Introduction
The strongyloid nematode subfamily Cloacininae occurs exclusively in the stomach and oesophagus of macropodid marsupials and is currently represented by 39 genera and 285 species, with significant numbers of species as yet undescribed (Spratt & Beveridge, Reference Spratt and Beveridge2016). This subfamily is possibly the largest strongyloid nematode radiation in mammals (Beveridge & Chilton, Reference Beveridge and Chilton2001). The evolution of complex strongyloid radiations in both eutherian and marsupial herbivores has been a relatively long-standing area of research and speculation (Inglis, Reference Inglis1971; Chabaud & Durette-Desset, Reference Chabaud and Durette-Desset1978; Kennedy & Bush, Reference Kennedy and Bush1992). However, co-phylogenetic studies of these nematodes and their macropodid marsupials are lacking, as this requires molecular phylogenies for both the parasites and their hosts.
Kangaroos and wallabies represent a speciose group (63 species) that are parasitized by a large number of described nematode species (285) (Spratt & Beveridge, Reference Spratt and Beveridge2016). These hosts therefore provide significant opportunities to test hypotheses concerning the evolution of complex nematode communities, provided that a sound molecular phylogeny exists for the nematodes. Here we examine the phylogenetic relationships of three tribes of cloacinine nematodes from macropodids.
Four tribes were recognized within the Cloacininae by Lichtenfels (Reference Lichtenfels, Anderson, Chabaud and Willmott1980); namely, the Cloacininea, Macropostrongylinea, Pharyngostrongylinea and Zoniolaiminea. Beveridge (Reference Beveridge1983) subdivided the Zoniolaiminea, erecting the new tribe Labiostrongylinea, and subsequently (Beveridge, Reference Beveridge1986a) subdivided the Macropostrongylinea, erecting another new tribe, Coronostrongylinea, thus resulting in six tribes. The tribes Macropostrongylinea and Coronostrongylinea are each defined by morphological synapomorphies (Beveridge, Reference Beveridge1986a). The former tribe is defined by the possession of a poorly sclerotized buccal capsule with prominent surrounding musculature, whereas the latter is defined by a buccal capsule consisting of a reduced outer, sclerotized layer and an inner non-sclerotized layer (Beveridge, Reference Beveridge1986a). Explicit morphological arguments have not been advanced for the remaining tribes. In the case of the Zoniolaiminea, Beveridge (Reference Beveridge1983) indicated that the division between the Pharyngostrongylinea and the Zoniolaiminea was not clear. These two tribes are distinguished by the presence (Pharyngostrongylinea) or absence (Zoniolaiminea) of a transversely striated buccal capsule, and differences in the morphology of the labial structures (Beveridge, Reference Beveridge1982, Reference Beveridge1983). The monophyly of tribes within the Cloacininae has not been tested using molecular data. Recent molecular studies of the tribe Labiostrongylinea (Chilton et al., Reference Chilton2011), of three genera within the tribe Pharyngostrongylinea (i.e. Cyclostrongylus, Pharyngostrongylus and Rugopharynx) (Chilton et al., Reference Chilton2016a, Reference Chiltonb, Reference Chiltonc), and within the genus Cloacina (tribe Cloacininea) (Chilton et al., Reference Chilton2017), have demonstrated the utility of the internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA for determining the relationships between nematode species belonging to this subfamily.
In the present study, we use the same approach for testing the monophyly of three tribes, Coronostrongylinea, Macropostrongylinea and Zoniolaiminea, and the genera within them, as well as comparing the host ranges of the parasites. Due to uncertainties regarding the relationships of the Zoniolaiminea and Pharyngostrongylinea (Beveridge, Reference Beveridge1983), representative genera of the latter tribe were also included in the analysis.
Materials and methods
Nematodes were obtained from the stomachs of kangaroos and wallabies that had been shot commercially, collected as fresh road-kills or from road-kills frozen prior to examination. Nematodes were washed in saline and then frozen in liquid nitrogen and stored at −80° prior to examination. Additional nematode samples from each host were fixed in Berland's fluid (glacial acetic acid and formalin) (Gibson, Reference Gibson1979) for morphological examination.
Frozen nematodes were thawed, and the head and tail were removed from individuals, fixed in lactophenol and either mounted permanently in polyvinyl lactophenol or returned to ethanol as voucher specimens, with the mid-body region being used for genetic analyses. Nematodes were identified following Beveridge (Reference Beveridge1981a, Reference Beveridgeb, Reference Beveridge1982, Reference Beveridge1983, Reference Beveridge1985, Reference Beveridge1986b, Reference Beveridgec, Reference Beveridged, Reference Beveridge2002), Beveridge & Johnson (Reference Beveridge and Johnson1981), Chilton et al. (Reference Chilton2002) and Huby-Chilton et al. (Reference Huby-Chilton2002). Voucher specimens (hologenophores) were deposited in the South Australian Museum (SAM), Adelaide (table 1). In some instances, the unique specimen used for genetic studies (the hologenophore) was not preserved. In these instances, fixed specimens of the same species from the same host individual (paragenophores) were deposited in SAM (table 1). Some species included in this study have relatively broad host ranges (Spratt & Beveridge, Reference Spratt and Beveridge2016). Only the host species from which the parasite was collected are considered here.
Table 1. New sequences of ITS-1 and ITS-2 of cloacinine nematodes included in the current study, with host, locality, coordinates, SAM registration numbers of voucher specimens and GenBank registration numbers for sequences. Abbreviations of host generic names: D., Dendrolagus, M., Macropus; P., Petrogale; T., Thylogale; W., Wallabia. Australian state names: NSW, New South Wales; Qld, Queensland; SA, South Australia; Tas, Tasmania; Vic, Victoria.
More than one specimen of each nematode species was examined if material was available from different host species, from different host sub-species, or from different geographical regions of the continent, particularly from the island state of Tasmania. Host nomenclature follows van Dyck & Strahan (Reference van Dyck and Strahan2008), as the use of subgenera in this classification is potentially more informative in demonstrating host relationships than the more recent classification of Jackson & Groves (Reference Jackson and Groves2015).
Genomic DNA was isolated from the remaining part of each nematode using a small-scale sodium-dodecyl-sulphate/proteinase K extraction procedure (Gasser et al., Reference Gasser1993), followed by purification using a mini-column (Wizard™ Clean-Up, Promega, Madison, USA). The region of rDNA comprising the ITS-1, 5.8S rRNA gene, ITS-2 and flanking sequences (= ITS+) was amplified by polymerase chain reaction (PCR) using primers NC16 (forward; 5′-AGTTCAATCGCAATGGCTT-3′) and NC2 (reverse; 5′-TTAGTTTCTTTTCCTCCGCT-3′). PCRs were performed in 50 μl volumes using the following conditions: 30 cycles at 94°C for 30 s (denaturation), 55°C for 30 s (annealing) and 72°C for 30 s (extension), followed by one cycle at 72°C for 5 minutes (final extension). Negative (no-DNA) controls were included in each set of reactions. Amplicons were purified using mini-columns (using Wizard™ PCR-Preps, Promega, Madison, USA), and the ITS+ sequenced in both directions using the primers NC16 and NC2 in separate reactions. The sequences generated in the present study have been deposited in GenBank (table 1). Additional sequences already present in the GenBank database (table 2) were also utilized.
Table 2. Sequences of nematodes used from GenBank.
Sequences were initially aligned using Muscle (Edgar, Reference Edgar2004) and alignments were adjusted manually using the program Mesquite v. 3.03 (Maddison & Maddison, Reference Maddison and Maddison2015). Analyses of the ITS-1 and ITS-2 sequence data (i.e. excluding the 5.8S rRNA gene and other flanking regions) were conducted by Bayesian inference (BI) using Monte Carlo Markov Chain analysis in the program MrBayes v. 3.2.3 (Ronquist & Huelsenbeck, Reference Ronquist and Huelsenbeck2003). The likelihood parameters set for the BI analysis of sequence data were based on the Akaike Information Criterion test in jModeltest v. 2.1.7 (Posada, Reference Posada2008). The number of substitutions was set at six, with a gamma-distribution. For the trees, posterior probability (pp) values were calculated by running 2,000,000 generations with four simultaneous tree-building chains. Trees were saved every 100th generation. At the end of each run, the standard deviation of split frequencies was < 0.01, and the potential scale reduction factor approached one. A 50% majority rule consensus tree was constructed based on the final 75% of trees produced by BI. Analyses were run three times to ensure convergence and insensitivity to priors. The same unpartitioned data were also subjected to the neighbour joining (NJ) distance method (Saitou & Nei, Reference Saitou and Nei1987) in MEGA v. 7.0.20 (Kumar et al., Reference Kumar, Stecher and Tamura2016). Evolutionary distances were computed using the number of differences (Nei & Kumar, Reference Nei and Kumar2000), including transitions and transversions among nematode species. Rates among sites were considered uniform and gaps were treated using pairwise deletion. A total of 2000 bootstrap replicates were performed and are reported as bootstrap (bs) values. The ITS-1 and ITS-2 sequences of Arundelia dissimilis, a species within a related genus in a related tribe, Cloacininea, was used as the outgroup in the phylogenetic analysis. The BI and NJ trees had similar topologies and, when in agreement, both pp and bs values are indicated (fig. 1).
Fig. 1. Phylogram of the genetic interrelationships of genera of the Coronostrongylinea (C), Macropostrongylinea (M) and Zoniolaiminea (Z). Numbers above branches indicate posterior probabilities obtained in the Bayesian analysis; those below branches indicate bootstrap values from the Neighbour Joining method. Branch lengths indicate genetic distances. * indicates members of the Pharyngostrongylinea (P).
The host associations of the parasites were examined by comparison with a molecular phylogeny of the hosts based primarily on Meredith et al. (Reference Meredith, Westerman and Springer2008), with additions for the genus Thylogale based on Macqueen et al. (Reference Macqueen2010). As there is no comprehensive molecular phylogeny for the Macropodidae, any taxa missing from the above studies (i.e. M. (N.) dorsalis) were interpolated based on the comprehensive dataset of Cardillo et al. (Reference Cardillo2004) and the resultant tree is therefore presented as a cladogram (fig. 2), as are the parasite data. Some of the nematode species are known to occur in more than one host species (Spratt & Beveridge, Reference Spratt and Beveridge2016). In the cladogram, only the host from which the nematode was sequenced is shown.
Fig. 2. Comparison of the phylogenetic relationships of genera and species of the Coronostrongylinea (C), Macropostrongylinea (M) and Zoniolaiminea (Z), presented as a cladogram, with a composite cladogram of their macropodid hosts. Numbers above branches in the nematode tree indicate posterior probabilities obtained in the Bayesian analysis; those below branches indicate bootstrap values from the Neighbour Joining method. Branch lengths indicate genetic distances. * indicates members of the Pharyngostrongylinea (P), for which host associations are not shown. Abbreviations: M, Macropus; N, Notamacropus; O, Osphranter.
Results
This molecular study included 41 of the 63 known species of the tribes Coronostrongylinea (12/23), Macropostrongylinea (12/16) and Zoniolaiminea (18/24). The parasite genera included in this study have never previously been tested for monophyly using molecular data. However, of the nematode genera included in the study, the monophyly of Alocostoma, Coronostrongylus, Macroponema, Thallostonema (with novel additions), Thylonema and Zoniolaimus (to the exclusion of Z. dendrolagi) was highly supported with BI values of > 0.97 (fig. 1). Species of Macropostrongylus and Popovastrongylus each formed a cluster in the phylogenetic analyses but with low nodal support. Macropostrongylus species are parasites of M. (Notamacropus) and Petrogale, thus being widely distributed among host species, while Popovastrongylus is distributed among species of Macropus (Macropus), Macropus (Osphranter), Petrogale and Thylogale, thus also exhibiting a broad host range (fig. 2).
Three genera, Monilonema, Wallabinema and Papillostrongylus, appeared to be paraphyletic in this analysis. Monilonema ochetocephalum from Wallabia bicolor clustered with species of Macropostrongylus, although with low nodal support, within the tribe Macropostrongylinea. Two species, M. lacunosum and Foliostoma macropodis, both found in Thylogale stigmatica, clustered with species of Thallostonema, which are also primarily parasites of Thylogale species. Wallabinema appeared to be paraphyletic. There was high nodal support for a sister taxa relationship between W. australe, with a host range including M. (Notamacropus), Wallabia and Thylogale, and W. cobbi from M. (Osphranter). These two species belonged to a group, with total nodal support, that included two species of Thylonema. This group did not include W. labiatum or W. tasmaniense. There was total nodal support for W. labiatum, found primarily in M. (N.) rufogriseus and W. bicolor, and W. tasmaniense, a parasite of T. billardieri, representing sister taxa.
Paraphyly was also indicated within Papillostrongylus, with P. papillatus, from M. (N.) dorsalis, clustering, but with low nodal support from P. barbatus, which is a parasite of M. (O.) rufus.
Tribes
The current analysis provides only limited support for the composition of the three cloacinine tribes as currently defined. Monilonema lacunosum and Foliostoma macropodis, both currently placed within the Macropostrongylinea, clustered with species of Thallostonema (Zoniolaiminea) (fig. 1). Popovastrongylus, currently placed within the Coronostrongylinea, clustered strongly with the genera of the Macropostrongylinea, as did Papillostrongylus. Current members of the Zoniolaiminea were split between two highly supported clades. The first included Thallostonema, but with the addition of Monilonema lacunosum and Foliostoma. The second clade included Zoniolaimus, Cassunema and Wallabinema, but also included Thylonema (currently included in the Coronostrongylinea), as well as three genera currently included in the Pharyngostrongylinea (Pararugopharynx, Thylostrongylus and Woodwardostrongylus). Parapharyngostrongylus and Rugostrongylus (Pharyngostrongylinea) clustered together to the exclusion of all other genera and to the exclusion of other genera of the Pharyngostrongylinea (Pararugopharynx, Thylostrongylus and Woodwardostrongylus).
Species
The opportunity was taken, where possible, to include representatives of the same nematode species from different host species or from widely different geographical localities. Alocostoma clelandi from two different host species in Queensland were identical genetically, as were A. propinquum from Victoria and northern New South Wales. Similarly identical or near identical DNA sequences were obtained for Thylonema barkeri from northern and southern Queensland, and Popovastrongylus pearsoni from mainland and island sub-species of Macropus fuliginosus (M. f. fuliginosus and M. f. ocydromus).
Slight genetic differences were detected between Popovastrongylus thylogale from two different host species, Petrogale persephone and Thylogale stigmatica in northern Queensland, and between specimens of Thallostonema setiferum from Queensland, New South Wales and Tasmania, in Coronostrongylus coronatus from Queensland and Tasmania, and in Macroponema beveridgei from two different host species from the same locality in Queensland. In each instance, sequences of the same nematode species formed a highly supported clade.
Discussion
Cloacinine nematodes belonging to the tribes Coronostrongylinea, Macropostrongylinea, Zoniolaiminea and Pharyngostrongylinea are common parasites of the gastrointestinal tracts of Australian macropodid marsupials (Beveridge & Spratt, Reference Spratt and Beveridge2016). These tribes and the genera within them are defined primarily on a phenetic, morphological basis. There are proposed morphological synapomorphies for the Coronostrongylinea and Macropostrongylinea, but not for the Zoniolaiminea and Pharyngostrongylinea (Beveridge, Reference Beveridge1983). The primary aim of this study was to test current morphologically based hypotheses of the phylogenetic relationships of cloacinine nematodes within the Coronostrongylinea, Macropostrongylinea and Zoniolaiminea using a molecular approach. Some representatives of the Pharyngostrongylinea were also included in this study. The nematode genera (including the three principal tribes) included in this study have not previously been tested for monophyly based on analyses of ITS-1 and ITS-2 sequence data.
The results showed that there was strong support for the monophyly of the genera Coronostrongylus, Thylonema, Macroponema, Alocostoma, Thallostonema (with the unexpected addition of two genera) and Zoniolaimus (with the exclusion of a single species). Two other nematode genera, Macropostrongylus and Popovastrongylus, each formed a monophyletic clade; however, there was low nodal support for such groups, even though each genus is well defined morphologically (Beveridge, Reference Beveridge1983, Reference Beveridge1985). In contrast, three genera, Wallabinema, Monilonema and Papillostrongylus, were paraphyletic (figs 1 & 2).
Host ranges of the various genera varied significantly. Macropostrongylus, Monilonema, Papillostrongylus, Popovastrongylus, Coronostrongylus, Thallostonema, Wallabinema and Zoniolaimus occur in a wide range of hosts, including Macropus (subgenera: Macropus, Osphranter and Notamacropus), Petrogale and Thylogale. By contrast, species within the genus Thylonema are restricted to the host genus (Thylogale), whereas species of Macroponema and Alocostoma are found only in hosts of two closely related subgenera of Macropus (i.e. Macropus and Osphranter).
A formal co-phylogenetic analysis is not possible at this time because of a lack of a definitive molecular phylogeny of the Macropodidae (see Materials and methods). However, visual inspection of the host–parasite relationships (fig. 2) provides little evidence of co-speciation. These host–parasite associations are potentially best explained in evolutionary terms by a process of host colonization, as proposed in previous molecular studies of related tribes of the Cloacininae (Chilton et al., Reference Chilton2011, Reference Chilton2016a, Reference Chiltonb, Reference Chiltonc, Reference Chilton2017), but this should be considered as a preliminary hypothesis pending the possibility of more formal analyses. In several instances, genetically identical (based on ITS-1 and ITS-2 sequences) individuals of a nematode species (e.g. Alocostoma clelandi) were found in different host species. This clearly indicates the possibility of nematodes switching hosts without undergoing genetic differentiation. The suggestion made here of the significance of host switching accords with a recent overview of the processes of speciation in parasites that gives overwhelming support to the priority of host switching over co-speciation (Nylin et al., Reference Nylin2017).
Genera such as Thylonema, Thallostonema and one clade of Wallabinema have diversified primarily in the host genus Thylogale. In the case of species of Wallabinema, W. australe and W. cobbi (which form a clade in this molecular study) differ distinctively in oesophageal morphology from W. labiatum and W. tasmaniense (also included in this study) (Beveridge, Reference Beveridge1983) and may therefore warrant generic recognition. The latter species, W. labiatum and W. tasmaniense, represent species present in M. (N.) rufogriseus (W. labiatum) as well as a series of morphologically similar species (W. tasmaniense, W. gallardi, W. parvispiculare) parasitic in the genus Thylogale (Beveridge, Reference Beveridge1983). The host distribution of this clade is thus similar to that of Thallostonema, with most species occurring in hosts belonging to the genus Thylogale, but with additional species in M. (N.) rufogriseus. Within the genus Thylogale, parasite species associations are complicated as there are distinctive sub-species of T. stigmatica present in Queensland (Macqueen et al., Reference Macqueen2010), although there is some evidence of the sub-species interbreeding in central Queensland (Eldridge et al., Reference Eldridge2011). In addition, T. stigmatica occurs in sympatry with T. thetis in south-eastern Queensland, with some evidence of introgression between these two species (Eldridge et al., Reference Eldridge2011). Consequently, additional analyses are required to determine the evolutionary relationships between nematodes parasitic in species of Thylogale. Thylogale, together with Petrogale and Dendrolagus, belong to a clade that separated from Macropus and Wallabia c. 10 mya (Meredith et al., Reference Meredith, Westerman and Springer2008). This host genus also has a remarkable number of nematode genera that are either entirely specific to it or occur primarily within it (i.e. Cassunema, Foliostoma, Thylonema, Trigonostonema, Thylostrongylus, Thallostonema, Tethystrongylus and Wallabinema) suggesting that an investigation into their relationships might provide critical insights into the evolution of the Cloacininae.
Although the present study has highlighted the radiation of some nematode genera within a particular host genus (e.g. Thylonema, Trigonostonema and Thallostonema in species of Thylogale, and Alocostoma and Zoniolaimus in species of Macropus), relatively few instances of within-host speciation were identified, a phenomenon occurring in the related genus Cloacina (Chilton et al., Reference Chilton2017). As indicated above, associations within Thylogale spp. are difficult to interpret. Zoniolaimus mawsonae and Z. latebrosus co-occur commonly in the stomach of M. (O.) rufus (see Huby-Chilton et al., Reference Huby-Chilton2002), but the current analysis does not provide any evidence for within-host speciation in this case.
The data presented here also provide a basis for future studies. The tribes of the Cloacininae are currently phenetically based (Lichtenfels, Reference Lichtenfels, Anderson, Chabaud and Willmott1980), with only two (i.e. Macropostrongylinea and Coronostrongylinea) proposed on the basis of morphological phylogenetic hypotheses (Beveridge, Reference Beveridge1986a). Clearly, all of these taxonomic hypotheses warrant testing using molecular methods, as the current study did not support the tribal associations examined here.
Apart from the current molecular analyses raising concerns as to the validity of the current distribution of cloacinine genera among tribes, in particular the genera of the Pharyngostrongylinea, the apparent paraphyly of Wallabinema and Monilonema also needs to be addressed. The present study did, however, identify five highly supported clades that could warrant recognition at tribal level. The first is the Coronostrongylinea, which includes a single genus, Coronostrongylus. The second is the Macropostrongylinea, which includes Alocostoma, Macroponema, Macropostrongylus, Papillostrongylus, Popovastrongylus, Trigonostonema and Monilonema (in part). The third is the Pharyngostrongylinea, currently restricted to Parapharyngostrongylus and Rugostrongylus but with additional genera potentially to be added. The fourth clade is the Zoniolaiminea, with the current genera Zoniolaimus, Wallabinema and Cassunema, but would also include Thylonema as well as three currently pharyngostrongylidean genera, Pararugopharynx, Thylostrongylus and Woodwardostrongylus. A fifth, novel clade includes species of Thallostonema together with Monilonema lacunosum and Foliostoma, for which there is no current taxonomic identity.
Therefore, the current molecular-based study has contributed to our understanding of the phylogenetic relationships of nematodes within genera from four nominal tribes of the Cloacininae. It has provided insights into inadequacies in the current taxonomy of these nematodes at the generic and tribal levels, which will need to be addressed.
Acknowledgements
We wish to thank numerous colleagues who assisted in the collection of specimens.
Financial support
The study was funded by the Australian Research Council and by the Australian Biological Resources Study.
Conflict of interest
None.
Ethical standards
Specimens were collected under the following permits: Victorian Department of Sustainability and Environment 90-053, Queensland National Parks and Wildlife Service T00436, T1131, New South Wales National Parks and Wildlife Service A68, and the South Australian National Parks and Wildlife Service EO7358, G24351.
