Heth impalutiensis n. sp. (Nematoda: Ransomnematoidea: Hethidae) a millipede parasite from Central Mindanao, Philippines Author Malysheva, Svetlana V. Author Mohagan, Alma B. Author Spiridonov, Sergei E. text Zootaxa 2015 3926 1 100 110 journal article 10.11646/zootaxa.3926.1.4 3679af09-ebf2-4ff3-9f4d-8a52d60863b3 1175-5326 242166 CD8D2D62-6743-4E20-BB28-647C4B3ADC90 Heth impalutiensis n. sp. ( Figs 1 , 2 , 3 , 4 ) Type host and locality. Female spirostreptid millipede ( Spirostreptida : Harpagophoridae ) collected 23 November 2013 by Mohagan A.B. and Spiridonov S.E. near Dila Falls in Impalutao barangay of Bukidnon Province, Central Mindanao, Philippines . Coordinates: 8° 14' N and 125° 02' E , 800 m above sea level (a.s.l.). Also, representatives of described species were found 22 November 2013 in same millipede host not far from Mountain View College of Bukidnon Province. Coordinates: 7° 58' N and 125° 00' E , 680 m a.s.l. Type material. Holotype female (No. 1250) and paratype male (No. 1251) deposited in the collection of the Centre of Parasitology, Institute of Ecology and Evolution, Russian Academy of Sciences. Paratype male (No. UGMD 104302) and paratype female (No. UGMD 104303) from the type population also deposited in the nematological collection of the Museum voor Deirkunde, Ghent University (Ghent, Belgium ). Etymology. The specific epithet is derived from Impalutao barangay (the administrative division in the Philippines ) and refers to the collection site of the type specimens’ host. Measurements. See Table 1 . TABLE 1. Morphometrics of Heth impalutiensis n. sp. All measurements are in µm and in the form: mean±standard deviation (range). Character Dila Falls population Mountain View College population Description. Female. Cephalic extremity broad, body widening gradually behind the collar level reaching maximum diameter at mid-body then tapering posteriorly towards anus and terminating with long, subulate tail appendage ( Fig.1 , a). Body cuticle with fine annulation 1.5 µ m apart, becoming indistinct behind vulva. Surface of each annuli bears tiny cuticular knobs ca 0.5 µ m in diam. Lateral alae absent. Head with two well developed lateral pseudolabia forming the specific shape of anterior end ( Fig.1 , d). Free edges of each pseudolabia bear comb-like bristles reaching in length up to 5–6 µ m along dorsal and ventral sides and about 1 µ m along central slit between two pseudolabia ( Fig.3 , c). Each subventral and subdorsal flange of cephalic plate bearing two, posteriorly directed, recurved spines. Amphidial apertures rounded, surrounded by swollen cuticular rim. Cervical collar present, comprising 48–52 posteriorly directed cuticular spines sharing a common base ( Fig. 3 , b). Spines of unequal size increasing in length from center to the lateral edges. Collar continuous, except laterally where it extends posteriorly forming pairs of cuticular lappets with serrate outer margins ( Fig. 1 , b, c; Fig. 3 , a). Inner margins of lappets delimit trapezium-shaped region of smooth cuticle. Two lateral pairs of large, posteriorly directed spines on each side of body arranged in tandem posterior to lappets. Lateral spines 28–32 µ m long, divided by area of wrinkled cuticle. Bases of posterior pair are most approximated. Numerous somatic papillae present on the body cuticle.

Females	Males	Females	Males
Holotype Paratypes	Paratypes	Vouchers	Vouchers
n	– 8	6	9	8
L	3215 3464 ± 618 (2610–4225)	1923 ± 255 (1595–2200)	3182 ± 132 (3050–3450)	1739 ± 109 (1550–1875)
L ′	2015 2494 ± 481 (1860–3115)	1764 ± 248 (1425–2000)	2247 ± 57 (2165–2350)	1590 ± 111 (1375–1725)
a	16 16.8 ± 3 (15.4–24)	13 ± 1.8 (10.4–16)	12.2 ± 2.1 (13–20)	11.3 ± 1.3 (10.2–14.2)
b	7.4 8 ± 1.3 (7–10.3)	3.3 ± 0.5 (2.7–4)	7.7 ± 0.4 (7.2–8.4)	3.1 ± 0.2 (2.7–3.4)
c	2.6 4 ± 0.3 (3.2–4.2)	13 ± 2.3 (9.3–16.4)	3.4 ± 0.2 (3–3.5)	12 ± 1.9 (8.8–15)
c ′	13.3 13.1 ± 3.3 (8–19)	3.3 ± 0.6 (2.8–4.3)	13 ± 1.4 (10–15)	2.6 ± 0.6 (1.7–3.5)
V	60 68 ± 2.6 (67–74)	–	67.1 ± 1.4 (65–69)	–
V ′	96 95.2 ± 0.9 (94–97)	–	95 ± 0.4 (95–96)	–
Pharynx length	430 442 ±36.1 (400–500)	579 ± 25 (550–600)	411 ± 23 (380–460)	553 ± 48 (465–625)
Head to vulva	1930 2379.3 ± 475 (1750–2965)	–	2136 ± 54.1 (2050–2225)	–
Max. body diam.	200 198 ± 40.2 (140–250)	149 ± 31 (130–210)	200 ± 22.4 (175–240)	154 ± 19 (121–175)
Anal/cloacal body diam.	90 80 ± 30 (40–125)	47.2 ± 7.8 (40–60)	75.2 ± 6.2 (70–90)	59 ± 11 (43–75)
Spicule length (arc)	– –	138.3 ± 10.7 (128–158)	–	133 ± 6.4 (123–140)
Gubernaculum length	– –	62 ± 6.2 (55–73)	–	63 ± 7 (55–75)
Tail	1200 970 ± 172.4 (750–1300)	154 ± 19.1 (125–170)	947 ± 84 (850–1100)	149 ± 21 (113–175)

Buccal cavity 61–63 µ m long, divided into three parts ( Fig. 1 , e, f). Anterior part, situated between pseudolabia, is followed by middle one 14–15 µ m long provided with three cuticular plates. Plates are dorsal and subventral in position, bearing minute bristles at its inner side. Posterior part of buccal cavity ca 31–32 µ m long with distinctively striated walls. Pharynx divided into slender, cylindrical corpus, clearly delimited isthmus and rounded basal bulb ( Fig. 1 , g). Nerve ring 146 ± 15 (125–163) µ m from anterior end. Excretory pore 235 ± 28 (185–275) µ m from anterior extremity followed by sinuous duct composed by at least four cells and leading into transverse sinus located posteriorly ( Fig. 1 , h). Two excretory ducts extend laterally from each side of the sinus toward both ends of the body becoming undistinguishable under light microscope. Intestine long, straight, filled with food content. Vulva slightly salient, 116 ± 15 (100–150) µ m anterior to anus, body narrowing abruptly posterior to its level. Anterior anal lip strongly developed, overlapping anal aperture ( Fig. 1 , i). Genital tract didelphic, prodelphic. Muscular vagina leads to thin-walled uteri filled with a mass of filiform spermatozoa ( Fig. 1 , j) and containing up to 10 eggs . Eggs 177 ± 11.2 (155–190) × 82 ± 8 (70–90) µ m in size, with thin flexible shell. Tail very long, subulate. Male. Cephalic extremity narrow, body reaching its maximum diameter at mid-body and tapering posterior to ventral sucker gradually moving into tail appendage ( Fig. 2 , d). Body cuticle finely annulated, annules 1.2 µ m apart ( Fig. 2 , c). Lateral alae absent. Oral opening situated apically, surrounded by thin cuticular band projecting from the lumen ( Fig. 3 , d). Amphidial apertures rounded, surrounded by thin cuticular rim. Numerous somatic papillae present on the body cuticle. Buccal cavity ca 32 µ m long, consisting of two parts: anterior part lined by thin cuticule and thick-walled posterior part with one dorsal and two subventral cuticular formations projecting into buccal cavity ( Fig. 2 , a, b). Each cuticular formation consists of a central stalk surrounded by cuticular lamellae resembling rose petals. Pharyngeal corpus long, fusiform with max. diam. of 33 µ m at its middle. Isthmus well defined, basal bulb rounded with strongly cuticularised valves. Nerve ring 173 ± 28 (125–212) µ m from anterior end. Excretory pore 277 ± 25.4 (250–312) µ m from anterior extremity, arranged similarly to that of female. Intestine straight, tapering posterior to basal bulb. Testis flexure 196 ± 59 (135–300) µ m posterior to basal bulb. Vas deferens subdivided into three regions: anterior with granular content; swollen middle region and posterior mostly composed of polygonal cells and connected with cloaca. Ventral sucker rounded, 30–35 µ m diam., situated 175–205 µ m anterior to cloacal aperture. Narrow channel located at the base of sucker chamber leads into spherical cavity with light, fibrous content ( Fig. 2 , e). Spicule single, canoe-like, with two equal heads ( Fig. 2 , i, h; Fig. 3 , f). Spicule axis slightly curved close to distal end. Gubernaculum trowel-like, with curved lateral edges ( Fig. 2 , j). Seven pairs of genital papillae present: one pair of mammiform papillae situated subventrally, posterior to ventral sucker; two pairs precloacal, of which posterior-most located on the sides of cloaca aperture; one pair lateral at cloaca level and three pairs postcloacal, of which two pairs situated ventrally and one pair dorsolaterally ( Fig. 2 , f, g; Fig. 3 , e). Cuticularised median duct is well defined on anterior cloacal lip. Low bursa-like fold of cuticle extends from behind the cloaca level to base of filiform tail appendage. Tail initially conoid, rapidly attenuating into filiform tip. Diagnosis and relationships. Heth impalutiensis n. sp. is characterized by the presence of a cervical collar consisting of unequal sized spines, the shape of the lateral lappets and form of anterior anal lip. Males possess a low bursa-like extension around the cloacal aperture and a mammiform pair of precloacal papillae situated directly behind the ventral sucker. FIGURE 1. Heth impalutiensis n. sp. , female. a , total view; b , anterior end, lateral view; c , anterior end, ventral view; d , pseudolabia, apical view; e , f , optical section through buccal cavity; g , optical section through anterior end; h , anterior part of excretory system; i , vulva and anal region; j , posterior section of uterus filled with numerous sperm content. (Scale bar for a = 250 µ m, b , c , e , f = 40 µ m, d = 60 µ m, g = 85 µ m, h = 30 µ m, i = 100 µ m, j = 75 µ m). FIGURE 2. Heth impalutiensis n. sp. , male. a , b , optical section through the buccal cavity; c , anterior end, lateral view; d , total view; e , optical section through ventral sucker, lateral view; f , posterior end, ventral view; g , h , posterior end, lateral view; i , j , spicule and gubernaculum. (Scale bars for a–c , e = 30 µ m, d = 200 µ m, f , g = 40 µ m, h = 45 µ m, i , j = 75 µ m). FIGURE 3. SEM micrographs of Heth impalutiensis n. sp. a , female anterior end, lateral view; b , female anterior end, ventral view; c , female pseudolabia, apical view; d , male anterior end, apical view; e , male posterior end, subventral view; f , distal spicule end, lateral view. (Scale bar for a–c , e = 30 µ m, d , f = 10 µ m). FIGURE 4. Phylogenetic relationships of Heth impalutiensis n. sp. as inferred from Baysian analysis of partial sequences of SSU and LSU rDNA. a , the phylogram of relationships of new species with wide set of ascaridid-spirurid-oxyurid species, based on partial SSU rDNA sequences (scale = 0.1 expected changes per site); b , part of total SSU rDNA phylogram presented as cladogram with the level of support for separate nodes indicated (posterior probability). Only studied species of Ransomnematoidea are included. c , relationships with other Ransomnematoidea based on partial LSU rDNA sequences (D2- D3 segment). Twelve Heth species have been described to date from the Australasian ( Australia , New Zealand ) and Asia- Pacific region ( Papua New Guinea , Philippines , Sumatra, Vietnam ). The morphology of the cervical collar, lateral lappets and cuticle ornamentation of the species described herein is mostly related to the following 11 species: Heth juli Cobb, 1898 , Heth costata Hunt, 1994 , Heth dimorphum Chitwood, 1935 , Heth xaniophora Hunt, 1994 , Heth sutherlandi Hunt, 1994 , Heth ortonwilliamsi Hunt, 1994 , Heth vietnamensis Malysheva & Spiridonov, 2010 , Heth taybaci Malysheva & Spiridonov, 2010 , Heth taynguyeni Malysheva & Spiridonov, 2010 , Heth tonkinensis Malysheva & Spiridonov, 2010 and Heth baudini Malysheva & Cribb, 2012 . Females of H . impalutiensis n. sp. can be easily differentiated from H. costata and H. xaniophora by the absence of longitudinal cuticular ridges and lateral alae, respectively. The bases of lateral spines are widely spaced in H . impalutiensis n. sp. , whereas fused in H. juli and H. tonkinensis . Heth impalutiensis n. sp. is close to H. sutherlandi and H. ortonwilliamsi in terms of the closer spaced bases of the posterior pair of lateral spines, but in H. impalutiensis n. sp. the spines are of equal size while in H. sutherlandi the posterior pair is longer but shorter in H. ortonwilliamsi . Heth impalutiensis n. sp. can be differentiated from H. baudini by bigger size of lateral spines (28–32 µ m vs 13–15 µ m, respectively) and the overall shape of the lateral lappets. Body size (L) in females of H. impalutiensis n. sp. is significantly larger than that of H. taybaci and H. taynguyeni (2610–4225 µ m (in Dila Falls population) and 3050–3450 µ m (in Mountain View College population) vs 2150, 1905 µ m and 1365–2075 µ m, respectively). Females of H. impalutiensis n. sp. are relatively similar to females of H. dimorphum and H. vietnamensis in body size and form of lateral lappets, but can be distinguished by the longer tail (750–1300 µ m vs 470–670 1 µ m and 463–510 µ m, respectively). The presence of a bursa-like fold of cuticle in males H. impalutiensis n. sp. makes them similar to the following representatives of the genus: H. xaniophora , H. ortonwilliamsi , H. taybaci , H. taynguyeni , H. baudini and two New Guinean species, described as Heth sp. ‘A’ and Heth sp. ‘B’ ( Hunt 1994 ). The bursa-like fold in H. impalutiensis n. sp. is formed only by the body cuticle, whereas in H. baudini it has some embedded somatic papillae. Heth impalutiensis n. sp. can be also differentiated from the latter species by the rounded form of the ventral sucker vs elliptical. In Heth sp. ‘A’, Heth sp. ‘B’ and H. taybaci the bursa-like fold extends from the anterior pair of precloacal papillae, whereas in the present species it begins behind cloaca level. Heth impalutiensis n. sp. also varies from Heth sp. ‘A’ by presence vs absence of cuticular formations inside the buccal cavity. Males of H. impalutiensis n. sp. can be easily distinguished from those of H. ortonwilliamsi and H. taynguyeni by spicule shape (canoe-like with pointed distal tip vs boat-like in H. ortonwilliamsi and distally bifurcate in H. taynguyeni ). The combination of such morphological features as presence of a bursa-like cuticular fold, the form of spicules and development of mammiform papillae make H. impalutiensis n. sp. mostly similar to H. xaniophora , which can be distinguished by having one fewer pair of genital papillae. Heth impalutiensis n. sp. can be distinguished from all nominal species described from this geographical region by hypertrophy of the anterior anal lip which overlaps the anal aperture. Phylogenetic relationships. The phylogenetic analysis of the 18S rDNA sequence of H. impalutiensis n. sp. was used to define the phylogenetic links of this new species ( Fig. 4 , a). An undescribed Heth sp. ( JX987087 ) from Indonesia is the closest to the species described herein with only 4 bp nucleotide differences present. Two other representatives of Heth with known 18S rDNA data differ from described the Philippine specimens by 11 and 14 bp ( Heth sp. ( JX987086 ) from Australia and H. taybaci ( JX987085 ) from Vietnam , respectively). In the 18S rDNA tree H. impalutiensis n. sp. forms a cluster with other Heth species with 100% posterior probability ( Fig. 4 , b). A high level of support (99%) is also observed in the cladogram for the same four Heth species obtained with Bayesian analysis of partial 28S rDNA sequence (D2-D3 expansion segment) ( Fig. 4 , c). The only difference is in the position of H. taybaci ( JX946704 ), which is situated in a basal position in the 28S tree for the genus, with H. impalutiensis n. sp. forming a single, 99%-supported, group with the remaining two species. Heth sp. ( JX443484 ) from Australia is the closest with 19 bp difference with H. impalutiensis n. sp. , while the other two species differ by 28 and 38 bp ( Heth sp. ( JX 443483 ) and H. taybaci ( JX946704 ), from Indonesia and Vietnam , respectively).