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     

Intraclass diversification of immunoglobulin heavy chain genes in the African lungfish

Lungfish (Dipnoi) are the closest living relatives to tetrapods, and they represent the transition from water to land during vertebrate evolution. Lungfish are armed with immunoglobulins (Igs), one of the hallmarks of the adaptive immune system of jawed vertebrates, but only three Ig forms have been characterized in Dipnoi to date. We report here a new diversity of Ig molecules in two African lungfish species (Protopterus dolloi and Protopterus annectens). The African lungfish Igs consist of three IgMs, two IgWs, three IgNs, and an IgQ, where both IgN and IgQ originated evidently from the IgW lineage. Our data also suggest that the IgH genes in the lungfish are organized in a transiting form from clusters (IgH loci in cartilaginous fish) to a translocon configuration (IgH locus in tetrapods). We propose that the intraclass diversification of the two primordial gnathostome Ig classes (IgM and IgW) as well as acquisition of new isotypes (IgN and IgQ) has allowed lungfish to acquire a complex and functionally diverse Ig repertoire to fight a variety of microorganisms. Furthermore, our results support the idea that “tetrapod-specific” Ig classes did not evolve until the vertebrate adaptation to land was completed ~360 million years ago.     

The colonization of land by animals: molecular phylogeny and divergence times among arthropods

Background  

The earliest fossil evidence of terrestrial animal activity is from the Ordovician, ~450 million years ago (Ma). However, there are earlier animal fossils, and most molecular clocks suggest a deep origin of animal phyla in the Precambrian, leaving open the possibility that animals colonized land much earlier than the Ordovician. To further investigate the time of colonization of land by animals, we sequenced two nuclear genes, glyceraldehyde-3-phosphate dehydrogenase and enolase, in representative arthropods and conducted phylogenetic and molecular clock analyses of those and other available DNA and protein sequence data. To assess the robustness of animal molecular clocks, we estimated the deuterostome-arthropod divergence using the arthropod fossil record for calibration and tunicate instead of vertebrate sequences to represent Deuterostomia. Nine nuclear and 15 mitochondrial genes were used in phylogenetic analyses and 61 genes were used in molecular clock analyses.     

That awkward age for butterflies: insights from the age of the butterfly subfamily Nymphalinae (Lepidoptera: Nymphalidae)

The study of the historical biogeography of butterflies has been hampered by a lack of well-resolved phylogenies and a good estimate of the temporal span over which butterflies have evolved. Recently there has been surge of phylogenetic hypotheses for various butterfly groups, but estimating ages of divergence is still in its infancy for this group of insects. The main problem has been the sparse fossil record for butterflies. In this study I have used a surprisingly good fossil record for the subfamily Nymphalinae (Lepidoptera: Nymphalidae) to estimate the ages of diversification of major lineages using Bayesian relaxed clock methods. I have investigated the effects of varying priors on posterior estimates in the analyses. For this data set, it is clear that the prior of the rate of molecular evolution at the ingroup node had the largest effect on the results. Taking this into account, I have been able to arrive at a plausible history of lineage splits, which appears to be correlated with known paleogeological events. The subfamily appears to have diversified soon after the K/T event about 65 million years ago. Several splits are coincident with major paleogeological events, such as the connection of the African and Asian continents about 21 million years ago and the presence of a peninsula of land connecting the current Greater Antilles to the South American continent 35 to 33 million years ago. My results suggest that the age of Nymphalidae is older than the 70 million years speculated to be the age of butterflies as a whole.     

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.     

Allopolyploid evolution in Geinae (Colurieae: Rosaceae) – building reticulate species trees from bifurcating gene trees

A previous phylogenetic study of paralogous nuclear low-copy granule-bound starch synthase (GBSSI) gene sequences from polyploid and diploid species in Geinae indicated that the clade has experienced two major allopolyploid events in its history. These were estimated to have occurred several million years ago. In this extended study we test if the reticulate phylogenetic hypothesis for Geinae can be maintained when additional sequences are added. The results are compatible with the hypothesis and strengthen it in minor aspects. We also attempt to identify extant members of one of the inferred ancestral lineages of the allopolyploids. On the basis of previous molecular phylogenies, one specific group has been proposed to be the descendants of this taxon. However, none of the additional paralogues belong to this ancestral lineage. A general method is proposed for converting a bifurcating gene tree, with multiple paralogous low-copy gene sequences from allopolyploid taxa, into a reticulate species tree.     

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.     

Rodents of the Caribbean: origin and diversification of hutias unravelled by next-generation museomics

The Capromyidae (hutias) are endemic rodents of the Caribbean and represent a model of dispersal for non-flying mammals in the Greater Antilles. This family has experienced severe extinctions during the Holocene and its phylogenetic affinities with respect to other caviomorph relatives are still debated as morphological and molecular data disagree. We used target enrichment and next-generation sequencing of mitochondrial and nuclear genes to infer the phylogenetic relationships of hutias, estimate their divergence ages, and understand their mode of dispersal in the Greater Antilles. We found that Capromyidae are nested within Echimyidae (spiny rats) and should be considered a subfamily thereof. We estimated that the split between hutias and Atlantic Forest spiny rats occurred 16.5 (14.8–18.2) million years ago (Ma), which is more recent than the GAARlandia land bridge hypothesis (34–35 Ma). This would suggest that during the Early Miocene, an echimyid-like ancestor colonized the Greater Antilles from an eastern South American source population via rafting. The basal divergence of the Hispaniolan Plagiodontia provides further support for a vicariant separation between Hispaniolan and western islands (Bahamas, Cuba, Jamaica) hutias. Recent divergences among these western hutias suggest Plio-Pleistocene dispersal waves associated with glacial cycles.     

Novel evolutionary relationship among four fish model systems

Knowledge of the correct phylogenetic relationships among animals is crucial for the valid interpretation of evolutionary trends in biology. Zebrafish, medaka, pufferfish and cichilds are fish models for development, genomics and comparative genetics studies, although their phylogenetic relationships have not been tested rigorously. The results of phylogenomic analysis based on 20 nuclear protein-coding genes confirmed the basal placement of zebrafish in the fish phylogeny but revealed an unexpected relationship among the other three species, contrary to traditionally held systematic views based on morphology. Our analyses show that medaka (Beloniformes) and cichlids (Perciformes) appear to be more closely related to each other than either of them is to pufferfish (Tetraodontiformes), suggesting that a re-interpretation of some findings in comparative biology might be required. In addition, phylogenomic analyses show that fish typically have more copies of nuclear genes than land vertebrates, supporting the fish-specific genome duplication hypothesis.     

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.      

Classification and phylogenetic relationships of African tilapiine fishes inferred from mitochondrial DNA sequences

African cichlid fishes are composed of two major lineages, the haplochromines and the tilapiines. Whereas the phylogenetic relationships of the haplochromines have been studied extensively, primarily because of their spectacular adaptive radiations in the Great Lakes of East Africa, little is known about the relationships among the tilapiine species, despite the fact that they have become an important component of African, indeed world, aquaculture. To remedy this situation, molecular phylogenetic analysis of tilapiine fishes was undertaken. A segment of mitochondrial DNA encompassing the terminal part of the tRNA(Pro) gene and the most variable part of the control region was amplified by the polymerase chain reaction with DNA samples isolated from 42 tilapiine species, and the amplification products were subjected to heteroduplex analysis and sequencing. Phylogenetic trees based on 68 sequences revealed the existence of 11 sequence groups and 11 single-sequence branches. The groups, designated Ti1 through Ti11, were distinguished by specific combinations of diagnostic substitutions, formation of monophyletic clusters, and separation by genetic distances in excess of 0.04. Although the relationships among the groups could not be resolved, the sequences separated Oreochromis and Sarotherodon from Tilapia, as defined by Trewavas. The Oreochromis sequences clustered with the Sarotherodon sequences and thus supported the hypothesis that the mouthbrooding behavior of the tilapiine fishes evolved only once from the substrate-spawning behavior. Since on phylogenetic trees the O. alcalicus (sub)species were always separated from O. amphimelas by other Oreochromis species, it was concluded that the adaptation to life in water with a high salt concentration and high pH values evolved independently at least twice in the tilapiine fishes. The tilapiines diverged from the haplochromines more than 8 million years ago; most of the intragroup divergences among the tilapiines took place an estimated 1.1 to 6 million years ago.     

Phylogenetics of the grass genus Ehrharta: evidence for radiation in the summer-arid zone of the South African Cape

Abstract Climatic and geological change may play a key role in stimulating biological radiations. Here, we use phylogenetic data to test whether the comparatively high diversity of ehrharteoid grasses in the Cape region of South Africa is the result of rapid radiation associated with the onset of a seasonally arid climate during the late Miocene. A phylogenetic hypothesis based on morphological and nucleotide sequence (nuclear ITS1 and plastid trn L-F) data confirms the monophyly of the African Ehrharta species and shows that the diversification of this lineage was centered in the Cape region. Sequence divergence data (ITS1 + trn L-F) indicate a pulse of rapid speciation, which may explain poor phylogenetic resolution within the African Ehrharta clade. Alternative calibrations yield a broad range of time estimates for the start and end of this radiation, most of which indicate a radiation inside the last 11 million years. A calibration based on the age of Ehrhartoideae suggests that radiation started 9.82 ± 0.20 million years ago and ended 8.74 ± 0.21 million years ago. Under alternative calibrations, estimated speciation rates during the period of radiation range between 0.87 and 4.18 species per million years. Parsimony optimization of habitat parameters reveals that radiation was correlated with the occupation of seasonally arid succulent karoo environments, wet heathy (fynbos) environments being ancestral. These data support earlier suggestions that late Miocene climatic change stimulated floristic radiation at the Cape, and highlight the potential importance of environmental change in powering diversification in continental floras.     

Molecular phylogenetic relationships of the deep-sea fish genus Coryphaenoides (Gadiformes: Macrouridae) based on mitochondrial DNA

In order to characterize the phylogenetic relationship and deep-sea adaptation process of the deep-sea fish genus Coryphaenoides, the nucleotide sequences of the mitochondrial (mt) 12 S rRNA and COI gene sequences for seven Coryphaenoides species were analyzed. Our molecular phylogenetic tree shows a new arrangement of seven Coryphaenoides species, which form two distinct groups, abyssal and nonabyssal species, and differs from the results of previous taxonomic studies. Using the mutation rate of mitochondrial genes, the divergence time between abyssal and nonabyssal Coryphaenoides was found to be 3.2-7.6 million years ago. Our study suggests that hydraulic pressure plays an important role in the speciation process in the marine environment.     

Are all fishes ancient polyploids?

Euteleost fishes seem to have more copies of many genes than their tetrapod relatives. Three different mechanisms could explain the origin of these 'extra' fish genes. The duplicates may have been produced during a fish-specific genome duplication event. A second explanation is an increased rate of independent gene duplications in fish. A third possibility is that after gene or genome duplication events in the common ancestor of fish and tetrapods, the latter lost more genes. These three hypotheses have been tested by phylogenetic tree reconstruction. Phylogenetic analyses of sequences from human, mouse, chicken, frog (Xenopus laevis), zebrafish (Danio rerio) and pufferfish (Takifugu rubripes) suggest that ray-finned fishes are likely to have undergone a whole genome duplication event between 200 and 450 million years ago. We also comment here on the evolutionary consequences of this ancient genome duplication.     

Molecular evidence for the common origin of snap-traps among carnivorous plants

The snap-trap leaves of the aquatic waterwheel plant (Aldrovanda) resemble those of Venus' flytrap (Dionaea), its distribution and habit are reminiscent of bladderworts (Utricularia), but it shares many reproductive characters with sundews (Drosera). Moreover, Aldrovanda has never been included in molecular phylogenetic studies, so it has been unclear whether snap-traps evolved only once or more than once among angiosperms. Using sequences from nuclear 18S and plastid rbcL, atpB, and matK genes, we show that Aldrovanda is sister to Dionaea, and this pair is sister to Drosera. Our results indicate that snap-traps are derived from flypaper-traps and have a common ancestry among flowering plants, despite the fact that this mechanism is used by both a terrestrial species and an aquatic one. Genetic and fossil evidence for the close relationship between these unique and threatened organisms indicate that carnivory evolved from a common ancestor within this caryophyllid clade at least 65 million years ago.     

True enamel covering in teeth of the Australian lungfish Neoceratodus forsteri

Lungfish are a unique order of sarcopterygian fish cleidographically positioned between tetrapods and fish. An uninterrupted 400-million-year-old fossil record has documented lungfish skeletal elements to remain virtually unchanged since the Early Devonian. In the current study we investigated the enamel layer of lungfish teeth in order to determine whether there was evidence for higher vertebrate "true" enamel in the Australian lungfish. Juvenile lungfish from the Brisbane River were processed for light and electron microscopy and analyzed for parameters indicative of true enamel formation. Using anti-amelogenin primary antibodies for immunodetection and Western blots, enamel protein epitopes were detected in developing lungfish teeth. Using transmission electron microscopy and electron diffraction analysis, long and parallel-oriented hydroxyapatite crystals were observed in lungfish outer tooth coverings. Our findings indicate that Australian lungfish teeth are covered by a layer of true enamel. Based on the lungfish fossil record we conclude that features of true enamel formation may be as old as 400 million years. Based on taxonomic classification we confirm that true enamel is found not only in tetrapods but also in the sarcopterygian clade of the Gnathostomata.     

Evolutionary history of the Rh blood group-related genes in vertebrates

Rh and its homologous Rh50 gene products are considered to form heterotetramers on erythrocyte membranes. Rh protein has Rh blood group antigen sites, while Rh50 protein does not, and is more conserved than Rh protein. We previously determined both Rh and Rh50 gene cDNA coding regions from mouse and rat, and carried out phylogenetic analyses. In this study, we determined Rh50 gene cDNA coding regions from African clawed frog and Japanese medaka fish, and examined the long-term evolution of the Rh blood group and related genes. We constructed the phylogenetic tree from amino acid sequences. Rh50 genes of African clawed frog and Japanese medaka fish formed a cluster with mammalian Rh50 genes. The gene duplication time between Rh and Rh50 genes was estimated to be about 510 million years ago based on this tree. This period roughly corresponds to the Cambrian, before the divergence between jawless fish and jawed vertebrates. We also BLAST-searched an amino acid sequence database, and the Rh blood group and related genes were found to have homology with ammonium transporter genes of many organisms. Ammonium transporter genes can be classified into two major groups (amt alpha and amt beta). Both groups contain genes from three domains (bacteria, archaea, and eukaryota). The Rh blood group and related genes are separated from both amt alpha and beta groups.