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Analysis of the complete mitochondrial DNA sequence of the Terebratulina retusa places Brachiopoda within the

Article in Proceedings of the Royal Society B: Biological Sciences · November 1999 DOI: 10.1098/rspb.1999.0885 · Source: PubMed

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The user has requested enhancement of the downloaded file. Analysis of the complete mitochondrial DNA sequence of the brachiopod Terebratulina retusa places Brachiopoda within the protostomes

Alexandra Stechmann* and Martin Schlegel UniversitÌt Leipzig, Institut fÏr Zoologie/Spezielle Zoologie,Talstr. 33, 04103 Leipzig, Germany

Brachiopod phylogeny is still a controversial subject. Analyses using nuclear 18SrRNA and mitochondrial 12SrDNA sequences place them within the protostomes but some recent interpretations of morphological data support a relationship with . In to investigate brachiopod a¤nities within the metazoa further,we compared the gene arrangement on the brachiopod mitochondrial genome with several metazoan taxa. The complete (15 451bp) mitochondrial DNA (mtDNA) sequence of the articulate brachiopod Terebratulina retusa was determined from two overlapping long polymerase chain reaction products. All the genes are encoded on the same strand and gene order comparisons showed that only one major rearrangement is required to interconvert the T.retusa and Katharina tunicata (: Polyplaco- phora) mitochondrial genomes. The partial mtDNA sequence of the prosobranch mollusc Littorina saxatilis shows complete congruence with the T.retusa gene arrangement with regard to the ribosomal and coding genes. This high similarity in gene arrangement is the ¢rst to be reported within the protostomes. Sequence analyses of mitochondrial protein coding genes also support a close relationship of the brachiopod with molluscs and ,thus supporting the . Though being highly informative,sequence analyses of the mitochondrial protein coding genes failed to resolve the branching order within the lophotrochozoa. Keywords: brachiopod phylogeny; mitochondrial genome; gene order; sequence analysis; Lophotrochozoa

share a common ancestry with those , 1. INTRODUCTION implying an ancestral metamerous body plan for both of are a group of marine them. Segmentation must then have been secondarily lost comprising around 350 extant and 12 000 fossil species. in brachiopods,but so far there is no clear evidence for Their fossil record dates back to the Lower . any segmentation in brachiopods. Together with the Phoronida and they have been With the ¢rst partial brachiopod 18SrRNA (small classi¢ed as Tentaculata (or ) because they subunit ribosomal RNA) sequence (Field et al. 1988) used possess a similar suspension feeding apparatus,the lopho- in a molecular analysis of di¡erent metazoan taxa,new phore,which is a horseshoe-shaped structure surrounding emphasis was put on the idea of a a¤nity of the with ciliated tentacles arranged on it (Emig the Lophophorata. Although the method and interpreta- 1976). Because they show both protostome and deutero- tion of this work was criticized,a reanalysis led to the stome features,the Lophophorata have been seen as same result (Lake 1990). Analyses of complete 18SrRNA members of the protostomes,as deuterostomes or as inter- sequences from di¡erent lophophorate taxa demonstrated mediates. More recently the lophophorates have been that all three groups (Phoronida,Bryozoa and Brachio- classi¢ed as deuterostomes on the basis of morphological poda) always cluster within the protostomes along with and larval features (Salvini-Plawen 1982; Emig 1984; molluscs and annelids (Conway-Morris 1995; Halanych et Brusca & Brusca 1990; Eernisse et al. 1992; Backeljau et al. al. 1995; Conway-Morris et al. 1996; Mackey et al.1996; 1993; Nielsen 1995). Evidence from the fossil record gave Cohen & Gawthrop 1997) in a clade named Lophotro- rise to speculations about protostome a¤nities of the chozoa (Halanych et al. 1995). Analyses using partial brachiopods. It has been suggested that the setae of mitochondrial 12SrDNA (small subunit ribosomal RNA) brachiopods are derived from the of the fossil sequences (Cohen et al. 1998b) con¢rmed the clustering of Halkieriids,which are equivalent to the setae of annelids brachiopods within the protostomes. (Conway-Morris 1995,1998; Conway-Morris & Peel Toaddress the question of brachiopod a¤nity within the 1995). This would mean that brachiopods and annelids metazoa further,we determined the complete mitochon- drial DNA (mtDNA) sequence of Terebratulina retusa.Inthe *Author for correspondence ([email protected]). last few years mitochondrial gene order comparisons have

Proc. R. Soc. Lond. B(1999)266,2043^2052 2043 & 1999 The Royal Society Received 28 June 1999 Accepted 23 July 1999 2044 A. Stechmann and M. Schlegel Mitochondrial genome of the brachiopod T. retusa become a promising new tool for investigating phylo- overlapping mode. The 5'- and 3'-ends of this 4.7 kb fragment genetic relationships within the major metazoan groups were identical to the COI and 16SrRNA genes of Terebratulina that (see Boore & Brown 1998; Boore 1999). Several features of had been obtained previously. From this 4.7 kb sequence the mitochondrial genomes support the idea of investigating following two primers were designed to amplify the remaining deep level phylogenies in the metazoa. The gene content of mitochondrial genome: 5'-GTATCGTCGAGGTATCCCTCC- metazoan mtDNA is almost invariant,thus a comparable TA ATCCTAG -3 ' (Tr/LCOX) and 5'- CGA ATA AGGAGATTG - data set is available for all taxa (Moritz et al. 1987; Wolsten- CGACCTCGATGTTGG-3' (Tr/L16S). Both primers were used holme 1992). Stable structural gene rearrangements are in a long PCR with a combination of Taq- and Pwo-poly- rare events due to the fact that functional genomes have to merases (ExpandTM Long Template PCR System,Boehringer, be maintained. It is rather unlikely that two or more taxa Mannheim,Germany) and cycling conditions as recommended will independently converge on the same gene order,thus by the manufacturer. Electrophoretic analysis of the reaction an identical gene order strongly supports a common showed a single product of ca. 11.5 kb length. This product was ancestry of those taxa. Furthermore,reversals of gene digested with the following restriction to generate over- order are presumed to be unlikely. Thus,there is little lapping fragments: ApaI, BamHI, EcoRI, HindIII, PstI, SacI, danger of homoplastic events hiding the true phylogeny SmaIandXbaI. Double digests were carried out to create (Smith et al. 1993;Boore&Brown1994a,b;Booreet al. smaller fragments if necessary. All restriction fragments were 1995; Lee & Kocher 1995). Studies on the complete cloned into the vectors pUC18,pUC19 (Boehringer,Mannheim, mtDNA of T.retusa have already been carried out byJacobs Germany) and pBluescript SK + (Stratagene,Amsterdam,The et al. (1988) using blot hybridizations to determine the rela- Netherlands). Sequencing was performed as already described tive positions of three genes on the mitochondrial genome. for the 4.7 kb fragment. Cohen et al. (1991) investigated the population genetic structure between Scottish and Canadian Terebratulina (b) Sequence analysis and phylogenetic populations using,among other methods,restriction frag- reconstruction ment length polymorphism (RFLP) analysis of the Gene sequences were identi¢ed as follows. Ribosomal genes mtDNA. However,the complete mtDNA sequence was not were determined by their similarity to corresponding genes in determined in any of these works. alignments with other metazoan mtDNAs. Open reading frames (ORFs) were determined with the program DNASIS for Windows,v. 2 using the genetic code for mtDNA. 2. MATERIAL AND METHODS ORFs were identi¢ed according to the sequence similarity of (a) DNA extraction, cloning, polymerase chain their inferred amino-acid sequence in alignments using reaction and sequencing BLASTX (Altschul et al. 1995). In the case of ND4L and ND6 Single specimens of T.retusa (Linne 1758) were collected by (NADH dehydrogenase subunits 4L and 6) and AT P 8 (ATP divers in the Gullmarnfjord near Kristineberg,Sweden. Five synthetase subunit 8),which show only weak amino-acid specimens were shipped alive in cold seawater to Leipzig, sequence similarities,hydropathy pro¢les (Kyte & Doolittle Germany. Total genomic DNA was isolated from the gonads 1982) were calculated and compared with Katharina tunicata and muscles of a single specimen using standard phenol/chloro- (Boore & Brown 1994b), Drosophila yakuba (Clary & Wolsten- form protocols (Sambrook et al. 1989). The entire mtDNA was holme 1985), Lumbricus terrestris (Boore & Brown 1995) and Bala- ampli¢ed by two overlapping polymerase chain reactions noglossus carnosus (Castresana et al. 1998) (data not shown). (PCRs). The PCR strategy is described in the following. Two Transfer RNA (tRNA) genes were identi¢ed manually by their short fragments of the 16SrRNA and cytochrome c oxidase potential to be folded into the stems and loops characteristic of subunit I (COI) genes were obtained using standard PCR mitochondrial tRNAs and speci¢ed by the triplet code in their conditions with slightly changed universal primers from putative anticodon loop. Palumbi et al. (1991): 5'-CGCCTGTTTATCAAAAACAT-3' The gene arrangement of T.retusa was compared manually (16Sar-L),5 '-CCGGTCTGAACTCAGATCAYGT-3' (16Sbr-H), with di¡erent protostome and mitochondrial 5'-CCTGCAGGAGGAGGAGAYCC-3' (COIf-L) and5'-AGTAT- genomes to assess brachiopod a¤nity within the metazoa. Using AAGCATCTGGGTARTC-3' (COIa-H). Both gene fragments the criterion of parsimony the least number of steps necessary to were cloned,sequenced and compared to the existing database interconvert two mitochondrial genomes was interpreted as (BLASTN; Altschul et al. 1995) to ensure that no contaminants more probable for a common ancestry. were ampli¢ed. In order to amplify larger fragments of the To elucidate brachiopod relationships within the metazoa mitochondrial genome the 16SrRNA and COI primer pairs were further,phylogenetic trees were reconstructed from alignments of used in all four combinations. The combination of COIf-L and amino-acid sequences. Since the prosobranch Littorina saxatilis 16Sbr-H yielded a single product of ca. 4.7 kb length. This plays a major role in brachiopod a¤nities within the protostomes, product was digested with EcoRI and XbaI,generating three only those amino-acid sequences which have already been deter- and four fragments,respectively. A double digest with both mined for this mollusc were used. Furthermore,the alignments of restriction enzymes was also carried out. All restriction the COI and Cytb (cytochrome b apoenzyme) amino-acid fragments were cloned into pUC18 and pUC19 (Boehringer, sequences were adjusted with respect to the sequenced portion of Mannheim,Germany) and sequenced using dye-labelled Littorina. Alignments were carried out with CLUSTAL W,v. 1.74 universal M13 forward and reverse sequencing primers. Sequen- (Thompson et al. 1994) using default parameters. Amino-acid cing reactions were performed using Thermo Sequenase sequences from di¡erent metazoan taxa (see table 1) of the (Amersham,Freiburg,Germany) and separated on an auto- following protein coding genes were obtained from GenBank: mated DNA sequencer (LI-COR). Using restriction fragments AT P 6 , AT P 8 , COI, COII, Cytb, ND1 and ND6. Alignment of the from single and double digests and vectors with complementary AT P 8 amino-acid sequence produced only a poor ¢t and was orientated polylinkers,both strands were sequenced in an excluded from subsequent analyses. Regions with di¤cult

Proc. R. Soc. Lond. B(1999) Mitochondrial genome of the brachiopod T. retusa A. Stechmann and M. Schlegel 2045

Table 1. Specimens and mtDNA sequences used for et al. (1991). The complete sequence has an ATcontent of phylogenetic analysis 57.2% and consists of all 37 genes usually found in (Sequences of the following specimens representing di¡erent metazoan mitochondrial genomes (see also ¢gure 1): 13 phyla were taken from GenBank and EMBL for alignments of protein coding genes (COI^COIII, Cytb, ATP6, ATP8, mitochondrial amino-acid sequences to be used in ND1^ND6 and ND4L),two genes for subunits of the ribo- phylogenetic analysis.) somal RNA,a small subunit ( 12SrRNA) and a large subunit (16SrRNA),and 22 tRNA genes. The extreme species accession economic arrangement of genes typical of metazoan mito- chondrial genomes also applies in T.retusa. No introns are Chordata Xenopus laevis M10217 found and the longest region between two adjacent genes Petromyzon marinus U11880 spans 12 bp (COIII ^tRNAThr). Most of the genes abut Branchiostoma lanceolatum Y16474 directly or overlap by one to seven nucleotides. All genes Hemichordata carnosus AF051097 Echinodermata Paracentrotus lividus J04815 are transcribed from the same strand. As already shown Strongylocentrotus purpuratus X12631 by Jacobs et al. (1988) the COI and COII genes are adjacent Asterina pectinifera D16387 to each other,but the relative location of the 16SrRNA Florometra serratissima AF049132 gene is somewhat di¡erent (cf. ¢gure 10.3 in Jacobs et al. Arthropoda Artemia franciscana X69067 (1988)). These inconsistencies are not unexpected,consid- Locusta migratoria X80245 ering the methods used: Southern blot hybridization Drosophila yakuba X03240 versus complete sequence analysis (B. L. Cohen,personal Mollusca Katharina tunicata U09810 Littorina saxatilis AJ132137 communication). The Terebratulina mitochondrial genome Annelida Lumbricus terrestris U24570 contains one unassigned region of 794 bp length between Nematoda X54252 the ND1 and ND6 genes with a slightly higher ATcontent Ascaris suum X54253 (61.7%) as compared to the rest of the genome. The 5'- Metridium senile AF000023 part of this segment consists of six copies of a 68 bp tandem repeat followed by a seventh incomplete copy which stops after 32 bp. A repeat region of 13 guanines is alignments were detected manually and removed,as well as gaps. located 56 bp upstream from the inferred translation initia- All aligned amino-acid sequences were then concatenated to be tion of the ND6 gene. The exact number of nucleotides in used in phylogenetic analyses (alignment 1). Phylogenetic trees this homopolymer run still needs to be determined. From were obtained using di¡erent programs. Neighbour-joining trees reports of other metazoan mtDNAs this could possibly were inferred with the program TREECON for Windows,v. 1.3b represent the origin of replication (Clary & Wolstenholme (Van de Peer & De Wachter 1994). Distance estimations were 1985; Boore & Brown 1995) but,as long as this has not calculated using the Kimura (1983; Hasegawa & Fujiwara 1993) been shown experimentally,the assumption remains spec- and the Tajima & Nei (1984) models. Bootstrap analysis was done ulative. We generated a computer-based restriction frag- with 500 replicates to the robustness of the phylogenetic trees. ment pattern using the same enzymes as in the study of Maximum-likelihood analyses were done with PUZZLE,v. 4.0.2 Cohen et al. (1991) and compared this pattern to the (Strimmer & Von Haeseler 1996) using the mtREV24 model di¡erent mitotypes given there. We could determine the (Adachi & Hasegawa 1996) of amino-acid substitution. Amino- equivalent mitomorph for six enzymes; although the frag- acid frequencies were calculated by the program from the respec- ments sometimes di¡ered in size,in general they resembled tive data sets. The robustness of the inferred phylogenetic trees the Scottish samples. Whether these size di¡erences origi- was tested with likelihood mapping (Strimmer & Von Haeseler nate from a varying copy number of the tandem repeats in 1997) as implemented in PUZZLE,v.4.0.2 and by subjecting the long non-coding region remains unknown. Only alternative tree topologies to the Kishino & Hasegawa (1989) test. complete sequences of otherTerebratulina mtDNAs will help Maximum-likelihood analyses were done with 10 000 puzzling clarify this question. A more detailed description of the steps to test the reliability of internal branches. The variability of mtDNA of T.retusa will be given elsewhere. sites was estimated with PUZZLE,v. 4.0.2 which introduced eight gamma-distributed rates,where each position in the alignment is (b) Comparison of gene arrangements with given one of eight rate categories according to Felsenstein & mitochondrial genomes of di¡erent metazoan Churchill (1996). In three successive analyses those positions that phyla were most variable (category 8),positions with the two most vari- Once the gene order of T. r e t u s a was established the able (category 7 + 8),and three most variable (category 6^8) sites most striking feature was its similarity to the gene were removed. The resulting data sets (alignments 2^4) were arrangement of the polyplacophoran mollusc K. tunicata used for phylogenetic analyses as described above. (Boore & Brown 1994b). Only one major rearrangement The mtDNA sequence of T.retusa is deposited in EMBL is required to interconvert the brachiopod and polyplaco- (accession number: AJ245743). Sequence alignments are phoran gene order (see ¢gure 1). This involves an inver- available on request from the authors or from http://www.uni- sion of a fragment ranging from the 12SrRNA gene to the leipzig.art/bde/fak/biowiss.htm. ND5 gene with the inclusion of some of the adjacent tRNA genes (tRNAPhe, tRNATyr , tRNACys and tRNAMet). Of the 22 tRNA genes,ten keep their absolute and four their 3. RESULTS AND DISCUSSION relative positions when comparing both genomes, (a) Gene content whereas the positions of eight tRNA genes have changed. The complete mtDNA sequence of T. retusa is 15 451bp However,when we compared the brachiopod gene order long which is slightly less than the values given in Cohen with the partial mtDNA of the prosobranch gastropod

Proc. R. Soc. Lond. B(1999) 2046 A. Stechmann and M. Schlegel Mitochondrial genome of the brachiopod T. retusa

ATP8 S(UCN) L(CUN) MYQE KRI S(AGN)

COI COII ND5 ND4 Cytb ND1 16S 12S COIII ND2 Katharina ND3 ND6 ATP6 ND4L DFH T P L(UUR) V CWG AN

ATP8 C V A P N W E R S(AGN)

COI COII 12S 16S ND1 Cytb ND4 ND5 COIII ND2 Terebratulina ND6 ND3 ATP6 ND4L

D YM L(CUN) L(UUR) KS(UCN) QH FG T I

ATP8 MCQE L(UUR) P

COI COII 12S 16S ND1 Cytb Littorina ND6 ATP6

D YWG V L(CUN) Figure 1. Gene organization of the T. retusa (Brachiopoda) mitochondrial genome compared to K. tunicata and L. saxatilis (Mollusca). Protein coding genes and ribosomal genes are designated by their abbreviations as given in the text (see } 3(a)). tRNA genes are indicated by the one letter code of the amino acid they specify. Genes coding on the opposite strand are underlined. Unassigned regions are marked by a shaded box. Double arrows indicate the rearrangements required to interconvert the genomes of Terebratulina and Katharina with respect to protein coding and ribosomal genes. Rearrangements of tRNA genes are not shown. The region spanning the major rearrangement between the Terebratulina and Katharina mtDNA sequence is underlined. Note that the mitochondrial genome of L. saxatilis is only partial.

Littorina saxatilis (Wilding et al. 1999) we found that both Comparisons of the Terebratulina mitochondrial gene genomes show complete congruence in gene order with order with di¡erent revealed that three respect to the ribosomal and protein coding genes. Out of rearrangement events of protein coding and ribosomal the 12 tRNA genes determined so far in the Littorina genes are necessary to interconvert these genomes (e.g. mtDNA,two retain their absolute and ¢ve their relative Limulus polyphemus; Staton et al. 1997).The L. terres- positions,whereas ¢ve tRNA genes have changed posi- tris (Boore & Brown 1995) requires at least four rearran- tions compared to Terebratulina (¢gure 1). The partial gement events to produce an identical gene order to that mtDNA sequence of four genes of another prosobranch of the brachiopod (data not shown). mollusc, Plicopurpura columellaris (Boore et al. 1995),shows Comparisons of protein coding and ribosomal genes on complete congruence with the Terebratulina mtDNA gene the brachiopod mtDNA sequence with di¡erent deutero- order. The recently published partial mtDNA sequence of stome sequences revealed the following results (see ¢gure a (Loligo bleekeri;Sasugaet al. 1999) requires 2). In the three di¡erent arrangements of only one rearrangement event concerning the protein the mtDNA sequence have so far been found,which can coding genes when compared to the Terebratulina mito- be related to di¡erent lineages in this group (Smith et al. chondrial gene arrangement. This involves the transloca- 1993; Asakawa et al. 1995; A. Scouras and M. J. Smith, tion and inversion of the ND5^ND4^ND4L protein gene unpublished data). The brachiopod gene order shows only block. Out of the 14 tRNA genes determined so far,two little similarity to any of the arrangements, keep their positions,whereas 12 have changed their requiring a minimum of six rearrangement events. At positions when compared to Terebratulina (not shown). least four rearrangement events are needed to convert Other complete molluscan mtDNA sequences from the Terebratulina gene order into the Mytilus edulis (Ho¡mann et al. 1992) and pulmonate (B. carnosus; Castresana et al. 1998) or gastropods (Hatzoglou et al.1995;Terrettet al. 1996; (B. lanceolatum;Spruytet al. 1998) gene orders. These Yamazaki et al. 1997) show a rather di¡erent arrangement involve translocations of the COIII/ND3 and 12SrRNA/ to the Terebratulina, Katharina and Littorina gene order 16SrRNA/ND1 blocks,translocations of the ND6 and (data not shown). Cytb genes and an inversion of the ND6 gene (¢gure 2).

Proc. R. Soc. Lond. B(1999) Mitochondrial genome of the brachiopod T. retusa A. Stechmann and M. Schlegel 2047

D ATP8 G R H EP V L(CUN) QM NAY

COI COII COIII ND4 ND5 Cytb 12S 16S ND1 ND2 Balanoglossus ND3 ND6 ATP6 ND4L

S(UCN) K S(AGN) T F L(UUR) I WC

ATP8 C V A N W E R S(AGN)

COI COII 12S 16S ND1 Cytb ND4 ND5 COIII ND2 Terebratulina ATP6 ND6 ND3 ND4L D YM L(CUN) L(UUR) P K S(UCN) QH FG TI

ATP8 H PNAT CM F G Y I

COI COII COIII ND4 ND5 Cytb 12S 16S ND2 ND1 Florometra ND6 ND3 ND4L ATP6 RK S(UCN) S(AGN) QVELW D L(UUR)

Figure 2. Gene organization of the T. retusa (Brachiopoda) mitochondrial genome compared to B. carnosus (Hemichordata) and F. serratissima (Echinodermata). Abbreviations are as in ¢gure 1. Double arrows indicate the rearrangements required to convert the brachiopod mtDNA sequence into the hemichordate and echinoderm sequence. Regions of rearrangements are underlined. Rearrangements of tRNA genes are not shown.

With respect to the tRNA genes numerous rearrangements The several independent rearrangement events are required: in the Balanoglossus and Branchiostoma mito- required to interconvert the Terebratulina and any of the chondrial genomes 17 and 18 tRNA genes,respectively, deuterostome gene arrangements imply that a close have changed their positions compared to the brachiopod relationship with any of the deuterostome brachiopod gene arrangement. phyla seems highly unlikely. In contrast,only one major The relatively high number of rearrangement events rearrangement event separates the brachiopod from the concerning the tRNA genes conforms with the view that K. tunicata (Mollusca: Polyplacophora) gene order. In these genes are more mobile on the mitochondrial addition,the gene order of L. bleekeri (Mollusca: genome (Cantatore et al. 1987; Moritz et al. 1987). Cephalopoda),as determined so far,requires one rear- rangement event when compared to Terebratulina. (c) Phylogenetic implications from gene order However,with the exclusion of tRNA genes,the gene comparisons order of the partial mtDNA sequence of L. saxatilis Analysis of mitochondrial gene order can be done (Mollusca: Prosobranchia) is completely congruent with using the overall similarity in gene order,i.e. similar or the brachiopod. This high degree of similarity in gene identical gene arrangements imply common ancestry. order is the ¢rst to be reported between di¡erent proto- Because rearrangements have to be considered as rare stome phyla. In contrast,the gene arrangements found events,the most parsimonious explanation is favoured. in the bivalve M. edulis and the pulmonate mtDNAs are Those genomes requiring the least number of rearrange- rather di¡erent from Terebratulina. These di¡erences are ments when compared are likely to indicate a close rela- interpreted to represent highly derived states within tionship. Since tRNA genes seem to rearrange at a much those lineages (Sasuga et al. 1999; Wilding et al. 1999,see higher rate than ribosomal and protein coding genes, also Boore 1999). they are probably more informative for within-phyla rela- With the mtDNA data available so far,we cannot tionships (see Boore et al. 1995) and,therefore,not very completely rule out an independent evolution of the useful for deep-level phylogenetic relationships. For this lineages leading to brachiopods and molluscs. However, reason,the phylogenetic implications from comparisons the nearly identical gene order in T.retusa and the three of the gene order on the mitochondrial genome will be molluscs Katharina, Loligo and Littorina suggests a close restricted to the ribosomal and protein coding genes. a¤nity and perhaps even common ancestry of both

Proc. R. Soc. Lond. B(1999) 2048 A. Stechmann and M. Schlegel Mitochondrial genome of the brachiopod T. retusa

Table 2. Phylogenetic analysis of six amino-acid sequences encoded on mtDNA

%of ((prot,brach), (prot,(brach, (chor,((echi, (chor,((hemi, type of number constant lophotrochozoa deut)c deut)) hemi),brach)) brach),echi)) alignmenta of sites sites ln Lb Áln L Æ s.e. Áln L Æ s.e. Áln L Æ s.e. Áln L Æ s.e.

1 (full) 1143 28.3 À21 125.48* 33.25 Æ 14.09 79.55 Æ 20.19 154.64 Æ 26.26 189.37 Æ 28.01 2(Àcategory 8) 1005 32.1 À15 480.30* 34.62 Æ 11.02 75.52 Æ 16.73 135.20 Æ 22.26 175.64 Æ 23.98 3(Àcategory 8 + 7) 854 37.8 À10 610.60* 43.17 Æ 15.20 91.38 Æ 20.65 127.57 Æ 23.68 153.48 Æ 24.53 4(Àcategory 8 + 7 + 6) 691 46.7 À6554.41* 43.78 Æ 12.83 80.08 Æ 17.34 100.37 Æ 19.78 117.92 Æ 20.74 a Di¡erent alignments containing all eight gamma-distributed rate categories (alignment 1,full),exclusion of the most variable category, (alignment 2, Àcategory 8),exclusion of the two most variable categories,(alignment 3, Àcategory 8 + 7),exclusion of the three most variable categories,(alignment 4, Àcategory 8 + 7 + 6). b Log-likelihood values of the preferred topologies (ln L); di¡erences in the log likelihood (Áln L) and standard error of this value in relation to the preferred topolgy. An asterisk indicates the preferred maximum-likelihood topology. c Five di¡erent topologies relating the brachiopod (brach) with protostomes (prot),deuterostomes (deut),echinoderms (echi) and hemi- (hemi); lophotrochozoa: brachiopods + molluscs + annelids. phyla. This is further supported by 18SrRNA sequence analyses (Field et al. 1988; Lake 1990; Halanych et al. 1995; Mackey et al. 1996; Cohen & Gawthrop 1997; 33.9% Cohen et al. 1998a) and fossil evidence (e.g. Conway- Morris 1998). If it holds true that the represents the simultaneous diversi¢cation of three already long-existing stem lineages,i.e. Lophotrochozoa, 0.3% and Deuterostomia (Balavoine & Adoutte 0.3% 1998),with brachiopods having a close phylogenetic rela- 0.1% tionship to molluscs,we can infer the following hypothesis concerning mtDNA gene arrangements in these lineages. We interpret the gene order of the prosobranch L. saxatilis to represent the ancestral molluscan state. In each of the 31.5% 0.3% 33.7% lineages leading to the polyplacophorans and cephalo- Figure 3. Likelihood mapping analysis of the original pods a rearrangement must have occurred resulting in alignment (alignment 1) represented as a triangle. Values at the apomorphic gene order of K. tunicata and L. bleekeri, the corners show the percentages of well-resolved phylogenies respectively. The brachiopod Terebratulina has kept the for all possible quartets,values at the lateral region show the ancestral state and,therefore,both the brachiopod and percentages of unresolved quartets and the value in the centre Littorina represent the plesiomorphic state. Following the shows the percentage of completely unresolved quartets. The criteria of phylogenetic systematics (Hennig 1966) plesio- high number of all corner values (99.1%) indicates that the morphic features do not count in phylogenetic reconstruc- data set is phylogenetically informative. tions. Thus,we are not able to make any inferences about brachiopod and mollusc phylogenetic relationships since analyses (see table 2). The phylogenetic content of these they both share primitive characters. More data are four alignments was tested using the likelihood mapping needed,such as,for example,from inarticulate brachio- analysis as implemented in PUZZLE,v.4.0.2 (see pods and more basal branching articulate brachiopods,to ¢gure 3). The percentage of well-resolved phylogenies for see whether they share the same gene order with Terebratu- all possible quartets decreased only slightly with the lina or whether the Terebratulina gene order is derived reduced alignments,from 99.1% in the original alignment within the brachiopod lineage. In addition,data from (alignment 1) to 94.2% in the shortest alignment (align- more basal branching molluscs could help to assess the ment 4) (cf. table 2). Correspondingly,unresolved and ancestral gene order of all molluscs reliably. To test starlike phylogenies increased slightly from 0.9 and 0.1% whether the brachiopods share a close relationship with to 3.1 and 2.6%,respectively. Still,those values indicate a annelids,as suggested by 18SrRNA sequence comparisons, high phylogenetic content in all four data sets. The neigh- additional data from mitochondrial genomes of oligo- bour-joining analyses inferred from the four di¡erent chaetes and would be helpful. alignments showed that the brachiopod T.retusa always emerged within the protostomes along with the two (d) Phylogenetic trees inferred from amino-acid molluscs K. tunicata and L. saxatilis and the annelid sequence alignments L. terrestris with bootstrap support of 597%. The The original alignment of the six concatenated amino- maximum-likelihood analyses also showed the brachiopod acid sequences with 1143 sites (alignment 1) was tested for T.retusa within the Lophotrochozoan clade with quartet rate heterogeneity introducing eight categories of variable puzzling support of 589% (¢gure 4). Di¡erences sites with PUZZLE,v.4.0.2. In subsequent analyses three emerged in resolving the phylogeny within the Lophotro- di¡erent alignments (alignments 2^4) were produced by chozoa. In all neighbour-joining analyses the two molluscs stepwise omission of variable sites (see } 2). In all,four clearly branch o¡ together,but the branching of the di¡erent alignments were used for the phylogenetic brachiopod and the annelid could not be resolved and is

Proc. R. Soc. Lond. B(1999) Mitochondrial genome of the brachiopod T. retusa A. Stechmann and M. Schlegel 2049

Katharina tunicata 80/100 97/99 Littorina saxatilis 89/94 97/98 Terebratulina retusa PROTOSTOMIA Lumbricus terrestris

Ascaris suum 98/100 99/100 100/100 93/96 Caenorhabditis elegans

Drosophila yakuba 94/100 98/100 88/99 Locusta migratoria 84/100 Artemia franciscana

Paracentrotus lividus 99/100 99/100 83/99 Strongylocentrotus purpuratus 83/99 DEUTEROSTOMIA 82/100 Asterina pectinifera 99/100 79/100 Florometra serratissima 89/99 Balanoglossus carnosus 61/94 83/99 Xenopus laevis 93/100 99/100 97/100 Petromyzon marinus 75/97 Branchiostoma lanceolatum

Metridium senile

Figure 4. of protostome and deuterostome taxa inferred from alignments 1^4 (for more information see } 2). Phylogenetic inference was performed with maximum likelihood and neighbour joining. Upper numbers indicate quartet puzzling support values from 10 000 puzzling steps in the maximum-likelihood analyses of all four alignments (lowest and highest value). Lower numbers represent bootstrap values calculated with 500 replicates in the neighbour-joining analyses. Di¡erences emerged in the branching order of Lumbricus and Terebratulina,depending on the data set used for phylogenetic analysis (tree drawn as a multifurcation). In the maximum-likelihood and neighbour-joining analysis of alignment 4 the two taxa branch o¡ as sister groups with support values of 83 and 45%. In the neighbour-joining analyses the branching order of Lumbricus and Terebratulina varied and the bootstrap support was always below 50%. Since the branching of the two within the protostome clade could not be resolved in the di¡erent analyses it is also drawn as a multifurcation. therefore drawn as a multifurcation. Maximum-likelihood annelid branching o¡ with 83% followed by Terebratulina. analysis of the original alignment (alignment 1) shows But in the shortest alignment consisting of the most Lumbricus to emerge as the ¢rst taxon in the Lophotro- conserved sites (Àcategory 6^8; alignment 4),the annelid chozoa followed by Terebratulina with quartet puzzling and the brachiopod branch o¡ as sister- with 83% support of 78% (¢gure 5). In the following maximum-like- support. Obviously,the data set does not contain enough lihood analysis,reducing the alignment by the most vari- information to resolve the ingroup relationship of the able site (Àcategory 8; alignment 2) produced a lower Lophotrochozoa,although likelihood mapping analyses quartet support value for the branching of Lumbricus and indicated that the data set is phylogenetically informative. Terebratulina within the Lophotrochozoa,leaving their Another problem concerns the placement of the two nema- branching order unresolved. Further reduction of the todes C. elegans and A. suum. Recent analyses of 18SrRNA alignment (Àcategory 7 + 8; alignment 3) shows the sequences showed that the nematodes branch o¡ together

Proc. R. Soc. Lond. B(1999) 2050 A. Stechmann and M. Schlegel Mitochondrial genome of the brachiopod T. retusa

0.1 LOPHOTROCHOZOA Katharina tunicata 100

78 Littorina saxatilis

94 Terebratulina retusa

Lumbricus terrestris

100 Ascaris suum 99 Caenorhabditis elegans ECDYSOZOA 52 Drosophila yakuba 94

88 Locusta migratoria

Artemia franciscana

Paracentrotus lividus 100

99 Strongylocentrotus purpuratus

100 Asterina pectinifera DEUTEROSTOMIA

100 Florometra serratissima

Balanoglossus carnosus 94 Xenopus laevis 93

97 Petromyzon marinus

Branchiostoma lanceolatum

Metridium senile

Figure 5. Maximum-likelihood tree calculated from alignment 1 (1143 sites) with 10 000 puzzling steps showing maximum-likelihood branch lengths. The branch leading to the nematodes A. suum and C. elegans is around 4.3 times longer as shown. All bifurcations are supported with values above 50%,which correspond to the bootstrap values. In this analysis three groups emerge in the bilaterian clade: Lophotrochozoa,Ecdysozoa and Deuterostomia. The scale bar represents a distance of 0.1 substitutions per site. with the arthropods forming a clade named Ecdysozoa sister-group relationship with protostomes or deuteros- (Aguinaldo et al. 1997; Ruiz-Trillo et al. 1999). This could tomes,a sister-group relationship of brachiopods with a only be con¢rmed in one analysis with a su¤ciently high clade including and echinoderms and a quartet support value (see ¢gure 5). All other analyses sister-group relationship of brachiopods and hemichor- failed to resolve the branching of the nematodes with su¤- dates. The latter has been discussed since pterobranchs ciently high support (see also ¢gure 4). However,they and brachiopods both possess (Nielsen 1987; branch within the protostomes in all analyses and do not Halanych 1993,1995). Tests were carried out from all emerge at the base of bilaterian . alignments and the results are shown in table 2. The To test for various hypotheses concerning brachio- di¡erences in log likelihood had to exceed the standard pod relationships within the metazoa we compared error 1.96 times before being signi¢cant (p50.05) maximum-likelihood tree topologies with alternative (Strimmer & Von Haeseler 1996). In all tests the signi¢- topologies using the Kishino & Hasegawa (1989) test. cantly best tree was the one showing the brachiopod The alternative tree topologies included a brachiopod branching o¡ with the annelid and the molluscs,

Proc. R. Soc. Lond. B(1999) Mitochondrial genome of the brachiopod T. retusa A. Stechmann and M. Schlegel 2051 supporting the Lophotrochozoa concept. Di¡erent place- Altschul,S. F.,Gish,W.,Miller,W.,Myers,E. W. & Lipman, ments of the brachiopod were signi¢cantly worse and D. L. 1995 Basic local alignment search tool. J. Mol. Biol. 215, therefore rejected by the test. 403^410. Asakawa,S.,Himeno,H.,Miura,K. & Watanabe,K. 1995 Nucleotide sequence and gene organization of the star¢sh Asterina 4. CONCLUSIONS pectinifera mitochondrial genome. Genetics140,1047^1060. Backeljau,T.,Winnepenninckx,B. & De Bruyn,L. 1993 Both gene order comparisons and amino-acid sequence Cladistic analysis of metazoan relationships: a reappraisal. comparisons strongly support a protostome relationship of Cladistics 9,167^181. T.retusa. It becomes more evident that the long-held view Balavoine,G. & Adoutte,A. 1998 One or three Cambrian of a deuterostome relationship of brachiopods could be radiations? Science 280,397^398. wrong,at least this is evident from the molecular view- Boore,J. L. 1999 mitochondrial genomes. Nucl. Acids point. Furthermore,the new concept of the clade Lopho- Res. 27,1767^1780. within the protostomes becomes more and more Boore,J. L. & Brown,W. M. 1994 a Mitochondrial genomes and acceptable with the accumulating evidence from indepen- the phylogeny of mollusks. Nautilus 2(Suppl.),61^78. dent markers. This work now provides new evidence from Boore,J. L. & Brown,W. M. 1994 b Complete DNA sequence of the mitochondrial genome of the black Katharina tuni- mtDNA sequences. Gene order comparisons suggest a cata. Genetics 138,423^443. protostome a¤nity of brachiopods. Further,sequence Boore,J. L. & Brown,W. M. 1995 Complete sequence of the comparisons of six protein-coding genes support a closer mitochondrial DNA of the annelid worm Lumbricus terrestris. relationship of brachiopods,annelids and molluscs. The Genetics 141,305^319. close a¤nity ofT.retusa and the polyplacophoran K. tunicata Boore,J. L. & Brown,W. M. 1998 Big tress from little genomes: is consistent with the results of Cohen & Gawthrop (1997) mitochondrial gene order as a phylogenetic tool. Curr. Opin. and Cohen et al.(1998a),who found the mollusc to be the Genet. Dev. 8,668^674. most proximal outgroup to brachiopods using 18SrRNA Boore,J. L.,Collins,T. M.,Stanton,D.,Daehler,L. L. & sequences. Still,we are not able to resolve the detailed rela- Brown,W. M. 1995 Deducing the pattern of phylo- tionships within the clade Lophotrochozoa. Most studies geny from mitochondrial DNA rearrangements. Nat u re 376, 163^165. concerning complete mitochondrial genomes have been on Boore,J. L.,Lavrov,D. V. & Brown,W. M. 1998 Gene trans- chordates and arthropods while the mitochondrial location links and . Science 392,667^668. genomes of many invertebrate phyla are not yet known.We Brusca,R. C. & Brusca,G. J. 1990 Invertebrates. 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