Concatenated ScaA and TSA56 Surface Antigen Sequences Reflect Genome-Scale Phylogeny of Orientia tsutsugamushi: An Analysis Including Two Genomes from Taiwan

Orientia tsutsugamushi is an obligate intracellular bacterium associated with trombiculid mites and is the causative agent of scrub typhus, a life-threatening febrile disease. Strain typing of O. tsutsugamushi is based on its immunodominant surface antigen, 56-kDa type-specific antigen (TSA56). However, TSA56 gene sequence-based phylogenetic analysis is only partially congruent with core genome-based phylogenetic analysis. Thus, this study investigated whether concatenated surface antigen sequences, including surface cell antigen (Sca) proteins, can reflect the genome-scale phylogeny of O. tsutsugamushi. Complete genomes were obtained for two common O. tsutsugamushi strains in Taiwan, TW-1 and TW-22, and the core genome/proteome was identified for 11 O. tsutsugamushi strains. Phylogenetic analysis was performed using maximum likelihood (ML) and neighbor-joining (NJ) methods, and the congruence between trees was assessed using a quartet similarity measure. Phylogenetic analysis based on 691 concatenated core protein sequences produced identical tree topologies with ML and NJ methods. Among TSA56 and core Sca proteins (ScaA, ScaC, ScaD, and ScaE), TSA56 trees were most similar to the core protein tree, and ScaA trees were the least similar. However, concatenated ScaA and TSA56 sequences produced trees that were highly similar to the core protein tree, the NJ tree being more similar. Strain-level characterization of O. tsutsugamushi may be improved by coanalyzing ScaA and TSA56 sequences, which are also important targets for their combined immunogenicity.

It was not until 1995 that O. tsutsugamushi was demarcated from Rickettsia spp. with recognition of its distinct cellular envelope, surface antigens, growth characteristics, and divergent 16S rRNA gene (rrs) sequences [35,36].Today, the genetic basis of these differences has largely been elucidated [37], but the function of some surface antigens remains unknown.Orientia and Rickettsia have distinct autotransporter domain-containing surface cell antigen (Sca) proteins with secreted or surface-displayed passenger domains [38].Sca genes are among those used for the speciation of Rickettsia (superseded by genome-based similarity measures [39]), based on pairwise nucleotide sequence homologies of sca0 (ompA), sca4 (gene D), and sca5 (ompB), in addition to rrs and gltA (citrate synthase gene) [40].Some Sca proteins are absent in certain Rickettsia clades (e.g., Sca0 in the typhus group) [40], and such variation is observed at the strain level for O. tsutsugamushi [41].Intact genes encoding ScaA, ScaC, ScaD, and ScaE are present in all complete O. tsutsugamushi genomes sequenced to date (i.e., core genes), while genes encoding ScaB and ScaF are only present in a subset of strains (i.e., accessory genes).Among core Sca proteins, only ScaA and ScaC are of known function and are involved in cellular attachment, binding to an isoform of the mixed-lineage leukemia 5 protein [42] and fibronectin [43], respectively.Like TSA56, Sca proteins (particularly ScaA) are immunogenic [44] yet reveal different phylogenetic relationships [41].
Initial core genome-based phylogenetic analysis of O. tsutsugamushi revealed incongruencies with analysis based on tsa56 and other targets [31].Given that whole-genome sequencing is not widely available for strain-level characterization of O. tsutsugamushi, it is desirable to improve upon single locus genotyping.Thus, this study investigated whether concatenated surface antigen sequences, including Sca proteins, can reflect the genome-scale phylogeny of O. tsutsugamushi.

Cultivation, Purification, and Genomic DNA Isolation
Two representative O. tsutsugamushi strains in Taiwan, TW-1 and TW-22, were selected for whole-genome sequencing (WGS).Clinical O. tsutsugamushi isolates LC0708a (TW-1) and KHC0708a (TW-22) (originally reported in [25]) were recovered in mouse (Mus musculus) fibroblast-like L929 cells (BCRC # RM60091) from frozen stocks at the Taiwan CDC Laboratory of Vector-borne Viral and Rickettsial Diseases.LC0708a was isolated from a 19-year-old male who presented with fever and rashes in Lienchiang County (Matsu Islands) in August 2007, and KHC0708a was isolated from a 45-year-old female who presented with fever, headache, malaise, lymphadenopathy, and an eschar in Kaohsiung City in August 2007.L929 cells were maintained in a 75 cm 2 (T75) flask using MEM (Gibco, Grand Island, NY, USA) supplemented with 4% fetal bovine serum (FBS) (Gibco) and 1% Antibiotic-Antimycotic (Gibco) at 37 • C with 5% CO 2 .Frozen O. tsutsugamushi stocks (0.5 mL) were rapidly thawed, resuspended to disrupt host cells, and used to inoculate L929 cells at ~60% confluence in a 25 cm 2 flask with a small volume of serum-free MEM and incubated at 32 • C with 5% CO 2 for 60-90 min, followed by the addition of MEM containing 2% FBS and 1% Antibiotic-Antimycotic. Media was changed within 24 h and again within 72 h (on day 3 or 4, depending on cell health).Bacterial load was monitored with semiquantification of O. tsutsugamushi DNA extracted from culture supernatant with SYBR Green-based quantitative PCR (qPCR) targeting a 120 bp fragment of the single-copy 47-kDa gene (tsa47) [45], examining changes in cycle threshold (C T ) values.This assay was performed in 20 µL reactions with 1X KAPA SYBR FAST qPCR Master Mix (Roche, Basel, Switzerland), 0.2 µM of each primer (synthesized by Mission Biotech, Taipei, Taiwan), and 2 µL of DNA template or water (no template control), and qPCR was performed using a MyiQ2 thermal cycler (Bio-Rad, Hercules, CA, USA) at 95 • C for 3 min and 40 cycles of 95 • C for 3 s and 60 • C for 20 s followed by a dissociation curve analysis from 65 • C to 95 • C with 0.5 • C increments.Once bacterial growth reached the late exponential phase, cells were harvested for preservation at −80 • C via gentle scraping, and remaining cells were disrupted with 0.5 mm glass beads to release intracellular O. tsutsugamushi for passage.Briefly, the cell suspension was diluted in serum-free MEM (1:10 to 1:20 based on relative load) to infect fresh L929 cells as before, except in a T75 flask.This process was repeated until passage 8, upon which 5 to 8 flasks were inoculated to harvest O. tsutsugamushi for purification and genomic DNA extraction.
Filter purification and DNA isolation were performed using a similar approach to Batty et al. [31].Once O. tsutsugamushi growth was in the stationary phase, host cells were disrupted by gently agitating the flask containing 0.5 mm glass beads with a small volume of spent media, and the lysate, recovered using spent media, was filtered through a 2 µm pore size Puradisc 25 syringe filter (Whatman, Maidstone, UK).Filtered O. tsutsugamushi cells were pelleted at 14,000× g for 10 min, the supernatant was discarded, and cells were resuspended using 380 µL RDD Buffer (Qiagen, Hilden, Germany) and divided into two equal volumes for further processing.Residual host cell genomic DNA was depleted by adding 2.5 µL Benzonase nuclease (Qiagen) to each tube, with incubation in a 37 • C water bath for 30 min, followed by enzyme inactivation with the addition of 20 µL Proteinase K (Qiagen) and incubation at 56 • C for 30 min.O. tsutsugamushi cells were pelleted as before, the supernatant was discarded, and cells were resuspended in Dulbecco's phosphate-buffered saline.DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer's protocol, proceeding with the addition of 20 µL Proteinase K and 200 µL Buffer AL (Qiagen), gentle mixing, and incubation at 56 • C for 10 min.DNA was eluted using 100 µL 10 mM Tris-Cl (pH 8.5) per column, stored at 4 • C, and quantified using a Qubit fluorometer (dsDNA HS Assay Kit; Invitrogen, Waltham, MA, USA) and Fragment Analyzer 5200 (DNF-464 Kit; Agilent, Santa Clara, CA, USA).

Quantitative PCR
SYBR Green-based qPCR targeting tsa47 and a 108 bp fragment of the single-copy mouse adipsin gene (cfd) [46] was performed to evaluate the depletion of host cell genomic DNA.Triplicate 20 µL reactions were performed, each containing 1X iTaq Universal SYBR Green Supermix (Bio-Rad), 0.5 µM of each primer, and 2 µL of DNA template or water, and qPCR was performed using an ABI 7300 thermal cycler (Applied Biosystems, Foster City, CA, USA) at 95 • C for 5 min and 40 cycles of 95 • C for 15 s and 60 • C for 60 s followed by a dissociation curve analysis (system default).Copy number was determined based on calibration curves constructed using pCR2.1-TOPOvector (Thermo Fisher Scientific, Waltham, MA, USA) containing target gene fragments (tsa47 from Taiwan CDC Karp and cfd from L929) with 10 9 to 10 4 and 10 7 to 10 2 copies per reaction (serially diluted in 10-fold increments) for tsa47 and cfd assays, respectively.Linear regression was performed in R 4.3.0(https://www.r-project.org/,accessed on 25 April 2023), and ggpubr 0.6.0[47] was used for data visualization.The percentage of residual host cell genomic DNA was calculated referencing a genome size of 2.7 Gbp for M. musculus (reference assembly GRCm39; RefSeq GCF_000001635.27) and 2 Mbp for O. tsutsugamushi (reference assembly Ikeda; RefSeq GCF_000010205.1).

Whole Genome Sequencing, Assembly, and Annotation
WGS was performed by Genomics Bioscience and Technology Co., Ltd.(New Taipei City, Taiwan) using the PacBio Sequel sequencing platform (Pacific Biosciences, Menlo Park, CA, USA).Briefly, genomic DNA was sheared using a g-TUBE (Covaris, Woburn, MA, USA) and purified with AMPure PB beads (Beckman Coulter, Brea, CA, USA) for ~10 kbp libraries.SMRTbell libraries were sequenced using a SMRT Cell 1M v3 (Sequel Sequencing Kit 3.0; Pacific Biosciences).

Core Genome Phylogeny of O. tsutsugamushi
A core of 691 CDSs was identified among 11 O. tsutsugamushi strains, resulting in a concatenated amino acid alignment with 243,706 positions, which was reduced to 235,464 positions with 91.91% invariant sites after removal of positions containing gaps.Phylogenetic analysis based on 691 concatenated core protein sequences (235,464 positions) produced identical tree topologies with ML and NJ methods (Figure 1).TW-1 and TW-22 were in separate clades with Karp and Kato, respectively; TW-1 most related to Wuj/2014, UT76, and then UT176 and Karp; and TW-22 related to Ikeda and Kato, while TA686 and Gilliam were on separate ancestral branches with Boryong forming an outgroup.positions with 91.91% invariant sites after removal of positions containing gaps.Phylogenetic analysis based on 691 concatenated core protein sequences (235,464 positions) produced identical tree topologies with ML and NJ methods (Figure 1).TW-1 and TW-22 were in separate clades with Karp and Kato, respectively; TW-1 most related to Wuj/2014, UT76, and then UT176 and Karp; and TW-22 related to Ikeda and Kato, while TA686 and Gilliam were on separate ancestral branches with Boryong forming an outgroup.
Among individual surface antigens, TSA56 trees had the highest congruence with the core tree, while ScaA trees had the lowest (Table 2; Figures S5 and S6).Concatenated ScaA and TSA56 trees were highly congruent with the core tree, and the NJ tree had higher congruence than the ML tree (Table 2; Figure 2 and Figure S7).Concatenated ScaC and TSA56 produced an ML tree with similar congruence to the core tree as the ML tree for TSA56 alone, though with a different topology, and an NJ tree with lower congruence (Table 2; Figures S7 and S8).Concatenation of ScaD or ScaE with TSA56 also produced trees with higher congruence to the core tree than TSA56 trees (Table 2; Figures S7 and S8).produced an ML tree with similar congruence to the core tree as the ML tree for TSA56 alone, though with a different topology, and an NJ tree with lower congruence (Table 2; Figures S7 and S8).Concatenation of ScaD or ScaE with TSA56 also produced trees with higher congruence to the core tree than TSA56 trees (Table 2; Figures S7 and S8).

Discussion
This study found that phylogenetic analysis based on concatenated ScaA and TSA56 sequences produces trees highly similar to core protein-based phylogeny despite a >100fold difference in the number of aligned amino acid positions analyzed.TSA56-based trees were most similar to the core tree among the surface antigens examined in this study but still had many incongruencies between phylogenies, and ScaA-based trees were highly dissimilar.This suggests that ScaA possesses phylogenetically informative sites subject to different evolutionary pressures than TSA56, which may be clarified by characterizing their protein-protein interactions.Sca proteins translocate via type V secretion [38], and while this system has not been characterized for Orientia, it likely involves a β-barrel assembly machine complex and other periplasmic chaperons similar to Rickettsia [73].The translocation mechanism of TSA56 has not been described, but it possesses an N-terminal signal peptide that appears to be cleaved [10].ScaA requires a conserved block (CB2, Boryong aa 843 to 875) and involves its flanking regions (fragments F4 and F5, Boryong aa 607 to 994 and 867 to 1241) for attachment, with F5 exhibiting the highest immunogenicity (i.e., anti-ScaA IgG titer) [42].TSA56 primarily binds fibronectin at its surface-exposed antigen domain III and adjacent C-terminal region (Boryong aa 312 to 341) [11], which is relatively conserved [74] and may work in concert with ScaC [43].TSA56 produces a robust humoral response [75] with multiple B-cell epitopes [10,76]; as such, recombinant protein-based enzyme-linked immunosorbent assays have been developed detecting anti-TSA56 antibodies for clinical diagnosis [77][78][79].Neutralizing antibodies are important for protective immunity, but cellular immunity is also necessary to mount an effective immune response against intracellular pathogens [80].TSA56 elicits limited T-cell responses compared to other immunoprevalent antigens, including TSA22 [81], which remains uncharacterized, and TSA47 [75], a periplasmic serine protease involved in cellular exit [82].Notably, coimmunization of mice with ScaA and TSA56 provided enhanced protection against lethal challenge with heterologous strains compared to immunization with either antigen alone [44]; however, even in natural infection, ScaA-and TSA56-directed B-and T-cell immunity rapidly declines after one year [80], though multidose vaccines may overcome this shortcoming.Recently, nanoparticle vaccines have demonstrated enhanced immunogenicity with ScaA, TSA56, and TSA47 subunits, with enhanced protection provided by dual-layered antigen nanoparticles [83,84].In the future, enhanced heterologous protection could be provided via nanoparticle vaccines that combine antigens from multiple strains, similar to what has been implemented for influenza viruses [85] and, importantly, may be tailored for different geographic regions.To this end, the determination of ScaA and TSA56 sequences is indispensable to identify representative antigen sequences for vaccine development.
Phylogenetic trees produced using ScaA and TSA56 were not perfectly congruent with the core protein-based tree.Gilliam and TA686 were not placed on separate branches due to their high phylogenetic relatedness for ScaA.Additionally, using ML, TW-1 and Wuj/2014 were placed on different branches, though poorly supported with bootstrap replicates (<50%), whereas NJ, which is computationally much less intensive than ML, produced a topology that was more similar to the core protein-based tree with a higher level of bootstrap confidence across nodes (>75%).Even so, for core trees, both methods had nodes with low bootstrap support (<75%) but only consistently for the node separating the group containing TW-1, Wuj/2014, and UT76 and the group containing UT176 and Karp.This could be due to geographic relatedness between Thai strains UT76 and UT176 for CDSs other than ScaA and TSA56.Boryong formed an outgroup in core phylogenetic analyses and was also found to be ancestral in a preliminary phylogenetic analysis for Orientia spp.based on core CDSs with the inclusion of a partial assembly of Orientia chuto Dubai (RefSeq GCF_000964595.1) (findings not shown).ScaB was only identified in Boryong, which has been implicated in adherence to and invasion of nonphagocytic cells [86].ScaB has also been detected in TA686 [86] but has a gene sequence below the minimum identity threshold used to identify core CDSs in this study, which was relaxed from 80%, as used in the previous core phylogenetic analysis of O. tsutsugamushi with 657 core genes [31], to 70% in order to include tsa56 as a core CDS.The expression of this core proteome still needs to be verified, including in its natural host, while 599 of the previous 657 core genes were found to be transcribed in Karp and UT176 infecting human umbilic vein endothelial cells [87].Among other Sca proteins, ScaF was only identified in TA686 and Karp, which were clearly separated in the core tree, suggesting that ScaF has evolved multiple times, though its function remains unknown.TA686 and Karp also possessed similar ScaC, and whether ScaF is also involved in adherence in these strains should be determined.Most studies on adherence have been conducted using nonphagocytic cells [11,42,43,86], but O. tsutsugamushi also infects monocytes and antigen-presenting cells at the site of inoculation [88].It has yet to be determined whether variation in core Sca proteins or the presence of accessory Sca proteins control cellular tropism, which could explain variation in strain-level virulence among mice strains and nonhuman primates [89].In systemic infection, O. tsutsugamushi infects endothelial cells with the highest bacterial loads found in the lungs [90] and interstitial pneumonitis is commonly observed in severe cases which can progress to fatal acute respiratory distress syndrome [91,92], with macrophages playing a key role in pathogenesis [93].
Phylogenetic clustering did not consistently correspond with geographic origin for the 11 strains examined in this study.TW-1 was highly similar to Wuj/2014, which was isolated in Zhejiang, China (near Taiwan).TW-1 is the predominant strain isolated from scrub typhus patients in the offshore islands near China (Kinmen, Matsu, and Penghu) [25].TW-22 was most related to Ikeda and Kato, isolated in Japan to the north.However, TW-22 is predominantly isolated in southern Taiwan [25], which has a tropical climate.Ancestral to the aforementioned strains, TA686 and Gilliam were isolated in neighboring countries in Southeast Asia (Thailand and Burma), but TA686 was not found to cluster with other Thai strains (UT76 and UT176) in the Karp clade.Phylogenetic placement of Boryong (isolated in Korea), ancestral to TA686 and Gilliam, further obfuscates the phylogeographic picture.Thus, additional genomes of geographically diverse isolates (with adequate representation for each tsa56 genotype) are needed to clarify the phylogeography of O. tsutsugamushi.To this end, an effort should be made to obtain complete genomes for all described tsa56 genotypes in Taiwan.Studies are also needed to investigate the mite fauna of migratory birds, which have long been thought to play an important role in the dissemination of O. tsutsugamushi [94] and have been implicated in the spread of other acarids [95].There are at least 47 trombiculid mite species throughout Taiwan [96], but the association between mite species and O. tsutsugamushi strains remains unclear, and mite host-O.tsutsugamushi interactions remain poorly characterized.A single mite colony may be coinfected with O. tsutsugamushi [97], facilitating intragenic recombination [28].Competition with cocirculating Rickettsiaceae also needs to be clarified; for example, Rickettsia felis-like organisms that have been found to infect Leptotrombidium deliense in Taiwan [98].Globally, no complete genome sequences have been made publicly available for recently described divergent Orientia spp., including Orientia chuto (endemic in the Middle East) [99] and Candidatus Orientia chiloensis (endemic in South America) [100], and no criteria have been established for delineation of novel Orientia species.These taxa appear to be ancestral to O. tsutsugamushi and may shed light on the evolutionary origins of Sca proteins in Orientia, which has yet to be elucidated [73].
There are still methodological limitations in the ability to amplify and sequence complete tsa56 and scaA, as they are large (1.6 kbp and 4.3 to 4.6 kbp, respectively), and this is particularly challenging for culture-independent studies yielding small amounts of fragmented DNA.Nonetheless, long-range high-fidelity PCR can be used to amplify complete or nearly complete tsa56 [23] and scaA [44], though additional sequencing primers are required.For large-scale culture-independent studies, smaller fragments containing immunogenic epitopes may be prioritized; however, partial sequences will invariably exclude important phylogenetic signals and reduce congruence with core genome-based phylogeny.
TW-1 and TW-22 genomes had acceptable sequencing coverage (>100×), and no assembly errors were identified using Pilon.Among the genomes examined in this study, TA686 was an outlier in that it possesses >1000 pseudogenes (representing >40% of CDSs), leading to questioning of its assembly accuracy and whether it is accurately placed in phylogenetic analyses.Contaminant reads that map to the host cell genome should be removed before genome assembly, including mitochondrial DNA, which was not depleted with filtration and nuclease treatment.
In conclusion, phylogenetic analysis based on concatenated ScaA and TSA56 sequences offers a substantial improvement over TSA56-based analysis in its ability to reflect genomescale phylogeny, and future studies should prioritize their sequencing for O. tsutsugamushi isolates or clinical specimens if WGS-based methods are not available.ScaA and TSA56 sequences are also valuable to inform antigen selection for vaccine development.
TW-22, Table S1: Pairwise amino acid alignments of Orientia tsutsugamushi surface antigens, Table S2: Summary of NCBI locus tags for surface antigens, Figure S5 Informed Consent Statement: Not applicable.

Figure 1 .
Figure 1.Phylogenetic analysis of 11 Orientia tsutsugamushi strains based on 691 concatenated core protein sequences (235,464 positions without gaps) based on (a) maximum likelihood with RAxML-NG v1.2.0 [66], performed using the JTT + I + G4 + F model substitution (tree with the highest loglikelihood is shown) and (b) neighbor-joining with MEGA11 [64] based on evolutionary distances computed using the JTT matrix with 4 discrete gamma categories (optimal tree is shown).Scale branch lengths represent the number of amino acid substitutions per site, and the percentage of replicate trees in which the associated taxa clustered together in 1000 bootstrap replicates are shown above the branches.

Figure 1 .
Figure 1.Phylogenetic analysis of 11 Orientia tsutsugamushi strains based on 691 concatenated core protein sequences (235,464 positions without gaps) based on (a) maximum likelihood with RAxML-NG v1.2.0 [66], performed using the JTT + I + G4 + F model substitution (tree with the highest log-likelihood is shown) and (b) neighbor-joining with MEGA11 [64] based on evolutionary distances computed using the JTT matrix with 4 discrete gamma categories (optimal tree is shown).Scale branch lengths represent the number of amino acid substitutions per site, and the percentage of replicate trees in which the associated taxa clustered together in 1000 bootstrap replicates are shown above the branches.

Figure 2 .
Figure 2. Neighbor-joining-based phylogenetic analysis of 11 Orientia tsutsugamushi strains based on concatenated ScaA and TSA56 amino acid sequences (1910 positions without gaps) with MEGA11 [64] based on evolutionary distances computed using the JTT matrix with 4 discrete gamma categories.The optimal tree is shown (scale branch lengths represent the number of amino acid substitutions per site), and the percentage of replicate trees in which the associated taxa clustered together in 1000 bootstrap replicates are shown above the branches.

Figure 2 .
Figure 2. Neighbor-joining-based phylogenetic analysis of 11 Orientia tsutsugamushi strains based on concatenated ScaA and TSA56 amino acid sequences (1910 positions without gaps) with MEGA11 [64] based on evolutionary distances computed using the JTT matrix with 4 discrete gamma categories.The optimal tree is shown (scale branch lengths represent the number of amino acid substitutions per site), and the percentage of replicate trees in which the associated taxa clustered together in 1000 bootstrap replicates are shown above the branches.

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Maximum likelihood phylogenetic trees based on individual amino acid sequences, Figure S6: Neighbor-joining phylogenetic trees based on individual amino acid sequences, Figure S7: Maximum likelihood phylogenetic trees based on concatenated amino acid sequences, Figure S8: Neighbor-joining phylogenetic trees based on concatenated amino acid sequences.Author Contributions: Conceptualization, N.T.M. and K.-H.T.; methodology, N.T.M. and K.-H.T.; software, N.T.M.; validation, N.T.M.; formal analysis, N.T.M.; investigation, N.T.M. and K.-H.T.; resources, Y.-L.L.G., P.-Y.S. and K.-H.T.; data curation, N.T.M.; writing-original draft preparation, N.T.M.; writing-review and editing, N.T.M.; visualization, N.T.M.; supervision, T.-Y.Y., Y.-L.L.G., P.-Y.S. and K.-H.T.; project administration, T.-Y.Y.; funding acquisition, K.-H.T.All authors have read and agreed to the published version of the manuscript.Funding: This research was supported by the Ministry of Science and Technology, Taiwan (MOST 107-2314-B-002-279-MY2) and in part by the Taiwan CDC (MOHW113-CDC-C-315-144315).Institutional Review Board Statement: This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Taiwan CDC, Ministry of Health and Welfare (IRB No. 106111).

Table 2 .
[72]ruence between phylogenetic trees based on the quartet similarity measure implemented in R package Quartet (normalized scores are shown)[72].

Table 2 .
[72]ruence between phylogenetic trees based on the quartet similarity measure implemented in R package Quartet (normalized scores are shown)[72].