MORPHOLOGY, SYSTEMATICS, EVOLUTION
Amblyomma yucumense n. sp. (Acari: Ixodidae), a Parasite
of Wild Mammals in Southern Brazil
FELIPE S. KRAWCZAK,1 THIAGO F. MARTINS,1 CAROLINE S. OLIVEIRA,2 LINA C. BINDER,1
0
FRANCISCO B. COSTA,1 PABLO H. NUNES,3 FABIO GREGORI,1 AND
1,4
MARCELO B. LABRUNA
J. Med. Entomol. 52(1): 28–37 (2015); DOI: 10.1093/jme/tju007
ABSTRACT During 2013–2014, adult ticks were collected on the vegetation and subadult ticks were
collected from small mammals [Didelphis aurita Wied-Neuwied, Sooretamys angouya (Fischer), Euryoryzomys russatus (Wagner), Akodon montensis Thomas, Oxymycterus judex Thomas] in an Atlantic
rainforest reserve in southern Brazil. Analyses of the external morphology of the adult ticks revealed that
they represent a new species, Amblyomma yucumense n. sp. Partial 16S rRNA sequences generated
from males, females, and nymphs were identical to each other and closest (95% identity) to corresponding sequences of Amblyomma dubitatum Neumann. A. yucumense is morphologically and genetically
closest related to A. dubitatum. Dorsally, male of these species can be separated by major longitudinal
pale orange stripes associated with a pseudoscutum indicated by a pale stripe in A. yucumense, in contrast to pale creamy longitudinal stripes and absence of pseudoscutum in A. dubitatum. Ventrally, male
coxal I spurs are separated by a space narrower than external spur width in A. yucumense, and wider than
external spur width in A. dubitatum. Females of the two species can be separated by coxal I spurs, longer
in A. yucumense than in A. dubitatum. In addition, the adult capitulum and ventral idiosoma of A. yucumense are generally dark brown colored, while A. dubitatum is yellowish or light brown colored. The
nymph of A. yucumense differs from A. dubitatum by the scutal cervical groove length, slightly shorter in
the former species. Currently, A. yucumense is restricted to southern Brazil.
KEY WORDS Amblyomma, new species, description, Brazil
Introduction
The Brazilian tick fauna is currently composed by 66
species, 45 Ixodidae, and 21 Argasidae (Martins et al.
2014, Nava et al. 2014a). The genus Amblyomma is the
most numerous, with 31 species (Dantas-Torres et al.
2009, Nava et al. 2014a), comprising 46% of the Brazilian tick fauna. In the world, the genus Amblyomma is
currently composed of 136 valid species, distributed in
all continents except for Europe and Antarctica
(Guglielmone and Nava. 2014, Nava et al. 2014b). During the past 10 yr, eight new Amblyomma species were
described or resurrected, all from South America,
where nearly half of world Amblyomma species occur
(Labruna et al. 2005; Nava et al. 2009, 2014a,b). From
the medical stand point, the genus Amblyomma is the
most important in South America, where human diseases caused by tick-borne agents, namely, Rickettsia
rickettsii, Rickettsia parkeri, and Rickettsia sp. strain
1
Department of Preventive Veterinary Medicine and Animal
Health, Faculty of Veterinary Medicine, University of Sa˜o Paulo, Sa˜o
Paulo, SP, Brazil.
2
Department of Preventive Veterinary Medicine, Federal University of Santa Maria, Santa Maria, RS, Brazil.
3
Institute of Biosciences, Sa˜o Paulo State University, Rio Claro, SP,
Brazil.
4
Corresponding author, e-mail: [email protected].
Atlantic rainforest, are transmitted to humans by different Amblyomma species (reviewed by Parola et al.
2013). Herein, we describe an additional new
Amblyomma species from South America, based on
specimens collected in southern Brazil.
Materials and Methods
From June 2013 to January 2014, ticks were collected in the “Parque Estadual do Turvo”, an Atlantic
rainforest Reserve located in Derrubadas Municipality,
state of Rio Grande do Sul, southern Brazil, as part of
an ongoing study on the ecology of tick-borne spotted
fever in Rio Grande do Sul. Questing ticks were collected in the vegetation by dragging or by visual search
in animal trails, as previously described (Terassini et al.
2010). Ticks were also collected on small mammals that
were collected by Sherman and Tomahawk traps alongside the same animal trails.
Collected ticks were brought alive or in absolute ethanol to the laboratory, where they were separated for
morphological and molecular analyses; engorged larvae
were kept alive in an incubator (25 C, 95% relative humidity) to molt to nymphs. Unfed specimens, comprising 10 males, 9 females, and 10 nymphs were
measured using the Image-Pro Plus 5.1 program for
analysis of images and morphometry, fitted to an
C The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America.
V
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KRAWCZAK ET AL.: PARASITE OF WILD MAMMALS IN SOUTHERN BRAZIL
Olympus SZX stereoscope microscope (Olympus Corporation, Tokyo, Japan). In the description that follows,
all measurement are in mm; a range is given, with a
mean 6 standard deviation in parentheses. Three specimens of each stage (male, female and nymph) were
prepared for scanning electron microscopy (SEM) following techniques described by Corwin et al. (1979).
Light-microscopy photographs of live adult ticks were
prepared to show the natural scutal ornamentation
pattern.
Representative tick specimens were used for molecular analysis. For this purpose, each of the 2 males and
2 females had its leg segments collected in a sterile
microtube containing 50 ml of Tris-EDTA buffer, subsequently subjected to DNA extraction with DNeasy Tissue Kit (Qiagen, Valencia, CA), and processed by
polymerase chain reaction (PCR) with the use of primers targeting a 460-bp fragment of the tick 16S
rDNA mitochondrial gene, as previously described
(Mangold et al. 1998). The same procedure was performed with two nymphs, but in this case, the whole
tick body was individually used for DNA extraction.
PCR products of the expected size were sequenced in
an ABI automated sequencer (Applied Biosystems/
Thermo Fisher Scientific, model ABI 3500 Genetic
Analyser, Foster City, CA) with the same primers used
for PCR. Generated sequences were compared to each
other and submitted to BLAST analyses (www.ncbi.
nlm.nih.gov/blast) to infer closest similarities available
in GenBank. The consensus sequence obtained was
aligned with the corresponding 16S rRNA sequences
from 12 different sequences of the genera Amblyomma
retrieved from GenBank (including the sequences that
matched closest BLAST similarities to the present new
species) using Clustal/W v.1.8.1 (Thompson et al.
1994). Neighbor-joining and maximum likelihood phylogenetic trees using T92þG (Tamura 3-parameter
with Gamma: 0.17) substitution model were generated
using Mega 6.06 software (Tamura et al. 2013) with
1,000 bootstrap replicates. The substitution model was
selected using Mega 6.06 software (Tamura et al. 2013)
according to the lowest Bayesian Information Criterion
(BIC) score. The sequence of Amblyomma latum Koch
was used as outgroup.
For morphological comparisons, the following specimens of Amblyomma dubitatum Neumann available at
the tick collection “Colec¸a˜o Nacional de Carrapatos”
(CNC) at the Faculty of Veterinary Medicine of the
University of Sa˜o Paulo, Sa˜o Paulo, SP, Brazil, were examined in the present study: 53 males, 87 females ex
vegetation, Itu Municipality, state of Sa˜o Paulo, Brazil,
23 January to 07 February 2006 (accession number
CNC 958); 6 males, 8 females ex vegetation, Serra da
Canastra National Park, Sa˜o Roque de Minas Municipality, state of Minas Gerais, Brazil, 14 August 2007
and 08 March 2008 (CNC 1046, 1099); 16 males, 24 females ex vegetation, Intervales State Park, Ribeira˜o
Grande Municipality, state of Sa˜o Paulo, Brazil, 18 September 2005 (CNC 927). In addition, we also examined
the following specimens collected in Brazil, which were
used by Estrada Pen˜a et al. (2002) for the redescription
and revalidation of the species A. dubitatum: 10 males
29
ex Hydrochoerus hydrochaeris (L.), Sa˜o Bernardo do
Campo, state of Sa˜o Paulo, 29 November 1999 (CNC
263); 2 females ex vegetation, Arac¸ariguama, state of
Sa˜o Paulo, 27 March 1998 (CNC 129); 5 males and 3
females (collected as nymphs) ex Myocastor coypus
(Molina), Jundiai, state of Sa˜o Paulo, 17 August 1999
(CNC 288).
Three males and three females of A. dubitatum
collected ex vegetation, Intervales State Park, Ribeira˜o Grande Municipality, state of Sa˜o Paulo, Brazil,
18 September 2005 (CNC 927), were used for SEM.
One live and 1 live female of A. dubitatum, collected
ex vegetation, Pirassununga, state of Sa˜o Paulo,
Brazil, 10 March 2014, were used for light-microscopy photographs to show the natural scutal ornamentation pattern.
Results
Analyses of the external morphology of the adult
ticks collected in the present study revealed that they
were morphologically distinct from any other known
Amblyomma species, justifying their description as a
new species. Partial 16S rRNA sequences (404 bp) generated from 2 males, 2 females, and 2 nymphs were
100% identical to each other, and by BLAST analysis,
were closest (95% identity) to several sequences of
A. dubitatum from the states of Sa˜o Paulo (GU301914)
and Rio Grande do Sul (GU301913), Brazil, and from
Uruguay (DQ858954, DQ858955, and GU301912) and
Argentina (GU301910 and GU301911). Because all
nymphs examined in the present study had the same
external morphology, the above molecular results confirm that they represent the nymphal stage of the present new Amblyomma species.
Amblyomma yucumense Krawczak, Martins &
Labruna n. sp. (Figs. 1-4)
Male. (Figs. 1A and 2): Ten unfed specimens measured. Length from apices of scapulae to posterior body
margin 4.13–5.36 (4.70 6 0.36), breadth 3.17–4.04
(3.56 6 0.29). Outline oval, broadest at level of spiracular plates (Fig. 1A). Genital aperture broadly U-shaped
(Fig. 2D). Spiracular plate comma-shaped with elongate macula and numerous minute globlets. Eyes flat.
Basis capituli rectangular and triangular cornua on
posterior margin; numerous small punctations dorsally
(Fig. 2A). Length of palpal apices to cornua apices
0.95–1.32 (1.09 6 0.11), breadth 0.76–0.99 (0.83 6
0.07). Length of palpal article I 0.03–0.11 (0.07 6 0.02);
length of palpal article II 0.32–0.54 (0.46 6 0.07);
length of palpal article III 0.15–0.26 (0.21 6 0.04),
suture between II and III distinct. Presence of marked
ventral prolongation on palpal article I, as illustrated
(Fig. 2B). Hypostome elongate, broadly rounded apically with corona of fine denticles. Total length of hypostome 0.63–0.78 (0.71 6 0.04); length of toothed
portion 0.34–0.42 (0.38 6 0.02). Hypostomal dentition
3/3, with six to eight teeth per complete row (Fig. 2B);
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Fig. 1. Dorsal view of the adult stages of A. yucumense [(A) Male. (B) Female] and A. dubitatum [(C) Male. (D) Female]
by light microscopy.
Fig. 2. SEM of A. yucumense male. (A) Dorsal capitulum (scale bar: 200 mm). (B) Ventral capitulum (scale bar: 200 mm).
(C) Scutum (scale bar: 400 mm). (D) Coxae I–IV (scale bar: 400 mm).
in some specimens hypostomal dentition were 4/4
posteriorly.
Scutum ornate with pale orange markings (stripes)
over a dark brown background; major longitudinal
stripes starting at the cervical area, diverging posteriorly at the level of first festoon; numerous large and
deep punctations uniformly distributed, interposed by
dark brown elevated spots lacking punctations as follows: median-lateral spots, postero-lateral spots, and a
postero-median spot; limits of pseudoscutum indicated
by a pale stripe (Fig. 1A), clearly observed by naked
eyes. Cervical grooves short, deep, comma-shaped.
Marginal groove complete, deep posteriorly, marked
with numerous deep punctations anteriorly (Fig. 2C).
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Fig. 3. SEM of A. yucumense female. (A) Dorsal capitulum (scale bar: 200 mm). (B) Ventral capitulum (scale bar:
200 mm). (C) Dorsal idiosoma (scale bar: 400 mm). (D) Coxae I–IV (scale bar: 200 mm).
Punctations present on all festoons (Fig. 2C), festoons
4 and 6 with less ornamentation than remaining festoons (Fig. 1A). Short or indistinct ventral plates
present on all festoons, length 0.05–0.08 (0.06 6 0.01).
Coxa I with two moderately long, parallel, and robust
spurs of similar length; external spur slightly longer
than internal in some specimens; external thinner than
internal. Presence of an accessory spur situated anterior to the two regular spurs, as illustrated (Fig. 2D).
Coxae II–III each with triangular rounded external
spur and fine salient concave ridge extending from
spur to postero-internal angle of coxa, where a shorter
internal spur is evidenced in most specimens; a single
long, stout spur on coxa IV, spur length similar to the
coxa (Fig. 2D). Trochanters without spurs (Fig. 2D).
Tarsus I 0.73–1.06 (0.89 6 0.10) in length, 0.20–0.29
(0.26 6 0.03) in breath. Tarsus IV 0.73–0.83 (0.79 6
0.03) in length, 0.23–0.31 (0.27 6 0.03) in breath.
Female. (Figs. 1B and 3). Nine unfed specimens
measured. Length from apices of scapulae to posterior
body margin 3.18–4.79 (4.30 6 0.56), breadth
2.65–3.74 (3.41 6 0.39). Outline elliptical, broadest just
anterior to the level of spiracular plates (Fig. 1B). Genital aperture broadly V-shaped, with short and bulging
postero-lateral flaps (Fig. 3D). Spiracular plate subtriangular with rounded angles, elongate macula
surrounded by numerous minute globlets. Eyes flat.
Festoons without tubercles.
Basis capituli and palpi brown; if ornate, only spiked
by rare pale yellow marking. Basis capituli rectangular;
length of palpal apices to cornua apices 0.92–1.40
(1.14 6 0.16), breadth 0.71–1.05 (0.93 6 0.12). Cornua
faint or absent, posterior margin slightly concave (Fig.
3A). Porose areas deeply depressed, diameter of one
area 0.08–0.17 (0.12 6 0.03), interporose area
0.07–0.15 (0.11 6 0.03). Length of palpal article
I 0.08–0.13 (0.10 6 0.01); length of palpal article II
0.38–0.58 (0.48 6 0.07); length of palpal article III
0.20–0.37 (0.27 6 0.05), suture between II and III
distinct. Presence of marked ventral prolongation on
palpal article I, as illustrated (Fig. 3B). Hypostome
elongate, apically rounded with corona of fine denticles. Total length of hypostome 0.64–0.95 (0.84 6
0.11); length of toothed portion 0.34–0.50 (0.44 6 0.05).
Hypostomal dentition 3/3 anteriorly, 4/4 posteriorly (in
one specimen it was 3/3 posteriorly) with 7 to 10 teeth
per complete row.
Scutum length 1.61–2.62 (2.20 6 0.30), breadth
1.84–2.74 (2.39 6 0.29), ornamentation consisting primarily of pale yellow large spots on cervical and postero-lateral fields, separated by dark brown central
stripe ending at the posterior margin (Fig. 1B).
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Fig. 4. SEM of A. yucumense nymph. (A) Dorsal capitulum (scale bar: 30 mm). (B) Ventral capitulum (scale bar: 30 mm).
(C) Scutum (scale bar: 100 mm). (D) Coxae I–IV (scale bar: 200 mm).
Numerous large and deep punctations concentrated in
the lateral fields. Cervical grooves deep, converging
anteriorly then diverging as shallow depressions at scutal median third (Fig. 3C).
Coxa I with two moderately long and robust spurs of
similar length, the external thinner; presence of an
accessory spur situated anteriorly to the two regular
spurs, as illustrated (Fig. 3D). Coxae II–IV each with
small short external spur and fine salient ridge extending from spur to postero-internal angle of coxa, occupying position of missing internal spur. This external spur
broader than longer on coxae II–III; pointed, as long as
broad on coxa IV. Trochanters without spurs (Fig. 3D).
Tarsus I 0.78–1.28 (0.99 6 0.18) in length, 0.21–0.36
(0.28 6 0.05) in breath. Tarsus IV 0.57–0.86 (0.76 6
0.11) in length, 0.17–0.27 (0.22 6 0.03) in breath.
Nymph. (Fig. 4). Ten unfed specimens measured.
Length from apices of scapula to posterior body margin
1.18–1.37 (1.28 6 0.06), maximum breadth 0.93–1.09
(1.02 6 0.05); outline oval, with 11 festoons without
tubercles. Scutal length 0.61–0.66 (0.64 6 0.02),
breadth 0.71–0.78 (0.75 6 0.02), breadth/length ratio
1.14–1.22 (1.18 6 0.03); inornate, deep punctations
evenly distributed, larger laterally, smaller centrally
(Fig. 4C). Eyes large, not orbited, at lateral scutal
angles at the level of scutal midlength. Cervical grooves
long and deep reaching the scutal posterior third;
length 0.31–0.34 (0.33 6 0.01); cervical groove length/
scutal length ratio: 0.50–0.52 (0.51 6 0.01). Spiracular
plate rounded with an evident dorsal prolongation;
length 0.18–0.23 (0.21 6 0.02), breadth 0.11–0.16
(0.13 6 0.02). Gnathosoma length from palpal apices to
posterior margin 0.26–0.32 (0.28 6 0.02); breadth
0.26–0.29 (0.28 6 0.01). Basis capituli slightly hexagonal, posterior margin straight, without cornua (Fig. 4A);
posterior margin convex ventrally, without auriculae.
Palpi length 0.21–0.25 (0.23 6 0.02), article I with vestigial ventral prolongation; article II 0.13–0.16 (0.14 6
0.01) in length, article III 0.06–0.08 (0.07 6 0.01) in
length. Hypostome rounded apically; length 0.22–0.25
(0.23 6 0.01); length of toothed portion 0.11–0.13
(0.13 6 0.01); dentition 2/2 with seven to eight teeth
per row (Fig. 4B); row pairs diverge towards the anterior extremity. Coxa I with two pointed spurs, the external longer; coxae II-IV with a small triangular spur
(Fig. 4D). Trochanters without spur; tarsus I 0.29–0.34
(0.33 6 0.02) in length, 0.08–0.12 (0.09 6 0.01) in
breath; tarsus IV 0.24–0.28 (0.26 6 0.02) in length,
0.06–0.09 (0.08 6 0.01) in breath.
TYPES. Holotype male, Allotype female, nine Paratype males, eight Paratype females, all unfed specimens, ex vegetation at “Parque Estadual do Turvo”
(27 140 2600 S; 53 580 1900 W; altitude 390 m), Derrubadas Municipality, state of Rio Grande do Sul, Brazil, 11
January 2014; collectors: M.B. Labruna, F. Krawczak.
Legs of two Paratypes of each sex were processed for
DNA extraction. Thirty-tree Paratype nymphs, all
molted from engorged larvae collected on Didelphis
aurita Wied-Neuwied, 12 January 2014. Holotype,
Allotype and 32 Paratypes (5 males, 4 females, 25
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KRAWCZAK ET AL.: PARASITE OF WILD MAMMALS IN SOUTHERN BRAZIL
nymphs) deposited in the CNC tick collection under
accession numbers CNC 2784–2787. Two Paratypes of
each sex from lot CNC 2785 and two Paratype nymphs
from CNC 2787 were used for SEM. Paratypes deposited in other tick collections: 1 male, 1 female, 3
nymphs deposited at the Acari Collection of the Instituto Butantan (Sa˜o Paulo, Brazil) under accession number IBSP 11716–11717; 3 males, 3 females, 3 nymphs
deposited at the United States National Tick Collection
(Statesboro, Georgia) under accession number USNMENT00861423, CEN/RML 125117 and USNMENT00861424, CEN/RML 125118; 1 male, 1
female, 2 nymphs deposited at the INTA tick collection
(Rafaela, Santa Fe, Argentina).
Additional Material Collected. The following
additional specimens of A. yucumense were collected at
the “Parque Estadual do Turvo” (27 140 2600 S, 53
580 1900 W, 390 m; and 27 130 5100 S, 53 510 0400 W,
430 m), Derrubadas Municipality, state of Rio Grande
do Sul, Brazil: 2 nymphs (1 molted to a male) ex D.
aurita, 05 July 2013 (CNC 2791); 1 nymph ex Sooretamys angouya (Fischer), 05 July 2013 (CNC 2789); 5
nymphs ex vegetation, 03 July 2013 (CNC-2790); 37
nymphs (3 molted to 1 male and 2 females) ex D.
aurita, 09 October 2013 (CNC 2792); 6 nymphs ex
D. aurita, 10 October 2013 (CNC 2793); 8 nymphs ex
D. aurita, 09 October 2013 (CNC 2794); 3 nymphs
ex vegetation, 10 October 2013 (CNC 2797); 1 nymph
ex Euryoryzomys russatus (Wagner), 11 October 2013
(CNC 2796); 1 nymph ex Akodon montensis
Thomas, 09 October 2013 (CNC 2795); 8 nymphs (2
destroyed for DNA extraction in the present study) ex
Oxymycterus judex Thomas, 13 January 2014 (CNC
2798); 3 males ex vegetation, 11 January 2014 (CNC
2788).
Hosts and Distribution. Nymphs (and engorged
larvae from at least one host species) of A. yucumense
were collected from five species of small mammals (D.
aurita, S. angouya, E. russatus, A. montensis, and O.
judex), suggesting that small mammals are important
hosts for subadult ticks of A. yucumense. Because adult
ticks were found only on vegetation, hosts for the adult
stage remain unknown. However, it is noteworthy that
most of these adults were collected by the visual search
method. In this case, ticks were found questing at a stationary position on the vegetation (ambushing), at
heights between 50 and 80 cm. Many of these ticks
were found together with adults of Amblyomma incisum Neumann, a typical parasite of tapirs [Tapirus terrestris (L.)]. Because tapirs are large animals
(with heights over 80 cm), and are known to frequently
use the trails where ticks were collected, it is possible
that tapirs are major hosts for adults of A. yucumense, a
condition yet to be demonstrated. Until now, the
distribution of A. yucumense is restricted to its type
locality, an Atlantic forest Reserve in southern Brazil.
Because this Reserve is contiguous on the west to a
much larger Atlantic forest Reserve in Argentina, it is
possible that A. yucumense also occurs in this later
country.
Etymology. The new species is named yucumense
in reference to the Yucuma˜ Falls, at Uruguay River, in
33
the “Parque Estadual do Turvo” Atlantic Forest
Reserve, where the new species was found.
Phylogenetic Analysis. In the phylogenetic tree
inferred by partial sequences of the mitochondrial 16S
rRNA gene (Fig. 5), A. yucumense grouped in a branch
with Amblyomma coelebs Neumann and several
sequences of A. dubitatum from Brazil, Uruguay, and
Argentina. This topology was observed either by
neighbor-joining or maximum likelihood analysis.
Although these three species formed a group, their 16S
DNA sequences were relatively distantly to each other.
In this regard, the sequence of A. yucumense was 4.9
to 5.4% divergent from the A. dubitatum sequences
(DQ858954, DQ858955, GU301910–GU301914), and
6.6% divergent from A. coelebs (FJ424408). The
genetic divergences between A. dubitatum and A. coelebs were 8.6–9.4%. Finally, the intraspecific divergences among the A. dubitatum sequences, representing
populations from geographically separated areas of Brazil, Uruguay, and Argentina were in all cases <1%. The
partial sequence of the mitochondrial 16S rRNA gene
generated in this study for A. yucumense has been
deposited in GenBank under the accession number
KJ914670.
Discussion
A. yucumense is morphologically and genetically closest related to A. dubitatum (Figs. 1, 6, and 7). These
two species are unique in combining the following morphological characters: males with basis capituli rectangular and triangular cornua on posterior margin, coxa I
with two spurs of similar size, coxal IV spur much longer than coxal II–III spurs, marginal groove complete,
scutum with major longitudinal stripes starting at the
cervical area and numerous large and deep punctations
uniformly distributed, interposed by dark brown elevated spots lacking punctuations, festoon ventral
plates not incised; females with basis capituli rectangular, coxa I with two spurs of similar size, coxa II–IV with
a single spur, scutum with a dark brown stripe at the
posterior field, and genital aperture broadly V-shaped,
with short and bulging postero-lateral flaps.
Dorsally, male of these species can be separated by
major longitudinal pale orange stripes associated with a
pseudoscutum indicated by a pale stripe in A. yucumense (Fig. 1A), in contrast to pale creamy longitudinal
stripes and absence of pseudoscutum in A. dubitatum
(Fig. 1C). Ventrally, male coxal I spurs are separated by
a space narrower than external spur width in A. yucumense (Fig. 2D), and wider than external spur width in
A. dubitatum (Fig. 6D). Females of the two species can
be separated by the scutal large and deep punctations
evenly distributed in A. dubitatum (Fig. 7C), concentrated in the lateral fields in A. yucumense (Fig. 3C).
Ventrally, female coxal I spurs are unequivocally longer
in A. yucumense (Fig. 3D) than in A. dubitatum
(Fig. 7D), when the two species are observed side by
side. Finally, the adult capitulum and ventral idiosoma
of A. yucumense are widely dark brown colored (Fig
1A and B), while A. dubitatum is yellowish or light
brown colored (Fig. 1C and D).
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Fig. 5. Neighbor-joining phylogenetic tree of 16S rRNA partial sequences (393 nt) of A. yucumense and closely related
Ixodidae. Numbers on the nodes indicate bootstrap values from 1,000 replicates. Only bootstrap values >50 are shown.
Numbers in brackets are GenBank accession numbers. The corresponding 16S rRNA partial sequences of A. latum was used as
outgroup. Scale bar: number of substitutions per site.
Fig. 6. SEM of A. dubitatum male. (A) Dorsal capitulum (scale bar: 100 mm). (B) Ventral capitulum (scale bar: 100 mm).
(C) Scutum (scale bar: 400 mm). (D) Coxae I–IV (scale bar: 200 mm).
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35
Fig. 7. SEM of A. dubitatum female. (A) Dorsal capitulum (scale bar: 200 mm). (B) Ventral capitulum (scale bar: 200 mm).
(C) Dorsal idiosoma (scale bar: 400 mm). (D) Coxae I–IV (scale bar: 200 mm).
Recently in Argentina, Nava et al. (2014a,b)
described Amblyomma hadanii, a species closest
related to A. dubitatum and A. coelebs. Based on the
examination of two paratypes (1 male and 1 female) of
A. hadanii (CNC 1435), we could verify that this species is morphologically very closely related to A. yucumense, although males could be distinguished by the
scutal dark brown elevated spots lacking punctuations,
larger in A. yucumense (Fig. 2C) than in A. hadanii
[Figure 1 of Nava et al. (2014a,b)]. Females of these
two species can be separated by the genital aperture,
broadly V-shaped with short and bulging postero-lateral
flaps in A. yucumense (Fig. 3D), and U-shaped without
bulging postero-lateral flaps in A. hadanii [Figure 9 of
Nava et al. (2014a,b)]. A. coelebs are readily distinguished from A. yucumense though distinct scutal ornamentation patterns, due to the presence of large bright
red-orange patches in the scapular area and absence of
pseudoscutum in males of A. coelebs, and absence of a
dark brown central stripe ending at the posterior margin of the scutum in females of A. coelebs.
The nymphs of A. yucumense and A. dubitatum are
morphologically very similar to each other. The only
difference we could assign to separate them was the
extension of cervical groove, which is slightly longer in
A. dubitatum. For the present study, we re-examined
the same 10 A. dubitatum nymphal specimens recently
described by Martins et al. (2010), and determined
their cervical groove length: 0.49–0.55 (0.52 6 0.03).
Considering the scutal length values of these A. dubitatum nymphs (Martins et al. 2010), the cervical groove
length/scutal length ratio is 0.63–0.67 (0.65 6 0.02) for
A. dubitatum, higher than the ratio values here
reported for A. yucumense nymphs: 0.50–0.52
(0.51 6 0.01). In the dichotomous identification key
proposed for taxonomic identification of the nymphal
stage of 27 Amblyomma species from Brazil (Martins
et al. 2010), the nymph of A. yucumense would key
with A. dubitatum; therefore, these two species should
be separated through the cervical groove length/scutal
length ratio. In addition, these identifications should be
preferably associated with the identification of the adult
stage from the same collection site, as A. yucumense
and A. dubitatum seem to have distinct ecological
preferences. The nymph of A. hadanii is distinguished
from A. yucumense through the cervical groove,
which ends as shallow depression at the posterior
divergent half of the former species, in contrast to the
narrower and deeper posterior half in the later. The
nymph of A. coelebs is readily distinguished from A.
yucumense through scutal large and deep puncations,
present in both lateral and central fields of the former
species, while concentrated in the lateral fields of the
later.
36
JOURNAL OF MEDICAL ENTOMOLOGY
As recently reviewed by Guglielmone and Nava
(2014), the list of synonyms of A. dubitatum include
four names: Amblyomma lutzi Araga˜o (collected in the
state of Minas Gerais, Brazil), Amblyomma cooperi
Nuttall and Warburton (collected in Paraguay),
Amblyomma ypsilophorum Schulze (collected in the
state of Rio de Janeiro, Brazil) and Amblyomma cajennense chacoensis Ivancovich (collected in Chaco, northern Argentina). Although type specimens of A. lutzi, A.
cooperi, and A. cajennense chacoensis were not available for examination in the present study, we discard
the possibility that any of them could represent A.
yucumense because they were described as having typical features that separate A. dubitatum from A.
yucumense, e.g., female scutum with large and deep
punctations evenly distributed, female coxal I spurs
short, distance between male coxa I spurs wider than
the external spur width (Araga˜o 1908, Nuttall and Warburton 1908, Ivancovich 1973). In the description of A.
ypsilophorum, the drawings provided by Schulze
(1941) showed coxa I spurs compatible with A. yucumense, namely, distance between male coxa I spurs narrower than the external spur width. Owing to this
¨ bersee-Museum Bremen,
reason, we requested the U
where the types of A. ypsilophorum are deposited in
Germany, to provide us pictures of the type specimens.
By examining high-resolution pictures of A. ypsilophorum provided to us, our conclusions on coxa I spurs
were inconclusive, as the space between coxa I spurs
was nearly the same as the external spur width. On the
other hand, other features that separate A. dubitatum
from A. yucumense were observed, namely, absence of
pseudoscutum in the male scutum, and adult capitulum
and ventral idiosoma yellowish colored (Supp Figure 1
[online only]).
Adults of A. yucumense were observed inside the
Atlantic forest, questing on the vegetation 50–80 cm
above the soil in animal trails. These trails were not
used by capybaras (H. hydrochaeris) (data not shown),
which are the only known host species capable of sustaining A. dubitatum populations within its distribution
area (Nava et al. 2010). In a long-term study conducted
in another Atlantic forest reserve in southeastern Brazil
(Szabo´ et al. 2009), large numbers of A. incisum and A.
dubitatum adult ticks were collected from the vegetation; however, while the former was collected within
the dense forest (where there was no capybaras), the
later was collected exclusively in an open area characterized by few small trees, interspersed by bushes,
grasses and a swamp surrounding a dam, and with the
presence of capybaras. In addition, A. incisum was
found ambushing on vegetation mostly within
50–80 cm above soil, while A. dubitatum was never
found ambushing; it was collected only by dry ice traps.
Voucher specimens of these A. dubitatum ticks were
examined in the present study (CNC 927) and confirmed to represent typical morphotypes of A. dubitatum. Because the adult specimens of A. yucumense
were found ambushing on vegetation together with A.
incisum (sometimes the two species questing on the
same plant leaf) inside the forest, these two species
seem to have very similar ecological preferences. These
Vol. 52, no. 1
facts indicate that although A. yucumense and A. dubitatum are morphologically and genetically closely
related, they are distinct regarding their ecological
preferences.
Acknowledgments
We are very grateful to M. Haase and V. Lohrmann
¨ bersee-Museum Bremen, Germany) for providing high(U
resolution pictures of the type specimens of A. ypsilophorum,
to the staff of the “Parque Estadual do Turvo” for providing
invaluable logistic support for our field work, and to Gustavo
Fernochi for his technical support in editing photographs.
This work was financially supported by the Fundac¸a˜o de
Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico
(CNPq), and Coordenadoria de Apoio a Pesquisa e Desenvolvimento (CAPES), and has been authorized by the “Instituto
Chico Mendes de Conservac¸a˜o da Biodiversidade” (authorization SISBIO 38502-1) and the Ethical Committee in Animal Research of the Faculty of Veterinary Medicine of the
University of Sa˜o Paulo (protocol 2908/2013).
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Received 31 May 2014; accepted 2 October 2014.