The phylogenetic position of Ixodes stilesi Neumann, 1911 (Acari: Ixodidae): morphological and preliminary molecular evidences from 16S rDNA sequences

Springer 2006

Systematic Parasitology (2006) 65:1–11 DOI 10.1007/s11230-005-9024-4

The phylogenetic position of Ixodes stilesi Neumann, 1911 (Acari: Ixodidae): morphological and preliminary molecular evidences from 16S rDNA sequences Alberto A. Guglielmone1,*, Jose´ M. Venzal2, Daniel Gonza´lez-Acun˜a3, Santiago Nava1, Ana Hinojosa4 & Atilio J. Mangold1 1

Instituto Nacional de Tecnologı´a Agropecuaria, CC 22, CP 2300 Rafaela, Santa Fe, Argentina Departamento de Parasitologı´a Veterinaria, Facultad de Veterinaria, Universidad de la Repu´blica, Av. Alberto Lasplaces 1550, Montevideo, Uruguay 3 Facultad de Medicina Veterinaria, Universidad de Concepcio´n, Casilla 537, Chilla´n, Chile 4 Facultad de Ciencias Naturales y Oceanogra´ficas, Departamento de Zoologı´a, Universidad de Concepcio´n, Casilla 160C, Concepcio´n, Chile 2

Accepted for publication 29th August, 2005

Abstract The female of Ixodes stilesi Neumann, 1911 (Acari Ixodidae) is redescribed and the male and nymph are described from specimens collected from Pudu puda (Molina) (Artiodactyla: Cervidae) in Chile. Both sexes of I. stilesi have characteristics of the subgenera Ixodes Latreille, 1795 and Ixodiopsis Filippova, 1957. The females of I. stilesi are peculiar in having the combination of the sinuous scutum outline, rounded porose areas with distinct borders separated by the width of one area, slender and long palpi, and two subequal spurs on coxa I. The male is unique in having a combination of a posteriorly wrinkled marginal folder, a basis capituli longer than wide, a non-crenulate hypostome toothed portion, two spurs on coxa II to IV and the presence of a pseudoscutum. The nymph of I. stilesi has blunt anterior and posterior processes on palpal article I (characteristics of the subgenus Ixodiopsis and some Pholeoixodes Schulze, 1942) and a wingshaped basis capituli with a prominent triangular cornua. Phylogenetic analyses based on 16S mitochondrial rDNA sequences of 12 Neotropical and two Australian Ixodes species, plus three argasids, were carried out to clarify the position of I. stilesi. The results of phylogenetic analyses and morphological characters indicate a close relationships between I. stilesi and two other Neotropical species of uncertain subgeneric status, I. neuquenensis Ringuelet, 1947 and I. sigelos Keirans, Clifford & Corwin, 1976.

Introduction Barker & Murrell (2004) have recently proposed a list of valid tick taxa containing 249 species of Ixodes Latreille, 1795 worldwide. Nevertheless, the list includes, for example, I. uruguayensis Kohls & Clifford, 1967 distinct from I. longiscutatus Boero, 1944, when in fact they are synonyms (Venzal et al., 2001). We consider that there are a total of 243 species of Ixodes; 241 listed in Horak et al. *Author for correspondence (E-mail: aguglielmone@rafaela. inta.gov.ar)

(2002) plus I. paranaensis Barros-Battesti, Arzua, Pichorim & Keirans, 2003 and I. dicei Keirans & Ajohda, 2003. Among the 45 species recorded in the Neotropical region, 37 are endemic (Guglielmone et al., 2003). The immature stages of 25 Neotropical species remain unknown and several others need redescription. The southern Neotropical fauna of Ixodes (Argentina, Chile and Uruguay in the context of this study) is characterised by distinctive species found nowhere else, such as I. chilensis Kohls, 1956, I. longiscutatus Boero, 1944, I. neuquenensis Ringuelet, 1947, I. sigelos Keirans, Clifford &

2 Corwin, 1976, I. stilesi Neumann, 1911, I. taglei Kohls, 1969 and, if valid, I. abrocomae Lahille, 1916 (Guglielmone et al., 2003). The larva and the nymph of I. longiscutatus and I. neuquenensis, and the nymph of I. stilesi, were recently recognised (Venzal et al., 2001; Guglielmone et al., 2004; this study) but the immature stages of I. chilensis and I. taglei remain unknown. The only one of those species with a solid subgeneric position is I. longiscutatus, which belongs to the monotypic subgenus Haemixodes Kohls & Clifford, 1967 characterised by a Haemaphysalis-like shape of the palpi of the larva and the nymph, including a well-developed ventral spur on the palpal apex (Kohls & Clifford, 1967) (see Table 1). However, Clifford et al. (1973) included I. chilensis and I. taglei in the subgenus Pholeoixodes Schulze, 1942, whereas I. stilesi was considered a member of the subgenus Ixodes Latreille, 1795. Keirans et al. (1976) considered uncertain the subgeneric position of I. sigelos, but Xu et al. (2003) included I. sigelos in the Haemixodes, which is unjustified because the larva and the nymph of this species bear no morphological relationship with I. longiscutatus (see Guglielmone et al., 2005). Camicas et al. (1998) allocated I. longiscutatus,

I. neuquenensis, I. stilesi and I. taglei to the subgenus Amerixodes Morel, 1998, but Marques et al. (2004) rejected this classification because there is no morphological definition of the subgenus, which is considered a nomen nudum. Camicas et al. (1988) regard I. sigelos as a synonym of I. abrocomae, which is unjustified, as discussed by Guglielmone et al. (2003), and included these species in the subgenus Alloixodes Cerny´, 1969, again with no obvious justification for this unexplained arrangement. Guglielmone et al. (2004) described the larva and the nymph, redescribed the female and obtained a 16S rDNA sequence of I. neuquenensis but consider it premature to assign this species to a particular subgenus. In this paper, we redescribe the female and describe the nymph and the male of I. stilesi based on newly collected specimens. The 16S rDNA sequences were obtained for the female and the nymph of I. stilesi and for the only representative of the subgenus Haemixodes, I. longiscutatus. Neighbour-joining (NJ) and maximum parsimony (MP) analyses based on 16S mitochondrial rDNA sequences of 12 Neotropical and two Australian Ixodes species, plus three argasids, were prepared to clarify the phylogenetic position of I. stilesi. The

Table 1. Species of Ixodes, subgeneric status and the corresponding references, distribution and GenBank (GB) accession numbers for 16S rDNA sequences. Species

Subgenus

References

Distribution

GeneBank No.

lasallei longiscutatus loricatus

Ixodes Haemixodes Ixodes Uncertain but not Ixodes Ixodes Uncertain but not Ixodes Uncertain Ixodes (ricinus complex) Haemixodes Uncertain Ixodes Ixodes (ricinus complex) Ixodes (ricinus complex)* Multidentatus Ceratixodes Sternalixodes Endopalpiger

Clifford et al. (1973) Venzal et al. (2001) Clifford et al. (1973) Klompen (1999) Clifford et al. (1973) Klompen (1999) Guglielmone et al. (2004) Keirans et al. (1999) Xu et al. (2003) Guglielmone et al. (2005) Clifford et al. (1973) Keirans et al. (1999) Xu et al.(2003) Clifford et al. (1973) Clifford et al. (1973) Clifford et al. (1973) Clifford et al. (1973)

Neotropical Neotropical Neotropical

AF549850 DQ061294 AY510268

Neotropical

AF549851

Neotropical Neotropical Neotropical

AY254393 AF549855 AF549858

Neotropical Neotropical, Nearctic Neotropical, Nearctic Neotropical, Australian, Ethiopian Circumpolar** Australian Australian

DQ061292 AF549834 AF549841 AF549845 AF549859 AB051845 U95906

luciae neuquenensis pararicinus sigelos stilesi affinis minor auritulus uriae holocyclus tasmani

* Not included in the ricinus complex by Keirans et al. (1999). ** The distribution includes records in the southern Neotropical, Nearctic, Australian, Ethiopian and northern Palaearctic Zoogeographical Regions.

3 data obtained will be useful for future investigations of the phylogenetic relationships between Ixodes species, including the allegedly basal Australian Prostriata lineage (Klompen, 1999).

Materials and methods Tick specimens were preserved in 70% ethanol and deposited in the tick collection of Departamento de Ciencias Pecuarias, Laboratorio de Zoologı´ a, Facultad de Medicina Veterinaria in Chilla´n and in the Departamento de Parasitologı´ a, Facultad de Veterinaria, Montevideo, Uruguay. The female and the nymph of I. stilesi were stored at )20C until used for DNA extraction and polymerase chain reaction (PCR) amplification as described by Mangold et al. (1998). PCR conditions included an initial denaturation step at 94C for 2 min followed by 35 cycles for 45 s at 94C, 45 s for primer annealing and 45 s for primer extension at 72C. The annealing temperature of the first 7 cycles was increased by 0.3C every second cycle from 47 to 48.8C, followed by 28 cycles using an annealing temperature of 50C. A final extension step was carried out for 7 min at 72C. The primers used for the amplification and sequencing of circa 460 bp fragment of the 16S rRNA were: forward, 5¢-CTG CTC AAT GAT TTT TTA AAT TGC TGT GG-3¢ (16S + 1, Black & Piesman, 1994); reverse, 5¢-CCG GTC TGA ACT CAG ATC AAG T- 3¢ (16S - 1, Black & Piesman, 1994). All PCR reactions were performed in a100 ll volume. Negative controls (no template) were always run simultaneously, and entire reaction mixtures were discarded if a product appeared in the negative control. A10 ll volume of the reaction mixture was examined by 1% agarose-gel electrophoresis followed by staining with ethidium bromide. The amplified DNA was purified using Wizard SV Gel and PCR Clean-Up (Promega) according to the manufacturer’s protocol. Purified PCR products were sequenced using ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit and an Applied Biosystem 373A gene sequencer. The same technique was applied to a nymph of I. longiscutatus, (ex Scapteromys tumidus (Waterhouse) (Rodentia: Muridae), La Coronilla, 33
530 S, 53
300 W, Uruguay, November 2004, coll. A. Olmos & F. Acha´val). The DNA sequence of 16S rDNA were almost identical (99.8%

concordance) for the female and the alleged I. stilesi nymph (GenBank accession numbers DQ061292 for the female and DQ061293 for the nymph), supporting the conspecificity of these stages. The only difference was a transversion (T fi A) in position 199. The male was considered to be I. stilesi due to its association with the females and nymphs found on the same host and to morphological differences in relation to the males of I. taglei and I. montoyanus, the only others known species of ticks found on Pudu. A male, a female and a nymph of I. stilesi were prepared for scanning electron microscopy following Corwin et al. (1979). Mitochondrial 16S rDNA sequences of Neotropical Ixodes species available in GenBank plus sequences of the Australian I. holocyclus Neumann, 1899 and I. tasmani Neumann, 1899 were used for pairwise comparisons. Detail of the species studied, their subgenera, distribution and GenBank accession numbers are presented in Table 1. Aligned sequences (ALIGN_000915, available at: ftp://ftp.ebi.ac.uk/pub/databases/embl/align) were examined using MEGA version 3.0 (Kumar et al., 2004). Phylogenetic relationships were analysed using distance and maximum parsimony methods. A NJ tree was generated from the Kimura two parameter distance and gaps were excluded in the pairwise comparation. Support for the NJ topology was tested by bootstrapping over 1,000 replications. MP analysis was performed using Mega 3.0 and heuristic search procedure (Minmini algorithm option) was used to find the MP tree. In this analysis gaps were excluded. Relative support for internal nodes was assessed using bootstrap analysis with 1,000 replications. Sequences of Argas persicus (Oken, 1818) (GenBank L34321), Ornithodoros moubata (Murray, 1877) (GenBank L34328) and Otobius megnini (Duge`s, 1883) (GenBank L34325) were used as outgroups. Ixodes stilesi Neumann, 1911 Syn. Ixodes elegans Neumann, 1910 (pre-occupied) Material examined One female from Pudu puda, Chile, Chilean Region VIII, Coelemu (36
290 S, 72
420 W), 26 March, 2003, coll. D. Gonza´lez-Acun˜a. 1 male, 2 females and 2 nymphs from the same host, Chile,

4 Chilean Region VIII, Ninhue (36
230 S, 72
240 W), April 2003, coll. A. Hinojosa.

Redescription of the female and description of the male and nymph (Figures 1–13)

hypostome to Ph1 setae 0.67, breadth 0.23; dental formula 3/3 for almost all hypostomal length, then 2/2 to base, with c.14 teeth in files 1 and 2, c.10 in file 3. Lateral denticles much larger than medians. Scutum (Figure 3). Elongate-oval sinuous outline, slightly longer than broad, with length 1.42, breadth 1.1. Lateral carinae and scapulae distinct, pointed; cervical grooves converge then diverge towards junction with lateral carinae forming depression on each side of scutum; small punctations and setae scattered over scutum but much less distinct in central field; few larger punctuations present on lateral carinae. Spiracular plate. Oval outline with 3 to 4 files of goblets and central macula. Coxa I with 2 subequal medium-sized spurs; internal spur slightly longer than external spur, none reaching coxa II; coxa II-IV each with single small but distinctive triangular spur (Figure 4). Trochanters lacking spurs. Alloscutum. With several, short, whitish, dorsal and ventral inconspicuous setae; ventrally without chitinous plaque medial to coxa I. Genital aperture situated between coxae II and III. Anal groove: an inverted U-shape, reaches posterior margin of body.

Female (Figures 1–4). Measurements (mm) from best preserved slightly engorged specimen. Body. Length from apices of hypostome to posterior body margin 5.34, breadth 3.10. Outline oval, widest at level posterior to spiracular plates. Capitulum (Figures 1–2). Length from palpal apices to cornua apices 1.04. Basis capituli (Figure 1– 2) broad dorsally, 0.54 in width, subrectangular, with small blunt-ended cornua in form of small rectangles; posterior margin between cornua straight; lateral margins converge posteriorly. Large rounded porose areas (0.10 in diameter) with distinct borders and separated by interporose area 0.10 wide; ventrally with 2 pairs of posthypostomal setae; basis slightly constricted posterior to mid-length; posterior margin curved; auriculae in form of small protuberances; transverse suture faint. Palpi long and slender, 0.78 long, 0.19 broad; article II (femur), 0.45, more 1.3 times longer than article III (genu), 0.27 long; articulation between these segments distinct. Article IV (tibio-tarsus) recessed within ventrolateral surface of article III. Article I (trochanter) ventrally with posteriorly directed spur, 3 regular and 1 very long (ventral) setae. Hypostome long and slender, rounded apically, length from apex of

Male (Figures 5–9). Measurements (mm) from single male collected. Body (Figures 5–6). Length from apices of hypostome to posterior body margin 2.70, breadth 1.20. Outline oval, widest near mid-length. Capitulum (Figures 7–8). Length from palpal apices to cornua apices 0.44. Basis capituli (Figures 7–8) 0.29 broad dorsally at level of palpal insertion; shape broadly pentagonal, widest anteriorly; cornua small, triangular; posterior margin between cornua slightly concave. Surface with punctuations at centro-lateral levels; ventrally 2 pairs of hypostomal setae; posterior margin slightly convex; auriculae in form of triangular lateral extensions at mid-level. Palpi clavate, short; articles II and III subequal in length; their combined length 0.28, breadth 0.11. Article II with dorsal depression near base and long, conspicuous postero-internal seta; articulation between segments II and III discrete. Hypostome stout, notched, length from apex of hypostome to Ph1 setae 0.22; dental formula mostly 2/2 but 2 lateral rows with 3 denticles each; anterior denticles from first 2 rows smaller than subsequent denticles. Scutum (Figure 5). Elongate-oval in outline, length 2.1, breadth 0.8, with pseudoscutum which is more easily observed

Hosts and distribution This Chilean Ixodes is known from the original collection of 14 females from an unspecified locality (Neumann, 1910), five females from the Province of Valdivia (Region IX) (Kohls, 1969), and Osorio (2001) recorded the collection of ‘‘adults’’ I. stilesi from Concepcio´n (Region VIII). All these specimens were collected on the endangered small deer Pudu puda (Molina), which inhabits southern Chile and south-western Argentina (Wilson & Reeder, 1993). Pudu puda is a small deer, less than 45 cm high, with diurnal and nocturnal activity in the temperate, humid forests of Chile and Argentina, which is seldom observed in nature because of its shy temperament. This deer has quite sedentary habits and is considered as a generalist herbivore (Parera, 2002).

5

Figures 1–4. Ixodes stilesi, female. 1. Basis capituli dorsal view. 2. Basis capituli ventral view. 3. Scutum. 4. Coxae. Scale-bars: 1– 2,200 lm; 3–4,500 lm.

under stereo-microscopy that in scanning photomicrographs. Lateral carinae faint, inconspicuous; cervical grooves very shallow, slightly converging; few small punctations and whitish short setae scattered over scutum but absent on centrolateral fields; several larger punctations on antero-lateral part of scutum; lateral body fold with few very small punctations containing small setae on 2/3 of its length, thereafter disappearing as

surface becomes increasingly rugose towards posterior end. Ventral plates as figured, with several short setae more common at end of median plate and almost absent on anal plate. Genital aperture situated between coxae II. Spiracular plate. Oval in outline, with off-centre macula and 3–5 files of goblets. Coxa I-IV (Figure 9) with 2 spurs; internal spur on coxa I reaches coxa II and larger than external spur; spurs on coxa II almost

6

Figures 5–9. Ixodes stilesi, male. 5. Dorsal view. 6. Ventral view. 7. Basis capituli dorsal view. 8. Basis capituli ventral view. 9. Coxae. Scale-bars: 5–6, 1 mm; 7–8,100lm; 9,200 lm.

7

Figures 10–13. Ixodes stilesi, nymph. 10. Scutum. 11. Basis capituli dorsal view. 12. Basis capituli ventral view. 13. Coxae. Scalebars:100 lm.

equal in length; external spurs on coxae III and IV larger than internal spurs. Trochanters lacks spurs. Nymph (Figures 10–13). Measurements (mm) from best preserved, slightly engorged specimen. Body. Length from palpal apices to posterior body margin 2.31, breadth 1.31. Outline oval, broadest at level of spiracular plate. Capitulum (Figures 11– 12). Length from apex of hypostome to cornua apices 0.33, breadth 0.31. Basis capituli triangular shape dorsally with prominent triangular cornua, blunt ended; posterior margin between cornua straight; external margin diverges then converges giving appearance of winged basis capituli with cornua not positioned at extremes of posterior margin; ventrally with 2 pairs of post-hypostomal

setae, constricted posterior to auriculae; auriculae small, blunt, triangular, inconspicuous; posterior border convex. Palpi long and slender, length 0.23; twisted appearance resulting from oblique dorsal indent on article II and because article III is obviously broader than article II; breadth 0.04 and 0.06 for articles II and III, respectively; suture between articles II and III indistinct; setae number 14 on articles II and III, combined, c.12 on IV and 0 on I; palpal article I with very large ventral anterior and posterior processes which end bluntly at both extremties. Hypostome elongate, rounded apically, with length from apex to first pair of hypostomal setae 0.22 and breadth 0.06; dental formula 2/2; 8–9 denticles per file. Scutum (Figure 10). Longer than broad, Length 0.64, breadth 0.52. Faint lateral carinae. Cervical grooves

8 shallow but distinct, converging then diverging to reach posterolateral scutal margin. Scutal surface rather smooth, with small punctations present in lateral fields. Coxae (Figure 13). Coxa I with 2 spurs; external spur just reaching coxa II; internal spur small and inconspicuous; coxa II-IV with small triangular external spur and lacking internal spur. Trochanters lacking spurs. Alloscutum. Ventrally without small chitinous plaques located medially to coxa I; with scattered setae more abundant posterior to anal level; anal groove, with branches slightly divergent then slightly convergeny; arms not reaching posterior margin of body; spiracular plate circular in outline, with macula in centre. Morphological comparison The female of Ixodes stilesi can be easily distinguished from other tick species found on deer of the genus Pudu. The I. montoyanus female has one spur on coxa I and none on coxa II-IV, a 4/4 dental formula and numerous setae on the ventral surface. The female of I. taglei has shorter and less slender palpi and hypostome than the I. stilesi female, and rectangular porose areas separated by less than a width of one area. I. nuttalli Lahille, 1913 (a parasite of rodents in Peru and Argentina, considered to belong to the subgenus Pholeoixodes Schulze, 1942 along with the Chilean I. chilensis and I. taglei by Clifford et al., 1973) and several representatives of the subgenus Ixodiopsis have a basis capituli that resembles the corresponding anatomical area of I. stilesi but the porose areas in I. nuttalli are triangular and separated by less than the width of an area, and ventrally there is an evident constriction after the mid-length. Species of Ixodiopsis are characterized by the porose areas with indistinct margins and absence of auriculae. The outline of the scutum of I. stilesi is peculiar, and a useful character for further separating this from the former species. The diagnostic characters unique to the female of I. stilesi are a combination of the sinuous scutal outline, the rounded porose areas with definite borders separated by the width of one area, cornua small with an extremely blunt end, long slender palpi with a length/width ratio >3, small auriculae and two subequal spurs on coxa I. The male of I. stilesi can be separated from the male of I. montoyanus because the latter is heavily punctate and bears one spur on coxa I and none

on coxa II-IV. The male of I. nuttalli is characterised by a notorious pseudoscutum and an unwrinkled marginal fold, while I. stilesi has a faint pseudoscutum and wrinkled marginal fold. As in the case of female, the male of I. stilesi broadly resembles a representative of the subgenus Ixodiopsis, but crenulations characterise the arrangement of the teeth of this subgenus, whereas I. stilesi has separated denticles; additionally, I. stilesi has a basis capituli longer than wide, two spurs on coxa I to IV and a posteriorly wrinkled marginal fold. The male most closely related to I. stilesi is that of I. taglei, which also presents a posteriorly wrinkled marginal fold but with short hairs in I. taglei and glabrous in I. stilesi. Additionally, they can be separated by a delicate oval outline in I. stilesi, a basis capituli longer than wide in I. stilesi and almost as wide as large in I. taglei, which also bear long hairs over the last two-thirds of the scutum. Moreover, I. stilesi has two spurs on coxae II to IV in comparison with just one in I. taglei. The diagnostic characters unique to the male of I. stilesi are a combination of a posteriorly wrinkled marginal fold, basis capituli obviously longer than wide with small cornua and a slightly concave posterior border, a non-crenulate toothed portion of the hypostome and two spurs on coxa II to IV, and the presence of a pseudoscutum. The nymph of I. stilesi has a greatly enlarged palpal article I with both anterior and posterior processes. This feature of palpal article I is also found in nymphs of the Ixodes subgenera Endopalpiger Schulze, 1935 (Australian), Exopalpiger Schulze, 1935 (Neotropical, Palaearctic, Afrotropical, Australian), Partipalpiger Hoogstraal, Clifford, Saito & Keirans, 1973 (Palaearctic, Oriental), in most representatives of the Holarctic subgenus Ixodiopsis, in some members of the Nearctic-Neotropical-Palaearctic subgenus Pholeoixodes and in the Neotropical species Ixodes sigelos and I. neuquenensis, both of uncertain subgeneric status. The nymph of one of the alleged Neotropical species of Pholeoixodes, I. nuttalli, is known (Nuttall, 1916), but not the larva and nymph of I. taglei and I. chilensis. The enlarged article I of the palpi of the nymphs of the subgenera Endopalpiger, Exopalpiger and Partipalpiger are partly fused with the basis capituli, while this does not occur in Ixodiopsis, Pholeoixodes, I. sigelos and I. neuquenensis.

9 72 96

I. affinis I. pararicinus I. minor I. auritulus I. lasallei I. loricatus

97 100

I. luciae

96 60 72

I. sigelos I. stilesi I. neuquenensis I. longiscutatus I. tasmani I. uriae I. holocyclus

99 100 97

O. moubata O. megnini A. persicus

Figure 14. Neighbour-joining condensed tree using the Kimura two-parameter distance. Numbers on the branches represent bootstrap support (more than 50%) generated from 1,000 replications.

The nymph of I. stilesi has a characteristic winged appearance of the basis capituli, a feature not shared with any other species in Ixodiopsis or Ixodes neuquenensis. The nymph of I. sigelos also has a winged basis capituli, but the external margin of the basis diverges to form distinct lateral protuberances. Additionally, the posterior process in palpal article I ends in a bifurcate formation, while it is bluntly ended in I. stilesi. The nymph of I. nuttalli also has a winged basis capituli but this and the cornua are smaller than in I. stilesi; besides, the process in palpal article I is large anteriorly in I. stilesi, reaching the last third of toothed portion of the hypostome, while it does not reach the toothed portion of the hypostome in I. nuttalli; the twisted appearance of the palpi of I. stilesi is not shared with I. nuttalli. The diagnostic characters unique to the nymph of I. stilesi are a combination of a basis capituli which is wingshaped with prominent triangular cornua and massive, blunt anterior and posterior processes on palpal article I that reach the posterior third of the toothed portion of the hypostome. The palpi are long and slender, twisted in appearance and with article III obviously broader than article II. Thus, the female and male of I. stilesi appear to resemble a member of the subgenus Ixodes, as

broadly defined by Clifford et al. (1973). However, both sexes of I. stilesi also have characters typical of the Holarctic subgenus Ixodiopsis, such as the slender and distally tapered palpi, with a length/ width ratio >3, subequal spurs on coxa I in females, and palpal articles II and III about equal in length with a distinct intersegmental suture and spurs on coxa I in the males (Robbins & Keirans, 1992). These characters, along with the presence of the prominent anterior and posterior processes on palpal article I of the nymph, are shared by Ixodes stilesi and species of the subgenus Ixodiopsis. Nevertheless, the porose areas of the females and the teeth and spurs on coxa II to IV of the males of I. stilesi are not typical for Ixodiopsis.

Analyses based on 16S mitochondrial rDNA sequences The rooted NJ tree is presented in Figure 14. It consists of four groups. Three of them were confirmed by the MP analysis; one was formed by the Australian Ixodes holocyclus and the circumpolar I. uriae White, 1852 with 99% bootstrap support (BS), and a second (96% BS) comprised species which exclusively inhabit the

10 Neotropical Region. The latter group is divided into the I. loricatus Neumann, 1899–I. luciae Se´nevet, 1940 clade (100% BS), and the I. sigelos (I. neuquenensis–I. stilesi) clade (72% BS). The third group confirmed by NJ tree and MP analysis is formed by the Neotropical I. pararicinus Keirans & Clifford, 1985 and the Nearctic-Neotropical I. affinis Neumann, 1899–I. minor Neumann, 1902 (96% BS), all from the subgenus Ixodes and within the ricinus complex, if I. minor is included in this complex, as proposed by Xu et al. (2003). The fourth group in the NJ analysis is formed by I. (Multidentatus) auritulus known from Australia, Ethiopia and the Neotropics, and the Neotropical I. (Ixodes) lasallei Me´ndez Arocha & Ortiz, 1958 (97% BS), but it was not confirmed by the MP analysis. The strictly Neotropical I. longiscutatus and Australian I. tasmani are not bound to any group. The position of I. longiscutatus indicates that it may represent a different lineage of Neotropical Ixodes. The group formed by strict Neotropical species, where I. stilesi belongs, is of special interest for this preliminary study. The I. loricatus–I. luciae clade represents species which are morphologically close (the best criterion to differentiate them is the size of the spurs on coxa I), the adult ticks of both species feed on didelphines and they have partly sympatric distributions. The I. sigelos (I. neuquenensis–I. stilesi) clade contains species that feed on phylogenetically and ecologically distant hosts: I. neuquenensis is a parasite of the Microbiotheriidae (Microbiotheria); I. sigelos parasitises rodents; and all specimens of I. stilesi were found on cervids. The known immature stages of the three species (the larva of I. stilesi is unknown) have anterior and posterior processes on palpal article I and all of them are restricted to south-western South America (Guglielmone et al., 2004, 2005, this article). They probably radiated from a common (hypothetical) Neotropical ancestor shared with I. loricatus and I. luciae.

Conclusion It is clear that Ixodes stilesi does not belong to the subgenus Ixodes, as proposed by Clifford et al. (1973), and the same may be true for many Neotropical species currently considered to belong to this subgenus. The results of the 16S rDNA

sequences and some morphological similarities (especially in the immature stages) appear to indicate that I. neuquenensis, I. sigelos and I. stilesi may belong to the same natural group or subgenus. However, strong differences exist between these three species established in the southern Neotropics, especially in adult ticks, and we regard their subgeneric status as uncertain. Strong efforts are needed to obtain new material of ticks established in southern South America for both descriptions and redescriptions and for DNA analysis in order to acquire further insights into the relationship between these unique species of Ixodes.

Acknowledgements We are grateful to Dr A.V. Bochkov for his critical review of the manuscript. We are also grateful to Alejandro Ma´rquez and Jorge Troccoli from the Unidad de Microscopı´ a Electro´nica, Facultad de Ciencias, Montevideo (Uruguay) for their assistance in obtaining scanning electron micrographs. We acknowledge the support of INTA and the Fundacio´n ArgenINTA to AAG and AJM, and CONICET (Proyecto de Investigacio´n Plurianual 020508) to AJM.

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