Journal of Parasitology The Status of Heligmosomoides americanus, Representative of an American Clade of Vole-Infecting Nematodes --Manuscript Draft-Manuscript Number:
13-455R1
Full Title:
The Status of Heligmosomoides americanus, Representative of an American Clade of Vole-Infecting Nematodes
Short Title:
The Status of Heligmosomoides americanus
Article Type:
Short Communications
Corresponding Author:
Anna Paziewska-Harris Royal Tropical Institute, KIT Biomedical Research Amsterdam, Amsterdam NETHERLANDS
Corresponding Author Secondary Information: Corresponding Author's Institution:
Royal Tropical Institute, KIT Biomedical Research
Corresponding Author's Secondary Institution: First Author:
Philip D Harris
First Author Secondary Information: Order of Authors:
Philip D Harris Grzegorz Zaleśny Joanna Hildebrand Anna Paziewska-Harris Jerzy M Behnke Vasyl Tkach Yeen-Ten Hwang John M Kinsella
Order of Authors Secondary Information: Abstract:
Heligmosomoides americanus is shown by molecular phylogenetic analysis of two nuclear (28S, ITS-1 and ITS-2) and two mitochondrial (cytochrome oxidase 1 and cytochrome b) loci to be a distinct species of heligmosomid nematode with a long independent evolutionary history, and not a subspecies of H. polygyrus. Rather than being a recent arrival in North America, the species probably originated as a Beringian immigrant with the host vole Phenacomys, approximately 2 MYA.
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RH: SHORT COMMUNICATIONS The Status of Heligmosomoides americanus, Representative of an American Clade of Vole-Infecting Nematodes P. D. Harris, G. Zaleśny*, J. Hildebrand†, A. Paziewska-Harris‡, J. M. Behnke§, V. Tkach||, Y.-T. Hwang#, and J. M. Kinsella¶, Section for Research and Collections, Natural History Museum, University of Oslo, P.O.Box 1172, Oslo N-0316 Norway; *Department of Invertebrate Systematics and Ecology, Institute of Biology, Wrocław University of Environmental and Life Sciences, ul. Kożuchowska 5b, 51-631 Wrocław, Poland; †Department of Parasitology, Institute of Genetics and Microbiology, University of Wrocław, ul. Przybyszewskiego 63, 51-148 Wrocław, Poland; ‡KIT Biomedical Research, Royal Tropical Institute, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands; §School of Life Sciences, University of Nottingham, Nottingham NG2 7RD; ||Department of Biology, University of North Dakota, Grand Forks, North Dakota 58202-9019; #Department of Veterinary Pathology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B4, Canada; ¶HelmWest Laboratory, 2108 Hilda Avenue, Missoula, Montana 59801. Correspondence should be sent to:
[email protected] ABSTRACT: Heligmosomoides americanus is shown by molecular phylogenetic analysis of 3 nuclear (28S, ITS1 and ITS2) and 2 mitochondrial (cytochrome oxidase 1 and cytochrome b) loci to be a distinct species of heligmosomid nematode with a long independent evolutionary history, and not a subspecies of Heligmosomoides polygyrus. Rather than being a recent arrival in North America, the species probably originated as a Beringian immigrant with the host vole Phenacomys, approximately 2 million years ago (MYA). Heligmosomid nematodes form an important clade of gastro-intestinal (GI) nematodes infecting the duodenum of Holarctic voles and mice, which, because of their immunosuppressive properties, may act to modify the structure of rodent parasite communities (Behnke et al., 2005; Jackson et al., 2009). The best known species of the group,
frequently referred to as Heligmosomoides polygyrus bakeri (Maizels et al., 2011) but perhaps more realistically considered a separate species (Cable et al., 2006; Behnke and Harris, 2010), is Heligmosomoides bakeri (Durette-Desset et al., 1972; Tenora and Barus, 2001). This widely used laboratory model of chronic GI nematode infection was first reported from North America in the 1950s (Ehrenford, 1954) but probably originated as a recent invasion with the house mouse (Mus musculus Linn, 1758) during the late 19th Century, possibly via California (Behnke and Harris, 2010). The taxonomic status of the North American forms is confused and confusing, because of initial pre-molecular assumptions concerning their phylogeographic origin (Durette-Desset, 1968). In particular, the presumed trans-beringian migration of North American species in the relatively recent past (Durette-Desset, 1967) led to North American forms being described as subspecies of Eurasian taxa. This is particularly clear for the species considered in the present note, first described by Durette-Desset et al. (1972) as Heligmosomoides polygyrus americanus, from the western heather vole Phenacomys intermedius. At the time of the original description, the specific identity of this worm was subsumed within the catch-all Eurasian taxon H. polygyrus. Subsequently the taxon was raised to a full species by Tenora and Barus (2001) but without significant new evidence or analysis. In the interim, a distinctive North American heligmosomid fauna has been described, including the unusual hind-gut infecting H. hudsoni and H. johnsoni (Rausch and Rausch, 1973; Rausch and Johnson, 1983), a species infecting Peromyscus (H. vandegrifti, DuretteDesset and Kinsella, 2007) and species of Ohbayashinema infecting pikas (Durette-Desset et al., 2010), and, as noted by Durette-Desset and Kinsella (2007), the time is ripe for a reappraisal of American heligmosomids using molecular methodologies. This work presents a molecular analysis of H. americanus from the type host Phenacomys intermedius Merriam, 1889, and establishes this species as a unique and distinctive representative of the American heligmosomid fauna, which is not closely related to any Eurasian species, and especially not to H. polygyrus.
Specimens of Phenacomys intermedius were live trapped in 2007 in Kootenay National Park, British Columbia (50°68'N, 115°93'W) and snap trapped by Rafal Zwolak in 2001 from Lewis and Clark County, Montana (46°52'N, 112°29'W). The Montana voles were necropsied by Jonathan Vaughn, and the recovered nematodes collected preserved in 90% ethanol and stored at -20 C until utilized for sequencing. Voucher specimens are deposited in the U.S. National Parasite Collection, Beltsville, Maryland (accession number 108042 [specimen from Canada]), and in the Natural History Museum Oslo (accession number NHMO C5972). Extracted DNA is deposited in the Natural History Museum Oslo (accession numbers NEM020 [specimen from Canada] and NEM021 [specimen from the USA]). Worms were homogenized and DNA extracted using the E.Z.N.A.® Tissue DNA Kit (Omega BioTek, Norcross, Georgia) and 5 loci (3 nuclear and 2 mitochondrial) amplified using amplification conditions and primers described in Zaleśny et al. (2014). The loci were nuclear 28S rDNA, ITS1 and ITS2 and the mitochondrial cytochrome oxidase I (CO1) and cytochrome b (cytb). Species identity was confirmed microscopically; cross-sections of the synlophe were cut at 2 μm thickness and stained with 0.1% methylene blue after embedding in methacrylate resin. A comparative cross section of the synlophe of H. polygyrus from Apodemus sylvaticus from Jar, Norway (59°55'15"N, 10°37'46"E; see Zaleśny et al., 2014) was processed in an identical manner. DNA sequences for the respective loci for H. neopolygyrus from Poland (GenBank accession numbers KF765451, KF765455, KF765458, KF765463, KF765468), H. polygyrus from Norway (KJ994543, KJ994553, KJ994557, KJ994560, KF765469), H. glareoli from Edinburgh (KF765457, KF765453, KF765460, KF765465, KF765470), the laboratory isolate H. bakeri (DQ408627, DQ408624, AM039747 and 959 Nematode Genome project [http://www.nematodes.org/nematodegenomes/index.php/959_Nematode_Genomes] for cytb), H. polygyrus corsicum from Turkey (KJ994540, KJ994547, KJ994556, KJ994559, KJ994539), Heligmosomum mixtum from Poland (DQ408635, KF765454, KF765461,
KF765466, KF765471), H. kurilensis kobayashii from Japan (AJ971146, AM409077, AM409093) and Ohbayashinema erbaevae from Eurasian pikas (AY332647, AY333381, AF210038, AF210014, AF209991) were included for comparison with H. americanus. Novel sequences for H. americanus were deposited in GenBank (KF921074-KF921078). Alignments based on those used by Zaleśny et al. (2014) were produced using ClustalX within the Mega 5.0 package (Tamura et al., 2011) followed by visual inspection. Phylogenetic analysis was conducted using a Maximum Likelihood algorithm implemented in RaxML vs 8.0 (Stamatakis, 2014) via the CIPRES Science Gateway portal (Miller et al., 2010). Nuclear ribosomal analysis was conducted on 1712bp concatenated sequences partitioned into 28S, ITS1and ITS2 genes. Outgroups (Nematodirus battus for nuclear markers [AF194138, AM039752], and N. spathiger for mitochondrial markers [NC_024638]) were drawn from GenBank. Each concatenated alignment included sequences represented at only 1 or 2 of the loci included in the alignment, but overall each nucleotide site was represented by between 66% (28S) and 100% (central regions of ITS1 and ITS2) of the aligned sequences. At all sequenced loci H. americanus proved entirely distinct from H. polygyrus, the taxon with which it was first associated as a subspecies. Using the concatenated 28S/ITS1/ITS2 tree (Fig. 1A), only 2 clades were supported with better than 60% bootstrap support. One consisted of the 2 Heligmosomum species, H. costellatum and H. mixtum. The other was composed of H. polygyrus polygyrus, associated with high bootstrap support (97%) with H. polygyrus corsicum and H. bakeri, thought to have originated in the Middle East following a host switch from Apodemus to Mus (Nieberding et al., 2008; Behnke and Harris, 2010). H. americanus was never associated with this latter clade, indicating that it should not be regarded as a subspecies of H. polygyrus. Instead H. americanus associated weakly with H. glareoli, a Eurasian species from voles, with less than 60% bootstrap support. Heligmosomoides glareoli was basal to all other Heligmosomoides and Heligmosomum
species, but with less than 60% bootstrap support. As in the study of Zaleśny et al. (2014), Ohbayashinema (from pikas) and Heligmosomum (from voles) lay internal to Heligmosomoides clades, indicating the paraphyly of the latter genus. The concatenated phylogenetic tree of mitochondrial markers (CO1 and cytb) showed 2 distinct clades, 1 grouping H. polygyrus, H. bakeri, and H. glareoli with better than 80% bootstrap support, and the other joining H. americanus, H. neopolygyrus and Heligmosomum mixtum with 60% bootstrap support (Fig. 1B). According to this analysis H. kobayashii kurilensis was the most divergent Heligmosomoides species included. As with the nuclear markers, there was no evidence to support a grouping of H. americanus with H. polygyrus, rejecting the hypothesis that the former is a subspecies of the latter, or that they are sister clades. It is clear from the present analysis that H. americanus, rather than being part of the H. polygyrus species complex as originally suggested (Durette-Desset et al., 1972) represents a unique North American Heligmosomoides clade, distantly related to forms infecting Eurasian voles. The worm is morphologically distinct also; the synlophe shows a smaller number of less distinct crêtes, which are less enlarged in the bottom left quadrant of the worm relative to that of H. polygyrus (Fig. 2). The status of Heligmosomum as a separate genus has been commented on by Zaleśny et al. (2014) who consistently found that this taxon clustered within Heligmosomoides, suggesting that the stout, straight body form of this genus is derived secondarily from the tightly coiled slender body form. Zaleśny et al. (2014) further suggested that the vole-infecting forms could be derived from the Apodemus-infecting taxa, counter to the hypothesis of Durette-Desset (1967) that the vole-infecting forms are primitive. The present analysis of H. americanus may help to test this hypothesis; the host, P. intermedius, is amongst the oldest vole lineages, with a Beringian ancestry dating back to the beginning of the Pliocene (Repenning et al., 1987), some 5 million years ago (MYA) and the radiation into North America has been dated to c. 2 MYA (Repenning et al., 1987). Zaleśny et al. (2014)
suggest the split between H. polygyrus and H. neopolygyrus (the oldest of the mouse-infecting forms) dates back to c. 6-8 MYA, predating the radiation into voles. The long branch lengths between H. americanus and the other Heligmosomoides taxa suggest that this species represents an early colonization of voles, with a long independent history in North America, consistent with Repenning et al.’s (1987) dating for Phenacomys. Moreover, Phenacomys subsequently became isolated within mountain ranges in North America (Chavez and Kenagy, 2010), an isolation that also dates back to nearly 1.5 MYA. The current study utilized H. americanus from Phenacomys from British Columbia and Montana, within Chavez and Kenagy’s (2010) northern and interior population block, and worms from Montana were among paratypes in the original description (Durette-Desset et al., 1972). However, the holotype of the original description (Durette-Desset et al., 1972) was collected from further south, within the Washington Phenacomys population block, isolated from Montana for c. 1.5 million years (Chavez and Kenagy, 2010). The existence of the same nematode within 2 isolated highland population blocks of Phenacomys can only be explained parsimoniously by fragmentation of a previously continuous range, which in itself testifies to the long independent evolutionary history of H. americanus in North America. This explanation for the origin of H. americanus was first put forward by Rausch and Rausch (1973) but generally discounted in favor of the Durette-Desset et al. (1972) hypothesis. Molecular analysis clearly confirms Rausch and Rausch’s (1973) suspicion that H. americanus is indeed a long-standing member of the North American parasite fauna, and not a form recently diverged from H. polygyrus. PDH and GZ gratefully acknowledge a Zoologica Scripta/Research Council of Norway grant, which allowed completion of the molecular work for this paper in Oslo. LITERATURE CITED Behnke, J. M., F. S. Gilbert, M. A. Abu-Madi, and J. W. Lewis. 2005. Do the helminths of wood mice interact? Journal of Animal Ecology 74: 982-993.
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Figure 1. Phylogenetic trees of concatenated a) 28S, ITS1 and ITS2 nuclear genomic markers, and b) cytb and CO1 mitochondrial markers, constructed using maximum likelihood (500 bootstraps) within the RaxML package. Figure 2. Methacrylate embedded cross sections of a) Heligmosomoides americanus showing the synlophe The crêtes are sparse, and show relatively little asymmetry to the ventral left hand quadrant (boxed area) of the worm, and b) H. polygyrus (from Apodemus sylvaticus). The crêtes are frequent and increase in size to the ventral left hand quadrant (boxed area) where they are particularly prominent.
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