The mitochondrial genomes of the iguana (Iguana iguana) and the caiman (Caiman crocodylus): implications for amniote phylogeny

The complete mitochondrial genomes of two reptiles, the common iguana (Iguana iguana) and the caiman (Caiman crocodylus), were sequenced in order to investigate phylogenetic questions of tetrapod evolution. The addition of the two species allows analysis of reptilian relationships using data sets other than those including only fast-evolving species. The crocodilian mitochondrial genomes seem to have evolved generally at a higher rate than those of other vertebrates. Phylogenetic analyses of 2889 amino-acid sites from 35 mitochondrial genomes supported the bird-crocodile relationship, lending no support to the Haematotherma hypothesis (with birds and mammals representing sister groups). The analyses corroborated the view that turtles are at the base of the bird-crocodile branch. This position of the turtles makes Diapsida paraphyletic. The origin of the squamates was estimated at 294 million years (Myr) ago and that of the turtles at 278 Myr ago. Phylogenetic analysis of mammalian relationships using the additional outgroups corroborated the Marsupionta hypothesis, which joins the monotremes and the marsupials to the exclusion of the eutherians.     

Phylogenetic Analysis of 18S rRNA and the Mitochondrial Genomes of the Wombat, <Emphasis Type="BoldItalic">Vombatus ursinus,</Emphasis> and the Spiny Anteater, <Emphasis Type="BoldItalic">Tachyglossus aculeatus:</Emphasis> Increased Support for the Marsupionta Hypothesis

The monotremes, the duck-billed platypus and the echidnas, are characterized by a number of unique morphological characteristics, which have led to the common belief that they represent the living survivors of an ancestral stock of mammals. Analysis of new data from the complete mitochondrial (mt) genomes of a second monotreme, the spiny anteater, and another marsupial, the wombat, yielded clear support for the Marsupionta hypothesis. According to this hypothesis marsupials are more closely related to monotremes than to eutherians, consistent with a basal split between eutherians and marsupials/monotremes among extant mammals. This finding was also supported by analysis of new sequences from a nuclear gene—18S rRNA. The mt genome of the wombat shares some unique features with previously described marsupial mtDNAs (tRNA rearrangement, a missing tRNA^Lys, and evidence for RNA editing of the tRNA^Asp). Molecular estimates of genetic divergence suggest that the divergence between the platypus and the spiny anteater took place ≈34 million years before present (MYBP), and that between South American and Australian marsupials ≈72 MYBP. Received: 28 October 2000 / Accepted: 23 March 2001     

Mitochondrial genomes of a bandicoot and a brushtail possum confirm the monophyly of australidelphian marsupials.

Recent molecular analyses suggest that the position of bandicoots is the major difficulty in determining the root of the tree of extant marsupials. To resolve this, we analyse mitochondrial genome sequences of a bandicoot (Isoodon macrourus) and a brushtail possum (Trichosurus vulpecula) together with the previously available marsupial mitochondrial genomes, the Virginia opossum (Didelphis virginiana) and the wallaroo (Macropus robustus). Analyses of mitochondrial protein-coding and RNA genes strongly support the bandicoot as sister to the wallaroo and the brushtail possum. This result, combined with other recent molecular analyses, confirms the monophyly of Australidelphia (Australasian marsupials plus Dromiciops from South America). Further, RY coding was found to nullify AGCT coding nucleotide composition bias.     

Relationships Among the Families and Orders of Marsupials and the Major Mammalian Lineages Based on Recombination Activating Gene-1

Controversies remain over the relationships among several of the marsupial families and between the three major extant lineages of mammals: Eutheria (placentals), Metatheria (marsupials), and Prototheria (monotremes). Two opposing hypotheses place the marsupials as either sister to the placental mammals (Theria hypothesis) or sister to the monotremes (Palimpsest or Marsupionta hypothesis). A nuclear gene that has proved useful for analyzing phylogenies of vertebrates is the recombination activation gene-1 (RAG1). RAG1 is a highly conserved gene in vertebrates and likely entered the genome by horizontal transfer early in the evolution of jawed vertebrates. Phylogenetic analyses were performed on RAG1 sequences from seven placentals, 28 marsupials, and all three living monotreme species. Phylogenetic analyses of RAG1 sequences support many of the traditional relationships among the marsupials and suggest a relationship between bandicoots (order Peramelina) and the marsupial mole (order Notoryctemorphia), two lineages whose position in the phylogenetic tree has been enigmatic. A sister relationship between South American shrew opossums (order Paucituberculata) and all other living marsupial orders is also suggested by RAG1. The relationship between the three major groups of mammals is consistent with the Theria hypothesis, with the monotremes as the sister group to a clade containing marsupials and placentals.     

Evolution and molecular characterization of a beta-globin gene from the Australian Echidna Tachyglossus aculeatus (Monotremata).

Coinciding with a period in evolution when monotremes, marsupials, and eutherians diverged from a common ancestor, a proto-beta-globin gene duplicated, producing the progenitors of mammalian embryonic and adult beta-like globin genes. To determine whether monotremes contain orthologues of these genes and to further investigate the evolutionary relationships of monotremes, marsupials, and eutherians, we have determined the complete DNA sequence of an echidna (Tachyglossus aculeatus) beta-like globin gene. Conceptual translation of the gene and sequence comparisons with eutherian and marsupial beta-like globin genes and echidna adult beta-globin indicate that the gene is adult expressed. Phylogenetic analyses do not clearly resolve the branching pattern of mammalian beta-like globin gene lineages and it is therefore uncertain whether monotremes have orthologues of the embryonic beta-like globin genes of marsupials and eutherians. Four models are proposed that provide a framework for interpreting further studies on the evolution of beta-like globin genes in the context of the evolution of monotremes, marsupials, and eutherians.     

The platypus is in its place: nuclear genes and indels confirm the sister group relation of monotremes and Therians

Morphological data supports monotremes as the sister group of Theria (extant marsupials + eutherians), but phylogenetic analyses of 12 mitochondrial protein-coding genes have strongly supported the grouping of monotremes with marsupials: the Marsupionta hypothesis. Various nuclear genes tend to support Theria, but a comprehensive study of long concatenated sequences and broad taxon sampling is lacking. We therefore determined sequences from six nuclear genes and obtained additional sequences from the databases to create two large and independent nuclear data sets. One (data set I) emphasized taxon sampling and comprised five genes, with a concatenated length of 2,793 bp, from 21 species (two monotremes, six marsupials, nine placentals, and four outgroups). The other (data set II) emphasized gene sampling and comprised eight genes and three proteins, with a concatenated length of 10,773 bp or 3,669 amino acids, from five taxa (a monotreme, a marsupial, a rodent, human, and chicken). Both data sets were analyzed by parsimony, minimum evolution, maximum likelihood, and Bayesian methods using various models and data partitions. Data set I gave bootstrap support values for Theria between 55% and 100%, while support for Marsupionta was at most 12.3%. Taking base compositional bias into account generally increased the support for Theria. Data set II exclusively supported Theria, with the highest possible values and significantly rejected Marsupionta. Independent phylogenetic evidence in support of Theria was obtained from two single amino acid deletions and one insertion, while no supporting insertions and deletions were found for Marsupionta. On the basis of our data sets, the time of divergence between Monotremata and Theria was estimated at 231-217 MYA and between Marsupialia and Eutheria at 193-186 MYA. The morphological evidence for a basal position of Monotremata, well separated from Theria, is thus fully supported by the available molecular data from nuclear genes.     

Phylogenetic analysis of 1.5 Mbp and platypus EST data refute the Marsupionta hypothesis and unequivocally support Monotremata as sister group to Marsupialia/Placentalia

The extant mammalian groups Monotremata, Marsupialia and Placentalia are, according to the 'Theria' hypothesis, traditionally classified into two subclasses. The subclass Prototheria includes the monotremes and subclass Theria marsupials and placental mammals. Based on some morphological and molecular data, an alternative proposition, the Marsupionta hypothesis, favours a sister group relationship between monotremes and marsupials to the exclusion of placental mammals. Phylogenetic analyses of single genes and even multiple gene alignments have not yet been able to conclusively resolve this basal mammalian divergence. We have examined this problem using one data set composed of expressed sequence tags (EST) and another containing 1 510 509 nucleotide (nt) sites from 1358 inferred cDNA genomic sequences. All analyses of the concatenated sequences unambiguously supported the Theria hypothesis. The Marsupionta hypothesis was rejected with high statistical confidence from both data sets. In spite of the strong support for Theria, a non-negligible number of single genes supported either of the two alternative hypotheses. The divergence between monotremes and therian mammals was estimated to have taken place 168–178 Mya, a dating compatible with the fossil record. Considering the long common evolutionary branch of therians, it is surprising that sequence data from many thousand amino acid sites were needed to conclusively resolve their relationship to monotremes. This finding draws attention to other mammalian divergences that have been taken as unequivocally settled based on much smaller alignments. EST data provide a comprehensive random sample of protein coding sequences and an economic way to produce large amounts of data for phylogenetic analysis of species for which genomic sequences are not yet available.     

Brain gangliosides in monotremes, marsupials and placentals: phylogenetic and thermoregulatory aspects

The concentration and composition of brain gangliosides of 17 mammalian species belonging to the subclasses of Prototheria (monotremes), Metatheria (marsupials), and Eutheria (placentals) were investigated. The mean concentration of brain gangliosides ranges from 525 to 610 micrograms NeuAc/g wet wt in monotremes, 445-900 micrograms in marsupials and from 630 to 1130 micrograms in the placentals. In the phylogenetic series of mammals, a decrease in the complexity of brain ganglioside composition becomes obvious: a drastic reduction in the number of individual ganglioside fractions particularly those of the c-pathway of biosynthesis, took place from the level of monotremes to that of the marsupials and placentals. In monotremes, marsupials and "lower" placentals (insectivores) the percentage of alkali-labile gangliosides is relatively low (between traces and 5%), whereas in the higher evolved mammals it amounts to about 20% of all gangliosides. The ratio of the contents of the two major mammalian ganglioside fractions GD1a and GT1b is generally in the range of 1.0 and even higher; in the heterothermic platypus from the monotremes and in hibernators among the placental mammals, however, it is much lower (about 0.8). These data support the hypothesis that the brain ganglioside composition not only depends on the phylogenetic level of nervous organization (cephalization) but is additionally correlated with the state of thermal adaptation.     

Evolution of the shell coat and yolk in amniotes: a marsupial perspective

Two characters distinguish oogenesis and early development in marsupials and monotremes: (1) the shell coat that persists from the zygote to somite stages in marsupials or until hatching in monotremes; and (2) the numerous, apparently almost empty vesicles that appear in primary oocytes, increase during oogenesis in marsupials and monotremes before being shed into the cleavage cavity and are preferentially distributed to the trophoblast lineage in marsupials, but comprise the latebra in monotremes. Analysis of these unusual characters used Southern analysis of genomic DNA dot blots and histology and electron microscopy. The evidence suggests that the marsupial shell coat protein, CP4, was probably characteristic of the egg of the mammalian ancestor. Further, the vesicles, present in marsupials during oogensis and cleavage and in eutherian mammals during blastocyst formation are the residual elements of white yolk present in the larger yolky eggs of monotemes and sauropsids. By comparison with the function of the vesicle components in marsupials, it is suggested that one role for the white yolk in monotremes and the sauropsids is to provide extracellular matrix (ECM), especially hyaluronan containing stabilizing proteins, for epithelial construction. Thus, as oviparity was replaced by viviparity, egg size was reduced, the germinal cytoplasm was retained, and yellow yolk was markedly reduced or lost in marsupials and eutherians. The white yolk was retained in monotremes and marsupials where blastocyst epithelial construction requires ECM support, and its appearance is heterochronously shifted to after compaction, when blastocyst formation and expansion occurs, in eutherian mammals.     

Evolution of mammalian X-chromosome inactivation: sex chromatin in monotremes and marsupials

The inactive mammalian X-chromosome is always late-replicating, and in eutherian mammals it is heterochromatic and hypermethylated. We propose that this multistep system has evolved from a more primitive system, remnants of which may be found in marsupials and monotremes. The heterochromatic X (sex-chromatin body) is a distinctive feature of interphase cells of certain tissues in eutherian females but not males. Thus we have searched for a sex-specific chromatin body in these same tissues in marsupials (brush-tail possum, Trichosurus vulpecula) and monotremes (platypus, Ornithorynchus anatinus), using classical histological techniques. A female-specific chromatin body was observed at low frequency in nuclei of possum corneal epithelium, but not in any other tissues. No sex difference was observed in any monotreme tissue. These data suggest that stabilization of X-chromosome inactivation by heterochromatinization is tissue-specific in marsupials and absent in monotremes.     

The genomes of the South American opossum (Monodelphis domestica) and platypus (Ornithorhynchus anatinus) encode a more complete purine catabolic pathway than placental mammals

The end product of purine catabolism varies amongst vertebrates and is a consequence of independent gene inactivation events that have truncated the purine catabolic pathway. Mammals have traditionally been grouped into two classes based on their end product of purine catabolism: most mammals, whose end product is allantoin due to an ancient loss of allantoinase (ALLN), and the hominoids, whose end product is uric acid due to recent inactivations of urate oxidase (UOX). However little is known about purine catabolism in marsupials and monotremes. Here we report the results of a comparative genomics study designed to characterize the purine catabolic pathway in a marsupial, the South American opossum (Monodelphis domestica), and a monotreme, the platypus (Ornithorhynchus anatinus). We found that both genomes encode a more complete set of genes for purine catabolism than do eutherians and conclude that a near complete purine catabolic pathway was present in the common ancestor of all mammals, and that the loss of ALLN is specific to placental mammals. Our results therefore provide a revised history for gene loss in the purine catabolic pathway and suggest that marsupials and monotremes represent a third class of mammals with respect to their end products of purine catabolism.     

Phylogenetic relationships within the class mammalia: A study using mitochondrial 12S RNA sequences

The interrelationships of the three mammalian groups, Monotremata, Marsupialia, and Eutheria, have been studied using DNA sequences from the mitochondrial 12S ribosomal RNA gene. The results suggest that the monotremes diverged from the living therians only shortly before eutherians and marsupials separated from each other, although there is some evidence for a slowdown in rate of base change in the monotreme lineage. Whtin the Monotremata, the two extant species of tachyglossids show a very close genetic relationship and the data suggest a very recent divergence. We have also confirmed that the Patagonian Monito del Monte,Dromiciops australis, is more closely related to the australidephian marsupials than it is to other South American species.     

Combined mitochondrial and nuclear DNA sequences resolve the interrelations of the major Australasian marsupial radiations

Australasian marsupials include three major radiations, the insectivorous/carnivorous Dasyuromorphia, the omnivorous bandicoots (Peramelemorphia), and the largely herbivorous diprotodontians. Morphologists have generally considered the bandicoots and diprotodontians to be closely related, most prominently because they are both syndactylous (with the 2nd and 3rd pedal digits being fused). Molecular studies have been unable to confirm or reject this Syndactyla hypothesis. Here we present new mitochondrial (mt) genomes from a spiny bandicoot (Echymipera rufescens) and two dasyurids, a fat-tailed dunnart (Sminthopsis crassicaudata) and a northern quoll (Dasyurus hallucatus). By comparing trees derived from pairwise base-frequency differences between taxa with standard (absolute, uncorrected) distance trees, we infer that composition bias among mt protein-coding and RNA sequences is sufficient to mislead tree reconstruction. This can explain incongruence between trees obtained from mt and nuclear data sets. However, after excluding major sources of compositional heterogeneity, both the "reduced-bias" mt and nuclear data sets clearly favor a bandicoot plus dasyuromorphian association, as well as a grouping of kangaroos and possums (Phalangeriformes) among diprotodontians. Notably, alternatives to these groupings could only be confidently rejected by combining the mt and nuclear data. Elsewhere on the tree, Dromiciops appears to be sister to the monophyletic Australasian marsupials, whereas the placement of the marsupial mole (Notoryctes) remains problematic. More generally, we contend that it is desirable to combine mt genome and nuclear sequences for inferring vertebrate phylogeny, but as separately modeled process partitions. This strategy depends on detecting and excluding (or accounting for) major sources of non-historical signal, such as from compositional non-stationarity. [Base composition; combined data; marsupial; mitochondrial genome; phylogeny.].     

Molecular Phylogeny and Evolution of the Neurotrophins from Monotremes and Marsupials

We have investigated the phylogenetic relationships of monotremes and marsupials using nucleotide sequence data from the neurotrophins; nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). The study included species representing monotremes, Australasian marsupials and placentals, as well as species representing birds, reptiles, and fish. PCR was used to amplify fragments encoding parts of the neurotrophin genes from echidna, platypus, and eight marsupials from four different orders. Phylogenetic trees were generated using parsimony analysis, and support for the different tree structures was evaluated by bootstrapping. The analysis was performed with NGF, BDNF, or NT-3 sequence data used individually as well as with the three neurotrophins in a combined matrix, thereby simultaneously considering phylogenetic information from three separate genes. The results showed that the monotreme neurotrophin sequences associate to either therian or bird neurotrophin sequences and suggests that the monotremes are not necessarily related closer to therians than to birds. Furthermore, the results confirmed the present classification of four Australasian marsupial orders based on morphological characters, and suggested a phylogenetic relationship where Dasyuromorphia is related closest to Peramelemorphia followed by Notoryctemorphia and Diprotodontia. These studies show that sequence data from neurotrophins are well suited for phylogenetic analysis of mammals and that neurotrophins can resolve basal relationships in the evolutionary tree. Received: 27 January 1997 / Accepted: 20 March 1997     

Evolution of the Monotremes: Phylogenetic Relationship to Marsupials and Eutherians, and Estimation of Divergence Dates Based on α-Lactalbumin Amino Acid Sequences

The amino acid sequences of the -lactalbumins of the echidna, Tachyglossus aculeatus, and the platypus, Ornithorhynchus anatinus, were compared with each other and with those of 13 eutherian and 3 marsupial species. Phylogenetic parsimony analyses, in which selected mammalian lysozymes were used as outgroups, yielded trees whose consensus indicated that the two monotremes are sister taxa to marsupials and eutherians and that the latter two clades are each other's closest relatives. The data do not support the notion of a Marsupionta (monotreme–marsupial) clade. Pairwise comparison between the -lactalbumins yielded maximum-likelihood distances from which divergence dates were estimated on the basis of three calibration points. The distance data support the view that the echidna and platypus lineages diverged from their last common ancestor at least 50 to 57 Ma (million years ago) and that monotremes diverged from marsupials and eutherian mammals about 163 to 186 Ma.     

A mitochondrial genome phylogeny of Diptera: whole genome sequence data accurately resolve relationships over broad timescales with high precision

Abstract Mitochondrial genomes provide a promising new tool for understanding deep‐level insect phylogenetics, but have yet to be evaluated for their ability to resolve intraordinal relationships. We tested the utility of mitochondrial genome data for the resolution of relationships within Diptera, the insect order for which the most data are available. We sequenced an additional three genomes, from a syrphid, nemestrinid and tabanid, representing three additional dipteran clades, ‘aschiza’, non‐heteroneuran muscomorpha and ‘basal brachyceran’, respectively. We assessed the influence of optimality criteria, gene inclusion/exclusion, data recoding and partitioning strategies on topology and nodal support within Diptera. Our consensus phylogeny of Diptera was largely consistent with previous phylogenetic hypotheses of the order, except that we did not recover a monophyletic Muscomorpha (Nesmestrinidae grouped with Tabanidae) or Acalyptratae (Drosophilidae grouped with Calliphoridae). The results were very robust to optimality criteria, as parsimony, likelihood and Bayesian approaches yielded very similar topologies, although nodal support varied. The addition of ribosomal and transfer RNA genes to the protein coding genes traditionally used in mitochondrial genome phylogenies improved the resolution and support, contrary to previous suggestions that these genes would evolve too quickly or prove too difficult to align to provide phylogenetic signal at deep nodes. Strategies to recode data, aimed at reducing homoplasy, resulted in a decrease in tree resolution and branch support. Bayesian analyses were highly sensitive to partitioning strategy: biologically realistic partitions into codon groups produced the best results. The implications of this study for dipteran systematics and the effective approaches to using mitochondrial genome data are discussed. Mitochondrial genomes resolve intraordinal relationships within Diptera accurately over very wide time ranges (1–200 million years ago) and genetic distances, suggesting that this may be an excellent data source for deep‐level studies within other, less studied, insect orders.     

Marsupials and Eutherians reunited: genetic evidence for the Theria hypothesis of mammalian evolution

The three living monophyletic divisions of Class Mammalia are the Prototheria (monotremes), Metatheria (marsupials), and Eutheria (`placental' mammals). Determining the sister relationships among these three groups is the most fundamental question in mammalian evolution. Phylogenetic comparison of these mammals by either anatomy or mitochondrial DNA has resulted in two conflicting hypotheses, Theria and Marsupionta, and has fueled a ``genes versus morphology' controversy. We have cloned and analyzed a large nuclear gene, the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R), from representatives of all three mammalian groups, including platypus, echidna, opossum, wallaby, hedgehog, mouse, rat, rabbit, cow, pig, bat, tree shrew, colugo, ringtail lemur, and human. Statistical analysis of this nuclear gene unambiguously supports the morphology-based Theria hypothesis that excludes monotremes from a clade of marsupials and eutherians. The M6P/IGF2R was also able to resolve the finer structure of the eutherian mammalian family tree. In particular, our analyses support sister group relationships between lagomorphs and rodents, and between the primates and Dermoptera. Statistical support for the grouping of the hedgehog with Feruungulata and Chiroptera was also strong. Received: 8 December 2000 / Accepted: 01 February 2001