* Departamento de Xenética, Facultade de Bioloxía, Universidade de Santiago de Compostela, Spain; Departamento de Bioloxía Celular e Molecular, Universidade de A Coruña, Spain; and
Unidade de Medicina Molecular, INGO, Complexo Hospitalario Universitario de Santiago de Compostela, Spain
Correspondence: E-mail: bfcostas{at}usc.es.
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Abstract |
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Key Words: Ty3/gypsy retrotransposon Anopheles gambiae Drosophila melanogaster
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Introduction |
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With the availability of an increasing number of eukaryotic genomic sequences, a primary task in studies of transposon evolution is the characterization of the full transposon complement of sequenced genomes (Holmes 2002; Kaminker et al. 2002). The recently released genome of the Diptera A. gambiae (Holt et al. 2002) offers an extraordinary opportunity for comparative studies of TEs diversity and evolutionary dynamics between two related species, taking advantage of the existing information from D. melanogaster (Kaminker et al. 2002; Lerat, Rizzon, and Biémont 2003).
The most abundant type of TEs in Drosophila is the Ty3/gypsy group of long-terminal repeat (LTR) retrotransposons, also referred to as Metaviridae according to virus taxonomy (Boeke et al. 2000). Nine different lineages of this group have been so far identified in different organisms, based on the phylogenetic analysis of their reverse transcriptase (RT), ribonuclease H (RNaseH), and integrase (INT) amino acid domains (Malik and Eickbush 1999; Bae et al. 2001). However, so far, only six of them have been identified in insects, namely CsRn1, Gypsy, Mag, Mdg1, Mdg3, and Osvaldo. All but Mag have been previously detected in D. melanogaster (Bae et al. 2001; Kaminker et al. 2002; Kapitonov and Jurka 2003).
Our analysis of the Mdg1 lineage of A. gambiae revealed the existence of 10 different families, mainly consisting of degenerate copies and solitary LTRs (solo LTRs), although some of them also contain very recent, putatively active, insertions (Tubío, Costas, and Naveira 2004). Three additional Ty3/gypsy elements have been partially characterized previously; two of them (referred to as A. gambiae retrotransposon 1 and A. gambiae retrotransposon 2 [Volff et al. 2001]) belong to the Mag lineage, whereas the other, Ozymandias (Hill et al. 2001), has been assigned to the CsRn1 lineage (Tubío, Costas, and Naveira 2004). Here, we report our findings on the diversity of the Ty3/gypsy group of LTR-retrotransposons in A. gambiae. In addition to the recently published study focused on the non-LTR retrotransposons (Biedler and Tu 2003), this work represents an important step towards the characterization of the full set of TEs within the genome of the African malaria mosquito.
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Materials and Methods |
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All the family consensus sequences first reported in this paper have been deposited in the A. gambiae section of Repbase Update (http://www.girinst.org/Repbase_Update.html [Jurka 2000]). They were named from GYPSY18_AG to GYPSY72_AG. The previously discovered element Ozymandias (Hill et al. 2001) and the elements A. gambiae retrotransposon 1 and A. gambiae retrotransposon 2 (Volff et al. 2001) have been renamed as GYPSY50_AG, GYPSY28_AG, and GYPSY55_AG, respectively, after its full characterization, according to Repbase terminology. The families for which no consensus could be obtained are reported in this paper with the name of the contig or scaffold where a representative sequence was identified.
Characterization of Insertions
Putative open reading frames (ORFs) were found by sorted three-frame translation of each TE insertion with the aid of BioEdit version 5.0.9. The primer-binding site (PBS) of each element was localized by searching the compilation of tRNA sequences of Sprinzl et al. (1999), using sliding windows of 9 bp at 1-bp steps as probes, starting 1 bp relative to the 5' LTR end. Individual insertions were considered putatively active if they contained intact ORFs (i.e., without any frameshift or nonsense mutation) and two nontruncated LTRs (i.e., LTRs without indels >10 bp, as compared with the consensus sequence). Those insertions with frameshift mutations, nonsense mutations, or truncated LTRs were classified as inactive insertions. Those insertions with unsequenced gaps but meeting the criteria to be regarded as putatively active based on the analysis of the available sequence were not assigned to a specific activity status. Those insertions bearing identity exclusively to the LTR of a family consensus sequence were considered solo LTRs. Average pairwise divergence between both LTRs from the same element copy and between different copies of the same family were obtained as the proportion of nucleotide differences with the aid of MEGA version 2.1 (Kumar et al. 2002), using the pairwise deletion option.
Multiple Sequence Alignments and Phylogenetic Analyses
Our phylogenetic analyses were based on the alignment of the seven amino acid domains of the RT defined by Xiong and Eickbush (1990) and the RNaseH and INT domains defined by Malik and Eickbush (1999). The general alignment, available as Supplementary Material online, was obtained in two steps. First, we generated an alignment for each one of the Ty3/gypsy lineages present in insects using the multiple-alignment mode of ClustalX (Thompson et al. 1997). Each one of the alignments included the consensus sequences of the A. gambiae elements of the lineage, the available representative sequences of D. melanogaster elements of the lineage, and representative sequences of all the lineages (Cer1, CsRn1, Cyclops, Gypsy, Mag, Mdg1, Mdg3, Osvaldo, and Ty3). Second, these different alignments were joined together manually, using as guide the representative sequences for each one of the Ty3/gypsy lineages, common to all the lineage-specific alignments, with the help of BioEdit. For the purpose of phylogenetic analyses, the amino acid motifs of the D. melanogaster insertions at genomic sequences AC016130 and AE003787, corresponding to elements belonging to the Mag and CsRn1 lineages, respectively, have been reconstructed by the introduction of gaps to compensate for frameshift mutations.
Phylogenetic relationships between different retrotransposons based on this general alignment were obtained both by distance (neighbor-joining [NJ]) and maximum-parsimony (MP) methods, as implemented in MEGA version 2.1, using the pairwise deletion option. The amino acid distances were computed using the Poisson correction for multiple substitutions and assuming equality of substitution rates among sites. In MP analyses, we searched for the best tree using the close-neighbor interchange, with default parameter values and random addition of sequences to produce the initial trees. In both MP and NJ analyses, bootstrapping was performed (1,000 replicates) to assess the support for each internal branch of the tree.
Statistical Analysis of the Distribution of Insertions in the X Chromosome Versus the Autosomes
The equiproportional hypothesis of Montgomery, Charlesworth, and Langley (1987) postulates that the turnover of insertions should occur at equal rates on the X chromosome and the autosomes. Under this hypothesis, the expected ratio of haploid mean copy number of any given family in the X chromosome and the autosomes (HX/HA) at equilibrium can be obtained by solving the quadratic in X = HX/HA, after assigning numerical values to the constants in equation (2) of Montgomery, Charlesworth, and Langley (1987), corrected after Langley et al. (1988). We followed the statements of Krzywinski et al. (2004) and assumed that the Y chromosome is entirely heterochromatic, that it constitutes 10% of the haploid genome of a male, and that 975 out of the 8,845 total unmapped scaffolds of the A. gambiae genome are most likely to be linked to this chromosome. We also followed Holt et al. (2002) for assumptions on the relative size of each chromosome. All unmapped scaffolds, amounting to roughly 44 Mb, were pooled into a separate category, conceptually equivalent to part of the "heterochromatin" in the models of Montgomery, Charlesworth, and Langley (1987). Finally, after assuming that transposition rates per copy per generation do not differ either between sexes or between heterochromatic and euchromatic insertions, a value of 8.0% was obtained for the expected proportion of elements on the X chromosome under this equiproportional hypothesis. Observed and expected frequencies were compared by means of 2 tests.
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Results |
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Figure 1 shows the phylogenetic relationships of all the well-characterized families from D. melanogaster and A. gambiae belonging to the Ty3/gypsy group of LTR retrotransposons, based on the alignment of the conserved amino acid domains of RT, RNaseH, and INT; in addition to D. melanogaster elements belonging to the Mag (AC016130) and CsRn1 (AE003787) lineages, as well as representatives from other species of each one of the six lineages of the Ty3/gypsy group without well-characterized sequences in D. melanogaster genome.
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In contrast to A. gambiae, the CsRn1 lineage appears to be poorly represented in the D. melanogaster genome. Our Blast searches to evaluate this observation revealed the existence of only one family (referred to as AE003787DM in the phylogeny) with five insertions in the fly genome: three solo LTRs (located at genomic scaffolds AE003522, from 83073 to 83278; AE003526, from 203102 to 203307; and AE003784, from 304622 to 304425), one partial insertion (genomic scaffold AE03843; from 326893 to 322718) and one complete insertion bearing inactivating mutations in the INT domain (genomic scaffold AE003787; from 212564 to 208162).
The Gypsy Lineage
We have identified 24 putative new families belonging to the Gypsy lineage, but we were only able to obtain a consensus sequence for nine of them. Neither of the previously described Anopheles elements belonging to this lineage (Afun1 from A. funestus and Aste11 from A. stephensi [Cook et al. 2000]) were identified in A. gambiae. As shown in figure 2, we have identified an env-like ORF3 in three of the nine families, conforming to a R-X2-R-X4-5-6-G-X3-K-X3-G-X2-D-X2-D rule, which is slightly different from the general pattern proposed as a specific probe for the in silico detection of insect endogenous retroviral envelop protein (Terzian, Pélisson, and Bucheton 2001). The 18 insertions of families GYPSY41_AG, GYPSY42_AG, and GYPSY43_AG, where a target-site duplication (TSD) could be identified, showed preferential insertion at ATAT sites. Two other families of the Gypsy lineage (GYPSY44_AG and GYPSY45_AG) also showed preferential insertion at C(G/T)CG, based on 12 individual members.
The Mag Lineage
Two A. gambiae elements of the Mag lineage had already been partially characterized (referred to as A. gambiae retrotransposon 1 and A. gambiae retrotransposon 2 [Volff et al. 2001]). We have identified 53 putative families in the A. gambiae genome belonging to this lineage, but it has only been possible to characterize in detail 30 of them, representing 48% of all the characterized Ty3/gypsy families in A. gambiae. A. gambiae retrotransposon 1 and A. gambiae retrotransposon 2 have been identified as members of families GYPSY28_AG and GYPSY55_AG, respectively.
So far, no Mag-like TEs had been identified in the genus Drosophila. To confirm the absence of the Mag lineage from Drosophila, we carried out Blast searches of the genome of D. melanogaster, using the pol region of different A. gambiae families as queries. This search led to the detection of an insertion within a 2R centromeric heterochromatin sequence (AC016130.13, unfinished sequence; pol region around nucleotide positions 89502 to 91566), most similar to elements from the Mag lineage. The insertion bears several inactivating mutations. Additional hits related to this element were identified in unfinished genomic sequences. Phylogenetic analyses revealed that this element and other Mag families cluster together with high bootstrap values, representing and old branch of the lineage (fig. 1).
The Mdg1 lineage
Most mosquito members of this lineage have been described elsewhere (Tubío, Costas, and Naveira 2004). Here, we show the existence of a basal member of this lineage (GYPSY54_AG). Six additional putative families related to this one had to be excluded from the analysis (table 1 in Supplementary Material online). All the other A. gambiae families of this lineage are more related to D. melanogaster families than to this novel family (fig. 1). Nevertheless, both the phylogenetic relationships (well-supported by bootstrap values in the case of NJ) and the structural characteristics of this family are consistent with its classification within the Mdg1 lineage. Namely, the element lacks a CCHC domain, contains a GPY/F domain, and the translation of the pol ORF requires a frameshift of 1 bp as in the remaining elements of the lineage (fig. 2 [Tubío, Costas, and Naveira 2004]), although we were not able to identify any tRNA complementary to the PBS. Blast searches failed to identify elements closely related to this one in other genomes.
The Mdg3 Lineage
We have identified 25 putative new families in the A. gambiae genome belonging to this lineage, but it has only been possible to offer a full description of 16 of them. No Mdg3 lineage elements had been described in A. gambiae before this work.
Analysis of Individual Insertions
Table 1 shows the total number of insertions, classified as putatively active insertions, inactive insertions, and solo LTRs, belonging to each one of the families, as well as the chromosomal distribution of all the insertions. The most abundant family is GYPSY50_AG, containing 28 members. Five additional families are constituted by more than 20 members. We have identified putatively active members for 47 of the 63 characterized families. Nevertheless, it must be pointed out that at least some of the unclassified members (those meeting the criteria to be considered active but with short stretches of unfinished sequence) are most likely to be active. For 71 of the 85 putatively active elements, belonging to 40 different families, the two LTRs are identical in sequence. In addition, all families present inactive members, which have accumulated several indels and/or nonsense mutations. In general, the number of putatively active members per family is lower than that of inactive members. It was possible to calculate the average pairwise identity between putatively active copies and between inactive copies for 17 families (table 2). In all but one case, the identity was higher between putatively active copies. This difference is highly significant (Student's t-test = 6.319, P < 0.001). The average pairwise identity between putatively active copies was higher than 99% in the 17 families.
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Discussion |
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The other two mosquito lineages are almost absent from D. melanogaster but abundant in A. gambiae. The most extreme case is that of the Mag lineage. Thus, whereas we have identified for the first time a family of Mag-like elements in D. melanogaster, most probably consisting of old inactive members, A. gambiae contains at least 30 families, most of them putatively active, with an extraordinary structural diversity, accounting for approximately 48% of all the characterized Ty3/gypsy families in A. gambiae (fig. 2). Interestingly, 13 of the 30 families arose in a short period of evolutionary time (cluster J in figure 1). A similar situation occurs in the case of the CsRn1 lineage. We have characterized seven families belonging to this lineage in A. gambiae, whereas the D. melanogaster genome bears only a single family (Bae et al. 2001) with just two full-length members bearing inactivating mutations. In summary, our data reveal a rich diversity of LTR retrotransposons in A. gambiae, clearly greater than in D. melanogaster. A similar situation has been recently described in the case of non-LTR retrotransposons (Biedler and Tu 2003).
The seven new CsRn1 families characterized for the first time in this paper constitute a significant contribution to the total number of known families of this lineage, detected mainly in Trematoda (Bae et al. 2001; Copeland et al. 2003). This fact allows us to confirm the general distinctive characteristics of the CsRn1 lineage, such as a PBS complementary to tRNATrp, the unusual CHCC gag motif instead of the typical CCHC motif, and the existence of the GPY/F motif at the 3' end of the INT gene (Bae et al. 2001). In a similar way, each lineage shows distinctive features (or combination of features), as clearly shown in figure 2 from the A. gambiae representatives. Thus, members of the related lineages Gypsy and Mdg1 are characterized by a frameshift of 1 bp at the gag-pol boundary and the absence of the CCHC motif at the C-terminal end of gag. They differ in the presence (Mdg1) or absence (Gypsy) of the GPY/F motif. Members of the Mdg3 lineage are the only ones with a single gag-pol ORF bearing the CCHC gag motif and a GPY/F domain at the C-terminal end. Members of the Mag lineage also have the conventional CCHC gag motif, but they lack the GPY/F motif at the 3' end of the INT gene. In addition, it is the unique lineage that causes a TSD at the insertion site of 5 bp, instead of the typical 4 bp.
Several characteristics, although in general are conserved within members from the same lineage, evolved in some specific branches of the phylogenetic tree. One example of this is the PBS. Thus, four closely related Mdg3 families from A. gambiae (AAAB01008445, GYPSY37_AG, GYPSY38_AG, and GYPSY71_AG), clustered together with a strong bootstrap support (cluster F in figure 1), seem to shift from a PBS complementary to tRNALeu, common to all the other elements from the Mdg3 lineage, to another complementary to tRNAPr°. A similar situation has been detected in the case of the Mag-like elements GYPSY24_AG and GYPSY66_AG (cluster I in figure 1). These elements contain a PBS complementary to tRNALeu instead of one complementary to tRNASer as the other members of the Mag lineage, with the exception of GYPSY68_AG, which contains a PBS complementary to tRNAArg. Members of the Gypsy lineage may be further split into two groups, based on the presence of a PBS complementary to tRNASer or to tRNALys, as pointed out previously in the case of Drosophila elements (Terzian, Pélisson, and Bucheton 2001). The phylogenetic tree of figure 1 strongly suggests that the PBS complementary to tRNALys arose later in a specific branch of the tree (cluster A in figure 1). Interestingly, this acquisition predated the split of Diptera.
Another clear example of distinctive characteristics evolving at specific branches is the TRS at the gag-pol boundary. Thus, a wide variety of strategies have been identified within the Mag lineage. Although most elements contain a single ORF, there is one element presenting a 1 frameshifting (characteristic of other lineages) and a cluster of three elements (cluster H in figure 1) showing two nonoverlapping ORFs separated by more than 100 bp. A similar TRS has been previously observed in several plant retrotransposons, but the mechanism to express pol in these cases is not clear, although splicing, internal ribosomal entry, or a bypass mechanism have been suggested (Gao et al. 2003). Furthermore, there is a mosquito family of the Mag lineage characterized by a long ORF encoding all the protein domains with the exception of INT, which is encoded by a different overlapping ORF, requiring a frameshift of 2 to be translated. The reasons for this particular structure are unknown.
The CsRn1 lineage also shows different TRS. While some elements show a conventional 1 frameshifting, there is a cluster (cluster G in figure 1) that has a stop codon at the gag-pol boundary. Stop codon readthrough has been previously described in a few elements, such as the Kamikaze element from B. mori, the RIRE2 element from rice and several mammalian retroviruses (revised in Gao et al. [2003]). Interestingly, the LTRs termini of members of this cluster are TG...AA, instead of the expected TG...CA. Thus, all Drosophila retrotransposons have the TG...CA termini except those from the Gypsy lineage that show AG...YT at LTR ends (Kapitonov and Jurka 2003) and the Drosophila family from this lineage (AE003787) that shows TG...TA (Bae et al. 2001).
We have also identified two clear cases of acquisition of preferential insertion in specific sequences. Thus, those elements belonging to the cluster formed by GYPSY41_AG, GYPSY42_AG, and GYPSY43_AG (cluster C in figure 1) are inserted at ATAT sites and those belonging to the related families GYPSY44_AG and GYPSY45_AG are inserted at C(G/T)CG sites (cluster B in figure 1). This preferential insertion might play an important role in host-retrotransposon coevolution (SanMiguel et al. 1996; Voytas 1996).
Finally, the env ORF present in some members of the Gypsy lineage deserves more attention. It has been shown that this lineage has acquired its env gene from a class of insect baculoviruses early in its evolution (Malik, Henikoff, and Eickbush 2000). Later, a few Drosophila elements have lost the env gene, such as Burdock (Terzian, Pélisson, and Bucheton 2001). Our survey of elements of the Gypsy lineage in A. gambiae revealed the existence of nine families. Only three of them (namely GYPSY41_AG, GYPSY46_AG, and GYPSY47_AG) conserve the env gene. Taking into account the phylogenetic relationships shown in figure 1, this fact implies three independent losses of the env ORF during the evolution of these elements (branches B, D, and E in figure 1). The role of the env gene in the life cycle of the elements from the Gypsy lineage remains enigmatic. It has been shown that the env protein of Gypsy may confer infectious properties to the element (Song et al. 1994), leading to the suggestion of a mechanism for Gypsy mobilization through infection of the germline by retroviral particles produced in the follicle cells (Song et al. 1997). Nevertheless, amplification of Gypsy of D. melanogaster may occur in an env-independent manner in the female germline (Chalvet et al. 1999). In a similar way, the Drosophila Zam element, which contains an env ORF, enters the oocyte via the vitelline granule traffic with no apparent need for its env protein, after expression in follicle cells surrounding the oocyte (Leblanc et al. 2000).
Turnover of LTR Retrotransposons in A. gambiae
We have identified 47 families of the Ty3/gyspy group in mosquito containing putatively active elements (table 1). The average pairwise identity between the putatively active elements of each one of the families is always higher than 99% (table 2). Furthermore, 83.5% of the putatively active elements have identical flanking LTRs (table 1). Thus, the genome of the PEST strain of A. gambiae, the strain selected by the Anopheles genome project, presents clear evidence of recent activity for around 75% of the LTR retrotransposon families characterized in this work.
Lerat, Rizzon, and Biémont (2003) have recently shown that, in general, the TE families of D. melanogaster are characterized by a high degree of homogeneity and a lack of divergent elements. By contrast, all the families of the Ty3/gypsy group in A. gambiae contain a significant proportion of inactive degenerated elements, bearing indels and/or nonsense mutations and showing an average pairwise divergence significantly higher than that between active members (tables 1 and 2). Thus, there are around 40% of inactive degenerated elements within the sequenced genome of A. gambiae but only around 13% of active copies. Eighteen percent of the insertions (117/642) correspond to elements without obvious inactivating mutations but with unsequenced gaps, precluding their classification as either active or inactive. The significant overrepresentation of inactive elements within the unmapped scaffolds strongly indicates that heterochromatin is a shelter for these degenerate copies because of lower selection against inserted elements in heterochromatin. Bearing in mind that natural selection acts against inserted elements mainly because of insertional mutations and to chromosomal rearrangements generated by ectopic exchange between different insertions (Charlesworth and Langley 1989; Charlesworth, Sniegowski, and Stephan 1994), this lower selection is easily explained by the reduced gene density and recombination rates in heterochromatic regions. The high frequency of inactive copies is a strong indication of a slower turnover rate of Ty3/gypsy retrotransposons within the genome of A. gambiae than within that of D. melanogaster, most probably reflecting a reduced efficacy of selection against insertions of Ty3/gypsy retrotransposons in A. gambiae. The weakening of the efficacy of selection against TE insertions might be related to the complex genetic population structure of A. gambiae sensu stricto (della Torre et al. 2002). This species is composed of different isolated or semiisolated genetic units. There are different chromosomal and molecular forms showing incomplete premating barriers. This complex structure might give rise to a reduced effective population size and/or a reduced recombination rate, both features leading to a reduced efficiency of selection against TE insertions (Charlesworth and Langley 1989; Charlesworth, Sniegowski, and Stephan 1994).
In addition to the high frequency of inactive members, we have noted a strong excess of solo LTRs in A. gambiae in comparison with D. melanogaster, confirming our previous observation from the Mdg1 lineage (Tubío, Costas, and Naveira 2004). Thus, 176 of the 642 insertions (27%) identified in the present work correspond to solo LTRs versus 58 of 740 insertions (7.8%) reported by Kaminker et al. (2002) in D. melanogaster. This feature is in agreement to the above-mentioned slower turnover rate of retrotransposons in Anopheles. As a consequence, each individual insertion remains within the genome for a longer period of time, increasing the probability of exchange between the two LTRs flanking an element, giving rise to solo LTRs.
Finally, we have found a significant overrepresentation of insertions of LTR-retrotransposons on the X chromosome in comparison with the autosomes. Taking into account that A. gambiae exhibits comparable recombination frequencies in both sexes but males are hemizygous (Zheng et al. 1996), the overrepresentation of insertions on the X chromosome might be simply explained by a stronger selective pressure against autosomal insertions, because of their higher opportunity of ectopic recombination, according to theoretical expectations (Charlesworth and Langley 1989; Charlesworth, Sniegoski, and Stephan 1994). Nevertheless, the chromosomal distribution of TEs depends on a series of complex interacting factors in addition to recombination rates such as gene density, chromatin structure, transposition mechanisms, or interactions between TEs and host genes (Carr et al. 2002; Rizzon et al. 2002). Thus, another possibility to explain the underrepresentation of autosomal insertions might be, for instance, a lower transposition rate per copy per generation for overall male genomes. It is interesting to note that different transposition rates between sexes have been detected for specific elements (Pasyukova et al. 1997).
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Supplementary Material |
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Supplementary Alignment. Text file in fasta format. Insertions not assigned to families.
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Acknowledgements |
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Footnotes |
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References |
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