Torquenema n. g., Wallabicola n. g., and Macropostrongyloides phascolomys n. sp.: New Genera and a New Species of Nematode (Strongylida: Phascolostrongylinae) Parasitic in Australian Macropodid and Vombatid Marsupials

Simple Summary This article describes two new genera of parasitic nematodes, Torquenema n. g. from the eastern grey kangaroo and Wallabicola n. g. from the swamp wallaby, and a new species, Macropostrongyloides phascolomys n. sp. from the caecum of the common wombat. Abstract The strongyloid nematodes belonging to the subfamily Phascolostrongylinae occur primarily in the large intestines of macropodid and vombatid marsupials. Current molecular evidence suggests that the two nematode species, Macropostrongyloides dissimilis and Paramacropostrongylus toraliformis, from macropodid marsupials are distant from their respective congeners. Furthermore, specimens of Macropostrongyloides lasiorhini from the large intestines of the southern hairy-nosed wombat (Lasiorhinus latifrons) and the common wombat (Vombatus ursinus) are genetically distinct. This study aimed to describe the new genera Torquenema n. g. (with T. toraliforme n. comb. as the type species) from the eastern grey kangaroo, Wallabicola n. g. (with W. dissimilis n. comb. as the type species) from the swamp wallaby and a new species Macropostrongyloides phascolomys n. sp. from the common wombat, using light and scanning electron microscopy.


Introduction
The strongyloid nematode subfamily, Phascolostrongylinae Lichtenfels, 1980 is currently represented by seven genera and 19 species found in kangaroos, wallabies, and wombats (Macropodidae and Vombatidae) [1]. The Phascolostrongylinae has been subdivided into three tribes based on morphological features [1]. One of the tribes is the Macropostrongyloidinea, which comprises the genera Macropostrongyloides Yamaguti, 1961 and Paramacropostrongylus Johnston and Mawson, 1940, and they are generally characterised by small cylindrical buccal capsules with a distinctive Y-shaped dorsal gutter and the presence of peri-oral teeth or denticles [1]. Although the majority of species within the Phascolostrongylinae are restricted to the large intestines of their hosts, several exceptional species, all belonging to the tribe Macropostrongyloidinea, have been found within the stomachs of macropodid marsupials [2].
A recent molecular study based on the first and second internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA revealed that the genera Macropostrongyloides and Paramacropostrongylus were paraphyletic, with Macropostrongyloides dissimilis Johnston and Mawson, 1939 and Paramacropostrongylus toraliformis Beveridge and Mawson, 1978 distinct from their respective congeners [3]. The study found that M. dissimilis, a dark-red nematode from the stomach of the swamp wallaby Wallabia bicolor (Desmarest) is a sister taxon to the only other stomach-inhabiting phascolostrongyline species Paramacrop-

Collection of Specimens
Specimens were borrowed from the Australian Helminthological Collection, South Australian Museum (SAM), Adelaide and the Australian National Wildlife Collection (ANWC), Commonwealth Scientific and Industrial Research Organisation (CSIRO), Canberra, or collected from road killed hosts and stored in ethanol at −80 • C until required. These latter specimens have now been deposited in SAM.

Light Microscopy
Specimens were mounted on glass slides in lactophenol and examined under a compound microscope (Olympus BH-2, Olympus Optical CO., LTD., Tokyo, Japan). Illustrations were made using a drawing tube attached to a BH-2 Olympus microscope and digitised in Adobe Photoshop CC 2019. Measurements are presented in millimetres as ranges followed by the mean in parenthesis. The lengths and widths of the buccal capsule were taken from the external surface.
Morphological terminology follows that of Beveridge and Mawson [2] except for the papillae of the genital cone which follows the numerical system of Chabaud et al. [6]. Host nomenclature follows Jackson and Groves [7]. In instances where collection localities were within approximately 50 km of one another, records of occurrences were combined.

Scanning Electron Microscopy
Specimens for scanning electron microscopic (SEM) examination were dehydrated in an ethanol series with concentrations beginning at 70%, followed by 1:1 ratio of ethanol and hexamethyldisilazane (HMDS) (ProSciTech Pty. Ltd., Queensland, Australia) and finally with pure HMDS. The dehydrated specimens were air-dried and mounted onto stubs with double-sided carbon conductive tape, coated in gold and observed in a FEI XL30 FEG scanning electron microscope (Thermo Fisher Scientific Inc., Massachusetts, MA, USA).
Etymology: The new generic name is derived from 'torquem' which means collar in Latin and refers to the prominent cervical collar of Torquenema n. g. The specific name has been changed to toraliforme n. comb. in agreement with the neuter generic name.

Redescription
General (Figure 1a-d, Figures 2a-g and 3a,b). Robust nematodes, red or pink in colour when fresh. Body covered with numerous transverse annulations, c. 0.005 apart, with gaps between annulations along lateral surfaces of body. Cephalic collar enlarged, discshaped, extending peripherally beyond body; anterior surface divided by fine sutures visible with SEM, one surrounding cephalic papillae and amphids, outer suture close to peripheral margin (Figure 3a,b); outer region of collar with internal cavity, semi-lunar in shape in transverse section; inner region of collar pierced by four sub-conical, sub-medial papillae and two lateral amphids; mouth opening circular; buccal capsule sub-cylindrical, heavily sclerotised, longer than wide, with numerous inconspicuous internal longitudinal ridges, most readily visible in lateral views towards base of buccal capsule; anterior extremity bears c. 25 triangular projections forming leaf crown; teeth absent in buccal capsule; wall of buccal capsule complex with internal structure sub-divided; duct of dorsal oesophageal gland pierces wall of buccal capsule at mid-length, divides internally to form Y-shaped dorsal gutter, with branches passing internally near anterior margin of buccal capsule, joining ventrally. Oesophagus clavate; ventral sectors of oesophagus project prominently into base of buccal capsule; dorsal sector projects slightly into base of buccal capsule; nerve ring in anterior third of oesophagus; deirid papillate, posterior to nerve ring; excretory pore at about same level as deirid. The geographic distribution of T. toraliforme n. comb. corresponds to some degree with the distribution of its host, M. giganteus ( Figure 4). Collection records show that T. toraliforme n. comb. is most commonly encountered in Victoria and some parts of New South Wales. However, T. toraliforme n. comb. appears to be absent from Queensland except for areas around Inglewood, close to the New South Wales border ( Figure 4).

Generic Definition
Robust nematodes. Anterior extremity deviated slightly ventrally; cephalic collar prominent, pierced by four sub-conical, sub-median papillae and two lateral amphids; mouth opening elongated laterally; four triangular sub-median teeth arise from anterior wall of buccal capsule; buccal capsule sub-cylindrical, heavily sclerotised; duct of dorsal oesophageal gland runs within wall of buccal capsule but at mid-length, divides internally forming Y-shaped dorsal gutter. Oesophagus clavate; nerve ring in anterior third of oesophagus; deirid papillate, at level of nerve ring; excretory at pore same level as deirid and nerve ring. Spicules elongate, alate. Vulva immediately anterior to anus; ovejector Jshaped. Parasitic in macropodid marsupials.  Bursa short, stout; lateral and ventral lobes fused, ventral lobes joined ventrally; dorsal lobe shorter than ventro-lateral lobes, with slight median indentation. Ventro-ventral and ventro-lateral rays apposed, reaching margin of bursa; externo-lateral ray divergent from lateral trunk, not reaching margin of bursa, terminating in slight elevation on surface of bursa; medio-lateral and postero-lateral rays apposed, reaching margin of bursa; externolateral ray between lateral and dorsal trunks, not reaching margin of bursa, terminating in slight elevation on surface of bursa; dorsal ray slender at origin, dividing at mid-length; external branchlets directed postero-laterally, not reaching margin of bursa; internal branchlets arcuate, directed posteriorly, not reaching margin of bursa; spicules 2.95-3.45 (3.25) long, alate; alae with numerous transverse striations; anterior extremity of spicules irregularly knobbed; distal extremity curved, terminating in blunt tip; ala diminishes gradually towards distal tip of spicule, losing transverse striations; ventral lip of genital cone conical, bearing papilla 0; dorsal lip with paired appendages (papillae 7) closely applied to cloaca; dorsal to each of them, distally trifid projection; 3-4 elongate, simple projections lateral to each side of central projections; gubernaculum prominent, ovoid and laterally elongated in dorso-ventral view 0.13-0.15 (0.14) long.

Remarks
Torquenema n. g. is distinguishable from all other phascolostrongyline genera by the presence of a large disc-shaped cephalic collar which protrudes beyond its body margin. Torquenema n. g. differs from Paramacropostrongylus by the Y-shaped ovejector and its occurrence within the caecum of the host.
The current re-description of T. toraliforme n. comb. largely supports that of Beveridge and Mawson [2] and Beveridge [1], while providing additional details, particularly those shown by SEM. Beveridge and Mawson [2] reported the mouth opening of P. toraliformis as being surrounded by about 20 'denticles'. Their apical view of the mouth opening ( Figure 59 in Beveridge and Mawson [2]) shows 23 'denticles' which is closer to the 25 estimated in the current description. However, the current SEM observations of Torquenema n. g. (Figure 3a,b) suggest that these 'denticles' are not separate entities but form a continuous structure around the anterior margin of the buccal capsule, resembling a leaf crown. Consequently, they have here been described as such. The extent to which this situation differs from Paramacropostrongylus will require additional SEM studies of this genus.
One of the principal morphological features used to separate the Phascolostrongylinae from the Cloacininae has been the origin of the externo-dorsal ray, arising from the dorsal trunk in the Phascolostrongylinae and from the lateral trunk in the Cloacininae. However, in Torquenema n. g., the externo-dorsal ray arises between the lateral and dorsal trunks, a feature partially recognised by Beveridge and Mawson [2] in their definition of Paramacropostrongylus in which they state 'externo-dorsal ray arising from dorsal ray or separately'. Although uncommon within the Cloacininae, Beveridge [8] described two species of Cloacina in which the externo-dorsal ray arises from the dorsal ray. The current description of the genital cone differs slightly from that illustrated by Beveridge and Mawson [2] in that their Figure 62 lacks the actual papillae 7 on the dorsal lip, which are overshadowed by the paired appendages dorsal to them.
The geographic distribution of T. toraliforme n. comb. corresponds to some degree with the distribution of its host, M. giganteus (Figure 4). Collection records show that T. toraliforme n. comb. is most commonly encountered in Victoria and some parts of New South Wales. However, T. toraliforme n. comb. appears to be absent from Queensland except for areas around Inglewood, close to the New South Wales border ( Figure 4).   Wallabicola n. g. Type species: Wallabicola dissimilis (Johnston and Mawson, 1939) n. comb.

Generic Definition
Robust nematodes. Anterior extremity deviated slightly ventrally; cephalic collar prominent, pierced by four sub-conical, sub-median papillae and two lateral amphids; mouth opening elongated laterally; four triangular sub-median teeth arise from anterior wall of buccal capsule; buccal capsule sub-cylindrical, heavily sclerotised; duct of dorsal oesophageal gland runs within wall of buccal capsule but at mid-length, divides internally forming Y-shaped dorsal gutter. Oesophagus clavate; nerve ring in anterior third of oesophagus; deirid papillate, at level of nerve ring; excretory at pore same level as deirid and nerve ring. Spicules elongate, alate. Vulva immediately anterior to anus; ovejector J-shaped. Parasitic in macropodid marsupials. Site in host: Stomach. Representative DNA sequences: Ribosomal DNA first and second internal transcribed spacers sequences (Genbank Accession MK842126) from W. bicolor, Kamarooka, Victoria, Australia [5].
Etymology: The new generic name Wallabicola n. g. (masculine) is derived from 'incola' in Latin meaning inhabitant and Wallabia referring to the swamp wallaby, Wallabia bicolor, the host in which the new genus occurs.

Redescription
General (Figures 3c-f and 5a-l). Robust nematodes, red in colour when fresh. Body covered with numerous, fine transverse annulations, c. 0.001 apart. Anterior extremity deviated slightly ventrally; cephalic collar prominent, pierced by four sub-conical, submedial papillae and two lateral amphids; mouth opening elongated laterally; cephalic collar projects into mouth opening adjacent to amphids, with apical inflection, membrane surrounding teeth joined; four triangular sub-median teeth arise from anterior wall of buccal capsule, project into lumen; membrane between teeth and wall of buccal capsule continuous with internal projections from amphidial region of cephalic collar and bifid dorsal and ventral projections on anterior margin of buccal capsule; buccal capsule subcylindrical, heavily sclerotised, longer than wide; duct of dorsal oesophageal gland runs within wall of buccal capsule but at mid-length, divides internally to form Y-shaped dorsal gutter, with branches passing internally near anterior margin of buccal capsule, joining ventrally. Oesophagus clavate; nerve ring in anterior third of oesophagus at slight oesophageal constriction; deirid papillate, at level of nerve ring; excretory pore at about same level as deirid and nerve ring. ls 2021, 11, x 11 of 17  Ventro-ventral and ventro-lateral rays apposed, reaching margin of bursa; externo-lateral ray divergent from lateral trunk, not reaching margin of bursa, terminating in slight elevation on surface of bursa; medio-lateral and postero-lateral rays apposed, reaching margin of bursa; externo-lateral ray originating at base of dorsal trunk, not reaching margin of bursa, terminating in slight elevation on surface of bursa; dorsal ray slender at origin, dividing at mid-length; external branchlets directed postero-laterally, not reaching margin of bursa; internal branchlets directed posteriorly, not reaching margin of bursa; spicules 2.40-2.78 (2.53) long, alate; ala with numerous transverse striations; anterior extremity of spicules irregularly knobbed; distal extremity blunt; ala diminishes gradually towards distal tip of spicule, losing transverse striations; ventral lip of genital cone conical, bearing papilla 0; dorsal lip with paired appendages (papillae 7) closely applied to cloaca; dorsal to them array of elongate papillae; gubernaculum poorly sclerotised, ovoid and laterally elongated in dorso-ventral view 0.030-0.0.035 (0.032) long, slender in lateral views, not clearly visible in all specimens.

Remarks
Wallabicola n. g. most closely resembles Macropostrongyloides, differing in the J-shaped ovejector and its occurrence within the stomach of its host. The new genus is differentiated from other phascolostrongyline genera (except for Hypodontus and Corollostrongylus) by its ventrally deviated head.
The current re-description of this species confirms most of the observations of Beveridge and Mawson [2] and Beveridge [1] while adding additional details of the spicule tip and cephalic structures. The description of the cephalic structures presented here differs significantly from that of Beveridge and Mawson [2], particularly in the position of the amphids. The cephalic extremity of W. dissimilis is extremely small and difficult to study under the light microscope. However, the scanning electron photomicrographs presented here provide novel information (Figure 3c-f). As with species of Macropostrongyloides, the buccal capsule contains four sub-median teeth with a flap-like structure resembling a leaf crown element arising between the tooth and the margin of the buccal capsule. Sukee et al. [10] speculated that these, along with the 'denticles' might constitute a leaf crown if they were continuous. The comparable structure in W. dissimilis is continuous although denticles are lacking. It differs however in that in W. dissimilis the structure includes bifid projections on the dorsal and ventral aspect of the margin of the buccal capsule, which are absent in species of Macropostrongyloides, and the lateral tridents seen in Macropostrongyloides are replaced by internal projections of the cephalic collar. The peri-oral structures of W. dissimilis are therefore quite distinct from those found in Macropostrongyloides baylisi, M. mawsonae, M. spearei and M. woodi, but the observations presented here support the suggestion of Sukee et al. [10] that an unusual form of leaf crown is present at the anterior margin of the buccal capsule in these species. The female genital system of W. dissimilis was re-described by Beveridge ( Figure 3A, [1]), but was not shown it in its entirety. The entire system is shown in Figure 5j. The vagina is slightly convoluted with the degree of convolution differing between specimens.
The new genus is predominantly distributed in Victoria and New South Wales ( Figure 6). Although the distribution of W. bicolor is continuous along the eastern coast of Australia from Victoria to far north Queensland, W. dissimilis is has rarely been encountered in Queensland [2,11].  [9]. Open circles represent locations in which hosts have been examined but W. dissimilis was absent. Solid circles represent locations in which W. dissimilis were found. Abbreviations; Qld = Queensland, NSW = New South Wales, Vic = Victoria.

Site in host: Colon.
Representative DNA sequence: Ribosomal DNA first and second internal transcribed spacers sequences (Genbank Accession MK842123) from V. ursinus, Bullengarook, Victoria, Australia [5].  [9]. Open circles represent locations in which hosts have been examined but W. dissimilis was absent. Solid circles represent locations in which W. dissimilis were found. Abbreviations; Qld = Queensland, NSW = New South Wales, Vic = Victoria. Site in host: Colon. Representative DNA sequence: Ribosomal DNA first and second internal transcribed spacers sequences (Genbank Accession MK842123) from V. ursinus, Bullengarook, Victoria, Australia [5].

Description of a New
Etymology: The new species is named after the former generic name of the host, Phascolomys.

Description
General (Figure 7a-k). Robust nematodes, pink in colour when fresh. Body without transverse annulations. Anterior extremity with prominent cephalic collar, pierced by four sub-conical, sub-medial papillae and two lateral amphids; mouth opening circular; four triangular sub-median teeth arise from anterior wall of buccal capsule, project into lumen; petaloid membrane present between teeth and wall of buccal capsule; bifid or trifid membranous structure present at anterior margin of buccal capsule, internal to each amphid; paired denticles present on anterior margin of buccal capsule dorsally and ventrally; buccal capsule sub-cylindrical, heavily sclerotised, longer than wide; duct of dorsal oesophageal gland runs within wall of buccal capsule but at mid-length, divides internally to form Y-shaped dorsal gutter, with branches passing internally near anterior margin of buccal capsule, joining ventrally. Oesophagus clavate; nerve ring in anterior third of oesophagus at slight oesophageal constriction; deirid papillate, at level of nerve ring; excretory pore at about same level as deirid and nerve ring.
Female (measurements of 10 specimens). tween the two species, in M. lasiorhini the striation patterns change orientation further from the spicule tip and diminishes completely (Figure 8a,b) whereas in M. phascolomys n. sp., after the change in orientation the striations continue until almost reaching the end of the ala (Figure 8c).
The wombat host species of M. phascolomys n. sp. and M. lasiorhini also differ, with the former species occurring in V. ursinus and the latter in L. latifrons.

Remarks
The new species is very similar to M. lasiorhini with which it has been confused in the past. The females of the two species are indistinguishable and the only character separating the males is the morphology of the genital cone which has a ridge ventral to the ventral lobe of the cone and a pair of lateral projections on the two sides of the base of the dorsal part of the cone. In M. lasiorhini, the dorsal lobe of the cone is surrounded by numerous projections, some of which are bifid terminally. The spicule tip of M. lasiorhini has not been described in detail previously but it is distinguishable from the spicule tip of M. phascolomys n. sp. The comparisons between the spicule tips of M. lasiorhini and M. phascolomys n. sp. are shown in Figure 8a-c. There is a slight bend in the spicule tip of M. phascolomys n. sp. compared to the almost 45 degree bend in the spicule tip of M. lasiorhini (Figure 8b,c). The spicule ala in M. phascolomys terminates abruptly prior to reaching the end of spicule tip whereas in M. lasiorhini, the ala continues toward the end (Figure 8a) and wraps around the spicule tip in oblique views (Figure 8b). The striation patterns also differ between the two species, in M. lasiorhini the striation patterns change orientation further from the spicule tip and diminishes completely (Figure 8a,b) whereas in M. phascolomys n. sp., after the change in orientation the striations continue until almost reaching the end of the ala (Figure 8c).

Discussion
Examination of specimens previously identified as P. toraliformis and M. dissimilis revealed differential morphological features which warrant the transfer of these species into two new genera, Torquenema n. g. and Wallabicola n. g., respectively. The study also found that M. lasiorhini from V. ursinus varied from specimens in the type-host, L. latifrons; therefore, permitting the description of a new species M. phascolomys n. sp., which is restricted to V. ursinus. The new genera and species described here are supported by previous molecular data [3,5].
Whilst the current descriptions of the genera Torquenema and Wallabincola are largely consistent with previous work by Beveridge and Mawson [2], additional informative char- The wombat host species of M. phascolomys n. sp. and M. lasiorhini also differ, with the former species occurring in V. ursinus and the latter in L. latifrons.

Discussion
Examination of specimens previously identified as P. toraliformis and M. dissimilis revealed differential morphological features which warrant the transfer of these species into two new genera, Torquenema n. g. and Wallabicola n. g., respectively. The study also found that M. lasiorhini from V. ursinus varied from specimens in the type-host, L. latifrons; therefore, permitting the description of a new species M. phascolomys n. sp., which is restricted to V. ursinus. The new genera and species described here are supported by previous molecular data [3,5].
Whilst the current descriptions of the genera Torquenema and Wallabincola are largely consistent with previous work by Beveridge and Mawson [2], additional informative characters have been added to the current descriptions. The structure of the deirids is relatively uniform throughout the species of the Phascolostrongylinae and is short and papillate in shape. In the Cloacininae, the deirid is elongate and hair-like, and this difference may provide a means of separating the two sub-families. The structure was overlooked in previous taxonomic descriptions. The use of scanning electron microscopy has enabled detailed observations of the peri-oral structures, including the flap-like projections associated with the teeth of W. dissimilis and denticles of T. toraliforme. A previous study of Macropostrongyloides spp. observed similar structures and speculated that the peri-oral projections may be leaf-crowns given that they extend the full length of the buccal capsule [10]. The SE photomicrographs of the mouth opening of T. toraliforme (Figure 3b) and W. dissimilis (Figure 3c,e,f) show that these peri-oral structures in both species appeared to be continuous around the buccal capsule rather than projecting as individual elements, suggesting that the structures could potentially be considered leaf-crowns. The presence of leaf-crown elements is characteristic of some genera of the subfamily Cloacininae and has only been observed in the subfamily Phascolostrongylinae in the genera Phascolostrongylus and Oesophagostomoides from wombats [1]. Whether this is also the case in other genera and species within the Phascolostrongylinae is not known and additional SEM examination is required, particularly of Paramacropostrongylus, Macropostrongyloides yamagutii and M. lasiorhini which possess both teeth and denticles.
The shapes of the ovejector were used to differentiate the genera Torquenema n. g. and Wallabincola n. g. from their former genera. The presence of a Y-shape muscular ovejector is considered a plesiomorphic character within the Strongyloidea [12,13]. The genus Torquenema n. g. is differentiated from the genus Paramacropostrongylus by the presence of a Y-shaped ovejector and the genus Wallabincola is distinguished from Macropostrongyloides based on its J-shaped ovejector. In this study, the shape of the ovejector has been used as a generic character. However, the shape of the ovejector is not always reliable as a generic character. In the genus Cloacina for instance, species possess either a Y-shaped or J-shaped ovejector [14].
The geographic distribution of T. toraliforme and W. dissimilis appears to be primarily in the southern region of the distributions of their hosts (Figures 4 and 6). Similar patterns have been observed in Macropostrongyloides mawsonae from M. giganteus which is predominantly encountered in the southern region of Australia but rarely detected in the northern part of Australia despite the continuous distribution of the host [10]. Previous molecular studies [3,5] have indicated genetic differences between specimens of W. dissimilis from Queensland and those collected from Victoria. No morphological differences were detected between these two genotypes in the current study, but the existence of a cryptic species cannot be eliminated. Torquenema n. g. occurs primarily in M. giganteus in Victoria whereas Paramacropostrongylus iugalis occurs in the same host in northern New South Wales and central Queensland [15].
Macropostrongyloides phascolomys n. sp. occurs in V. ursinus while M. lasiorhini, occurs in L. latifrons. Fossil records show that the wombat genus Lasiorhinus once had a wider distribution and was present in Victoria, New South Wales in Queensland between the late-Miocene to mid-Pliocene period [16]. This suggests that in the past, Lasiorhinus and Vombatus may have been sympatric while the current geographic distribution of M. lasiorhini and V. ursinus is allopatric. Therefore, the two species of Macropostrongyloides may have undergone co-speciation with their respective hosts.

Conclusions
The present study described two new genera and a new species from the subfamily Phascolostrongylinae supporting previous molecular evidence [3,5,17]. Future studies utilising the nuclear and mitochondrial DNA sequence data could provide better insights into the evolution of this highly diverse group of parasitic nematodes from herbivorous Australian marsupials.