Abursanema iranicum, Yaghoubi, Ali, Pourjam, Ebrahim, Pedram, Majid, Siddiqi, Mohammad Rafiq & Atighi, Mohammad Reza, 2014

Yaghoubi, Ali, Pourjam, Ebrahim, Pedram, Majid, Siddiqi, Mohammad Rafiq & Atighi, Mohammad Reza, 2014, Description of Abursanema iranicum n. gen., n. sp. (Nematoda: Hexatylina, Sphaerularioidea) from Iran and its phylogenetic relationships, Zootaxa 3826 (2), pp. 301-314 : 305-309

Abursanema iranicum n. gen., n. sp.

( Figs 1 & 2 )

Measurements. See Table 3 .

Description. Male. Free-living. Body slightly ventrally arcuate after heat relaxation. Inner cuticle finely annulated, outer cuticle smooth throughout body length, lateral field with two incisures. Deirid at the level of excretory pore. Lip region low, rounded and slightly flattened, continuous with body contour. Stylet short, weak, 6.5–10.5 Μm, without basal knobs, dorsal arm of the shaft slightly longer than the ventral. Orifice of dorsal gland 1–2 Μm behind stylet base. Corpus cylindroid, metacorpus weakly developed, spindle-shaped, without a valvular apparatus, isthmus narrow, 35–47 Μm long. Basal bulb large, pyriform, with stem-like extension projecting into the lumen of intestine. Excretory pore opening at the level of middle of basal bulb, 86–107 Μm from anterior end. Hemizonid located just anterior to excretory pore. Nerve ring enveloping isthmus in its posterior half. Testis single, anteriorly outstretched, gonoduct 159–252 Μm long; sperm spheroid. Spicules paired, ventrally arcuate, with rounded manubrium and pointed distal tip, gubernaculum arcuate. Bursa absent. Phasmid hardly visible, located at 0.75–1.0 times body width posterior to cloaca. Tail long, with narrowly rounded or pointed tip.

Female. Free-living (infective entomoparasitic female not found). General morphology similar to that of male, except for character states associated with sexual differences. Reproductive system monodelphic-prodelphic, ovary with single row of oocytes, usually reflexed two times at proximal end, spermatheca rounded to ellipsoid, filled with spheroid sperm, crustaformeria short, ca 1.7–2.1 body widths long, vulva a transverse slit, vagina extending into the body a little less than half body width, postvulval uterine sac less than corresponding body width. Vulva-anus distance 46–69 Μm, ca 0.3–0.6 of the tail length.

Etymology. The name of the genus is Greek meaning ‘nematode without a bursa’ and is neuter in gender. Specific epithet refers to the country of occurrence.

Molecular phylogenetic relationships. Partial 18S rDNA phylogeny. Our 942 bp partial SSU rDNA sequence, GenBank accession number KF885743 , was used to determine the phylogenetic relationships of Abursanema n. gen. with other tylenchid nematodes. A BlastN search of the GenBank sequence database was performed and the closest sequences/species, along with sequences of genera with morphological similarity, were selected for inclusion in the analyses. The dataset was composed of 2493 total characters, of which 2118 characters were variable and 1824 characters were parsimony-informative after manual editing. The average nucleotide composition was as follows: 25.7% A, 20.6% C, 26.4% G and 27.2 % T. Figure 3 presents a Bayesian phylogenetic tree inferred from the multiple alignment of partial SSU sequences of 23 tylenchid taxa, one outgroup taxon and one isolate of the newly recovered/sequenced genus. The BPP and ML BS values are given on the appropriate clades in the form BPP/ML BS. In this tree, the new genus forms a clade with one species of Deladenus Thorne, 1941 (saccession number EU306345 ), and two species of Sphaerularia (accession numbers AB 300595 and AB 250213 ), members of the superfamily Sphaerularioidea (see Siddiqi, 2000). The monophyly of this clade is well supported in both BI and ML methods (1.00 and 0.96% respectively). The other species of Deladenus used in our analyses ( D. proximus Bedding, 1974 , accession number JF304744 ) does not share a common ancestor with Deladenus sp. ( EU306345 ) and occupies another position in the tree. This clade forms a sister clade to the remaining members of Hexatylina with low BPP (0.55) and no support under ML. Based on the current paucity of data and the restricted number of Hexatylina species that have been sequenced, the relationships among these taxa cannot be clearly resolved.

D2/D3 segment of 28S rDNA phylogeny. To reconstruct the 28S rDNA tree, a 706 bp partial sequence of D2/ D3 region with accession number KF885742 was used. Figure 4 presents the phylogenetic tree of 37 tylenchid taxa, one outgroup and one isolate of Abursanema iranicum n. gen., n. sp. The dataset was composed of 941 total characters of which 887 characters were variable and 827 characters were parsimony-informative after manual editing, with an average nucleotide composition of 23.7 % A, 19.6 % C, 29.8 % G and 26.9 % T. As in the 18S tree, the new genus formed a clade with two species of Sphaerularia ( AB 300595 and AB733665 ) with high BPP and ML BS support. This makes sense, considering the morphological similarities of these taxa, mostly in the shape of the pharynx. According to Siddiqi (2000), the families Sphaerulariidae and Paurodontidae are synonymous and the grouping of Sphaerularia , as a member of Sphaerulariidae , and the new genus, as a member of Paurodontidae , in our tree supports his opinion.

Discussion. Our phylogenetic study, using both partial 18S rDNA and D2/D3 segment of 28S rDNA, reveals new insights on the molecular phylogenetic affinities of members of Hexatylina , including Abursanema n. gen. In constructed trees using partial sequences of both SSU ( Fig. 3 ) and LSU ( Fig. 4 ), Abursanema n. gen. ( Paurodontidae ) clusters with sequenced species of Sphaerularia (Sphaerularidae) , supporting the synonymy of Paurodontidae and Sphaerulariidae ( Siddiqi, 2000, Chizhov, 2004 and Andrássy, 2007). In the partial 18S tree, Deladenus sp. ( EU306345 ) forms a clade with Abursanema n. gen. but D. proximus ( JF304744 ) is placed in a clade with Howardula spp. ( AF519234 , JX 291137 and AY146451 ) and Bradynema listronoti Zeng, Giblin-Davis, Ye, Belair, Boivin & Thomas, 2007 ( DQ915805 ). No free-living mycetophagous generation is known for the latter two genera, but one occurs in Deladenus . Based on our result, using of this biological character to classify genera in Hexatylina is questionable. The inferred tree using partial sequences of D2/D3 of 28S rDNA shows the same result as in the SSU tree. In our 28S tree, Howardula phyllotretae Oldham, 1933 ( DQ328728 ) and Parasitylenchus sp. ( DQ328729 ) form a highly supported clade in both BI and ML methods (1.00/99). These genera are currently classified in different families, Allantonematidae Pereira, 1931 and Parasitylenchidae Siddiqi, 1986 respectively, assigned to different superfamilies, Sphaerularioidea and Iotonchioidea Goodey, 1953 ( Siddiqi, 1986), respectively. Members of both genera are insect parasites. Thus, parasitism of the insect haemocoel may have evolved independently in both superfamilies ( Siddiqi, 2000). We believe that additional molecular data from other genera may give a clearer picture of the relationships among members of Hexatylina . Both partial 18S and 28S rDNA trees confirm that Howardula Cobb, 1921 is polyphyletic (see various papers, including Ye et al., 2007). Abursanema n. gen. and Sphaerularia spp. form a highly supported clade in both ML and BI methods, but again these genera have been assigned to different families ( Paurodontidae and Sphaerulariidae , respectively), albeit families that may in fact be synonymous. In Paurodontidae , a fungus-feeding generation is well-known but nothing is known of entomoparasitic forms, while in Sphaerulariidae a free-living generation may be present ( Siddiqi, 2000). Accordingly, and as noted above for the partial 18S tree, life cycle should not be considered a basic character for classification of members of the Hextaylina. This proposal conflicts with what was noted by Chizhov et al. (2012) who emphasized the importance of life cycle characteristics in the systematics of hexatylenchid nematodes. The results of our molecular phylogenetic studies with partial sequences of both 18S rDNA and especially the D2/D3 expansion segment of 28S rDNA show a close relationship of members of Paurodontidae and Sphaerulariidae , and also highlight the need for data from additional representatives of both families so that their relationships could be more clearly elucidated.

Figures

FIGURE 1. Abursanema iranicum n. gen., n. sp. A: Anterior body region, with details of pharynx; B: Head region, showing annulated inner cuticle; C: Female reproductive system; D, E: Male and female entire body; F: Male cloacal region showing absence of bursa; G: Female, cross section; H: Anterior end of female reproductive system, showing extension over the posterior part of the pharynx.

FIGURE 2. Abursanema iranicum n. gen., n. sp. A: Anterior body region showing annulated inner cuticle and details of stylet; B: Female tail end; C: Extension of pharynx; D: Lip region, en – face view; E: Lateral field; F: Part of female reproductive system showing spermatheca; G & H: Male cloacal region, ventral and lateral view; I: Anterior end of female reproductive system, showing extension over the posterior part of the pharynx and excretory pore (arrow); J: Phasmid (arrow) (scale bars: D = 5 µm, all the rest = 10 µm).

FIGURE 3. Bayesian 50 % majority rule consensus tree inferred from 25 sequences of partial SSU rDNA under the GTR + I + G model. Posterior probabilities and ML BS values> 50 % are given on appropriate clades in the shape BPP / ML BS. The new genus is in bold.

FIGURE 4. Bayesian 50 % majority rule consensus tree inferred from 39 sequences of D 2 / D 3 expansion segment of 28 S rDNA under the GTR + I + G model. Posterior probabilities and ML BS values> 50 % are given on appropriate clades in the shape BPP / ML BS. The new genus is in bold.

Tables

TABLE 3. Morphometrics of Abursanema iranicum n. gen., n. sp. All measurements are in μm and in the form: mean ± s. d. (range).

Characters Male		Female
Holotype	Paratypes	Paratypes
n –	16	26
L 586	561.0 ± 20.7 (525 – 593)	684.0±40.6 (585 – 740)
L' 462	437 ± 14 (412 – 462)	540 ± 36.3 (465 – 595)
a 34.5	33.6 ± 1.9 (31 – 37)	32.0 ± 1.2 (29 – 34)
b 4.2	4.3 ± 0.2 (4 – 5)	4.8 ± 0.4 (4.1 – 5.4)
c 4.7	4.6 ± 4.0 (4.0 – 5.6)	4.8 ± 0.5 (3.9 – 5.6)
c' 7.8	8.1 ± 1.1 (6.3 – 9.8)	8.8 ± 1.2 (6.8 – 11.6)
V or T 40.4	41.0 ±4.9 (31 – 48)	70.7 ± 1.9 (66 – 75)
V' –	–	89.6 ± 0.9 (88.0 – 91.5)
Head height 2.5	2.1 ± 0.3 (1.5 – 2.5)	2.3 ± 0.3 (2 – 3)
Head width 6	5.8 ± 0.2 (5.5 – 6.0)	6.8 ± 0.5 (6 – 8)
Stylet 9	8.5 ± 1.2 (6.5 – 10.5)	8.9 ± 0.7 (8.0 – 10)
Conus 3	3.3 ± 0.6 (2 – 4)	3.7 ± 0.6 (3 – 5)
m 33	40.5 ± 5.5 (33 – 50)	41.0 ± 6.5 (30 – 50)
Anterior end to centre of median bulb 57	52.0 ± 4.2 (45 – 58)	51.7 ± 4.0 (43 – 58)
MB 41	40.1 ± 3.2 (34.6 – 45.2)	36.3 ± 3.5 (32.5 – 46.0)
Excretory pore 107	99.0 ± 6.3 (86 – 107)	108.7 ± 6.0 (98 – 121)
Pharynx 139	130.0 ± 6.4 (111 – 139)	144.0 ± 10.2 (123 – 163)
Head – vulva –	–	484.0 ± 32.6 (415 – 531)
Max. body diam. 17	16.8 ± 0.9 (15 – 18)	21.4 ± 1.2 (18 – 23)
PUS –	–	7.8 ± 1.3 (5 – 10)
PUS/BW –	–	0.4 ± 0.1 (0.3 – 0.5)
Anal/cloacal body diam. 16	15.3 ± 0.7 (13.5 – 16.0)	16.6 ± 1.7 (13 – 20)
Vulva – anus –	–	56.0 ± 6.3 (46 – 69)
Tail 124	123.0 ± 14.3 (97 – 142)	144.3 ± 16.0 (109 – 174)
Spicule 22	20.6 ± 1.4 (18 – 23)	–
Gubernaculum 6	6.1 ± 0.8 (4 – 7)	–