Evolutionary Lineages of RT1.Ba in the Australian Rattus

J. M. Seddon1,Go,* and P. R. Baverstock{ddagger}

*Centre for Animal Conservation Genetics, Faculty of Resource Science and Management, and
{dagger}Graduate Research College, Southern Cross University, Lismore, New South Wales, Australia

Abstract

In this study, the evolutionary history of the variable second exon of RT1.Ba and its adjoining intron b are compared across a number of species and subspecies of the Australian Rattus. Three lineages are identified in the second intron across a range of Rattus species. Two of these lineages, separated by the insertion of a probable rodent short interspersed nucleotide element and by point mutations outside the indel region, are both found in each of the major clades of the endemic Australian Rattus. This pattern of ancestral polymorphism is reflected in the adjoining exon 2 sequences, although phylogenetic constraints confirm that the clustering is not identical to that of the associated intron sequences. In addition, the coding sequences show evidence of the retention of ancestral polymorphism, with identical exon sequences found in two divergent species, and some indication of gene conversion detected for the exon sequences.

Introduction

The major histocompatibility complex (Mhc) is a multigene family identified across a range of vertebrate species. Many Mhc loci show high levels of polymorphism, which are thought to derive in part from the retention of ancestral polymorphisms (Klein 1987Citation ), leaving some Mhc alleles with similarity to those in otherwise distant species. Gene conversion or intragenic segmental transfer has also been postulated as a means of increasing the level of polymorphism of Mhc alleles (Hedrick et al. 1991Citation ). In addition, an observed increase in the number of nonsynonymous over synonymous changes in regions associated with peptide binding suggests that balancing selection is involved in the maintenance of polymorphism of Mhc alleles (Hughes and Nei 1989Citation ).

These processes of selection, the retention of ancestral polymorphisms, and gene conversion have uncoupled the phylogenetic history of the coding regions of Mhc loci from that of the remaining genome and from that of the species in which they are found. The strength of the evolutionary forces shaping the phylogeny of the Mhc coding regions makes the Mhc a good model for examining the interplay between the evolution of an exon and that of its adjoining intron.

Empirical studies present examples from both extremes of linkage for Mhc loci. Cosegregations of exon and intron segments have been identified in several class II Mhc loci (e.g., Ammer et al. 1992Citation ), and such tight linkage has led to the suggestion that intron polymorphisms, which often include microsatellite or retroposon variation, can be used as markers for haplotype differences (Ellegren, Davies, and Andersson 1993Citation ). In contrast, poor correlation between the evolutionary history of an exon and intron of mouse H-2 Ab was found (Lu et al. 1996Citation ) and attributed to intraexonic recombination. Between these extremes, an intron repeat in DRB of artiodactyls was found to evolve with the ß-sheet–encoding region of an exon but not with the {alpha}-helix–encoding region (Schwaiger et al. 1993Citation ).

In this study, the evolutionary history of the second intron of RT1.Ba was examined across a number of species and subspecies of Australian Rattus and compared with that of its adjoining exon to determine the degree to which their evolutionary histories have become uncoupled.

Materials and Methods

Tissues were obtained from six species and eight subspecies of Australian Rattus, namely, R. colletti, R. fuscipes assimilis, R. fuscipes coracius, R. fuscipes fuscipes, R. fuscipes greyii, R. leucopus cooktownensis, R. leucopus leucopus, R. sp., R. tunneyi culmorum, R. tunneyi tunneyi, and R. villosissimus. These samples provide representatives for six of the eight species of endemic Australian rats. Genomic DNA (gDNA) was isolated using a standard proteinase K digestion and phenol/chloroform extraction procedure (Bothwell, Yancopoulos, and Alt 1990Citation ). Exon 2 was amplified by the polymerase chain reaction (PCR), using primers and procedures described previously (Seddon and Baverstock 1998Citation ). Intron b was amplified under similar conditions, but with the primers RT1.Ba 612C (5'-ATGAAGAGGTCAAATTCAACTCCAGCTA-3') and RT1.Ba 1263NC (5'-GCTGACCCAGCAGCACAGGAGACTT-3').

Because this study compared the evolutionary history of the second exon and its adjoining intron, it was essential that the chromosomal pair of the exon and intron sequences could be deduced without doubt. To simplify this procedure, individuals were chosen on the basis of homozygosity at the intron using temperature gradient gel electrophoresis (TGGE). Consequently, exon sequences determined for these individuals, whether present in homozygous or heterozygous form, could be assigned unequivocally to their coevolving introns.

Both strands of the PCR products were sequenced manually using the ThermoSequenase Cycle Sequencing kit (Amersham Life Science) or were sequenced using the Applied Biosystems 373A autosequencer. Homozygous individuals were sequenced directly. For heterozygous samples, the two homoduplex bands on TGGE were cut separately from the gel and reamplified by PCR (Meyer et al. 1991Citation ) prior to sequencing.

The sequences were aligned by eye with assistance, for the intron sequences, from CLUSTAL W (Thompson, Higgins, and Gibson 1994Citation ). Tajima-Nei distances were calculated to account for a nucleotide base frequency bias (A, 33.4%; C, 19.6%; G, 22.3%; T, 24.7%) in the exon sequences, and Jukes-Cantor distances were calculated for the intron sequences. To calculate synonymous and nonsynonymous distances, peptide-binding regions (PBRs) of the exons were identified by comparison with the postulated PBR sites for other species (Brown et al. 1988Citation ). Phylogenetic analyses were performed using the neighbor-joining distance algorithm in MEGA (Saitou and Nei 1987Citation ) and using a maximum-likelihood approach in DNAML of the PHYLIP package (Felsenstein 1993Citation ).

The strength of association between the intron and exon sequences was tested by applying topological constraints during phylogeny reconstruction. The intron sequences were used to construct a maximum-likelihood tree under the constraints of the exon phylogeny and of two species phylogenies compatible with that of Baverstock, Adams, and Watts (1986)Citation . One species phylogeny is the most parsimonious tree when constrained to the phylogeny of Baverstock, Adams, and Watts (1986)Citation and the second places fuscipes and leucopus as sister taxa and the sordidus group and tunneyi as sister taxa. The degree of congruence between the unconstrained trees and the constrained trees was assessed in a maximum-likelihood analysis by the change in log likelihood ({Delta}lnL) of the tree, with the level of significance assigned by the method of Kishino and Hasegawa (1989)Citation as implemented in PHYLIP (Felsenstein 1993Citation ).

Results and Discussion

Exon
Twenty-one nucleotide sequences of the second exon of RT1.Ba were recovered from 18 individuals (accession numbers AF145094–AF145102), and of these, 12 sequences have previously been presented (Seddon and Baverstock 1998Citation ; Seddon 1998Citation ). Of the 249 sites, 56 (22.5%) are variable and 32 are informative under parsimony. As anticipated for variable Mhc loci, there is a high level of nucleotide divergence, with Tajima-Nei distances reaching 11.7%.

Identical exon 2 nucleotide sequences were found in two divergent species, R. t. culmorum (R132A) and R. colletti, two species whose distributions are widely separated and distinct. Although the reporting of shared motifs is relatively common, the sharing of a complete exon sequence between two species has been reported infrequently, for example, in Mhc-E for two closely related chimpanzee species (Suarez et al. 1997Citation ). Convergence and reticulate evolution cannot be dismissed as explanations; however, this is the first example of a shared Mhc exon sequence in two otherwise divergent species and is strong evidence of the retention of ancestral polymorphisms.

Such an evolutionary pattern is further supported by the presence of a codon deletion at positions 222–224 (corresponding to residue 80 of the first domain) in two sequences of R. t. culmorum (this study), in 17 exon 2 alleles of R. f. greyii (Seddon 1998Citation ), in R. colletti (Seddon and Baverstock 1998Citation ), and in R. norvegicus (Holmdahl et al. 1993Citation ). This codon deletion indicates an ancestry shared since the divergence of these species.

The influence of balancing selection on these exons is suggested by an increase in the level of nonsynonymous changes at sites postulated to be involved in peptide binding. The 12 PBR codons showed more nonsynonymous than synonymous changes (mean pn = 0.1723, mean ps = 0.1233), but for the 70 codons outside the PBR, there were fewer nonsynonymous than synonymous changes (mean pn = 0.0380, mean ps = 0.0652).

The phylogenetic relationships of the exon 2 sequences (fig. 1A ) clearly differ from the expected species phylogeny. This discrepancy is likely due to the retention of ancestral polymorphisms and the presence of balancing selection acting on the exon 2 sequences of RT1.Ba. However, the small number of nucleotides used to construct the tree may result in an inaccurate phylogeny, with few branches which have strong bootstrap support.



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Fig. 1.—A, Phylogenetic relationships of exon 2 sequences for Australian Rattus; neighbor-joining tree based on Tajima-Nei distances, rooted by the Rattus norvegicus (X07551) sequence. Bootstrap values (x500) are shown for values greater than 50. The alleles of heterozygous individuals are designated "A" and "B." Numbers on the right identify the associated intron lineage. B, Phylogenetic relationships of intron b sequences for Australian Rattus; neighbor-joining tree based on Jukes-Cantor distances with complete omission of indel sites and rooted by the R. norvegicus (X07551) sequence. Bootstrap values (x500) are shown for values greater than 50. The three intron lineages are indicated. Sequences sharing a symbol have identical exon sequences

 
In addition, gene conversion may result in an inaccurate species phylogeny. The alignment of seven amino acid sequences from this study with 11 sequences of R. f. greyii (Seddon 1998Citation ) in figure 2 identifies three sequence motifs among the species of Australian Rattus. A combination of the second and third motifs and, in an individual of R. f. greyii, a combination of the first and third motifs suggests the action of intraexonic recombination. The first and second motifs are precisely defined, but there are many variants by point mutations in the third motif. For this motif in particular, it is difficult to distinguish polymorphism derived through gene conversion from that derived from point mutations with homoplasy, including convergence (Edwards and Hedrick 1998Citation ). Most exon 2 sequences (14 of the 22 sequences in this study) do not share these motifs, suggesting that gene conversion, if present, has played a minor role in the generation of polymorphism.



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Fig. 2.—Sequence motifs in exon 2 sequences of Australian Rattus. Boxed regions are postulated sequence motifs shared between taxa for which both the nucleotide and amino acid sequences are identical. Not all exon 2 sequences are shown. Species are as follows: Rfg, Rattus fuscipes greyii; Rsp, Rattus sp.; Rv, Rattus villosissimus; Rtt, Rattus tunneyi tunneyi; Rfc, Rattus fuscipes coracius; Rfa, Rattus fuscipes assimilis.

 
Intron
Eighteen nucleotide sequences of intron b of RT1.Ba were obtained (accession numbers AF145103–AF145120). Of the 742 sites, 125 (16.8%) are variable positions with 54 sites informative under parsimony. A high level of divergence is found, with the Jukes-Cantor distances varying from 0.69% to 11.67%. The smaller distances were not necessarily found within species.

The evolutionary patterns of the exon have substantially influenced the evolution of its adjoining intron. The nucleotide divergence of the intron sequences approximate the high levels found for the Mhc exon sequences. The presence of balancing selection acting on the PBR of the second exon, together with hitchhiking, can be postulated to have increased the divergence in the adjoining intron sequences beyond neutral expectations (Hudson, Kreitman, and Aguade 1987Citation ; Hudson 1990Citation ). However, such an explanation requires that the recombination rate between these regions has been fairly small. Even a recombination rate of 10-5 per generation has been shown to be sufficient to substantially decrease the influence of balancing selection on the linked neutral sites (Takahata and Satta 1998Citation ).

The most interesting feature of the intron sequences is the pattern of large insertion/deletion events which give three lineages. The sequence of R. norvegicus (X07551) is designated lineage 1. In comparison with this sequence, all Australian rat sequences (lineages 2 and 3) show a deletion of 171 nt (positions 487–657). A large indel of approximately 107 nt separates lineages 2 and 3 (positions 334–440), with the proposed insertion defining lineage 2 in some species and subspecies of Australian rats. This insertion has a long run of thymidine bases, resulting in slippage of the Taq sequencing enzyme and leading to difficulty in verifying the exact number of thymidine bases. The insertion, defining lineage 2, shows a 93% similarity to a rodent short interspersed nucleotide element (SINE) (Pascale et al. 1993Citation ), identified using a FASTA search of GenBank sequences. The subdivision of the intron lineages on the basis of a probable rodent SINE is reminiscent of a similar lineage split reported in an intron of H-2 Ab in an extensive array of Mus species (Lu et al. 1996Citation ).

In addition to the insertion/deletion events, there are changes outside these regions which further define the lineages. A neighbor-joining tree calculated using Jukes-Cantor distances with complete omission of sites containing gaps (thus effectively removing insertion and deletion events) clearly shows the division of the 19 sequences into three lineages (fig. 1B ).

Importantly, both lineage 2 and lineage 3 do not occur in a species-specific manner. Both lineages are represented across a range of species and subspecies. For example, two individuals of R. f. greyii were sequenced, one showing the insertion characteristic of lineage 2 and the other not. This pattern is repeated for paired individuals of each of R. l. cooktownensis, R. t. culmorum, and R. villosissimus. The retention of ancestral polymorphisms and the presence of balancing selection, which led to a discrepancy between the exon 2 phylogeny and that of the expected species phylogeny, has clearly influenced this intron phylogeny.

There is remarkable similarity in the grouping of the exon sequences by intron-defined lineages, although the phylogenies of the intron and exon sequences have, to some extent, become uncoupled. The individuals with lineage 2 introns are joined in a clade with 94% bootstrap support in the intron neighbor-joining tree (fig. 1B ) and are again united in a phylogenetic tree constructed from the exon sequences (fig. 1A ). However, the linkage is not complete. In the exon tree, this clade also contains R. f. greyii (KA3) and one exon of the heterozygote R. l. cooktownensis (D71B), supported by a moderate (68%) bootstrap value. Further support for the absence of complete linkage between the exon and intron sequences is shown by two individuals of R. l. cooktownensis (D72 and D71B) that share an identical exon sequence, yet one has a lineage 2 intron and the other has a lineage 3 intron. The application of topological constraints during the re-creation of phylogenetic relationships allows an assessment of the strength of hitchhiking between the intron and exon sequences. Such an analysis shows that an intron tree constrained to either the exon phylogeny ({Delta}lnL = -589.45) or one of two species phylogenies ({Delta}lnL = -527.37, -527.69) is significantly different from the unconstrained tree (lnL = -2,080.40).

The patterns of evolution acting on the exon, such as balancing selection and the retention of ancestral polymorphisms, have clearly influenced the evolutionary history of the intron. Therefore, in this example of RT1.Ba in species and subspecies of endemic Australian Rattus, a low level of recombination can be postulated for this locus, allowing the footprints of the evolutionary forces acting on the coding region to be detected in the adjacent noncoding region.

Furthermore, the presence of two clear lineages in the intron of the Australian Rattus species and subspecies gives some indication of the speciation history of this group of endemic rats. The large size of the insertion into the intron suggests that it represents a single evolutionary event. Therefore, the presence of both lineages in each major clade of the Australian rats (fig. 3 ) indicates that the insertion predates the rapid divergence of Australian Rattus that followed their movement into Australia during the Pleistocene (Taylor, Calaby, and Smith 1983Citation ). The origin of the insertion cannot be determined without further study of the Asian species of Rattus. In addition, because the continuation of ancestral polymorphisms through speciation events requires a sufficiently large population to prevent the loss of variation through genetic drift or bottlenecks (Vincek et al. 1997Citation ), we can infer that each speciation event within the divergence of the Australian Rattus lineage occurred with a relatively large founding population.



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Fig. 3.—Phylogeny of the Rattus species showing the three intron lineages; phylogeny from Baverstock, Adams, and Watts (1986). Lineages are marked as follows: lineage 1, dark block; lineage 2, light hatch; lineage 3, dark hatch; unknown, light block

 

Acknowledgements

We thank the South Australian Museum for providing tissue from their collection, and Martin Elphinstone, Fiona Harriss, and the South Australian National Parks and Wildlife Service for the collection of samples for this study. Financial support was provided by the Australian Research Council and the School of Resource Science and Management, Southern Cross University.

Footnotes

Eleptherios Zouros, Reviewing Editor

1 Present address: School of Biological Sciences, University of East Anglia, Norwich, England. Back

2 Keywords: MHC RT1.Ba, Australian Rattus. Back

3 Address for correspondence and reprints: J. M. Seddon, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom. E-mail: j.seddon{at}uea.ac.uk Back

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Accepted for publication January 17, 2000.





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