To infer the presence of paralogous sequences, the actin sequences (obtained by direct sequencing of PCR products) were examined for the presence of ambiguous sites.
Ambiguous sites were detected in 17 out of 24 achatinoid taxa and all three streptaxid outgroups (Appendix 3.7, p. 434), indicating the presence of more than one copy of the actin gene in the Achatinoidea and the Streptaxoidea.
Clones derived from three representative taxa, which had varied numbers of ambiguous sites in their direct sequences (Coeliaxis blandii, 21 ambiguities; Cochlitoma granulata, 9 ambiguities; Leptinaria lamellata, 0 ambiguities), were examined to assess
the ability of the PCR-direct sequences to detect variation among actin copies as ambiguities in the direct sequence. (See details of actin clones in Appendix 3.8, pp.
435-437). The three taxa were selected to represent those with many, few and no ambiguous sites, respectively and were chosen from a batch of actin sequences processed early on, with their cloned sequences used as guides to assign ambiguities in direct sequences. Other actin sequences obtained later yielded considerably more ambiguous sites after subsequent processing, as in the case of Bocageia sp. with 101 ambiguous sites. Although the sequences of the clones would be subject to Taq error, a reasonably close correlation would be expected between the amount of variation detected among clones and the amount of ambiguity detected in the direct sequences.
However, for C. blandii, a total of 163 variable sites were detected among the 4 clones obtained, as opposed to only 21 ambiguities in the direct sequence (Appendix 3.8A, p.
435). Of these variable sites, 132 were attributed to a single clone (Clone 3) that was clearly not picked up in the direct sequence (see Appendix 3.8B-1, p. 436). Likewise for C. granulata, 72 variable sites were detected among the 7 clones obtained as
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opposed to only 9 ambiguities in the direct sequence (Appendix 3.8A). The majority of these variable sites (55) were attributed to Clones 3 and 6 that were not picked up in the direct sequence (see Appendix 3.8B-2, p. 437). For L. lamellata, six clones were obtained, with three clones (Clones 1, 3 and 5) bearing sequences identical to the direct sequence (Appendix 3.8A). Clones 2 and 4 had one variable site each while Clone 6 had two; none of the four variable sites were picked up in the direct sequence (see Appendix 3.8B-3, p. 437). The very small number of variable sites in the L. lamellata clones and their absence in the direct sequence would suggest that these variable sites were probably due to Taq sequencing error. The cloned sequences therefore confirmed the presence of multiple actin genes in at least some achatinoid taxa, with some clones exhibiting highly divergent sequences when compared to other clones derived from the same taxon. Moreover, not all of the variation observed in the clones was detected in the PCR-direct sequences. The reason for the failure of the direct sequences to identify all of the variation among the clones is not clear. One explanation may lie in the fact that the PCR-direct sequence is effectively a consensus of the different actin copies within an individual in which rare copies of the gene might reasonably be expected to be averaged out.
To assess the utility of the actin gene for phylogenetic analysis of the Achatinoidea, four approaches were undertaken to determine whether the observed paralogy in actin was likely to mislead phylogeny. First, a neighbor-joining tree was constructed for all actin sequences for the Achatinoidea as well as all clones obtained for C. blandii (4 clones), C. granulata (7 clones) and L. lamellata (6 clones) in order to determine whether the clones for each species were monophyletic. Monophyly would suggest that the gene duplication event that led to the actin paralogs detected was recent relative to the date of species divergence. Moreover, if all copies of the gene were
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monophyletic, the organismal phylogeny would not be misled. However, monophyly was not observed for the clones of either C. blandii or C. granulata, with the most divergent clones (Clone 3 for C. blandii and Clones 3 and 6 for C. granulata) falling separately from both the PCR direct sequence and the other clones from the species (see Appendix 3.9, p. 438). This suggested a high probability that the organismal phylogeny may be misled. Nevertheless, the most divergent sequences for C. blandii and C.
granulata (that fell separately from the other C. blandii and C. granulata sequences in
the neighbor-joining tree) were not represented as ambiguities in the PCR-direct sequences and would therefore not affect the phylogenetic signal of the direct sequences.
Second, the number of ambiguous sites within a taxon was compared with the total number of nucleotide differences between the taxon and its closest relative (based on actin sequence identity). The number of ambiguous sites within a taxon should not exceed the number of differences between this taxon and its closest relative if the time of divergence between the two taxa were to be earlier than the evolution of the different actin genes within these taxa. With the exception of Pyrgina umbilicata and Thyrophorella thomensis, a lower number of ambiguous sites was observed within each
taxon when compared to the number of nucleotide differences between the taxon and its closest relative. Thus, based on the number of ambiguous sites observed from the direct sequences, in most cases the divergence among taxa was deeper than the divergence among the different copies of the actin gene, suggesting that some phylogenetic signal could be derived from actin at and above the genus level (see Table 3.4). However, the presence of divergent actin gene sequences falling deeper than the divergence among taxa should not be ruled out, as some divergent sequences, which were not picked up as ambiguities in the direct sequences, were detected by cloning; these sequences did not
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cluster with the other clones or the direct sequence from that species (i.e. Coeliaxis blandii and Cochlitoma granulata). It should also be noted that since the closest relative was identified based on actin sequence identity, such a relationship was not always found to be associative. For instance, Achatina achatina is most similar to A.
fulica with 65 nucleotide differences, and yet the latter is most similar to C. ustulata
with only 46 differences.
Third, a partition homogeneity test (see Section 2.9.10, p. 86-88) was undertaken to determine whether or not the sequences from the actin gene exhibited a significant difference in terms of phylogenetic signal compared to the other genes being evaluated (see results in Section 3.3.2.3, p. 161-165). The test revealed that the actin dataset was not too divergent in terms of its evolutionary history relative to the other datasets and that the presence of multiple copies of the actin gene was not having a significant effect on phylogeny.
Lastly, the phylogenetic tree obtained from the actin dataset was checked for concordance with the phylogeny obtained from the rRNA cluster. The actin phylogeny showed concordance with the rRNA phylogeny with respect to many well-supported groups (see results in Section 3.3.3, pp. 162-163 and 169-171), suggesting that the presence of multiple copies of the actin gene was not having a significant effect on phylogeny.
Thus, despite serious reservations over the utility of the actin gene in phylogenetic anaysis of the Achatinoidea, it seems that some useful phylogenetic signal could be gleaned from the gene. Actin was therefore utilised in phylogenetic analyses of the Achatinoidea though its shortcomings suggest that its findings should be interpreted with extreme caution.
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Table 3.4. Comparison of the actin ambiguous sites and nucleotide differences for each achatinoid and its closest relative. The closest relative for each taxon is determined based on the actin sequence identity (least number of nucleotide differences). Note that in scoring for the total differences, comparison between an ambiguous site and an unambiguous site is counted as different even if one of the possible nucleotides for the ambiguous site is the same as that found in the unambiguous site being compared (e.g.
A vs. R, which is either A or G). See also Table 2.8 of p. 73 for assignment of
Achatina achatina (45) Achatina fulica (18) 65 Achatina fulica (18) Cochlitoma ustulata (0) 46 Achatina stuhlmanni (2) Cochlitoma ustulata (0) 28 Cochlitoma ustulata (0) Achatina stuhlmanni (2) 28 SUBULINIDAE
Allopeas clavulinum (0) Eutomopeas layardi (0) 36 Bocageia sp. (101) Rumina decollata (29) 126 Eutomopeas layardi (0) Allopeas clavulinum (0) 36 Leptimnaria lamellata (0) Allopeas clavulinum (0) 104
Paropeas clavulinum (1) Allopeas clavulinum (0) 46 Riebeckia sp. (76) Coeliaxis blandii (21) 107 Rumina decollata (29) Zootecus insularis (20) 55
Subulina octona (0) Subulina striatella (26) 63 Subulina striatella (26) Subulina octona (0) 63 Subulina vitrea (37) Coeliaxis blandii (21) 87
Subulona sp. (0) Achatina stuhlmanni (2) Cochlitoma ustulata (0)
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Tortaxis erectus (32) Rumina decollate (29) Zootecus insularis (20)
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Xerocerastus sp. (0) Zootecus insularis (20) 60 Zootecus insularis (20) Rumina decollata (29) 55 COELIAXIDAE
Coeliaxis blandii (21) Zootecus insularis (20) 82 Pyrgina umbilicata (88) Thyrophorella thomensis (62) 77 THYROPHORELLIDAE
Thyrophorella tomensis (62) Pyrgina umbilicata (88) 77 GLESSULIDAE
Glessula ceylanica (53) Cochlitoma ustulata (0) 149 FERUSSACIIDAE
Cecilioides gokweanus (4) Xerocerastus sp. (0) 81 Ferussacia folliculus (4) Xerocerastus sp. (0) 99 STREPTAXIDAE (OUTGROUP)
Gibbulinella dewinteri (6) Gonaxis quadrilateralis (55) 94 Gonaxis quadrilateralis (55) Gibbulinella dewinteri (6) 94 Gonospira sp. (77) Gonaxis quadrilateralis (55) 117