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

Systematic Parasitology 51: 95–106, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands. 95 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. S. Patricia Stock1∗, Christine T. Griffin2&AnnM.Burnell 2 1Department of Nematology, University of California Davis. One Shields Ave., Davis, CA 95616-8668, USA 2Institute of Bioengineering and Agroecology, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland ∗Present address: Department of Plant Pathology, University of Arizona, 1140 E. South Campus Drive, Tucson, AZ 85721-0036, USA Accepted for publication 19th March, 2001 Abstract The morphological variation of three representative isolates of the ‘Irish group’ of Heterorhabditis was examined. First generation hermaphrodites were characterised by having a blunt and mucronate tail. Females (second generation) and third-stage infective juveniles were also distinguished by the morphology of the tail and the presence of a refractile projection in the tail tip. Males were characterised by the position of the excretory pore and by the value of ratio SW. These morphological features do not fit the description of currently recognised Heterorhabditis species, and provide additional evidence in support for the consideration of the Irish group as a new species. A description of this species, as H. downesi n. sp., is provided. Introduction Entomopathogenic nematodes of the family Heterorhabditidae Poinar, 1976 are obligate and lethal parasites of insects. The only free-living stage is the third-stage infective juvenile (IJ), which is symbiotically associated with enteric bacteria of the genus Photorhabdus Boemare et al., 1993. The IJs penetrate into the haemocoel of the host either from the gut or by abrading the intersegmental membranes of the insect by using a dorsal tooth. Once in the haemocoel of the insect, the IJs release the bacterial cells, which multiply and digest the host tissues, thereby providing suitable nutrient conditions for nematode growth and development. The insect host dies rapidly, usually within 2 days of the initial penetration of the nematodes. The nematodes resume development, moult to the J4 stage and reach adulthood within three days (in H. bacteriophora Poinar, 1976) when cultured in vivo in larvae of the greater wax moth Galleria mellonella at 23 ◦C (Wang & Bedding, 1996). Nematode reproduction continues for at least two generations until the nutrient status of the cadaver deteriorates, whereupon adult development is suppressed and IJs accumulate and emerge into the soil where they may survive for several months in the absence of a suitable host. Heterorhabditids are effective biological control agents of several insect pests (Berry et al., 1997; Gerritsen et al., 1998; Shields et al., 1999; Long et al., 2000; Mannion et al., 2000). Thus, interest in studying these nematodes has increased dramatically in recent years, not only to address aspects related to biological control, but also regarding basic research areas such as ecology, biodiversity, evolution, biochemistry, symbiosis and molecular genetics (review by Burnell & Stock, 2000). The Heterorhabditidae currently comprise one genus, Heterorhabditis Poinar, 1976, with nine recognised species, H. bacteriophora Poinar, 1976 (typespecies); H. megidis Poinar, Jackson & Klein, 1987; H. zealandica Poinar, 1990; H. indicus Poinar, Karunakar & David, 1992; H. argentinensis Stock, 96 1993; H. hawaiiensis Gardner, Stock & Kaya, 1994; H. brevicaudis Liu, 1994; H. marelatus Liu & Berry, 1996 and H. poinari Kakulia & Mikaia, 1997. However, the taxonomic status of some of these species is currently being reviewed (Stock et al., unpublished). Surveys for heterorhabditids, which have been conducted in many parts of the world, indicate that these nematodes have a global distribution (Hominick et al., 1996; Griffin et al., 1999; Stock et al., 1999). Studies conducted in Europe indicate that three Heterorhabditis types are present in this continent, H. bacteriophora, the north-west European (NWE) (considered to be H. megidis) and the ‘Irish group’ (Smits et al., 1991; Griffin et al., 1994). H. bacteriophora isolates have been recovered in Spain, southern France, Italy, central Germany, Hungary and Moldavia, suggesting this species is dominant in southern and central Europe (Smits et al., 1991; De Doucet & Gabarra, 1994; Grenier et al., 1996; Griffin et al., 1999). NWE type isolates have been reported from the Netherlands, Poland, the south of England, Belgium, northern Germany, Denmark and Estonia, (Smits et al., 1991; Hominick et al., 1995; Miduturi et al., 1996; Griffin et al., 1999). Irish type isolates were originally recovered in Ireland (Griffin et al., 1991, 1994), but were subsequently reported in Britain (Griffin et al., 1994; Hominick et al., 1995), Hungary and Denmark (Griffin et al., 1999). Additionally, anewHeterorhabditis species, H. poinari, has been reported from Georgia (Kakulia & Mikaia, 1997). Molecular evidence (RFLP profiles and IEF protein electrophenograms) strongly indicates the Irish type isolates represent a separate species (Smits et al., 1991; Joyce et al., 1994a, b). Moreover, crosshybridisation tests have demonstrated that the Irish type isolates are reproductively isolated from other Heterorhabditis species (Dix et al., 1994; Griffin et al., 1994; Joyce et al., 1994b). Likewise, a molecular phylogenetic study, based on ITS-1 rDNA sequence data, by Adams et al. (1998) indicated the Irish type of Heterorhabditis (isolate K122) was a distinct species. In spite of this cumulative evidence, the taxonomic status of the Irish group of Heterorhabditis has not yet been implicitly settled. No studies have been carried out to describe the morphological variability (if any) among these isolates and their differences to known Heterorhabditis species. In this study we examined the morphological variation of three representative isolates of the Irish type of Heterorhabditis, providing additional evidence in support for their consideration as a distinct species. A description of this new species is herein presented. Materials and methods Three isolates representing the Irish type of Heterorhabditis were selected for this study. One of these isolates, K122, was the first isolate recovered in this group; therefore, it will be considered as the type isolate. This isolate was recovered from grasslands in Wexford, Ireland (Griffin et al., 1991, 1994). The remaining isolates studied are from continental Europe. Isolate EU94 was collected from an open coniferous forest with ground oak, roses and grass in Hornbaek, Denmark (Griffin et al., 1999), and isolate EU349 was collected from a roadside verge with trees in Kesckemet, Hungary (Griffin et al., 1999). All isolates were propagated in vivo with last instar larvae of Galleria mellonella at 22 ±3◦C. Insect cadavers were dissected on days 3-5 to recover first generation hermaphrodites and on days 7-9 to recover second generation adults, following Kaya & Stock (1997). Third-stage infective juveniles were harvested during the first 2 days after initial emergence (approximately days 13-18) from the cadaver, as suggested by Nguyen & Smart (1996). For each of the studied isolates, 20 specimens from each stage (first-generation hermaphrodites, secondgeneration males, second-generation females and third-stage infective juveniles) were randomly collected from 10 G. mellonella cadavers (Hominick et al., 1997). Nematodes were examined live or heatkilled in 60 ◦C Ringer’s solution. Nematodes were fixed in triethanolamine formalin (TAF) (Courtney et al., 1955) and processed to anhydrous glycerine for mounting (Seinhorst, 1959). Specimens were mounted on glass slides supported with glass rods to avoid their flattening. Observations were made from live and mounted specimens using a Nikon Eclipse E600 microscope equipped with differential interference contrast optics. Specimen measurements were made using Scion Image software (Frederick, Maryland, USA) that was calibrated using a stage micrometer. Selection of morphometric characters was done according to Stock & Kaya (1996) and Stock (1997). Illustrations were prepared from digitised camera lucida images. The following abbreviations have been used in the text or tables: L =total body length; ABW =anal or cloacal body width; EP =excretory pore position; ES =oesophagus length; GS =GuL/SpL; GuL =gu- 97 bernaculum length; MBW =maximum body width; ML =mucro length; NR =nerve-ring position; ratio a=L/MBW; ratio b =L/ES; ratio c =L/TL; ratio d=EP/ES; ratio e =EP/TL; SpL =spicule length (measured along the curvature in a line along the centre of the spicule); StL =stoma length; StW =stoma width; SW =SpL/ABW; TL =tail length (measured without considering the extra cuticular sheath of the second stage juvenile); TRL =testis reflexion length; V=vulva position from anterior end expressed as percentage of L. Heterorhabditis downesi n. sp. Type-host: Unknown. Nematodes recovered by soil baiting technique (Bedding & Akhurst, 1975). Type-locality: Grassland at Curracloe, Co. Wexford, Ireland. This type-locality corresponds to the sampling site where isolate K122 (the type-isolate) was recovered (see Griffin et al., 1994). Type-specimens: Holotype, second generation male (UCDNC 3816), allotype second-generation female (UCDNC 3817), 15 paratype hermaphrodites (UCDNC 3818-3820), 15 males (UCDNC 38213823), 15 third-stage infective juvenile (UCDNC 3824-3826) deposited at the University of California Davis Nematode Collection. Additional paratype material (5 hermaphrodites, 4 second-generation males, 4 second-generation females and 5 third-stage infective juveniles deposited at CABI Bioscience Nematode Collection, UK Centre (Egham), UK. Etymology: This species is named for Professor Martin J. Downes, a colleague and active collaborator in the isolation of the Irish type of Heterorhabditis. Description (Figures 1-3) The following description is based on the examination of morphological and morphometric features of 3 Heterorhabditis isolates considered in this study. Adults. Head truncated or slightly rounded (Figure 1A,F). Six distinct protruding pointed lips surrounding oral aperture. Each lip bears one labial papilla. Amphidial apertures inconspicuous, pore-like. Stoma short, wide. Cheilostom short with refractile rhabdions; gymnostom and stegostom reduced (Figure 2A). Oesophagus rhabditoid. Corpus cylindrical; metacorpus not differentiated. Isthmus short. Basal bulb pyriform with reduced valve. Nerve-ring located in middle of isthmus. Excretory pore located at level of basal bulb (Figure 1A). Hermaphrodite (first generation). With ovotestis. Vulva located near middle of body. Vulval lips protruding, with anterior lip larger than posterior lip (Figures 1B, 2B). Postanal swelling well developed (Figures 1C, 2C). Tail terminus blunt and mucronate (Figures 1C, 2C). Female (second generation). Amphidelphic. Vulva situated near middle of body. Vulval lips slightly protruding (Figures 1D, 2D). Tail conoid, narrowing towards end into refractile spike-like structure (Figures 1E, 2E). Postanal swelling slightly developed (Figure 1E). Male (second generation). Monorchic. Testis anteriorly reflexed. Spicules paired, symmetrical, straight or arcuate, with pointed tips (Figures 1G, 2F,H). Manubrium (head) square, well set off from lamina. Calamus distinct. Lamina with one internal rib. Velum absent. Gubernaculum thin, about half size of spicules. Bursa open, peloderan, attended by complement of 9 pairs of bursal rays (papillae). Arrangement of papillae isasfollows:12()33(Figure1G).Pairs4and7 bent dorsally. Pair 8 does not reach rim of bursa (Figure 2G). Third-stage infective juvenile (IJ). Ensheathed in cuticle of second-stage juvenile (J2) (Figure 3A). Cuticle of J2 with longitudinal ridges throughout most of body length and tessellate pattern in most anterior part of body (seen with DIC light microscopy). Body slender, gradually tapering posteriorly. IJ with prominent cuticular dorsal tooth (seen with DIC light microscopy) (Figure 3B,C). Amphidial opening small, pore-like. Nerve-ring located in middle of isthmus. Excretory pore located posteror to basal bulb (Figure 1H). Lateral field with 2 ridges (as seen with DIC microscopy and confirmed by cross-sections) (Figure 3D). Tail short, conoid, tapering to small spike-like tip (Figures 1I-K, 3E-G). Morphology of tail tip variable, with most characteristic shape (80% of examined IJs from 3 studied isolates) shown in Figures 1I,J, 3E,F. Dimensions Holotype male (isolate K122): L =877 µm; MBW = 33 µm; StL =7µm; StW =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 Figure 1. Heterorhabditis downesi n. sp. A. Anterior end of hermaphrodite; B. Vulva of hermaphrodite; C. Tail of hermaphrodite in lateral view; D. Vulva of female; E. Tail of female in lateral view; F. Male; entire worm; G. Male tail in lateral view; H. Anterior end of third-stage infective juvenile; I-K. Tail of third-stage infective juvenile. Scale-bars:A,D-F,50µm; B,C,G,H, 25 µm; I-K, 18 µm 99 Figure 2. Adult stages of Heterorhabditis downesi n. sp. A. Anterior region of first generation hermaphrodite showing stoma region; B. Tail of first generation hermaphrodite showing mucro (arrow); C. Vulval lips of second generation female; D. Tail of male in lateral view; E. Tail of male in ventral view showing arrangement of bursal rays; F. Spicule. Scale:1cm=A, 8 µm; B, 2 µm; C, 8 µm; D-F, 1 µm. 100 Figure 3. Third-stage infective juvenile of Heterorhabditis downesi n. sp. A-C. Anterior end showing cuticular tooth (arrows); D. Cuticle showing lateral field pattern (lf); E-F. Tail showing most typical shape of the ‘spike-like’ tip (arrows). G. Atypical tail tip. Scale-bars: A-C,E-G, 15 µm; D, 10 µm. 101 Table 1. Morphometric characters (in µm) of adults of Heterorhabditis downesi n. sp. Means and standard deviation are given in parentheses. Paratypes 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)Hermaphrodite (first generation). (2)Male (second generation). (3)Female (second generation). 102 Allotype second-generation female (isolate K122): L =1,500 µm; MBW =102 µm; StL =9µm; StW = 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. Paratypes (hermaphrodites, males and females, and third-stage infective juvenile): See Tables 1 and 2. Diagnosis and relationships Heterorhabditis downesi n. sp. can be separated from other species in the genus by a combination of morphological and morphometric traits of all life stages (Tables 3, 4). Hermaphrodites of H. downesi n. sp. can be distinguished from all currently recognised Heterorhabditis species by having a blunt and mucronate tail. Males of the new species can be separated from other Heterorhabditis species by having the narrowest body width. Additionally, males of this species are characterised by the position of the excretory pore, which is more anteriorly located than other described Heterorhabditis species. The spicules of H. downesi n. sp. resemble those of H. marelatus and H. megidis in having a square-shaped manubrium (head), which is well set off from the lamina by a thin calamus (neck), but can be separated from these species by the value of ratio SW (mean: 1.8) (Table 3). The tail of the females (second generation) of H. downesi n. sp. resembles that of H. marelatus. However, females of the new species can be distinguished by the presence of a conspicuous refractile mucronated process. The body length of third-stage infective juveniles of H. downesi n. sp. (mean: 637 µm) is practically indistinguishable from H. argentinensis (657 µm) and H. marelatus (640 µm). The average tail length of H. downesi n. sp. (68 µm ) is similar to that of H. marelatus (65 µm). H. brevicaudis is also characterised by having a short tail (75.5 µm). However, comparisons between the new species and H. brevicaudis are difficult because the original description does not provide details of how the tail length of the third-stage infective juveniles was measured (i.e. considering or not the extra cuticular sheath from the second-stage juvenile). But the infective juveniles of the new species can be distinguished from these and the rest of the recognised Heterorhabditis species by the morphology of the tail (which is pointed and tapers to a short spike-like tip) and/or the value of the ratio c (Table 4). Table 2. Morphometric characters (in µm) of third-stage infective juveniles of Heterorhabditis downesi n. sp. Means and standard deviation are given in parentheses. Paratypes 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) Ratio a 29-42 26-38 25-37 (35 ±4) (33 ±4) (31 ±3) Ratio 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) Ratio c 8.5-10.5 8-11 7.6-11 (9.5±0.5) (9 ±1) (9.5±1) Ratio 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) Ratio 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 Heterorhabditis species have essentially been delimited on the basis of Linnaean species concepts. Morphological and morphometric characters of adult stages (specifically second generation males) and third-stage infective juveniles, have been used for diagnostic purposes and to assess their identity as morphospecies (Poinar, 1976; Poinar 1990; Stock, 1993; Liu & Berry, 1996; Stock et al, 1996; Stock & Kaya, 1996). However, because of the paucity of morphological traits in the Heterorhabditidae, other approaches have been applied for their delimitation and diagnosis at the species level. For instance, different molecular methods, such as starch gel electrophoresis, restriction enzyme analyses (RFLPs), isoelectric focussing (IEF), random amplified polymorphic DNA (RAPD) markers and sequence data, have been successfully used to identify and infer phylogenetic relationships 103 Table 3. Comparison of morphometric characters (in µm) of males of Heterorhabditis species. Means and standard deviation are given in parentheses. Species L MBW AE-ES AE-NR AE-EP TL ABW SpL GuL SW GS H. bacteriophora∗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. argentinensis∗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. marelatus∗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. brevicaudis∗∗ 840-950 40-48 104-112 80-88 92-100 not not 44-48 18-22 not not (900) (43) (108) (82) (95) available available (47) (19 ±2) available available H. poinari∗∗∗ 970-1,100 43-70 150-170 not not not not 43-55 24-32 not not available available available available available available 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) ∗After Hominick et al. (1997). ∗∗After Liu (1993). ∗∗∗ After Kakulia & Mikaia (1997).