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Revision of Macroponema Mawson, 1978 (Nematoda: Strongylida) from macropodid marsupials with the description of two new species

Background Species of Macroponema Mawson, 1978 are strongyloid nematodes which occur in the stomachs of macropodid marsupials in Australia. In this study, the genus Macroponema is revised, redescriptions of the two known species are provided, and two new species are added to the genus. Methods A molecular characterisation of the internal transcribed spacers of the nuclear ribosomal DNA of representative specimens of Macroponema from all known host species was undertaken to confirm the status of M. cf. comani. This resulted in the identification of a further new species within the genus. Consequently, a review of all available material in museum collections was undertaken. Results The two known species M. beveridgei Mawson, 1978 from Osphranter antilopinus (Gould) and O. robustus (Gould), and M. comani Mawson, 1978 from Macropus giganteus Shaw are re-described and their geographical distributions expanded. Two new species added to the genus are M. arundeli n. sp. from Ma. giganteus found in Queensland and the north east of New South Wales, and M. obendorfi n. sp. from O. antilopinus and O. robustus in the Northern Territory, the Kimberley Division of Western Australia and eastern Queensland. The latter species was formerly identified as M. cf. comani based on molecular studies. The specific identification of both of the new species is supported by ribosomal DNA sequence data. Conclusions Based on the morphological and molecular characterisation of nematodes, this study has revealed the existence of four species within the genus Macroponema. The current phylogenetic data suggest that Macroponema spp. plausibly evolved by host switching; however, further studies are required to test this hypothesis.

host, Macropus giganteus Shaw and based on differences in sequences of the internal transcribed spacers of the nuclear ribosomal DNA suggested that their specimens represented a cryptic species within M. comani, designated as M. cf comani.
In the present study, a molecular examination of representative specimens of Macroponema from all known host species was undertaken to confirm the status of M. cf comani. However, this resulted in the identification of a further new species within the genus. Consequently, a review of all available material in museum collections was undertaken, resulting in re-descriptions of the two known species, the description of M. cf comani as a new species as well as the description of another new species.

Collection of samples
Specimens of nematodes were collected opportunistically from: (i) fresh, road-killed kangaroos; (ii) kangaroos killed by commercial shooters; (iii) kangaroo culls; or (iv) animals used in related parasitological studies. In some instances, entire gastrointestinal tracts were preserved in formalin; in other instances, stomach contents were preserved in either formalin or 70% ethanol. Nematodes retrieved from these sources were stored in 70% ethanol. When fresh material was available, samples for morphological analysis were preserved in formalin or ethanol, while those required for molecular analysis were either frozen immediately in liquid nitrogen or were fixed in 70% ethanol and all were stored at − 80 °C until use.

Morphological studies
The anterior and posterior extremities of nematodes were excised and cleared in lactophenol for identification while the mid-section of the body was stored in 70% ethanol for molecular studies. The anterior and posterior extremities of specimens used for molecular studies were then stored in 70% ethanol and deposited in the Australian Helminthological Collection (AHC) of the South Australian Museum, Adelaide (SAM). Collection localities are listed under states with individual localities listed in order of increasing latitude ( Table 1).
All specimens present in the Australian Helminthological Collection of the South Australian Museum, Adelaide, were examined. Nematodes were cleared in lactophenol and examined using an Olympus BH-2 microscope. Drawings were made with a drawing tube attached to the microscope and measurements, made with an ocular micrometer are presented in millimetres as the range followed by the mean in parentheses. Type-specimens have been deposited in SAM. Higher nematode taxonomic categories follow Beveridge et al. [5]. Host nomenclature follows Jackson & Groves [6]. The geographical distribution of M. giganteus in maps  [8], details of the new species have been submitted to ZooBank. The Life Science Identifier (LSID) of the article is urn:lsid:zoobank. org:pub: 8C095D38-A90D-441B-878C-83AC1D0E595C.
Amplicons were purified using shrimp alkaline phosphate and exonuclease I [10] prior to automated Sanger DNA sequencing (96-capillary 3730xl DNA Analyser, Applied Biosystems, Foster City, CA, USA) at Macrogen Incorporation, South Korea. The primers NC16 and NC2 were used in separate reactions. Assessment of sequencing quality was conducted in the software Geneious Prime 2019.0.4 (http://www.genei ous. com). Sequences were aligned using MUSCLE v.3.8.31 [11] and manually adjusted in the program MEGA X [12].
Phylogenetic analysis of the aligned ITS+ sequences was conducted by Bayesian inference (BI) in MrBayes [13]. The most appropriate partition scheme and the evolutionary model were determined using PartitionFinder V. 2.0 [14] under the AICc criterion. The data were partitioned into subset 1 (ITS1 and ITS2) and subset 2 (5.8S). The evolutionary models assigned were nst = 6 with a proportion of invariable sites for subset 1 and nst = 1 for subset 2. The BI analysis was conducted with the Markov chain Monte Carlo (MCMC) with three heated and one cold chain for 20 million generations sampled every 1000th generations for three runs to ensure convergence and calculate posterior probabilities (pp). At the end of each run, the standard deviation of split frequencies was < 0.01, and the PSRF (Potential Scale Reduction Factor) equals one. For each analysis, a 50%-majority rule consensus tree was constructed based on the final 75% of trees. The ITS+ sequence of Monilonema ochetocephalum (GenBank: HE775537) was included as the outgroup. Tree topology was visualised using the software Figtree v1.4.4 (http://tree.bio.ed.ac.uk/softw are/figtr ee/) and iTOL [15].

Molecular characterisation
The phylogenetic tree generated from the BI analysis included three clades ( Fig. 1), each with a maximum support (pp = 1.0). One of these clades included specimens of the previously described species M. beveridgei. Specimens of M. comani were placed in a second clade and specimens previously identified as M. cf. comani by Tan et al. [4], redescribed here as M. obendorfi n. sp. were positioned as a sister taxon with maximum nodal support (pp = 1.0). A third clade included specimens of M. arundeli n. sp., which clustered with M. comani and M. cf. comani with limited support (pp = 0.70). dorso-ventrally elongated; 2 lateral amphids and 4 submedian papillae, each papilla with 2 setae; buccal capsule poorly sclerotised, with anterior band of sclerotisation; buccal capsule supported externally by sets of radial muscles; oesophagus elongate, lining of corpus with sclerotised thickenings; oesophageal bulb elongate; anterior extremity of intestine enlarged; deirids in region of buccal capsule. Male. Bursa unornamented; ventroventral and ventro-lateral rays apposed; externo-lateral ray divergent; medio and postero-lateral rays apposed; externo-dorsal ray arising from lateral trunk; dorsal ray with 4 branches; external branchlets very short, recurved, terminating in elevations on internal surface of bursa; gubernaculum absent; spicules elongate, alate. Female. Tail conical; vulva immediately anterior to anus; vagina straight; ovejector J-shaped. Type-species: Macroponema beveridgei Mawson, 1978.

Remarks
Macroponema arundeli n. sp. is readily distinguishable from congeners based on the ornamentation of the oesophagus, with a unique arrangement of arcuate sclerotised formations in each dorsal and two subventral sectors of the oesophagus. This species was found only in Ma. giganteus in north-eastern New South Wales and Queensland ( Fig. 14) and commonly co-occurred with M. comani.
The new species can be readily distinguished from M. comani by its larger size, the pattern of oesophageal ornamentation, the termination of the spicular ala (abrupt in M. comani; gradual in the new species) and the fact that the enlarged anterior intestinal cells do not envelope the posterior region of the oesophageal bulb, as is the case in M. comani.

Remarks
The present re-description of M. beveridgei confirms that of Mawson [1] and provides additional details. The species can be readily distinguished from all congeners by its oesophageal ornamentation, with multiple transverse sclerotisations of the oesophageal lining.
Mawson [1] nominated Osphranter antilopinus (syn. Macropus antilopinus) as the type-host and Elizabeth Downs Station as the type-locality. It seems likely therefore that the specimens deposited as SAM AHC 6083 from the same host and locality represent paratypes, even though they were not designated as such in the original description. Mawson [1] also reported the species from Notamacropus agilis (syn. Macropus agilis) from Marrakai Plains (as Merridai Plains), but this appears to be an error. The material from this locality in SAM (SAM AHC 22992) is labelled as being from O. antilopinus. The records of this species occurring in N. agilis in Speare et al. [16] and in Spratt & Beveridge [17], based on the report by Mawson [1], are therefore suspect, with no supporting material from a wide range of collections from the same host species [16]. A further record of this species from Wallabia bicolor on Stradbroke Island, Queensland in Mawson [1] was again not substantiated based on collections in SAM and was not included in the parasites of this species of wallaby by Beveridge [18]. The validity of this record consequently needs to be confirmed.
In the present collections, M. beveridgei was most commonly encountered in O. robustus, probably because this species has been more intensely sampled than O. antilopinus. The molecular data suggest that nematodes from these two closely related macropodid species are identical. Also included in the molecular component of this study were specimens from Notamacropus dorsalis (Gray). This association was not reported by Beveridge et al. [19] based on an examination of 39 N. dorsalis from Queensland. The host from which the molecular specimens were obtained was collected at the same time as a specimen of O. robustus at the same locality, and while specimens were frozen for molecular studies, it appears that none were preserved for morphological study. Assuming that there were no errors in the labelling of specimens at the time of collection, this is the first record of M. beveridgei in N. dorsalis, but it requires confirmation. Mawson, 1978

Description
General. Robust, whitish nematodes. Cephalic collar absent; mouth dorso-ventrally elongate; elevation on each side of mouth opening bears lateral amphid and 2 dome-shaped sub-median papillae; papillae with 2 short, anteriorly-directed setae. Buccal capsule elongate, dorsoventrally elongate, poorly sclerotised, with partially sclerotised annulus in anterior half; shelf projects into buccal capsule at anterior extremity of buccal capsule, not visible in all specimens; buccal capsule supported externally by strong radial musculature in posterior half; sclerotised annulus at junction of buccal capsule with oesophagus; oesophagus elongate; corpus widening posterior to nerve-ring; posterior half of corpus with c.20 sets of bead-like sclerotised projections in lining, one arising from each sector of oesophagus; isthmus narrow, elongate; bulb elongate; intestinal cells enlarged at anterior extremity, surrounding posterior part of oesophageal bulb. Nerve-ring in anterior oesophageal region; excretory pore at level of oesophageal bulb; deirids at level of buccal capsule. . Bursal lobes poorly separated; lateral lobes slightly longer than ventral and dorsal lobes; no median indentation in dorsal lobe; ventro-ventral and ventro-lateral rays apposed, reach margin of bursa; externo-lateral ray divergent from lateral trunk, not reaching margin of bursa; medio-lateral and posterolateral rays apposed in distal half, fused in proximal half, reaching margin of bursa; externo-dorsal ray arising from lateral trunk, stout, not reaching margin of bursa; dorsal ray slender at origin, divides at mid-length; branches arcuate, internal branchlets reaching margin of bursa; external branchlets very short, arise soon after principal bifurcation, terminate in elevations on internal surface of bursa. Spicules elongate, alate; alae with numerous, fine, transverse striations; anterior extremity irregularly knobbed; distal extremity blunt-tipped; alae terminate abruptly anterior to spicule tip, lose striations prior to termination; spicule length 1.70-1.98 (1.81); gubernaculum absent; central cordate and paired lateral thickenings of spicule sheaths present. Ventral lip of genital cone large, conical, bearing papilla 0; dorsal lip with paired bifid appendages.

Remarks
The present redescription confirms that of Mawson [1] but provides additional details including egg sizes and a line drawing of the genital cone, which was illustrated by a scanning electron micrograph only by Mawson [1]. The transverse section of the oesophagus illustrated by Mawson [1] (figure 16 in [1]) suggests that the beadlike thickenings lining the oesophagus are arranged in pairs on each segment (dorsal and two subventral) of the oesophagus. This is not the case (Fig. 36) and a single structure is present for each segment of the oesophagus at each level. Mawson [1] commented on the plasticity of the buccal capsule providing ventral and sublateral views with the buccal capsule in collapsed and expanded positions. Similar variation was observed in the specimens here, but the illustrations presented in the redescription are limited to a ventral view with the buccal capsule collapsed. Mawson [1] did not illustrate or describe the complex array of muscles running between the wall of the buccal capsule and the longitudinal musculature shown here. They are similar in antilopinus; open squares indicate locations at which antilopine wallaroos have been examined but the nematode has not been found their arrangement to those described by Beveridge [21]. The illustrations of Beveridge [21] complement those presented here and have not been included in the present redescription along with the projections into the lumen of the buccal capsule at its anterior extremity. Beveridge [21] considered the host of his specimens of M. comani to be O. robustus which he noted was unusual. However, examining the range of nematodes collected from this individual host and the fact that it was collected at the same time as the material from Ma. giganteus, it seems most likely that the original host identification was in error and consequently the material (SAM AHC 13354) has been included above under Ma. giganteus.
Although a wide range of specimens was available, very few were gravid, probably accounting for the lack of any description of the egg by Mawson [1]. However, in some of the new specimens, the entire female reproductive tract was visible (Fig. 44) and indicated that unlike in species of other genera of the Cloacininae, the distal uterus is narrow and tubular, eventually becoming saccate anteriorly, with eggs largely restricted to the saccate region. In addition, a seminal receptacle is detectable at the proximal extremity of the uterus (Fig. 44). The central cordate and paired lateral thickenings of the spicule sheaths characteristic of the family [22], have not been reported previously for this genus, but are here illustrated in Figs. 38 and 39.
The current data confirm the restriction of this species to a single host, Ma. giganteus, but indicates that its distribution extends from Victoria to northern Queensland (Fig. 45).

Description
General. Robust, whitish nematodes; cephalic collar absent; mouth dorso-ventrally elongate; elevation on each side of mouth opening bears lateral amphid and 2 dome-shaped sub-median papillae; papillae with 2 short, anteriorly-directed setae; buccal capsule elongate, dorsoventrally elongate, poorly sclerotised, with partially sclerotised annulus in anterior half; buccal capsule supported externally by strong radial musculature in posterior half; non-sclerotised annulus at junction of buccal capsule with oesophagus; oesophagus elongate; corpus widening posterior to nerve-ring; posterior half of corpus with c.12-14 sets of bead-like sclerotised projections in lining, one arising from each sector of oesophagus; isthmus narrow, elongate; bulb elongate; intestinal cells enlarged at anterior extremity, surrounding posterior extremity of oesophageal bulb. Nerve-ring in anterior oesophageal region; excretory pore at level of oesophageal bulb or anterior to it; deirids at level of buccal capsule. ; nerve-ring from anterior extremity 0.45-0.63 (0.58); excretory pore from anterior extremity 1.98-2.45 (2.28); deirid from anterior extremity 0.08-0.14 (0.11). Bursal lobes poorly separated; lateral lobes slightly longer than ventral and dorsal lobes; no indentation in dorsal lobe; ventro-ventral and ventro-lateral rays apposed, reaching margin of bursa; externo-lateral ray divergent from lateral trunk, not reaching margin of bursa; medio-lateral and posterolateral rays apposed in distal half, fused in proximal half, reaching margin of bursa; externo-dorsal ray arising from lateral trunk, slender, not reaching margin of bursa; dorsal ray slender at origin, divides at mid-length; branches arcuate, internal branchlets reaching margin of bursa; external branchlets very short, arise soon after principal bifurcation, terminate in elevations on internal surface of bursa. Spicules elongate, alate; alae with numerous, fine, transverse striations; anterior extremity irregularly knobbed; distal extremity blunt tipped; alae terminate abruptly anterior to spicule tip, lose striations prior to

Remarks
This species was initially identified as M. cf. comani by Tan et al. [4] based on molecular differences and a difference in host distribution with M. comani restricted to Ma. giganteus and M. cf. comani occurring in O. r. woodwardi. The metrical morphological data included in that study [4] indicated no obvious differences between the populations of M. comani distinguishable using molecular methods. In the present morphological study, a few features were identified to separate M. comani occurring in Ma. giganteus from the closely related species, here identified as M. obendorfi n. sp. occurring in O. robustus. The simplest feature to observe is the number of groups of bead-like sclerotised projections in the oesophagus which range from 12 to 14 in M. obendorfi n. sp. compared with 20 in M. comani. There are slight differences in the mean spicule length (1.66 mm in M. obendorfi n. sp. vs 1.81 mm in M. comani) but there is considerable overlap. The two species also share the unusual feature of having the medio-lateral and postero-lateral rays fused in the proximal region and apposed only in their distal regions, in contradistinction to the two rays being apposed along their entire lengths in the remaining species. The two species appear to be very similar morphologically with no additional differences noted apart from the numbers of groups of sclerotised beads lining the oesophagus and the anomalous host and geographical distribution (Fig. 55).

Discussion
The present review of the genus Macroponema using both molecular and morphological approaches has established the presence of four species within the genus. The previously identified species, M. beveridgei and M. comani, are re-described and their host and geographical ranges defined in greater detail. Macroponema cf. comani of Tan et al. [4] was confirmed as a distinct species and, with its description as M. obendorfi n. sp., its differential morphological features were defined for the first time.
In addition, a fourth species, M. arundeli n. sp. was  10,24) while in the latter pair, the ala terminates abruptly anterior to the spicule tip (Figs. 37, 52). The genital cone of M. beveridgei differs from all congeners in having multiple appendages surrounding the dorsal lip (Fig. 25).
Three of the species appear to be highly host-specific with M. arundeli n. sp. and M. comani present only in Ma. giganteus and M. obendorfi n. sp. found only in O. robustus woodwardi. There is a single report of M. comani from O. robustus robustus in north-eastern Queensland [21], but other associated nematodes suggest that this is a case of host misidentification, particularly since specimens of Ma. giganteus were collected at the same locality on the same date and consequently, the host associations of this collection have been changed.
Macroponema beveridgei is primarily a parasite of O. robustus, and although found in all three subspecies, O. r. robustus (eastern New South Wales and Queensland), O. r. erubescens (north-western Queensland) and O. r. woodwardi (Northern Territory and north-western Western Australia), appears to be limited to the east and north of the continent (Fig. 28). It has also been found in the closely related and sympatric species, O. antilopinus (the type-host), although this is not surprising as O. robustus and O. antilopinus share approximately 55% of their helminth species in northern Queensland [19]. Notamacropus dorsalis was reported for the first time as a host of M. beveridgei by Chilton et al. [23], but the record requires confirmation as a previous survey of the helminths of this host species did not include this nematode species [19].
The generic independence of Macroponema within the tribe Macropostrongylinea is strongly supported by molecular studies, including M. beveridgei and M. comani, by Chilton et al. [23]. Morphologically, its key distinguishing features are the dorso-ventrally elongated mouth opening, the laterally compressed buccal capsule (Figs. 5, 18, 32, 49) and the ornamentation of the lining of the oesophagus. Mawson [1] utilised a different series of morphological characters apart from the laterally compressed buccal capsule which she considered to occur also in Macropostrongylus Yorke & Maplestone, 1926 and Papillostrongylus Johnston & Mawson, 1939. She noted differences in the ventral lobes of the bursa which she considered to be joined in Macropostrongylus and Popovastrongylus Mawson, 1977, while separate in Macroponema and Papillostrongylus, as well as Macropostrongylus having "longitudinal ridges" in the buccal capsule which were absent in Macroponema [1]. The present descriptions and redescriptions of species of Macroponema suggest that the ventral lobes are joined ventrally in all species, thereby invalidating this feature as a generic character. This feature is not clearly visible unless apical views of the bursa are illustrated, and such views are presented here for the first time. The buccal capsule is not laterally compressed in all species of Macropostrongylus [24] and the "longitudinal ridges" of the buccal capsule of Macropostrongylus are present in the type-species, M. macropostrongylus Yorke & Malpestone, 1926 (see figure 1F in [24]), in M. yorkei Baylis, 1927 (see figure 3E in [24]) and in M. macrostoma Davey & Wood, 1938 (see figure 5H in [24]), but not in the remaining species [24]. Consequently, this character should not be used in the discrimination of the two genera. In summary, the lateral compression of the mouth opening and the oesophageal ornamentation appear to be the two defining morphological characteristics of the genus.
Few of the female nematodes reported in this study were gravid, such that details of the eggs were not available for M. arundeli n. sp. and M. obendorfi n. sp. This may be due to the seasonal development of the nematodes. In the related genera Labiosimplex Smales, 1995 andLabiomultiplex Smales, 1994, in species such as Labiosimplex longispicularis Wood, 1929 and Labiomultiplex eugenii Johnston & Mawson, 1940, gravid females are only present during the months of the year that survival of eggs and larval stages is likely to be optimum [25][26][27]. Similar phenomena may be operating in the case of species of Macroponema, but this remains to be investigated.
While the currently available phylogenetic data suggest that M. obendorfi n. sp. has arisen by a host switch from Ma. giganteus to O. robustus, the relationships of M.  (Figs. 14, 45), suggest that the evolution of the genus has occurred primarily in eastern and northern Australia. Beveridge [20] noted the unusual distribution of M. beveridgei in O. robustus, being absent in South and much of Western Australia. The currently available but limited data, therefore, suggest the evolution of M. beveridgei in O. robustus and M. arundeli n. sp. in Ma. giganteus, followed by the evolution of M. comani within the same host species and of M. obendorfi n. sp. by a host switch into O. robustus. These are clearly tentative hypotheses suggested by the available data which necessitate more rigorous testing.

Conclusions
Based on morphological and molecular characterisation of nematodes, this study revealed the existence of at least four species within the genus Macroponema. The morphology of two previously described species, M. beveridgei and M. comani, was redescribed whereas Macroponema cf. comani of Tan et al. [4] was confirmed as a distinct species as M. obendorfi n. sp. In addition, a fourth species, M. arundeli n. sp. was described from Ma. giganteus. The current phylogenetic data suggest that Macroponema spp. plausibly evolved by host switching; however, further studies are required to test this hypothesis.