Dating of the human-ape splitting by a molecular clock of mitochondrial DNA

Summary A new statistical method for estimating divergence dates of species from DNA sequence data by a molecular clock approach is developed. This method takes into account effectively the information contained in a set of DNA sequence data. The molecular clock of mitochondrial DNA (mtDNA) was calibrated by setting the date of divergence between primates and ungulates at the Cretaceous-Tertiary boundary (65 million years ago), when the extinction of dinosaurs occurred. A generalized leastsquares method was applied in fitting a model to mtDNA sequence data, and the clock gave dates of 92.3±11.7, 13.3±1.5, 10.9±1.2, 3.7±0.6, and 2.7±0.6 million years ago (where the second of each pair of numbers is the standard deviation) for the separation of mouse, gibbon, orangutan, gorilla, and chimpanzee, respectively, from the line leading to humans. Although there is some uncertainty in the clock, this dating may pose a problem for the widely believed hypothesis that the bipedal creatureAustralopithecus afarensis, which lived some 3.7 million years ago at Laetoli in Tanzania and at Hadar in Ethiopia, was ancestral to man and evolved after the human-ape splitting. Another likelier possibility is that mtDNA was transferred through hybridization between a proto-human and a protochimpanzee after the former had developed bipedalism.     

Biochemical and molecular characterisation of hemocyanin from the amphipod <Emphasis Type="Italic">Gammarus roeseli</Emphasis>: complex pattern of hemocyanin subunit evolution in Crustacea

Hemocyanin is a copper-containing respiratory protein that is widespread within the arthropod phylum. Among the Crustacea, hemocyanins are apparently restricted to the Malacostraca. While well-studied in Decapoda, no hemocyanin sequence has been known from the ’lower’ Malacostraca. The hemocyanin of the amphipod Gammarus roeseli is a hexamer that consists of at least five distinct subunits. The complete cDNA sequence of one subunit and a tentative partial sequence of another subunit have been determined. The complete G. roeseli hemocyanin subunit comprises 2,150 bp, which translates in a protein of 672 amino acids with a molecular mass of 76.3 kDa. Phylogenetic analyses show that, in contrast to previous assumptions, the amphipod hemocyanins do not belong to the α-type of crustacean hemocyanin subunits. Rather, amphipod hemocyanins split from the clade leading to α and γ-subunits most likely at the time of separation of peracarid and eucarid Crustacea about 300 million years ago. Molecular clock analyses further suggest that the divergence of β-type subunits and other crustacean hemocyanins occurred around 315 million years ago (MYA) in the malacostracan stemline, while α- and γ-type subunits separated 258 MYA, and pseudohemocyanins and γ-subunits 210 million years ago.     

Hominid and gelada baboon evolution: Agreement between molecular and fossil time scales

The time of origin of the hominid lineage has long been debated. Macromolecular studies have consistently shown genetic distances between living humans and African apes to be quite small. The molecular clock hypothesis proposes that the time of separation of these lineages is relatively recent (in the range of 4–8 million years ago) and not 15 million years or more ago as usually suggested. Three independent molecular comparisons yield a mean estimate of 4.6 million years for the hominid-African pongid divergence. The relationship of Theropithecusand Papiois a parallel case within Primates of two taxa which are quite similar at the molecular level, but which are usually thought to have separated relatively long ago. The two cases of seeming discordance between different lines of evidence are analogous. Each involves a speciation event which eventually resulted in one substantially derived lineage and one or more relatively unchanged lineages. In each case, claims of the antiquity of the divergence event extend to at least twice the age of the first certain appearance of the more derived lineage in the fossil record. Finally, in each case, the molecular clock model suggests a range of possible divergence times that overlaps with the first appearances of undoubted hominids and Theropithecusin the fossil record. This test involving paleontological evidence supports the molecular clock hypothesis.     

Maximum-likelihood models for combined analyses of multiple sequence data

Models of nucleotide substitution were constructed for combined analyses of heterogeneous sequence data (such as those of multiple genes) from the same set of species. The models account for different aspects of the heterogeneity in the evolutionary process of different genes, such as differences in nucleotide frequencies, in substitution rate bias (for example, the transition/transversion rate bias), and in the extent of rate variation across sites. Model parameters were estimated by maximum likelihood and the likelihood ratio test was used to test hypotheses concerning sequence evolution, such as rate constancy among lineages (the assumption of a molecular clock) and proportionality of branch lengths for different genes. The example data from a segment of the mitochondrial genome of six hominoid species (human, common and pygmy chimpanzees, gorilla, orangutan, and siamang) were analyzed. Nucleotides at the three codon positions in the protein-coding regions and from the tRNA-coding regions were considered heterogeneous data sets. Statistical tests showed that the amount of evolution in the sequence data reflected in the estimated branch lengths can be explained by the codon-position effect and lineage effect of substitution rates. The assumption of a molecular clock could not be rejected when the data were analyzed separately or when the rate variation among sites was ignored. However, significant differences in substitution rate among lineages were found when the data sets were combined and when the rate variation among sites was accounted for in the models. Under the assumption that the orangutan and African apes diverged 13 million years ago, the combined analysis of the sequence data estimated the times for the human-chimpanzee separation and for the separation of the gorilla as 4.3 and 6.8 million years ago, respectively.     

DNA evidence for morphological and cryptic Cenozoic speciations in the Anaspididae, ‘living fossils’ from the Triassic

The speciation history of Anaspides tasmaniae (Crustacea: Malacostraca) and its close relatives (family Anaspididae) was studied by phylogenetic and molecular clock analyses of mitochondrial DNA sequences. The phylogenetic analyses revealed that the Anaspides morphotype conceals at least three cryptic species belonging to different parts of its range. The occurrence of multiple cryptic phylogenetic species within one morphological type shows that substantial genetic evolution has occurred independently of morphological evolution. Molecular clock dating of the speciation events that generated both the cryptic and the morphological species of Anaspididae indicated continuous speciation within this group since the Palaeocene ~55 million years ago. This relatively constant rate of recent morphological and cryptic speciation within the Anaspididae suggests that the speciation rate in this group does not correlate with its low extinction rate or morphological conservatism.     

Assessing divergence time of Spirulida and Sepiida (Cephalopoda) based on hemocyanin sequences

The phylogenetic position of the mesopelagic decabrachian cephalopod Spirula is still a matter of debate. Since hemocyanin has successfully been used to calibrate a molecular clock for many molluscan species, a molecular clock was calculated based on this gene with special attention to the cephalopod genera Spirula and Sepia. The obtained partial sequence comprising ca., one third (3567 bp) of the complete gene is similar to that of Sepia officinalis. The molecular clock was calibrated using the splits of Gastropoda-Cephalopoda (ca. 550 ± 50 mya) and Heterobranchia-Vetigastropoda (ca. 380 ± 10 mya). The resulting hemocyanin-based molecular clock is stable, and the estimated divergence time of Spirulida and Sepiida, some 150 ± 30 million years ago, can be deemed reliable.     

PHYLOGEOGRAPHY OF THE TRUMPETFISHES (AULOSTOMUS): RING SPECIES COMPLEX ON A GLOBAL SCALE

Abstract.— The distribution of circumtropical marine species is limited by continental boundaries, cold temperate conditions, and oceanic expanses, but some of these barriers are permeable over evolutionary time scales. Sister taxa that evolved in separate ocean basins can come back into contact, and the consequences of this renewed sympatry may be a key to understanding evolutionary processes in marine organisms. The circumtropical trumpetfishes (Aulostomus) include a West Atlantic species (A. maculatus), an Indian‐Pacific species (A. chinensis), and an East Atlantic species (A. strigosus) that may be the product of a recent invasion from the Indian Ocean. To resolve patterns of divergence and speciation, we surveyed 480 bp of mitochondrial DNA cytochrome b in 196 individuals from 16 locations. Based on a conventional molecular clock of 2% sequence divergence per million years, the deepest partitions in a neighbor‐joining tree (d= 0.063‐0.082) are consistent with separation of West Atlantic and Indian‐Pacific species by the Isthmus of Panama, 3–4 million years ago. By the same criteria, trumpetfish in the East Atlantic were isolated from the Indian Ocean about 2.5 million years ago (d= 0.044‐0.054), coincident with the advent of glacial cycles and cold‐water upwelling around South Africa. Continental barriers between tropical oceans have only rarely been surmounted by trumpetfishes, but oceanic barriers do not appear to be substantial, as indicated by weak population partitioning (ø_ST= 0.093) in A. chinensis across the Indian and Pacific Oceans. Finally, morphological and mitochondrial DNA data indicate hybridization of A. strigosus and A. maculatus in Brazil. After 3–4 million years and a globe‐spanning series of vicariant and dispersal events, trumpetfish lineages have come back into contact in the southwest Atlantic and appear to be merging. This ring species phenomenon may occur in a broad array of marine organisms, with clear implications for the production and maintenance of biodiversity in marine ecosystems.     

Tempo and Mode of Spliceosomal Intron Evolution in Actin of Foraminifera

Spliceosomal introns are present in almost all eukaryotic genes, yet little is known about their origin and turnover in the majority of eukaryotic phyla. There is no agreement whether most introns are ancestral and have been lost in some lineage or have been gained recently. We addressed this question by analyzing the spatial and temporal distribution of introns in actins of foraminifera, a group of testate protists whose exceptionally rich fossil record permits the calibration of molecular phylogenies to date intron origins. We identified 24 introns dispersed along the sequence of two foraminiferan actin paralogues and actin deviating proteins, an unconventional type of fast-evolving actin found in some foraminifera. Comparison of intron positions indicates that 20 of 24 introns are specific to foraminifera. Four introns shared between foraminifera and other eukaryotes were interpreted as parallel gains because they have been found only in single species belonging to phylogenetically distinctive lineages. Moreover, additional recent intron gain due to the transfer between the actin paralogues was observed in two cultured species. Based on a relaxed molecular clock timescale, we conclude that intron gains in actin took place throughout the evolution of foraminifera, with the oldest introns inserted between 550 and 500 million years ago and the youngest ones acquired less than 100 million years ago. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Debashish Bhattacharya]     

Genetic differentiation of Macrognathus siamensis within the Mekong River between Laos and Cambodia

This study investigated the genetic variation in the Mastacembelidae fish Macrognathus siamensis by sequencing 1047 bp of the mitochondrial ND2 gene of 48 fish samples collected in the Mekong River. They were divided into 15 mtDNA haplotypes. The haplotype network and Bayesian tree revealed striking genetic differences between the Laos and Cambodian samples. The high pairwise F statistic values (0.93–0.97) also confirmed this genetic differentiation. Analysis of the molecular clock indicated that these two samples separated approximately four million years ago. Most likely, the genetic variation was influenced by immigration and isolation events as well as by environmental factors. Furthermore, the present study results could be valuable in identifying the conservation and resource management units of this species.     

The Phylogenetic Informativeness of Nucleotide and Amino Acid Sequences for Reconstructing the Vertebrate Tree

To aid in future efforts to accurately reconstruct the vertebrate tree, a quantitative measure of phylogenetic informativeness was applied to nucleotide and amino acid sequences for a set of 11 genes. We identified orthologues and assembled published fossil-calibrated divergence times between taxa that had been sequenced for each gene. Rates of molecular evolution for each site were estimated to characterize the molecular evolutionary pattern of genes and to calculate the phylogenetic informativeness. The fast-evolving gene albumin yielded the highest informativeness over the period from 60 million years ago to 500 million years ago. In contrast, calmodulin yielded the lowest informativeness, presumably because functional constraint minimized substitutions in the amino acid sequence. The gene c-myc showed an intermediate level of informativeness. The nucleotide sequence of cytochrome b showed extremely high utility for recent epochs, but low utility for times before 100 million years ago. We ranked nine other genes for their utility during the epochs of the divergence of the muroid rodents, early placental mammals, early vertebrates, and early metazoa, yielding results consistent with, but more precise than, previous studies. Interestingly, DNA sequence always exceeded amino acid sequence in informativeness over all time scales, yet support values were at best moderately higher. For epochs not subject to strong phylogenetic conflict due to convergence, we advocate gleaning the additional power of the threefold increase in number of characters that is present for DNA sequences over resorting to the less noisy but less informative amino acid sequences.     

Phylogenetic relationships within the New Zealand frog genus Leiopelma — immunological evidence

Abstract

Albumin evolution is examined within the anuran genus Leiopelma using the quantitative immunological technique of micro-complement fixation (MC'F). The albumins of L. archeyi and L. hamiltoni are relatively similar to each other, yet both differ greatly from the albumin of L. hochstetteri. Using albumin as a molecular clock, we estimate that L. hochstetteri diverged from the lineage leading to L. archeyi and L. hamiltoni during the Miocene, about 15 million years ago. The divergence of L. archeyi and L. hamiltoni appears to have been a Pliocene event (about 3 million years ago).     

The Central American Isthmus: Implications for Intraspecific Phylogeny and Biogeography of a Pantropical Green Alga

An intraspecific phylogenetic study was undertaken to resolve the evolutionary relationship of isolates of the green alga Phyllodictyon anastomosans (Harv.) Kraft et Wynne that occur on Atlantic and Pacific coasts of the Central American Isthmus. Patterns of vicariance related to the emergence of the Central American Isthmus were evident, but numerous examples of recent trans‐oceanic and trans‐isthmian dispersal obscured the underlying pattern. This study, one of the first studies to assess the impact of the emergence of the Central American Isthmus in seaweed phylogeography, provided an ideal opportunity to estimate the rate of sequence evolution using a single time point. Using this newly calibrated molecular clock the timing of an historical introduction across the isthmus is shown to be concordant with a shallow water breach of the isthmus dated at approximately 2.3–2.0 million years ago. This work contributes to a growing body of literature that suggests marine algae are fairly successful at dispersing over long distances in recent times.     

Evolution of Phosphagen Kinase VII. Isolation of Glycocyamine Kinase from the Polychaete Neanthes diversicolor and the cDNA-Derived Amino Acid Sequences of α and β Chains

Glycocyamine kinase (GK) was isolated from the marine polychaete Neanthes diversicolor by gel filtration, DEAE-cellulose chromatography, butyl-Toyopearl hydrophobic chromatography, and chromatofocusing. The GK was eluted as a single peak on the latter three chromatographies, and the molecular mass for the native GK was estimated to be about 80 kDa. The SDS–PAGE showed that the isolated GK consists of two distinct subunits in equal proportion, α and β chains, with molecular masses of 42.2 and 43.8 kDa, respectively. The present results suggest that the Neanthes GK has a heterodimeric structure. The cDNAs for α and β chains of Neanthes GK were amplified by PCR and their cDNA-derived amino acid sequences were determined. The α and β chains are composed of 374 and 390 amino acids, and the molecular masses were calculated to be 42,392 and 43,966 Da, respectively, in good agreement with the apparent masses on SDS–PAGE. The β chain has a characteristic N-terminal extension of 15 amino acids, and all of the sequence differences between α and β chains were restricted in the N-terminal region of 50 residues. The overall sequence identity was 92%. The occurrence of heterodimeric nature in Neanthes GK is of great interest from the evolutionary point of view, because the heterodimeric structure is only known for creatine kinase MB-isozyme specific for mammalian heart muscle among phosphagen kinases.     

Hemoglobins of baboons, Papio cynocephalus, P. gelada and P. hamadryas

Major hemoglobins of adult Papio cynocephalus, P. gelada, and P. hamadryas and of newborn P. cynocephalus were purified; globins were prepared; and α and β or α and γ chains were separated. The amino acid compositions and aminoterminal groups were determined. These were compared with analogous data for human hemoglobins, other baboon hemoglobins and macaque hemoglobins.     

Molecular evolution and phylogeny of theDrosophila virilis species group as inferred by two-dimensional electrophoresis

Summary Systematic relationship among the 12 species of theDrosophila virilis species group, andDrosophila robusta, were investigated by the use of two-dimensional electrophoresis (2-DE). A total of 389 protein characters (about 200 loci) were scored and analyzed both phylogenetically and phenetically. The resulting phylogeny was found to be largely concordant with the current views of evolution among these species based on other independent morphological, chromosomal, electrophoretic, and immunological data sets, although some notable differences were observed. The 2-DE data also appeared to be useful for constructing a molecular clock to date the absolute times of divergence among the species. It appears from this analysis that the evolution of the major clades within the species group occurred about 20 million years ago. Previous suggestions that the rate of molecular evolution was different between the virilis and montana phylads was not confirmed. The technique of 2-DE seems to be an excellent tool for reconstructing phylogenies and should be particularly valuable for examining relatively closely related species.     

The age of the angiosperms: a molecular timescale without a clock

The age of the angiosperms has long been of interest to botanists and evolutionary biologists. Many early efforts to date the age of the angiosperms and evolutionary divergences within the angiosperm clade using a molecular clock have yielded age estimates that are grossly inconsistent with the fossil record. We investigated the age of angiosperms using Bayesian relaxed clock (BRC) and penalized likelihood (PL) approaches. Both of these methods allow the incorporation of multiple fossil constraints into the optimization procedure. The BRC method allows a range of values for among-lineage rate of substitution, from a nearly clocklike behavior to a condition in which each branch is allowed an optimal substitution rate, and also accounts for variation in molecular evolution across multiple genes. A topology derived from an analysis of genes from all three plant genomes for 71 taxa was used as a backbone. The effects on age estimates of different genes, single-gene versus concatenated datasets, and the inclusion and assumptions of fossils as age constraints were examined. In addition, the influence of prior distributions on estimates of divergence times was also explored. These results indicate that widely divergent age estimates can result from the different methods (198-139 million years ago), different sources of data (275-122 million years ago), and the inclusion of temporal constraints to topologies. Most dates, however, are between 180-140 million years ago, suggesting a Middle Jurassic-Early Cretaceous origin of flowering plants, predating the oldest unequivocal fossil angiosperms by about 45-5 million years. Nonetheless, these dates are consistent with other recent studies that have used methods that relax the assumption of a strict molecular clock and also agree with the hypothesis that the angiosperms may be somewhat older than the fossil record indicates.     

Embryonic β-globin in the non-Antarctic notothenioid fish Cottoperca gobio (Bovichtidae)

Haemoglobins are sensitive to temperature and their properties mirror the thermal conditions encountered by species during their evolutionary histories. This paper provides data on molecular phylogeny of the haemoglobin chains of Cottoperca gobio, a notothenioid fish of sub-Antarctic latitudes, belonging to the basal family Bovichtidae. Unlike most Antarctic notothenioids, C. gobio has two major haemoglobins sharing the β chain. In the molecular phylogenetic analysis, the β chain is included in the clade of the “embryonic” or minor Antarctic globins. Although, in the majority of notothenioids, “embryonic” (minor) α and β globins are expressed in traces or small amounts in the adult stage, in C. gobio the present analysis supports the occurrence of a complete “switch” to exclusive expression of the embryonic β-globin gene in adult fish. The α and β chains sequences have been used to expand our knowledge of the evolution of notothenioid haemoglobins.The protein sequence data reported in this paper will appear in the UniProt Knowledge base under the accession number: P84652 (β chain), P84653 (α ¹ chain).     

Molecular Clock Calibrations and Metazoan Divergence Dates

It has recently been argued that living metazoans diverged over 800 million years ago, based on evidence from 22 nuclear genes for such a deep divergence between vertebrates and arthropods (Gu 1998). Two ``internal' calibration points were used. However, only one fossil divergence date (the mammal–bird split) was directly used to calibrate the molecular clock. The second calibration point (the primate–rodent split) was based on molecular estimates that were ultimately also calibrated by the same mammal–bird split. However, the first tetrapods that can be assigned with confidence to either the mammal (synapsid) lineage or the bird (diapsid) lineage are approximately 288 million years old, while the first mammals that can be assigned with confidence to either the primate or the rodent lineages are 65 million years old, or 85 million years old if ferungulates are part of the primate lineage and zhelestids are accepted as ferungulate relatives. Recalibration of the protein data using these fossil dates indicates that metazoans diverged between 791 and 528 million years ago, a result broadly consistent with the palaeontological documentation of the ``Cambrian explosion.' The third, ``external' calibration point (the metazoan–fungal divergence) was similarly problematic, since it was based on a controversial molecular study (which in turn used fossil dates including the mammal–bird split); direct use of fossils for this calibration point gives the absurd dating of 455 million years for metazoan divergences. Similar calibration problems affect another recent study (Wang et al. 1999), which proposes divergences for metazoans of 1000 million years or more: recalibrations of their clock again yields much more recent dates, some consistent with a ``Cambrian explosion' scenario. Molecular clock studies have persuasively argued for the imperfection of the fossil record but have rarely acknowledged that their inferences are also directly based on this same record. Received: 26 January 1999 / Accepted: 14 April 1999     

Highly conserved D-loop-like nuclear mitochondrial sequences (Numts) in tiger (Panthera tigris)

Using oligonucleotide primers designed to match hypervariable segments I (HVS-1) ofPanthera tigris mitochondrial DNA (mtDNA), we amplified two different PCR products (500 bp and 287 bp) in the tiger (Panthera tigris), but got only one PCR product (287 bp) in the leopard (Panthera pardus). Sequence analyses indicated that the sequence of 287 bp was a D-loop-like nuclear mitochondrial sequence (Numts), indicating a nuclear transfer that occurred approximately 4.8–17 million years ago in the tiger and 4.6–16 million years ago in the leopard. Although the mtDNA D-loop sequence has a rapid rate of evolution, the 287-bp Numts are highly conserved; they are nearly identical in tiger subspecies and only 1.742% different between tiger and leopard. Thus, such sequences represent molecular ‘fossils’ that can shed light on evolution of the mitochondrial genome and may be the most appropriate outgroup for phylogenetic analysis. This is also proved by comparing the phylogenetic trees reconstructed using the D-loop sequence of snow leopard and the 287-bp Numts as outgroup.     

Determination of the primary sequence of the duck αD globin mRNA and comparison of all adult duck and chick globin mRNA sequences

The nucleotide sequence of the duck α^D globin mRNA was determined. Its main feature is an exceptionally short 3′ non-coding segment of only 46 nucleotides, placed after the coding sequence of 141 codons. The last of the 6 adult globin mRNA of duck and chicken being thus sequenced, a comparison of all their features has become possible. Comparing the duck α^D mRNA to the related sequence in the chicken, we found greater homology than comparing it to the linked α^A globin sequence in the same species. Extensive homology can be found for a same globin chain α^A, α^D or β in between different avian species including also the goose and the ostrich; the avian α globin chains show a lower degree of sequence conservation in between species than the β chains. In contrast, within one species the three globin sequences have further diverged. The divergence between the α^A and α^D globin within a same species point to individual functional specificity and hence independent evolution and suggest that a mechanism of ‘gene conversion’ did not operate in between the avian α globin genes. Two segments of the amino acid sequence which we named ‘A^α’ and ‘B^α’ remain homologous in all avian α globins; two other regions ‘A^β’ and ‘B^β’ are identical in between the β globins. Segment A is placed at the 5′ end of exon II, and segment B at the 3′ end of the same exon; some amino acids in those segments are involved in the Heme binding site. Being almost identical in all know mammalian and avian globins of the α respectively the β type, regions A and B seem to represent the best conserved sequences in adult globin mRNA maintained during the divergence of species.