Introduction

Ticks from echidnas were first described by Lucas (1878), when he described Ixodes acanthoglossi (Lucas, 1878), collected from what was then called Acanthoglossus bruijni, the New Guinean long-beaked echidna, now Zaglossus bruijni (Peters & Doria). Unfortunately, his description is rather unspecific and was not accompanied by detailed diagnostic illustrations. Referring to the note published by Lucas, Oudemans (1906) included I. acanthoglossi in the recognised ixodid fauna of New Guinea. I. acanthoglossi was, however, listed as a rejected tick name by Camicas, Hervy, Adam & Morel (1998), probably because its description did not correspond to any known tick and because the Lucas types, to the best of our knowledge, were unavailable for comparison.

Neumann (1910) described and illustrated Aponomma oudemansi (Neumann, 1910), based on male specimens collected from the skin of a specimen of “Zaglossus (Proechidna) bruyni nigroaculeatus”. These ticks are deposited in the Zoological Museum of Amsterdam (ZMAM) of Amsterdam University, The Netherlands. Among other features, Neumann (1910) described the presence of spiracular plates extruding from the lateral border of the body anterior to the first festoon and a 2 ½/2 ½ hypostomal dental formula.

Schulze (1936) described female ticks deposited in the Berlin Museum, Germany, which had been collected from Echidna aculeata, now Tachyglossus aculeatus (Shaw), from an unknown geographical region. Because his specimens had a similar hypostomal dentition (2 ½/2 ½) and host preference, he deduced that these were the missing A. oudemansi females. He later subdivided the taxon into two subspecies, A. o. oudemansi (Neumann, 1910) (the nominate subspecies), for the specimens he described in 1936, and A. oudemansi galactites Schulze, 1941, for two female ticks collected from an unknown animal at Sydney, Australia, which were characterized by the presence of heavy white markings on the scutum (Schulze, 1941). Much later (Kaufmann, 1972), A. oudemansi was synonymised with Aponomma concolor Neumann, 1899. However, after examining a syntype male of A. oudemansi (Nuttall No. 2878, RML111755) in the Nuttall Collection of the Natural History Museum [then the British Museum (Natural History)] in London, Keirans (1985) noted that this tick did not have ventral lobes on palpal articles II, which are typical for A. concolor. Dias (1993), after examining the specimens at the ZMAM, concurred with Keirans (1985) in considering A. oudemansi to be a valid tick name. Later, Camicas et al. (1998) again included A. oudemansi in the synonymy list of A. concolor, but did not provide any justification for doing so.

Based on molecular data and larval morphology, the genus Aponomma Neumann, 1899 has now been split in two groups of taxa (Klompen, Dobson & Barker, 2002). The newly elevated genus Bothriocroton Keirans, King & Sharrad, 1994, originally described as a subgenus of Aponomma by Keirans, King & Sharrad (1994) includes the so-called “Australian endemic” former Aponomma species, B. auruginans (Schulze, 1936), B. concolor (Neumann, 1899), B. hydrosauri (Denny, 1843), B. glebopalma (Keirans, King & Sharrad, 1994) and B. undatum (Fabricius, 1775). All remaining Aponomma species were reassigned to Amblyomma Koch, 1844, partly confirming Koch’s (1844) opinion that Aponomma were simply eye-less Amblyomma. Klompen, Oliver, Keirans & Homsher (1997) and Klompen et al. (2002) also found a synapomorphic character that distinguishes members of Bothriocroton: all Bothriocroton larvae, with the exception of B. undatum larvae which were not available for examination, have three pairs of large wax glands (formerly known as sensilla sagittiformia) on each side of the idiosoma near setae s6 anterior to the first festoon. Aponomma oudemansi was recently transferred to Amblyomma and not included in Bothriocroton (see Horak, Camicas & Keirans, 2002; Barker & Murrell, 2004).

Eye-less ticks from long-beaked echidnas in Papua New Guinea were recently collected by one of us (MDO) as part of an ecological study of this monotreme. Herein, we describe the results obtained by comparing these ticks with former Aponomma species based on morphological analyses. Because we lack larval specimens of B. oudemansi to check for the morphology of large wax glands, we performed molecular phylogenetic analyses to establish the systematic position of this tick species.

Moreover, the name Aponomma tachyglossi Roberts, 1953, formerly treated as a junior synonym of B. hydrosauri, was recently resurrected for ticks collected from echidnas (Tachyglossus aculeatus) along the eastern coast of Queensland, Australia (Andrews, Beveridge, Bull, Chilton, Dixon & Petney, 2006). Therefore, in order to verify the identification of Bothriocroton ticks from echidnas maintained in the US National Tick Collection (USNTC), we reexamined all our Bothriocroton holdings and include a synoptic list.

Materials and methods

Preparation of specimens for morphological examinations

All measurements (8 males, 5 females and 2 nymphs) in the following descriptions are in micrometres and given as the range is followed by the mean in parentheses. Specimens were prepared for scanning electron microscopy using the method of Corwin, Clifford & Keirans (1979).

Material studied

RML numbers refer to USNTC accession numbers. All fresh samples were collected from Zaglossus bruijni in Papua New Guinea (Fig. 1). Eight males and 1 nymph (RML123103) were collected in Chimbu Province, north of Haia. Additional specimens, all collected in the Crater Mountain Wildlife Management Area in Eastern Highlands Province, included: 1 female (RML123610); 2 males, 7 females and 1 nymph (RML123611); 2 females (RML123612); 2 females (RML123613); and 2 males and 7 females (RML123614). The male lectotype and 13 male paralectotypes, designated in 1982 by Dias (1993), of Neumann’s Aponomma oudemansi, were borrowed from the ZMAM for comparison with our specimens. The females described by Schulze in 1936 are deposited in the USNTC (RML49311). These and all other USNTC collections re-examined in this study and their associated collection data are listed in Table 1.

Fig. 1
figure 1

Geographical map of Papua New Guinea showing the Crater Mountain Area (cross) where the Bothriocroton oudemansi specimens were collected. Black circles correspond to locality and white triangles to mountains

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Table 1 Taxonomic status and collection data for Bothriocroton spp. examined during this study
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Molecular analysis

DNA was extracted using DNAeasy tissue kits (Qiagen, Valencia, CA) and a previously described modified protocol, which is particularly effective when working with alcohol-preserved, blood-fed arthropods (Beati & Keirans, 2001; Beati, Cáceres, Galati, Lee & Munstermann, 2004). A portion of the 18S rDNA gene was amplified and sequenced from a nymph (RML123611) in order to establish whether this tick is a Bothriocroton or an Amblyomma. The 18SrRNA gene fragment was amplified using the primer pair NS1 and NS8, and sequenced with primers NS1, NS8, NS4 and NS 58.1 (Black, Klompen & Keirans, 1997). The 18S rDNA gene sequence was manually aligned with homologous sequences from Genbank by using McClade4 (v. 4.07) (Maddison & Maddison, 2000). Phylogenetic reconstructions were inferred by using PAUP (v. 4.0b10) with Maximum Parsimony (MP) and maximum likelihood (ML) (Swofford, 2000). Sequence divergence values were calculated based on the model evaluated by maximum likelihood. Node support was assessed by bootstrap analysis (MP with 1,000 replicates). The Genbank accession numbers of all 18S rDNA sequences included in the analysis are listed in Fig. 5 with the tick names. Fragments of the 12S rDNA and of the 28S rDNA genes of one male (RML123614), one female (RML123612) and one nymph (RML123611) of the New Guinea ticks, one B. glebopalma (RML121855), one B. undatum (RML83244) and one B. concolor (RML100403) were amplified and sequenced by using primers TIB and T2A (Beati & Keirans, 2001) and 28SV and 28SX, respectively (Hillis & Dixon, 1991). Other sequences used for comparisons are 12S rDNA fragments of B. hydrosauri (U95860) and B. glebopalma (U95858) from Genbank.

Bothriocroton oudemansi (Neumann, 1910) n. comb.

Syns Aponomma oudemansi Neumann, 1910; Amblyomma oudemansi (Neumann, 1910) Horak, Camicas & Keirans, 2002

Comments on synonymy

For lack of conclusive proof that Ixodes acanthoglossi (Lucas, 1878) is the same tick species, and in agreement with Camicas et al. (1998), we will not include this name in this synonymy and consider it a nomen dubium. Aponomma concolor was not included in the synonymy list because Kaufmann (1972) synonymised A. oudemansi and A. concolor while looking at what Schulze thought were female A. oudemansi (1936), but were in fact female B. concolor. Morphological comparisons of our tick specimens with the lecto-and paralectotypes from the ZMAM confirm that our male ticks are identical to Neumann’s (1910) A. oudemansi (Fig. 2A-E). Schulze’s (1936) A. oudemansi females (RML49311) were all characterised by having ventral finger-like processes on palpal articles II, round and deep porose areas located close to the posterior margin of the basis capituli, and alloscutal punctation typical of B. concolor (see Roberts, 1970; Kaufmann, 1972). These features were not found in our females (Fig. 3A-F), which also differed from all known former Australasian Aponomma. While re-examining all Bothriocroton holding of the USNTC, we discovered an additional collection (RML88313) of ticks (males and nymphs) from the eastern long-beaked echidna Zaglossus bartoni (Thomas), collected in the Indonesian Province of Papua, which were identical to our samples from Papua New Guinea (Table 1).

Fig. 2
figure 2

Bothriocroton oudemansi (male, RML123103): A. Scutum; B. Coxae I-IV, ventral view; C. Spiracular plate; D. Capitulum, dorsal view; E. Capitulum, ventral view. Scale-bars: A,B,D,E, 500 μm; C, 250 μm

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Fig. 3
figure 3

Bothriocroton oudemansi (female, RML123614; two specimens): A. Scutum; B. Coxae I-IV, ventral view; C. Scutum; D. Spiracular plate; E. Capitulum, dorsal view; F. Capitulum. Scale-bars: A,B, 550 μm; C,E,F, 500 μm; D, 60 μm

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Descriptions of the female and the nymph and redescription of the male (Figs. 2–4)

Male (Fig. 2A-E)

[Measurements made from 8 males.] Body. Small, pale yellow, subcircular, slightly longer than wide. Length from scapular apices to posterior body margin 2,342–2,718 (2,513), breadth 2,153–2,370 (2,243). Capitulum (Fig. 2C, E). Basis capituli 236–330 (280) long, 523–604 (568) broad, with rounded lateral margins; cornua present, 42–66 (53) long. Palps elongate, 595–772 (679) long, 177–230 (209) broad; average length of palpal article I 89, II 337 and III 259. Length of the spatulate hypostome 507 with 2 ½ : 2 ½ dentition; length of toothed portion 244–338 (294); apex of hypostome slightly narrower with numerous fine denticles. Conscutum (Fig. 2A). Inornate, with mid-gut typically visible through cuticle in fed specimens (Neumann, 1910); eyes absent. Regularly distributed small punctations, slightly deeper along margins and on festoons, giving scutum smooth overall appearance. Cervical grooves absent; marginal grooves barely distinguishable as shallow depressions. Venter with uniformly distributed punctations each with a fine seta; genital opening at level of coxae II and III (Fig. 2B); anal groove present, posterior to anus, broadly U-shaped. Large spiracular plates (Fig. 2C) anterior to first festoon, extruding from lateral body margin, 532–655 (591) long, 232–401 (319) broad. Legs. Coxa I (Fig. 2B) with 2 equally short rounded spurs; all other coxae with single stout, rounded external spur. Tarsus I (not shown) gradually tapered distally, 477–571 (51) long, 175–217 (194) broad; tarsus IV (not shown) with subapical hump, 469–590 (527) long, 140–173 (159) broad.

Female (Fig. 3A-F)

[Measurement from 5 partly engorged specimens.] Body. Length from apices of scapulae to posterior body margin 2,818–3,512 (3,097), breadth 2,473–3,076 (2,718), small, pale yellow, subcircular; gut visible through cuticle in fed specimens. Large spiracular plates (Fig. 3D) extruding from body external margins, 544–612 (571) long, 403–607 (531) broad. Genital aperture U-shaped at level of coxae II and III; anal groove present, posterior to anus and broadly U-shaped. Deep alloscutal punctuations homogeneously distributed dorsally and ventrally. Scutum (Fig. 3A,C). Length 1,417–1,530 (1,435), breadth 1,659–1,737 (1,697). Subtriangular, inornate, covered with fine punctations, slightly coarser along margins and in lateral fields; eyes absent. Cervical grooves very shallow if present at all. Capitulum (Fig. 3E,F). Basis capituli 265–501 (399) long, 707–756 (744) broad, trapezoid, with rounded lateral margins; cornua absent. Porose areas well delimited, depressed, pyriform, slightly divergent anteriorly, 93–143 (114) in diameter; interporose distance 52–110 (93). Palps elongate, 910–959 (933) long, 190–264 (226) broad; length of palpal segment I 117–138 (128), II 448–585 (503), and III 258–287 (270). Hypostomal dentition as in males, but hypostome anteriorly more rounded, with fine apical denticles. Hypostome 655–721 (693) long, length of toothed portion 360–421 (386). Legs. Coxa I (Fig. 3B) with 2 short, rounded spurs; all other coxa with single external rounded protuberance. Tarsi I (not shown) 741–892 (824) long, 194–230 (217) broad; Tarsi IV (not shown) 690–872 (790) long, 163–204 (188) broad.

Nymph (Fig. 4A-E)

[Measurement made from 2 partly engorged specimens.] Body. Length from apices of scapulae to posterior body margin 1,616–2,403 (2,010), breadth 1,390–1,781 (1,586), pale yellow, subcircular, with uniform punctations; gut visible through cuticle in fed specimens. Spiracular plates extruding from lateral body margins, 274 long, 181–208 (195) broad (Fig. 4C). Scutum (Fig. 4E) broader – 785–915 (850) than long – 598–661 (630), subtriangular, with few scattered punctations. Cervical grooves short, shallow, slightly chagrined; eyes absent. Capitulum (Fig. 4A,B). Basis capituli subtriangular, laterally rounded, length 126–128 (127), breadth 332–360 (346); cornua absent. Palps 357–379 (368) long, 97–111 (104) broad; length of article I 37–45 (41), II 123–185 (154) and III 120–123 (122). Hypostome 315–326 (321) long; length of toothed portion 164–175 (170); hypostomal dentition 2/2. Legs. Coxa I (Fig. 4D) with 2 short rounded spurs; coxae II-IV with single short rounded external spurs. Tarsi I (not shown) 307–375 (341) long, 117–130 (124) broad; tarsi IV (not shown) 261–375 (318) long, 83–97 (90) broad.

Fig. 4
figure 4

Bothriocroton oudemansi (nymph, RML123103). A. Capitulum, dorsal view; B. Capitulum, ventral view; C. Spiracular plate; D. Coxae I-IV, ventral view; E. Scutum. Scale-bars: A,B, 100 μm; C, 50 μm; D, 400 μm; E, 200 μm

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Species relationships

Molecular analyses

The 18S rDNA sequence alignment of the 29 selected taxa included 1,719 characters, 142 of which were parsimony informative. The MP heuristic search found 80 trees with identical length (342 steps). The topology of their strict consensus tree and the ML trees were totally congruent with each other and, therefore, only the MP reconstruction is shown in Fig. 5. The inferred tree shows that the New Guinea echidna tick belongs to Bothriocroton. Among Bothriocroton species, B. concolor constitutes the basal lineage and B. oudemansi clusters with B. hydrosauri, B. undatum and B. glebopalma. For comparison, 18S rDNA sequence divergence between Bothriocroton species varied from 0.3 to 1.9%, whereas divergence of Bothriocroton taxa from the other considered genera varies from 5.7 to 8.7% (Ixodes), 2.5 to 4.4% (Haemaphysalis) and 3.6 to 6.0% (Amblyomma) (Table 2).

Fig. 5
figure 5

Strict consensus maximum parsimony tree based on the alignment of 18S rDNA partial sequences of 29 tick species. The heuristic search found 80 best trees: length = 342; CI = 0.708; RI = 0.883; RC = 0.625 and HI = 0.292. Bootstrap values are based on 1,000 replica and are only shown above supported branches (>78%)

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Table 2 Pairwise distances (%) between the 18S rDNA sequences included in our phylogenetic analysis. The ML pairwise distances were calculated based on the substitution model estimated from the MP tree with the best ML score by PAUP
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The 349 bp fragment of the small-subunit ribosomal mitochondrial gene sequences of the female – RML123612 (GenBank accession number: DQ668031) and the nymph – RML123611 (DQ668030) were identical and differed from the male – RML123614 sequence (DQ668032) by only 2 bp (0.3% sequence divergence). The B. oudemansi sequences differed from homologous sequences of B. hydrosauri (U95860), B. glebopalma (U95858 and EF173724), B. undatum (EF173726) and B. concolor (EF173725) by 11.9–16.9%. The 12S rDNA sequence of B. glebopalma (RML121855; EF173724) differed from the Genbank sequence (U95858) by 0.3%. The 28S rDNA sequences (EF064255, EF064256, and EF064257) of the female, male and nymph B. oudemansi were identical and differed from the homologous fragment of B. concolor (EF173727) by 0.7%, of B. undatum (EF173729) by 0.7% and of B. glebopalma (EF173728) by 1.44%. Comparing the 12S and 28S rDNA sequences of the specimens from New Guinea with the available homologous Bothriocroton sequences confirms that the New Guinea male, female and nymph all belong to a single species. Bothriocroton currently contains five species (Roberts, 1953, 1964, 1970; Keirans et al., 1994; Klompen et al., 2002). Our molecular results show that Amblyomma oudemansi is definitively not an Amblyomma and should be assigned to Bothriocroton with all the endemic Australian former Aponomma species.

Morphological comparisons

B. oudemansi can be distinguished from other Bothriocroton species by the presence of its unusually extruding and large spiracular plates and by its geographical distribution. B. oudemansi is not an ornamented tick and can, therefore, be readily differentiated from both B. undatum and B. glebopalma (see Neumann, 1899; Roberts, 1970; Keirans et al., 1994). Both sexes of B. glebopalma are much smaller than B. oudemansi and are covered with deep punctations, each with stout short setae, which give them an unmistakable appearance. Their hypostomal dentition is 2/2 and all coxae bear a single spur. Furthermore, B. glebopalma is associated with varanid lizards in northwestern Australia and not with echidnas. B. undatum is a reddish-brown tick, which is also found exclusively on reptiles. In both sexes, this tick is characterised by homogeneously distributed coarse punctations, which contrast with the smooth overall appearance of B. oudemansi. Male B. undatum also present distinct non-continuous lateral grooves marked by confluent deep punctations (Neumann, 1899; Roberts, 1970). Among the echidna-associated Bothriocroton ticks, B. oudemansi can easily be differentiated from B. concolor and B. hydrosauri [or Aponomma tachyglossi, according to Andrews et al. (2006)]. B. hydrosauri is a dark brown tick, the males of which are larger than B. oudemansi and are characterised by a heavily punctated conscutum with continuous lateral grooves. The scutum of the female B. hydrosauri is dark brown, with a clearer median area, sometimes with a pale spot in the posterior angle, and with numerous punctations which are coarser laterally; the alloscutum is covered with deep punctations each with stout whitish setae. Porose areas are large and oval, almost contiguous with the posterior margin of the basis capituli (Roberts, 1970). A. tachyglossi is distinguished from B. hydrosauri only by the presence of larger punctated areas contiguous to the lateral grooves in males and by smaller porose areas in females (Andrews et al., 2006). None of these characters should constitute a problem when identifying B. oudemansi. Both sexes of B. concolor have prominent ventral finger-like processes on palpal article II, and females have small round and deep-set porose areas located close to the posterior margin of the basis capituli. The female scutum is punctate and has deep cervical grooves. Males are characterised by having a continuous lateral groove on a heavily punctate scutum (Schulze, 1936, 1941; Roberts, 1970). B. auruginans is generally larger than B. oudemansi and is a tick strictly associated with wombats. Males are oval in shape and narrower anteriorly, with a hypostomal dentition of 2/2, stout palps with palpal article II one third as wide as long and about twice as long as article III, and lateral grooves ending at the anterior margin of the external festoon. The female scutum is almost rugose, and the porose areas are very large and encroach on the posterior margin of the basis capituli (Schulze, 1936; Roberts, 1970).

Host, distribution and ecology

B. oudemansi has been collected from two of the long-beaked echidna species known to occur in New Guinea, Zaglossus bruijni and Z. bartoni (some consider Z. bartoni to be a synonym of Z. bruijni), but Groves (2005) treated these taxa as distinct species. These monotremes are largely insectivorous and, unlike their close relative T. aculeautus, which mainly feeds on termites and ants, they prefer to eat insect larvae and earthworms. Z. bruijni is usually found in the highland forests of New Guinea from 1,300–4,000 m, whereas Z. bartoni is known to occur along the central cordillera of New Guinea from its Indonesian region to the Huon Peninsula in Papua New Guinea (Flannery & Groves, 1998; Musser, 2003). Because long-beaked echidnas are endangered species, B. oudemansi should, therefore, also be included in endangered tick species lists (see Durden & Keirans, 1996).

Discussion

Morphological characters typical, if not unique, for Bothriocroton had been listed by Kaufmann (1972), who stated: “conceivably, the indigenous Australian species and the ‘primitive’ species may represent new genera”. None of these morphological features clustering endemic Australian taxa together are, however, synapomorphic for Australian taxa and can also be found in some species of Amblyomma. As larval specimens of B. oudemansi were not available, a molecular phylogenetic analysis of its relationships with other former Aponomma species appeared to be the easiest way of establishing its taxonomic status. All molecular data confirm that this tick is in fact a Bothriocroton and allow us to determine with confidence that adult and nymphal stages in our recent collection all belong to the same species. The female specimens were, however, morphologically very different from Schulze’s (1936) description of ‘A. oudemansi’. Our direct examination of Schulze’s specimens confirms that they are all B. concolor. Schulze did not have access to female B. concolor, which was not fully recognised and described until much later (Roberts, 1964, 1970). Nevertheless, his misidentification is certainly at the heart of most of the taxonomic contradictions found in the literature. Kaufmann (1972) did not have access to the types, and probably based his conclusions only on the examination of Schulze’s illustrations of the females, but stated that Aponomma oudemansi was a synonym of A. concolor. He neglected to compare males of B. concolor to Neumann’s illustrations (1910) and was, therefore, unable to realise how different these two species in fact are. Because both Keirans (1985) and Dias (1993) had access to Neumann’s types (all males), they did not agree with Kaufmann (1972) and considered A. oudemansi to be a valid tick species.

In recently published lists of valid tick names (Horak et al., 2002; Barker & Murrell, 2004), this tick was included in Amblyomma. The decision of reassigning this tick to Amblyomma can be explained by the fact that all ‘non-endemic Australian’ Aponomma species had then been transferred to Amblyomma (see Klompen et al., 2002). The use of the term ‘non-endemic Australian’ species proved to be an unfortunate choice, because the Australasian ecozone, delimited from the Indomalayan ecozone by Wallace’s line, extends well beyond Australia (Brown & Lomolino, 1998). It also includes the island of New Guinea, the eastern part of the Indonesian archipelago and several additional Pacific Islands and archipelagos. Australia and New Guinea were contiguous from about 5 million to 8,000 years ago (Pliocene to Pleistocene) across the shallow Sahul continental shelf (now occupied by the Torres Strait) when eustatic sea levels were c.30–200 m lower than at present (Parsons, 1998). Monotremes are endemic species of the Australasian ecozone and occur (as different species) as part of the native fauna in both Australia and New Guinea.

In conclusion, based on our molecular results, Bothriocroton now includes six recognised species: B. auruginans, B. concolor, B. glebopalma, B. hydrosauri, B. oudemansi n. comb. and B. undatum. The taxonomic status of Amblyomma tachyglossi (see Andrews et al., 2006) as a distinct species from B. hydrosauri is not totally convincing, particularly because there does not seem to be any morphological difference between immature stages of the two species. However, if this hypothesis is confirmed in future by molecular data, A. tachyglossi will certainly be added to the list of Bothriocroton species. This genus is now known to occur in the Australasian ecological zone and is not limited to the Australian continent.