Open Access
How to translate text using browser tools
1 December 2006 Time Scale for Cyclostome Evolution Inferred with a Phylogenetic Diagnosis of Hagfish and Lamprey cDNA Sequences
Shigehiro Kuraku, Shigeru Kuratani
Author Affiliations +

The Cyclostomata consists of the two orders Myxiniformes (hagfishes) and Petromyzoniformes (lampreys), and its monophyly has been unequivocally supported by recent molecular phylogenetic studies. Under this updated vertebrate phylogeny, we performed in silico evolutionary analyses using currently available cDNA sequences of cyclostomes. We first calculated the GC-content at four-fold degenerate sites (GC4), which revealed that an extremely high GC-content is shared by all the lamprey species we surveyed, whereas no striking pattern in GC-content was observed in any of the hagfish species surveyed. We then estimated the timing of diversification in cyclostome evolution using nucleotide and amino acid sequences. We obtained divergence times of 470–390 million years ago (Mya) in the Ordovician–Silurian–Devonian Periods for the interordinal split between Myxiniformes and Petromyzoniformes; 90–60 Mya in the Cretaceous–Tertiary Periods for the split between the two hagfish subfamilies, Myxininae and Eptatretinae; 280–220 Mya in the Permian–Triassic Periods for the split between the two lamprey subfamilies, Geotriinae and Petromyzoninae; and 30–10 Mya in the Tertiary Period for the split between the two lamprey genera, Petromyzon and Lethenteron. This evolutionary configuration indicates that Myxiniformes and Petromyzoniformes diverged shortly after the common ancestor of cyclostomes split from the future gnathostome lineage. Our results also suggest that intra-subfamilial diversification in hagfish and lamprey lineages (especially those distributed in the northern hemisphere) occurred in the Cretaceous or Tertiary Periods.


Extant agnathans, the cyclostomes, comprise the hagfishes (Hyperotreti; order Myxiniformes) and lampreys (Hyperoartia; order Petromyzoniformes [often misspelled as “Petromyzontiformes”]) (Hardisty and Potter, 1971; Forey and Janvier, 1993; Jørgensen, 1998; see also Ota and Kuratani, 2006) (Fig. 1). After a long-standing controversy on the phylogenetic positions of hagfishes and lampreys, the monophyly of cyclostomes has been unequivocally supported by molecular phylogenetics using a triad of molecules frequently used for reconstruction of species phylogeny, namely, mitochondrial genes (mtDNA), nuclear ribosomal RNA genes (rDNA), and nuclear protein-coding genes (nuDNA) (Fig. 1; Stock and Whitt, 1992; Mallatt and Sullivan, 1998; Kuraku et al., 1999; Delarbre et al., 2002; Furlong and Holland, 2002; Takezaki et al., 2003; Blair and Hedges, 2005; Delsuc et al., 2006). Therefore, our interest in cyclostome evolution has shifted to the topological and temporal aspects of divergence patterns within this animal group.

Fig. 1.

Taxonomy and molecular phylogeny of cyclostomes. Taxonomic information was based on NCBI Entrez Taxonomy, which is mainly consistent with the classification by Renaud (1997) and Fernholm (1998). Taxonomic ranks are indicated in the top row. References that suggest particular phylogenetic relationships are listed near the corresponding nodes in gray shades, with the type of molecule (nuDNA, rDNA, or mtDNA) employed in their analyses. Note that branch lengths do not correspond to evolutionary times. The numbers of nucleotide and amino acid sequences, respectively, found in NCBI Entrez Nucleotide and NCBI Entrez Protein (as of February 15, 2006) are shown in parentheses beside genus names. The hagfish genera Eptatretus and Paramyxine are not monophyletic to each other according to the molecular phylogenetic tree of the 16S rRNA gene (Kuo et al., 2003; Chen et al., 2005). The lamprey genera Entosphenus, Lampetra, and Lethenteron are not monophyletic to one another according to the molecular phylogenetic tree of the cytochrome b and NADH dehydrogenase 3 genes (Docker et al., 1999; see DISCUSSION for details).


The order Myxiniformes is thought to be monophyletic, based on molecular phylogenetic studies using mitochondrial 16S rDNA (Kuo et al., 2003; Chen et al., 2005). This order is divided into the two subfamilies Myxininae and Eptatretinae, based on morphological features (Fig. 1; Fernholm, 1998). The subfamily Myxininae consists of four genera, Myxine and three genera intrinsic to the southern hemisphere (Neomyxine, Nemamyxine, and Notomyxine). The other subfamily, Eptatretinae, consists of three genera, Eptatretus, Paramyxine, and Rubicundus. In contrast, the order Petromyzoniformes is composed of three subfamilies, Mordaciinae, Geotriinae, and Petromyzoninae, in accordance with morphology such as dentition (Hubbs and Potter, 1971; Gill et al., 2003). The subfamilies Mordaciinae and Geotriinae are endemic to the southern hemisphere, and each comprises a single genus, Mordacia and Geotria, respectively (Potter and Strahan, 1968). The subfamily Petromyzoninae is composed of at least six genera (Fig. 1; Hardisty and Potter, 1971; Hubbs and Potter, 1971; Potter and Gill, 2003). However, there are few detailed reports of molecular approaches to estimate divergence times in the cyclostome lineage. To address questions regarding the temporal pattern of cyclostome evolution, the accumulating nucleotide and amino acid sequences of hagfishes and lampreys will provide novel information.

In this study, we analyzed the GC-content in cDNA sequences of hagfishes and lampreys and calculated the divergence times of several branching points in cyclostome phylogeny using nucleotide and amino acid sequences, based on an updated version of vertebrate phylogeny representing the monophyly of cyclostomes.


GC4 calculation

Currently available annotated nucleotide sequences (as of February 15, 2006) were retrieved for each cyclostome species from NCBI Entrez Nucleotide ( Redundant sequences were manually removed. To avoid biased gene selection, in which a large proportion of the sequence population for one species is occupied by members of a limited number of gene families, cDNAs derived from variable leukocyte receptor genes in the sea lamprey, Petromyzon marinus (accession numbers CK988414-CK988652 in NCBI dbEST; Pancer et al., 2004a), and the inshore hagfish, Eptatretus burgeri (accession numbers AY964719-AY965612; Pancer et al., 2005), were excluded from our sequence collection. The nucleotide sequences were used to calculate the GC-content at four-fold degenerate sites (GC4) with the Perl script, in which an open reading frame is automatically detected with an alignment generated by BLASTX (Altschul et al., 1997). Sequences of mtDNA and nuclear rRNA genes were excluded from this GC calculation.

Molecular phylogenetic tree inference

Sequences that showed significant similarity to a query in a BLASTP search (Altschul et al., 1997) were retrieved from databases: GenBank (release 151), NCBI-refseq (release 06-02-16), SWISSPROT (release 49.0), and PIR (release 80.0). An optimal multiple alignment of these amino acid sequences was constructed using the alignment editor XCED implemented in the MAFFT program (Katoh et al., 2002) in combination with manual inspection. Molecular phylogenetic trees were inferred by the neighbor-joining method (Saitou and Nei, 1987) with XCED and the maximum-likelihood method (Felsenstein, 1981; Kishino et al., 1990) with PAML 3.1 (Yang, 1997), using amino acid sites at which the alignment was unambiguous with no gaps, with among-site rate heterogeneity taken into account (Yang, 1994).

Estimation of number of synonymous and nonsynonymous substitutions per site

Nucleotide sequences were prepared as described above in the procedures for GC4 calculation. By inferring molecular phylogenetic trees, we selected genes with a homologue present as a single orthologue in a pair of species in question. Nucleotide sequences of the selected genes were aligned based on an alignment generated for their deduced amino acid sequences. The number of synonymous and nonsynonymous substitutions per site (Ks and Ka, respectively) was calculated with the codon-based maximum-likelihood method (Goldman and Yang, 1994). Computation was accomplished using PAML 3.1 (Yang, 1997).

Amino acid sequence-based divergence time estimation

Estimation of divergence times was processed without assuming a global molecular clock, using the MULTIDIVTIME program in which Markov-chain Monte-Carlo (MCMC) procedures for Bayesian analysis are implemented (Kishino et al., 2001). The upper and lower limits of divergence times outside the cyclostomes were preset by referring to a set of fossil records (Young, 1962) used by Dickerson (1971), or by referring to molecular dating (Kumar and Hedges, 1998; Blair and Hedges, 2005). To estimate divergence times using mitochondorial genes, we used a modified version of the MULTIDIVTIME program as instructed on the developers' web page ( Results were confirmed with the program R8S, which enables penalized rate smoothing (data not shown; Sanderson, 2002, 2003).


GC-content in cyclostome cDNAs

GC4 was calculated for cDNA sequences derived from nuclear protein-coding genes for each cyclostome species. The GC4 of annotated cDNAs exhibited a unimodal distribution, with peaks at 40–60% in hagfish species (Figs. 2A–C) and at 70–90% in lamprey species (Figs. 2D–F). Non-annotated abundant cDNAs of Eptatretus burgeri (Suzuki et al., 2004) and Petromyzon marinus (Pancer et al., 2004b) showed similar GC4 distributions to those of annotated cDNAs for hagfish and lamprey species, respectively (Fig. 2G). The results of the GC4 calculation for genera or species with a small number of available cDNAs were as follows: Paramyxine, 41–61% (n=5); Ichthyomyzon, 73–87% (n=7); Entosphenus, 75–83% (n=2); Mordacia mordax, 72–82% (n=3); Geotria australis, 73–90% (n=9).

Fig. 2.

Distribution of GC4 for hagfish and lamprey cDNAs. Histograms representing the fraction of cDNAs for each GC4 value are shown for three hagfish species, (A) Myxine glutinosa, (B) Eptatretus stoutii, and (C) Eptatretus burgeri, and three lamprey species, (D) Petromyzon marinus, (E) Lethenteron japonicum, and (F) Lampetra fluviatilis. (G) The distributions of GC4 in assembled expressed sequence tags (ESTs) of Eptatretus burgeri and Petromyzon marinus, also shown as histograms. ESTs were retrieved from NCBI dbEST for Eptatretus burgeri (accession numbers, BJ644497-BJ668380; Suzuki et al., 2004) and Petromyzon marinus (CO542795-CO553159; Pancer et al., 2004b). These ESTs were automatically assembled with Phrap (, so that one cDNA sequence represents one gene. The assembled sequences are available upon request. In analyses using the GC-content of the third positions (GC3), we obtained similar results for all the graphs in this Fig.


Estimated number of synonymous substitutions

We selected genes that existed as single orthologues in a pair of species in question with more than 600 bp of aligned nucleotide stretches. Estimation of Ks was processed using the maximum-likelihood method (Goldman and Yang, 1994). The average Ks between the two hagfish genera, Myxine and Eptatretus, was 0.24 (standard deviation (SD), 0.11; n=11; Table 1). In contrast, the average Ks for Petromyzon–Lethenteron and Geotria–Lethenteron pairs was 0.15 (SD, 0.09; n=19; Table 2A) and 1.03 (SD, 0.39; n=2; Table 2B), respectively. Sequence comparison between a hagfish species and a lamprey species always yielded an apparently saturated Ks (>3; data not shown).

Table 1.

Estimated numbers of synonymous and non-synonymous substitutions between Myxininae and Eptatretinae.


Table 2.

Estimated numbers of synonymous and non-synonymous substitutions between lamprey species.


We also estimated the Ks of mitochondrial protein-coding genes in the Myxine–Eptatretus and Petromyzon–Lampetra pairs for which the sequences were available in public databases (Lee and Kocher, 1995; Rasmussen et al., 1998; Delarbre et al., 2000; Delarbre et al., 2001; Delarbre et al., 2002). The average Ks for the Petromyzon–Lampetra pair was 1.19 (SD, 0.57; n=13; Supplemental Table S1,, whereas the Myxine–Eptatretus pair yielded an apparently saturated Ks (>2; data not shown). The difference in Ks values between mitochondrial and nuclear genes (7.9-fold for the Petromyzon–Lampetra pair) was roughly consistent with previous observations in mammals and amphibians (Miyata et al., 1982; Crawford, 2003).

Rough estimation of divergence times based on number of synonymous substitutions

The number of synonymous substitutions per site has been reported for some pairs of chordate species (Table 3). In the present study, to supplement pre-existing data, we preliminarily estimated the Ks between two species of the cephalochordate genus Branchiostoma (Supplemental Table S2, The divergence times and Ks values in Table 3, including the data for Branchiostoma, were plotted two-dimensionally in Fig. 3. The overall rate of this putative clock was 2.4×10−9/site/year (Clock A; Fig. 3A), whereas the rate of the clock for selected species pairs (for details, see legend for Fig. 3) was 1.9×10−9/site/year (Clock B; Fig. 3B). By applying these clocks tentatively to cyclostome taxon pairs, we obtained divergence times of the inter-subfamilial split between Myxininae and Eptatretinae in the hagfish lineage at 93–28 Mya, the inter-generic split between Petromyzon and Lethenteron at 57–15 Mya, and the inter-subfamilial split between Geotriinae and Petromyzoninae in the lamprey lineage at 383–136 Mya (Table 4).

Table 3.

Divergence times and numbers of synonymous substitutions reported for closely related organism pairs.


Fig. 3.

Divergence times and number of synonymous substitutions per site. (A) The tentative clock calibrated by all of the divergences of species pairs (black circles) in Table 3. (B) The tentative clock calibrated by nodes 3, 5, 6, and 8 in Table 3. In this “Clock B”, nodes 1 and 2 were excluded because of an elevated evolutionary rate in the rodent lineage. Node 4 was excluded because of its unreliably high Ks value (>1). Node 7 was excluded because Ks for this species pair was estimated with a different method from the others. In both clocks, regression lines indicated as broken lines pass through the origin. For nodes 6 and 7, the intermediate values of estimated ranges of divergence times in Table 3 were used as divergence times in this Fig. Divergences in cyclostomata (e–g) that were dated with these clocks are indicated as open circles.


Table 4.

Divergence times estimated with tentative synonymous substitution clocks.


Fig. 4.

Molecular phylogenetic tree of a guanine nucleotide-binding protein, beta polypeptide 2-like 1 (GNB2L1) protein. The tree was inferred with the neighbor-joining method using 300 amino acid residues and assuming amongsite rate heterogeneity (shape parameter α=0.64; see MATERIALS AND METHODS for details). The maximum-likelihood method yielded similar tree topologies, although they are not statistically significant. For amino acid-based divergence time estimation, we selected 10 genes with no gene duplication, as shown in this tree.


Fig. 5.

A tree topology preset for divergence time estimation. Letters at nodes correspond to those in Tables 6 and 7.


Table 5.

Genes on nuDNA used for amino acid-based estimation of divergence times.


Table 6.

Divergence times for constrained nodes.


Table 7.

Divergence times estimated with amino acid sequences.


Estimation of divergence times using amino acid sequences

We selected 10 nuclear protein-coding genes with relatively long alignment lengths (>150 amino acids; total length, 2947 amino acids; Table 5) in which no gene duplication was detected in major vertebrate lineages, as shown in Fig. 4 for the gene GNB2L1. The upper and lower limits of divergence times for branching points outside the cyclostomes were preset as shown in Table 6, and the tree topology shown in Fig. 5 was assumed. By executing the MULTIDIVTIME program (Kishino et al., 2001), we obtained divergence times for Myxiniformes and Petromyzoniformes at 671–391 Mya, Myxininae and Eptatretinae at 162–63 Mya, and Petromyzon and Lethenteron at 30–2 Mya (Table 7).

In addition, the timing of the above branching points was estimated using 12 mitochondrial protein-coding genes that had relatively long alignment lengths (total length, 3320 amino acids). ATP synthase F0 subunit 8 was excluded from this analysis because of its short alignment length. As a result, divergence times were estimated to be 728–459 Mya for the Myxiniformes–Petromyzoniformes split, 72–39 Mya for the Myxininae–Eptatretinae split, and 37–18 Mya for the PetromyzonLampetra split (Table 7).


GC4 as a reflection of base composition in cyclostome genomes

The GC-content at synonymous sites in a protein-coding gene is expected to positively correlate with the global GC-content of the genomic region where the gene is located (Clay et al., 1996; Musto et al., 1999; Kuraku et al., 2006). Therefore, we focused on GC4 in currently available cDNAs reported for cyclostomes (Fig. 2). Hagfish and lamprey cDNAs show similar levels of heterogeneity in GC-content (Fig. 2). However, there is a striking difference in the level of GC-content between hagfish and lamprey: every lamprey species we analyzed showed a high GC4 (70–90%; Figs. 2A–C), whereas every hagfish species we analyzed showed a relatively moderate GC4 (40–60%; Figs. 2D–F).

Cytogenetic studies have revealed that hagfishes have a relatively moderate number of relatively moderate-sized chromosomes compared to other vertebrates (2n=14–36 in somatic cells), whereas lampreys possess a much greater number of small, dot-like chromosomes (2n=76–178; Potter and Rothwell, 1970; Potter and Robinson, 1971; Robinson et al., 1975; Nakai et al., 1995; Animal Genome Size Database, The contrast in chromosome size, chromosome number, and GC-content between hagfishes and lampreys is reminiscent of the intra-genomic difference between macrochromosomes and microchromosomes seen in sauropsids (Burt, 2002; Kuraku et al., 2006). Further investigation will be required to understand the putative relationships among these genomic features.

Ks as a tool to standardize evolutionary distances

The number of synonymous substitutions per site in a protein-coding region serves as an ideal standard for evolutionary distance when comparing closely related species, as long as the gene in question has evolved in a neutral manner (Miyata and Yasunaga, 1980; Perler et al., 1980). In this study, we estimated Ks with the maximum-likelihood method (Goldman and Yang, 1994) because this method is expected to produce relatively appropriate estimates, even in species with highly biased base compositions, such as lampreys (Fig. 2). For all the genes analyzed in this study, Ks was larger than Ka, indicating that these genes have evolved neutrally without experiencing positive selection. In selecting pairs of cyclostome species for Ks estimation, we treated the lamprey genus Lampetra as having the same distance the genus Lethenteron has from Petromyzon. This is based on a previous phylogenetic study using the cyto-chrome b and NADH dehydrogenase subunit 3 (ND3) genes (Docker et al., 1999), which is consistent with the classification by Potter (1980), who formerly considered that the three subgenera, Entosphenus, Lethenteron, and Lampetra, compose the single genus Lampetra. Despite the small number of genes sampled, similar Ks values were consistently obtained for Petromyzon–Lampetra and Petromyzon–Lethenteron pairs (Table 2). In contrast to inter-generic and inter-subfamilial comparisons in cyclostomes, our preliminary Ks estimates between two tunicate species in the same genus (Ciona intestinalis and C. savignyi) and two amphioxus species in the same genus (Branchiostoma belcheri and B. floridae) yielded relatively larger Ks values (see Supplemental Table S2, 1053 for Branchiostoma; Ks>3 for Ciona; data not shown), despite their close taxonomic distances.

Theoretically, the Ks value is determined by the time that has elapsed since the divergence of two species in question, as long as neutrality holds. This feature of Ks is useful in judging orthology between genes (especially members of a gene family prone to gene duplications) of closely related species. If this idea is tentatively applied to previous crossspecies comparisons in lamprey studies, for example, orthology between L. japonicum Hox6w and L. fluviatilis HoxL6 is again confirmed with an extremely low Ks value (Ks=0.039), a reasonable estimate for intrasubfamilial comparison, as suggested previously (Takio et al., 2004).

Methodological aspects of molecular dating

Proposing a constant rate of nucleotide and amino acid substitutions was one of the milestones for molecular dating of divergence times (“molecular clock”; Zuckerkandl and Pauling, 1962, 1965; also see Donoghue et al., 2003; Bromham and Penny, 2003; and Kumar, 2005, for review). As shown in Fig. 3, the numbers of synonymous substitutions per site and divergence times behave in a clock-like manner, at least within the chordates, indicating that this may serve as a rough molecular clock for species pairs with an unsaturated Ks. However, this clock was calibrated by branching points in different lineages outside the cyclostomes (Table 3), because no branching point with a known divergence time was available within cyclostomes. In addition, variation of evolutionary rate among lineages, such as rate elevation in rodents (Kikuno et al., 1985; Wu and Li, 1985; Rat Genome Sequencing Project Consortium, 2004), may confuse divergence time estimation. Therefore, our results need to be verified by refinement of this silent clock with reexamination of divergence times and estimation of Ks for more pairs of organisms.

We utilized amino acid sequences to obtain more robust estimates. Especially in our Ks analysis, synonymous substitutions between Myxiniformes and Petromyzoniformes were apparently saturated (data not shown), suggesting that this split would need to be dated with amino acid sequences rather than nucleotide sequences. However, as exemplified in Fig. 4, phylogenetic trees including cyclostome species often show a high degree of rate heterogeneity because of the accelerated evolutionary rate in hagfishes. To minimize the undesirable influence of this rate heterogeneity on divergence time estimation, we employed a non-parametric molecular dating method that does not assume rate constancy (Kishino et al., 2001; Thorne and Kishino, 2002; see also Hasegawa et al., 2003). Moreover, we paid close attention to the orthologous/paralogous relationships of multiple members of gene families between hagfishes, lampreys, and gnathostomes, because putative genome duplications in early vertebrate evolution (Ohno, 1970; McLysaght et al., 2002) often confuse orthology identification; Kuraku et al. (1999) provided an example in the enolase gene family. In addition, inclusion of genes prone to gene duplications may also result in misleading estimates of divergence time, possibly because of an accelerated evolutionary rate caused by neofunctionalization or subfunctionalization of duplicates. For these reasons, we deliberately selected genes for which no gene duplication was detected in major vertebrate lineages (Table 5).

As calibration points outside the cyclostomes, we used three sets of divergence time constraints (Table 6). One of the three constraint sets was based on the fossil records used by Dickerson (1971) (constraint set I), whereas the other two were based on previous studies using molecular data (constraint set II, Kumar and Hedges, 1998; constraint set III, Blair and Hedges, 2005). However, these molecular studies included genes that underwent duplication events early in vertebrate evolution and do not represent a 1:1 relationship between a cyclostome gene and a gnathostome counterpart (e.g., bone morphogenetic protein (BMP) 2/4, enolase-2). Although fossil records inherently tend to yield more recent divergence times because of potential incomplete fossil sampling in more ancient eras, it is possible that inappropriate gene selection might have yielded much more ancient estimates in these studies (constraint sets II and III) compared with commonly accepted fossil records (constraint set I). This discrepancy emphasizes again that precision in gene selection cannot be sacrificed, even in the name of high-throughput analysis using large data sets. For this reason, we summarize our results below, along a temporal axis based on the fossil records.

Temporal reconstruction of cyclostome phylogeny

The monophyly of cyclostomes has resulted in a dispute over when hagfishes and lampreys split from each other in the cyclostome lineage. To answer this question, our analyses using mitochondrial and nuclear genes consistently showed that Myxiniformes and Petromyzoniformes diverged from each other 30–110 million years after the cyclostome lineage split from the future gnathostome lineage (Table 7). When calibration by fossil records was applied, this Myxiniformes–Petromyzoniformes split dated back to 470–390 Mya in the Ordovician–Silurian–Devonian Periods (Fig. 6), when fossil agnathans are thought to have diversified (Forey and Janvier, 1993; Janvier, 1996). Although we still do not know the precise branching pattern among these agnathans, hagfishes and lampreys represent two distinct agnathan groups that diverged early in vertebrate evolution and have survived thereafter for more than 400 million years. Our results indicate that, although both groups are classified as cyclostomes, the distance between hagfishes and lampreys is similar to that between humans and cartilaginous fishes, in terms of the geological time that has elapsed since their divergence.

Fig. 6.

Hypothesized time scale of cyclostome evolution. The phylogeny and timing of cyclostome diversification are illustrated so that branch lengths correspond to evolutionary times (black solid lines). Numerical time scales corresponding to constraint sets I–III in Table 6 are indicated in the upper rows. The geological time scale based on the chart provided by the Geological Society of America ( is added to the time scale based on constraint set I. Black circles indicate branching points whose divergence times were estimated in this study. The divergence time of the split between Myxininae and Eptatretinae and between Petromyzon and Lethenteron/Lampetra is based on both synonymous substitution clocks and amino acid-based relaxed clocks using mtDNA and nuDNA. The divergence time of the split between Myxiniformes and Petromyzoniformes is solely based on a relaxed clock analysis using amino acid sequences encoded by mtDNA and nuDNA. The divergence time of the split between Geotriinae and Petromyzoninae is estimated based synonymous substitution clocks. Fossil records are indicated by solid grey lines, with the putative phylogenetic relationships proposed by Janvier (1997).


Later in the hagfish lineage, our molecular dating with amino acid-based relaxed clocks and synonymous substitution clocks indicated that there was no branching of extant taxa until the two hagfish subfamilies, Myxininae and Eptatretinae, split from each other 90–60 Mya in the Cretaceous–Tertiary Periods (Fig. 6). However, there is a paleontological report of a fossil species, Myxinikela siroka, from the Carboniferous fauna (~300 Mya) that is regarded as a putative outgroup of extant hagfishes (Bardack, 1991). In our divergence time estimate using relaxed molecular clocks, even when we tentatively constrained the upper limit of the divergence time of the Myxininae–Eptatretinae split to 300 Mya, we obtained an identical result (data not shown), suggesting that this fossil species, Myxinikela siroka, actually is an outgroup of extant hagfishes (Fig. 6).

All the three subfamilies (Mordaciinae, Geotriinae, and Petromyzoninae) in the lamprey lineage are thought to have diverged from one another in a considerably short period of time (Conlon et al., 2001; Gill et al., 2003), as indicated by ambiguous phylogenetic relationships between these three taxa in recent molecular studies (Baldwin et al., 1988; Silver et al., 2004; Takahashi et al., 2006). Our synonymous substitution clock indicates that the Geotriinae–Petromyzoninae split occurred in the Permian–Triassic Periods (280–220 Mya; Fig. 6). Although this estimate needs to be reinforced with more robust analyses using amino acid sequences, this divergence time may coincide with the break-up of Gondwana (inhabited by species in Mordaciinae and Geotriinae) from Laurasia (inhabited by species in Petromyzoninae). Later, in the lineage of Petromyzoninae, an inter-generic split between Petromyzon and Lethenteron/Lampetra occurred 30–10 Mya in the Tertiary Period (Fig. 6). This result is consistent with the rough estimate by Docker et al. (1999), who simply assumed that a 2% divergence in mtDNA sequence corresponds to one million years (Brown et al., 1979). To further confirm phylogenetic relationships in Petromyzoninae, reported previously based on morphological features (Gill et al., 2003), molecular sequence data from other genera need to be included. Fossils of the lampreys Hardistiella montanensis, Mayomyzon pieckoensis, and Pipiscius zangerli were found in the Carboniferous fauna (~280 Mya) and have been treated as outgroups of all extant lampreys (Bardack and Zangerl, 1971; Bardack and Richardson, 1977; Janvier and Lund, 1983). In nuDNA-based and mtDNA-based analyses using relaxed molecular clocks, adding these paleontological data did not produce any substantial differences in results (data not shown), indicating that these fossil lampreys should still be regarded as outgroups of all extant lamprey species in Petromyzoniformes (Fig. 6).


Thanks to the efforts of researchers in various fields of biology (e.g., Kuratani et al., 2002), nucleotide and amino acid sequences of hagfishes and lampreys are accumulating in public databases. However, information at the molecular level is still far from satisfactory for cyclostomes (Fig. 1), in two aspects. First, in terms of the coverage of species diversity, there is a paucity of molecular data for southern hemisphere species, and these data are crucial for inferences of phylogenetic relationships and divergence times. For example, in Myxiniformes, no molecular sequence data have been reported for Notomyxine, Nemamyxine, or Neomyxine, which are thought to belong in the subfamily Myxininae (Jørgensen, 1998). Similarly, in lampreys, the unavailability of appropriate nucleotide sequences for Mordacia hindered inclusion of Mordaciinae in our silent clock analysis, and the unavailability of appropriate amino acid sequences for Mordacia and Geotria did not allow us to include Mordaciinae and Geotriinae in our amino acid-based relaxed clock analysis.

Second, there are few reports of genomic DNA sequences for hagfishes and lampreys, and most of the reported nucleotide sequences are derived from mRNAs. In this study, based on the nucleotide sequences of proteincoding exons, we estimated accumulated levels of synonymous substitutions for inter-subfamilial and inter-generic species pairs, which highlighted a relatively low level of neutral nucleotide changes in the Myxine–Eptatretus and Petromyzon–Lethenteron pairs (Tables 1 and 2A). If the synonymous substitution rate in coding regions roughly corresponds to the neutral substitution rate in intergenic or intronic regions without regulatory functions, our estimates imply that these taxon pairs might be too phylogenetically close to discern potentially functional sequences, such as cis-regulatory elements or non-coding genes.

This principle is referred to as “phylogenetic footprinting” (Gumucio et al., 1992; see also Zhang and Gerstein, 2003, for review), and selecting multiple species with appropriate levels of nucleotide substitution facilitates an efficient in silico detection of potentially functional genomic sequences (Uchikawa et al., 2003; Johnson et al., 2004; Kusakabe, 2005). In this context, using lampreys as an example, comparison of non-coding genomic sequences among multiple species in Petromyzoninae alone will not provide a sufficient level of resolution to highlight functional fractions. Instead, judging from the almost saturated Ks level between Geotriinae and Petromyzoninae (Table 2B), inclusion of southern hemisphere lampreys (Mordacia or Geotria) would be highly promising in comparisons with species in the northern hemisphere subfamily Petromyzoninae, such as Petromyzon marinus, whose genome sequencing project is now underway.

Despite the scarcity of sequence information, we attempted to overview the general features of base composition and evolutionary distance in cyclostomes, and obtained results that will serve as standards for future evolutionary and genomic studies of cyclostomes. We propose that, in any taxa, this sort of succinct evolutionary analysis should be a prerequisite for any biological studies involving multispecies comparisons. Even if the amount of available sequence information is limited, general trends embedded in sequence information can be extracted in light of the theories of molecular evolution.


We are very grateful to Kazutaka Katoh for computational advice and to Kinya Ota, David McCauley, and Yoichi Matsuda for insightful discussion. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.



S. F. Altschul, T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman . 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. Google Scholar


E. Axelsson, M. T. Webster, N. G. Smith, D. W. Burt, and H. Ellegren . 2005. Comparison of the chicken and turkey genomes reveals a higher rate of nucleotide divergence on microchromosomes than macrochromosomes. Genome Res 15:120–125. Google Scholar


J. Baldwin, K. Mortimer, and A. Patak . 1988. Evolutionary relationships among lamprey families: amino acid composition analysis of lactate dehydrogenase. Biochem Syst Ecol 16:351–353. Google Scholar


D. Bardack 1991. First fossil hagfish (Myxinoidea): a record from the Pennsylvanian of Illinois. Science 254:701–703. Google Scholar


D. Bardack and E. S. Richardson . 1977. New agnathous fishes from the Pennsylvanian of Illinois. Fieldiana Geol 33:489–510. Google Scholar


D. Bardack and R. Zangerl . 1971. Lampreys in the fossil record. In “The Biology of Lampreys”. Ed by M. W. Hardisty and I. C. Potter , editors. Academic Press. London & New York. pp. 67–84. Google Scholar


M. J. Benton 1993. The Fossil Record 2. 1st edPalaeontological Association, Royal Society (Great Britain), Linnean Society of London, Chapman and Hall. London. Google Scholar


J. E. Blair and S. B. Hedges . 2005. Molecular phylogeny and divergence times of deuterostome animals. Mol Biol Evol 22:2275–2284. Google Scholar


L. Bromham and D. Penny . 2003. The modern molecular clock. Nat Rev Genet 4:216–224. Google Scholar


W. M. Brown, M. George Jr, and A. C. Wilson . 1979. Rapid evolution of animal mitochondrial DNA. Proc Natl Acad Sci USA 76:1967–1971. Google Scholar


D. W. Burt 2002. Origin and evolution of avian microchromosomes. Cytogenet Genome Res 96:97–112. Google Scholar


Y. Chen, H. Chang, and H. Mok . 2005. Phylogenetic position of Eptatretus chinensis (Myxinidae: Myxiniformes) inferred by 16S rRNA gene sequence and morphology. Zool Stud 44:111–118. Google Scholar


O. Clay, S. Caccio, S. Zoubak, D. Mouchiroud, and G. Bernardi . 1996. Human coding and noncoding DNA: compositional correlations. Mol Phylogenet Evol 5:2–12. Google Scholar


J. M. Conlon, Y. Wang, and I. C. Potter . 2001. The structure of Mordacia mordax insulin supports the monophyly of the Petromyzontiformes and an ancient divergence of Mordaciidae and Geotriidae. Comp Biochem Physiol B Biochem Mol Biol 129:65–71. Google Scholar


A. J. Crawford 2003. Relative rates of nucleotide substitution in frogs. J Mol Evol 57:636–641. Google Scholar


T. Crnogorac-Jurcevic, J. R. Brown, H. Lehrach, and L. C. Schalkwyk . 1997. Tetraodon fluviatilis, a new puffer fish model for genome studies. Genomics 41:177–184. Google Scholar


C. Delarbre, H. Escriva, C. Gallut, V. Barriel, P. Kourilsky, P. Janvier, V. Laudet, and G. Gachelin . 2000. The complete nucleotide sequence of the mitochondrial DNA of the agnathan Lampetra fluviatilis: bearings on the phylogeny of cyclostomes. Mol Biol Evol 17:519–529. Google Scholar


C. Delarbre, A. S. Rasmussen, U. Arnason, and G. Gachelin . 2001. The complete mitochondrial genome of the hagfish Myxine glutinosa: unique features of the control region. J Mol Evol 53:634–641. Google Scholar


C. Delarbre, C. Gallut, V. Barriel, P. Janvier, and G. Gachelin . 2002. Complete mitochondrial DNA of the hagfish, Eptatretus burgeri: the comparative analysis of mitochondrial DNA sequences strongly supports the cyclostome monophyly. Mol Phylogenet Evol 22:184–192. Google Scholar


F. Delsuc, H. Brinkmann, D. Chourrout, and H. Philippe . 2006. Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 439:965–968. Google Scholar


R. E. Dickerson 1971. The structures of cytochrome c and the rates of molecular evolution. J Mol Evol 1:26–45. Google Scholar


D. E. Dimcheff, S. V. Drovetski, and D. P. Mindell . 2002. Phylogeny of Tetraoninae and other galliform birds using mitochondrial 12S and ND2 genes. Mol Phylogenet Evol 24:203–215. Google Scholar


M. F. Docker, J. H. Youson, R. J. Beamish, and R. H. Devlin . 1999. Phylogeny of the lamprey genus Lampetra inferred from mitochondrial cytochrome b and ND3 gene sequences. Can J Fish Aquat Sci 56:2340–2349. Google Scholar


P. C. J. Donoghue, M. P. Smith, and I. J. Sansom . 2003. The origin and early evolution of chordates: molecular clocks and the fossil record. In “Telling the Evolutionary Time”. Ed by P. C. J. Donoghue and M. P. Smith , editors. Taylor & Francis. London. pp. 190–223. Google Scholar


J. Felsenstein 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376. Google Scholar


B. Fernholm 1998. Hagfish systematics. In “The Biology of Hagfishes”. Ed by J. M. Jørgensen, J. P. Lomholt, R. E. Weber, and H. Malte , editors. Chapman & Hall. London. pp. 33–44. Google Scholar


P. Forey and P. Janvier . 1993. Agnathans and the origin of jawed vertebrates. Nature 361:129–134. Google Scholar


R. F. Furlong and P. W. H. Holland . 2002. Bayesian phylogenetic analysis supports monophyly of ambulacraria and of cyclostomes. Zool Sci 19:593–599. Google Scholar


H. S. Gill, C. B. Renaud, F. Chapleau, R. L. Mayden, and I. C. Potter . 2003. Phylogeny of living parasitic lampreys (Petromyzontiformes) based on morphological data. Copeia 4:687–703. Google Scholar


N. Goldman and Z. Yang . 1994. A codon-based model of nucleotide substitution for protein-coding DNA sequences. Mol Biol Evol 11:725–736. Google Scholar


D. L. Gumucio, H. Heilstedt-Williamson, T. A. Gray, S. A. Tarle, D. A. Shelton, D. A. Tagle, J. L. Slightom, M. Goodman, and F. S. Collins . 1992. Phylogenetic footprinting reveals a nuclear protein which binds to silencer sequences in the human gamma and epsilon globin genes. Mol Cell Biol 12:4919–4929. Google Scholar


M. W. Hardisty and I. C. Potter . 1971. The Biology of Lampreys. Academic Press. London & New York. Google Scholar


M. Hasegawa, J. L. Thorne, and H. Kishino . 2003. Time scale of eutherian evolution estimated without assuming a constant rate of molecular evolution. Genes Genet Syst 78:267–283. Google Scholar


C. L. Hubbs and I. C. Potter . 1971. Distribution, phylogeny, and taxonomy. In “The Biology of Lampreys”. Ed by M. W. Hardisty and I. C. Potter , editors. Academic Press. London & New York. pp. 1–66. Google Scholar


International Chicken Genome Sequencing Consortium (ICGSC) 2004. Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432:695–716. Google Scholar


O. Jaillon, J. M. Aury, F. Brunet, J. L. Petit, N. Stange-Thomann, E. Mauceli, L. Bouneau, C. Fischer, C. Ozouf-Costaz, A. Bernot, et al . 2004. Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 431:946–957. Google Scholar


A. Janke and U. Arnason . 1997. The complete mitochondrial genome of Alligator mississippiensis and the separation between recent archosauria (birds and crocodiles). Mol Biol Evol 14:1266–1272. Google Scholar


P. Janvier and R. Lund . 1983. Hardistiella montanensis n. gen. et sp. (Petromyzontida) from the Lower Carboniferous of Montana, with remarks on the affinities of the lampreys. J Vert Paleont 2:407–413. Google Scholar


P. Janvier 1996. The dawn of the vertebrates: characters versus common ascent in the rise of current vertebrate phylogenies. Paleontology 39:259–287. Google Scholar


P. Janvier 1997. The Tree of Life Project (URL: Google Scholar


D. S. Johnson, B. Davidson, C. D. Brown, W. C. Smith, and A. Sidow . 2004. Noncoding regulatory sequences of Ciona exhibit strong correspondence between evolutionary constraint and functional importance. Genome Res 14:2448–2456. Google Scholar


J. M. Jørgensen 1998. The Biology of Hagfishes. 1st edChapman and Hall. London. Google Scholar


F. G. Jørgensen, A. Hobolth, H. Hornshoj, C. Bendixen, M. Fredholm, and M. H. Schierup . 2005. Comparative analysis of protein coding sequences from human, mouse and the domesticated pig. BMC Biol 3:2. Google Scholar


K. Katoh, K. Misawa, K. Kuma, and T. Miyata . 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30:3059–3066. Google Scholar


R. Kikuno, H. Hayashida, and T. Miyata . 1985. Rapid rate of rodent evolution. Proc Jpn Acad 61:153–156. Google Scholar


H. Kishino, T. Miyata, and M. Hasegawa . 1990. Maximum likelihood inference of protein phylogeny and the origin of chloroplasts. J Mol Evol 30:151–160. Google Scholar


H. Kishino, J. L. Thorne, and W. J. Bruno . 2001. Performance of a divergence-time estimation method under a probabilistic model of rate evolution. Mol Biol Evol 18:352–361. Google Scholar


S. Kumar 2005. Molecular clocks: four decades of evolution. Nat Genet Rev 6:654–662. Google Scholar


S. Kumar and S. B. Hedges . 1998. A molecular timescale for vertebrate evolution. Nature 392:917–920. Google Scholar


C. H. Kuo, S. Huang, and S. C. Lee . 2003. Phylogeny of hagfish based on the mitochondrial 16S rRNA gene. Mol Phylogenet Evol 28:448–457. Google Scholar


S. Kuraku, D. Hoshiyama, K. Katoh, H. Suga, and T. Miyata . 1999. Mono-phyly of lampreys and hagfishes supported by nuclear DNA-coded genes. J Mol Evol 49:729–735. Google Scholar


S. Kuraku, J. Ishijima, C. Nishida-Umehara, K. Agata, S. Kuratani, and Y. Matsuda . 2006. cDNA-based gene mapping and GC3 profiling in soft-shelled turtle suggest chromosome size-dependent GC bias shared by sauropsids. Chromosome Res 14:187–202. Google Scholar


S. Kuratani, S. Kuraku, and Y. Murakami . 2002. Lamprey as an evo-devo model: lessons from comparative embryology and molecular phylogenetics. Genesis 34:175–183. Google Scholar


T. Kusakabe 2005. Decoding cis-regulatory systems in ascidians. Zool Sci 22:129–146. Google Scholar


W. J. Lee and T. D. Kocher . 1995. Complete sequence of a sea lamprey (Petromyzon marinus) mitochondrial genome: early establishment of the vertebrate genome organization. Genetics 139:873–887. Google Scholar


J. Mallatt and J. Sullivan . 1998. 28S and 18S rDNA sequences support the monophyly of lampreys and hagfishes. Mol Biol Evol 15:1706–1718. Google Scholar


A. McLysaght, K. Hokamp, and K. H. Wolfe . 2002. Extensive genomic duplication during early chordate evolution. Nat Genet 31:200–204. Google Scholar


T. Miyata and T. Yasunaga . 1980. Molecular evolution of mRNA: a method for estimating evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences and its application. J Mol Evol 16:23–36. Google Scholar


T. Miyata, H. Hayashida, R. Kikuno, M. Hasegawa, M. Kobayashi, and K. Koike . 1982. Molecular clock of silent substitution: at least six-fold preponderance of silent changes in mitochondrial genes over those in nuclear genes. J Mol Evol 19:28–35. Google Scholar


H. Musto, H. Romero, A. Zavala, and G. Bernardi . 1999. Compositional correlations in the chicken genome. J Mol Evol 49:325–329. Google Scholar


Y. Nakai, S. Kubota, Y. Goto, T. Ishibashi, W. Davison, and S. Kohno . 1995. Chromosome elimination in three Baltic, south Pacific and north-east Pacific hagfish species. Chromosome Res 3:321–330. Google Scholar


M. Nohara, M. Nishida, V. Manthacitra, and T. Nishikawa . 2004. Ancient phylogenetic separation between Pacific and Atlantic cephalo-chordates as revealed by mitochondrial genome analysis. Zool Sci 21:203–210. Google Scholar


S. Ohno 1970. Evolution by Gene Duplication. Springer-Verlag. Berlin. Google Scholar


K. Ota and S. Kuratani . 2006. The history of scientific endeavors towards understanding hagfish embryology. Zool Sci 23:403–418. Google Scholar


Z. Pancer, C. T. Amemiya, G. R. Ehrhardt, J. Ceitlin, G. L. Gartland, and M. D. Cooper . 2004a. Somatic diversification of variable lymphocyte receptors in the agnathan sea lamprey. Nature 430:174–180. Google Scholar


Z. Pancer, W. E. Mayer, J. Klein, and M. D. Cooper . 2004b. Prototypic T cell receptor and CD4-like coreceptor are expressed by lymphocytes in the agnathan sea lamprey. Proc Natl Acad Sci USA 101:13273–13278. Google Scholar


Z. Pancer, N. R. Saha, J. Kasamatsu, T. Suzuki, C. T. Amemiya, M. Kasahara, and M. D. Cooper . 2005. Variable lymphocyte receptors in hagfish. Proc Natl Acad Sci USA 102:9224–9229. Google Scholar


F. Perler, A. Efstratiadis, P. Lomedico, W. Gilbert, R. Kolodner, and J. Dodgson . 1980. The evolution of genes: the chicken preproin-sulin gene. Cell 20:555–566. Google Scholar


I. C. Potter 1980. The Petromyzoniformes with particular reference to paired species. Can J Fish Aquat Sci 37:1595–1615. Google Scholar


I. C. Potter and H. S. Gill . 2003. Adaptive radiation of lampreys. J Great Lakes Res 29:95–112. Google Scholar


I. C. Potter and E. S. Robinson . 1971. The chromosomes. In “The Biology of Lampreys”. Ed by M. W. Hardisty and I. C. Potter , editors. Academic Press. London. pp. 279–293. Google Scholar


I. C. Potter and B. Rothwell . 1970. The mitotic chromosomes of the lamprey, Petromyzon marinus L. Experientia 26:429–430. Google Scholar


I. C. Potter and R. Strahan . 1968. The taxonomy of the lampreys, Geotria and Mordacia, and their distribution in Australia. Proc Linn Soc Lond 179:229–240. Google Scholar


A. S. Rasmussen, A. Janke, and U. Arnason . 1998. The mitochondrial DNA molecule of the hagfish (Myxine glutinosa) and vertebrate phylogeny. J Mol Evol 46:382–388. Google Scholar


Rat Genome Sequencing Project Consortium (RGSPC) 2004. Genome sequence of the brown Norway rat yields insights into mammalian evolution. Nature 428:493–521. Google Scholar


C. B. Renaud 1997. Conservation status of northern hemisphere lampreys (Petromyzontidae). J Appl Ichthyol 13:143–148. Google Scholar


E. S. Robinson, I. C. Potter, and N. B. Atkin . 1975. The nuclear DNA content of lampreys. Experientia 31:912–913. Google Scholar


N. Saitou and M. Nei . 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. Google Scholar


M. J. Sanderson 2002. Estimating absolute rates of molecular evolution and divergence times: a penalized likelihood approach. Mol Biol Evol 19:101–109. Google Scholar


M. J. Sanderson 2003. r8s: inferring absolute rates of molecular evolution and divergence times in the absence of a molecular clock. Bioinformatics 19:301–302. Google Scholar


M. R. Silver, H. Kawauchi, M. Nozaki, and S. A. Sower . 2004. Cloning and analysis of the lamprey GnRH-III cDNA from eight species of lamprey representing the three families of Petromyzoniformes. Gen Comp Endocrinol 139:85–94. Google Scholar


M. S. Springer, W. J. Murphy, E. Eizirik, and S. J. O'Brien . 2003. Placental mammal diversification and the Cretaceous-Tertiary boundary. Proc Natl Acad Sci USA 100:1056–1061. Google Scholar


D. W. Stock and G. S. Whitt . 1992. Evidence from 18S ribosomal RNA sequences that lampreys and hagfishes form a natural group. Science 257:787–789. Google Scholar


T. Suzuki, I. T. Shin, Y. Kohara, and M. Kasahara . 2004. Transcriptome analysis of hagfish leukocytes: a framework for understanding the immune system of jawless fishes. Dev Comp Immunol 28:993–1003. Google Scholar


A. Takahashi, O. Nakata, S. Moriyama, M. Nozaki, J. M. P. Jos, S. A. Sower, and H. Kawauchi . 2006. Occurrence of two functionally distinct proopiomelanocortin genes in all modern lampreys. Gen Comp Endocrinol 148:72–78. Google Scholar


N. Takezaki, F. Figueroa, Z. Zaleska-Rutczynska, and J. Klein . 2003. Molecular phylogeny of early vertebrates: monophyly of the agnathans as revealed by sequences of 35 genes. Mol Biol Evol 20:287–292. Google Scholar


Y. Takio, M. Pasqualetti, S. Kuraku, S. Hirano, F. M. Rijli, and S. Kuratani . 2004. Evolutionary biology: lamprey Hox genes and the evolution of jaws. Nature 429:1. p following 262. Google Scholar


J. L. Thorne and H. Kishino . 2002. Divergence time and evolutionary rate estimation with multilocus data. Syst Biol 51:689–702. Google Scholar


M. Uchikawa, Y. Ishida, T. Takemoto, Y. Kamachi, and H. Kondoh . 2003. Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mammals. Dev Cell 4:509–519. Google Scholar


C. I. Wu and W. H. Li . 1985. Evidence for higher rates of nucleotide substitution in rodents than in man. Proc Natl Acad Sci USA 82:1741–1745. Google Scholar


Z. Yang 1994. Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: approximate methods. J Mol Evol 39:306–314. Google Scholar


Z. Yang 1997. PAML: a program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci 13:555–556. Google Scholar


J. Z. Young 1962. The Life of Vertebrates. 2nd edOxford Univ Press. Oxford. Google Scholar


Z. Zhang and M. Gerstein . 2003. Of mice and men: phylogenetic footprinting aids the discovery of regulatory elements. J Biol 2:11. Google Scholar


E. Zuckerkandl and L. Pauling . 1962. Molecular disease, evolution and genetic heterogeneity. In “Horizons in Biochemistry”. Ed by M. Kasha and B. Pullman , editors. Academic Press. New York. pp. 189–225. Google Scholar


E. Zuckerkandl and L. Pauling . 1965. Evolutionary divergence and convergence. In “Evolving Genes and Proteins”. Ed by V. Bryson and H. J. Vogel , editors. Academic Press. New York. pp. 97–166. Google Scholar
Shigehiro Kuraku and Shigeru Kuratani "Time Scale for Cyclostome Evolution Inferred with a Phylogenetic Diagnosis of Hagfish and Lamprey cDNA Sequences," Zoological Science 23(12), 1053-1064, (1 December 2006).
Received: 1 May 2006; Accepted: 1 July 2006; Published: 1 December 2006
molecular clock
synonymous substitution
Back to Top