The ABC transporter gene family of Caenorhabditis elegans has implications for the evolutionary dynamics of multidrug resistance in eukaryotes
Genome Biology volume 5, Article number: R15 (2004)
Many drugs of natural origin are hydrophobic and can pass through cell membranes. Hydrophobic molecules must be susceptible to active efflux systems if they are to be maintained at lower concentrations in cells than in their environment. Multi-drug resistance (MDR), often mediated by intrinsic membrane proteins that couple energy to drug efflux, provides this function. All eukaryotic genomes encode several gene families capable of encoding MDR functions, among which the ABC transporters are the largest. The number of candidate MDR genes means that study of the drug-resistance properties of an organism cannot be effectively carried out without taking a genomic perspective.
We have annotated sequences for all 60 ABC transporters from the Caenorhabditis elegans genome, and performed a phylogenetic analysis of these along with the 49 human, 30 yeast, and 57 fly ABC transporters currently available in GenBank. Classification according to a unified nomenclature is presented. Comparison between genomes reveals much gene duplication and loss, and surprisingly little orthology among analogous genes. Proteins capable of conferring MDR are found in several distinct subfamilies and are likely to have arisen independently multiple times.
ABC transporter evolution fits a pattern expected from a process termed 'dynamic-coherence'. This is an unusual result for such a highly conserved gene family as this one, present in all domains of cellular life. Mechanistically, this may result from the broad substrate specificity of some ABC proteins, which both reduces selection against gene loss, and leads to the facile sorting of functions among paralogs following gene duplication.
ATP-binding cassette (ABC) transporters are one of the largest families of transport proteins constituting the single largest gene family, comprising about 5% of the genome, in Escherichia coli . ABC transporters are grouped into several structural classes, or subfamilies, on the basis of amino acid sequence and domain organization  (Figure 1). The presence of a strongly conserved ATP-binding motif defines membership in the family and the basic functional organization of an ABC transporter in the membrane is the same from bacteria to humans, and in all subclasses [3–5]. A complex of at least two ATP-binding domains, coupled to two blocks of membrane-spanning helices, appears to be the minimum requirement for a functional transporter. Often these domains are found in tandem within a single molecule, but in many cases are distributed across separate proteins that must then assemble in the membrane. ABC transporters are collectively able to accommodate an unusually large array of different substrates. This diversity of function is manifest at the family level, but also in individual members of the family, for example those associated with multidrug resistance (MDR).
Decottignies and Goffeau  catalogued the entire ABC transporter family of the yeast Saccharomyces cerevisiae and in so doing delineated six of the major subgroups of eukaryotic ABC transporters. Allikmets et al.  catalogued all the then known 33 human ABC transporters, including those known only from partial expressed sequence tag (EST) sequences, and divided these into seven subfamilies. This scheme has been adopted, with a revised nomenclature, by the Human Genome Organisation (HUGO)  in order to provide a unified nomenclature for both human and mouse ABC transporters. Of these seven subfamilies, one, ABCA, has no exact equivalent in the yeast genome [9, 10]. Genes considered to be part of subfamily ABCA have been identified in the slime mold Dictyostelium discoideum, as well as in malaria parasites  and Caenorhabditis elegans (this paper). With the completion of the human and Drosophila melanogaster genomes, a joint summary of the ABC transporter complements of both genomes was published . This identified a new subfamily, ABCH, which appears to be the most divergent yet. One, previously unclassified yeast ABC gene, YDR061w , appears to be a structurally aberrant member of subfamily H.
The phenotypes of five ABC transporter knockouts have been reported in C. elegans. Four of these involve genes expected, by homology to mammalian genes, to be involved in drug resistance: three P-glycoproteins (Pgp-1, Pgp-3 and Pgp-4) (subfamily B) and one multi-drug resistance protein (MRP) [14, 15] (subfamily C). These ABC transporter mutants are associated with sensitivity to environmental insult . Pgp-3 mutant strains of C. elegans are more sensitive to the drugs chloroquine and colchicine. Pgp-1 and mrp-1 strains are hypersensitive to toxic pigments produced by some bacteria . All the nematode P-glycoproteins examined so far seem to be highly expressed in intestinal cells , and in the excretory cell, which functions somewhat like a kidney in C. elegans. The mrp-1, pgp-1 and pgp-3 mutant strains have been reported to be hypersensitive to the heavy metals cadmium and arsenite . The fifth reported knockout is of the product of the ced-7 gene . Mutant alleles of ced-7 cause a defect in engulfment of the cell corpses left behind by apoptosis. ced-7 is a member of the ABCA subfamily, and has a similar phenotype to the abca1 gene in humans. ABCA1 protein is required for engulfment of apoptotic cells by macrophages and is thought to regulate membrane fluidity through an increase in phosphatidylserine exposure on the outer leaflet of the cell membrane .
The term orthology is used to describe genes separated from one another by speciation events while paralogy describes those separated by gene duplication events . Of particular interest, from the point of view of functional annotation, are the cases where a pair of genes, one from each of a pair of organisms, are found. In these cases it is reasonable to presume that the orthologous genes may share a conserved function retained from the same single gene present in the common ancestor of the two organisms. However, where a single gene (or set of duplicated genes) in one genome is most closely related to a set of duplicated (paralogous) genes in another genome this is sometimes termed co-orthology , and then no particular orthologous pair can be unambiguously specified. In the case of co-orthologs the argument for retention of analogous functions between members of the sets of descendant genes is much weaker. Comparison of two complete genomes, those of C. elegans and S. cerevisiae , demonstrated a high fraction of ortholog pairs in gene families involved in core biological functions. Specifically, Chervitz et al.  found, when pairing conserved yeast genes with their most similar worm homologs (subject to a BLAST score cut-off of < 10-100), 57% of these highly conserved gene pairs involved orthologous, rather than paralogous, pairs of genes. In this category of core functions they included trafficking, and, as possibly the largest family of trafficking genes in animal genomes, ABC transporters should be expected to share in this high level of one-to-one correspondence between genomes. We expected therefore that this would allow us to assign predicted functions to newly discovered C. elegans ABC proteins on the basis of their already-characterized mammalian orthologs. Following a comprehensive phylogenetic analysis of ABC transporters from four eukaryote genomes, we found that the frequency of orthologous pairs among ABC transporters was substantially lower than we expected. Particular domain organizations and substrate specificities seem to have evolved independently several times in multiple lineages. This is expected to complicate the functional analysis of ABC transporter function in newly characterized genomes.
Results and discussion
Here we present a classification of all ABC transporters encoded in the C. elegans genome, based on a phylogenetic analysis which includes the 49 currently known human ABC proteins for which there are reliable, public, sequence data. We took the approach of analyzing primarily the conserved ATP-binding cassettes from each protein, regardless of the structural class from which the domain is drawn. This allows evaluation of the evolutionary history of each protein in the family, without biases that might result from gene-fusion events resulting in convergent acquisition of similar domain structures by distantly related proteins. In addition, we re-evaluated the relationships of transporters within statistically reliable clusters whose members are closely related enough that structural variations do not lead to errors in alignment. We did this to capture additional phylogenetic information, which may be apparent in the less conservative transmembrane domains, at a level of analysis where it is less likely to be misleading.
An example of our first-pass approach is given in Figure 2, which shows an analysis of isolated ATP-binding cassette domains from the human ABC transporters only. In particular, we find that all seven subfamilies recognized by Allikmets et al.  are recovered with significant bootstrap support. Their finding, that subfamily B is more closely related to the carboxy-terminal component of subfamily C than the two halves of ABCC molecules are with one another, is supported by our results.
A collection of transporters
We found a total of 60 confirmed ABC transporters in the annotated protein set derived from the C. elegans genome sequence. This represents approximately 0.3% of the total number of genes (approximately 19,000) in the worm genome. Only 8 of the 60 predicted genes lack any corresponding mRNA (Table 1), and only one (F56F4.6) is structurally aberrant in a way that would suggest it is likely to be a pseudogene.
Thirty ABC transporters are described in the yeast genome, or approximately 0.5% of its approximately 6,000 proteins . At present 49 human ABC transporters have been identified and, at least partially, cloned. They are included here (Figures 3,4,5,6,7 and Table 2) to illustrate their relationships with nematode proteins, which might then shed light on their biological roles. Inclusion of human as well as D. melanogaster ABC transporters in our tree allows us to explicitly classify C. elegans ABC transporters according to the current eight-subfamily taxonomic scheme for ABC transporters .
Typing ABCs to subfamily
We define membership of a particular gene in an ABC transporter subfamily primarily on the basis of the position of its ATP-binding domains in our first phylogenetic tree (not shown). Genes that fell unambiguously within a clade containing genes already assigned to given subfamily, were included in that subfamily. Where we could not assign a gene to a particular clade with a significant bootstrap value, the assignment was made on the basis of which subfamily's members scored highest when that gene was used as query in a BLAST search. The subfamilies are sometimes named according to the well-characterized mammalian genes that typify each of them, for example, P-gp (P-glycorprotein), MRP, White gene homologs, RNAse L inhibitor, GCN20 homologs, ABC1 and ALDP . These correspond to the HUGO-defined subfamilies B, C, G, E, F, A and D, respectively. Re-analysis of the full-length sequences confirmed the placement all C. elegans genes within the preexisting subfamilies, with substantial bootstrap support (Figures 3,4,5,6,7).
Instances of orthology
In the set of worm and human ABC transporters, only 8 of 49 possible pairs (16%) of sister genes contained a single human protein and a nematode homolog (Table 3). Similarly, 10% of ABC transporters were found in orthologous pairs when the comparison is made between yeast and worm genomes. A more comprehensive comparison of worm and yeast genomes  came to the overall conclusion that 57% of genes in highly conserved gene families were found in orthologous pairs, and the study suggested that such gene families provide a conserved core proteome which forms the basis of eukaryote biochemistry. ABC transporters are conserved in all eukaryotic and prokaryotic genomes, so it is interesting to note that they are found in orthologous pairs much less frequently than most gene families that are roughly as well conserved. Clearly, ABC transporter evolution has not been typical of strongly conserved gene families, and while we might have inferred that ABC-transporter-mediated metabolism differs radically among eukaryotes, this seems improbable, given the broadly comparable set of substrates associated with ABC transporters in all eukaryotes where they have been studied.
Within the P-gp-related ABCB subfamily, the only one-to-one pairings found between C. elegans and human genes are those of W09D6.6 (Haf-5) and MTABC3 (B6), and Y48G8AL.11 (Haf-6) and MABC1 (B8). These are both half-transporters localized to the mitochondria. MTABC3 (B6) is involved in iron homeostasis  and its rat ortholog, PRP, is overexpressed during hepatocarcinogenesis . Two other mitochondrial ABC transporters in humans, MABC2 (B10) and ABCB7, have orthologs in flies and/or yeast, but not nematodes.
Among ABCC molecules, whose range of functions broadly overlaps with P-gps, only C18C4.2 (Cft-1) and CFTR (C7) are indicated as orthologs in our analysis. However, the bootstrap value on this pairing is very low (51%, see Figure 5), so we cannot attach much confidence to this observation. It may simply be that C18C4.2 (Cft-1) is a highly divergent member of subfamily C, and does not bear much functional similarity to CFTR (C7). Although not forming simple pairs with any nematode gene, human MRP5 (C5), a transporter of nucleotide analogs [26, 27], and ABCC11 and ABCC12 appear to be co-orthologous to worm F14F4.3 (Mrp-5), which may provide some hint as to the function of the latter.
All four of the C. elegans members of subfamilies E and F (Figure 6) form strongly supported and unambiguous pairs with their homologs in D. melanogaster, Homo sapiens, and yeast. This unusually strong conservation, compared to the other subfamilies of ABC genes, argues for involvement in something indispensable, at least on an evolutionary timescale. The three genes in subfamily F, which lack transmembrane domains, are generally regarded as forming ribosome associated proteins involved in regulation of mRNA translation, rather than transporters. The RNase L inhibitor (E1), also known as the oligoadenylate-binding protein (OABP), is thought to be involved in the regulation of the interferon-induced antiviral response  that bears some similarities to the mechanism thought to underlie the now common molecular biology technique of double-stranded RNA-directed interference (RNAi). It also seems to have a role in muscle differentiation  in mammals. The critical role of the RNase L inhibitor is underlined by its conservation even in a highly reduced genome. In the rather minimal genome of the endosymbiotic Guillardia theta nucleomorph (302 genes) the RNase L inhibitor is the only ABC protein found . The yeast ortholog of the RNase L inhibitor protein, YDR091c, is essential for growth, as is YER036c, the yeast ortholog of T27E9.7/ABCF2 . On the other hand GCN20, the yeast version of F42A10.1/ABCF3, is not essential, although mutants do have specific defects in translation.
Processes of gene duplication and loss
While the conservation of simple orthologous gene pairs is a rare observation in our study, the numbers of genes in most ABC transporter subfamilies are about the same, despite numerous instances of gene duplication and loss. For example, within ABCB the number of half-transporters in each genome is almost identical. Furthermore, most mammalian half-transporters in subfamily B are found in clusters of functionally related, or at least co-localized, genes (the TAP (B2 and B3) genes, and the four mitochondrial ABCB genes, MABCs1 and 2 (B8 and B10), MTABC3 (B6) and ABCB7 ), paired with similarly sized groups of C. elegans genes. Likewise the number of genes in subfamilies A, C and D is much the same between genomes. However, it does appear that C. elegans, relative to humans, has undergone a massive expansion in the P-gp (full or pseudo-dimer configuration) subclass of subfamily B, and subfamily G, the 'White-like' genes. The likelihood that ABC transporter lineages have been lost repeatedly in evolution is evident from the phylogeny. The single group of P-gps in mammals contains only four members, while C. elegans has 15 P-gps, of which only three are closely related to their mammalian homologs. A literal reading of the tree (Figure 4) would suggest the presence of five additional P-gp lineages in the common ancestor of nematodes, flies and mammals that have been lost, independently, in both mammals and flies. These losses, and the species-specific expansion of the remaining lineages of genes, underlines the peculiarly dynamic composition of this group of multifunctional transport proteins.
The completion of the C. elegans and D. melanogaster genome projects [33, 34] make it possible to analyze entire gene families in metazoans. The advantage of performing a combined analysis of all known ABC proteins from two organisms is that it allows unambiguous identification of orthologous pairs of genes, as well as allowing the pattern of evolution by a process of gene duplication, lineage sorting, and functional convergence to be explicitly modeled.
Saurin et al.  surveyed the ABC transporters, considering both eukaryotic and prokaryotic systems, and found that there is a fundamental phylogenetic division among ABC transporters involved in import versus export processes. The importer class of ABCs is found only in prokaryotes, whereas exporters are found in all domains of life . However, that survey, while covering all classes of ABC transporter, was not comprehensive with respect to any of the organisms surveyed. Most recently, Schriml and Dean  compared the human ABC family to that of the mouse Mus musculus, and found almost perfect identity between the two genomes. We have integrated previous information with the complete inventory of ABC transporters from the genome of the nematode worm C. elegans. We find that most of the ABC transporters in the worm can be classified into the existing human transporter taxonomy. We find 60 ABC transporters in the worm genome, representing an overall doubling in size of the ABC transporter family relative to yeast, whose genome contains one third as many protein-coding genes. No ABC genes were found that could be classified among the bacterial import proteins.
At least three subfamilies of ABC transporter contain members capable of a conferring an MDR phenotype, and transporters from at least two different subfamilies cause MDR in human tumors . A multi-drug transporter is a single protein capable of specifically recognizing several structurally distinct classes of compounds, and which catalyzes their efflux from the cell or sequestration in a subcellular compartment. Proteins of the P-glycoprotein (P-gp) group (ABCB) transport hydrophobic compounds and function in transport of lipids and bile from the liver as well as generally defending the body from toxic natural products in the diet . P-gps are also a component of the blood-brain barrier and function in tolerance of drugs normally minimally toxic to mammals, such as ivermectin . Multi-drug resistance mediated by MRP group (ABCC) proteins depends on a slightly different mechanism. MRPs seem to function by co-transporting toxic compounds with glutathione, or as glutathione conjugates . An MDR phenotype is also associated with some members of the ABCG group of transporters, in both yeast  and humans . The MDR phenotype appears to have evolved not just once, but at least three times in the history of ABC transporters. Given the distribution of MDR-causing and non-MDR genes among mammalian P-gps; it seems reasonable to infer that MDR genes may well have arisen more than once among the P-gps themselves. It has been observed [41, 42] that the entire ABC transporter family is characterized by a highly adaptable common mechanism for coupling substrate binding to ATP hydrolysis and extrusion. It has been pointed out that, because P-gp recognizes substrate directly within the cytoplasmic leaflet of the plasma membrane , it does so at a much higher effective substrate concentration than would be the case if it recognized aqueous substrate. As a result, P-gp drug-binding sites can operate at relatively low affinity, and this, in turn, facilitates recognition of multiple substrates. This flexibility may be the key to explaining the range of tasks performed by ABC transporters, but also their apparently anomalous evolutionary history.
The mammalian P-gps include proteins capable of producing an MDR phenotype (MDR1 (B1)), as well as members with, apparently, specificity restricted to single physiological substrates such as phosphatidylcholine (MDR3 (B4)). As none of these have simple, orthologous, relationships with any of the C. elegans P-gps, no detailed predictions of function in nematode P-gps can be drawn on the basis of phylogeny alone. C. elegans P-gps do differ from one another in their ability to cause resistance to various environmental toxins , with no apparent correlation between phenotype and genetic distance from their mammalian homologs. Both human abca1 and nematode ced-7 mutants present similar apoptotic phenotypes, despite their rather distant relationship (Figure 3). ABCA1 mutations also cause defects in high-density lipoprotein cholesterol transport, and it is still an open question as to whether the analogous function of these two homologs in apoptosis accurately predicts a sharing of other functions. Similar limitations on the extent to which function may be predicted from sequence alone are likely to obtain in those subfamilies whose members are noted for variability and multiplicity of function, that is, subfamilies A, B, C and G.
Schriml and Dean  speculated that the distinct clustering of amino- and carboxy-terminal halves of ABCA proteins suggests that full ABC transporters have generally evolved from half-transporters. The pattern of structural change within the closely related subfamilies ABCD, ABCC and ABCB does suggest that the half-transporter configuration was the ancestral one for at least these three subfamilies (Figure 2). It also reveals instances where half-transporters have evolved from duplicated genes, as in the origination of ABCB from a fragment of an ABCC gene, and that, in turn, some ABCB genes have duplicated again, in giving rise to the P-gp genes.
A comprehensive comparison of worm and yeast genomes  noted that while most of the nematode genome did not closely resemble that of yeast, there was a strongly conserved 20% of the nematode genome that had a high degree of homology to a corresponding 40% of the yeast genome. Within this highly conserved subset of genes, there was a very frequent finding of orthology between members of the two genomes. As many as 57% of the most closely related gene pairs contained exactly one worm and one yeast gene. The obvious inference is that one corresponding gene was present in the common ancestor of the two species. Their overall picture of genome evolution is one in which a conserved cadre of proteins performs core biological functions required by all eukaryotes. These would remain essentially invariant throughout eukaryotes, and one expects analogous functions to be carried out by orthologous genes across large evolutionary distances. These gene families are presumably protected over the long run by their essential and irreplaceable roles in basic biochemical functions required by all organisms. However, as Chervitz et al.  point out, only a minority of gene families fit this mode, with most genes belonging to poorly conserved or taxonomically restricted families.
We expected that the frequency of simple orthologous gene pairs typical of highly conserved gene families shared by both yeast and worm would hold true for our comparison between nematode and human versions of such a highly conserved gene family as ABC transporters. However, this generality clearly does not apply to ABC transporters, despite their strong conservation across all domains of life. It seems reasonable to suppose that the rather loose relationship between substrate specificity and amino acid sequence that characterizes ABC transporters allows for much more potential exchange and sorting of biological functions among homologous genes than is typical. In turn, this pervasive pre-adaptation for functional overlap enables organisms to survive the occasional loss of substantial numbers of ABC transporters and to rapidly re-evolve lost functionality by co-opting homologous genes.
The evolutionary dynamic we propose here is reminiscent of an explanation put forward by Huynen et al.  to explain a pattern observed in a comparative analysis of 11 microbial genomes. They found that the frequency distribution of gene-family sizes within each completely sequenced genome tended to follow a power-law distribution across a 30-fold range of genome sizes. Their model is one in which genes are duplicated or deleted randomly in time, but the gene families are coherent with respect to the probability of duplication or deletion in each time unit in the simulation. In other words, the probability of duplicating or deleting a gene may change over time, but every member of a gene family always has the same probability of duplication or deletion as every other member of the family. So, whereas a given family can be either favored for expansion or targeted for deletion in a given time period, all members of the family are equally favored or disfavored by selection at the same time. Huynen et al. argued that this property of 'dynamic coherence' in a gene family could arise if all gene-family members have more or less the same function, so that they are all favored or disfavored by selection at the same time, depending on how much that function is needed.
Under a power-law distribution, gene families would tend to be subject to fluctuations of a size on the same order as the gene-family size itself . We should then expect that typical gene families will have undergone very substantial episodes of expansion and near-extinction, and in Huynen et al.'s model all gene families do become extinct within a finite time. It is evident that ABC transporters are highly atypical for a strongly conserved gene family, in that the family as a whole is highly conserved across genomes despite being subject to the same large fluctuations in size, which would tend to eventually eliminate gene families whose members are not individually indispensable. It should be noted that the ABC family does not seem uniformly subject to one or the other mode of evolution. Subfamilies E and F, which are not involved with transport, but rather have roles in translation and gene regulation, fit the 'strongly conserved'  model very well, retaining simple orthologous relationships over long spans of time. Only the transporter subfamilies themselves, because of their highly adaptable substrate-recognition capability, are subject to large fluctuations in size. We propose that finding large sets of paralogous genes, and infrequently conserved orthologs, in a gene family reflects ongoing cycles of gene loss and reacquisition of analogous functions in distantly related, newly expanded, lineages. Furthermore, we suggest that this is in fact the expected outcome of dynamic coherence, a mode shared, perhaps, by most of the less-conservative gene families, as well as the ABC genes.
We expect that future functional studies, to determine the extent of parallel and convergent evolution among ABC transporters, will eventually allow us to discern the fundamental roles of ABC transporters that ensure their long-term survival as a group. Also of interest will be whether the functional suites of genes fulfilling these roles are bounded in any way that resembles the phylogenetic subdivisions into which we presently categorize these proteins.
Materials and methods
Identification of ABC transporter genes
A computer file, WormPep16 , containing 16,332 protein sequences predicted from the completed C. elegans genome was searched using the FASTA program . Our initial query sequences were those of known C. elegans ABC proteins (for example, Pgp-1, the D. melanogaster white gene homolog T26A5.1, and so on). Matching protein sequences returned by FASTA were checked by BLAST , using either the NCBI  or Baylor College of Medicine (BCM) servers . Only those with highly significant matches to annotated ABC proteins in the sequence database were retained. The most poorly matched, verified ABC protein from each FASTA run was used as the query sequence for an additional FASTA search, and this process was repeated until no new ABC proteins were found. At a later stage in the analysis, representative members of different ABC transporter subfamilies were used as query sequences to search the updated WormPep81 file using a BLAST server at the Sanger Centre . Searches were conducted using multiple queries until all proteins already included in our dataset were found. No additional ABC proteins were identified, though some sequences were found to have been included in our dataset twice under different names. These redundant sequences were eliminated. FASTA searches were run on a SUN Microsystems UltraSPARC 5 computer. All other computer operations were carried out on an Apple Power Macintosh G3. Yeast and human ABC transporter sequences were obtained from NCBI and are described in the literature [10, 13].
Identification of ABC protein features
BLAST + Beauty searches on the BCM server identified the location of the conserved Walker A and ABC signature motifs (Prosite motifs  PS00017 and PS00211, respectively) associated with the ATP-binding cassette(s) of each protein. The number and positions of transmembrane domains in each ABC protein were predicted by using TopPred II v1.3  and then vetting the program's results by eye to exclude spurious transmembrane segments. Chromosomal locations of each ABC protein in the C. elegans genome were looked up in the C. elegans database AceDB .
Using the information derived from each protein sequence (as above) we extracted only the sequence of each predicted ATP-binding cytoplasmic domain. These domains were assembled into a single file using the SeqApp1.9 multiple sequence editor , and aligned using ClustalX . In those cases where two ATP-binding cassettes (ABCs) are present in a single protein with no intervening transmembrane domains (Subfamilies E and F, see Figure 1), the entire sequence was divided into two at an arbitrary point halfway between the two predicted ABC domains. As a result, 'two-domain' proteins are represented twice in our initial analysis. Once this approach had been used to assign genes to particular well-supported subgroups, we realigned the sequences and reanalyzed the relationships within each group using full-length amino acid sequence data.
Aligned sequences were used to generate matrices of mean distances between proteins, and these matrices were used to generate phylogenetic trees according to the neighbour-joining algorithm , refined using the SPR branch-swapping technique under the minimum evolution criterion, implemented by PAUP*4.0b10 . Bootstrapping (1,000 replicates) was done according to the method of Felsenstein , using the same parameters described above. Phylogenetic trees were visualized and manipulated using TreeView 1.6.2  and MacClade 3.0.4 .
Additional data files
The following additional data are included with the online version of this article: the protein sequence alignments for the ABCA subfamily (Additional data file 1), the ABCB subfamily (Additional data file 2), the ABCC subfamily (Additional data file 3), the ABCD subfamily (Additional data file 4), the ABCE and ABCF subfamilies (Additional data file 5), the ABCG subfamily (Additional data file 6), the ABCH subfamily (Additional data file 7), and the protein sequences from the nucleotide-binding folds only (Additional data file 8). In addition to the four genomes discussed in this paper, mouse (M. musculus) ABC transporter genes are included in some of these alignments. All eight files are in Nexus format, which is a plain-text format designed for use with the programs PAUP  and MacClade . A Nexus Data Editor for Windows is also available .
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We thank Fang Zhang, whose insight and curiosity were essential, on more than one occasion, to the initiation and completion of this work. We are grateful to Yuji Kohara for the elucidation of C. elegans cDNAs. The helpful comments of anonymous reviewers made a substantial contribution to the final draft.
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Sheps, J.A., Ralph, S., Zhao, Z. et al. The ABC transporter gene family of Caenorhabditis elegans has implications for the evolutionary dynamics of multidrug resistance in eukaryotes. Genome Biol 5, R15 (2004). https://doi.org/10.1186/gb-2004-5-3-r15