Howe e , emales o he new species can be dis in- guished by he p esence o a conspicuous e ac ile muc ona ed p ocess. The body leng h o hi d-s age in- ec i e ju eniles o H. downesi n. sp. (mean: 637 µm) is p ac ically indis inguishable om H. a gen inensis (657 µm) and H. ma ela us (640 µm). The a e age ail leng h o H. downesi n. sp. (68 µm ) is simila o ha o H. ma ela us (65 µm). H. b e icaudis is also cha ac e ised by ha ing a sho ail (75.5 µm). Howe e , compa isons be ween he new species and H. b e icaudis a e di icul because he o iginal de- sc ip ion does no p o ide de ails o how he ail leng h o he hi d-s age in ec i e ju eniles was measu ed (i.e. conside ing o no he ex a cu icula shea h om he second-s age ju enile). Bu he in ec i e ju eniles o he new species can be dis inguished om hese and he es o he ecognised He e o habdi is species by he mo phology o he ail (which is poin ed and ape s o a sho spike-like ip) and/o he alue o he a io c (Table 4). Table 2. Mo phome ic cha ac e s (in µm) o hi d-s age in- ec i e ju eniles o He e o habdi is downesi n. sp. Means and s anda d de ia ion a e gi en in pa en heses. Pa a ypes K122 EU94 EU349 (n =20) (n =20) (n =20) L 588-692 521-691 520-741 (637 ±32) (607 ±51) (597 ±54) MBW 15-22 14-25 15-24 (18 ±2) (19 ±4) (20 ±3) NR 96-105 74-115 82-108 (101 ±3) (92 ±9) (92 ±6) EP 96-128 88-123 97-123 (115 ±8) (107 ±9) (106 ±9) ES 126-141 121-136 115-143 (135 ±4) (128 ±4) (127 ±8) TL 62-74 54-78 51-83 (68 ±4) (65 ±9) (64 ±10) ABW 9-14 9-14 10-15 (12 ±1) (12 ±1) (12 ±2) Ra io a 29-42 26-38 25-37 (35 ±4) (33 ±4) (31 ±3) Ra io b 4.4-5.3 4.3-5.2 4.3-5.2 (4.7±0.3) (4.7±0.3) (4.7±0.3) Ra io c 8.5-10.5 8-11 7.6-11 (9.5±0.5) (9 ±1) (9.5±1) Ra io d 0.76-0.98 0.66-0.97 0.8-0.90 (0.85 ±0.05) (0.84 ±0.1) (0.85 ±0.04) Ra io e 1.6-1.8 1.2-2.1 1.3-2.2 (1.7±0.1) (1.7±0.3) (1.7±0.2) Discussion He e o habdi is species ha e essen ially been de- limi ed on he basis o Linnaean species concep s. Mo phological and mo phome ic cha ac e s o adul s ages (speci ically second gene a ion males) and hi d-s age in ec i e ju eniles, ha e been used o diagnos ic pu poses and o assess hei iden i y as mo - phospecies (Poina , 1976; Poina 1990; S ock, 1993; Liu & Be y, 1996; S ock e al, 1996; S ock & Kaya, 1996). Howe e , because o he pauci y o mo pholog- ical ai s in he He e o habdi idae, o he app oaches ha e been applied o hei delimi a ion and diagnosis a he species le el. Fo ins ance, di e en molecula me hods, such as s a ch gel elec opho esis, es ic- ion enzyme analyses (RFLPs), isoelec ic ocussing (IEF), andom ampli ied polymo phic DNA (RAPD) ma ke s and sequence da a, ha e been success ully used o iden i y and in e phylogene ic ela ionships 103 Table 3. Compa ison o mo phome ic cha ac e s (in µm) o males o He e o habdi is species. Means and s anda d de ia ion a e gi en in pa en heses. Species L MBW AE-ES AE-NR AE-EP TL ABW SpL GuL SW GS H. bac e iopho a∗620-940 38-64 95-130 68-83 106-136 26-43 23-34 31-41 14-21 1.20-1.34 0.45-0.51 (831 ±75) (51 ±9) (125 ±14) (76 ±10) (125 ±16) (36 ±3.5) (28 ±2.5) (36 ±2) (18 ±2) (1.28 ±0.01) (0.48 ±0.1) H. a gen inensis∗1,000-2,000 42-70 103-121 75-88 145-172 28-49 28-36 35-49 20-25 1.75-1.95 0.38-0.51 (1,445 ±55) (56 ±11) (115 ±9) (79 ±8) (160 ±15) (40 ±4) (23 ±3) (45 ±3) (22 ±1.5) (1.80 ±0.02) (0.48 ±0.1) H. indicus∗573-800 35-47 93-109 69-84 110-138 24-33 19-24 34-48 18-23 1.7-1.9 0.43-0.48 (702 ±33) (42 ±2.5) (100 ±4) (78 ±6) (124 ±10) (28 ±3) (21 ±3) (42 ±4) (20 ±2) (1.8±0.10) (0.47 ±0.2) H. hawaiiensis∗864-1130 50-83 100-150 67-112 71-146 26-40 21-34 41-49 18-26 1.44-1.95 0.44-0.53 (998 ±33) (64 ±12) (119 ±9) (86 ±8) (130 ±17) (34 ±11) (26 ±5) (45 ±3) (21 ±2) (1.73 ±0.05) (0.46 ±0.2) H. ma ela us∗800-1,010 49-97 114-139 83-114 102-130 36-49 24-30 42-52 17-24 1.73-1.78 0.4-0.46 (910 ±35) (68 ±23) (125 ±11) (101 ±9) (115 ±10) (39 ±8) (27 ±2) (48 ±4) (22 ±2) (1.75 ±0.01) (0.42 ±0.1) H. megidis∗780-1,001 46-65 116-140 75-110 128-147 32-48 25-37 39-49 16-24 1.35-1.45 0.45-0.5 (892 ±45) (53 ±14) (131 ±9) (95 ±11) (136 ±8) (45 ±7) (33 ±4) (42 ±3) 19 (1.4±0.01) (0.47 ±0.1) H. zealandica∗958-1,693 46-53 157-197 79-110 132-148 30-46 26-36 36-47 16-24 1.3-1.4 0.44-0.51 (1209 ±186) (48 ±3) (175 ±9) (98 ±9) (135 ±8) (40 ±3) (30 ±2) (42 ±2.5) (19 ±2) (1.35 ±0.2) (0.45 ±0.01) H. b e icaudis∗∗ 840-950 40-48 104-112 80-88 92-100 no no 44-48 18-22 no no (900) (43) (108) (82) (95) a ailable a ailable (47) (19 ±2) a ailable a ailable H. poina i∗∗∗ 970-1,100 43-70 150-170 no no no no 43-55 24-32 no no a ailable a ailable a ailable a ailable a ailable a ailable H. downesi n. sp. (K122) 669-876 33-40 97-106 62-78 86-91 29-34 21-28 41-47 17-19.5 1.7-2.2 0.36-0.47 (800 ±76) (36 ±3) (101 ±3) (70 ±7) (89 ±2) (32 ±2) (24 ±2) (43 ±2) (18 ±1) (1.8±0.2) (0.43 ±0.04) ∗A e Hominick e al. (1997). ∗∗A e Liu (1993). ∗∗∗ A e Kakulia & Mikaia (1997).