A Novel Mitochondrial Gene Order in the Crinoid Echinoderm Florometra serratissima

Andrea Scouras and Michael J. Smith

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 literature cited
 
The complete nucleotide sequence of the mitochondrial genome of the crinoid Florometra serratissima has been determined. It is a circular DNA molecule, 16,005 bp in length, containing the genes for 13 proteins, small and large ribosomal RNAs, and 22 transfer RNAs (tRNAs). Three regions of unassigned sequence (UAS) greater than 73 bp have been located. The largest, UAS I, is 432 bp long and exhibits sequence similarity to the putative mitochondrial control regions seen in other animals. UAS II (77 bp) and UAS III (73 bp) are located between the 5' ends of coding sequences and may play roles as bidirectional promoters. Analyses of nucleotide composition revealed that the major peptide-encoding strand is high in T and low in C. This bias is reflected in a specific pattern of codon usage. Molecular phylogenetic analyses based on cytochrome c oxidase (COI, COII, and COIII) amino acid and nucleotide sequences did not resolve all the relationships between echinoderm classes. The overall animal mitochondrial gene content has been maintained in the crinoid, but there is extensive rearrangement with respect to both the echinoid and the asteroid mtDNA gene maps. Florometra serratissima has a novel genome organization in a segment containing most of the tRNA genes, large and small rRNA genes, and the NADH dehydrogenase subunit 1 and 2 genes. Potential pathways and mechanisms for gene rearrangements between mitochondrial gene maps of echinoderm classes and vertebrates are discussed as indicators of early deuterostome phylogeny.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 literature cited
 
The complete DNA sequences of mitochondrial genomes are now available from a variety of different animals. Most of these genomes are circular DNA molecules that range in size from 14 to 17 kb. Linear mitochondrial genomes are found in some cnidarians (the cubozoans, scyphozoans, and hydrozoans) (Bridge et al. 1992Citation ). There is a notable conservation of gene content in animal mitochondrial genomes but gene maps vary between taxa (Cantatore et al. 1989Citation ; Desjardins and Morais 1990Citation ; Pääbo et al. 1991Citation ; Okimoto et al. 1992Citation ; Wolstenholme 1992Citation ; Boore and Brown 1994a, 1994b, 1995Citation ; Yamazaki et al. 1997Citation ). The animal mitochondrial genome contains genes for 13 proteins, the oxidative phosphorylation system cytochrome c oxidase subunits I, II, and III (COI, COII, and COIII) and cytochrome b (Cyt b), the ATPase complex subunits 6 and 8 (ATPase 6 and ATPase 8), and the respiratory chain NADH dehydrogenase subunits 1–6 and 4L (ND1ND6 and ND4L); 22 transfer RNA (tRNA) genes; and small and large ribosomal RNA genes (srRNA and lrRNA). In a limited number of cases, sequences associated with the initiation of replication and transcription of the mitochondrial DNA (mtDNA) have been demonstrated (Walberg and Clayton 1981Citation ; Clayton 1982, 1984, 1991Citation ; Montoya et al. 1982Citation ; Montoya, Gaines, and Attardi 1983Citation ; Brown et al. 1986Citation ).

Among vertebrates, the mtDNA gene maps are relatively stable, although rearrangements have been reported for a sea lamprey (Lee and Kocher 1995Citation ), frogs (Yoneyama 1987Citation ; Macey et al. 1997Citation ), reptiles (Seutin et al. 1994Citation ; Kumazawa and Nishida 1995Citation ; Quinn and Mindell 1996Citation ; Macey et al. 1997Citation ), birds (Desjardins and Morais 1990, 1991Citation ; Quinn and Wilson 1993Citation ; Härlid, Janke, and Arnason 1997, 1998Citation ; Mindell, Sorenson, and Dimcheff 1998Citation ), and marsupials (Pääbo et al. 1991Citation ; Janke et al. 1994Citation ; Janke, Xu, and Arnason 1997Citation ). In the phylum Echinodermata, complete mitochondrial genome sequences are available for the echinoids (order Camarodonta) Strongylocentrotus purpuratus (Jacobs et al. 1988bCitation ), Paracentrotus lividus (Cantatore et al. 1989Citation ), and (order Stirodonta) Arbacia lixula (De Giorgi et al. 1996Citation ), as well as the asteroid (order Valvatida) Asterina pectinifera (Asakawa et al. 1995Citation ). Partial sequence data from a second asteroid, Pisaster ochraceus (order Forcipulatida), is also available (Jacobs et al. 1989Citation ; Smith et al. 1990Citation ).

The analysis of the complete DNA sequence of the S. purpuratus mitochondrial genome (Jacobs et al. 1988bCitation ) demonstrated a novel gene map for echinoderms in comparison to vertebrates and Drosophila. Considering only protein and rRNA genes, the echinoid mitochondrial map can be related to the vertebrate pattern with only two rearrangements, the transpositions of lrRNA and ND4L (Jacobs et al. 1988bCitation ; Cantatore et al. 1987b, 1989Citation ). Among echinoderms, a major inversion event has been described in sea stars (class Asteroidea) in comparison to sea urchins (class Echinoidea). The asteroid mtDNA inversion contains a 4.6-kb segment encompassing a 13-tRNA-gene cluster, ND1, ND2, and lrRNA (Jacobs et al. 1989Citation ; Smith et al. 1989, 1990Citation ; Asakawa et al. 1991, 1995Citation ). This inversion was seen in two asteroid orders (Forcipulatida and Valvatida). PCR and partial sequence data indicated that the sea cucumbers (class Holothuroidea) maintained the echinoid mitochondrial gene order, but the brittle stars (class Ophiuroidea) demonstrated the basic asteroid pattern (Smith et al. 1993Citation ). This 4.6-kb inversion supports phylogenies that have two major echinoderm lineages: an echinoid/holothuroid lineage and an asteroid/ophiuroid one. Subsequent investigations have demonstrated the presence of a novel tRNA duplication event within the mtDNA of the holothuroid genus Cucumaria with respect to other holothuroids (Arndt and Smith 1998Citation ) and multiple inversion events succeeding the original 4.6-kb inversion in the ophiuroids (unpublished data).

This study reports the mtDNA sequence and genome map for a member of the echinoderm class Crinoidea, or feather stars, that is generally considered to represent a primitive echinoderm lineage. Crinoidea is the only extant class of the stalked echinoderm subphylum Pelmatozoa; the Asteroidea, Echinoidea, Holothuroidea, and Ophiuroidea classes belong to the free-living subphylum Eleutherozoa. The overall mitochondrial gene content has been maintained in the crinoid Florometra serratissima, but the mitochondrial genome demonstrates a unique gene order with respect to both the echinoid and the asteroid mtDNA gene maps. The complete crinoid mtDNA sequence allows the examination of echinoderm phylogenies by comparison of gene sequences and maps between echinoderm classes. With knowledge of the mtDNA gene order for five echinoderm classes, both potential pathways and mechanisms of echinoderm mitochondrial genome rearrangements may be inferred.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 literature cited
 
mtDNA Isolation
Florometra serratissima specimens were obtained by SCUBA diving at Bowen Island, B.C., or were supplied by the Bamfield Marine Station, Bamfield, B.C. Florometra mtDNA was extracted by two methods. To generate a partial mtDNA genomic library, mtDNA was isolated according to Barriga Sosa et al. (1995)Citation , followed by CsCl isopycnic centrifugation. The purified mtDNA was restriction-digested with HindIII according to the supplier's specifications (Bethesda Research Labs) and cloned into the plasmid vector pUC19. mtDNA was also obtained for direct PCR amplification from isolated Florometra eggs. The eggs were digested in a final volume of 10 ml Protease K buffer (100 mM Tris-Cl [pH 8], 100 mM NaCl, 50 mM EDTA, 1% SDS, 200 µg/ml Protease K) for 2 h at 65°C. The digests were extracted with an equal volume of 1 x TE equilibrated phenol (pH 8.0) until the interface was clean, and then they were extracted with an equal volume of 1:1 phenol/sevag (24:1 chloroform/isoamyl alcohol). The phases were separated by centrifugation in a Beckman JS-13.1 rotor at 20°C for 10 min at 8,000 rpm. The aqueous layer was extracted with CTAB/NaCl (10% [w/v] cetyl trimethyl ammonium bromide in 0.7M NaCl); a one-sixth volume of 5 M NaCl was added, followed by the addition of a one-eighth (original) volume of CTAB/NaCl. The sample was incubated at 65°C for 35 min, mixed occasionally, and extracted with 0.5 volumes of sevag and centrifuged as before. The aqueous phase was precipitated with 2.5 volumes of 95% ethanol at -20°C overnight. The DNA was pelleted using a Beckman JA-13.1 rotor at 13,000 rpm, 4°C, for 20 min, rinsed in 70% ethanol, and recentrifuged for 5 min. The dry pellet was taken up in 1 ml of 1 x TE.

PCR and DNA Sequencing
Florometra-specific oligonucleotide primers that span gene junction regions were designed from sequence obtained from partial F. serratissima mtDNA genomic HindIII clones. Internal gene primers were designed from consensus echinoderm sequences, from the mtDNA sequence of the holothuroid Cucumaria miniata (A. Arndt, personal communication), or from published sequences. A complete list of the primer sequences and relative positions used in this analysis may be obtained from the authors on request. Circularity of the mtDNA genome was indicated by the successful PCR amplification and sequencing across all genes and gene junctions.

Florometra mtDNA was amplified in a Genetic Thermal Cycler (GTC-2) (GL Applied Research Inc.) under the following conditions: an initial cycle of denaturation at 94°C for 90 s, annealing between 50°C and 58°C (depending on the primer utilized) for 30 s, and extension at 72°C for 1–3 min (approximately 45 s/kb amplified product), followed by 29 cycles of 94°C for 30 s, 50–58°C for 30 s, and 72°C for 1–3 min, with the final cycle extension time being lengthened to 10 min. The amplification products were electrophoresed in 0.7% agarose gels and stained with ethidium bromide. The agarose gel slices containing the product band were excised under long wavelength UV (360 nm), and the DNA was purified from the agarose plug by sedimentation through silanized glass wool plugs for 20 min at 6,000 rpm in a desktop microcentrifuge, followed by an ethanol precipitation. Amplified products were either sequenced directly or cloned using one of two methods: the TA cloning method of Marchuk et al. (1990)Citation utilizing the plasmid vector pUC19 or pBluescript II KS(+), or use of the pCR-Script Amp SK(+) cloning kit (Stratagene) according to the manufacturer's protocol.

All DNA sequences were obtained by the chain termination method of Sanger, Nicklen, and Coulson (1977)Citation using the Sequenase version 2.0 DNA sequencing kit, the Sequenase PCR product sequencing kit, or the Thermo Sequenase radiolabeled terminator cycle sequencing kit (all from U.S. Biochemicals/Amersham). Alternatively, sequences were obtained by automated ABI (Applied Biosystems) sequencing protocols. Sequenase version 2.0 sequencing reaction mixtures with recombinant plasmid template included 10% DMSO (T. Snutch, personal communication). Standard plasmid primers or specific PCR amplification primers were used for DNA sequencing reactions. All reported sequences resulted from two or more sequence determinations of overlapping independent PCR amplifications, cloned PCR fragments, or mtDNA genomic clones. Equivocal segments were resequenced from novel PCR sites either in the same strand or in the opposite strand.

Phylogenetic Methods and Data Analyses
Ribosomal RNA and tRNA genes were identified by sequence comparison to known echinoderm genes. In addition, tRNA genes were folded to verify their secondary structures (Sprinzl et al. 1989Citation ; Steinberg and Cedergren 1994Citation ; Steinberg, Gautheret, and Cedergren 1994Citation ). Unassigned sequence (UAS) regions were examined for stable stem-loop structures using the PCFOLD program (Zuker and Stiegler 1981Citation ).

Florometra DNA sequence encoding mtDNA proteins was translated using the echinoderm mtDNA genetic code (Himeno et al. 1987Citation ; Jacobs et al. 1988bCitation ; Cantatore et al. 1989Citation ; Asakawa et al. 1995Citation ; De Giorgi et al. 1996Citation ). Since complete sequence data were not available for all of the protein genes in every echinoderm class, the phylogenetic analyses were restricted to the genes for cytochrome c oxidase subunits I, II, and III. COI, COII, and COIII are all transcribed from the same DNA strand. Florometra nucleotide and predicted amino acid sequences were aligned with homologous genes from the following deuterostome species (GenBank accession numbers in parentheses): the sea lamprey Petromyzon marinus (U11880) (Lee and Kocher 1995Citation ); the amphioxus Branchiostoma lanceolatum (Y16474) (Spruyt et al. 1998Citation ); the hemichordate Balanoglossus carnosus (AF051097) (Castresana et al. 1998Citation ); the echinoids Strongylocentrotus purpuratus (X12631) (Jacobs et al. 1988bCitation ), Paracentrotus lividus (J04815) (Cantatore et al. 1989Citation ), and Arbacia lixula (X80396) (De Giorgi et al. 1996Citation ); the asteroids Asterina pectinifera (D16387) (Asakawa et al. 1995Citation ) and Pisaster ochraceus (X55514) (Smith et al. 1990Citation ); the holothuroid Cucumaria miniata; and the ophiuroid Ophiopholis aculeata (unpublished data). In addition, the three genes (COI, COII, and COIII) were combined to form concatenated nucleotide (2,982 nt) and predicted amino acid (994 aa) sequences. Alignments were constructed as follows: the individual translated COI, COII, and COIII genes (start and stop codons removed) were aligned manually using ESEE, version 3.2 (Cabot and Beckenbach 1989Citation ), and truncated at the 5' and 3' ends to the first conserved amino acid. The three truncated amino acid sequences were joined to form the concatenated COI-COII-COIII amino acid segments. The individual and concatenated nucleotide sequences were adjusted to reflect these amino acid alignments. Protein and nucleotide maximum-likelihood trees were prepared using the PROTML and NUCML programs in the MOLPHY package, version 2.3 (Adachi and Hasegawa 1996aCitation ), and with PUZZLE, version 4.0.2 (Strimmer and von Haeseler 1996Citation ). The mtREV24 model for mtDNA-encoded proteins (Adachi and Hasegawa 1996bCitation ) was employed for amino acid sequences, and the HKY85 model (Hasegawa, Kishino, and Yano 1985Citation ) was used for nucleotide sequences, excluding third codon positions. Differing tree topologies were compared using the Kishino-Hasegawa test (Kishino and Hasegawa 1989Citation ) at a 95% confidence interval. Nucleotide sequences were analyzed in two ways: first and second nucleotide positions were considered separately and their log likelihood values totaled, and first and second nucleotide positions were analyzed together. The nucleotide sequences of the individual and combined cytochrome c oxidase genes were also analyzed using a LogDet paralinear distance (Lockhart et al. 1994Citation ) implemented in the PAUP* program, version 4.0b2a (Swofford 1998Citation ), excluding the third nucleotide position of each codon.

Mitochondrial gene order rearrangements were analyzed directly by visually comparing proposed rearrangement steps between each mtDNA genome and by utilizing the rearrangement program DERANGE II (Blanchette, Kunisawa, and Sankoff 1996Citation ) to explore the spectrum of potential rearrangements. The mitochondrial gene orders for all five echinoderm classes and the consensus nonavian vertebrates were compared.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 literature cited
 
General Features of the F. serratissima Mitochondrial Genome
The complete mtDNA sequence has been determined for F. serratissima (GenBank accession number AF049132). A genetic map of the crinoid mitochondrial genome is shown in figure 1 . It is a circular DNA molecule, 16,005 bp in length, and it encodes the complete sequences of 13 protein genes, 22 tRNA genes, and 2 ribosomal RNA genes as found in other animal mtDNA (Jacobs et al. 1988bCitation ; Cantatore et al. 1989Citation ; Asakawa et al. 1995Citation ; De Giorgi et al. 1996Citation ; reviewed in Wolstenholme 1992Citation ). There are three regions of UAS greater than 73 bp and 14 short intergenic UAS segments (1–16 bp in length: see fig. 1 for map locations).



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Fig. 1.—The mitochondrial genome map of the crinoid Florometra serratissima. Arrows indicate the transcriptional polarity of each gene. Names of protein-coding and ribosomal RNA genes are abbreviated as in the text. tRNA genes are represented by their single-letter codes, and the arrowheads indicate their transcriptional polarity. Serine and leucine tRNA genes are also identified by codon family (in parentheses). Unassigned sequence regions UAS I, UAS II, and UAS III are shown as crosshatched areas. The lengths (bp) and positions of the intergenic UAS regions are indicated inside the circular map

 
All of the Florometra mtDNA protein genes begin with ATG, except for ND4L, which appears to begin with GTG. This use of GTG as an initiation codon for Florometra ND4L is unique among echinoderm ND4L genes. The ND4L genes of three echinoids and two asteroids utilize the initiation codons ATC or ATT (Jacobs et al. 1988bCitation ; Cantatore et al. 1989Citation ; Smith et al. 1990Citation ; Asakawa et al. 1995Citation ; De Giorgi et al. 1996Citation ), whereas the ophiuroid uses the standard ATG (unpublished data). GTG initiation codons have been reported in prokaryotes (Stormo, Schneider, and Gold 1982Citation ; reviewed in Osawa et al. 1992Citation ), as well as in the mtDNA protein genes of other echinoderms, vertebrates, Drosophila (reviewed in Wolstenholme 1992Citation ), and Mytilus edulis (Hoffmann, Boore, and Brown 1992Citation ).

The Florometra mtDNA protein genes all contain full termination codons, except COI, which terminates with TA, and ND1 and ND2, which end with a T. In all three cases, the 3' ends of the genes are punctuated with a tRNA gene (fig. 1 ). A complete termination codon may be created through posttranscriptional polyadenylation, as suggested for other organisms (Anderson et al. 1981Citation ; Ojala, Montoya, and Attardi 1981Citation ; Clayton 1991Citation ). Animal mtDNA protein-coding genes with incomplete stop codons TA or T are common (Attardi 1985Citation ; Wolstenholme 1992Citation ).

Apart from the small numbers of base pairs between identified genes, three significant regions of unassigned sequence (UAS I, UAS II, and UAS III) have been found within the F. serratissima mtDNA genome (fig. 1 ). The largest of these (UAS I) is 432 bp and is located between tRNAAsp and tRNAThr (fig. 1 ). UAS II (77 bp) is located between the 5' ends of ND1 and COI (fig. 1 ), and UAS III (73 bp) is located between the 5' termini of Cyt b and ND6 (fig. 1 ). In echinoids, the origin of replication has been shown to occur between tRNAThr and tRNAPro (Jacobs, Herbert, and Rankine 1989Citation ). In asteroids, a putative control region is located between tRNAThr and lrRNA (Smith et al. 1990Citation ; Asakawa et al. 1995Citation ). The Florometra UAS I region contains sequence similarities to the putative control regions in these other echinoderms. Adjacent to tRNAThr, there is a high pyrimidine containing segment that is followed by a G-rich region (13 out of 17 nucleotides are G) and an A+T-rich region. UAS I can be folded into a complex stem-loop structure of high thermal stability (-82.7 kcal/mol) if G-T pairing is allowed.

UAS III contains only A and T nucleotides, including a TTATATATAA motif that is frequently conserved in other intergenic regions of echinoderm mtDNAs (Jacobs et al. 1989Citation ). In S. purpuratus, P. lividus, A. lixula, A. pectinifera, and A. amurensis, variations of this sequence motif occur between the 5' ends of oppositely transcribed Cyt b and ND6. Since mitochondrial genes are transcribed from both DNA strands in crinoids, these A+T-rich segments may function as bidirectional promoters (Elliot and Jacobs 1989Citation ; Jacobs et al. 1989Citation ).

The significance of the Florometra UAS II region is still unclear. No obvious TTATATATAA-like motif (Jacobs et al. 1989Citation ) is seen in UAS II, although the region is A+T-rich (84.4% A+T). Florometra COI and ND1 flank the UAS II region and are transcribed in opposite directions (fig. 1 ). The location and A+T content of UAS II suggest that it may also act as a bidirectional promoter, such as has been proposed between the oppositely transcribed COI and tRNAPro genes in asteroids (Jacobs et al. 1989Citation ; Smith et al. 1990Citation ; Asakawa et al. 1995Citation ).

Nucleotide Bias and Codon Usage
Many animal mitochondrial genomes deviate from a random usage of nucleotides, particularly in the third positions of synonymous codons. These deviations are said to result from directional mutation pressure (Sueoka 1962Citation ; Asakawa et al. 1991Citation ; Jermiin et al. 1994, 1996Citation ; Jermiin, Graur, and Crozier 1995Citation ). In F. serratissima, the mtDNA major sense strand includes 10 protein and 10 tRNA genes, whereas the minor sense strand includes the remaining three protein (ND1, ND2, and ND6), 12 tRNA, and the 2 ribosomal RNA genes. The nucleotide composition of the major sense strand is 26.4% A, 46.4% T, 15.6% G, and 11.6% C. When compared with other echinoderm mtDNA sequences, a clear positive bias for T with an apparent loss of C is seen in Florometra. This T-bias is also seen in the nucleotide composition of the protein genes. The nucleotide compositions of all 13 protein genes (from both the major and minor sense strands) from representative echinoderms are reported in table 1 . The observed Florometra protein gene T-bias is substantially higher than the reported T-bias (with a decrease in C) for the echinoid A. lixula (De Giorgi et al. 1996Citation ) and for the other representative echinoderms (table 1 ). This T over C bias is particularly exaggerated in the third codon positions of the 10 F. serratissima protein genes encoded on the major sense strand (62.5% T vs. 1.9% C).


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Table 1 Nucleotide Composition Percentages of the 13 Mitochondrial Protein Genes from the Major and Minor Sense Strands of Representative Echinoderms

 
Several potential causes for nucleotide biases have been suggested, including reduced pools for specific nucleotides, a biased preference of the mitochondrial gamma DNA polymerase for specific nucleotides, and a propensity for a specific mutational direction for relatively long term exposed H-strands during replication (discussed in Asakawa et al. 1991Citation ). The immediate significance of these observed nucleotide biases is that extreme caution must be exercised in the use of conventional models for ascertaining molecular phylogenies that assume no bias in nucleotide replacements.

A corollary of nucleotide bias is clearly reflected in codon usage. In table 2 , we compare the four-codon family relative synonymous codon usage (RSCU) (Sharp, Tuohy, and Mosurski 1986Citation ) values for S. purpuratus, A. pectinifera, O. aculeata, and F. serratissima major and minor sense strands. RSCU is the observed frequency of a codon divided by the expected frequency if there is uniform usage of all codons for that amino acid. In the major protein-encoding strand of F. serratissima, T is used most frequently in the third codon position in every four-codon family. In the minor sense strand, encoding ND1, ND2, and ND6, A is the most recurrent third codon position nucleotide. The other echinoderms do not exhibit this same type of codon bias (table 2 ). Asterina pectinifera, S. purpuratus, and O. aculeata demonstrate a codon usage pattern that appears to be opposite to that of F. serratissima. Their major protein-encoding strands generally follow an L-strand pattern of codon usage (A or C in the third position) adopted by most animal major sense strands. However, the major protein-encoding strand of F. serratissima seems to have a codon usage pattern commonly adopted by H-strand proteins (T or G in the third position). The codon usage pattern of the echinoid A. lixula is more like that of F. serratissima, as there is similar usage of A and T in both strands (De Giorgi et al. 1996Citation ).


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Table 2 Relative Synonymous Codon Usage in Four-Codon Families

 
Molecular Phylogenies
Traditionally, echinoderm phylogenies have been based on larval and/or adult morphological characters and fossil evidence. More recently, DNA and protein sequence data have become central to the establishment of phylogenies. We analyzed both nucleotide and predicted amino acid sequence alignments of COI, COII, and COIII from representative echinoderm, hemichordate, and chordate species (see Materials and Methods). The maximum-likelihood and LogDet approaches were used primarily because of their resistance to artifacts resulting from nucleotide compositional biases. Parsimony analyses were consistent with both LogDet and maximum-likelihood results (data not shown).

The five highest scoring phylogenetic trees constructed from a PROTML maximum-likelihood analysis utilizing the combined cytochrome c oxidase amino acid sequences are shown in figure 2AE . The phylogenetic tree with the highest likelihood topology placed the crinoid as a sister group to clades containing echinoids, asteroids, and the holothuroid; however, the ophiuroid consistently coupled an extremely long branch length with a position closer to the chordates than the crinoid (fig. 2A ). This same tree topology was resolved by quartet puzzling using the PUZZLE program for both amino acid and nucleotide (first and second positions only) sequence analyses. Figure 2A is also the "best tree" generated in a NUCML maximum-likelihood analysis with the combined cytochrome c oxidase nucleotide sequences (first and second nucleotide positions only) and from a LogDet paralinear distance analysis on the same nucleotide data set. Of the remaining four top scoring trees (fig. 2BE ), none have a likelihood value that is significantly different than that of figure 2A. The fourth tree (fig. 2D ) places Florometra as a sister group to the eleutherozoan clades, but the holothuroid position is inconsistent with proposed echinoderm phylogenies (reviewed in Littlewood et al. 1997Citation ).



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Fig. 2.—Alternative maximum-likelihood trees for echinoderm phylogeny. Combined COI, COII, and COIII amino acid sequences were analyzed using the mtREV24 model of amino acid substitution. Petromyzon marinus (P.m.) and Branchiostoma lanceolatum (B.l.) were used as outgroups. AE, The five top scoring trees estimated by an exhaustive search using PROTML in the MOLPHY package. FH, User trees constructed to reflect proposed echinoderm phylogenies described by Littlewood et al. (1997)Citation . Comparative log likelihood values were generated with the Kishino-Hasegawa user specified tree option (Kishino and Hasegawa 1989Citation ). The lowest log likelihood value (ln L) is shown for tree A; the differences in log likelihood values ({Delta}ln L) from tree A and the corresponding standard errors (SE) are shown below trees BH. A.l. = Arbacia lixula; A.p. = Asterina pectinifera; B.c. = Balanoglossus carnosus; C.m. = Cucumaria miniata; F.s. = Florometra serratissima; O.a. = Ophiopholis aculeata; P.o. = Pisaster ochraceus; S.p. = Strongylocentrotus purpuratus.

 
The log likelihood values of several tree topologies that reflect a range of proposed echinoderm phylogenetic relationships were compared with that of the best tree. The three most prominent echinoderm tree topologies, proposed by Littlewood et al. (1997)Citation , are shown in figure 2FH . In all of the proposed phylogenies, the crinoids are positioned closer to the hemichordate/chordate clade than any of the eleutherozoan classes, including the ophiuroid. Tree topologies that place the crinoid as a sister group to other echinoderms reflect the general opinion that the crinoids and the eleutherozoans diverged very early in echinoderm evolution. All three alternative trees have log likelihood values that are significantly greater than that of the "best tree" (fig. 2A ).

Among trees constructed to compare the alternative proposed echinoderm phylogenies (fig. 2FH ), that with the lowest likelihood value places the holothuroid with the echinoids and the ophiuroid as a sister group to the rest of the eleutherozoans (fig. 2F ). Forcing the ophiuroid into the echinoid/holothuroid clade (fig. 2G ) or into a clade with the asteroids (fig. 2H ) does not result in significantly different likelihood values (table 3 ). However, the likelihood value is significantly different when the ophiuroid is grouped with the echinoids and the holothuroid is grouped with the asteroids (tree I in table 3 ).


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Table 3 Maximum-Likelihood Comparison of Proposed Echinoderm Phylogenetic Trees

 
The extremely long branch length of the ophiuroids may have distorted the phylogenetic analysis. Alternative phylogenetic trees were compared excluding the ophiuroid data set. Without any imposed restrictions, the best tree with the lowest likelihood value placed the crinoid as a sister group to the eleutherozoans, with the holothuroid diverging prior to the echinoid/asteroid node (fig. 3A ). An alternative tree that groups the holothuroid with the echinoids, separate from the asteroid clade (fig. 3B ), is not significantly different from figure 3A . However, placing the holothuroid with the asteroids (fig. 3C ) results in a tree with a significantly different likelihood value.



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Fig. 3.—Maximum-likelihood tree comparisons excluding the ophiuroid data set. Tree topologies were generated by an exhaustive PROTML maximum-likelihood analysis (the mtREV24 model of amino acid substitution) with the combined COI, COII, and COIII genes. A, The exhaustive search tree with the lowest log likelihood value (ln L). B, Alternative tree topology that places the holothuroid as a sister group to the echinoids. C, Alternative tree topology that places the holothuroid as a sister group to the asteroids. Comparative log likelihood values were generated with the Kishino-Hasegawa user-specified tree option (Kishino and Hasegawa 1989Citation ). The differences in log likelihood values ({Delta}ln L) and corresponding standard errors (SE) for trees B and C versus tree A are given. Species are abbreviated as described in figure 2

 
Several conclusions may be drawn from these types of amino acid and nucleotide analyses. Although the data resolve the monophyly of the echinoids and the asteroids, they do not definitively reconcile the relationships between the echinoderm classes. Phylogenetic proposals that place the holothuroid with the echinoids rather than with the asteroids (reviewed in Littlewood et al. 1997Citation ) are not rejected by our data. However, the placement of the ophiuroid branch with respect to the other echinoderms is still unresolved. Long branch lengths observed for the crinoid and especially the ophiuroid, resulting from either extreme nucleotide bias or differential evolutionary rates, may result in incorrect inferences of phylogenetic relationships. A detailed report on the mitochondrial genome and affinities of the ophiuroid will be discussed elsewhere (unpublished data).

Mitochondrial Gene Rearrangements in Echinoderms and Vertebrates
Recently, Boore and Brown (1998)Citation noted three reasons why incorrect phylogenies may result from morphological or molecular data: (1) a lack of a satisfactory number of shared features that support each branch, (2) errors in assessing homology among features of various organisms, and (3) convergent evolutionary changes being misinterpreted to support false relationships. To that list should be added long branch effects due to base composition biases or differential evolutionary rates such as may be the case with Florometra and Ophiopholis. Mitochondrial gene arrangement comparisons have been suggested as a more promising tool to resolve deeper phylogenies (Brown 1985Citation ; Jacobs et al. 1988aCitation ; Smith et al. 1993Citation ; Boore and Brown 1994a, 1995, 1998Citation ; Macey et al. 1997Citation ). It has been argued that the large number of possible gene arrangements would make it unlikely that the same gene order arose from convergent evolution (reviewed in Boore and Brown 1998Citation ). Therefore, shared gene arrangements would likely indicate a common ancestry. Recently, however, the utility of mitochondrial gene order as a phylogenetic tool has been questioned by Mindell, Sorenson, and Dimcheff (1998)Citation and Curole and Kocher (1999)Citation . These authors point out both the potential for convergent evolution of gene order and the limitations to forming phylogenetic conclusions with insufficient knowledge of the rate and mechanisms of gene rearrangement.

The mechanisms of gene rearrangement within animal mitochondrial genomes are restricted. There is no direct evidence that intermolecular recombination between animal mtDNA molecules occurs; however, intramitochondrial recombination has not been ruled out (Jacobs et al. 1988aCitation ; Boore and Brown 1998Citation ). Recently, Lunt and Hyman (1997)Citation presented evidence for the deletion of minicircles from the mtDNA genome of nematodes that is consistent with an excision-based model of recombination/transposition. Many protein and rRNA gene rearrangements between mtDNA genomes are thought to be the result of intramolecular inversions (Smith et al. 1993Citation ) or intramolecular duplications with subsequent deletions of gene duplicates (Moritz and Brown 1987Citation ). Since single tRNA genes may move more frequently and perhaps by mechanisms distinct from those of structural genes (Cantatore et al. 1987aCitation ; Jacobs et al. 1989Citation ; Voytas and Boeke 1993Citation ), they are generally excluded when calculating the number of rearrangement steps required to obtain the different structural gene orders found between mitochondrial genomes. However, pairs or groups of tRNA genes may be displaced by inversions or transpositions, as is seen in both the ophiuroids (Smith et al. 1993Citation ; unpublished data) and the crinoids, or by duplication in some holothuroids (Arndt and Smith 1998Citation ).

Clearly, there has been a complicated pattern of rearrangements between the mitochondrial genomes in the lineages leading to extant echinoderm classes and the vertebrates. The crinoid mitochondrial gene order encompassing COI to ND6 (fig. 1 ) is conserved across all five echinoderm classes, with the notable exception of a segment duplication in the holothuroid genus Cucumaria (Arndt and Smith 1998Citation ). Moreover, the genome from COI to Cyt b is conserved in four of five echinoderm classes, the exception being the Ophiuroidea. This region of the mtDNA genome (COI to Cyt b) is relatively stable when compared with the area around the putative control region. The more variable mitochondrial genome segments from Cyt b to COI (encompassing the putative control region) for the echinoid, asteroid, and crinoid are shown in figure 4 . Although most mitochondrial gene order rearrangements are found in this region, particular blocks of genes are conserved between echinoderms. The three complete echinoid mtDNA maps, from species representing two orders, all exhibit identical mtDNA gene arrangements (Jacobs et al. 1988bCitation ; Cantatore et al. 1989Citation ; De Giorgi et al. 1996Citation ). These two echinoid orders were reportedly distinct by 150–200 MYA (Smith, Lafay, and Christen 1992Citation ) or 155 MYA (De Giorgi et al. 1996Citation ), supporting mtDNA gene order stability within the class Echinoidea. Jacobs et al. (1989)Citation , Smith et al. (1989, 1990)Citation , and Asakawa et al. (1991, 1995)Citation reported that the asteroids contained a 4.6-kb inversion in comparison with the mtDNA of the echinoids. This inversion includes a 13-tRNA-gene cluster, ND1, ND2, and lrRNA (segments B and C in fig. 4 ). The inversion is present in asteroids from the orders Forcipulatida and Valvatida (Jacobs et al. 1989Citation ; Smith et al. 1989, 1990, 1993Citation ; Asakawa et al. 1991, 1995Citation ) that have been distinct for at least 225 Myr (Blake 1987Citation ). PCR amplification of mitochondrial gene junctions indicated that the holothuroid Parastichopus californicus has the basic echinoid mitochondrial gene orientation (Smith et al. 1993Citation ). Similar studies with the ophiuroids demonstrated that they displayed the basic asteroid pattern with respect to the 4.6-kb inversion event (Smith et al. 1993Citation ). However, multiple inversion events occurred in the ophiuroid subsequent to the characteristic 4.6-kb inversion (unpublished data). In addition, the ophiuroids (Smith et al. 1993Citation ; unpublished data), as well as holothuroid species from the genus Cucumaria (Arndt and Smith 1998Citation ), exhibit altered tRNA clusters that contain less than the 13 tRNA genes found in echinoids and asteroids.



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Fig. 4.—Mitochondrial gene order arrangements between 5'-Cyt b and COI-3' of the basic echinoid, asteroid, and crinoid patterns. The putative origin of replication is denoted in the echinoid as a displacement loop (dl) where the origin of replication has been demonstrated (Jacobs, Herbert, and Rankine 1989Citation ), and as unassigned sequence (uas) in the asteroid and the crinoid. The tRNA genes are represented by their single letter abbreviations. The leucine tRNA gene is further identified by codon family (Lc = tRNALeu(CUN) and Lu = tRNALeu(UUR)). Echinoids and asteroids contain three conserved blocks, boxed and labeled A, B, and C for clarity. Arrows indicate the relative orientation of the conserved blocks with respect to the echinoid map, and not the transcriptional polarity of the individual genes. In the crinoid, the A segment is maintained, but the B segment (tRNA cluster) is fragmented into B', containing 10 tRNA genes, B'', containing 2 tRNA genes, and tRNATyr (designated *), inserted between lrRNA and ND2 (C' segment)

 
Although there are highly modified tRNA gene arrangements among the echinoderms, the original 4.6-kb inversion supports an affinity of the echinoids with the holothuroids as distinct from the asteroids. The Asteroidea and Ophiuroidea classes have been distinct since the Ordovician (490–530 MYA), as have the Echinoidea and Holothuroidea classes (450–530 MYA) (Smith 1988Citation ). Fossil evidence suggests that the class Crinoidea was distinct from the rest of the echinoderms probably before the end of the Lower Cambrian (550–560 MYA) (Smith 1988Citation ). Since fossil evidence indicates a very early divergence of the crinoids from the eleutherozoans, knowledge of the F. serratissima mtDNA gene map might provide added insights into the structure of the ancestral echinoderm mtDNA genome arrangement.

The original 4.6-kb inversion has a conserved gene order and encompasses the region spanning from tRNAPro (of the basic echinoid/holothuroid/asteroid tRNA cluster) to lrRNA inclusive (segments B plus C in fig. 4 ). In the echinoid gene order, the tRNA gene cluster follows the putative control region (fig. 4 ). In the asteroid mtDNA pattern, the polarity of the 4.6-kb segment is inverted, which joins the reversed tRNA gene cluster to the 5' end of COI and an inverted lrRNA abutting the putative control region (fig. 4 ). In the crinoid, the integrity of the 4.6-kb segment is not maintained. Only part of the 4.6-kb segment, ND1 to lrRNA, is inverted in the Florometra mtDNA genome (segment C' in fig. 4 ). The majority of the crinoid modified tRNA cluster (10 out of 13 tRNA genes) is not inverted (segment B' in fig. 4 ). The terminal three tRNA genes seen in the 13-tRNA-gene cluster after tRNAAsp (5'-tRNATyr-tRNAGly-tRNALeu(UUR)-3') are displaced, with tRNAGly-tRNALeu(UUR) probably as a single event between the ribosomal RNA genes (segment B'' in fig. 4 ). tRNATyr is uniquely positioned, in comparison with other echinoderms, between the 3' end of ND2 and the 5' end of lrRNA (5'-ND2-tRNATyr-lrRNA-3') (figs. 1 and 4 ).

In the echinoids, the transcriptional order of the ribososmal RNA genes is srRNA preceding lrRNA (fig. 4 ). In vertebrates, srRNA and lrRNA are also transcribed in the same polarity, srRNA preceding lrRNA with an intervening tRNAVal. In the crinoid, the ribosomal RNA genes are transcribed from the same strand, but lrRNA precedes srRNA (figs. 1 and 4 ). The presence of tRNAPhe at the 5' end of srRNA is a conserved feature throughout the echinoderms and vertebrates.

In addition to this novel ribosomal RNA gene arrangement, there is a unique organization of Cyt b, the remaining Florometra tRNA cluster, and the putative control region (fig. 4 ). The Florometra Cyt b is directly adjacent to its modified tRNA cluster, and the putative Florometra control region is situated on the opposite side of the cluster. In echinoids, srRNA and the putative control region separate the original tRNA cluster from Cyt b (fig. 4 ). The unique gene order between Cyt b and the rRNA genes, including the modified tRNA cluster and the putative control region, supports the hypothesis that the 5'-ND1-ND2-lrRNA-3' Florometra inversion occurred independent of the Cyt b to srRNA gene order modifications. The class Crinoidea is the first of the extant echinoderms to appear in the fossil record (Paul and Smith 1984Citation ; Smith 1988Citation ). However, the variations in gene order seen between the echinoderm classes suggest that the mitochondrial gene arrangement in crinoids is most probably derivative of an ancestral pattern.

Phylogenetic Interpretations of Gene Orders
Several approaches, or "models," may be used to establish phylogenies based on gene orders. One could disregard potential rearrangement mechanisms and simply score the minimum number of steps required to derive each gene order. Alternatively, the mechanism of rearrangement could be considered and weighted for the type, i.e., inversion, transposition, or transversion (transposition plus inversion), as suggested in the rearrangement model of Blanchette, Kunisawa, and Sankoff (1996)Citation . We have no data on rates, frequency, or costs of various rearrangement mechanisms, so weighting is arbitrary. In addition, simply scoring the presence or absence of gene junctions is not strictly a valid approach because observed changes are not independent characters.

There is only one change, the 4.6-kb inversion, between the basic echinoid/holothuroid and asteroid mtDNA genome patterns. The ancestral polarity of the inversion could be either orientation. Considering protein-coding and ribosomal RNA genes only, we identify a minimum of one movement required to obtain the crinoid pattern from either the echinoid or the asteroid gene order. The crinoid mtDNA gene order can be reached from the echinoid order by means of the transversion of approximately two thirds of the echinoid genome (COI through Cyt b segment) to a position between srRNA and ND1 (fig. 5A ). Conversely, the echinoid gene order can be reached by the opposite transversion of the crinoid COI-Cyt b segment between srRNA and lrRNA. The crinoid gene order and that of the asteroid can be related through the inversion of srRNA (fig. 5A ). An alternative path to correlate the crinoid gene order with that of the echinoid is with two rearrangements: the inversion of srRNA and the inversion of 5'-ND1-ND2-lrRNA-3' (fig. 5B ).



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Fig. 5.—Minimal step models relating the crinoid mtDNA map to that of the echinoid and the asteroid patterns using protein-coding and ribosomal RNA genes only. All gene names are abbreviated as in the text. Gene orders start at COI and are arranged 5'–3'. Dark underlines indicate genes transcribed on the opposite strand. Shaded curved arrows indicate inversions, and shaded straight arrows indicate transversions. A, Outline of the minimum number of steps required to correlate the echinoid or the asteroid gene order with that of the crinoid. After the echinoid{leftrightarrow}crinoid gene order transversion, the crinoid map was reoriented to a map starting with 5'-COI-3'. The crinoid{leftrightarrow}asteroid rearrangement requires one inversion. B, An alternative pathway to reach the crinoid gene order from that of the echinoid, requiring two inversions

 
In order to establish a polarity in gene rearrangements for echinoderms, an outgroup is required. A likely candidate for such an outgroup could be the consensus nonavian vertebrate gene map. Jacobs et al. (1988b)Citation , excluding the tRNA genes, noted that the echinoid pattern and the vertebrate pattern could be related with one protein (ND4L) and one rRNA (lrRNA) gene transposition (fig. 6A ). Converting the asteroid gene order to that of the vertebrate requires a minimum of two steps as well: the ND4L transposition and the transversion of the large COI-srRNA asteroid genome segment between lrRNA and ND2. Correlating the crinoid gene order with that of the vertebrates requires a minimum of three steps: the ND4L transposition as in the echinoids and asteroids, the transversion of the large COI-srRNA fragment as in the asteroids, and the inversion of srRNA. Such COI-srRNA transversion events would require the unlikely excision and insertion of a segment consisting of approximately three fourths of the mitochondrial genome to the junction between the lrRNA and ND2 genes. Alternatively, the asteroid pattern can be converted to that of the vertebrate with three moves: the ND4L transposition and two independent inversions (lrRNA and ND1-ND2). The crinoid{leftrightarrow}vertebrate map conversion can also be achieved in four moves: the transposition of ND4L and three independent inversions (lrRNA, ND1-ND2, and srRNA).



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Fig. 6.—Rearrangement paths required to achieve the various mitochondrial gene orders based on protein-coding and ribosomal RNA genes and the tRNA gene cluster (tclus). Gene names are abbreviated as in the text. Gene orders are arranged 5'–3'. Dark underlines indicate genes transcribed on the opposite strand. Transpositions are indicated by white straight arrows, transversions by shaded straight arrows, and inversions by shaded curved arrows. A, Conversion steps between the consensus nonavian vertebrate and echinoid mtDNA gene orders, disregarding the tRNA gene cluster. B, Conversion pathways between the echinoid{leftrightarrow}crinoid{leftrightarrow}asteroid mitochondrial gene maps involving only the variable region (including the tRNA cluster and the ND1, ND2, srRNA, and lrRNA genes). C, Alternative conversion pathways between echinoid{leftrightarrow}asteroid{leftrightarrow}crinoid mitochondrial gene orders involving the variable region. In B and C, the Cyt b gene is included in the partial gene orders to provide a focal point for their orientations

 
Therefore, if we exclude the tRNA genes and extremely large segment transversions, there are fewer steps needed to convert the echinoid gene order to the vertebrate gene order than from any of the other echinoderms. This implies that the ancestral polarity of the 4.6-kb inversion is the echinoid pattern. The asteroid and crinoid patterns would then be derivative.

Superficially, these types of gene order analyses could also imply a closer relationship between the crinoid and the asteroid rather than between the crinoid and the echinoid. However, the 13-tRNA-gene cluster should be included in the analysis, as it is maintained in the echinoid, basic holothuroid, and asteroid genomes and is lacking only the three terminal tRNA genes, tRNATyr, tRNAGly, and tRNALeu(UUR), in the crinoid. Once included, the numbers and mechanisms of the rearrangement steps necessary to convert the crinoid pattern to that of either the echinoid or the asteroid are similar. Both require two moves: one transversion and one inversion to reach the crinoid pattern (fig. 6B and C ). Other models that adopt an inversion only mechanism of movement and include the tRNA gene cluster also result in an equal number of events required to correlate the echinoid{leftrightarrow}crinoid or the asteroid{leftrightarrow}crinoid mtDNA maps. Therefore, inclusion of the gene cluster does not support a closer relationship of the crinoids to either the echinoids or the asteroids.


    Conclusions
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 literature cited
 
The molecular data and mitochondrial map analyses, excluding the ophiuroid set, result in similar phylogenetic conclusions. First, both approaches support an echinoid/holothuroid clade; the echinoid and the basic holothuroid mitochondrial gene orders are identical. Second, the position of the asteroids as a sister clade to the echinoid/holothuroid clade is also reinforced by the conserved block of genes in the 4.6-kb segments.

We propose that the original 4.6-kb inversion that supports the separation of the echinoid/holothuroid and asteroid lineages must have occurred after the split of the Pelmatozoa, to which the crinoids belong, and the Eleutherozoa, which includes the other four classes. The relationship of the echinoid mtDNA pattern to that of the vertebrates and crinoids suggests that the echinoderm ancestor to these groups may have had an mtDNA gene order very similar to that of present day echinoids. Although extant crinoids represent the class of echinoderms that appeared first in the fossil record (Paul and Smith 1984Citation ; Smith 1988Citation ), all extant echinoderm classes are equally distant from the common echinoderm ancestor. Considering the position of the modified tRNA gene cluster in the crinoid (fig. 4 ), the apparent inversion event in the Florometra mtDNA genome from ND1 to lrRNA most likely happened in that lineage, independent of the inversion seen in the asteroids. Multiple independent origins of a novel mitochondrial gene order have also been demonstrated in birds (Mindell, Sorenson, and Dimcheff 1998Citation ). Furthermore, it is unclear if this mtDNA arrangement within F. serratissima is common to all crinoids or if there is variation within the class Crinoidea. Obtaining the mtDNA gene order from different species would establish whether the mtDNA gene order within the crinoids is stable. The variability in known echinoderm mitochondrial gene arrangements emphasizes the limitations of gene order as a phylogenetic tool. As noted by Curole and Kocher (1999)Citation , until a more complete sample of genomes is available and some measure of the probabilities of various gene rearrangement mechanisms is known, phylogenetic conclusions based on gene order are tenuous.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 literature cited
 
We would like to thank S. Gorski for her work in isolating some of the F. serratissima mitochondrial genomic clones, and K. Beckenbach for her technical assistance. We would also like to thank A. Arndt and B. Hartwick for collecting most of our animals. This work was supported by a National Science and Engineering Research Council grant to M.J.S.


    Footnotes
 
Stephen Palumbi, Reviewing Editor

1 Keywords: Florometra crinoid mtDNA mitochondrial gene rearrangements nucleotide composition bias echinoderm phylogeny Back

2 Address for correspondence and reprints: Michael J. Smith, Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Boulevard, Burnaby, British Columbia, Canada V5A 1S6. E-mail: smitha{at}sfu.ca Back


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Accepted for publication September 29, 2000.