Searching for the closest living relative(s) of tetrapods through evolutionary analyses of mitochondrial and nuclear data

The phylogenetic relationships of the African lungfish (Protopterus dolloi) and the coelacanth (Latimeria chalumnae) with respect to tetrapods were analyzed using complete mitochondrial genome DNA sequences. A lungfish + coelancanth clade was favored by maximum parsimony (although this result is dependent on which transition:transversion weights are applied), and a lungfish + tetrapod clade was supported by neighbor-joining and maximum-likelihood analyses. These two hypotheses received the strongest statistical and bootstrap support to the exclusion of the third alternative, the coelacanth + tetrapod sister group relationship. All mitochondrial protein coding genes combined favor a lungfish + tetrapod grouping. We can confidently reject the hypothesis that the coelacanth is the closest living relative of tetrapods. When the complete mitochondrial sequence data were combined with nuclear 28S rRNA gene data, a lungfish + coelacanth clade was supported by maximum parsimony and maximum likelihood, but a lungfish + tetrapod clade was favored by neighbor-joining. The seeming conflicting results based on different data sets and phylogenetic methods were typically not statistically strongly supported based on Kishino-Hasegawa and Templeton tests, although they were often supported by strong bootstrap values. Differences in rate of evolution of the different mitochondrial genes (slowly evolving genes such as the cytochrome oxidase and tRNA genes favored a lungfish + coelacanth clade, whereas genes of relatively faster substitution rate, such as several NADH dehydrogenase genes, supported a lungfish + tetrapod grouping), as well as the rapid radiation of the lineages back in the Devonian, rather than base compositional biases among taxa seem to be directly responsible for the remaining uncertainty in accepting one of the two alternate hypotheses.     

The complete mitochondrial DNA sequence of the shark Mustelus manazo: evaluating rooting contradictions to living bony vertebrates

A remarkable example of a misleading mitochondrial protein tree is presented, involving ray-finned fishes, coelacanths, lungfishes, and tetrapods, with sea lampreys as an outgroup. In previous molecular phylogenetic studies on the origin of tetrapods, ray-finned fishes have been assumed as an outgroup to the tetrapod/lungfish/coelacanth clade, an assumption supported by morphological evidence. Standard methods of molecular phylogenetics applied to the protein-encoding genes of mitochondria, however, give a bizarre tree in which lamprey groups with lungfish and, therefore, ray-finned fishes are not the outgroup to a tetrapod/lungfish/coelacanth clade. All of the dozens of published phylogenetic methods, including every possible modification to maximum likelihood known to us (such as inclusion of site heterogeneity and exclusion of potentially misleading hydrophobic amino acids), fail to place the ray-finned fishes in a biologically acceptable position. A likely cause of this failure may be the use of an inappropriate outgroup. Accordingly, we have determined the complete mitochondrial DNA sequence from the shark, Mustelus manazo, which we have used as an alternative and more proximal outgroup than the lamprey. Using sharks as the outgroup, lungfish appear to be the closest living relative of tetrapods, although the possibility of a lungfish/coelacanth clade being the sister group of tetrapods cannot be excluded.      

The Complete DNA Sequence of the Mitochondrial Genome of a ``living Fossil,' the Coelacanth (Latimeria Chalumnae)

The complete nucleotide sequence of the 16,407-bp mitochondrial genome of the coelacanth (Latimeria chalumnae) was determined. The coelacanth mitochondrial genome order is identical to the consensus vertebrate gene order which is also found in all ray-finned fishes, the lungfish, and most tetrapods. Base composition and codon usage also conform to typical vertebrate patterns. The entire mitochondrial genome was PCR-amplified with 24 sets of primers that are expected to amplify homologous regions in other related vertebrate species. Analyses of the control region of the coelacanth mitochondrial genome revealed the existence of four 22-bp tandem repeats close to its 3' end. The phylogenetic analyses of a large data set combining genes coding for rRNAs, tRNAs, and proteins (16,140 characters) confirmed the phylogenetic position of the coelacanth as a lobe-finned fish; it is more closely related to tetrapods than to ray-finned fishes. However, different phylogenetic methods applied to this largest available molecular data set were unable to resolve unambiguously the relationship of the coelacanth to the two other groups of extant lobe-finned fishes, the lungfishes and the tetrapods. Maximum parsimony favored a lungfish/coelacanth or a lungfish/tetrapod sistergroup relationship depending on which transversion:transition weighting is assumed. Neighbor-joining and maximum likelihood supported a lungfish/tetrapod sistergroup relationship.     

Complete Mitochondrial Genome Sequences of the South American and the Australian Lungfish: Testing of the Phylogenetic Performance of Mitochondrial Data Sets for Phylogenetic Problems in Tetrapod Relationships

We determined the complete nucleotide sequences (16403 and 16572 base pairs, respectively) of the mitochondrial genomes of the South American lungfish, Lepidosiren paradoxa, and the Australian lungfish, Neoceratodus forsteri (Sarcopterygii, Dipnoi). The mitochondrial DNA sequences were established in an effort to resolve the debated evolutionary positions of the lungfish and the coelacanth relative to land vertebrates. Previous molecular phylogenetic studies based on complete mtDNA sequences, including only the African lungfish, Protopterus dolloi, sequence were able to strongly reject the traditional textbook hypothesis that coelacanths are the closest relatives of land vertebrates. However, these studies were unable to statistically significantly distinguish between the two remaining scenarios: lungfish as the closest relatives to land vertebrates and lungfish and coelacanths jointly as their sister group (Cao et al. 1998; Zardoya et al. 1998; Zardoya and Meyer 1997a). Lungfish, coelacanths, and the fish ancestors of the tetrapod lineage all originated within a short time window of about 20 million years, back in the early Devonian (about 380 to 400 million years ago). This short divergence time makes the determination of the phylogenetic relationships among these three lineages difficult. In this study, we attempted to break the long evolutionary branch of lungfish, in an effort to better resolve the phylogenetic relationships among the three extant sarcopterygian lineages. The gene order of the mitochondrial genomes of the South American and Australian lungfish conforms to the consensus gene order among gnathostome vertebrates. The phylogenetic analyses of the complete set of mitochondrial proteins (without ND6) suggest that the lungfish are the closest relatives of the tetrapods, although the support in favor of this scenario is not statistically significant. The two other smaller data sets (tRNA and rRNA genes) give inconsistent results depending on the different reconstruction methods applied and cannot significantly rule out any of the three alternative hypotheses. Nuclear protein-coding genes, which might be better phylogenetic markers for this question, support the lungfish–tetrapod sister-group relationship (Brinkmann et al. 2004).This article contains online supplementary material.Reviewing Editor: Dr. Rafael Zardoya     

Gene Structure and Amino Acid Sequence of Latimeria chalumnae (Coelacanth) Myelin DM20: Phylogenetic Relation of the Fish

The structure of Latimeria chalumnae (coelacanth) proteolipid protein/DM20 gene excluding exon 1 was determined, and the amino acid sequence of Latimeria DM20 corresponding to exons 2–7 was deduced. The nucleotide sequence of exon 3 suggests that only DM20 isoform is expressed in Latimeria. The structure of proteolipid protein/DM20 gene is well preserved among human, dog, mouse, and Latimeria. Southern blot analysis indicates that Latimeria DM20 gene is a single-copy gene. When the amino acid sequences of DM20 were compared among various species, Latimeria was more similar to tetrapods than other fishes including lungfish, confirming the previous finding by immunoreactivity (Waehneldt and Malotka 1989 J. Neurochem. 52:1941–1943). However, when phylogenetic trees were constructed from the DM20 sequences, lungfish was clearly the closest to tetrapods. Latimeria was situated outside of lungfish by the maximum likelihood method. The apparent similarity of Latimeria DM20 to tetrapod proteolipid protein/DM20 is explained by the slow amino acid substitution rate of Latimeria DM20.     

The phylogenetic relationship of tetrapod, coelacanth, and lungfish revealed by the sequences of forty-four nuclear genes

The origin of tetrapods is a major outstanding issue in vertebrate phylogeny. Each of the three possible principal hypotheses (coelacanth, lungfish, or neither being the sister group of tetrapods) has found support in different sets of data. In an attempt to resolve the controversy, sequences of 44 nuclear genes encoding amino acid residues at 10,404 positions were obtained and analyzed. However, this large set of sequences did not support conclusively one of the three hypotheses. Apparently, the coelacanth, lungfish, and tetrapod lineages diverged within such a short time interval that at this level of analysis, their relationships appear to be an irresolvable trichotomy.     

A molecular phylogeny of <Emphasis Type="Italic">Haptoglossa</Emphasis> species,terrestrial peronosporomycetes (oomycetes) endoparasitic on nematodes

Phylogenetic relationships of seven Haptoglossa isolates were analyzed by using mitochondrial COII amino acid sequences with a data set of 34 peronosporomycetes. Haptoglossa isolates formed a single clade and appeared to be basal to the clade consisting of all other peronosporomycetes. The Haptoglossa clade was divided into two subclades: one clade consisted of five aplanosporic isolates and the other included one aplanosporic and one zoosporic isolate. These results indicate that the genus Haptoglossa is monophyletic, and patterns of infection cell formation reflect more the phylogenetic relationship between the species than patterns of sporogenesis.     

The Mitochondrial Sequences of Heptathela hangzhouensis and Ornithoctonus huwena Reveal Unique Gene Arrangements and Atypical tRNAs

We have sequenced the complete mitochondrial genomes of the spiders Heptathela hangzhouensis and Ornithoctonus huwena. Both genomes encode 13 protein-coding genes, 22 tRNA genes, and 2 ribosomal RNA genes. H. hangzhouensis, a species of the suborder Mesothelae and a representative of the most basal clade of Araneae, possesses a gene order identical to that of Limulus polyphemus of Xiphosura. On the other hand, O. huwena, a representative of suborder Opisthothelae, infraorder Mygalomorphae, was found to have seven tRNA genes positioned differently from those of Limulus. The rrnL–trnL1–nad1 arrangement shared by the araneomorph families Salticidae, Nesticidae, and Linyphiidae and the mygalomorph family Theraphosidae is a putative synapomorphy joining the mygalomorph with the araneomorph. Between the two species examined, base compositions also differ significantly. The lengths of most protein-coding genes in H. hangzhouensis and O. huwena mtDNA are either identical to or slightly shorter than their Limulus counterparts. Usage of initiation and termination codons in these protein-coding genes seems to follow patterns conserved among most arthropod and some other metazoan mitochondrial genomes. The sequences of the 3 ends of rrnS and rrnL in the two species are similar to those reported for Limulus, and the entire genes are shortened by about 100–250 nucleotides with respect to Limulus. The lengths of most tRNA genes from the two species are distinctly shorter than those of Limulus and the sequences reveal unusual inferred tRNA secondary structures. Our finding provides new molecular evidence supporting that the suborder Mesothelae is basal to opisthothelids.Reviewing Editor Dr. Rafael Zardoya     

Complete mitochondrial genome of Tubulipora flabellaris (Bryozoa: Stenolaemata): the first representative from the class Stenolaemata with unique gene order

Mitochondrial genomes play a significant role in the reconstruction of phylogenetic relationships within metazoans. There are still many controversies concerning the phylogenetic position of the phylum Bryozoa. In this research, we have finished the complete mitochondrial genome of one bryozoan (Tubulipora flabellaris), which is the first representative from the class Stenolaemata. The complete mitochondrial genome of T. flabellaris is 13,763 bp in length and contains 36 genes, which lacks the atp8 gene in contrast to the typical metazoan mitochondrial genomes. Gene arrangement comparisons indicate that the mitochondrial genome of T. flabellaris has unique gene order when compared with other metazoans. The four known bryozoans complete mitochondrial genomes also have very different gene arrangements, indicates that bryozoan mitochondrial genomes have experienced drastic rearrangements. To investigate the phylogenetic relationship of Bryozoa, phylogenetic analyses based on amino acid sequences of 11 protein coding genes (excluding atp6 and atp8) from 26 metazoan complete mitochondrial genomes were made utilizing Maximum Likelihood (ML) and Bayesian methods, respectively. The results indicate the monopoly of Lophotrochozoa and a close relationship between Chaetognatha and Bryozoa. However, more evidences are needed to clarify the relationship between two groups. Lophophorate appeared to be polyphyletic according to our analyses. Meanwhile, neither analysis supports close relationship between Branchiopod and Phoronida. Four bryozoans form a clade and the relationship among them is T. flabellaris + (F. hispida + (B. neritina + W. subtorquata)), which is in coincidence with traditional classification system.     

Origin of tetrapods inferred from their mitochondrial DNA affiliation to lungfish

Summary This paper shows that questions of an unexpected phylogenetic depth can be addressed by the study of mitochondrial DNA (mtDNA) sequences. For decades, it has been unclear whether coelacanth fishes or lungfishes are the closest living relatives of land vertebrates (Tetrapoda). Segments of mtDNA from a lungfish, the coelacanth, and a ray-finned fish were sequenced and compared to the published sequence of a frog mtDNA. A tree based on inferred amino acid replacements, silent transversions, and ribosomal RNA (rRNA) substitutions showed with statistical confidence that the lungfish mtDNA is more closely related to that of the frog than is the mtDNA of the coelacanth. This result appears to rule out the possibility that the coelacanth lineage gave rise to land vertebrates; hence, morphological characters that link the latter two groups are possibly due to convergent evolution or reversals and not to common descent. Besides supporting the theory that land vertebrates arose from an offshoot of the lineage leading to lungfishes, the molecular tree facilitates an evolutionary interpretation of the morphological differences among the living forms. It would appear that the common ancestor of lungfishes and tetrapods already possessed multiple morphological traits preadapting their locomotion, circulation, and respiration for life on land.