Pseudogenes Contribute to the Extreme Diversity of Nuclear Ribosomal DNA in the Hard Coral Acropora

Luis M. Márquez*,1, David J. Miller{dagger}, Jason B. MacKenzie*,{ddagger} and Madeleine J. H. van Oppen§,

* Biochemistry and Molecular Biology
{dagger} Comparative Genomics Centre
{ddagger} Marine Biology and Aquaculture, James Cook University, Townsville, Queensland, Australia
§ Australian Institute of Marine Science, Townsville, Queensland, Australia

Correspondence: E-mail: m.vanoppen{at}aims.gov.au.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
One characteristic of Indo-Pacific Acropora spp. is extremely high diversity in rDNA sequences at both the species and individual levels. In order to test the hypothesis that pseudogenes may contribute to this diversity, three kinds of analyses were conducted. First, for A. millepora (the species containing the most diverse suite of rDNA types), RT-PCR was used to determine which 5.8S rDNA types are expressed. Second, as previous studies have indicated that interspecific hybridization has occurred in the genus Acropora and silencing of rDNA loci via nucleolar dominance has been shown in some cases to involve methylation, patterns of variation were examined at methylation-susceptible sites. Third, patterns of substitution at conserved sites (including those that are likely to contribute to secondary structure in rRNA) in the 5.8S rDNA were examined. These analyses consistently indicated that one rDNA sequence type present in a broad range of Indo-Pacific Acropora species is likely to consist predominantly of pseudogenes. Patterns of variation also suggest that species may differ with respect to which rDNA sequence types have been silenced and which are active. These pseudogenes are likely to have arisen as a consequence of the introduction of highly divergent rDNA types into single genomes by interspecific hybridization events, and we attribute the extreme rDNA diversity characteristic of many Acropora species to both the independent evolution of these silenced rDNA types and to the suppressive effects of high sequence diversity on homogenization processes acting on functional loci.

Key Words: ribosomal DNA • pseudogenes • ITS • reticulate evolution • coral • Acropora


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
To date, the most frequently used DNA marker to study reef coral evolution is the internal transcribed spacer (ITS1-5.8S-ITS2) region of the nuclear ribosomal RNA (rRNA) transcription unit (Hunter, Morden, and Smith 1997; Lopez and Knowlton 1997; Odorico and Miller 1997; Medina, Weil, and Szmant 1999; van Oppen et al. 2000, 2002; Diekmann et al. 2001; Rodriguez-Lanetty and Hoegh-Guldberg 2002). rDNA constitutes a multigene family of tandem repeats, which evolve in a concerted fashion (Arnheim et al. 1980) through unequal crossing over and gene conversion (Dover 1982). As a result of concerted evolution, all copies of rDNA families are generally rapidly homogenized within individuals and species, but interspecific divergence can be high (Hillis and Dixon 1991).

The presence of highly divergent rRNA types within a single genome can, however, result in homogenization proceeding extremely slowly (Modrich and Lahue 1996), particularly if these are on different chromosomes (Arnheim et al. 1980). Hence, ancestral polymorphism at rDNA loci can be maintained for long periods of time relative to single-copy loci. Another possible consequence of combining diverse rDNA copies during hybridization is silencing of some loci. Failure of one of the parents' chromosomes to organize nucleoli in an interspecific hybrid may lead to nucleolar dominance—the selective silencing of the corresponding rRNA genes (Honjo and Reeder 1973; Durica and Krider 1977) by mechanisms that involve chromatin modification (Chen, Comai, and Pikaard 1998; Frieman et al. 1999; Pikaard 2000a, 2000b; Muir, Fleming, and Schlötterer 2001). In these cases, silenced rDNA loci can then evolve independently as pseudogenes (although concerted evolution is maintained within each locus), potentially complicating phylogenetic analyses. It is believed that this has happened in the hybridizing oak species Quercus petrea and Q. robur (Muir, Fleming, and Schlötterer 2001). These two species hybridize frequently and share three divergent rDNA types, two of which are believed to be pseudogenes whose origin predates the species divergence (Muir, Fleming, and Schlötterer 2001).

Extremely high levels of variability in the ITS1-5.8S-ITS2 region have been observed within and among several species of the hard coral Acropora (Odorico and Miller 1997; van Oppen et al. 2002). In Acropora, up to nine rDNA types can be present in a single colony (Odorico and Miller 1997; van Oppen et al. 2002), and distinct ITS types are often shared between species. These patterns are broadly consistent with the occurrence of interspecific hybridization, although alternative explanations such as incomplete lineage sorting cannot yet be completely ruled out (van Oppen et al. 2000, 2001, 2002).

To examine the possibility that some rDNA copies in Acropora are nonfunctional and constitute pseudogenes, three kinds of analyses were conducted. First, we used RT-PCR to determine which 5.8S rDNA types are expressed in A. millepora, the species containing the most diverse suite of rDNA types. Second, we examined patterns of methylation in the 5.8S rDNA that may indicate silencing caused by nucleolar dominance. Third, we examined the patterns of substitution at conserved sites in the 5.8S rDNA, looking for mutations that may disrupt the secondary structure and functionality of the rRNA. These studies indicate that pseudogenes are likely to constitute at least one of the major sequence types present in a broad range of Indo-Pacific Acropora species.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Sample Collection, PCR, Cloning, and Sequencing
In addition to 171 published sequences (Odorico and Miller 1997; van Oppen et al. 2000, 2002), 243 new sequences were obtained from 73 Acropora colonies (table 1) (GenBank accession numbers AF538378 toAF538600 and AF540600 to AF540620). Tissue samples were collected by snapping off small branches (2 to 5 cm) from individual colonies and stored in 70% to 90% EtOH. DNA extraction, PCR, cloning, and sequencing procedures followed van Oppen, Willis, and Miller (1999) and van Oppen et al. (2002).


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Table 1 Samples and rDNA Sequence Codes.

 
Examination of Expressed rDNA Types
Total RNA was extracted from three A. millepora individuals collected at Magnetic Island in the central Great Barrier Reef using the Choczynski and Sacchi (1987) method. The RNA was DNase-treated several times until no DNA contamination could be detected using PCR amplification of scnDNA Acropora loci. RT PCR was performed on 1.3 µg of RNA using the Amersham First Strand cDNA kit and the primer 5.8S_RP1 (5'-GACRSCCTYRCTCGCCTCG-3'). One µl of undiluted cDNA/RNA hybrid was used in the subsequent PCR reactions to amplify the partial 5.8S region (~120 bp in length) using the primers 5.8S_FP1 (5'-GGCTYGSGYATCGATGAAGA-3') and 5.8S_RP1. PCR products were run on a 1.5% TAE-agarose gel stained with EtBr and excised from the gel. Excised fragments were purified by spinning the gel slice through Whatman 1 filter paper. The spun-through fragments were cloned into pGEM-T (Promega) following the manufacturer's protocol. Ten, nine, and five clones of each of three individuals were sequenced as before.

Alignment
Sequences were aligned manually using Sequencher 3.0 (Gene Codes Corporation). The gene/spacer boundaries were identified using Odorico and Miller's (1997) alignment of Acropora rDNA sequences. The 5.8S region was easily alignable, but even following Odorico and Miller's (1997) alignment, it was impossible to align the ITS1 region objectively, and this region was therefore not used in subsequent analyses. However, the ITS2 region was relatively easy to align in a subset of 185 sequences corresponding to the largest clade in the 5.8S phylogeny (clade IVB, see below). Phylogenetic analysis of ITS2 was only performed on this subset of sequences.

Phylogenetic Analyses
Due to the difficulty in aligning the ITS regions (as discussed above), only the 5.8S gene was used for the phylogenetic analyses across the entire data set. The data set was reduced from 414 to 160 sequences, because identical sequences (table 2) were excluded from the analysis. A maximum-likelihood phylogeny was constructed using the HKY85 (Hasegawa, Kishino, and Yano 1985) substitution model in MOLPHY 2.3 (Adachi and Hasegawa 1996), which estimates approximate bootstrap probabilities (Felsenstein 1985) among trees by the RELL method (Kishino, Miyata, and Hasegawa 1990; Hasegawa and Kishino 1994). The tree was rooted using Acropora (Isopora) cuneata as an outgroup. The subgenus A. Isopora forms a sister group to A. Acropora in both cladistic analyses using morphology (Wallace 1999) and sequence analysis of cytochrome b and NADH dehydrogenase subunit 2 (van Oppen, Willis, and Miller 1999; Fukami, Omori, and Hatta 2000). Analysis of molecular variance (AMOVA) (Excoffier, Smouse, and Quattro 1992) was used to partition the genetic variance among species and among phylogenetic clades using the Kimura two-parameter distance (1980) in ARLEQUIN 2.0 (Schneider, Roessli, and Excoffier 2000). A Permutation Tail Probability (PTP) test was performed to examine whether a strong phylogenetic signal is present in the 5.8S data set by generating 100,000 random trees and comparing the lengths of these with a consensus tree obtained using parsimony analysis in PAUP* 4.0 beta (Swofford 1999). The phylogenetic signal was also assessed by evaluating the skewness in the distribution of the length of another 100,000 randomly generated trees (i.e., a strong phylogenetic signal is characterized by a left skew in the tree length distribution, caused by several trees having a reduced length due to phylogenetic signal in addition to those being short by chance).


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Table 2 Identical 5.8S Sequences in the Original Data Set of 416 Acropora Sequences.

 
Relative-Rate Test
To examine the evolutionary rate constancy among clades in the 5.8S phylogeny, a two-cluster relative-rate test (Takezaki, Rzhetsky, and Nei 1995) was implemented in Phyltest 2.0 (Kumar 1996) using the Kimura two-parameter distance. A representative of each clade was haphazardly chosen (from fig. 1 and supplement 1 in the online Supplementary Material) and used in the analysis. The test was only performed for the 5.8S region.



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FIG. 1. Rooted maximum-likelihood tree of the 5.8S in Acropora. Bootstrap values are above the branches (1,000 replicates). Major clades and subclades are indicated. Sample codes are as in table 1 in this manuscript and in Odorico and Miller (1997), van Oppen et al. (2000), and van Oppen et al. (2002). A dot and a number after the sample code indicate the clone that was sequenced. Sample names for the A. hyacinthus and A. aspera groups indicate geographic origin: GBR, Central Great Barrier Reef; TS, Torres Strait; and WA, Western Australia

 
Methylation-Related Substitutions
For representatives of each clade in molecular phylogenies (and using A. cuneata as a reference for the ancestral state), the number of cytosine sites showing at least one deamination-like substitution and those that did not contain such substitutions were compared with those of the most basal clade in the phylogeny (Muir, Fleming, and Schlötterer 2001) using a 2x2 contingency table. Given the small sample sizes, the asymptotic property of a {chi}2 distribution cannot be assumed. Instead, we performed an exact test that uses the random permutation procedure of Roff and Bentzen (1989). In this procedure, a contingency {chi}2 statistic is calculated and the probability of observing the exact test statistic or larger is generated using a random permutation procedure that maintains the marginals but simulates the null hypothesis of no association. The random permutation is implemented in Chiperm 1.2 (Chiperm is available at the Crandall lab Web site at http://bioag.byu.edu/zoology/crandall_labprograms.htm).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
The complete sequence data set consists of 414 sequences of ITS1-5.8S-ITS2 from a range of Acropora and one Isopora species. Many 5.8S sequences are identical (table 2), whereas ITS sequences are generally very different and sometimes almost impossible to align. Nevertheless, 54 ITS1 sequences are not unique. Most of these are observed only twice, but four sequences are observed 11, seven, six, and four times, respectively. Another five ITS1 sequences are found three times each within this data set. The maximum length of the sequences (table 3) is slightly larger than those reported previously (i.e., 166 and 200 for 5.8S and ITS2, respectively, as compared with 112 and 158 reported by Odorico and Miller [1997]), due to the inclusion of the Isopora sequences that contain inserts not present in the subgenus Acropora. The base composition of both regions (table 3) does not differ significantly among species, among subgenera, or in comparison with those previously reported (i.e., Odorico and Miller 1997).


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Table 3 Length, Mean Base Compositions (%), and Ranges of Kimura Two-Parameter Pairwise Sequence Distances (%) for the 5.8S and the ITS Regions of Acropora.

 
Levels of genetic diversity in the 5.8S gene within the subgenus Acropora are extremely high, with a maximum corrected distance between A. cerealis and A. gemmifera of 20.3% (table 3). The distance between subgenera is slightly higher, with a maximum of 22.8% between A. cuneata and A. gemmifera. The alignment of representatives from observed rDNA types is attached as supplement 1 in the online Supplementary Material. ITS2 distance estimates are very high (table 3), and the difficulty encountered in aligning these sequences suggests that they are saturated, although estimates of transition/transversion ratios do not indicate saturation (0.95 and 1.01 for ITS1 and ITS2, respectively).

Phylogenetic Analysis
The 5.8S alignment of 160 sequences used in the phylogenetic analysis consists of 166 positions of which 58 are constant, 33 are variable but parsimony uninformative, and 75 are parsimony informative. The phylogenetic signal in this alignment is high, as indicated by permutation tests (length of most parsimonious tree is 227 steps, shortest length of randomly generated tree is 606, P = 0.01), respectively, and a significantly left-skewed tree length distribution, which was tested by generating 100,000 random trees (g1 = -0.419) (Hillis and Huelsenbeck 1992).

Four major clades are resolved in the 5.8S phylogeny (fig. 1), corresponding to clearly distinct ITS2 types (see supplement 1 in the online Supplementary Material). The base of the tree forms a trichotomy leading to the following clusters: clade I, clades II and III, and the major clade IV. Clade I consists only of two A. millepora and eight A. cerealis sequences (fig. 1 and table 2); note that the only other A. cerealis sequence obtained falls into clade IVC, whereas A. millepora sequences are scattered throughout the tree. As in the case of scnDNA and mtDNA data (van Oppen et al. 2001), sequences from the Caribbean species constitute a clade (II) that is well separated from all Indo-Pacific species. The composition of clade III is also broadly consistent with mtDNA and scnDNA trees, except that a few A. spicifera, A. cytherea, and A. hyacinthus sequences cluster in clade III based on 5.8S sequences, whereas the corresponding clades in scnDNA and mtDNA trees contain no sequences from these species (Márquez et al. 2002b). The major derived clade IV contains sequences from a wide range of species and shows clear substructuring. Most A. aspera and all A. florida sequences fall in clade IVB, which is again consistent with previous analyses based on scnDNA and mtDNA (van Oppen et al. 2001; Márquez et al. 2002). In some cases, clones from single individuals occur in completely different clades (e.g., A. spicifera 79 subclade IIIA and IVB). The AMOVA results are concordant with this in that the variance in the data is higher between clades than between species but at the same time is very high within species (table 4).


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Table 4 Results of AMOVA Between Acropora Species and Acropora Clades in the Maximum-Likelihood Phylogeny.

 
A phylogenetic analysis (not shown) of the largest subclade IVB was also performed based only on the ITS2 region, which was unambiguously alignable, but found no evidence for clustering based on either species identity or geographic localities.

Expressed 5.8S Sequences
To examine whether all or only a subset of rDNA types are expressed in Acropora, RT PCR was used to determine 5.8S rRNA sequences from three colonies of A. millepora, the species that contains the most diverse suite of rDNA types. Twenty-two of the 24 cDNA sequences obtained were identical; the remaining two sequences had single substitutions at different positions, which are likely to represent PCR errors. The maximum number of substitutions observed in this 81-bp region of the 5.8S gene (~120 bp minus the primers), among all pairs of sequences in the A. aspera group (of which A. millepora is a member), is five. These expressed sequences are identical to the corresponding 81 bp of the sequences in clades IVA and IVB and some sequences in IVE, they differ by one substitution from the sequences in clade I and some sequences in clade IVE, by two to four substitutions from sequences in clade IVD, and up to five substitutions from those in clade IVC. Hence, it appears that in A. millepora, only rDNA genes falling into clades IVA and B (and possibly IVE) are likely to be functional.

Relative-Rate Test
Rate constancy may differ between different groups of species, as they are not necessarily subjected to the same evolutionary constraints, whereas substitution rates are expected not to differ significantly within species. With rate constancy rejected at the 5% level (table 5), the relative-rate test indicates that some clades and subclades in the 5.8S phylogeny have different rates of evolution. Clade I evolves more slowly than clades II, III, and IV, and clades II and III have a faster substitution rate compared with clade IV (table 5). Moreover, subclade IVB evolves significantly slower than clades IVC and IVD, although the latter two clades do not differ from one another. Note that sequences of some species (e.g., A. millepora and A. pulchra) are spread across subclades that exhibit different substitution rates (table 5).


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Table 5 Relative-Rate Test Results for Acropora 5.8S Clades in Maximum-Likelihood Phylogeny.

 
Methylation Mutation Analysis
Gene silencing in animals frequently involves DNA methylation at cytosine residues in CpG motifs. Because the resulting methylcytosine residues are prone to spontaneous deamination (resulting in mutation to thymine), silenced chromatin typically drifts towards higher (A+T) content relative to active regions. Methylation has been implicated in nucleolar dominance phenomena, and the resulting rDNA pseudogenes therefore typically show patterns of deamination-like substitutions (C->T and G->A) at methylation sensitive sites relative to their active counterparts. Therefore, following Muir et al. (2001) and using A. cuneata as a reference for the ancestral state, we counted the number of cytosine sites (only CpG, because animals are not known to methylate CpNpG) showing at least one deamination-like substitution and those that did not contain any of such substitutions for each clade. Table 6 shows that the number of deamination-like substitutions at methylation sites in the 5.8S region (in comparison to the number of mutations at nonmethylation sites) is not significantly higher in any clade or subclade compared with clade I, which is considered the ancestral state. However, the number of deamination-like substitutions at methylation sites per sequence in subclade IVC is surprisingly high (20 in 14 sequences) and significantly larger than in subclade IVB (10 in 57 sequences), in relation to the number of substitutions in nonmethylation sites (4 and 7 respectively) ({chi}2 = 29.7, P = 0.001).


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Table 6 Deamination-Like Susbtitutions in 68 Methylation and 21 Nonmethylation Sites of the 5.8S in Acropora.

 
Analysis of Conserved Sites and Secondary Structure
Pseudogenes are expected to mutate freely because, unlike their active counterparts, they are not under selective constraints. Therefore, mutations are expected to occur even at otherwise conserved positions. At active rDNA loci, some of these constraints result from the secondary structure of the resulting rRNA; mutation at sites critical for secondary structure is either selected against or accompanied by compensatory mutations at complementary sites. Conversely, in rDNA pseudogenes, selection is not imposed against mutations affecting the secondary structure of the rRNA, and noncompensatory mutations are tolerated.

Based on an alignment of 30 5.8S rDNA sequences from a broad range of eukaryotes including fungi, plants, invertebrates, and vertebrates, a total of 45 conserved sites were identified (the alignment is attached as supplement 2 in the online Supplementary Material). Patterns of mutation at these conserved sites were examined in sequences representative of each clade and subclade in the Acropora phylogeny. Although differences in at least one of those sites were present in each of the Acropora (sub)clades, the number of such differences was very small in all cases (table 7). In terms of substitutions at these conserved sites, the highest number (five) was observed in the A. cerealis 114.1 sequence, a member of clade IVC.


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Table 7 Number of Mutations in 45 Conserved Positions of the 5.8S.

 
A broad range of metazoan 5.8S sequences, containing annotations of the secondary structure derived by comparative sequence analysis, was obtained from the European Large Subunit Ribosomal RNA Database (Wuyts et al. 2001) (http://rrna.uia.ac.be/lsu). Representatives from each clade and subclade in the Acropora phylogeny were aligned with these data to identify noncompensatory mutations that may disrupt the formation of helices. This approach allowed the identification of three sequences in clade IVC containing noncompensatory mutations that potentially disrupt helices B6 and B9 (fig. 2). In these cases, maintenance of secondary structure would require the pairing of two guanine residues, which is energetically unfavorable.



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FIG. 2. Folding of 5.8S rRNA sequences from A. hyacinthus DOAht.1 (from clade IVB in fig. 2) and A. cerealis 114.1 (from clade IVC in fig. 1) following Odorico and Miller (1997). Helices numbers after Wuyts (2001). The arrows indicate noncompensatory mutations

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Characteristics of the rDNA Region in Acropora
Our results confirm the notion that the rDNA ITS regions of Acropora, including the subgenus Isopora, are among the shortest in corals (Takabayashi et al. 1998) and eukaryotes in general (Odorico and Miller 1997). To our knowledge, only the green alga Halimeda has shorter ITS regions (42 to 77 bp and 149 to 161 bp for ITS1 and ITS2, respectively [Kooistra, Coppejans, and Payri 2002]). The levels of variability in the 5.8S and ITS2 regions of Acropora are the highest reported for any coral, at the level of both the species and the individual. The complete ITS1-5.8S-ITS2 region exhibits only 2% variation among morphospecies in the Montastrea annularis complex (Lopez and Knowlton 1997; Medina, Weil, and Szmant 1999) and only up to 4.9 % between species of the genus Madracis (Diekmann et al. 2001). Takabayashi et al. (1998) reported only ITS1 variation, which was 15% in Goniopora tenuidens, 2% in Heliofungia actiniformis, 11% in A. longicyathus, and 31% in Stylophora pistillata. In Plesiastrea versipora, a species with an extreme latitudinal distribution range (40° N to 40° S) and accordingly expected to be genetically variable, populations across the whole range showed sequence variation of just 1.53% ± 0.43 and 3.15% ± 2.24 in ITS1 and ITS2, respectively (Rodriguez-Lanetty and Hoegh-Guldberg 2001). Maximum pairwise distances in the ITS1-5.8S-ITS2 region between Porites species are approximately 11% (Hunter, Morden, and Smith 1997). Despite the 5.8S subunit being a coding region, we estimated corrected genetic divergences of approximately 20% within the subgenus Acropora, which is five times higher than the estimate of Odorico and Miller (1997) using a smaller sample size. These high levels of variability prompted us to question whether some of these 5.8S genes may have lost their function and are evolving as pseudogenes (see below).

Phylogeny
The topologies of all Acropora phylogenies obtained so far using scnDNA loci, mtDNA, and rDNA have several features in common, including the derived and distinct A. aspera clade with A. florida as a sister taxon, and the basal A. tenuisA. longicyathus clade (van Oppen et al. 2001; Márquez et al. 2002b). The 5.8S phylogeny resembles that of the mtDNA in that the Caribbean taxa are among the basal groups. However, in contrast to these other cases, the base of the 5.8S tree is unresolved and forms a trichotomy. Moreover, unlike phylogenies based on the Pax-C intron, A. aspera and A. tenuis are not strictly monophyletic in the 5.8S tree, with a small number of sequences from each species occurring in clades IVA and IVB. Several other species that are nonmonophyletic based on the 5.8S gene show the same pattern in the nuclear intron and mtDNA phylogenies. These patterns of species polyphyly and paraphyly may either result from recent speciation coupled with incomplete lineage sorting or from introgressive hybridization. Low rDNA diversity both in the three Caribbean Acropora species (van Oppen et al. 2000) and in the genetically distinct brooding species Acropora (Isopora) cuneata (Ayre, Veron, and Dufty 1991) (fig. 1) relative to most Indo-Pacific members of this genus, together with the fact that genetically distinct species tend to differ in the timing of release of their gametes, supports the hybridization hypothesis and is discussed in detail elsewhere (van Oppen et al. 2000, 2001, 2002; Márquez et al. 2002a, 2002b).

Although incongruences exist between the 5.8S phylogeny and the nuclear intron and mtDNA phylogenies (van Oppen et al. 2001), the 5.8S gene provides a high phylogenetic signal. Nevertheless, true phylogenetic relationships between specimens can be confounded by the presence of pseudogenes, and care needs to be taken in interpreting relationships from ribosomal DNA alone.

Identification of Possible Pseudogenes
Substitution rates vary between some clades that differ in species composition, possibly as a consequence of heterogeneity in the predominant evolutionary forces operating on different species. Subclade IVC is composed mainly of sequences from colonies (including A. millepora) that are also represented in other clades and shows an accelerated evolutionary rate compared with the clade to which the expressed A. millepora rDNA sequences belong (subclade IVB). Although the total numbers of substitutions per sequence at conserved sites are not atypically high, sequences in subclade IVC typically show large numbers of substitutions at potential methylation sites relative to other clades. Furthermore, A. cerealis 114.1, a member of subclade IVC, does show the highest number of mutations and the 5.8S gene sequences from clade IVC are characterized by noncompensatory mutations that can potentially disrupt the secondary structure of the large subunit of the rRNA. These results support the notion that at least some rDNA types (those of clade IVC) in Acropora are likely to be nonfunctional.

Neither the Caribbean Acropora nor the A. florida species is represented in subclade IVB, which contains the expressed A. millepora sequences. Similarly, very few of the A. aspera and A. tenuis sequences fall into this subclade. This suggests that rDNA types that have been silenced in some species may not necessarily have been silenced in others. Independent hybridization events could lead to different patterns of nucleolar dominance, possibly explaining this observation. Species such as A. tenuis and A. florida may have been reproductively isolated from their congeners for sufficient time to allow homogenization of rDNA units to approach completion, and in the case of A. tenuis, the small number of sequences outside clade II may reflect incomplete lineage sorting. Whether this is likely also in the case of A. aspera is unclear, as A. aspera appears to have a semipermeable species boundary with other members of its species group (van Oppen et al. 2002).

Origin of Extreme rDNA Diversity in Acropora
How did the unprecedented rDNA diversity within individual Acropora colonies and species originate? Interspecific hybridization may have combined divergent rDNA copies within single genomes. As the mismatch repair machinery seems sensitive to high numbers of mismatches, large sequence divergences in the spacer regions may have suppressed recombination across the entire rDNA array, impeding concerted evolution (Petit et al. 1991; Muir, Fleming, and Schlötterer 2001) and causing the persistence of divergent rDNA types within individuals. In contrast, when parental species have very similar sequence composition, such as Armeria villosa and Armeria colorata (Plumbaginaceae), where the ITS2 region differs in only six out of 245 sites, homogenization can occur in only two generations (Fuertes Aguilar, Roselló, and Nieto Feliner 1999). Asexual reproduction may also limit the effectiveness of concerted evolution to homogenize divergent rDNA copies because in asexual taxa, gene conversion and crossing over are restricted to mitotic divisions (Pringle, Moncalvo, and Vilgalys 2000). This may be the case in A. millepora, which has very high rates of asexual reproduction (Ayre and Hughes 2000) and exhibits the most extreme pattern of rDNA diversity of all species examined (fig. 2). Some of the rDNA types brought together by hybridization may have been silenced by nucleolar dominance, causing them to evolve as pseudogenes and sequence diversity to further increase. Alternatively, hybridization may have caused chromosomal rearrangements (Rieseberg, van Fossen, and Desrochers 1995), which relocated rDNA copies to different chromosomal positions, also reducing the homogenizing action of concerted evolution (Arnheim et al. 1980; Muir, Fleming, and Schlötterer 2001). In situ hybridization is required to determine whether the divergent sequences in Acropora are present in more than one nucleolus organizer region (NOR).

In conclusion, it is likely that the clade IVC rDNA type has been silenced by nucleolar dominance after a hybridization event and has since evolved neutrally as a pseudogene. These nonfunctional rDNA types are expected to show Mendelian inheritance and are subject to concerted evolution. Hence, the presence of pseudogenes does not affect inferences from these data about interspecific hybridization. However, inclusion of functional and nonfunctional copies in the same analysis may affect the resulting phylogenetic hypotheses, as mutation patterns are likely to differ between functional and nonfunctional copies. The data also imply that there may be differences between species with respect to which rDNA types are expressed and which are silent. Expression studies across a range of species are required to address this issue.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We thank Danielle de Jong and Lesa Peplow for obtaining the expressed A. millepora sequences. We are also thankful to Bette Willis and Jackie Wolstenholme for their help collecting and identifying samples. This project was supported by the Australian Research Council, the Australian Institute of Marine Science, and James Cook University. L.M.M. acknowledges receipt of a scholarship from the Venezuelan Council for Scientific Research.


    Footnotes
 
1 Present address: Centro de Ecología, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela. Back

Axel Mayer, Associate Editor Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
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Accepted for publication March 10, 2003.





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