Mitochondrial genes have been used extensively in population genetic and phylogeographical analyses, in part due to a high rate of nucleotide substitution in animal mitochondrial DNA (mtDNA). Nucleotide sequences of anthozoan mitochondrial genes, however, are virtually invariant among conspecifics, even at third codon positions of protein-coding sequences. Hence, mtDNA markers are of limited use for population-level studies in these organisms. Mitochondrial gene sequence divergence among anthozoan species is also low relative to that exhibited in other animals, although higher level relationships can be resolved with these markers. Substitution rates in anthozoan nuclear genes are much higher than in mitochondrial genes, whereas nuclear genes in other metazoans usually evolve more slowly than, or similar to, mitochondrial genes. Although several mechanisms accounting for a slow rate of sequence evolution have been proposed, there is not yet a definitive explanation for this observation. Slow evolution and unique characteristics may be common in primitive metazoans, suggesting that patterns of mtDNA evolution in these organisms differ from that in other animal systems.
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The animal mitochondrial genome exhibits several characteristics that make this molecule suitable for population genetic and phylogenetic analysis. First, substitution rates are generally high, with much polymorphism occurring as nucleotide substitutions at third codon positions (Brown et al. 1979). In mammals, substitutions accumulate up to 10 times faster in mitochondrial DNA (mtDNA) than in single-copy nuclear DNA (scnDNA; Brown et al. 1979, 1982). Rates of mtDNA evolution, however, have been shown to be higher in mammals than in fish, amphibians and a range of invertebrates (sea urchins, insects and nematodes; Lynch & Jarrell 1993), which sometimes exhibit substitution rates roughly similar between invertebrate mitochondrial and nuclear genomes (e.g. Vawter & Brown 1986; Sharp & Li 1989; Lynch & Jarrell 1993; DeGiorgi et al. 1996; Metz et al. 1998b). Second, animal mitochondrial genomes are generally maternally inherited and nonrecombining. Hence, the entire mitochondrial genome has the same historical pattern of common descent (Wilson et al. 1985). Moreover, because of uniparental inheritance and haploidy, mtDNA has a four-fold smaller effective population size compared with nuclear DNA (nDNA), leading to faster lineage sorting (Birky et al. 1983).
Although many mitochondrial genes are highly conserved at the amino acid level, third codon position substitutions are often silent and thus selectively neutral (Brown et al. 1979), providing many potentially phylogenetically informative characters. In most organisms, the slowest evolving mitochondrial genes are those encoding the two ribosomal RNAs (rRNAs) and the 22 transfer RNAs (tRNAs), while the protein-coding genes accumulate base changes at an intermediate pace (summarized in Brown 1985). The most rapidly evolving region is generally within the putative control region (also called the D-loop or AT-rich region; Pesole et al. 1999), where translation and transcription of the mitochondrial genome are initiated. When present, intergenic sequences may also show rapid sequence divergence (McKnight & Shaffer 1997). Most of the mitochondrial protein-coding genes, particularly cytochrome c oxidase subunit and cytochrome b, as well as rRNAs, have been used in phylogenetic or population genetic studies, many involving marine invertebrates (see References in Figs 1 and 2).
Mitochondrial sequences have been useful for macro-evolutionary studies in cnidarians (e.g. Bridge et al. 1992, 1995; France et al. 1996; Romano & Palumbi 1996, 1997; Cappola & Fautin 2000; Romano & Cairns 2000; Won et al. 2001). Other studies (e.g. McFadden et al. 2000; see below), however, have indicated that mtDNA from members of the class Anthozoa (sea anemones, corals and sea pens) often exhibits unexpectedly low sequence diversity within species. The intention of this review is to summarize the literature on the diversity and divergence of anthozoan mtDNA, and to address the use of mtDNA markers in evolutionary studies of this class of cnidarians. The literature to date demonstrates that low levels of mitochondrial sequence divergence are typical within and among anthozoan species. In this respect, the rate of anthozoan mtDNA evolution differs from that of other animals, including other marine invertebrates. The intrinsic and/or extrinsic factors contributing to the slow evolutionary rate of this group have been addressed, yet are not understood.
Intraspecific mtDNA Divergence in the Anthozoa
Despite the increased utilization of mitochondrial markers in studies of cnidarian evolution, very few studies have focused on intraspecific mitochondrial nucleotide variation. This is not due to a lack of interest in this area of research, but rather a lack of variation in these genetic markers. Mitochondrial nucleotide sequences are virtually invariant among conspecifics (Table 1), even geographically distant, potentially isolated populations up to 3000 km apart are invariant (France et al. 1996; Snell 1997; Snell et al. 1998; France & Hoover 2001a; Hellberg manuscript submitted for publication). If gene flow is restricted, it is expected that independent evolutionary processes, including genetic drift and local selective forces, would promote differentiation among these populations (Wright 1943; Kimura & Weiss 1964), particularly at synonymous third position sites of protein-coding genes. Despite the presumed silent, selectively neutral nature of such substitutions (Brown et al. 1979), virtually no substitutions are observed at third codon positions in these anthozoan species. This is in sharp contrast to results for corresponding regions from other marine invertebrates (Fig. 1). Unfortunately, sample sizes have been small for most species; in many cases only two sequences have been compared. Thus, the range of variation within a species has not been quantified accurately, with the exception of Balanophyllia elegans, which exhibits no variation within the cytochrome c oxidase subunit 1 (COI) gene among 67 individuals from 18 populations over a 3000-km range (Hellberg manuscript submitted).
Table 1. Observed mitochondrial sequence diversity within anthozoan species
Mitochondrial genes commonly utilized in phylogenetic analyses, such as COI, 16S rDNA and cytochrome b (cyt b), as well as other less commonly used genes, including 12S rDNA, ATPase 6 (ATP6) and NADH dehydrogenase subunits 2, 3, 4 and 6 (ND2, ND3, ND4L and ND6) have been used to infer phylogenetic relationships among various anthozoan species. Relative to many other marine invertebrates, each of these genes consistently exhibits low levels of congeneric divergence (Fig. 2). In fact, divergence within and among anthozoan families is often less than congeneric divergence within other marine invertebrate species (see Fig. 2B, C). These observations are not confined to one group of anthozoans, but are documented across multiple orders including hard corals, soft corals and anemones (Table 2).
Table 2. Observed mitochondrial sequence divergence among congeneric anthozoan species
Mitochondrial sequences usually offer sufficient polymorphism to provide robust inferences of higher level relationships. Caution, however, must be used in analysing phylogenies based on mitochondrial data as genetic variation among species can be disproportionate to the current taxonomic classification. For example, Diploria strigosa and Favia fragum of the scleractinian family Faviidae exhibit very low divergence in COI nucleotide sequences (0.16%), while amino acid sequences are identical (Snell 1997, 2000). This divergence is unusually low in light of a 2.4% divergence between congeners Montastraea cavernosa and M. annularis, also of the family Faviidae (Snell 1997, 2000; Medina et al. 1999). More surprisingly, six species (Merulina scabricula, Hydnophora rigida, Pectinia alcicornis, Caulastraea furcata, Cyphastraea ocellina and Echinopora lamellosa), which represent three scleractinian families, possess identical 16S rDNA sequences (Romano & Palumbi 1996, 1997). Whether these discrepancies are related to inappropriate morphological classifications or to the use of inappropriate genetic markers is unclear. It is clear that gene trees may not accurately reflect species trees (e.g. Moore 1995).
Micro-evolutionary processes involving the putative control region in some anthozoans may be acting in a different manner relative to other regions of the mtDNA, resulting in higher levels of polymorphism. As is characteristic of the control region in other organisms (Brown 1985), the putative control region in some scleractinians is more variable than protein-coding regions of the mitochondrial genome (Table 2, Fig. 3; van Oppen et al. 1999a, 2001; Vollmer & Palumbi 2002). Divergence of the putative control region is similar to that in sea stars (0.19–6.83%; Hrincevich & Foltz 1996). Preliminary analysis of the short putative control region of alcyonacean corals, however, reveals little sequence divergence among congeners (Tables 3, C. McFadden, pers. commun.). Hence, use of this region for population-level analyses may be limited to particular groups of anthozoans (i.e. scleractinians).
Table 3. Results of two-cluster relative rate tests based on the amino acid sequence of the 5′-end of the COI gene
Phylum Class Genus
Difference in evolutionary rates for that comparison statistically significant at the 5% level determined in PHYLTEST.
The tempo of evolution in anthozoan mitochondrial genes appears to be at least 10–20 times lower than the ‘standard’ mitochondrial clock based on vertebrate sequences, which averages a sequence divergence of 1–2%/Myr (Brown et al. 1979; Ferris et al. 1983; Higuchi et al. 1984; Wilson et al. 1985). In line with the increasing evidence of rate heterogeneity among lineages, the pattern in rate of anthozoan nucleotide substitution is an order of magnitude slower than the rate observed among most other marine invertebrate species.
The disparity between rates can be demonstrated using a region of the COI gene, for which homologous amino acid sequence data could be aligned for cnidarians, echinoderms, molluscs and arthropods. A comparison of relative rates of COI amino acid sequences using the two-cluster relative-rate test (Takezaki et al. 1995) in phyltest (Kumar 1996) reveals significant rate heterogeneity between anthozoans and other phyla (Table 3). Rate heterogeneity is also exhibited within the Anthozoa and Scyphozoa, however, among these classes, only the scyphozoan Cassiopea andromeda exhibits rates similar to that observed in other phyla. Overall, representatives of the cnidarian classes Anthozoa, Hydrozoa and Scyphozoa (except C. andromeda), exhibit significantly slower evolutionary rates in the COI gene than members of other phyla. The limited cnidarian data available for this analysis are insufficient to determine whether a slow evolutionary rate of the mitochondrial genome is a common characteristic of Hydrozoa and Scyphozoa. However, despite exhibiting slow evolutionary rates relative to other phyla, COI nucleotide divergence within the scyphozoan genus Aurelia (13–27%; Dawson & Jacobs 2001) is greater than that observed in any anthozoan and is comparable with conspecific divergence in other marine invertebrates. This, in addition to the relatively high evolutionary rate of C. andromeda, provides some indication that scyphozoans may not share this trait.
Vertebrate mtDNA generally evolves up to 10 times faster than single-copy nuclear genes (Brown et al. 1979, 1982; Vawter & Brown 1986), while invertebrates exhibit similar evolutionary rates between nDNA and mtDNA (Vawter & Brown 1986; Sharp & Li 1989; Lynch & Jarrell 1993; DeGiorgi et al. 1996; Metz et al. 1998b). Within the scleractinian coral genus Acropora, substitution rates of mitochondrial genes are lower relative to three types of coral nuclear genes (Fig. 3): two nuclear introns (Fukami et al. 2000), third codon positions of the single-copy mini-collagen nuclear gene (Fukami et al. 2000; van Oppen et al. 2001), and the hypervariable ITS1-5.8S-ITS2 region (van Oppen et al. 1999b, 2002a). Interspecific divergence among Acropora species reveals rates of mtDNA sequence evolution 2–3 times slower than the rate of evolution of a nuclear coding region and nuclear intron from the same species (Fukami et al. 2000; van Oppen et al. 2001). Cyt b and ATP6 sequence divergence between subgenera Isopora and Acropora is also 3 and 10 times smaller, respectively, than in the nuclear intron and coding region (Fukami et al. 2000). Although this pattern of evolution is not typical, it has been observed in other invertebrates (e.g. Palumbi & Wilson 1990; Palumbi & Metz 1991). The putative mitochondrial control region of Acropora species, however, exhibits a range of divergence similar to the Acropora nuclear coding region (third codon positions), yet much less than the ITS-5.8S-ITS2 region (Fig. 3). Slow evolutionary rates of Acropora mitochondrial vs. nuclear genes are likely to be representative of anthozoan patterns in general, due to the similarly low rates of nucleotide substitution in anthozoan mitochondrial genes despite high levels of polymorphism among nuclear markers (see above).
Factors Contributing to Slow Mitochondrial Evolution
A correlation between intraspecific diversity and interspecific divergence is a prediction of the neutral theory of molecular evolution (Hudson et al. 1987). It is therefore not surprising to observe low mitochondrial divergence among anthozoan species with low genetic diversity. Whether the same factors influence both low diversity within species and low sequence divergence among species is unclear. The following mechanisms, albeit a limited list, have been suggested to contribute to the apparently slow evolution in anthozoan mitochondrial genes, yet all remain untested.
Slow evolution observed in the anthozoan mitochondrial genes studied to date may result from a constraint on the ability of the mitochondrial genome to accumulate mutations or from diversity within the mitochondrial genome having been severely reduced in the recent history of these species. An unusually low mutation rate has been considered as an explanation for both low diversity and low divergence in anthozoans (France & Hoover 2001a), as in other organisms (e.g. snapdragons; Vieira & Charlesworth 2001); however, the cause for this low rate is not understood. The level of efficiency of mtDNA repair mechanisms has been implicated in influencing substitution rates. For example, the high rate of nucleotide substitution in the mitochondrial genome of other animals is believed to be a result of an apparent inefficiency in mtDNA repair (Brown et al. 1982; Clayton 1982; Moritz et al. 1987). In contrast, it appears that in yeast, nuclear-encoded proteins involved in DNA repair are imported into the mitochondria and may function in repairing DNA mismatches (Chi & Kolodner 1994). This may explain the slow mitochondrial gene evolution in yeast relative to that in nuclear genes (Clark-Walker 1991). A similar phenomenon may be occurring in some anthozoans, as a homologue of a component of the bacterial MutSLH mismatch repair pathway has been identified in the mitochondrial genome of octocorals (mtMSH; Pont-Kingdon et al. 1995, 1998; Beaton et al. 1998; France & Hoover 2001a,b). The functionality of this gene is, however, speculative (Wolstenholme 1992) as other required components of the repair pathway are not encoded in the mitochondrial genome and must be imported.
Low levels of genetic diversity within a species are often explained by a severe bottleneck during the history of the species (e.g. Avise et al. 1987; Palumbi & Wilson 1990; Palumbi & Kessing 1991). Genetic diversity in the mitochondrial genome is more drastically reduced in these situations relative to nDNA diversity because of the smaller effective population size of the maternally inherited, haploid mitochondrial genome (Nei et al. 1975; Barton & Charlesworth 1984; Wilson et al. 1985). This is consistent with the pattern of high diversity in nDNA relative to that in mtDNA observed in anthozoans, but it is unlikely that such a wide range of taxa have all experienced a recent bottleneck.
Factors such as time since divergence and hybridization may additionally reduce observed species divergence. Time since species separation may be insufficient for mutations to independently accumulate in the newly reproductively isolated species, resulting in low genetic diversity despite physiological or morphological differentiation. This hypothesis may be relevant to the Montastraea annularis species complex (Medina et al. 1999), in which sibling species may have recently diverged from an ancestor possessing little mitochondrial diversity.
Hybridizing species will not achieve genetic differentiation if introgression occurs. Similarity of mitochondrial genomes will result if hybridization is not reciprocal and cytoplasmic ‘capture’ occurs (see Avise 1994), because the mitochondrial genome is maternally inherited and nonrecombining (note that this has yet to be confirmed in the phylum Cnidaria). This leads to different species with similar mitochondrial gene sequences, and could potentially explain the similarity between 16S rDNA sequences in some Fungia species (Romano & Palumbi 1996).
These and other processes, including genetic hitchhiking (selection on a closely linked gene) and background selection (negative selection against deleterious alleles), may be influencing the apparent substitution rate within the anthozoan mitochondrial genome at different levels and controlling divergence among species. The mechanism and ultimate evolutionary explanation for the lack of variation at presumably silent sites is intriguing. Sampling error may contribute to the lack of sequence variation within species, however, owing to similar findings among many species and across subclasses (Table 1), this is an unlikely explanation.
The most parsimonious explanation for the rate difference between anthozoans (and poriferans, see below) and other animals is that the rate of mitochondrial sequence evolution of early metazoans was slow and accelerated near the origination of bilateral animals (R. Watkins, pers. commun.). An opposing hypothesis, that anthozoans experienced a deceleration in the rate of mitochondrial evolution, would be unlikely given that this characteristic would have arisen independently within each anthozoan lineage that was established. Despite the limited mitochondrial sequence data available for other cnidarians, there is evidence that some scyphozoans exhibit mtDNA substitution rates similar to those in other marine invertebrates (Table 3). If higher mitochondrial evolutionary rates are confirmed in other cnidarian classes, the accelerated rate character evolved near the origin of these other classes, as Anthozoa is ancestral to these groups (Bridge et al. 1995).
Interestingly, data on other primitive metazoans, sponges (Porifera), also suggest slow rates of mtDNA evolution in the few genes studied thus far. Nucleotide sequences of poriferan mitochondrial genes (cytochrome c oxidase subunits II and III and ATP6) appear to evolve 2–8 times slower than the mitochondrial sequences of triploblastic metazoans (R. Watkins, pers. commun.). Intraspecific variability and interspecific diversity of COI nucleotide sequences in some sponge species are low similar to anthozoans (Figs 1A and 2A). Low COI sequence diversity among conspecific sponges renders this gene inappropriate for intraspecific application; however, interspecific signals may be informative for higher level phylogenies in demosponges (Erpenbeck et al. 2002). In addition, reconstructing phylogenetic and phylogeographical relationships at the species and genus level has also been difficult in calcarean sponges (family Leucettidae) using a different mitochondrial gene (COII, Wörheide et al. 2000), but taxonomic and phylogeographical relationships of those species were clearly resolved using ITS sequences (Wörheide et al. 2002).
Low rates of substitution in the mitochondrial genome are not a novel phenomenon. Angiosperms in general exhibit low rates of synonymous substitution in mitochondrial genes (Wolfe et al. 1987; Palmer & Herbon 1989; Laroche et al. 1997; Palmer et al. 2000). Some vertebrates exhibit slowed evolutionary rates in mitochondrial genes relative to closely related taxa (sharks, Martin 1999; sturgeon and paddlefish, Krieger & Fuerst 2002). As is the case with the Anthozoa, the mechanisms controlling evolutionary rates in these examples remain to be unravelled.
The relative influence of the various mechanisms on evolutionary rates in the anthozoan mitochondrial genome are difficult to determine at this point. First, the data are fragmented: in species where nuclear data is abundant there is no corresponding mitochondrial data for comparison. In addition, as researchers determine that mitochondrial genes do not suit their needs owing to a lack of variation, the few invariant sequences (usually unpublished) are abandoned while other polymorphic markers are utilized. Second, differences in biological and genetic characteristics of species lead one to believe that the influences on evolutionary rates differ among groups of anthozoans. Rigorous testing is necessary to assess the influences governing the rate of evolution in anthozoan mitochondrial genes, keeping in mind that the primary mechanisms may differ among species. Understanding patterns and mechanisms of mtDNA evolution in anthozoans and poriferans is an interesting evolutionary story in itself, but will ultimately lead to a better understanding of the early radiation of metazoans.
Generalizations concerning the use of particular genetic markers for particular analyses are often made. For example, protein-coding mitochondrial genes are usually regarded as useful for population-level analyses because of the high rate of nucleotide substitutions, particularly at third-position sites. In order to resolve relationships within species or between closely related species, many researchers turn to mitochondrial markers as the abundance of genetic variation accumulated in this genome often reveals a phylogenetic signal. This is contrary to the situation with anthozoan mitochondria where evolution is so extremely slow. This does not mean that anthozoan species are not evolving, rather, polymorphisms within the nuclear genome are likely to reveal population and species divergence. For anthozoans, the genetic markers appropriate for deciphering phylogenetic relationships within and among some species are not mitochondrial in nature. However, mtDNA markers are useful in resolving higher level relationships.
The observation of slow evolutionary rates within the anthozoan mitochondrial genome supports evidence that rate heterogeneity can vary greatly among lineages. The difference in evolutionary rates in anthozoans from other marine invertebrates indicates that use of molecular clock estimates from other organisms cannot be applied to infer dates of evolutionary separation for these animals. Use of a nonanthozoan molecular clock would grossly overestimate time since divergence — using rates from other marine invertebrates could realistically be > 10 times the actual rate of evolution. Although error is inherent in all methods of estimating species divergence, dating species using the fossil record and/or vicariance events would likely provide a more accurate divergence estimate on which molecular clocks can be calibrated.
This review is an outcome of the mini-symposium on Molecular Phylogeny and Population Genetics in Coral Reefs held during the 9th International Coral Reef Symposium in Bali in October 2000. We would like to thank the organizers and presenters of this symposium. Thanks also to M Dawson, D Erpenbeck, S France, JB Geller, C McFadden, S Nichols and R Watkins for access to unpublished data and manuscripts and to the many reviewers of this manuscript. TLS acknowledges support from an NSF grant and a National Undersea Research Center grant, both to Dr Mary Alice Coffroth. MJHvO acknowledges receipt of a JCU postdoctoral fellowship (1997–2000). SLR acknowledges support from an NIH grant to Dr RH Richmond and an NIH-RISE grant to the University of the Virgin Islands. GW acknowledges funding from the German Academic Exchange Service (DAAD) and the Australian Biological Resources Study (ABRS), AstraZeneca R & D Griffith University and the Queensland Museum (Brisbane).
Tonya Shearer's research interests include population structure, systematics and evolution of scleractinian corals. Madeleine van Oppen's research focuses on the genetic aspects of coral bleaching, the use of genetics to aid in the design of Marine Protected Areas as well as on evolutionary and population genetics of marine organisms. Sandra Romano's research focuses on the evolution, ecology and systematics of scleractinian corals. Gert Wörheide's research interests focus on biodiversity (molecular) systematics and phylogeography of Indo-Pacific sponges, biodiversity, phylogeography and palaeobiogeography of relict faunas, and marine conservation.