*Instituto de Investigaciones Biomédicas CSIC-UAM, Madrid, Spain;
and
Alfred Wegener Institute, Bremerhaven, Germany
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Abstract |
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Introduction |
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Na/K-ATPase is a heterodimeric protein composed of two subunits: an subunit that is homologous to P-type ATPases and contains most of the active centers, and a smaller ß subunit (reviewed by Lingrel et al. 1990
). Two genes coding for different
subunit isoforms have been cloned from Artemia franciscana,
1 (cDNA 136; Macías, Palmero, and Sastre 1991
) and
2 (Baxter-Lowe et al. 1989
). A gene coding for the ß subunit has also been cloned (Bhattacharyya, Bergstrom, and Hokin 1990
). The proteins encoded by the
1 Na/K-ATPase and
2 Na/K-ATPase genes are 73.8% identical in their deduced amino acid sequences (Macías, Palmero, and Sastre 1991
). The
1 form is expressed in all of the proposed osmoregulatory organs of the larvae: the salt gland, the midgut, and the antennal gland (Escalante, García-Sáez, and Sastre 1995
). The level of
1 mRNA accumulation increases from cysts to larvae, when the resumption of development takes place after cryptobiosis (Escalante et al. 1994
). The other gene,
2, shows restricted expression to the salt gland in early larval stages (Guo and Hokin 1989
; Sun et al. 1992
; Escalante et al. 1994
; Escalante, García-Sáez, and Sastre 1995
). The broader temporal and spatial pattern of expression of the
1 Na/K-ATPase gene suggests that the corresponding
1 protein isoform is the main factor responsible for the osmoregulatory role of Na/K-ATPase in A. franciscana.
In an attempt to characterize the transcriptional regulation and the structure of this gene, genomic clones of 1 were isolated from A. franciscana. The length (approximately 41 kb) and exon-intron distribution (14 introns, 10 of them shared with mammals) were described previously (García-Sáez, Perona, and Sastre 1997
). An unexpected degree of heterogeneity was found in the restriction maps of the genomic clones and in their exon coding sequences, without parallel in other genes whose exon-intron structures have been characterized in this organism (Escalante and Sastre 1994
; Ortega, Díaz-Guerra, and Sastre 1996
; Sastre 1999
). The pairwise differences were of 0.4%3.5% in translated regions and of 1.6%8.2% in untranslated regions. There were also differences in the promoter regions of the genes. Based on these differences, two different clones were isolated; they showed 6% divergence in the nucleotide sequences, as well as different promoter activities when assayed in transfection experiments in cell culture. The introns were not sequenced, but their sizes also varied in some of the genomic clones isolated. Synonymous and nonsynonymous substitutions, insertions/deletions, and tandem duplications were found among the differences. It was shown, however, that the Na/K-ATPase
1 subunit is encoded by a single gene in A. franciscana, since restriction pattern analysis of DNAs from different individuals presented one or two fragments that hybridized to each
1 probe, in agreement with the expected distribution for different alleles of a single gene (García-Sáez, Perona, and Sastre 1997
).
In the present study, we investigated the polymorphism of the A. franciscana 1 Na/K-ATPase gene (hereafter called
1) in greater detail. We used Southern blotting to verify the broad degree of variation at the locus suggested by the genomic clones. Sequences of the previously described cDNA and genomic clones (Macías, Palmero, and Sastre 1991
; García-Sáez, Perona, and Sastre 1997
), as well as new cDNA and reverse transcriptionpolymerase chain reaction (RT-PCR) sequences, were used to assess the degree and distribution of intraspecific variability at the coding positions of the
1 gene. Given the putatively essential role of the Na/K-ATPase
1 subunit in the adaptation of Artemia populations to highly saline environments, it is tempting to relate the intraspecific variability of
1 in A. franciscana to the effect of natural selection. We used several statistical tests, for which the
1 gene from Artemia parthenogenetica was partially cloned and sequenced, to study whether there are departures in the distribution of DNA sequence variation from neutral evolution (Kimura 1983
). Since there were insufficient studies on nucleotide polymorphism at other A. franciscana loci for comparison, we also sequenced and analyzed the Actin 302 locus (Macías and Sastre 1990
) for intra- and interspecific DNA sequence variation.
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Materials and MethodsOrigin of A. franciscana Samples |
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RFLP Studies
Cysts were cultured for 20 h at 30°C in 0.25 M NaCl as previously described (Osuna and Sebastián 1980
), and DNA was extracted from the emerged nauplii (Cruces, Sebastián, and Renart 1981
). DNA was digested with EcoRI, HindIII, and the combination of both. Aliquots of 15 µg of DNA per lane were electrophoresed in 0.8% agarose gels, which were then transferred under alkaline conditions (0.4 N NaOH) to nylon membranes (Zeta-Probe, Bio-Rad, Richmond, Calif.). These membranes were prehybridized for 2 h in 7% SDS, 500 mM NaPO4 (pH 7.2), and 1 mM EDTA (pH 7.0) at 65°C (Church and Gilbert 1984
). Hybridization was performed for 15 h at 65°C with 106 cpm/ml of probe in the same buffer. Washes were repeated three times for 30 min each in highly stringent conditions (0.1 x SSC, 1% SDS at 65°C) or, when indicated, at low stringency (2 x SSC, 1% SDS at 65°C). Under these conditions, no cross- hybridization with the
2 gene was detected. The locations and approximate lengths of the
1 probes are shown in figure 1
. Probe E is the HindIII-HindIII fragment from clone gArATCa23, which contains most of exon 7 from the sarco/endoplasmic reticulum Ca-ATPase gene (Escalante and Sastre 1994
).
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The cysts used to construct the A. parthenogenetica genomic library contained a small proportion of A. franciscana animals (Ortega, Díaz-Guerra, and Sastre 1996
). For this reason, the species from which genomic clones Par3 and Par7 originated was confirmed by PCR using DNA from pure A. franciscana and A. parthenogenetica populations. The differences in intron regions of the genomic clones were used to design oligonucleotides specific for genomic clones Par3 or Par7 (5'-AGATGGCAAAAGGCAAGC-3' and 5'-GGGACTCAGATAAAGGAGAG-3') or for the previously characterized A. franciscana genomic clones (5'-AGATGGCAAAAGGCAAGC-3' and 5'-GGTACTGAGATACAGCAGAG-3'). The Par3- and Par7-specific oligonucleotides amplified DNA fragments of the predicted size from pure A. parthenogenetica DNA but not from A. franciscana DNA. Converse results were obtained with the oligonucleotides derived from A. franciscana genomic clones: amplification products were obtained from A. franciscana DNA but not from A. parthenogenetica DNA (data not shown).
Amplification of cDNA Fragments by RT-PCR
Cysts were cultured as above, and aliquots of more than 105 nauplii were obtained. RNA was purified from each aliquot with Trizol (GIBCO-Life Technologies, Gaithersburg, Md.). Five micrograms from each RNA aliquot were used as substrate in reverse transcriptase (RT) reactions using specific primers for 1 or Actin 302. One fifth of the RT reaction was used as substrate for PCR amplification. Ten independent RT-PCR reactions were performed for
1, and one to three clones obtained from each reaction were sequenced. Five different RT-PCR reactions were also carried out for Actin 302, and four clones from each reaction were sequenced. The low-error-rate Expand High Fidelity PCR System (Boehringer Mannheim, Germany) was used for these amplifications. PCR products were purified in agarose gels and ligated to the pGEMT easy vector (Promega Corporation, Madison, Wis.). The oligonucleotides used for RT and PCR priming were 5'-ATGGCAAAAGGCAAGCAAAAG-3' and 5'-GTTGGCAGAGTTGAATGG-3' for
1 Na/K-ATPase and 5'-GTTACTTCTTT-GATTGAGGCTCG-3' and 5'-CGGGCAAGTCATTTAGAATG-3' for Actin 302.
DNA Sequencing
The nucleotide sequences of both strands of the cDNA clones were determined using pUC/M13 universal sequencing primers after the generation of progressive deletions from both ends of each clone (Guo, Yang, and Wu 1983
). The coding-region sequences of the genomic clones were determined for both DNA strands using universal sequencing primers or specific primers complementary to the previously determined sequences of the cDNA clones. Nucleotide sequences were determined by the dideoxy chain-termination method (Sanger, Nicklen, and Coulson 1977
) as modified by Chen and Seeburg (1985)
for sequencing double-stranded DNA. The nucleotide sequences of the RT-PCR-amplified fragments were determined for one strand with the Taq Dye Deoxy Terminator Cycle Sequencing Kit and a 373A Sequencer (Applied Biosystems). All sequences were independently incorporated into the alignments. The differences detected were then visually confirmed in the chromatograms.
Statistical Analyses
DnaSP versions 2.82 and 2.90 (Rozas and Rozas 1997
) were used to calculate statistics of intra- (
,
, etc.) and interspecific differentiation (divergence) and the number of synonymous and nonsynonymous sites (Nei and Gojobori 1986
, eqs. 13) and to perform the neutrality tests (Hudson, Kreitman, and Aguadé 1987
; Tajima 1989
; Fu and Li 1993
). Only total number of segregation sites was considered, not total number of mutations. For the analyses of RT-PCR-amplified regions, for
1, we used the following samples:
121, composed of the colinear 20 RT-PCR clones plus the homologous region from cDNA 136;
121+102+83, composed of
121 plus the homologous available regions from cDNA 102 and genomic clone 83 (only partially overlapping among themselves and with the RT-PCR clones);
121 - (2+20), which is not a random sample but
121 without clones 2 and 20 (the most differentiated RT-PCR clones) to study the effect of their elimination from
121. For Actin 302, we used Act-21, made of the 20 RT-PCR clones plus the homologous region from actin cDNA 302. Sequence alignments are available at asaez@awi-bremerhaven.de.
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Results |
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Most of the differences observed are silent. Of 107 polymorphisms found among the genomic and cDNA clones (104 segregating sites), 12 imply amino acid replacements (11.2%) (fig. 4
). figure 5
shows the deduced amino acid sequences of the clones. The protein polymorphisms are conservative (e.g., Leu/Ile, Glu/Asp, Ser/Asn, and Cys/Ser), although their functional relevance cannot be excluded. To investigate whether the amino acid substitutions are restricted to particular domains of the protein, we have depicted the location of the putative transmembrane or catalytic domains of the Na/K-ATPase subunit (Serrano 1989
) (fig. 5
). There are 10 proposed transmembrane regions (H1H10); regions aj are considered important functional domains, as they are conserved among all P-type ATPases. The functional relevance of many of these domains has also been confirmed by mutagenesis experiments (Andersen and Vilsen 1995
). The replacements are apparently randomly distributed throughout the protein sequence, although just four of them (two marginally) are found in the proposed domains (which account for 47% of the protein sequence). To our knowledge, none of the replaced positions has been found to be critical for Na/K-ATPase activity in the extensive point mutation studies conducted so far (reviewed by Vasilets and Schwartz 1993
; Lingrel and Kuntzweiler 1994
). We notice, however, that one of the replaced amino acid sites (V/A in H4) has been reported to have functional effects in the homologous sarco/endoplasmic reticulum Ca-ATPase (Clarke et al. 1993
).
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We were particularly interested in comparing the ratio of synonymous versus replacement substitutions within A. franciscana and between A. franciscana and A. parthenogenetica. Different ratio for the within- and between-species comparisons would reveal nonneutral evolution for 1, suggesting a tendency to accumulate either replacement or synonymous substitutions in one linage relative to the other. An appropriate test to detect deviations from neutrality in the synonymous-versus-nonsynonymous ratio when comparing intraspecific and interspecific data was described by McDonald and Kreitman (1991)
. This test was applied to the 1,122 positions shared by the sequences from both species. The differences between them are shown (fig. 4
) with indications for each substitution of whether is fixed or polymorphic and whether it induces a silent or a replacement substitution. Thirty-three silent changes and 4 replacements were observed within A. franciscana (10.8% of replacements), whereas 41 silent and 4 fixed replacement substitutions were found between species (8.9%). A G-test of independence between these four values with Williams correction for continuity (Sokal and Rohlf 1995
) gave a G value of 0.08 (P > 0.5). This result failed to reject the null hypothesis of the test: that the proportion of replacement substitutions is independent of whether the substitutions are fixed or polymorphic. According to these analyses, there is thus no specific tendency in A. franciscana to accumulate either synonymous or nonsynonymous substitutions at
1 with respect to the evolutionary tendency between A. parthenogenetica and A. franciscana for the same locus.
The divergence values observed between A. franciscana and A. parthenogenetica at 1 are shown in table 2
. For comparison, we used the only other nuclear locus with available sequences from both species: Actin 302 (Ortega, Díaz-Guerra, and Sastre 1996
) (EMBL accession numbers AJ269682AJ269685). The Actin 302 alignment contains a coding fragment of 1,002 bp that comprises the region from codon 43 to the codon previous to the stop codon. The values in table 2
are the total numbers of synonymous and nonsynonymous sites. The percentage of fixed differences in Actin 302 is lower than that in
1, but not significantly lower in relation to synonymous positions; the value for
1 is 18.7% and that for Actin 302 16.6%, suggesting that the neutral mutation rates are similar in both loci. One amino acid substitution was found in Actin 302, whereas five were observed in
1 (4.5 times as many per site), probably due to the greater constraints in the evolution of Actin 302. The level of divergence in synonymous sites has been calculated for each exon in both loci and has been found statistically homogeneous in the six
1 exons compared (24, 68;
2 = 7.73, P > 0.1) and exons 25 in Actin 302 (
2 = 6.43, P > 0.05).
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Accordingly, the variation at 1 was further characterized by comparing the nucleotide sequences of 20 randomly chosen colinear fragments generated by RT-PCR (EMBL accession numbers AJ389864AJ389883). The amplified region was 1,362 nt, excluding the annealing primer regions, which code for 454 amino acids beginning at residue 8 of the Na/K-ATPase
1 subunit. For comparison, a fragment of 1,148 nt containing the complete coding region of the Actin 302 gene plus 17 nt of the 3' untranslated region was also amplified, and 20 sequences were obtained (EMBL accession numbers AJ269567AJ269586). The polymorphisms found at both the
1 and the Actin 302 loci are shown in figure 6
, and the parameters obtained after their analyses are shown in table 3
. Within the 20 RT-PCR clones plus the corresponding fragment from cDNA 136 (
121; for a description of the samples, see Materials and Methods), we found 54 segregating sites and 55 substitutions (site 902 has two independent substitutions). Since the length of the alignment is 1,362 bp, the number of polymorphisms per site is 0.0396. In contrast, for the Actin 302 gene, sample set Act-21 (the 20 RT-PCR fragments plus c302) contains only 13 polymorphisms along 1,148 bp (0.0113 polymorphisms per site). When
values from the two loci are compared, the difference is smaller:
121 has a
of 0.0049, and Act-21 has a
of 0.0027. According to these data, the
1 fragment is thus 3.5 times as polymorphic as the actin region, and 1.8 times as heterozygotic. The larger difference between both loci for the polymorphism (or
) than for heterozygosity (or
) is due to the abundance of polymorphisms found in only one sample (singletons) in
121 (45 singletons; 82% of the substitutions); Act-21 has eight singletons (61%). Interestingly, the excess of single substitutions in
1 derives from just two of the RT-PCR clones, 2 and 20. Without these two, the
for
1 is very similar (slightly lower) to that of Actin 302 (see table 3
), and the polymorphism rates are much closer between the two loci, although that for
1 is still higher (1.4 times as high). These results are equally supported when only synonymous sites are considered (table 3
), due to the relative abundance of synonymous versus nonsynonymous substitutions in both loci. The above-mentioned difference between
and
is corroborated by Tajimas test, which detects significant deviations from the equation
=
, an equivalence that is predicted in the neutral model (Tajima 1989
). If the corresponding D statistic is significantly different from zero, there is rejection of neutrality, as both values are significantly different; this is the case for the data set
121.
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The distribution of the polymorphisms in 121 and Act-21 has been compared among exons (fig. 6
). In the amplified regions from
1, exons 2 and 8 are incomplete; the actual lengths of these exons are 123 and 133 bp, respectively. There is an apparently heterogeneous distribution of the polymorphisms across the seven exons of
1 and the five exons of Actin 302. Two of the
1 exons, exons 4 and 8, present no polymorphism, although their interspecific differences are not particularly low compared with the other exons. The statistical significance of this heterogeneity in
1 exons has again been approached using the HKA test for the two more polymorphic exons, exons 2 and 6. All-site intraspecific variability in exon 2 has been compared with the intraspecific variability from the other six exons combined using the sample
121; the corresponding P value is 0.15. Similar analysis for exon 6 rendered a P value of 0.10. For synonymous sites, we obtained P values of 0.16 for exon 2 against the other six exons combined and 0.30 for exon 6 against all other exons from the RT-PCR fragments, respectively. The conclusion is that despite the heterogeneity in polymorphic levels among
1 exons, the data do not show significant intraspecific differences among them when the HKA test is applied (i.e., taking into account the interspecific variation).
RT-PCR Sequences Compared with Genomic and cDNA Sequences
To integrate the sequence information generated from the RT-PCR reactions with the previously studied 1 genomic or cDNA sequences, the four sequences with most overlap with the amplified region (cDNAs 130 and 102 and genomic clones 83 and 11; EMBL accession numbers AJ269664AJ269668 and AJ269669AJ269673) were included in the analyses shown in figure 6
. Most of the substitutions in these clones are shared with the RT-PCR sequences. In particular, clones c130 and g11 are similar to RT-PCR sequences 18 and 10; clone g83 is similar to RT-PCR sequence 2 and to the 5' region of PCR sequence 20, and clone c102 is identical to the most commonly found RT-PCR haplotype. The
121 sample size was increased with the addition of the two longest genomic or cDNA clones, c102 and g83, and the Tajima and HKA tests were performed again. The resultant sample,
121+102+83, can still be considered a random sample, yet the decision about the number of clones to add to sample
121 is somewhat arbitrary. For
121+102+83,
= 0.0078 and
= 0.0135 (table 3 ). Both values are higher than those for
121, but the relative increase in
is greater than that in
. This is due to the incorporation of clone g83 and its similarity to PCR sequence 2. The unequal increment in
and
made Tajimas D appear closer to zero in
121+102+83 than in
121 (table 3
); this is because the addition of g83 to
121 increases the average variability (
) more than the number of polymorphic sites (reflected in
). The HKA test between sample
121+102+83 and sample Act-21 was also performed, as for the data set
121. The test rendered P values just above (P = 0.052; all sites) or below (P = 0.047; synonymous sites) the 5% level of significance of this conservative test. Again, as with the Tajima test for the data set
121, the nucleotide variation at the
1 locus shows signs of deviation from a strictly neutral origin.
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Discussion |
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The distribution of intraspecific variation at the 1 locus was studied by RFLP (fig. 2
). Similar intraspecific variation levels were found throughout the entire
1 locus. For every probe assayed, we obtained a main hybridizing restriction fragment and one to three less abundant fragments. Also taking into account the results obtained on nucleotide sequence polymorphism, discussed later, these results could be interpreted by the existence of a majority of very similar alleles, with almost identical restriction maps, and a minor population of more divergent alleles.
We next attempted to quantify the amount of intraspecific variation at the A. franciscana 1 locus and to compare it with the interspecific variability. Genomic clones coding for approximately half of the gene were isolated from a close species, A. parthenogenetica. In addition, 20 independent, randomly generated RT-PCR clones coding for the same gene region were isolated and sequenced. Since there were no previous DNA variation studies for other A. franciscana loci, we also used the RT-PCR to amplify the Actin 302 locus, since this was the only locus with available sequences for interspecific comparisons between A. franciscana and A. parthenogenetica.
The 20 amplified clones for 1 Na/K-ATPase showed a
value of 0.0049 (
121), 1.8 times as high as that for Actin 302 (0.0027); the value for
was 0.0110 (
121), 3.4 times as high as that for Act-21 (0.0032). These results clearly demonstrate the higher variability of the
1 gene compared with that of the Actin 302 gene. This variability seems to be mainly due to the existence of some divergent alleles, such as those represented by RT-PCR clones 2 and 20. If these two clones are not considered (data set
21 - (2+20)), the results obtained,
= 0.003 and
= 0.0044, are very similar to those of Actin 302. Interestingly, the sequences of the divergent clones are very similar to those of previously isolated cDNA or genomic clones, such as c102 or g83 (fig. 6
). When these two clones are included in the comparison (from
121 to
121+102+83), the nucleotide diversity increases by 59%, although both samples can be considered random. The fact that clone g83 contains a large number of mutations increases
drastically in
121+102+83, although
does not increase in proportion, as g83 shares many of the substitutions with RT-PCR sequences 2 and 20. The presence of a large percentage of differences in a few sequences makes
more dependent on sample size. It might be considered that the variability described at the
1 locus could be due to a contamination in our batch of A. franciscana cysts with other Artemia species. The main argument against this possibility is that we have found recombinant haplotypes between differentiated and common sequences (e.g., RT-PCR clones 2 and 20; fig. 6
).
The sequence analyses of interspecific sequence variation showed a similar proportion of fixed differences for both genes, but a lower rate of amino acid replacement in Actin 302 than in 1. This result is not unexpected, since actins are highly conserved proteins: 249 out of 375 amino acid residues were identical in 95% of more than 100 actins that were analyzed by Sheterline and Sparrow (1994)
. Nonetheless,
1 and Actin 302 showed similar percentages of synonymous differences between the two Artemia species, albeit the Actin 302 synonymous rate was slightly lower, suggesting similar mutation rates for both genes. Therefore, the higher polymorphism of
1 is not just derived from a higher mutation rate, a finding that is statistically corroborated by the HKA test.
Neutral and Nonneutral Features at 1
To test for departures from neutral evolution at the 1 locus, we performed three types of neutrality tests. We first analyzed the rate of replacement vesus synonymous substitutions, comparing the ratio within A. franciscana to that between A. franciscana and A. parthenogenetica (McDonald and Kreitman 1991
). No signs of nonneutrality were found. The sequences of the genomic and cDNA clones were used for this test, since they span a longer region than do the 20 RT-PCR derived sequences. The fact that these clones are not completely overlapping should not affect the results, as we do not measure the overall level of variation, but, rather, the levels of silent variation within the observed differences. Another advantage of using the genomic and cDNA clones compared with the RT-PCR clones is that with the former, there is no risk of considering Taq polymerase errors; since the RT-PCRs were performed on pools of animals of heterogeneous genetic composition, we have no means of checking Taq polymerase errors, although a low-error-rate polymerase system was used. The hypothetical errors could affect the results of the McDonald and Kreitman test more than those of other tests, because nonsynonymous positions are in the majority. In any case, all RT-PCR clones were also tested and showed neutral behavior (data not shown).
The second neutrality test employed was the Tajima (1989)
test. This test detected a significant deviation from the neutral expectation (
=
) in the data set
121 as a consequence of the high percentage of low-frequency substitutions in this sample (44 singleton sites of 54 segregating sites). When the
121 sample is augmented with clones c102 and g83, this significance is lost, as the similarity between clone g83 and RT-PCR clones 2 and 20 increases the frequency of many of the substitutions. We also used four similar tests from Fu and Li (1993)
to analyze our data and obtained results very similar to those of the Tajima test: there is also rejection of neutrality for
121, although the P values are lower using Tajimas test (data not shown). Simulations to estimate the relative power of these tests by Simonsen, Churchill, and Aquadro (1995)
showed better performance for Tajima tests than for Fu and Li tests, explaining the observed difference in P values. Both tests are considered useful for detecting selective sweeps or background selection, but we do not think that they reflect these events at
1. In such cases, a general deficiency of intraspecific variability would be found and, as discussed above,
1 has an excess rather than a deficiency of variability. The fact that the removal of sequences 2 and 20 from
121 transforms the statistics of variation of the remnant sample to values similar to those observed for data set Act-21 (table 3
) indicates that haplotypes 2 and 20 represent the "excess" of intraspecific variation at
1 and not the "remainder" of a lost variation.
The HKA test applied to 1 and Actin 302 also showed values that were significant or very close to significance (P = 0.05; table 3 ), again implying a surplus of polymorphism over the expectation of the neutral model after taking interspecific values into consideration. There is an excess of synonymous (88.8%) over nonsynonymous substitutions (11.2%), an indication of selective constrains acting at
1. Also, nonsynonymous substitutions seem to be subjected to strong selective constraints, since the observed amino acids replacements are chemically conservative. The number of amino acid substitutions present in the original population could be much larger, as the number of overlapping sequences per site is quite small. Nonconservative amino acid changes could be taking place. It is also conceivable that some of the observed substitutions in nonsynonymous or even in synonymous positions offer selective advantage to their carriers, but we would expect most, even the nonsynonymous changes, to be neutral because of their conservative nature. In such a case, the forces increasing the presence of synonymous and nonsynonymous substitutions at
1 would be the same.
The existence in a population of several alleles maintained by natural selection produces an increase in the neutral intraspecific variation surrounding (or linked to) the balanced polymorphism sites (Strobeck 1983
). A well-known case is that of Adh in Drosophila melanogaster. In the description of the HKA test, this locus was shown to present an abundance of polymorphisms when compared to its 5'-flanking region (Hudson, Kreitman, and Aguadé 1987
). This excess of polymorphism was subsequently localized in a ~400-bp region whose only nonsynonymous substitution is contained in its middle part (Kreitman and Hudson 1991
). If the excess of substitutions at A. franciscana
1 derives from the presence of one or more balanced polymorphisms at the locus or at a nearby one, we would expect an accumulation of substitutions around those polymorphisms. We have no candidates for such maintained polymorphisms, since none of the nonconservative changes appears to have a specific functional significance. The distribution of the substitutions among exons shows no clear sign of heterogeneity in intraspecific or interspecific comparisons, although an intriguing absence of polymorphisms was found in exons 4 and 8, two exons with interspecific substitution ratios close to and above the average value at
1, respectively.
An alternative explanation accounting for the polymorphic nature of 1 is that the population sampled in this study is derived from two or more previously isolated or semi-isolated subpopulations. It is not difficult to imagine how the subdivision of an A. franciscana population might take place: continental aquatic ecosystems, such as those in which Artemia lives, are more geologically dynamic than are terrestrial ecosystems, especially if they are coastal. In addition, it is known that A. franciscana habitats vary considerably in saline concentration or composition, not only between different ecosystems, but within a single one, both temporally and spatially (Lenz and Browne 1991
). A simple geographical subdivision that would isolate populations from A. franciscana, later mixed in our sample, is unlikely to produce the observed results, as a similar differentiation pattern would have been expected for
1 and Actin 302 (or other loci by RFLPs). Genetic isolation through a suppression of the recombination between genomic regions closely linked to
1 is possible, but some evidence of recombination exists at positions 202224 (RT-PCR clone 20), 398548 (RT-PCR clone 2), and 11571166 (RT-PCR clone 2) (fig. 6
). The existence of divergent haplotypes with a moderate degree of recombination among them suggest, rather, that populations divided geographically or ecologically in the past were subjected to environments that, at least in one subpopulation, imposed different selective pressure on the
1 locus. Environmentally driven positive selection acting specifically in one or more subpopulations can explain the abundance of synonymous substitutions that are strongly linked among themselves in a few haplotypes. During the hypothetical population subdivision, other loci would also be evolving independently between the subpopulations, but with no specific selective pressure acting on them, their evolution would be slower. A certain degree of differentiation would be expected, and, in fact, Act-21 shows a slight degree of linkage disequilibrium: 10 out of 78 pairwise comparisons show significant association (using the Fishers exact test or the chi-square method).
In summary, the data obtained in this study show increased polymorphism at the 1 Na/K-ATPase locus (as compared with Actin 302) in brine shrimp A. franciscana. The analyses of these data indicate that the polymorphism observed is larger than expected for neutral evolution. Given the importance of Na/K-ATPase in osmotic regulation, the adaptation to the salinity conditions of the media could be the selective force acting on this gene to generate the polymorphism observed. Transient physical or ecological isolation of some subpopulations temporally exposed to different environmental conditions could have induced an accelerated positive selection process on the
1 locus of A. franciscana in at least one of the subpopulations, increasing the intraspecific levels of variability at the locus. However, a more extensive and intensive survey should be conducted on
1 and other genes from natural Artemia populations to advance the knowledge of the nature of the
1 polymorphism and to confirm or reject the proposed hypothesis.
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Acknowledgements |
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Footnotes |
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1 Keywords: Artemia franciscana,
osmoregulation,
population genetics,
molecular evolution,
Na/K ATPase,
neutral evolution.
2 Address for correspondence and reprints: Leandro Sastre, Instituto de Investigaciones Biomédicas, Arturo Duperier, 4, 28029 Madrid, Spain. E-mail: lsastre{at}biomed.iib.uam.es
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