- Research article
- Open Access
Arsenophonus, an emerging clade of intracellular symbionts with a broad host distribution
© Nováková et al; licensee BioMed Central Ltd. 2009
- Received: 25 June 2009
- Accepted: 20 July 2009
- Published: 20 July 2009
The genus Arsenophonus is a group of symbiotic, mainly insect-associated bacteria with rapidly increasing number of records. It is known from a broad spectrum of hosts and symbiotic relationships varying from parasitic son-killers to coevolving mutualists.
The present study extends the currently known diversity with 34 samples retrieved mainly from hippoboscid (Diptera: Hippoboscidae) and nycteribiid (Diptera: Nycteribiidae) hosts, and investigates phylogenetic relationships within the genus.
The analysis of 110 Arsenophonus sequences (incl. Riesia and Phlomobacter), provides a robust monophyletic clade, characterized by unique molecular synapomorphies. On the other hand, unstable inner topology indicates that complete understanding of Arsenophonus evolution cannot be achieved with 16S rDNA. Moreover, taxonomically restricted Sampling matrices prove sensitivity of the phylogenetic signal to sampling; in some cases, Arsenophonus monophyly is disrupted by other symbiotic bacteria. Two contrasting coevolutionary patterns occur throughout the tree: parallel host-symbiont evolution and the haphazard association of the symbionts with distant hosts. A further conspicuous feature of the topology is the occurrence of monophyletic symbiont lineages associated with monophyletic groups of hosts without a co-speciation pattern. We suggest that part of this incongruence could be caused by methodological artifacts, such as intragenomic variability.
The sample of currently available molecular data presents the genus Arsenophonus as one of the richest and most widespread clusters of insect symbiotic bacteria. The analysis of its phylogenetic lineages indicates a complex evolution and apparent ecological versatility with switches between entirely different life styles. Due to these properties, the genus should play an important role in the studies of evolutionary trends in insect intracellular symbionts. However, under the current practice, relying exclusively on 16S rRNA sequences, the phylogenetic analyses are sensitive to various methodological artifacts that may even lead to description of new Arsenophonus lineages as independent genera (e.g. Riesia and Phlomobacter). The resolution of the evolutionary questions encountered within the Arsenophonus clade will thus require identification of new molecular markers suitable for the low-level phylogenetics.
- Maximum Parsimony
- Lateral Gene Transfer
- Basic Matrix
- Symbiotic Bacterium
- rRNA Operon
Since these descriptions, the number of Arsenophonus records has steadily been increasing, resulting in two important changes in knowledge of Arsenophonus evolution and roles in hosts. First, the known host spectrum has been considerably extended with diverse insect groups and even non-insect taxa. So far, Arsenophonus has been identified from parasitic wasps, triatomine bugs, psyllids, whiteflies, aphids, ticks, ant lions, hippoboscids, streblids, bees, lice, and two plant species [4, 7–23]. Second, these recent studies have revealed an unsuspected diversity of symbiotic types within the genus. This dramatically changes the original perception of Arsenophonus as a bionomically homogeneous group of typical secondary ("S-") symbionts undergoing frequent horizontal transfers among phylogenetically distant hosts. For example, recent findings indicate that some insect groups harbor monophyletic clusters of Arsenophonus, possibly playing a role of typical primary ("P-") symbionts. These groups were reported from the dipteran families Hippoboscidae and Streblidae  and most recently from several lice species [18, 24, 25]. Such a close phylogenetic relationship of different types of symbiotic bacteria is not entirely unique among insect symbionts. With the increasing amount of knowledge on the heterogeneity and evolutionary dynamics of symbiotic associations, it is becoming clear that no distinct boundaries separate the P- and S-symbionts. Thus, in their strict meaning, the terms have recently become insufficient, especially for more complex situations, such as studies exploring bacterial diversity within a single host species [14, 17]. Furthermore, these terms have been shown not to reflect phylogenetic position; remarkable versatility of symbiotic associations can be observed in the Gammaproteobacteria overall, as well as within the individual clusters, such as Arsenophonus or Sodalis [16, 26].
The genus Arsenophonus is striking in the diversity of symbiont types represented. Apart from many lineages with typical S-symbiont features, this genus has given rise to several clusters of P-symbionts [18, 20, 24]. Unfortunately, this heterogeneity introduced an annoying degree of phylogenetic instability and nomenclatory confusion. Because P-symbionts show accelerated evolutionary rates, they form long branches in phylogenies, leading to unstable patterns of clustering as observed for P-symbionts within Enterobacteriaceae . The same behavior can be seen in the louse-specific clade of Arsenophonus, which are consequently originally described as a new bacterial genus Riesia . In addition, the Arsenophonus cluster is the only monophyletic group of symbiotic bacteria currently known to possess at least four highly different phenotypes, including son-killing , phytopathogenicity , obligate association with bacteriocytes in the host [18, 20, 24], and apparently non-specific horizontally transmitted bacteria that are possibly mutualistic . These characteristics indicate that the genus Arsenophonus represents an important and widespread lineage of symbiotic bacteria that serves as a valuable model for examining molecular evolution of bacteria-arthropod associations.
In this study, we add 34 new records on symbionts to the known spectrum of Arsenophonus lineages. We explore and summarize the current picture of Arsenophonus evolution by analyzing all sequences available for this clade. To investigate the phylogenetic position, stability and evolutionary trends of the Arsenophonus cluster, we complete the sample with related symbionts and free-living bacteria. Finally, we explore molecular characteristics and informative value of the 16S rRNA gene as the most frequently used phylogenetic marker.
Sequences and alignments
All phylogenetic analyses of the Basic matrix yielded a monophyletic Arsenophonus clade (Figure 2). The new 34 sequences (Figure 2, arrows), identified by BLAST as putative members or relatives of the genus Arsenophonus, always clustered within the Arsenophonus clade. Their precise position was only partially correlated with host taxon. Some of the Arsenophonus sequences from hippoboscoid hosts clustered within monophyletic host-specific groups (Figure 2, printed in red) while others were scattered across the tree as isolated lineages (Figure 2, printed in dark orange). Two distinct sequences were determined from each individual specimen of O. avicularia; these clustered at distant positions within the tree (Figure 2, numbers with asterisks).
The most typical lineages display short-branches with low divergence and unstable positions within the Arsenophonus clade (Figure 2, printed in dark orange). At the opposite extreme are well supported host-specific clusters exhibiting long branches, such as the louse symbiont Riesia or the symbionts described from several streblid species. An intermediate situation is found in putatively host-specific but less robust clusters, such as the Arsenophonus lineages from triatomine bugs, some hippoboscoids or homopterans (Figure 2). In an analogy to previously analyzed symbiotic bacteria [e.g. [28, 29]], the phylogenetic properties of the sequences were also reflected in their GC contents. In the short-branched taxa, the GC content of the 16S rRNA sequence varies from 51.72 to 54.84%, the values typical for S-symbionts and free-living bacteria . In contrast, the 16S rRNA sequences with low GC content, varying between 46.22 and 51.93%, were found in the long-branched taxa clustering within the host-specific monophyletic lineages (e.g. the symbionts from Ornithomyia, Lipopten a, Trichobius, and the Riesia clade).
Considerable loss of phylogenetic information was observed in the Conservative matrix. In this case, the relationships among individual Arsenophonus lineages were highly unstable, resulting in large polytomies of many short-branched taxa within the consensus trees (see Additional file 1). Also the relationships among the long-branched lineages, although resolved, differ sharply from those derived from the Basic matrix data, and the genus Proteus was not positioned as the closest relative of Arsenophonus. Thus, the information contained in the Conservative matrix (restricted to one fourth of Basic dataset, i.e., 284 bp) is insufficient for reliable phylogenetic placement of closely related taxa.
The calculation of divergence times yielded substantially different results depending on the choice of calibration points. Use of the Riesia diversification as a reference point suggested a recent origin of the triatomine-associated Arsenophonus branch; the median value of the estimate distribution was 2.6 mya. In contrast, the calibration by Escherichia-Salmonella returned considerably higher dates with the median at 24.5 mya.
Phylogenetic patterns and the stability of the information
The low resolution and instability of the trees inferred from the Conservative matrix suggest that a substantial part of the phylogenetic information is located within the "ambiguously" aligned regions that were removed by the GBlocks procedure. This fact is particularly important when considering the frequent occurrence of insertions/deletions within the sequences (see Additional file 3). This may lead to deletion of these critical fragments in many phylogenetic analyses. Interestingly, the monophyletic nature of Arsenophonus was preserved even in this highly Conservative matrix. This indicates that within the complete data set, the phylogenetic information underlying the Arsenophonus monophyly is sufficiently strong and is contained in the conservative regions of the sequences. In accordance with this presumption, several molecular synapomorphies can be identified in the Basic and Conservative matrices. The most pronounced is the motif GTC/GTT located in positions 481–483 and 159–161 of Basic matrix and Conservative matrix, respectively.
Relevance of the sampling
To test an effect of sampling on the phylogenetic inference within Arsenophonus, we examined five Sampling matrices with different taxa compositions (see the section Methods). In addition to the MP, ML, and Bayesian analyses, we performed an ML calculation under the nonhomogeneous model of the substitutions, designated as T92 [31, 32]. This model was previously used to test the monophyly/polyphyly of the P-symbiotic lineages and brought the first serious evidence for a possible independent origin of major P-symbiotic taxa . We were not able to apply the same approach to the Basic and Conservative matrices since the program Phylowin failed to process these large datasets under the ML criterion. The analyses of several taxonomically restricted Sampling matrices proved the sensitivity of phylogenetic signal to the sampling. In the most extreme case, shown in Figure 3A, even the monophyly of the Arsenophonus clade was disrupted by other lineages of symbiotic bacteria. Considering the results of the extensive analysis of the Basic matrix, this arrangement is clearly a methodological artifact. Since both Riesia and the P-symbiont lineage are long-branched taxa with rapid evolution of 16S rDNA, their affinity is very likely caused by Long Branch Attraction (LBA; for review see ) within the taxonomically compromised matrix. It is symptomatic that this topology was inferred by MP, the method known to be particularly prone to the LBA. To further test this distortion, one of the long-branched taxa was removed from the data set (matrix Sampling4). This approach restored the Arsenophonus monophyly and confirmed the effect of LBA phenomenon (see Additional file 2).
The aim of these taxonomically restricted analyses was to "simulate" phylogenetic placement of newly determined symbionts. In such casual studies, the symbiotic lineages are rarely represented by all available sequences in the way we composed the Basic matrix. Rather, each symbiotic lineage is represented by few randomly selected sequences. Under such circumstances, incorrect topologies (e.g. the Sampling5-derived topology on the Figure 4) can be obtained due to various methodological artifacts. This situation can be illustrated by empirical data: at least in two studies, the louse-associated lineage of Arsenophonus was not recognized as a member of the Arsenophonus clade [25, 34]. Consequently, when more recent studies, based on better sampling, proved the position of Riesia within the Arsenophonus cluster [18, 24] the genus Arsenophonus became paraphyletic (see the section Conclusion for more details).
Interestingly, topologies inferred by likelihood analyses using the T92 evolution model  were influenced neither by the compromised sampling nor by the removal of unreliably aligned regions.
Cophylogeny vs. horizontal transfers: possible sources of phylogenetic incongruence
The phylogenetic tree of all Arsenophonus sequences exhibits both patterns, the parallel evolution of symbionts and their hosts and the haphazard association of symbionts from different host taxa. Coincidentally, both arrangements can be demonstrated on the newly sequenced symbionts from various hippoboscoid species. Some of hippoboscoid-associated Arsenophonus show possible host specificity; in a few analyses they cluster within several monophyletic short-branched groups. Since relationships among the short-branched taxa are generally not well resolved, these lineages are scattered throughout the whole topology (Figure 2). In contrast, relationships within the long-branched clusters of hippoboscoid-associated taxa are in agreement with the host phylogeny (the Arsenophonus clusters strictly reflecting the host phylogeny are designated by solid circle in the Figure 2). Interestingly, a coevolutionary pattern was also identified for streblids of the genus Trichobius and their symbionts. In the original study published by Trowbridge et al. , the distribution of Trichobius symbionts was apparently not consistent with the host phylogeny. Our analysis in a broad context indicates that this discrepancy might have been caused by different bacterial sampling and particularly by aberrant behavior of the sequence from Trichobius yunkeri [GenBank: DQ314776]. This sequence is likely to be an artificial chimerical product of at least two distant lineages; according to our BLAST tests it shares 100% identity with S-symbiont of Psylla pyricola [GenBank: AF286125] along a 1119 bp long region. Removal of this sequence from the dataset restored a complete phylogenetic congruence between Trichobius, based on the phylogeny of this genus published by Dittmar et al. , and its symbionts. This finding exemplifies the danger of chimeric sequences in studies of symbiotic bacteria, obtained by the PCR on the sample containing DNA mixture from several bacteria. The presence of several symbiotic lineages within a single host is well known [e.g. [14, 36–38]]. In this study, we demonstrate a possible such case in O. avicularia. From three individuals of this species we obtained pairs of different sequences branching at two distant positions (labelled by the numbers 1* to 3* in Figure 2). The identical clustering seen in all three pairs within the tree shows that they are not chimeric products but represent two different sequences.
While the identity between symbiont relationships and the host phylogeny is apparently a consequence of host-symbiont cophylogeny, the interpretation of the randomly scattered symbionts is less obvious. Usually, such an arrangement is explained as result of transient infections and frequent horizontal transfers among distant host taxa. This is typical, for example, of the Wolbachia symbionts in wide range of insect species . Generally, the capability to undergo inter-host transfers is assumed for several symbiotic lineages and has even been demonstrated under experimental conditions [40, 41]. Since the Arsenophonus cluster contains bacteria from phylogenetically distant insect taxa and also bacteria isolated from plants, it is clear that horizontal transfers and/or multiple establishments of the symbiosis have occurred. However, part of the incongruence could be caused by methodological artifacts. A conspicuous feature of the Arsenophonus topology is the occurrence of monophyletic symbiont lineages associated with monophyletic groups of insect host but without a co-speciation pattern. Although our study cannot present an exhaustive explanation of such a picture, we want to point out two factors that might in theory take part in shaping the relationships among Arsenophonus sequences, lateral gene transfer (LGT) and intragenomic heterogeneity. Both have previously been determined as causes of phylogenetic distortions and should be considered in coevolutionary studies at a low phylogenetic level.
Incongruence due to LGT and intragenomic heterogeneity
An apparently "mosaic" structure of the Arsenophonus alignment (for example see Additional file 4) raises the question of whether various regions of this sequence could have undergone different evolutionary histories. Recombination of 16S rDNA genes were previously identified in some other bacteria [42–44]. In actinomycetes, the occurrence of short rDNA segments with high number of non-random variations was attributed to the lateral transfer as the most parsimonious explanation . Later, Gogarten et al.  suggested that, analogously to an entire bacterial genome, 16S rDNA possesses a mosaic character originated by LGT, respectively by transfer of gene subunits.
As bacterial genomes often carry more than one rRNA operon, intragenomic heterogeneity of the rDNA copies is occasionally found to blur the phylogenetic picture [47–50]. Although there is no direct information on the number of rRNA gene copies in Arsenophonus genomes, Stewart and Cavanaugh  showed bacterial genomes to encode in average five rRNA operons. The most closely related bacterium of which the complete genome has recently been sequenced, Proteus mirabilis, carries seven copies [GenBank: AM942759]. Arsenophonus-focused studies indicate that two different forms of the rRNA operon are present in its genome, as is typical for Enterobacteraceae [23, 52]. Furthermore, Šorfová et al.  suggest that the variability among individual copies may cause the incongruence observed between triatomines and their Arsenophonus lineages. They point out that this process could, in principle, explain an otherwise problematic observation: in some hosts, such as triatomines or some homopterans, the hosts and the Arsenophonus bacteria create reciprocally monophyletic clusters but do not show any cospeciation pattern. In the symbionts of grain weevils, divergence between rRNA sequences within a genome was shown sometimes to exceed divergence of orthologous copies from symbionts from different hosts; this unusual situation was hypothesized to reflect loss of recombinational repair mechanisms from these symbiont genomes .
Estimates of the divergence time
With the present incomplete knowledge of the Arsenophonus genome, it is difficult to assess whether and how deeply rRNA heterogeneity affects phylogenetic reconstruction. Trying to find alternative solution, Šorfová et al.  attempted to use the estimation of divergence times as a guide for deciding between different coevolutionary scenarios. They used the Escherichia-Salmonella divergence [54, 55] as a calibration point for calculating the divergence time among various Arsenophonus lineages from triatomine bugs. Applying the Multdiv method , they placed the ancestor of triatomine-associated symbionts into a broad range of approx. 15 – 40 mya and concluded that this estimate is compatible or even exceeds the age estimates available for the tribe triatomine (according to Gaunt and Miles ). Here, we took advantage of a new age-estimate for closely related bacteria, namely the louse-associated symbionts of the genus Riesia . Comparing the estimates based on the two calibration methods (Escherichia-Salmonella and Riesia), we found that due to the variability of evolutionary rates among the lineages, the results may differ by an order of magnitude. Such marked variance among different bacterial lineages (including different symbiotic bacteria from the same host species) was previously reported for many bacterial groups [29, 30, 37, 39, 58–63]. Most recently, Allen et al.  reported an extremely high evolutionary rate for the young symbiotic lineage Riesia, and suggested that the evolutionary tempo changes with the age of the symbiotic lineage. We therefore conclude that this method cannot be directly used to assess the effect of intragenomic heterogeneity on our reconstruction of Arsenophonus relationships.
With more than one hundred records, the genus Arsenophonus represents one of the richest and most widespread clusters of insect symbiotic bacteria. Considering its broad host spectrum and apparent ecological versatility, Arsenophonus should play an important role in studies of evolutionary trends in insect intracellular symbionts. Due to this fact, Arsenophonus is likely to attract a growing attention, and the number of the records may rapidly be increasing during the next years. For example, 7 new sequences were deposited into the GenBank since the completion of this study [, and unpublished record FJ388523]. However, since these new Arsenophonus records originated in screening rather than phylogenetic study, they are only represented by short DNA fragments (approx. 500 bp). Preliminary analyses of these fragments together with our complete datasets confirmed a limited informative value of such short sequences and they were not included into the more exhaustive phylogenetic procedures.
The analysis of 110 available sequences of Arsenophonus 16S rDNA from 54 host taxa revealed several interesting evolutionary patterns. In particular, this clade includes at least two transitions from S-symbiont, with ability to invade new host lineages, to P-symbiont, showing obligate relationship to hosts and a strict pattern of maternal transmission. Thus, it is a promising system for exploring the genomic and biological changes that accompany the shift from facultative to obligate symbiont. Arsenophonus is also one of the few groups of insect symbionts for which strains have been grown in pure culture [4, 7, 16], a feature that further enhances its potential as a model for symbiont research.
Our results also indicate that a complete understanding of the Arsenophonus phylogeny cannot be achieved with 16S rDNA genes alone. A similar situation is, for example, found in another large symbiotic group, the genus Wolbachia, where other genes are often used as alternative sources of phylogenetic information [66, 67]. Identification of suitable low-level-phylogeny marker(s) is thus one of the most crucial steps in the further research on Arsenophonus evolution. The sequencing of the complete Arsenophonus genome, which is currently under the process http://genomesonline.org/gold.cgi?want=Bacterial+Ongoing+Genomes&pubsort=Domain, will provide a valuable background for such enterprise.
Based on the presented analyses, we also want to point out that the genus Arsenophonus is currently paraphyletic due to the two lineages described as separate genera Riesia and Phlomobacter but clustering within the Arsenophonus group (e.g. Figure 2). Two procedures can, in principle, solve this undesirable situation, splitting of the Arsenophonus cluster into several separate genera or classification of all its members within the genus Arsenophonus. Taking into account the phylogenetic arrangement of the individual lineages, the first approach would inevitably lead to establishment of many genera with low sequence divergences and very similar biology. The second option has been previously mentioned in respect to the genus Phlomobacter , and we consider this approach (i.e. reclassification of all members of the Arsenophonus clade within a single genus) a more appropriate solution of the current situation within the Arsenophonus clade.
The host species used in this study were acquired from several sources. All of the nycteribiid samples were obtained from Radek Lučan. Most of the hippoboscids were provided by Jan Votýpka. Ant species were collected by Milan Janda in Papua New Guinea. All other samples are from the authors' collection. List of the sequences included in the Basic matrix is provided in the Additional file 5.
DNA extraction, PCR and sequencing
The total genomic DNA was extracted from individual samples using DNEasy Tissue Kit (QIAGEN; Hilden, Germany). Primers F40 and R1060 designed to amplify approx. 1020 bp of 16S rDNA, particularly within Enterobacteriaceae , were used for all samples. PCR was performed under standard conditions using HotStart Taq polymerase (HotStarTaqi DNA Polymerase, Qiagen). The PCR products were analyzed by gel electrophoresis and cloned into pGEM-T Easy System 1 vector (Promega). Inserts from selected colonies were amplified using T7 and SP6 primers and sequenced in both directions, with the exception of 3 fragments sequenced in one direction only (sequences from Aenictus huonicus and Myzocalis sp.). DNA sequencing was performed on automated sequencer model 310 ABI PRISM (PE-Biosystems, Foster City, California, USA) using the BigDye DNA sequencing kit (PE-Biosystems). For each sample, five to ten colonies were screened on average. The contig construction and sequence editing was done in the SeqMan program from the DNASTAR platform (Dnastar, Inc. 1999). Identification of the sequences was done using BLAST, NCBI http://www.ncbi.nlm.nih.gov/blast/Blast.cgi.
To analyze thoroughly the behavior of Arsenophonus 16S rDNA and assess its usefulness as a phylogenetic marker, we prepared several matrices and performed an array of phylogenetic analyses on each of them.
The Basic matrix was composed of the 34 new sequences, all Arsenophonus sequences available in the GenBank and additional 45 sequences of various P-symbionts, S-symbiont and 5 free living bacteria (see Additional file 5). To show the impact of random or restricted sampling on the resulting topology, five different matrices labelled Sampling i (i.e. Sampling1, Sampling2, etc.) were prepared from Basic matrix by removing various taxa and including additional/alternative outgroups. The matrices Sampling1 to Sampling4 were composed of various numbers of non-Arsenophonus symbiotic taxa (ranging from 3 to 35), three sequences of free-living bacteria, and an arbitrarily selected set of all Arsenophonus lineages. Matrix designated as Sampling5 was restricted to a lower number of taxa, including 5 ingroup sequences and alternative lineages of symbiotic and free-living bacteria.
All matrices were aligned in the server-based program MAFFT http://align.bmr.kyushu-u.ac.jp/mafft/online/server/, using the E-INS-i algorithm with default parameters. The program BioEdit  was used to manually correct the resulting matrices and to calculate the GC content of the sequences.
To test an effect of unreliably aligned regions on the phylogenetic analysis, we further prepared the Conservative matrix, by removing variable regions from the Basic matrix. For this procedure, we used the program Gblocks  available as server-based application on the web page http://molevol.cmima.csic.es/castresana/Gblocks_server.html.
Finally, the Clock matrix, composed of 12 bacterial sequence (see Additional file 5), was designed to calculate time of divergence for several nodes within the Arsenophonus topology.
The matrices were analyzed using maximum parsimony (MP), maximum likelihood (ML) and Bayesian probability. For analyses, we used the following programs and procedures. The GTR+Γ+inv model of molecular evolution was determined as best fitting by the program Modeltest  and was used in all ML-based analyses. MP analysis was carried out in TNT program  using the Traditional search option, with 100 replicates of heuristic search, under the assumptions of Ts/Tv ratio 1 and 3. ML analysis was done in the Phyml program  with model parameters estimated from the data. Bayesian analysis was performed in Mr. Bayes ver. 3.1.2. with following parameter settings: nst = 6, rates = invgamma, ngen = 3000000, samplefreq = 100, and printfreq = 100. The program Phylowin  was employed for the ML analysis under the nonhomogeneous model of substitution .
A calculation of divergence time was performed in the program Beast  which implements MCMC procedure to sample target distribution of the posterior probabilities. The gamma distribution coupled with the GTR+invgamma model was approximated by 6 categories of substitution rates. Relaxed molecular clock (uncorrelated lognormal option) was applied to model the rates along the lineages. To obtain a time-framework for the tree, we used the estimate on louse divergence (approximately 5.6 mya ). Since the resulting estimate was considerably lower that that reported previously with Escherichia-Salmonella calibration , we prepared an additional matrix and used the Escherichia-Salmonella split [54, 55] as an alternative calibration; taxa included according to Šorfová et al. . All analyses were performed in three independent runs, each taking 5 million generations.
This work was supported by Ministry of Education, Czech Republic (grants LC06073 and MSM 60076605801), the Grant Agency of Academy of Sciences of the Czech Republic (Grant IAA601410708) and a National Science Foundation grant (0626716) to N.A. Moran (University of Arizona). We thank all of our collaborators for providing the samples.
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