Syst. Biol. 52(3):296–310, 2003
c Society of Systematic Biologists
Copyright °
ISSN: 1063-5157 print / 1076-836X online
DOI: 10.1080/10635150390196957
The Value of Idiosyncratic Markers and Changes to Conserved tRNA Sequences
from the Mitochondrial Genome of Hard Ticks (Acari: Ixodida: Ixodidae)
for Phylogenetic Inference
ANNA M URRELL, NICK J. H. CAMPBELL, AND S TEPHEN C. B ARKER
Department of Microbiology and Parasitology, Institute for Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia;
E-mail: [email protected] (A.M.)
Analyses of DNA sequences have revolutionized our
view of evolution at all levels of the tree of life.
These analyses have provided resolution of many longstanding phylogenetic questions. For example, the phylogenetic position of the phylum Hemichordata within
the deuterostomes was controversial until recently. Analyses of nucleotide sequences from 18S ribosomal RNA
(rRNA) genes (Halanych, 1995) and entire mitochondrial genomes (Castresana et al., 1998) have now shown
unambiguously that the hemichordates are the sister
group of the echinoderms. However, for many reasons,
DNA sequences have not been a panacea (for a summary, see Rokas and Holland, 2000). For example, despite the availability of entire sequences of mitochondrial genomes from all major lineages of vertebrates,
there are still conflicting hypotheses about the relationships among these lineages (Curole and Kocher, 1999).
Analyses of DNA sequences from lineages that diverged
recently have even been misleading in some cases. For
example, Black and Roehrdanz (1998) found that the
sequences of the mitochondrial genomes of two ticks
(Ixodida: Chelicerata) placed the ticks with the honeybee (Apis mellifera: Hymenoptera: Insecta) to the exclusion of other insects. This result was probably an artifact of bias in these genomes towards amino acids
encoded by AT-rich codons (Black and Roehrdanz,
1998).
Avise (1994) first used the term idiosyncratic molecular markers. We use this term to describe the markers
Avise referred to plus the rare genomic changes (RGCs)
of Rokas and Holland (2000). Idiosyncratic molecular
markers are a source of phylogenetic information free
from some of the problems associated with DNA sequences. Idiosyncratic molecular markers are so unusual
it is unlikely that they evolved more than once in a lineage. Thus, these markers almost invariably diagnose
monophyletic groups. In cladistic terms, idiosyncratic
markers are the derived character states of highly conserved molecular characters.
Rearrangements of genes in animal mitochondrial
genomes are perhaps the best known idiosyncratic
molecular markers (e.g., Boore and Brown, 1998). For example, the translocation of a mitochondrial tRNA gene
links crustaceans and insects to the exclusion of other
arthropods (Boore et al., 1998). Rearrangements within
genes can also be phylogenetically informative (e.g.,
Moum et al., 1994). Changes in the genetic code of mitochondrial genes are yet another type of idiosyncratic
marker. For example, in vertebrate mitochondrial DNA
(mtDNA), AGA and AGG are stop codons, whereas
in other bilateral animals these triplets code for serine
(Wolstenholme, 1992). Other types of idiosyncratic markers include changes in the secondary structure of tRNAs
and rRNAs and features of the noncoding sequences,
such as the presence of repeats (Wolstenholme, 1992;
Avise, 1994; Macey et al., 2000; Rokas and Holland, 2000).
Their rarity makes idiosyncratic molecular markers very
useful but, obviously, only in those clades in which they
occur.
Here, we explore the potential of candidate idiosyncratic mitochondrial markers and changes to typically
conserved nucleotides in tRNAs as an additional source
of information for phylogenetic inference in a major
group of arthropods, the hard ticks (Ixodidae: Ixodida:
Chelicerata). There are two main groups of hard ticks:
the Prostriata (one genus Ixodes, >240 species) and the
Metastriata (13 genera, >430 species) (Keirans, 1992;
Camicas et al., 1998). We chose hard ticks as a model
group because their phylogeny has been studied extensively with both morphological and nucleotide sequence
data sets (Black and Piesman, 1994; Crampton et al.,
1996; Klompen et al., 1996, 1997, 2000; Black et al., 1997;
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Abstract.— Idiosyncratic markers are features of genes and genomes that are so unusual that it is unlikely that they evolved
more than once in a lineage of organisms. Here we explore further the potential of idiosyncratic markers and changes to
typically conserved tRNA sequences for phylogenetic inference. Hard ticks were chosen as the model group because their
phylogeny has been studied extensively. Fifty-eight candidate markers from hard ticks (family Ixodidae) and 22 markers
from the subfamily Rhipicephalinae sensu lato were mapped onto phylogenies of these groups. Two of the most interesting
markers, features of the secondary structure of two different tRNAs, gave strong support to the hypothesis that species
of the Prostriata (Ixodes spp.) are monophyletic. Previous analyses of genes and morphology did not strongly support
this relationship, instead suggesting that the Prostriata is paraphyletic with respect to the Metastriata (the rest of the hard
ticks). Parallel or convergent evolution was not found in the arrangements of mitochondrial genes in ticks nor were there
any reversals to the ancestral arthropod character state. Many of the markers identified were phylogenetically informative,
whereas others should be informative with study of additional taxa. Idiosyncratic markers and changes to typically conserved
nucleotides in tRNAs that are phylogenetically informative were common in this data set, and thus these types of markers
might be found in other organisms. [Evolution; idiosyncratic markers; Ixodidae; phylogeny; ticks.]
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MURRELL ET AL.—IDIOSYNCRATIC MARKERS, CONSERVED T RNAS , AND TICK PHYLOGENY
M ATERIALS AND M ETHODS
Searching for Candidate Markers in Mitochondrial Genomes
In the mitochondrial genomes studied, we targeted
regions that contain tRNAs and noncoding sequences;
these regions occur between the larger protein-coding
and rRNA genes (Fig. 1). The mitochondrial genomes of
metastriate ticks have been rearranged substantially relative to the ancestral arrangement that persists in prostriate ticks and other chelicerates (Black and Roehrdanz,
1998; Campbell and Barker, 1998). Therefore, some of the
regions studied in metastriate ticks were different from
those studied in prostriate ticks and other chelicerates
(Fig. 1). The regions targeted in metastriate ticks were as
follows: 1 = ND3–ND1, 2 = ND1–16S, 3 = 12S–ND5, 4 =
cob–ND2, and 5 = ND2–CO1; regions targeted in prostriate ticks and the outgroups (other chelicerates) were
regions 2 and 5 plus two others: 6 = ND3–ND5 and 7 =
12S–ND2 (ND = NADH dehydrogenase subunit; CO1 =
cytochrome c oxidase subunit 1; cob = cytochrome b
apoenzyme; CR = noncoding control region; 12S = small
subunit rRNA; 16S = large subunit rRNA) (Fig. 1). We
looked for rearrangements of genes, unusual features
of secondary structure and sequences of tRNAs, repeated sequences, and large (>10 nucleotides) insertions
FIGURE 1. The seven mitochondrial regions searched for idiosyncratic markers and changes in typically conserved tRNA sequences in
metastriate and prostriate ticks and other chelicerate arthropods (outgroups). Single-letter amino acid codes indicate the genes of the tRNAs
that specify those amino acids (after Sprinzl et al., 1998). Genes are transcribed from left to right except those genes whose labels are underlined,
which are transcribed from right to left.
Downloaded from http://sysbio.oxfordjournals.org/ by guest on November 8, 2016
Mangold et al., 1998; Dobson and Barker, 1999; Klompen,
1999; Norris et al., 1999). We identified 58 candidate
markers from features of the mitochondrial genomes of
hard ticks and their relatives. We scored these markers for hard ticks (family Ixodidae) and outgroups and
then mapped the markers onto a phylogeny of the hard
ticks that was independently inferred from molecular
and morphological data sets (Klompen et al., 2000). To
examine the potential of these candidate mitochondrial
markers for recently diverged groups, we also studied
the distribution of 22 markers in a subfamily of hard
ticks, the Rhipicephalinae. The phylogeny of this group
has also been studied extensively (Zahler et al., 1997;
Borges et al., 1998; Crosbie et al., 1998; Murrell et al.,
1999, 2000, 2001a, 2001b). Our data suggest that like other
phylogenetic markers idiosyncratic mitochondrial markers and changes to typically conserved tRNA sequences
may undergo reversals or may evolve convergently or
in parallel; however, homoplasy is rare in these markers. Also phylogenetically informative idiosyncratic mitochondrial markers and changes to typically conserved
tRNA sequences might be found in any group of interest
if one looks for them. The same sequences can be used
to both infer sequence-based phylogenies and to look for
these markers.
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SYSTEMATIC BIOLOGY
and deletions (indels). Features that differed from the
putative ancestral condition for arthropods, i.e., that
found in most or all arthropods studied previously
(Sprinzl et al., 1998; Boore, 1999), were selected as candidate markers (idiosyncratic markers and changes to
typically conserved tRNA sequences). These candidate
markers were then scored for all available hard ticks and
outgroups.
DNA Methods
Ticks and outgroups were preserved in 70% ethanol.
We extracted DNA from ticks with standard phenol–
chloroform techniques (Hillis et al., 1996). Mitochondrial
region 2 (see Fig. 1) was amplified with the primers N1-J12571 (50 -TTT TAT TTG GCC CTT TTC GAA-30 ) and LRN-12951 (50 -WWG WTT GCG ACC TCG ATG TTG-30 ),
and region 5 was amplified with the primers TW-J-1302
(50 -TAA GTT AAC AAA ACT AGT AGC CTT CAA AG30 ) and C1-N-1540 (50 -TAC TTA ATC CCA ATA TTC CTG
CTC A-30 ). For the Metastriata, the following primers
were used: N3-J-5825 (50 -TTT TTW ATT TTT GAT GTW
GAA AT-30 ) and N1-N-11819 (50 -AGT TGA TAA TAW
TAG CWT GAA-30 ) for region 1, TI-J-36 (50 -AAA AAG
GAT TAT CTT GAT AGG-30 ) and N5-N-7060 (50 -TTG
GGG GGT TTT ATT CAA AGG AT-3) for region 3, and
CB-J-11338 (50 -TCA TAT CCA ACC TGA ATG ATA TTT30 ) and TM-N-193 (50 -TGG GGT ATG AAC CCA GTA
GC-30 ) for region 4. For the prostriate ticks and other chelicerates, we amplified region 6 with primers N3-J-5825
and TF-N-6385 (50 -AAT TAA ARA ARC ATT ACA CTG
AA-30 ) and region 7 with the primers SR-J-14766 (50 -GTA
TRA CCG CGR WTG CTG GCA C-30 ) and TM-N-193.
Primers were named according to where they anneal by
gene, strand, and 30 base number relative to the sequence
of Drosophila yakuba (after Simon et al., 1994). Target regions were amplified by PCR with Elongase (Gibco: BRL)
as recommended by the manufacturer, with the following cycling conditions: 94◦ C for 1 min, 35 cycles of 95◦ C
for 30 sec, 40◦ C for 30 sec, and 68◦ C for 1 min, and a final
stage at 68◦ C for 7 min.
PCR fragments were purified with Wizard columns
(Promega) and sequenced with the primers listed above
plus LR-N-12868 (50 -ACR WGA TYT GAG TTC AGA
CC-30 ), C1-N-1576 (50 -GGT TGA CCT AGY TCA RST
CGA AT-30 ), TN-J-6170 (50 -AGA GGC GTT TCA CTG
Data Analysis
Protein-coding and rRNA sequences were identified
by aligning them with sequences from B. microplus
(Campbell and Barker, 1999) and I. hexagonus (Black and
Roehrdanz, 1998) using Clustal W (Thompson et al.,
1994). The tRNA genes were identified from their secondary structure and anticodons: the secondary structure of the tRNA gene sequences was inferred by hand
according to the model of Kumazawa and Nishida (1993)
and are available on request (from A.M.). Idiosyncratic
markers and changes to typically conserved tRNA sequences were mapped onto phylogenies from morphology and DNA sequences (see Klompen et al., 2000, for
the family Ixodidae; see Murrell et al., 2001b, for the subfamily Rhipicephalinae) with MacClade 3.08 (Maddison
and Maddison, 1999). Klompen et al. (2000) inferred a
phylogeny using 106 informative morphological characters and 614 informative molecular characters (from
the 18S rRNA, 28S rRNA, and 16S rRNA genes), and
Murrell et al. (2001b) inferred a phylogeny using 30 informative morphological characters and 591 informative
molecular characters (from the 12S rRNA, CO1, internal
transcribed spacer 2, and 18S rRNA genes). We also used
MacClade to calculate consistency indices, retention indices, and rescaled consistency indices for the whole data
set. All indices were calculated after parsimony uninformative characters had been removed. Phylogenies were
inferred using maximum parsimony with PAUP 4.0b10
(Swofford, 2002).
R ESULTS
Overview of Candidate Mitochondrial Markers
We divided the candidate markers we found between
two matrices. The characters in data matrix A vary
among lineages of the family Ixodidae (hard ticks) and
outgroups (Table 2; Appendix 1; see Fig. 2). Data matrix
A has 58 candidate markers that can be viewed as 58 derived character states of 46 molecular characters. Of the
46 characters, 4 were features of the arrangement of genes
in mitochondrial genomes, 2 were features of the control
region, 1 was a repeated nucleotide sequence, 4 were
changes to the secondary structure of tRNAs, 20 were
correlated sequence changes in the secondary structure
of tRNAs, and 15 were changes at nucleotide positions
in tRNAs that are highly conserved among other arthropods (Table 2).
The characters in data matrix B vary among lineages of
the subfamily Rhipicephalinae and outgroups (Table 3;
Appendix 2; see Fig. 3). Data matrix B has 22 candidate
markers (derived character states) and 19 characters. Of
the 19 characters, 4 were repeated nucleotide sequences,
1 was a secondary structural change to a tRNA, 7 were
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Taxa Studied
We attempted to score each marker for at least one
representative of each lineage of hard ticks and each
lineage of the subfamily Rhipicephalinae, but this was
not always possible because we could not amplify
and sequence some regions for some species. Our aim,
therefore, was to represent each lineage, but not necessarily with the same species for each marker. Published data for hard ticks and other chelicerates were
also used (Table 1). Some large gene rearrangements,
which have been described before, were scored using
only polymerase chain reaction (PCR) results (Black and
Roehrdanz, 1998; Campbell and Barker, 1998).
TTA AT-30 ), and S1-J-6266 (50 -ATG AAT AAT TCA TTT
AAT CTT-30 ). We used 100–200 ng of purified PCR product in each cycle-sequencing reaction (DyeDeoxy terminator; Perkin Elmer–Applied Biosystems). An ABI
377 sequencer was used to resolve the sequencing
fragments.
2003
MURRELL ET AL.—IDIOSYNCRATIC MARKERS, CONSERVED T RNAS , AND TICK PHYLOGENY
299
TABLE 1. Species of ticks and other chelicerate arthropods (outgroups) studied, mitochondrial regions studied (see Fig. 1), and GenBank
accesssion numbers. Names of taxa in the Ixodidae follow the recommendations of Klompen et al. (2000) and Murrell et al. (2001b). Asterisk
indicates taxa listed in Tables 2 and 3 and Appendices 1 and 2.
Taxon
Arachnida
Acari (ticks and mites)
Parasitiformes
Ixodida (ticks)
Ixodidae (hard ticks)
Metastriata
∗
Rhipicephalinae s.l.b
∗
Boophilus
B. annulatus
B. decoloratus
∗
Dermacentor
D. andersoni
D. marginatus
D. reticulatus
D. variabilis
∗
Hyalomma
H. aegyptium
H. dromedarii
H. marginatum
H. truncatum
Nosomma monstrosum
Rhipicentor nuttalli
∗
Rhipicephalus
∗
Rhipicephalus appendiculatus group
R. appendiculatus
R. zambeziensis
R. compositus
∗
R. evertsi group
R. evertsi
∗
R. pulchellus group
R. maculatus
R. pulchellus
∗
R. pravus group
R. punctatus
R. pravus
∗
R. sanguineus group
R. sanguineus
R. turanicus
R. simus
R. zumpti
Haemaphysalinae
∗
Haemaphysalis
H. humerosa
H. inermis
H. leporispalustris
H. longicornis
Amblyomminae
∗
Amblyomma
A. americanum
A. cajennense
A. fimbriatum
GenBank no./Reference
1
(1, 3, 4)
(3, 4)
1, 2, 3
(3, 4)
1
1
1, 3, 4
2, 5
AY059196
Black and Roehrdanz (1998)
Campbell and Barker (1998)
AY059197–59199
Campbell and Barker (1998)
AY059200
AY059201
AF110618, AF110621, AF110612/Campbell and Barker (1998)
AF110619, AF110613/Campbell and Barker (1999)
(1, 3, 4)
1, 5
2
(3, 4)
1, 2
(1, 3, 4)
(3, 4)
Black and Roehrdanz (1998)
AY059251, 59252
AY059253
Campbell and Barker (1998)
AY059254, 59255
Black and Roehrdanz (1998)
Campbell and Barker (1998)
1, 2
(3, 4)
(1, 3, 4)
(3, 4)
1, 3, 4, 5
(3, 4)
(3, 4)
2
AY059264–59269
Campbell and Barker (1998)
Black and Roehrdanz (1998)
Campbell and Barker (1998)
AY059270–59273
Campbell and Barker (1998)
Campbell and Barker (1998)
AY059234
1, 2, 3, 4
(1, 3, 4)
(3, 4)
2
2
AY059214–59217
Black and Roehrdanz (1998)
Campbell and Barker (1998)
AY059225
AY059218
1, 2, 3
AY059219–59221
2
1, 2, 3
AY059222
AY059228–59230
1, 2, 3
3
AY059231–59233
AY059227
1, 2, 3, 4, 5
2
2
2
AF081829/Black and Roehrdanz (1998)
AY059224
AY059223
AY059226
1, 3, 4, 5
(3, 4)
(1, 3, 4)
(1, 3, 4)
1, 2, 3, 5
(3, 4)
AY059256–59259
Campbell and Barker (1998)
Black and Roehrdanz (1998)
Black and Roehrdanz (1998)
AY059260–59263
Campbell and Barker (1998)
(1, 3, 4)
(1, 3, 4)
2, 3, 4, 5
Black and Roehrdanz (1998)
Black and Roehrdanz (1998)
AY059189–59192
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B. geigyi
B. kohlsi
B. microplus
Regions studieda
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SYSTEMATIC BIOLOGY
TABLE 1. Continued
Regions studieda
Taxon
A. glebopalma
A. hebraeum
A. latum
A. maculatum
A. varanensis
A. variegatum
A. vikirri
∗
Bothriocroton
B. concolor
B. undatum
I. hexagonus
I. pilosus
I. uriae
∗
I. tasmani group
I. holocyclus
I. simplex
I. tasmani
∗
Argasidae (soft ticks)
Argasinae
Argas lagenoplastis
Ornithodorinae
Carios capensis
Otobius megnini
Holothyrida (mites)
Allothyrus sp.
∗
Mesostigmata (mites)
Cercomegistus sp.
Sejus sp.
Opilioacariformes (mites)
∗
Opilioacarida
Undescribed Opilioacarid species
Scorpiones (scorpions)
Liocheles waigiensis
∗
Araneae (spiders)
Nephila plumipes
Merostomata (horseshoe crab)
Limulus polyphemus
a
b
(1, 3, 4)
1, 2, 3, 4, 5
(3, 4)
(1, 3, 4)
(1, 3, 4)
(1, 3, 4)
(1, 3, 4)
1, 2, 3, 4, 5
(3, 4)
Black and Roehrdanz (1998)
AY059170–59174
Campbell and Barker (1998)
Black and Roehrdanz (1998)
Black and Roehrdanz (1998)
Black and Roehrdanz (1998)
Black and Roehrdanz (1998)
AY059177–59181
1, 2, 3, 5
(3, 4)
1, 2, 5
(3, 4)
AY059185–59188
Campbell and Barker (1998)
AY059193–59195
Campbell and Barker (1998)
(7)
2, 5, 6, 7
(7)
2, 5, 6, 7
(7)
(7)
Black and Roehrdanz (1998)
AY059274–59281
Campbell and Barker (1998)
AF081828/Black and Roehrdanz (1998)
Campbell and Barker (1998)
Black and Roehrdanz (1998)
5, 6, 7
(7)
(7)
(7)
(7)
5, 7
2, 6, 7
(7)
(7)
AY059282–59289
Black and Roehrdanz (1998)
Campbell and Barker (1998)
Campbell and Barker (1998)
Black and Roehrdanz (1998)
AY059175, 59176
AY059239–59247
Campbell and Barker (1998)
Campbell and Barker (1998)
5, 7
AY059182–59184
2, 7
2, 5
AY059248–59250
AY059235–59238
2, 5, 6
AY059206–59213
2, 7
(7)
AF059202–59205
Campbell and Barker (1998)
(7)
Campbell and Barker (1998)
2, 5, 6, 7
AF002650, AF002653, AF002646, AF002652,
AF002651/Staton et al. (1997)
Regions in parentheses were scored based on results of PCR analyses and were not actually sequenced.
Includes the genus Hyalomma; see Murrell et al. (2001b).
correlated sequence changes in the secondary structures
of the tRNAs, 5 were changes at nucleotide positions in
tRNAs that are highly conserved among other arthropods, and 2 were large insertions of nucleotides between
genes (indels). Six of these characters (5, 6, 8, 9, 10, and
14; Table 3) were also in data matrix A (characters 10, 13,
23, 25, 24, and 34, respectively; Table 2).
Phylogeny of Hard Ticks and the Evolution of Idiosyncratic
and Conserved tRNA Markers
Klompen et al. (2000) used a combined data approach
to infer a well-supported and well-resolved phylogeny
of the hard ticks (family Ixodidae), and this tree has
been widely accepted by workers in tick systematics. We
mapped our 58 candidate markers onto this phylogeny
(Fig. 2). Our unpublished analyses of the Klompen et al.
(2000) molecular data sets showed that there was not
significant heterogeneity of nucleotide composition (chisquare test) across taxa for the variable sites nor were
any other systematic biases apparent that might have affected the phylogenies and biased our analyses. Ten of
the 58 markers mapped to the base of the tree (branch 1;
Fig. 2; Table 2). These markers probably diagnose monophyletic groups (yet to be identified) that contain all
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Prostriata
∗
Ixodes s.s.
I. affinis
I. auritulus
GenBank no./Reference
2003
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MURRELL ET AL.—IDIOSYNCRATIC MARKERS, CONSERVED T RNAS , AND TICK PHYLOGENY
TABLE 2. The 46 characters from the mitochondrial genomes of ticks of the family Ixodidae and outgroups in data matrix A (Appendix 1).
Putative ancestral state (0) and derived states (1, 2, 3) for arthropods and region of the mitochondrial genome are indicated (see Fig. 1). Nucleotide
positions and the secondary structures of tRNAs are described with the terminology of Sprinzl et al. (1998).
Character no.
Character descriptiona
Region of genomeb
Gene arrangements
2
3
4
Position of ∼5-Kilobase block of genes, tRNA(F) to tRNA(S)(UCN): 0 = between tRNA(E) and
ND1; 1 = between tRNA(Q)and tRNA(L)(CUN) [7]
Position of tRNA(C): 0 = between tRNA(W) and tRNA(Y); 1, between CR2 and tRNA(M) (7)
Strand encoding C: 0 = majority; 1 = minority [7]
Position of tRNA(L)(CUN): 0 = between tRNA(L)(UUR) and 16S; 1 = between tRNA(S)(UCN) and CR2 [7]
5
6
Number of CRs: 0 = one; 1 = two [7]
Size of CR: 0 = long, ≥500 nucleotides; 1 = short, ∼300 nucleotides [1/2]
7
∼25-base pair (bp) repeated sequence at 50 end of ND1 and 50 end of 16S: 0 = absent; 1 = present [7]
1, 2
8
9
10c
11
tRNA structural changes
D-arm in tRNA(C): 0 = present; 1 = absent [7]
Number of nucleotides between D- and AC-arms in tRNA(C): 0 = one; 1 = two [26]
Length of T-stem in tRNA(N): 0 = long, ≥4 bp [22]; 1 = short, 2 or 3 bp [1]
Number of nucleotides between AA- and D-stems in tRNA(C): 0 = two [26]; 1 = one [3]
4, 5
4, 5
1, 6
2, 4
12
13c
14
15
16
17
18
19
20
21
22
23c
24c
25c
26
Changes to typically conserved nucleotides in tRNA
Position 32 in tRNA(I): 0 = T; 1 = C [17]
Position 8 in tRNA(Q): 0 = T; 1 = G [12, 15]
Position 22 in tRNA(F): 0 = G; 1 = A [1]
Position 65 in tRNA(I): 0 = C; 1 = T [15]
Position 33 in tRNA(A): 0 = T; 1 = C [16]
Position 11 in tRNA(I): 0 = C; 1 = T [21]
Position 26 in tRNA(L)(CUN): 0 = A; 1 = G [13, 26, 21]
Position 30 in tRNA(L)(CUN): 0 = A; 1 = G [19]
Position 4 in tRNA(N): 0 = A; 1 = G [25]
1st nucleotide of anticodon of tRNA(S)(AGN): 0 = G; 1 = T [5]
Position 10 in tRNA(Y): 0 = G; 1 = A [17, 23]
Position 48 in tRNA(L)(UUR): 0 = T; 1 = G [19]; 2 = A [22, 28]; 3 = C [22]
Position 23 in tRNA(N): 0 = A; 1 = C [21, 22, 25]
Position 46 in tRNA(Q): 0 = G; 1 = A [12]
Position 8 in tRNA(L)(CUN): 0 = T; 1 = A [28]
3, 7
3, 7
3, 6
3, 7
1, 6
3, 7
2, 4
2, 4
1, 6
1, 6
5
2
1, 6
3, 7
2, 4
27
28
29
30
31
32
33
34c
35
36
37
38
39
40
41
42
43
44
45
46
Correlated sequence changes in tRNAs
2nd bp (positions 11, 24) in D-stem of tRNA(F): 0 = C-G; 1 = T-A [1]
1st bp (positions 49, 65) in T-stem of tRNA(A): 0 = A-T; 1 = G-C [1]
1st bp (positions 27, 43) in AC-stem of tRNA(C): 0 = T-A; 1 = A-T [17]
6th bp (positions 6, 67) in AA-stem of tRNA(E): 0 = T-A [6]; 1 = A-T [1]
1st bp (positions 27, 43) in AC-stem of tRNA(L)(UUR): 0 = A-T; 1 = T T [26]
2nd bp (positions 28, 42) in AC-stem of tRNA(L)(UUR): 0 = T-A [26]; 1 = A-T [1]; 2 = G-C [3/4]
5th bp (positions 31, 39) in AC-stem of tRNA(L)(CUN): 0 = A-T; 1 = T-A [19]
2nd bp (positions 50, 64) in T-stem of tRNA(N): 0 = A-T; 1 = T-A [10]; 2, T-T [22]
5th bp (positions 5, 68) in AA-stem of tRNA(R): 0 = A-T; 1 = G-C [19]
1st bp (positions 10, 25) in D-stem of tRNA(R): 0 = G-C; 1 = A-T [19]
2nd bp (positions 50, 64) in T-stem of tRNA(R): 0 = G-C; 1 = A-T [5]
7th bp (positions 7, 66) in AA-stem of tRNA(R): 0 = G-C; 1 = A-T [5]
5th bp (positions 5, 68) in AA-stem of tRNA(Q): 0 = T-A; 1 = C-G [26]
3rd bp (positions 29, 41) in AC-stem of tRNA(S)(AGN): 0 = A-T [5]; 1 = G-T [1]
1st bp (positions 27, 43) in AC-stem of tRNA(Q): 0 = T-A; 1 = A-T [5]
2nd bp (positions 28, 42) in AC-stem of tRNA(Q): 0 = T-A; 1 = A-T [7]
6th bp (positions 6, 67) in AA-stem of tRNA(R): 0 = A-T; 1 = T-A [2/3/4]; 2, C-G [14]
4th bp (positions 30, 40) in AC-stem of tRNA(S)(AGN): 0 = A-T [16]; 1 = C-G [1]
5th bp (positions 31, 39) in AC-stem of tRNA(S)(AGN): 0 = G-C; 1 = A-T [6]
2nd bp (positions 28, 42) in AC-stem of tRNA(L)(CUN): 0 = T-A [7/8]; 1 = C-G [1]
3, 6
1, 6
4, 5
1, 6
2
2
2, 4
1, 6
1, 6
1, 6
1, 6
1, 6
3, 7
1, 6
3, 7
3, 7
1, 6
1, 6
1, 6
2, 4
1
(1, 3, 4, 6, 7)
(4, 5, 7)
4, 5
(2, 4)
Changes to the control region (CR)
(3, 4, 7)
(3, 4, 7)
a
Number in brackets at the end of each description identifies the branch(es) that the marker mapped to in the phylogeny of the hard ticks (Fig. 2). Where it is
unclear which branch a marker mapped to (because of missing data), the alternative branches are both given.
b
Regions in parentheses were scored based on results of PCR analyses.
c
Character also present in data matrix B (Table 3; Appendix 2).
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Gene duplications
FIGURE 2. Phylogeny of the major lineages of hard ticks and other chelicerate arthropods (outgroups) (after Klompen et al., 2000) with markers from data matrix A (Table 2; Appendix 1)
mapped onto it. Bold lines indicate relationships that had >70% bootstrap support in the Klompen et al. (2000) study (some tip branches are bold because those branches represent multiple
taxa). Tree length = 45; consistency index = 0.82; retention index = 0.89; rescaled consistency index = 0.73. Vertical lines labeled P and M indicate prostriate ticks and metastriate ticks,
respectively. Amblyomma s.l. = Amblyomma s.s. plus the non-Australian species of Aponomma (after Klompen et al., 2000, 2002). Numbers above branches and taxa identify those branches
and taxa in the text and tables. Because of missing data, in some cases it was unclear to which of two or more branches a marker mapped (see Appendix 1). Thus, the possible branches that
a marker mapped to are indicated with arrows.
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TABLE 3. The 19 characters from the mitochondrial genomes of ticks of the subfamily Rhipicephalinae in data matrix B (Appendix 2). Putative
ancestral state (0) and derived states (1, 2) for arthropods and region of the mitochondrial genome are indicated (see Fig. 1). Nucleotide positions
and the secondary structures of tRNAs are described with the terminology of Sprinzl et al. (1998).
Character no.
Character descriptiona
Region of genome
Duplications
1
2
3
4
126–131-nucleotide repeated sequence at the 30 end of ND1: 0 = absent; 1 = present [21]
2nd copy of 24 nucleotides at 30 end of E and 50 end of tRNA(E): 0 = absent; 1 = present [21]
174-nucleotide repeated sequence at junction of tRNA(E) and ND1: 0 = absent; 1 = present [21]
40-nucleotide repeated sequence at junction of tRNA(E) and ND1: 0 = absent; 1 = present [26]
5b
Length of T-stem in tRNA(N): 0 = long, ≥4 base pairs (bp) [16]; 1 = short, 2 or 3 bp
1
1
1
1
tRNA structural changes
1
Changes to typically conserved nucleotides in tRNAs
Position 8 in tRNA(Q): 0 = T; 1 = G [2/3]
Position 45 in tRNA(A): 0 = A; 1 = T [15]; 2 = G [17]
Position 48 in tRNA(L)(UUR): 0 = T; 1 = C [14]; 2 = A [16]
Position 46 in tRNA(Q): 0 = G; 1 = A [2/3]
Position 23 in tRNA(N): 0 = A; 1 = C [7]
3
1
2
3
1
11
12
13
14b
15
16
17
Correlated tRNA sequence changes
1st bp (positions 10, 25) in D-stem of tRNA(A): 0 = T-A; 1 = A-T [15]
5th bp (positions 31, 39) in AC-stem of tRNA(E): 0 = A-T; 1 = T-T [4]
1st bp (positions 27, 43) in AC-stem of tRNA(E): 0 = T-A; 1 = T-C [26]
2nd bp (positions 50, 64) in T-stem of tRNA(N): 0 = A-T; 1 = T-T [12]
3rd bp (positions 29, 41) in AC-stem of tRNA(S)(AGN): 0 = A-T; 1 = G-T [10]
4th bp (positions 13, 22) in D-stem of tRNA(Q): 0 = T-G; 1 = T-T [2/3]; 2 = T-C [21]
1st bp (positions 27, 43) in AC-stem of tRNA(S)(AGN): 0 = T-A; 1 = A-G [21]
1
1
1
1
1
3
1
18
19
24-nucleotide insertion between ND3 and tRNA(A): 0 = absent; 1 = present [26]
15-nucleotide insertion between tRNA(A) and tRNA(R): 0 = absent; 1 = present [15]
Indels
1
1
a
Number in brackets at the end of each description identifies the branch(es) that that marker mapped to in the phylogeny of the Rhipicephalinae (Fig. 2). Where
it is unclear which branch a marker mapped to (because of missing data), the alternative branches are both given.
b
Character also present in data matrix A (Table 2; Appendix 1).
FIGURE 3. Phylogeny of the major lineages of hard ticks of the subfamily Rhipicephalinae, after Murrell et al. (2001b), with markers from
data matrix B mapped onto it (Table 2; Appendix 1). Bold lines indicate relationships that had >70% bootstrap support in the Murrell et al.
(2001b) study (some tip branches are bold because those branches represent multiple taxa). Tree length = 15; consistency index = 0.86; retention
index = 0.91; rescaled consistency index = 0.78. Vertical line labeled Rp indicates the genus Rhipicephalus s.l. (=Rhipicephalus s.s. + Boophilus;
as recommended by Murrell et al., 2001b). Numbers above branches and taxa identify those branches and taxa in text and tables. Because of
missing data (see Appendix 2), in some cases it is unclear to which of two or more branches a marker mapped. Thus, the possible branches that
a marker mapped to are indicated with arrows.
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6b
7
8b
9b
10b
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SYSTEMATIC BIOLOGY
tRNAs (derived states of characters 30 and 45) from
two different tRNAs, tRNA-Glu and tRNA-Ser(AGN),
respectively. In contrast, the monophyly of the Prostriata had little support in the combined data analysis of
Klompen et al. (2000). Some of the data sets analyzed by
Klompen et al. (2000) indicate monophyly of the Ixodes
tasmani group plus the Metastriata, to the exclusion of the
other species of Ixodes. These two markers strengthen
the case for the conventional division of hard ticks
into two monophyletic lineages, the Prostriata and the
Metastriata.
One candidate marker is particularly noteworthy: a
change at a nucleotide position in tRNA-Gln that is
highly conserved among other arthropods (derived state
of character 13; Table 2). This change seems to have
occurred independently in two of the four main lineages of the Metastriata, the Rhipicephalinae s.l. (i.e.,
Rhipicephalinae s.s. + Hyalomma) and the Bothriocroton
species (branches 12 and 15, respectively; Fig. 2). This
marker may be a synapomorphy for the Rhipicephalinae
s.l. and the genus Bothriocroton, and this new hypothesis should be tested with sequences from a wider range
of ticks. If this marker is limited to a monophyletic subset of species in either or both of the Rhipicephalinae s.l.
and the Bothriocroton species, this finding would indicate
that this marker has evolved convergently. However, if
this marker is found in all ticks from Rhipicephalinae s.l.
and Bothriocroton, this finding would be evidence of their
monophyly to the exclusion of Haemaphysalis and Amblyomma. Klompen et al. (2000) found little support for any
of the possible arrangements of the four main lineages
of the Metastriata.
The candidate markers were used to infer a maximum
parsimony phylogeny (Fig. 4A). This phylogeny indicated support for monophyly of the Ixodida (1 change),
monophyly of the Prostriata (5 changes), monophyly
of the Metastriata (14 changes), and a sister-group
relationship between the genus Bothriocroton and the
Rhipicephalinae (1 change).
Phylogeny of Rhipicephaline Ticks and the Evolution
of Idiosyncratic and Conserved tRNA Markers
A recent combined data analysis of nuclear and mitochondrial DNA sequences and of morphology gave
a well-supported and well-resolved best phylogeny of
the Rhipicephalinae s.l. (Murrell et al., 2001b), and we
mapped our candidate markers onto this tree (Fig. 3).
Our unpublished analyses of the Murrell et al. (2001)
molecular data sets showed that there was not significant heterogeneity of nucleotide composition (chi-square
test) across taxa for the variable sites nor were any other
systematic biases apparent that might have affected the
phylogenies and biased our analyses. Five of the 22 candidate markers in data matrix B were found in species
from more than one lineage of rhipicephaline ticks. Thus,
excluding homoplasy, each of these five markers apparently diagnoses a monophyletic group (branches 2–8;
Fig. 3; Tables 1, 3). Seventeen candidate markers were
found in species from only one lineage; each of these
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of the lineages we studied plus other lineages. Some
of these markers, however, have undergone reversals.
Nineteen of the 58 markers were found in more than
one lineage. Thus, excluding convergence, each of these
markers diagnoses a monophyletic group of lineages
(branches 2–9; Fig. 2; Table 2). Twenty-nine of 58 markers were found in species from only one lineage; and
these markers diagnose either those lineages or clades
within them (branches 10–29; Fig. 2; Table 2); in most
cases these clades have not been identified yet. Only 5 of
58 markers (derived states of characters 13, 18, and 22–
24; Table 2) seem to have evolved more than once in the
lineages we studied. All of these markers were changes
at nucleotide positions in tRNAs that are usually highly
conserved in arthropods. Seven markers, all features of
the sequences and secondary structure of tRNAs, have
reversed to the putative ancestral condition for arthropods (ancestral states of characters 10, 11, 30, 32, 40, 44,
and 46; Table 2). Overall, 24 characters had phylogenetically informative markers, 7 characters had potentially
informative markers (these markers should be informative with the addition of lower taxonomic levels but are
not informative here), 7 characters had markers that had
reversed to the ancestral states, 5 characters had markers that had evolved in parallel, and 3 characters had
uninformative markers. The high values for the consistency index (CI = 0.82), retention index (RI = 0.89), and
rescaled consistency index (RCI = 0.73) reflect the low
level of homoplasy in data matrix A. By comparison, the
CIs for nucleotide, morphological, and combined data
sets (nucleotides and morphology) for these ticks and
outgroups ranged from 0.33 to 0.61, and the RIs ranged
from 0.51 to 0.83 (Klompen et al., 2000). However, the
higher values we found for these indices may also be
due in part to our smaller number of taxa.
Monophyly of the hard ticks (Ixodidae; branch 5, Fig.
2) was supported by five candidate markers (derived
states of characters 21, 37, 38, 40, and 41; Table 2; Fig. 2).
None of these markers were found in the other lineages we studied. All of these markers are from tRNAs:
one change in the anticodon of tRNA-Ser(AGN) (derived state of character 21) and four correlated sequence
changes in tRNAs (derived states of characters 37, 38, 40,
and 41). The branch that leads to the Metastriata (branch
7; Fig. 2) had the most support: eight candidate markers.
Again, none of these markers were found in the other
lineages we studied. Of these eight markers, four are
features of the arrangement of genes in the mitochondrial genome (derived states of characters 1–4; Table 2).
However, these four markers plus the duplication of the
control region (derived state of character 5) and the loss
of the D-arm from tRNA-Cys (derived state of character 8) may not be independent characters (Campbell and
Barker, 1999; Lavrov et al., 2000). Regardless of whether
we treat these six markers as independent or as one
complex marker, they probably evolved only once and
thus provide strong evidence for the monophyly of the
Metastriata.
Two features indicate monophyly of the Prostriata
(branch 6; Fig. 2): correlated sequence changes in
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305
markers diagnoses either that lineage or a clade within it
(branches 9–28; Fig. 2; Tables 1, 3). Overall, 8 characters
had phylogenetically informative markers, 10 characters
had potentially informative markers (these markers are
likely to be informative with the addition of lower taxonomic levels but are not informative here), and 1 character had a marker that had undergone a reversal.
None of our candidate markers have evolved independently in different lineages of rhipicephaline ticks (CI =
0.86, RI = 0.91, RCI = 0.78; Fig. 3). However one candidate marker, a change in the secondary structure of a
tRNA, has converged on the apparent ancestral condition (ancestral state of character 5; Table 3). Homoplasy
was much more common in the nucleotide and morphological data used to infer the rhipicephaline tick phylogeny (CIs of 0.32–0.67; RIs of 0.40–0.90; Murrell et al.,
2001b). However, the higher values for these indices may
also be due in part to the smaller number of taxa we
studied.
Three markers diagnose either the Rhipicephalinae
s.l. (Rhipicephalinae + Hyalomma; branch 2, Fig. 3) or a
clade of Rhipicephalus s.l. (Rhipicephalus + Boophilus) plus
Hyalomma to the exclusion of Dermacentor s.l. (see Murrell
et al., 2001b; branch 3, Fig. 3). All three of these markers
were from tRNA-Gln: a correlated sequence change and
two changes at nucleotide positions that are highly conserved among other arthropods (characters 16, 6, and
9, respectively; Table 3). In the combined data analyses
of Murrell et al. (2001b), Dermacentor s.l. (Dermacentor +
Anocentor) is the lineage that diverged earliest in the
Rhipicephalinae, but the branching order of the other
three lineages, Rhipicentor, Hyalomma + Nosomma, and
Rhipicephalus s.l., was unresolved. The monotypic genus
Cosmiomma, which was not in the combined data analyses of Murrell et al. (2001b), may also be an early diverging lineage of the Rhipicephalinae (Klompen et al., 1997).
The distribution of these three markers in Dermacentor,
Rhipicentor, and Cosmiomma may shed light on the phylogeny of these early diverging rhipicephaline lineages. It
would be particularly worthwhile to determine whether
one of these changes to typically conserved tRNA bases
that occurs in Bothriocroton (derived state of character 13,
Table 2; derived state of character 6, Table 3) also occurs
in the other early diverging rhipicephaline lineages (i.e.,
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FIGURE 4. (A) Consensus of 15 shortest maximum parsimony trees (length = 18; CI = 1; RI = 1; RCI = 1) for the Ixodida inferred from the
characters in Table 2 (matrix in Appendix 1). Numbers above branches are the number of changes. (B) Consensus of five shortest maximum
parsimony trees (length = 5; CI = 1; RI = 1; RCI = 1) for the Rhipicephalinae inferred with the characters from Table 3 (matrix in Appendix 2).
Numbers above branches are the number of changes.
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SYSTEMATIC BIOLOGY
Rhipicephalus–Boophilus clade (four changes) and a sistergroup relationship between the R. appendiculatus and
R. pulchellus groups (one change) in this phylogeny.
D ISCUSSION
Idiosyncratic features of genes and genomes have revolutionized our views of the evolution of some animal
groups (Rokas and Holland, 2000). The largest problem
with idiosyncratic molecular markers is that they are
rare; they have often been considered too rare to be worth
looking for. To address this problem, we targeted regions
that contain tRNAs and noncoding sequences because
these regions are rearranged more often in mitochondrial
than in protein-coding and rRNA genes. Moreover, novel
motifs often evolve in tRNAs and noncoding sequences.
This strategy allowed us to identify 58 candidate markers (idiosyncratic markers and sequence changes in typically conserved tRNAs) in data matrix A (46 characters)
and 22 candidate markers in data matrix B (19 characters: 9 markers that were also in data matrix A plus 13
additional markers). Sampling in other regions of the mitochondrial genome that contain tRNAs and noncoding
sequences would almost certainly reveal more candidate
idiosyncratic markers, we studied only 4 of the 10 regions that contain tRNAs. By mapping markers onto
well-resolved phylogenies of the family Ixodidae and
the subfamily Rhipicephalinae, we were able to assess
the degree of convergent evolution in these markers and
their utility for phylogenetic inference. These types of
markers probably are present in the published sequences
of other organisms in public databases.
There was little or no homoplasy in our data matrices
compared with the homoplasy in the morphology and
nucleotide data sets of Klompen et al. (2000) and Murrell
et al. (2001b). Some of these comparisons are conservative because the extent of homoplasy was calculated for
phylogenies (Figs. 2, 3) inferred without these characters (Tables 2, 3) whereas the indices for the other data
sets were calculated for phylogenies inferred from those
data sets (an “appropriate” influence; de Queiroz, 2000).
Moreover, almost all of the homoplasy found when characters were mapped onto preexisting phylogenies was in
lineages that are not closely related, according to analyses of morphology and nucleotide sequences. Thus,
homoplasy was easy to identify. For example, the adenine at position 10 in Y (derived state of character 22;
Table 2) evolved in both Allothyrus sp. (a mite, suborder Holothyrida; an outgroup) and Amblyamma hebraeum
(a metastriate tick, suborder Ixodida). Therefore, even
much patchier taxon sampling than that employed here
would have revealed the parallel evolution of this candidate marker in the two lineages.
Our results suggest that of the types of markers we
studied changes at nucleotide positions in tRNAs that are
usually highly conserved among other lineages of arthropods are most likely to evolve convergently or in parallel
(i.e., evolve independently in different lineages). Macey
et al. (2000) found that single base changes in tRNAs
have evolved in parallel in lizards. However, Macey et al.
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Dermacentor, Rhipicentor, and Cosmiomma). Perhaps this
marker unites the Rhipicephalinae s.l. and Bothriocroton
(Fig. 2).
A thymine–thymine mismatch in the AC-stem of
tRNA-Glu (derived state of character 12, Table 3) unites
species of Rhipicephalus s.l. to the exclusion of other rhipicephaline ticks (branch 4, Fig. 3). This relationship was
also supported strongly in the combined data analyses
of Murrell et al. (2001b). However, this marker might
actually diagnose a Rhipicentor + Rhipicephalus s.l. clade
(we did not study tRNA-Glu in Rhipicentor species).
A cytosine at position 23 in tRNA-Asn (derived state
of character 10, Table 3) unites the R. pulchellus and
R. appendiculatus species groups (branch 7, Fig. 3). This
sister-group relationship was found but not strongly supported in the combined data analyses of Murrell et al.
(2001b). However, the species in these two groups have
been placed in a single group by other authors (the
R. appendiculatus group sensu Pegram and Walker, 1988;
Walker et al., 1999). The marker identified here is additional evidence for a close relationship between the
R. pulchellus and R. appendiculatus groups.
Nine markers mapped to the terminal branches of
the phylogeny (Fig. 3). One of these, a cytosine at position 48 in tRNA-Leu(UUR), links the two species of the
R. sanguineus group that we studied, R. sanguineus and
R. turanicus (derived state of character 8; Table 3). In
the analyses of Murrell et al. (2001b), R. sanguineus and
R. turanicus were strongly supported as sister species,
but there was only weak support for a R. sanguineus +
R. turanicus + R. pumilio clade (R. pumilio also belongs
to the R. sanguinus group). Rhipicephalus sanguineus and
R. turanicus are obviously closely related (e.g., Zahler
et al., 1997; Murrell et al., 2001a, 2001b), but if all of the
species in the R. sanguineus group have this marker, and
species from closely related groups do not, this finding would be strong evidence for monophyly of the
R. sanguineus group. Conversely, if this marker is
found in other groups but not in all members of the
R. sanguineus group, this finding would suggest strongly
that the R. sanguineus group is paraphyletic with respect
to other Rhipicephalus groups.
Eight candidate markers mapped to the terminal taxa
of the rhipicephaline tree: five to Boophilus (branch 21)
and three to Dermacentor (branch 26, Fig. 3). Four of these
markers are repeated sequences (derived states of characters 1–4; Table 3), one is a large insertion of apparently
noncoding DNA between genes (derived state of character 18), and three are correlated sequence changes in
tRNAs (derived states of characters 13, 16, and 17). Another repeated sequence (derived state of character 1)
links B. microplus and B. annulatus to the exclusion of
the other three Boophilus species This B. microplus +
B. annulatus relationship was also supported in the combined data analysis (Murrell et al., 2001b) and has long
been suspected from morphological similarities between
B. microplus and B. annulatus (Feldman-Muhsam and
Shechter, 1970).
The candidate markers were used to infer a maximum
parsimony phylogeny (Fig. 4B). There was support for a
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Prostriata. In the combined analysis of Klompen et al.
(2000), when gaps were treated as missing the Prostriata
was monophyletic, but when gaps were treated as a
fifth base then the Prostriata was paraphyletic. Klompen
et al. noted that the only partition for which gap treatment made a difference was the 28S rRNA partition,
which had different topologies for the Prostriata taxa
depending on the way gaps were treated. They concluded (2000:87) that there was the “potential for ‘overweighting’ of shared deletions/insertions of several base
pairs” when gaps were treated as a fifth base. Partitioned
Bremer support (PBS) for the analysis with gaps treated as missing indicated that the 18S rRNA gene provided no or negative support, the 28S rRNA gene
provided no or positive support, the 16S rRNA gene provided positive support, and the morphological characters provided negative or a small amount of support for
monophyly of Prostriata. Our unpublished assessment
of the hidden conflict and support (see Gatesy et al., 1999)
in their data set showed five or six steps of hidden support for the monophyly of the Prostriata (hidden support
or conflict = PBS–BS). Thus, despite the fact that some
of the individual partitions did not support the monophyly of the Prostriata there was support from within
those partitions that remained hidden until a combined
analysis was done. In contrast, the combined analysis
with Prostriata as paraphyletic (gaps treated as a fifth
base) had a hidden support/conflict value of −0.89-2
steps, which indicates hidden conflict or very low hidden
support (Gatesy et al., 1999). Thus, the combined analyses of Klompen et al. (2000) when gaps were treated
as missing data were more robust than their analyses
when gaps were treated as a fifth base. The former indicated monophyly of Prostriata. Our unpublished analyses of the Klompen et al. (2000) molecular data sets
showed that there was not significant heterogeneity of
nucleotide composition (chi-square test) across taxa for
the variable sites nor were any other systematic biases
apparent that might have affected whether the Prostriata was monophyletic or paraphyletic. Our study of idiosyncratic markers and changes to typically conserved
sequences in tRNAs provides further evidence for the
monophyly of the Prostriata (the ∼240 Ixodes species).
Some of the markers we identified were uninformative for determining relationships among the lineages
studied here (Figs. 2, 3). Some of these markers will
probably be informative at lower phylogenetic levels
(e.g., markers that mapped to branches 10–29 [Fig. 2]
and branches 9–28 [Fig. 3]). Other markers were informative and either provided additional evidence for relationships inferred from sequences and morphology
(Klompen et al., 2000; Murrell et al., 2001b) or indicated
relationships not previously found, e.g., Bothriocroton
(=Bothriocrotoninae sensu Klompen et al., 2002) and the
Rhipicephalinae s.l. When the markers were used to infer
maximum parsimony phylogenies (Figs. 4A, 4B), these
phylogenies were congruent with the phylogenies of
Klompen et al. (2000) and Murrell et al. (2001b), with the
exception of the sister-group relationship between Bothriocroton and the Rhipicephalinae (Fig. 4A), which was
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concluded that these changes were useful at some of the
phylogenetic levels they examined.
Although, we found no evidence for convergent or
parallel evolution of repeated sequences, previous studies of noncoding mitochondrial repeats have indicated
that repeats can evolve in this way (e.g., Fumagalli et al.,
1996; Broughton and Dowling, 1997). We found no gene
arrangements that had evolved convergently or in parallel in different lineages, although the position of tRNACys next to tRNA-Met in metastriate ticks is a reversal
to the putative arrangement of these genes in an ancestral protostome (Campbell and Barker, 1999). However, gene arrangements have evolved in parallel in
other groups of animals. For example, the arrangement
tRNA-Asp–tRNA-Lys has evolved independently four
times in the Hymenoptera (Dowton and Austin, 1999)
and once in the Orthoptera (Flook et al., 1995) from the
tRNA-Lys–tRNA-Asp arrangement in the ancestor of all
insects. All studies of early diverging (basal) lineages
from the groups that have rearrangements have revealed
the homoplasy. These cases of homoplasy illustrate the
importance of wide taxon sampling.
Previous work has shown that rearrangements of the
mitochondrial protein-coding and rRNA genes are much
less common than rearrangements of tRNAs (Boore,
1999). In our study, all of the rearrangements of regions
including these large genes mapped to the branch that
leads to the Metastriata (branch 7); this finding alone
is suggestive of the nonindependence of these markers.
Thus, the mitochondrial genomes of the metastriate ticks
show that the arrangement of protein-coding and rRNA
genes in the phylum Arthropoda is not necessarily conserved, even within a family (Black and Roehrdanz, 1998;
Campbell and Barker, 1998; 1999; see also Shao et al.,
2001a, 2001b). A rearrangement of protein-coding genes
was discovered also in two species of Schistosoma, a genus
of parasitic flatworms (Le et al., 2000). These discoveries are timely reminders that each mitochondrial rearrangement began as a mutation in a single mitochondrial
genome in a single mitochondrion, and thus rearrangements may diagnose monophyletic groups at any level of
the taxonomic hierarchy.
Structural and correlated sequence changes in tRNAs
and changes at various nucleotide positions in tRNAs
that are usually highly conserved in arthropods apparently diagnose monophyletic groups at all taxonomic
levels in the phylogenies we studied (Tables 2, 3; Figs. 2,
3). In contrast, most repeated sequences and large insertions (>10 nucleotides) were found in only one or two
closely related species of ticks (Table 3; Fig. 3). However,
there is an exception to this trend: a 25-base pair repeated
sequence that diagnoses the Metastriata (derived state of
character 7; Table 2; Fig. 2). Data from this and other studies indicate that we should be cautious when generalizing about the phylogenetic level at which different types
of idiosyncratic and typically conserved tRNA markers
will be useful.
In contrast to Klompen et al. (2000:93), who stated
that there is “the real possibility that Ixodes (=Prostriata)
is paraphyletic,” we found support for monophyly of
307
308
SYSTEMATIC BIOLOGY
not congruent with the phylogenies of Klompen et al.
(2000).
Idiosyncratic markers and changes to typically conserved sequences in tRNAs should be instructive at
many different phylogenetic levels. Because these types
of markers are often present in nucleotide sequences collected for conventional phylogenetic studies, little or no
extra sequencing may be needed to collect markers that
are inherently interesting and far less likely than As,
Cs, Gs, and Ts to evolve through convergent or parallel
evolution.
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Funding was provided by an Australian Research Council grant to
S.C.B. and N.J.H.C. We thank the people who kindly gave us ticks:
P. Green, W. Mazhowu, D. Birkvens, R. Davey, H. Heyne, J. Keirans,
D. Kemp, G. Kolonin, G. Needham, J. Rehacek, M. Samish, R. de la
Vega, J. Walker, and H. Wassef. We also thank Mike S. Y. Lee for his
helpful comments on a draft of this manuscript and C. Simon, R. Page,
M. Wilkson, F. Frati, and two anonymous referees for valuable criticism
during the revision process.
VOL.
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First submitted 10 January 2001; reviews returned 17 April 2001;
final acceptance 7 February 2003
Associate Editor: Roderic Page
APPENDIX 1. Data matrix A: 46 characters from the mitochondrial genomes of ticks from the family Ixodidae (hard ticks) and outgroups
(Holothyrida, Mesostigmata, Opilioacarida). Characters are described in Table 2: 0 = ancestral character state; 1–3 = derived character states; ?
= unknown.
Characters
Taxon
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Ixodes tasmani group
Ixodes s.s.a
Bothriocroton
Haemaphysalis
Amblyomma
Rhipicephalinae s.l.b
Argasidae
Holothyrida
Mesostigmata
Opilioacarida
0
0
1
1
1
1
0
0
0
0
0
0
1
1
1
1
0
0
0
?
0
0
1
1
1
1
0
0
0
?
0
0
1
1
1
1
0
?
0
?
0
0
1
1
1
1
0
0
0
0
1
1
1
1
1
1
1
1
1
?
0
0
1
1
1
1
0
?
0
0
0
0
1
1
1
1
0
0
0
0
0
0
?
?
?
?
0/1
0
0
?
1
1
1
1
?
0/1
1
?
?
1
1
1
?
?
?
?
0/1
1
0
?
0
0
0
0
0
0
0
1
0
?
0
0
1
0
0
1
0
0
0
?
1
1
?
?
?
1
1
?
?
1
0
0
1
0
0
0
0
0
0
?
0
0
0
0
?
?
1
?
?
0
0
0/1
0
0
0
0
0
0
0
?
?
0
?
?
0
0
0/1
?
0
0
?
0
?
?
0
0
0
?
0
1
0
0
0/1
0
?
0
0
?
?
0
1
1
1
1
?
1
0
?
?
0
0
0
0
0
0/1
0
0
1
0
0
0
0
0
0
0
0/2/3
0
?
0/2
1
Characters
Taxon
Ixodes tasmani group
Ixodes s.s.a
Bothriocroton
Haemaphysalis
Amblyomma
Rhipicephalinae s.l.b
Argasidae
Holothyrida
Mesostigmata
Opilioacarida
a
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
0
0/1
0/1
0
?
0/1
0
?
?
0
0
0
0
0
0
1
0
0
0
?
?
0
?
?
0
0
0
?
0/1
0
1
1
?
?
?
1
1
?
?
1
1
1
1
1
?
1
1
?
?
1
0
0
?
?
0
0
0
1
0
?
0
0
1
1
1
1
1
?
?
1
0
0
0
0
0
0
0/1
?
0
0
2
2
2
2
2
2
0/2
?
1
1
?
0
?
?
0
0
0
?
0
1
1
0
0
0
?
0/2
0
?
?
0
0
0
0
0
?
0
0
?
?
1
0
0
0
0
?
0
0
?
?
1
1
1
1
1
?
1
0
?
?
0
1
1
1
1
?
1
0
?
?
0
0
0
0
0
0
0
0/1
0
0
?
0
0
0
0
?
0
1
?
?
1
1
1
1
1
1
1
0
0
0
?
0
0
1
1
1
1
0
0
0
?
1
1
1
2
?
1
1
?
?
0
1
1
1
1
?
1
0
?
?
1
1
1
0
0
?
0
0
?
?
0
?
1
?
?
0
0
1
?
1
1
Klompen et al. (2000).
Klompen et al. (2000) and Murrell et al. (2001b).
b
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M URRELL, A., N. J. H. CAMPBELL, AND S. C. B ARKER. 2001a. Recurrent
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R OKAS , A., AND P. W. H. HOLLAND . 2000. Rare genomic changes as a
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310
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SYSTEMATIC BIOLOGY
APPENDIX 2. Data matrix B: 19 characters from the mitochondrial genomes of ticks from the subfamily Rhipicephalinae s.l. (see Klompen
et al., 2000) and outgroups (Haemaphysalis spp., Amblyomma s.l.). Characters are described in Table 3: 0 = ancestral character state; 1,2 = derived
character states; ? = unknown.
Characters
a
Taxon
Boophilus
Rp. evertsi group
Rp. pravus group
Rp. sanguineus group
Rp. appendiculatus group
Rp. pulchellus group
Hyalomma
Dermacentor
Haemaphysalis
Amblyomma
a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
0/1
0
0
0
0
0
0
0
0
0
0/1
0
0
0
0
0
0
0
0
0
0/1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0/1
0
0
1
1
1
1
1
1
1
0
1
?
1
1
1
1
1
1
1
?
0
0
0
0
0
0
0
0
1
0
2
?
0
0
0
1
0
0
0
2
0
0
1
1
1
1
1
1
1
?
0
0
0
0
0
0
1
1
0
0
0
?
0
0
0
0
0
0
1
0
0
?
1
1
1
1
1
1
0
0
0
?
0
0
0
0
0
0
0
0/1
0
0
0
0
0
0
0
1
0
0
0
?
0
0
1
0
0
0
0
0
0
?
1/2
1
1
1
1
1
1
?
0
0
0/1
0
0
0
0
0
0
0
0
?
0
0
0
0
0
0
0
0/1
0
?
0
0
0
0
0
0
1
0
0
?
Rp. = Rhipicephalus.
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