Understanding the evolutionary structural variability and target specificity of tick salivary Kunitz peptides using next generation transcriptome data
© Schwarz et al.; licensee BioMed Central Ltd. 2014
Received: 17 June 2013
Accepted: 13 December 2013
Published: 7 January 2014
Ticks are blood-sucking arthropods and a primary function of tick salivary proteins is to counteract the host’s immune response. Tick salivary Kunitz-domain proteins perform multiple functions within the feeding lesion and have been classified as venoms; thereby, constituting them as one of the important elements in the arms race with the host. The two main mechanisms advocated to explain the functional heterogeneity of tick salivary Kunitz-domain proteins are gene sharing and gene duplication. Both do not, however, elucidate the evolution of the Kunitz family in ticks from a structural dynamic point of view. The Red Queen hypothesis offers a fruitful theoretical framework to give a dynamic explanation for host-parasite interactions. Using the recent salivary gland Ixodes ricinus transcriptome we analyze, for the first time, single Kunitz-domain encoding transcripts by means of computational, structural bioinformatics and phylogenetic approaches to improve our understanding of the structural evolution of this important multigenic protein family.
Organizing the I. ricinus single Kunitz-domain peptides based on their cysteine motif allowed us to specify a putative target and to relate this target specificity to Illumina transcript reads during tick feeding. We observe that several of these Kunitz peptide groups vary in their translated amino acid sequence, secondary structure, antigenicity, and intrinsic disorder, and that the majority of these groups are subject to a purifying (negative) selection. We finalize by describing the evolution and emergence of these Kunitz peptides. The overall interpretation of our analyses discloses a rapidly emerging Kunitz group with a distinct disulfide bond pattern from the I. ricinus salivary gland transcriptome.
We propose a model to explain the structural and functional evolution of tick salivary Kunitz peptides that we call target-oriented evolution. Our study reveals that combining analytical approaches (transcriptomes, computational, bioinformatics and phylogenetics) improves our understanding of the biological functions of important salivary gland mediators during tick feeding.
KeywordsRed Queen hypothesis Tick saliva Kunitz-domain proteins Cysteine motif Structural bioinformatics Antigenicity Protein disorder Molecular clock Evolution
Ticks have been successful as ectoparasites due to morphological and physiological adaptations . As obligate hematophagous (blood-sucking) arthropods, ticks transmit various bacterial and viral diseases, e.g., babesiosis, theileriosis, anaplasmosis, Lyme disease and tick-borne encephalitis, and, thus greatly impact human and animal health . To combat host defense mechanisms, ticks possess powerful pharmacological proteins in their saliva that are injected into the vertebrate host during blood feeding [3, 4]. Our current understanding of host-parasite interactions has been greatly impacted by the revolution in sequencing technologies that has provided a massive amount of data previously unimaginable . Among the various transcriptome studies to date, transcriptome projects from arthropod disease vectors are using Sanger or next generation sequencing techniques. These include several salivary gland (SG) transcriptome and proteome projects (also known as sialomes) from hard tick [6–18] and soft tick species [19–22]. Hundreds of novel SG transcripts that putatively encode proteins were discovered from these sialome projects, thus elucidating how ticks may complete a blood meal while providing insight towards their evolutionary expansion .
Available SG transcriptomes show that the most frequently secreted tick salivary protein families are lipocalins, enzymes, and protease inhibitors. Among protease inhibitors, Kunitz-domain transcripts are one of the most abundant protein families in tick SGs [9, 18, 19, 21]. The archetypal Kunitz fold is highly conserved, resembling the first Kunitz-domain protein, the bovine pancreatic trypsin inhibitor (BPTI) that was functionally described in 1936 by Moses Kunitz . To date, about 15 single Kunitz-domain peptides from ticks have been functionally characterized. Classical serine protease inhibitors were analyzed from the hard ticks Rhipicephalus microplus (BMCL) , Rhipicephalus appendiculatus (TdPI) , Rhipicephalus haemaphysaloides (Rhipilin-1) , Haemaphysalis longicornis (HlChl, HIMKI and Haemangin) [28–30], Amblyomma cajennense (Amblyomin-X) , and Ixodes scapularis (Tryptogalinin) . Protease inhibitors were also characterized from the soft ticks Ornithodoros moubata (TAP)  and Ornithodoros savignyi (FXaI) that mainly function as anti-clotting agents. Anti-platelet inhibitors were also identified as single Kunitz-domain inhibitors, such as the Monogrins (1A and 1B) from Argas monolakensis, and, their orthologs Disagregin  and Savignygrin from Ornithodoros spp. . Several tick salivary Kunitz-domain proteins that possess multiple domains (1–7 Kunitz-domains) were also characterized as serine protease inhibitors [37–39]. Of all the tick SGs Kunitz-domain proteins, however, single Kunitz-domain peptides are highly represented (we will refer to these single domains as Kunitz peptides, henceforth) [9, 12]. These Kunitz peptides vary in their cysteine (Cys) motifs (possessing more or less than 6 Cys residues) with some lacking the archetypal disulfide bonds causing a more flexible fold, therefore diversifying their inhibitory activity [26, 32, 40].
Kunitz SG peptides of I. scapularis were phylogenetically analyzed to uncover their emergence in ticks and the expression trends of these Kunitz peptides were also statistically analyzed . These Kunitz peptides were categorized in three different groups (groups I, II and III) based on their Cys motif. The I. scapularis Kunitz peptides belonging to group I were suggested to represent the ancestor of all tick Kunitz-domain family (single and multiple domains). Several Kunitz peptides seemed to have lost their ability to function as serine protease inhibitors and instead to block and/or modulate ion channels, possibly related to the tick’s necessity for prolonged feeding on the vertebrate host . The authors are aware of only one study that functionally and structurally characterized a tick Kunitz peptide as an ion channel effector, the maxiK channel modulator Ra-KLP from R. appendiculatus. This paucity must be remedied since Dai et al.  putatively characterize the majority of I. scapularis Kunitz peptides as ion channel blockers/modulators. Furthermore, Fry et al.  have argued that hematophagous secreted proteins, such as of the Kunitz family, should be classified as venomous. Most classified toxins are stabilized by their disulfide bridges and once these toxins become functionally essential as a venom, their adaptation is often reinforced by gene duplication . Gene sharing and gene duplication are the main mechanisms advocated to explain the functional heterogeneity of tick salivary Kunitz family proteins [23, 42].
In our study, we used computational, structural bioinformatics and phylogenetic methods to reevaluate tick salivary Kunitz peptides from a more in-depth structural point of view by analyzing the functional, antigenic, and evolutionary characteristics of Kunitz peptides from the recently annotated Ixodes ricinus SG transcriptome ; GenBank Bioproject PRJNA177622. Compared to classical biochemical analyses and classical Sanger sequencing techniques that revealed only a few thousands of sequences from tick transcriptome studies presented until today (with the exception of the A. maculatum transcriptome), the large amount of available data of the 454 pyrosequencing/Illumina I. ricinus SG transcriptome makes it feasible to thoroughly analyze multigenic protein families (i.e., the Kunitz-domain family). In the I. ricinus transcriptome, 4% of all Illumina reads were classified as transcripts encoding for Kunitz-domain proteins and of these, 1.4% accounted for single Kunitz-domain transcripts (that putatively encode archetypal Kunitz peptides possessing more or less 6 Cys residues) . Our approach demonstrates how to interpret next generation transcriptome data to expand our understanding of the molecular, structural and functional evolution of hematophagy in ticks. Our overall analysis developed a novel concept that elucidates the divergence and adaptation of I. ricinus salivary Kunitz peptides that we call target-oriented evolution.
Results and discussion
Organizing the different I. ricinusSG Kunitz peptides based on their Cys motifs facilitates functional predictions
Aligning a representative of each I. ricinus Kunitz peptide group (Figure 1B) in our study shows that specific residues coincide with the putative functions described by Dai et al. , classified as serine protease inhibitors or ion channel blockers/modulators. Groups G3, G6 and G11, however, do not contain these conserved residues (boxed in Figure 1B). With the exception of the annotated I. scapularis sialome [6, 9], both protein BLAST  and PSI-BLAST  searches do not reveal any known or functionally described single Kunitz-domain peptide with the Cys motif of G3, G6 and G11. The proteins that slightly resemble the Cys motif of G6, and lack the archetypal Cys2 and Cys4 disulfide bridge, are the I. scapularis salivary Kunitz proteins Ixolaris  and Penthalaris , both possessing multiple Kunitz domains, and some members of the Kunitz family found in R. microplus. An alignment using the classical Kunitz serine protease inhibitor BPTI (UniProt: P00974) with G1 and G7 shows the conserved positively charged arginine (Arg or R) or lysine (Lys or K), known as the P1 site that interacts with the negatively charged active site of serine proteases (highlighted blue in Figure 1C). Furthermore, the key residues for the ion channel blocker beta-bungarotoxin (UniProt: P00989), from the venomous snake Bungarus multicinctus, are also conserved in G2, G4, G5 and G8-G10 (highlighted green in Figure 1C).
We putatively classify G2, G4, G5 and G8-G10 as ion channel blockers and/or modulators (Figure 1C) and as Figure 2B shows they exhibit a separate profile than the serine protease inhibitors of Figure 2A (G1 and G7). These putative ion channel blockers/modulators show lower transcript reads during the nymphal stages of feeding (both early, EN, and late, LN), but the reads are increased in adult ticks during the early stage of blood feeding (EA). The profile then decreases during the late stages of feeding (Figure 2B). A possibility, as noted in mayfly nymphs SGs, for the expression of ion channel blockers/modulators in I. ricinus nymphs may partially be due to maintaining osmoregulatory function during molting . To resist desiccation, tick nymphs rely on both water vapor and water retention, while adult ticks only rely on enhanced water retention ; therefore, this may explain the increased transcript read encoding for ion channel blockers/modulators during adult feeding stages (compared with nymphs – Figure 2B). In Figure 2C we show that G6 deviates from the other two profiles, however, G3 and G11 have similar profiles to that of ion channel blockers/modulators (Figure 2B). The distinct profile and Cys motif G6 displays suggests that this group may belong to (a) a combination of serine protease inhibitors and ion channel blockers/modulators, (b) either serine protease inhibitors or ion channel blockers/modulators, or, (c) a completely new, undefined function – i.e., gene sharing (moonlighting).
The I. ricinusSG Kunitz groups possess intra-Cys residue variability, assemble into various secondary structure clusters, and vary in antigenicity and protein disorder
To demonstrate the structural outcome of these “frozen” and “melted” clusters, we predicted the secondary structure for each Kunitz peptide in all groups. We show that 22 models depict the variability of secondary structure among the 145 Kunitz peptides (excluding SK peptides) (Additional file 3). Five models in Additional file 3 (Representative models G1: Ir2:98; G1: Ir2:4878; G1: Ir2:19262; G6: Ir2:1982 and G7: Ir2: 4792) are combined β-strand and α-helical secondary structure models, differing in length, amount and order of α-helices and β-strand, and describe 81.6% of the secondary structure variation present in the I. ricinus Kunitz/BPTI family. All the groups contain different amounts of secondary structure models (Figure 3B and Additional file 3). It seems, however, that the secondary structure presented in Ir2-4878 and Ir2-98 of G1 is the structural skeleton for the remaining secondary structures; these structures are also present in 10 of the 11 groups and explain 50% of the secondary structures. The fact that G1 represents the structural skeleton may be since its Cys motif (as displayed in Figure 1A) is considered the ancestor of Kunitz peptides in I. scapularis. Structural convergence, however, has been reported for venoms, a general classification that the Kunitz protein family belongs to [Revised in 41].
When analyzing the aa variability (V/C or AVE) in relation to the secondary structure models per group (excluding G6) a high correlation is given (R2 = 0.78; Figure 3E); however, by including G6 in this analysis the correlation decreases (R2 = 0.18; Figure 3F). This dictates that the aa variability per group is partly justifiable for structural reasons (functionality), except in G6. Figure 3C shows that ratio V/C or AVE of aa variability are correlated, therefore, using V/C or AVE of aa variability is interchangeable for Figure 3E and F. The presence of different folds (i.e., secondary structures) suggests that gene sharing events may have occurred throughout the evolution of I. ricinus and may cause additional and unique functional properties. Such moonlighting proteins may explain the diverse inhibitory effect of tick salivary Kunitz peptides.
The I. ricinusSG Kunitz transcripts are under different selective pressures
The Red Queen hypothesis was proposed by Van Valen in 1973  to explain the molecular evolution through evolutive interactions of prey–predator or host-parasite. The hypothesis establishes “that a proportional amount of successful response by one species produces a total negative effect on other species[…] To maintain itself as before, the [other] species must increase its fitness [in a proportional amount]”. The I. ricinus SG Kunitz encoding transcripts may adhere to the Red Queen hypothesis, where inverse selection (positive or negative) is a series of one species adapting (the tick) and the other counteradapting (the host). To elucidate or validate the type of selective pressure (positive or purifying) each I. ricinus Kunitz peptide group has undergone throughout its evolution we used the Synonymous Non-synonymous Analysis Program (SNAP) (http://www.hiv.lanl.gov) and Datamonkey servers  (see Methods for further details).
Statistical analysis on nucleotide substitution
*d N /d S
*d N /d S
The evolution of I. ricinusSG Kunitz peptide groups
The second largest clade also contained only prostriate Kunitz peptides composed mainly of I. ricinus group G6. As in the ion channel blocker/modulator clade, no functionally described Kunitz appeared in this clade, therefore any possible function remains unknown. Functionally described Kunitz peptides were present in the remaining two largest clades. One of these clades was made up of only argasid Kunitz peptides that included anti-platelet inhibitors. This group seem to evolved independently from hard ticks as previously postulated by Mans et al. , possibly due to different blood feeding behavior compared with hard ticks. The other clade mainly contained I. scapularis peptides together with I. ricinus group G3 and anti-clotting inhibitors of Ornithodoros spp.. Our aa sequence and structural analyses of G3 did not reveal any putative function for this I. ricinus group, although the transcript profile of G3 was similar to that of ion channel blockers/modulators (as G11 in Figure 2C). However, since G3 clusters with anti-clotting inhibitors and not with G11 may suggest a different function during tick feeding. I. ricinus groups G1 and G7 clustered together with hard and soft tick Kunitz peptides, but their relationships are uncertain since they were not well supported in the ML and Bayesian trees (Additional file 4). Nevertheless, in our Bayesian analysis, I. ricinus groups G1 and G7 appeared with functionally described serine protease inhibitors and G7 proteins were also grouped together with G1 proteins in our ML analysis. Both our phylogenetic and structural analyses of the I. ricinus groups G1 and G7, suggests that these two groups may act as serine protease inhibitors. SK peptides appeared throughout the phylogram. Some SKs were similar in their aa sequence (Additional file 5) with the other I. ricinus Kunitz peptides assuming a similar function during blood feeding. Others SKs may be more functionally distinct, such as Ir2-24967-SK, since it differs in its aa sequence from all other I. ricinus Kunitz peptides.
Our molecular clock analysis of tick and spider Kunitz peptides recovered similar divergence times for the main splits of 1) spiders from ticks (426 MYA, 95% HPD: 389–462 MYA, node S), 2) soft ticks from hard ticks (233 MYA, 95% HPD: 198–266 MYA, node A) and 3) prostriate ticks within Ixodidae (221 MYA, 95% HPD: 181–254 MYA, node P) as calibrated for these taxon sets; the tree root was estimated as 436 MYA (95% HPD: 395–479 MYA) (Figure 6, Additional file 4B). Most of these splits were well supported in the chronogram. Metastriate Kunitz peptides (fourth taxon set), however, started to evolve earlier than calibrated. Nevertheless, this split is not well supported in the chronogram as previously stated (193 MYA, 85% HPD: 160–228 MYA, Figure 3, node M). Although I. scapularis homologs of I. ricinus group G1 Kunitz peptides are considered to be the ancestral form of all Kunitz-domain proteins , our phylogenetic reconstructions of I. ricinus Kunitz peptides cannot confirm these findings. Even though G1 is not well supported in the ML and Bayesian analyses, it can be assumed that this group of proteins evolved convergently in ticks as both phylogenetic trees indicate (Additional file 4). Expansion of protein families due to gene duplication events has been previously reported for ticks [23, 64] and can explain the convergent evolution of G1. Overall, the I. ricinus Kunitz peptides in our molecular clock analysis underlie recent gene duplication events that explains how several members of the I. ricinus groups and other tick Kunitz peptides do not strictly follow the calibrated divergence times within the taxon sets in the phylogenetic trees.
Target-oriented evolution, a model for I. ricinusSG Kunitz peptide evolution
The Red Queen hypothesis  explains that host-parasites interactions must be characterized by constant mutations in both systems in order for parasites to efficiently infect the host and for the host to survive or avoid the parasitic attack. We understand the evolution of I. ricinus salivary Kunitz family in this dynamic framework. In the following paragraphs we explain our basis to propose one model of target-oriented evolution for this family of proteins and briefly discuss our findings regarding the evolution of venomous proteins.
Our model considers G6, compared with all other I. ricinus Kunitz peptide groups, as a cornerstone in understanding the Kunitz family evolution of I. ricinus. Firstly, members of G6 show unique molecular, structural and evolutive properties by possessing the highest amount of intra-Cys residues between Cys2 and Cys4 (Figure 1), a distinct Illumina read profile (Figure 2C), a high aa variability (Figure 3A and D), a higher number of different secondary structures (Figure 3B), and an increased number of negatively selected sites (Figure 5 and Table 1). Secondly, in the phylogenetic analysis, the clade formed by G6 is mainly composed of I. ricinus Kunitz peptides (Figure 6 and Additional file 4), also representing the largest monophyletic clade suggesting a recent and specific evolution for this group in I. ricinus. Thirdly, based on the molecular clock analysis G6 is the fastest evolving group, diverging about 70 MYA ago and having the highest number of members. Taking into account the time of divergence and the number of members, we should also consider G6 as an example of possessing an accelerated rate of evolution that has been reported for several other families of venomous proteins . At this point we consider it important to make a distinction. As depicted in the phylogenetic tree (Figure 6) members of other groups appear to evolve around the same time point as G6. It is crucial to note here that even when some genes show the same time of evolutive divergence, as they belong to a distinct group, they do have different functional states at the moment of divergence. For example, our tree depicts a clade containing members of G8 and G11 that diverged 71 MYA ago (Figure 6, marked with a red star), but, as we previously showed (Figures 1, 2, 3B, 4A-B), these groups have different properties compared with G6. In this way we consider that molecular mechanisms working for the observed Kunitz family expansion (e.g., gene duplication) must be operating on the whole genome background, thus, functionally and structurally different gene(s) may show genetic amplification at similar evolutive times. But we consider that the genetic expansion must be occurring at different rates for different groups of genes, depending on the function of the gene and its importance in the interaction with the host – as previously reported for venomous protein families .
The Red Queen hypothesis is an arms race proposed as a model for co-evolution for host-parasite interactions . Today’s increasing knowledge about the interface between host-tick interactions has provided insight that ticks successfully feed to repletion by counteracting the host immune response, platelet aggregation, inflammation and vasoconstriction [4, 64, 66, 67]. We therefore infer that two main evolutionary pressures may be driving the evolution in I. ricinus Kunitz peptides – i.e., immune response and the necessity of functional diversity against the diverse host molecular targets. The former is in agreement with specific immune response against tick salivary proteins , and, specifically against Kunitz members . The pressure for functional diversity on tick Kunitz peptides may be that several targets have been associated with their inhibitory activity such as trypsin, elastase, kallikrein , chymotrypsin , tissue factor complex inhibitor  and ion channels . We consider our model of target-oriented evolution (Figure 7) to explain the evolutionary dynamics found in the I. ricinus SG Kunitz peptides in the framework of the Red Queen hypothesis.
Identifying single Kunitz-domain peptides from the I. ricinustranscriptome and peptide sequence alignments
We chose all full-length, secreted and non-truncated single Kunitz-domain peptide sequences from the SG I. ricinus transcriptome . We then submitted all sequences to the Pfam server  to verify and identify them as single Kunitz-domain peptides. To further verify that these sequences are classical Kunitz-domain peptides we performed a protein BLAST alignment using BPTI (GenBank accession no.: P00974) as the query. As a final filtering point we submitted the remaining sequences to SignalP 4.0 server , since we were only interested in secreted peptides. All signal peptides were removed from all sequences and this resulted in a total of about 200 mature, single Kunitz-domain peptide sequences.
Only sequences that possessed a minimum of 6 Cys residues and a maximum of 7 Cys residues were used for our analysis. The majority of functionally described tick Kunitz peptides possess 6–7 Cys and usually >7 Cys are proteins with more than a single Kunitz-domain – with a few documented exceptions [26, 40]. We specifically wanted to investigate the archetypal Kunitz-domain peptides of I. ricinus (hence, those possessing more or less 6 Cys residues). All peptides were divided into groups based on their Cys motif (i.e., the number intra-Cys residues) and a group was defined as whether there were two or more representative sequences for each motif. We chose a minimum of two sequences as a criterion since G3 has been previously reported by Dai et al.  and we only found two sequences for this group in the I. ricinus salivary transcriptome. This grouping resulted in a total of 145 sequences that formed eleven groups (G1-G11). We additionally found several Kunitz-domain peptides (n = 7) in the I. ricinus salivary transcriptome that possessed less than 6 Cys residues that were included in our phylogenetic analysis. These small peptides were defined as ‘simple Kunitz’ (SK) due their atypical Cys motif compared to archetypal Kunitz-domain peptides. Due to limitations of dealing with extremely small peptides and variations on their Cys motif, these SKs were used in only a few of our analyses. Thus, a total of 152 I. ricinus salivary Kunitz peptide sequences were used in our subsequent analyses.
Tertiary protein modeling and disulfide bridge predictions
To predict the disulfide bond patterns we first modeled a representative of each group (those indicated in Figure 1) using the evolutionary protein model building web-server Phyre2 . Using the Schrödinger’s Maestro software , each predicted 3D model was minimized, prepared, refined and re-minimized – minimization was used to remove steric clashes. Minimization uses a Truncated Newton algorithm , an OPLS-AA force field and a surface generalized Born (SGB) implicit solvent . The total CPU time for minimizing was 2–3 min. After minimization, Cys resides were connected, hydrogen atoms were added, and the overall hydrogen-bond network was optimized using the Schrodinger’s Protein Preparation Wizard , which optimizes the entire hydrogen bond network by means of side chain sampling. The Protein Preparation Wizard analyzes the structure then builds a cluster of hydrogen bonds and 100000 Monte Carlo orientations for each cluster. The algorithm determines the quality of the hydrogen-bond network to produce an optimized structure. The modeled structures were then re-minimized. The disulfide bond patterns were verified using the web-server BRIDGED [79, 80].
Amino acid (aa) variability (Shannon entropy) and secondary structure analysis
P i is the amount of each aa type (i), and M is the number of aa types (maximum 20 aa). When H ≥2.0 the site is considered variable, while H ≤1.0 count for highly conserved positions . Secondary structure was predicted using the position-specific scoring matrices method  from the PSIPRED server [82, 83]. The presence (or not) of β-strands and α-helices, their length, amount, and position in the sequences were used as criteria for 2D model differentiation.
Nucleotide substitution rate analysis (SNAP and Datamonkey server)
We then used d S and d N to identify whether a group has undergone positive (>1) or purifying selection (<1); where p S and p N are the synonymous differences per synonymous site and non-synonymous differences per non-synonymous site, respectively. Standard deviation (σ) and variance were also calculated by the SNAP server using the methods described by Nei and Gojobori .
Under the Datamonkey server  we used the algorithm PARRIS  to detect positive selection and the SLAC algorithm  was used to calculate the ratio d N /d S . A two-tailed extended binomial distribution set at p-value < 0.05 was used to assess significance of both algorithms. The SLAC and PARRIS algorithms use a neighbor-joining tree with a maximum likelihood for branch lengths and substitution rates. A few of our codon alignments contained multiple segments (i.e., recombination) as analyzed by GARD , therefore the base frequencies and substitution rates obtained from GARD were inferred together from the entire alignment, while branch lengths were fitted to each segment separately. Based on these analyses a global d N /d S ratio was obtained.
If dN < dS a codon was negatively selected and if dN > dS a codon was positively selected.
Phylogenetic analysis of Kunitz-domain peptides from hematophagous arthropods
All 152 I. ricinus Kunitz peptide sequences were used for a phylogenetic reconstruction of the Kunitz-domain family within ticks. Accordingly, we searched the NCBI database for Kunitz sequences from hard and soft ticks, and additionally spider sequences were included as outgroup. Similar to the I. ricinus Monolaris, only secreted and full-length Kunitz-domain proteins with up to 7 Cys residues in their aa sequence were chosen. All candidate proteins were re-confirmed as single Kunitz-domain peptides using the Pfam server . Additionally, sequences of single Kunitz-domain proteins that have been functionally described were also included into the phylogenetic analysis.
A codon alignment was constructed using TranslatorX  and then all mature 249 single Kunitz-domain protein sequences were aligned using the program MAFFT v7 . For the multiple sequence alignment we applied an iterative refinement method (L-INS-I) with the BLOSUM 62 matrix (New scoring scheme, gap opening penalty: 2.0, Offset value: 0.5). The protein alignment was then used in TranslatorX to align the corresponding Kunitz nucleotide sequences of ticks and spiders to create the final codon alignment. All unaligned, flanking sequence regions of the codon alignment were trimmed and all gaps were closed apart from gaps that were introduced by up to two sequences only (final sequence lengths: 126 bp, Additional file 5). The final alignment was analyzed using jmodeltest v2.1.2 in order to select the best-fit nucleotide substitution model for the phylogenetic reconstruction . A phylogenetic tree was constructed under the maximum likelihood (ML) optimality criterion and the GTR model with a gamma distribution of substitution rate and estimated base frequencies using the program RAxML (HPC v7.2.6) . Tree searching and bootstrapping were performed simultaneously (−f a, 1000 bootstrap replicates).
Divergence time estimation
The codon alignment of coding single Kunitz-domain proteins was also used to estimate divergence times among ticks using BEAST v1.7.5 . For this Bayesian analysis, the following four taxon sets and divergence times (unit: million years ago, MYA) were calibrated according to Jeyaprakash and Hoy  using normal prior settings: Araneae/Scorpions/Pycnogonida/Acari 459 ± 18 MYA (included taxa: all tick sequences), Argasidae 214 ± 28 MYA (included taxa: all argasid sequences), Prostriata 196 ± 27 MYA (included taxa: all prostriate sequences) and Metastriata (included taxa: all metastriate sequences) 134 ± 22 MYA. All tick Kunitz-domain sequences in the Araneae/Scorpions/Pycnogonida/Acari taxon set were enforced to be kept monophyletic. Default settings were used for all other prior and operator settings in all analyses. Similar to the ML analysis, a GTR substitution model with a gamma rate distribution across sites and estimated base frequencies were applied. Two independent Beast runs with four independent Markov-chain Monte Carlo (MCMC) chains and 100,000,000 generations were performed. The starting trees were randomly generated and all trees were sampled every 10,000 generations. Different molecular clock (uncorrelated exponential and lognormal relaxed clock)  and speciation models  (Yule process and birth-death process) were tested. A uniform prior distribution for the calculations of the mean branch rates under the uncorrelated lognormal or exponential relaxed molecular clock was set. All BEAST analyses were assessed if each parameter converges on the same posterior distribution in the MCMC runs and if an effective sample size was reached using Tracer v1.5 (http://tree.bio.ed.ac.uk/software/tracer). Additionally, the marginal likelihoods were estimated using path sampling and step stone sampling methods of each analysis in order to select the appropriate model for our analysis . According to our evaluation of the different BEAST runs, the uncorrelated relaxed lognormal molecular clock model was chosen with the Yule tree prior for speciation. The two tree files from the latter BEAST analyses were combined using LogCombiner v1.7.5 from the BEAST software package using a burn-in of 10% of all sampled trees was set. Finally, TreeAnnotator v1.7.5 was used to summarize all trees in order to obtain a single maximum clade credibility tree including mean node heights.
Alexandra Schwarz and Alejandro Cabezas-Cruz joint first authorship.
Jan Kopecký and James J Valdés joint senior authorship.
Bovine pancreatic trypsin inhibitor
Early feeding nymphs
Early feeding female adults
Late feeding nymphs
Late feeding female adults
Million years ago
Synonymous non-synonymous analysis program
- V/C or AVE:
Variable/conserved sites or average variability.
AS was funded by the Grant Agency of the Czech Republic (Grant No. P302/11/P798) and the Ministry of Education, Youth and Sports of the Czech Republic (KONTAKT II grant no. LH12002). ACC was funded by POSTICK ITN within the FP7-PEOPLE-ITN program (EU Grant No. 238511). JK was supported by the Grant Agency of the Czech Republic (project P302/12/2208). JJV was sponsored by project CZ.1.07/2.3.00/30.0032, co-financed by the European Social Fund and the state budget of the Czech Republic. We will also like to acknowledge the financial support of the Academy of Sciences of the Czech Republic (grant no. Z60220518).
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